Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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Revista de los másteres de Biotecnología de la UPOes-ESBiosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria2254-3821A protocol for expressing plants' chloride transporters into yeast cells.
https://www.upo.es/revistas/index.php/biosaia/article/view/8379
<p>In the plant kingdom, chloride (Cl-) was defined as an essential micronutrient and, according to the results recently reported from our research Group, it has been also described as a beneficial macronutrient (1,2). Due to the similarity of their physicochemical properties Cl- and nitrate (NO3-) share membrane transport mechanisms and present strong interactions in plant cells. For example, a reduction of nitrate transport in Arabidopsis thaliana mutants gives rise to increased root chloride uptake. We propose that these phenomena respond to a compensatory mechanism aimed to regulate chloride homeostasis, possibly optimizing nitrogen use efficiency in plants under low NO3- availability (1,2). Presently, a single Cl- uptake transporter has been described in plants: AtNPF6.3 and the corresponding orthologous from other spp (3). In addition, the molecular mechanisms that regulate Cl- nutrition in plants remain unknown. Furthermore, functional characterization of plant Cl- transporters is a difficult task since: (i) it requires complex electrophysiological procedures in both plants and heterologous (e.g. Xenopus laevi oocyte) systems; (ii) commonly, plant knockout mutations exhibit unclear phenotypes.<br>In order to easily detect and quantify the activity of Cl- transporters and their regulatory partners, we will take advantage of the model microorganism Saccharomyces cerevisiae, which has very low Cl- transport ability in the 1.0 - 10 mM range (4). We intend to obtain yeast lines expressing recombinant probes sensitive to Cl- and to pH, respectively. As a proof-of-concept, genes encoding the A. thaliana AtNPF6.3 (NO3-selective) and the Medicago truncatula MtNPF6.5 (Cl-selective) transporters, will be expressed in these yeast lines under the control of an inducible promoter when cloned in the Gateway® pYES-DEST52 vector. Using the Gateway clonase II system these transporters as well as the recombinant Cl- and pH-sensitive fluorescent probes will be co-expressed on yeast cells.<br>Once the system is set up, fluorescence assays and microelectrode analysis will be carried out to characterize these and other candidate Cl- transporters. In the near future this protocol will allow also a fast screening of Cl- transporters after yeast transformation with a plant cDNA library and selection through fluorescence-activated cell sorting (FACS) using a flow cytometry.</p>Adrián Perera-BonañoJosé Manuel Colmenero-Flores
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Optimization of brain organoids as models for the study of neurodegenerative diseases
https://www.upo.es/revistas/index.php/biosaia/article/view/9568
<p>ABSTRACT<br>Motivation: One of the current challenges faced by neuroscience is the limited availability of in vitro models of neurodegenerative diseases, as there are notable anatomical and molecular differences among murine and human brain. As a result, significant efforts have been made towards the development of new models based on human cells, with cerebral organoids standing out as a particularly promising approach. Brain organoids are 3D models usually developed from induced pluripotent stem cells (iPSCs) that simulate the composition and cytoarchitecture of different regions of the human brain. They may allow us to obtain in vitro information about the human brain, which makes them a valuable model for investigating neurodegenerative diseases. Nevertheless, they present disadvantages associated with the absence of essential components for their development and functionality. Therefore, we will investigate the effect of incorporating an extracellular matrix (ECM) from human and pig brain into the culture of these organoids, as it contains specific combinations of components that play a role in multiple neuronal processes. On the other hand, in order to find the optimal model for generating this organoids, two protocols, Lancaster (1) and Rosebrock (2), have been compared. Lancaster’s protocol is the most cited for brain organoids, but following it, other embryonic layers are developed. Rosebrock’s protocol, is a modification of the Lancaster’s protocol, in which SMAD pathway inhibitors are used to avoid the formation of non-ectodermal layers.<br>Methods: We generated brain organoids from iPSC, following two protocols: Lancaster (1) and Rosebrock (2). Two ECM conditions were used during cultures: Matrigel versus ECM obtained from pig brain. Subsequently, the addition of human ECM will be tested. Finally, organoids are being characterized using different techniques such as immunofluorescence, RT-PCR and expression arrays.<br>Results: We have found molecular differences among brain organoids obtained following the different protocols, as those obtained following Rosebrock’s protocol express fewer endodermal and mesodermal markers than those obtained with Lancaster's protocol. As well, we have identified different features between those organoids matured with ECM and those matured only with Matrigel, the former being larger than the latter. Further studies will discern whether there are other relevant differences between the models.</p>Celia Inmaculada Atalaya Rey, Beatriz Fernandez MuñozGarcía Delgado Ana Belén
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2024-01-292024-01-2912Involvement of sensor histidine kinases in general stress response of Sphingopyxis granuli strain TFA.
https://www.upo.es/revistas/index.php/biosaia/article/view/8403
<p>Motivation: In their natural habitat, bacteria face constantly changing conditions that can portraiy a challenge to their survival. To overcome this problem, bacteria are able to react to their medium and adapt accordingly to it. One of these responses is the so-called General Stress Response (GSR), an unspecific response to a myriad of different stress signals.<br>Sphingopyxis granuli strain TFA is an alphaproteobacteria with the ability to use tetralin, an organic solvent with industrial applications, as a carbon and energy source. It is also the first facultative anaerobe described within its genus. These characteristics made TFA an interesting strain from a biotechnological point of view.<br>In alphaproteobacterial, the regulatory network of the GSR is composed at its most basic level by a sigma factor (EcfG), an anti-sigma factor (NepR), an anti-anti-sigma factor (PhyR) and a sensor histidine kinase that targets the anti-anti-sigma factor. In TFA, EcfG, NepR and PhyR are duplicated and a total of four putative sensor histidine kinases have been found within TFA genome. Previous works by de Dios et al have partially described the regulatory network of the GSR at to the point of the histidine kinases. Therefore, in this work we aim to unravel role that two of these histidine kinases, SGRAN_1655 and SGRAN_2544, have in the regulation of the GSR in TFA.<br>Methods: The experimental approach for this work consist in the obtention of deletion mutants in the SGRAN_2544 and SGRAN_1165 by the method described by Martínez-García and de Lorenzo. Once the mutants were obtained, We examined their response to several stresses, like high sodium chloride concentration, high heavy metals concentration, hydrogen peroxide treatment and desiccation, by both checking the effect on their growth and quantifying the activity of lacZ fusions to a GSR reporter gene, such as nepR2.<br>Results: We have been able to succesfully obtain a deletion mutant of one of the two target genes, SGRAN_2544 gene.</p> Alberto Pires AcostaInmaculada García Romero Francisca Reyes Ramírez
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Optimization of a new protocol that allows speeding up the process of screening genes and candidate drugs for Spinal Muscular Atrophy in C. elegans.
https://www.upo.es/revistas/index.php/biosaia/article/view/8401
<p>Spinal Muscular Atrophy (SMA) is a rare genetic disease that affects 1 in 8,000 people. It is caused by a recessive mutation in the SMN1 gene, which produces the SMN protein and is highly conserved in invertebrates [1]. In humans, the SMN2 gene is also found, which differs by 5 nucleotides from SMN1, which means that only 10% of the translated proteins are complete [2]. For the study of SMA, a strain of C. elegans, developed by our group, is used, which presents the smn1 gene fused to the mCherry fluorescence marker, in such a way that it allows measuring the expression of SMN in response to different candidates (drugs and RNAi ) through fluorescence. These candidates have been chosen by using the ASACO bioinfomatic tool, which makes it possible to generate an expression profile opposite to SMN1. However, normally, to measure fluorescence, a confocal microscope is used, which slows down this procedure considerably and makes it difficult to carry out large screenings of candidates. For this reason, in this work a new method has been sought to measure fluorescence in a faster way and that, like the confocal method, gives reliable results. For this, tests have been carried out with two pieces of equipment: the Apotome microscope and a fluorimeter, and candidates were used, both drugs and RNAi, which had been confirmed by confocal microscopy as capable of increasing SMN levels. In this way, tests were carried out with both teams to see which of the two provides results of fluorescence levels more similar to the confocal one. The results show the fluorimeter as the most appropriate equipment for this task, since, in addition to presenting very similar results to the confocal microscope, it is a faster method that allows for large scrutinies of candidates. In the future, using the ASACO tool, new candidate genes and drugs will be selected and fluorescence levels will be measured using this new method.</p>Rafael De la Torre RomeroAndrés GarzónManuel Muñoz Ruiz Antonio J. Pérez PulidoAna María Brokate-Llanos
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Analisys of lactose-free samples by chromatographic methods.
https://www.upo.es/revistas/index.php/biosaia/article/view/8370
<p>Currently, lactose-free or lactose-free products are gaining importance in the daily consumption habits of many lactose intolerant people. Approximately 70% of the adult population in the world suffers from lactose intolerance due to the absence of lactase, which is caused by the absence of lactase in some people. o [1] For this reason, the food information provided to consumers regarding the use of some ingredients that can cause allergies or intolerances, such as lactose, must be adequate so that they can make the most appropriate choice for their consumption. The Ministry of Health differentiates between lactose-free and low-lactose products, the difference lying in the amount of measurable residual lactose. Thus, when the amount of this lactose is less than 1%, it is considered a low-lactose product and when it is less than 0.01%, it is considered a lactose-free product [2]. To ensure these detection levels, testing laboratories must use sufficiently sensitive and accurate methods. Conventionally, detection of lactose in milk and milk products has been carried out using different methods such as gravimetry, polarimetry, enzymatic methods or methods based on high performance liquid chromatography with refractive index detector (HPLC-IR, High Performance Liquid Chromatography). However, when high sensitivity and precision are required, other analytical techniques such as Ultra High Performance Liquid Chromatography (UHPLC- MS/MS) or High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD), which is also being used for the estimation of residual lactose with very high sensitivity and selectivity, should be used [3]. Therefore, in this Master Thesis we propose to evaluate the amount of lactose in different dairy products (cheese and milk) by HPLC-MS/MS depending on the type of sample, its fat content or its state at room temperature (liquid or solid). Initial results show high recoveries for all the tests performed.</p>David Guerrero CollanteMaria del Mar González PérezMenta Ballesteros
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Neuroprotective strategies against neonatal hypoxia-ischemia.
https://www.upo.es/revistas/index.php/biosaia/article/view/8397
<p>Motivation: Neonatal hypoxia ischemia (HI) is a brain damage caused by oxygen deprivation in newborns. Nowadays, it continues to be a major cause of neonatal mortality and lifelong neurodevelopmental disabilities worldwide.<br>Neonatal HI physiopathology includes oxidative stress, inflammation, and apoptosis. Some studies have shown that nutraceuticals that have antioxidant, anti-inflammatory or anti-apoptotic properties can prevent neonatal HI by reducing brain damage. Thus, the aim of this work is to evaluate the neuroprotective potential of a plant-derived phenolic compound (PDPC) administered as a pre-treatment before the HI event, with focus on myelination and astroglia activation.<br>Methods: Our group used the Rice-Vannucci mouse model of neonatal HI by ligating the left common carotid artery and then subjecting 7-day-old pups to hypoxia for 90 min. Pups were administered PDPC (at 20 or 100 mg/kg) or saline intraperitoneally 20 min before the intervention. Two days later, the brains were dissected, homogenized and stored at -80ºC for future experiments to evaluate the antioxidant potential of the PDPC. In a parallel study, seven days later, the brains were dissected to study myelination and astroglia activation by immunohistochemistry using MBP (myelin basic protein) and GFAP (glial fibrillary acidic protein) to assess the neuroprotective potential of PDPC.<br>Results: Preliminary results indicate a loss of myelination in the ipsilateral hemispheres (where the carotid artery was ligated) of mice subjected to HI, but this loss seemed to be significantly reduced in a dose dependent manner in those mice pretreated with the PDPC. In the case of astroglia staining, experiments are still ongoing but these suggest an overexpression of astroglia in certain areas of the affected hemisphere of the brain in HI mice, such as the cortex or thalamus. We are currently stablishing an image analysis protocol to evaluate the overexpression of GFAP+ cells and observe if it lowers in the treated versus the non-treated group.<br>Conclusions: To conclude, we can highlight that ongoing experiments suggest that pretreatment with PDPC, specially at 100 mg/kg, preserves myelination and may reduce astroglial activation. Future experiments will also evaluate the antioxidant potential of PDPC pretreatment by measuring the ROS production and the activity of antioxidant enzymes in brain homogenates.</p> Laura Gil GonzálezMarta Reyes CorralJavier Tovar Luzón Patricia Ybot González
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Study of water quality through microbiological and physico-chemical analysis of the Guadaira river.
https://www.upo.es/revistas/index.php/biosaia/article/view/8366
<p>Water is one of the most abundant and used resources in the world since ancient times, necessary for life on Earth (Díaz Peña, D. 2019). Its main purpose is to cover the basic needs of all living beings, and it is sometimes thought that this resource is unlimited, but it must be remembered that we are dealing with a limited and valuable resource that must be managed efficiently and prudently (Pardo, C.F. 2004). The problem of water and wastewater management is a global problem that has been around for a long time. (Satyendra, et al. 2023). The problem of water and wastewater management is a long-standing global problem, and with population growth and human modifications causing pollution of natural waters, water quality is getting worse and worse (Prato-Moreno, J.G., et al. 2020). The Guadaira River is a local river that runs through important towns in the province of Seville, such as Morón, Alcalá de Guadaira and Seville itself. The high number of inhabitants in these towns means that the wastewater treatment plants are sometimes very stressed and their sewage treatment capacity is limited. We have performed microbiological and physicochemical analyses at two critical locations in the Guadaira river to study water quality. The first sampling point is upstream of the treatment bridge at the discharge of the Dos Hermanas ETAP and the other downstream in the Arroyo Culebras in Bellavista. Samples collected in plastic bottles with thiosulphate were plated on different culture media depending on the microorganism to be studied. In addition, physicochemical analyses were carried out on samples collected in unsterilised plastic jars for further analysis in the laboratory equipment. The microbiological analysis was carried out in accordance with Royal Decree 817/2015, and showed high levels of contamination, both from a microbiological point of view and in terms of ammonium and phosphate levels in the water. The study concludes that the river Guadaíra is polluted but within the limits of the legislation, so that the problem of pollution of the river could be solved by means of a filtration mesh at the water outlet of the ETAP or by avoiding the dumping of solid urban waste into the river. We can include that the sampling and analysis of the waters of the Guadaíra river can be carried out again after the rainy season, in this way, we check if the level of contamination increases or decreases.</p>José Antonio Gallardo RodríguezInmaculada Otero CifuentesInés Canosa Perez-Fragero
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Evaluation of the looptail mutation efects on neural tube closure in early mouse embryo.
https://www.upo.es/revistas/index.php/biosaia/article/view/8415
<p>Motivation: Neural tube defects (NTDs), such as spina bifida, are defined as congenital malformations that impair proper neural tube closure in early embryonic development. There are deeply complex mechanisms behind NTDs, of which we can stand out the role of the Wnt/PCP pathway, that through cytoskeleton reorganization and cell-cell interactions drives tissue rearrangements which finally lead to neural tube closure. In this project, cellular morphology and dynamics in node cells, an essential organizer in early development, have been studied in both, its dorsal and ventral regions, to determine how affected it is in Wnt/PCP-mutant mouse embryos . It is the great importance of NTDs in current population what has motivated this study as it has been estimated that NTDs prevalence varies between 0.5-0.8/1000 births in most European countries and the USA, and it is even 20 times higher in countries such as China. Taking into account this data, it seems to exist a clear need to investigate the physiopathology of these malformations and initiate the run for further solutions.<br>Methods: In pursuit of valuable information, the looptail (Lp) mouse strain, carrying a mutation in one of the WNT/PCP pathway core proteins, Vangl2, has been selected as it is a great NTDs model. Whole-mount inmunohistochemistry against Zonulla Occludens-1 (ZO-1), a tight junction protein, as well as phalloidin staining, have been performed on mice embryos. Afterwards, z-images have been obtained through scannig laser confocal microscopy. Finally, segmentation has been done using the python software "SeedWaterSegmenter" and the "DVRosettes" and "CellRois" macros, both from Fiji/ImageJ.<br>Results: The aforementioned macros offer personalized morphological information for each cell and its interaction with neighbouring cells according to the pre-selected parameters: apical area, apical elongation, cell apical orientation and vertex order (number of cells sharing a vertex). In the previous months, this group has been working with CD1 mouse embryos in order to optimize the complex methodology and results concerning looptail embryos are expected to be available soon.<br>Conclusions: Morphological parameters will be crucial as we can estimate how apical constriction and cell deviation from the midline axis are affected in Lp embryos. As well as cell dynamics parameters, especially rosettes formation (5-cell unions), which play a key role in driving coordinated cell movements.</p>Alejandro Meléndez Araujo Patricia Ybot González
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Achievement of high-added value products from microalgae cultures in sludge-derived process water.
https://www.upo.es/revistas/index.php/biosaia/article/view/8360
<p>Motivation<br>Due to the increasing production of wastewater and leachate, the resulting environmental problems, such as the eutrophication of aquatic ecosystems where these pollutants may end up, are also increasing. The problems related to wastewater and leachate are caused by the high concentration of nutrients they contain, especially nitrogen and phosphorus, but also heavy metals and pesticides (Hernández-García et al., 2019).<br>Consequently, research is taking into consideration alternative solutions based on living organisms. Microalgae are gaining importance in the field of bioremediation, and there is a lot of previous research on the use of these organisms for their ability to remove nutrients and pollutants from the environment (Roccuzzo et al., 2016; Ye et al., 2019).<br>The company G2G Algae Solutions works with microalgae and they produce different products based on these organisms. It is interesting to look for an environmental solution that is also economically useful: we try to use wastewater and leachate to grow microalgae and, at the same time, transform these liquids into less polluted products, instead of using tap water.<br>Materials and Methods<br>Wastewater from an experimental process, in which the invasive macroalgae Rugulopterix okamurae is being utilized to make bricks, was used at different dilutions. The microalgae chosen were those belonging to the Trebuoxiophyceae family, added to the wastewater in a 1:2 ratio. The experiment was carried out with and without fertilizer for two weeks.<br>In another experiment, the conditions of the previous one were replicated, but leachate was employed instead of wastewater.<br>Every two days, some physicochemical properties were sampled: pH (using a pH meter), salinity (using a refractometer), cell density (using the Neubauer chamber), and chlorophylls a and b (using a spectrophotometer and the equations from Wellburn, 1994).<br>Results<br>Further experiments are needed in order to analyze the results, but the 75% dilution seems to be the best to achieve the desired results in terms of biomass production.<br>Conclusions:<br>Due to the high level of dilution (75%) of polluted water needed to reach the standard biomass production under the conditions studied, the economic viability of the project is in question. However, we are performing additional experiments to determine the bioremediation potential of Trebuoxiophyceae.</p>Paloma Cabezas BlancoAgustín González-FontesManuel Antonio González del Valle
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Immune dysregulation in children with Down Syndrome and Janus Kinase inhibition as targeted therapy.
https://www.upo.es/revistas/index.php/biosaia/article/view/8413
<p>Down Syndrome (DS), also known as Trisomy 21, is a genetic disorder that results from partial or complete third copy of chromosome 21. It is one of the most common genetic disorders worldwide affecting 6 million individuals. It is the leading cause of lifelong intellectual disability and is often associated with other clinical alterations [1], including cardiac and gastrointestinal anatomic defects, an increased incidence of Alzheimer's disease as well as a heightened vulnerability to infections.<br>DS has shown to be associated with inflammatory and autoimmune processes such as periodontitis, alopecia, dermatitis among others [2]. The fact that 4/6 Interferon (IFN) receptors are localized on chromosome 21 is currently an appealing hypotheses to explain the observed IFN hypersensitivity in DS population. A subset of DS individuals shares clinical manifestations with patients with Signal-Transducer and Activator of Transcription 1 gain-of-function (STAT1 GOF) patients, who also present IFN hypersensitivity and increased levels of STAT1 and its phosphorylated form (pSTAT1) [3]. Janus Kinase (JAK) inhibition has been a successful treatment for STAT1 GOF patients. However, its potential and effectiveness in Down Syndrome has not been explored yet. We here propose Baricitinib as a targeted therapeutic approach for a selected subgroup of DS patients with IFN hypersensitivity and clinical manifestations similar to the ones seen in patients with STAT1 GOF. Whole blood (WB) obtained from STAT1 GOF patients, DS individuals and healthy controls (HC) was stimulated with IFN αlpha/gamma in the presence of Baricitinib. Flow cytometry was performed using specific antibodies against CD14 (monocytes), CD3, STAT1 and pSTAT1. In parallel, PBMCs were stimulated with IFN alpha/gamma , and transcription levels of STAT1, CxCL10, SOCS1 and PD-L1 were evaluated by RT-PCR. According to previous studies [3], we found in DS individuals an increased gene dosage of IFNaR1 and IFNaR2. Furthermore, similar to STAT1 GOF patients some DS individuals present increased levels of STAT1 and pSTAT1 after stimulation compared to HC in CD14 and CD3 T cells. We also observed that ex vivo JAK inhibition was effective reducing this IFN hypersensitivity in DS cells similar to STAT1 GOF patients’ cells. Taken together, Baricitinib a JAK1/2 inhibitor might represent a promising targeted therapy for a subgroup of DS with inflammatory and/or infectious manifestations.</p>Paula Gilabert Prieto Pilar Blanco Lobo Olaf Olaf
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Food microblology analisys. Coagulase-positive Staphylococcus count method verification acording to ISO 16140.
https://www.upo.es/revistas/index.php/biosaia/article/view/8392
<p>Staphylococcus aureus and other coagulase-positive staphylococcus are bacteria with the capacity to produce staphylococcal enterotoxins, that can produce a lot of food poisonings manifested as nausea, vomits and diarroheas. The Spanish Association for Standardization (UNE in Spanish) has published some changes to the specifications that specify methods for coagulase-positive Staphylococcus count [1]. One of these rules, the UNE-EN ISO 6888-1:2021, is the one that has been followed in this Master's Thesis, together with the verification according to ISO 16140 [2].<br>UNE-EN ISO 6888-1:2021 details a horizontal method for counting coagulase-positive staphylococci by counting the colonies obtained on a solid medium (Baird-Parker) after an incubation period between 34 and 38 ºC. After this, the confirmation by coagulase takes place [1]. In this case, the test is carried out on 5 samples of 5 different foods: egg, meat, cheese, cake and cooked food.<br>Colonies suspected of being coagulase-positive staphylococci are black or gray, shiny, and convex and surrounded by a translucent halo that may be partially opaque. After 24 h of incubation, a count of those typical colonies is carried out. And, later, the confirmation is carried out. This is done by incubation of the colony in brain-heart medium and rabbit plasma, in which it is determined positive if a large compact clot is observed [1].<br>Moreover, during my Master's Thesis I have been involved in the analisys of many different food samples as well as water for the detection of different microorganisms like Enterobacer, Salmonella, etc.</p>María Rubio CumplidoVerónica Díaz Simón
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Micropropagación de plantas en biorreactores de inmersión temporal (BIT).
https://www.upo.es/revistas/index.php/biosaia/article/view/8336
<p>Micropropagation is a method to produce genetically identical plantlets (clones or also called microplants) by using tissue culture techniques. The aim of this work is to obtain a large number of potato microplants already rooted, free of pathogens and with genetic uniformity.<br>The optimization of this micropopagation process begins with the sterilization and stabilization of the material to be propagated, in our case potatoes. This process is carried out to eliminate any microbial contamination of the donor or parent plant and to reduce the risk of contamination during in vitro culture. First, several healthy tubers (free of apparent pathogens) will be selected, washed in water for 1 hour, internally disinfected with a solution of sodium hypochlorite and tween 20, and then rinsed with sterile water to remove the disinfectants. After that, the potato buds will be cut and placed in sterile culture medium. The in vitro culture will be carried out under controlled conditions for a period to stabilize plantlets before transplanting them to soil.<br>A fine-tuning process will be performed to optimize the micropropagation process from plantlets obtained from in vitro cultures. This fine-tuning process will consist of three phases:<br>In the first phase, it will be measured which type of cut on the node is the most optimal according to growth performance position of the node in the culture medium shall be checked, whether horizontally or inclined.<br>During the second phase, the yield between two of the most consumed potato varieties in Spain will be compared. During this phase, auxins and cytokinins will be added to the medium to check if there is noticeable improvement in rooting and plant growth [1]. Also, the optimal pH in the medium for potato growth will be analyzed.<br>A temporary immersion system will be carried out in the third phase, as it is an effective tool for micropropagation, because it increases the multiplication coefficient and improves the quality of regenerated material in vitro [2]. Therefore, during this phase, the efficiency of a robotic or automated process of temporary immersion in a bioreactor (BIT) will be compared with the manual process.</p>Francisco López DelgadoAndrés Bermúdez Luque
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Design of a new clean label vegan texturizing ingredient.
https://www.upo.es/revistas/index.php/biosaia/article/view/8389
<p>The general objective of this project is the design and development of a new clean label vegan texturising ingredient, with binding and gelling properties, based on tiger nut derivatives with the capacity to replace gelling/thickening E number additives in meat substitutes. This new intermediate product aims to replace the use of additives (hydrocolloids) in vegan products, giving rise to a clean-label end product, minimizing those additives with little or no nutritional value. The objective is to develop an intermediate product with a specific composition concentrated in starch and fiber, with a malleable structure and rheology, and adequate palatability. To achieve this, the tigernut raw materials will be subjected to the individual and synergic application of physical, chemical and/or enzymatic techniques, for the modification at the level of nutritional concentrations, lipid reduction, amylose/amylopectin ratio and size/morphology of the starch granules. By doing this we aim to favor and maximize the technological capacities of the tiger nut, allowing the functionality of the texturising additives to be emulated.<br>In addition, it must be guaranteed that the ingredient does not add any strange color, taste or odor, in order to maintain the same sensory and textural characteristics in vegan matrices with respect to their meat and/or vegan counterparts with additives. It should be noted that the development of this intermediate product opens up the possibility of taking advantage of those tiger nut peeling by-products of low commercial value originating in the company, giving them a new use of greater benefit. As for the tasks carried out, it is worth highlighting the technical study and characterisation of the tiger nut and its derivatives for the development of the intermediate product, as well as the design and development of the clean label texturising ingredient based on tiger nut derivatives.<br>To date, this project has not been completed yet, so alternatives are still being sought to improve the product through the company's internal research lines. The results obtained have provided sufficient information and data to rule out certain types of treatments and products used, but there is still room for improvement in order to fine-tune the final product to be obtained.</p>Sergio Vela Verdugo
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Characterization of the microbiome of sourdoughs from Andalusian artisan bakers.
https://www.upo.es/revistas/index.php/biosaia/article/view/8334
<p>The elaboration of products based on sourdough is becoming increasingly popular and successful in the market, due to consumer demand for more natural, healthy and good-tasting products. Defining sourdough as a mixture of flour and water, rich in microorganisms dominated by lactic acid bacteria (LAB), yeast and acetic acid bacteria, contributing to the production of organic acids, aromas and helping to improve stability, texture and freshness of the dough(1).<br>The present study intends to characterize the microbiota of this artisan sourdough from one of the most representative bakeries in Andalusia, in addition to analyzing the stability of its composition as a function of time, therefore three samples were subjected to the isolation and identification of lactic acid bacteria (LAB) and acetic bacteria themselves which were differentiated by their band pattern from a REP PCR(2), 16S ribosomal RNA gene DNA was amplified and purified from representatives of each pattern(3).<br>After sequencing the DNA of the candidates, the genera of lactic acid bacteria (LAB) Companilactobacillus and Lactobacillus dominate the sourdough population, Acetobacter and Gluconobacter were identified as representatives of acetic acid bacteria and the genus Saccharomyces was maintained for yeasts.<br>In conclusion, microorganisms representative of the sourdough have been identified at the genus and species level, which have maintained their stability over time, likewise, different microorganisms have been incorporated into this sourdough in the last sample taken from this bakery.</p>Karol Belén Carvajal Avalos
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Evaluation of the effects of a hydrogen sulfide donor on neural plasticity.
https://www.upo.es/revistas/index.php/biosaia/article/view/8408
<p>The aging brain can exhibit significant modifications related with a progressive atrophy. Previous studies have shown that this atrophy may result from a combination of dendritic regression and neuronal death (1). Age-related memory and cognitive decline have been shown to coincide frequently with morphological changes which affect the neural plasticity and number of dendritic spines in the brains of both humans and animals (2). Furthermore, many neuropathologic conditions and neurodegenerative diseases exhibit abnormalities in dendritic tree structure. Animal studies have shown that even mild prolonged stress has been observed to induce the shrinkage of dendritic fields and the loss of dendritic spines (3).<br>Recent evidence suggest that H2S is a gasotransmitter with neuroprotective properties. In addition, a few sulfur donors have shown beneficial therapeutic effects in experimental models of neurodegenerative diseases (4). Moreover, previous research in our lab suggests that a pharmacological treatment aimed at increasing intracellular H2S improves physical and metabolic health in mice. Nonetheless, the specific properties of these compounds maintaining neuron homeostasis and plasticity remain unknown.<br>Here we aim to investigate whether modulation of intracellular H2S by a pharmacological intervention can improve neuronal plasticity in terms of morphological changes at the level of dendritic arborization and dendritic spine density. To this purpose, we will perform analyses in murine primary neuron cultures that will be treated with increasing concentrations of drug “δ”. Experimental conditions will be: untreated (0, vehicle solution), 10 μM, 50 μM, and 100 μM. Cells will be maintained for 12-14 days in culture, and will be treated with compound “δ” for 48 hours. Then cells will be fixed and MAP2 immunocytochemistry analyses will be performed. Photos will be taken under a fluorescence microscope and analyzed using software ImageJ to determine the percentage of arborized area and the dendritic spine density. The results will provide us with an insight into the potential of drug “δ” as a neuroprotective agent to prevent age-related loss of neuroplasticity.</p> Ryan Conesa-Bakkali Ángela Vega-Blanco Daniel González-MoránNaym El Kharoubi-ZamudioAlejandro Sola-GarcíaMaría Ángeles Cáliz-MolinaRaúl López-Fernández Alejandro Martín-Montalvo Isabel Espadas
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Preclinical evaluation of the safety and potency of neural stem cells from the germinal zone (Gz-NSC) for the treatment of intraventricular hemorrhage (IVH) consequences.
https://www.upo.es/revistas/index.php/biosaia/article/view/8387
<p>Motivation: Intraventricular hemorrhage (IVH) is a common cause of morbidity and mortality in premature infants with no available treatment. After IVH, there is a rupture of the germinal zone into the ventricles that entail the loss of neural stem cells (Gz-NSC). These Gz-NSC can be retrieved from the cerebrospinal fluid (CSF) of IVH patients, obtained after the therapeutic neuroendoscopic lavage performed in these patients to decrease intracranial pressure and that is usually discarded. We have found that Gz-NSC have the potential to differentiate into neuroblasts, oligodendrocyte precursors and few astrocytes when grafted into human brain organoids from iPSCs and mouse brains (1,2). We are evaluating the safety and efficacy profile of CSF-derived Gz-NSC in order to develop a cell therapy for IVH patients.<br />Methods: To examine the differentiation potency of Gz-NSC, we used immunofluorescence assays, fluorescence microspy techniques and computer analysis (ImageJ) to expand previous data, increase sample size, and quantify cell differentiation of grafted Gz-NSC cells in mouse brains and human brain organoids derived from iPSCs.<br />To study the safety profile, flow cytometry assays were carried on to analyze Gz-NSC cell proliferation (Ki67) and immunogenicity (CD80,CD86,CD40, major histocompatibility complex class II (MHC-II)).<br />Results: Based on the immunofluorescence assays, we have found less cells expressing doublecortin, an immature neural protein, and more cells expressing parvalbumin, an interneuron marker, in human brain organoids compared to animal models, suggesting that host can influence cell fate.<br />On the other hand, in order to study the immunogenicity of the Gz-NSC (safety profile), we have analyzed the expression of MHC-II and co-stimulatory molecules (CD80, CD86, CD40) in Gz-NSC before and after in vitro differentiation. Flow cytometry assays revealed Gz-NSC do not express co-stimulatory molecules and express different levels of MHC-II that are reduced when differentiated in vitro, which decreases the probability of an immune response in a future Gz-NSC based cell therapy<br />Conclusions: Taking into account that Gz-NSC have the potency to differentiate to a wide range of cerebral cell linages in both, human organoids and animal models, and are weakly immunogenic, an autologous Gz-NSC cell therapy could be a promising opportunity for IVH patients to overcome some of the neurocognitive problems associated to their condition.</p>Marina Suárez-PizarroAna Belén García-Delgado, Rafael Campos-Cuerva Beatriz Fernández-Muñoz,
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Bioelectrochemical removal of indigo dye in wastewater.
https://www.upo.es/revistas/index.php/biosaia/article/view/8325
<p><strong>ABSTRACT</strong><br /><strong>Motivation:</strong> Textile sector is one of the main industrial consumers of water and, consequently, is generating a significant amount of wastewater loaded with metals, salts, acids, nitrate and organic pollutants such as dyes. Such dyes are carcinogenic and exhibit toxicity for plants and microorganism after disccharge [1]. Indeed, indigo dye is broadly commercialised as blue dye in the denim industry. It has a very strong blue colour and its complex structure make it difficult to degrade.<br />Microbial electrochemical technologies (MET) are a promising option for the treatment of different organic pollutants present in wastewater. These technologies exploit the ability of some microorganisms (electroactive bacteria) to exchange electrons with electroconductive material in order to stimulate the oxidative metabolism [2]. Among the electroactive bacteria reported so far, Purple Phototrophic Bacteria (PPB) are one of the most versatile and diverse group of microorganisms that are capable of tdegrading complex structures as well [3].<br />In this work we propose a electrobioremediation strategy based on activating PPB by means of electrodes to degrade indigo dye present in textile wastewater. Besides, the influence of the electrode in the microbial population will also be studied to evaluate the polarization effect in the wastewater treatment.<br /><strong>Methods</strong>: We used a PPB culture enriched from brewery wastewater. To simulate real wastewater conditions we made a<br />synthetic textile wastewater with indigo as the sole carbon source. The PPB were grown heterotrophically at 30ºC and illuminated with infrared light. Firstly, we study the response of PPBs to indigo dye in two different reactors with 0.1 g/L and 0.5 g/L of dye.<br />Secondly, we compared the impact of i) open circuit polarization (ocp) and ii) polarization at +0.4V (vs. Ag/AgCl) using a<br />electrochemical cell in media supplemented with 0.1 g/L of indigo. The working electrode was graphite, a platinized titanium was used as counter electrode and a Ag/AgCl as reference electrode. We carried a polarized negative control with the synthetic wastewater and 0,1 g/L of indigo without PPB.<br />We measured the optical density (A590) of the culture of PPB, variations in the colour of the indigo dye (A610) and the chemical oxygen demand (COD). The electrochemical analyses consisted in chronoamperometries and cyclic voltammetries during the course of the assay.</p>Raquel Arnal SierraFernando Muniesa-MerinoAbraham Esteve-Núñez
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Role of aak-2 in neuroprotective action in Alzheimer's and Parkinson's disease models in C. elegans.
https://www.upo.es/revistas/index.php/biosaia/article/view/8406
<p>Caenorhabditis elegans is an important model organism as it shares genetic and physiological similarities with humans.. Age-related diseases, such as Alzheimer's or Parkinson's, are caused by the progressive decline of proteostasis during the aging, characterized by the appearance of protein aggregates, which can cause cell function failure in addition to cell death (1).<br>An increase in sulfated steroid hormones has been shown to extend lifespan and ameliorate diseases caused by aging. For this purpose, sul-2 mutants were generated. Sul-2 is a sulfatase of steroid hormones. When the sulfatase gene is deleted, the ability to remove sulfate from hormones is lost and there is an increase in sulfated steroid hormones in the organism, and a reduction in symptoms of neurodegenerative diseases and an increase in longevity. Treatment with the drug STX64, a specific inhibitor of the steroid sulfatase enzyme, in C. elegans and in murine models has been shown to produce the same beneficial effects (2).<br>Recently in our laboratory, results obtained in a RNA seq show that sul-2 mutants share expression patterns with AMPK activation mutants. There are two AMPKα subunit homologs in C. elegans, aak-1 and aak-2. aak-2 is the homologous gene of AMPK in humans, a cellular fuel sensor that regulates cellular energy homeostasis and functions in stress resistance and to extend lifespan (3). The results of a research show that AMPK activation also has neuroprotective effects in Huntington's disease, its activation can preserve striatal neurons to combat the consequences of toxicity in murine models and protects C. elegans neurons from the dysfunction induced by human exon-1 huntingtin expression (4).<br>To know if aak-2 has a role in the neuroprotective effects of sul-2 mutants, we have constructed aak-2 mutants in Alzheimer's and Parkinson's models to check the effects of aak-2. The different strains are subjected to different assays, Alzheimer’s model show a paralysis phenotype when they are shifted to 25ºC and Parkinson’s models show slow movement in buffer, so we perform thrashing assays. In subsequent assays, an aak-2;sul-2 double mutant will be generated to test whether the neuroprotective action of sul-2 mutants is reversed. At the same time, we have started assays with STX64, another way of looking at the consequences of sul-2 deletion.<br>The aim of the project is to understand the role of aak-2 in proteostasis and neurodegenerative diseases and in this way provide a better understanding of the mechanism of action of the drug STX64.</p>Silvia Rodríguez Rodríguez M. Mercedes Pérez JiménezManuel J.J. Muñoz
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Fine-tuning a brain spheroid model as a screening platform for neuroprotective therapies against ischemic stroke.
https://www.upo.es/revistas/index.php/biosaia/article/view/8380
<p><strong>Motivation:</strong> Stroke is the second cause of death and a leading cause of disability worldwide. Ischemic stroke represents more than 80% of all events and it is due to the presence of a clot blocking the blood flow. European legislation has already assumed the goal of no longer needing to use animals for scientific research in the future. To improve current in vitro models, a two-stage 3D brain spheroid model was previously described to recreate the blood-brain barrier and serve as a tool to assess ischemic damage and neuroinflammation. Our objective is to fine tune this organoid model, so we can use it as a screening platform of different neuroprotective therapies against ischemic stroke.</p> <p><strong>Methods:</strong> Six different human brain cells were cultured following manufacturer´s recommendations. In order to be able to visualize each cell type in the 3D model, immunocytochemistry protocols were stablished. Cells were fixed and immunolabeled using a suitable primary antibody for each type of cell followed by the appropriate Alexa 568 secondary antibody. Negative controls were also prepared following the same protocol without the addition of the primary antibody. As a fist approach for spheroid formation, astrocytes, microglia, oligodendrocytes and cortical neurons were co-cultured in round bottom 96 well plates building the spheroid core. After 48h microvascular cells and pericytes were added to the culture creating the surface area. Finally, spheroids images were taken employing bright field microscopy and diameter was measured.</p> <p><strong>Results:</strong> Fluorescent images confirmed that the immunolabeling protocols for each cell type were set up appropriately. Red fluorescence staining indicated cell´s detection by their specific markers. Fluorescent signal was absent in negative controls, confirming the specific binding. Finally, two-stage spheroids (4.000 cells) were formed. After 48, one round-shaped spheroid per well was observed. Spheroid diameter was 313.9± 22 μm after 48 h and 690.9±64 μm after 4 days.</p> <p><strong>Conclusions:</strong> We have fine-tuned the immunocytochemical technique to identify each cell line that will be used to analyse brain spheroids. The first trial for spheroid formation showed homogenous shape and size. Next step would be testing smaller spheroids (2.000 cells) and verifying that each cell type is correctly located in the 3D structure. If results are satisfactory these spheroids could be used as a tool to test potential neuroprotective therapies.</p> Javier Aguilar Román Marina Romero Bernal, Ángela, González Díaz Carmen Del Río Mercado Joan Montaner Villalonga
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Effect of Epas1 and Pcx inactivation in pancreatic β-cell formation and function.
https://www.upo.es/revistas/index.php/biosaia/article/view/8404
<p>According to the consensus model for GSIS (Glucose-Stimulated Insulin Secretion), glucose is rapidly metabolized and coupled to insulin secretion involving a substantial number of cofactors and metabolites intermediates such as ATP, NADPH and citrate, among many others (1). In particular, pyruvate carboxylase (PC) is a fundamental enzyme in redox cycling between NADH and NADPH and also participates in an intricate process known as “pyruvate cycling” which allows the anaplerotic entry of pyruvate in the krebs cycle (2).<br>Pancreatic β-cells express abnormally high levels of pyruvate carboxylase (PC) and insignificant levels of phosphoenolpyruvate carboxylase, the enzyme necessary for gluconeogenesis. This implies that PC must play a different role in β-cells, such as insulin secretion, which is required for the "metabolic switch" from glycolytic to aerobic metabolism during β-cell maturation. It is also known that pyruvate carboxylase activity is elevated in mature β-cells but diminished under diabetic conditions. Recent studies have revealed that the Hypoxia Inducible factor (HIF) pathway plays an important role of in β-cell function (cita algun articulo nuestro). Both overexpression and inactivation of HIF-1α in β cells cause defects in insulin secretion. However, the role of HIF-2α in β-cell formation and function has been largely ignored despite been reported to be activated during diabetic conditions.<br>In this project, we hypothesize that HIF-2α and pyruvate carboxylase activity during late pancreatic HIF-2α formation is critical for the metabolic switch that ocurrs in β-cell during early postnatal development and thus for proper β-cell function.<br>In this study, we will analyze the expression of Epas1 (the gene encoding HIF-2α and Pcx) at different prenatal and postnatal stages by mRNA TaqMan essay. Using Cre/lox technology in mice, we will inactivate Epas1 and Pcx specifically in β-cells. Immunofluorescence and immunohistochemical assays will be carried out to determine specific markers of cell identity, vascularization, proliferation, and polarity in pancreatic tissue of Epas1- and Pcx-deficient mice. Finally, we will also evaluate the in vivo behavior of pancreatic β-cells in transgenic mice through glucose and insulin tolerance assays (GTT and ITT). This will help us understand the relationship between HIF-2 and PC activity and β-cell development and function, as well as whether HIF-2 and PC activity play a role in β-cell failure during diabetes.</p> Alejandro Calderón Villalba Alexia Barroso RomeroAnabel Rojas GonzálezDavid Cano González
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Verification of the alternative method COMPASS Listeria according to UNE-EN ISO 16140-3:2021 for detection of Listeria monocytogenes.
https://www.upo.es/revistas/index.php/biosaia/article/view/8377
<p>Listeria monocytogenes is a ubiquitous bacterium, able to survive in different environments. It is a facultative intracellular pathogen that can cause listeriosis in animals and humans. It is transmitted through the eating of contaminated food, after which the bacteria can cross the intestinal barrier and reach target tissues, via the bloodstream, such as the liver and spleen. Although the disease is usually asymptomatic, for some groups, including children, elderly or immunocompromised people, infection with the bacterium can be life-threatening and in the case of pregnant women it can cause miscarriage. Thus, listeriosis has one of the highest case fatality rates among foodborne diseases, with numerous outbreaks of listeriosis occurring in Europe and the United States in recent years. In Spain, there is a clear increasing trend of listeriosis infection, standing out among the European Union countries, indicating a greater need for prevention and control of this disease. The EN/ISO 11290-1 standard defines the standard methodology to carry out the detection of L. monocytogenes, but it takes up to seven days to obtain the results. Therefore, accreditation of alternative and rapid methods in laboratories is necessary.<br>The objective of this work was to verify the detection of Listeria monocytogenes using the COMPASS Listeria method, in order to incorporate it into the daily procedures in Laboratorios Vital. For this purpose, the estimated LOD50 was calculated in six different matrices to compare it to the LOD50 calculated in the validation of the method. The results obtained were positive, so the verification can be considered valid.</p>José Manuel Morales RodríguezFernando AndradaMaría de la Menta Ballesteros Martín
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Molecular analysis of prolactinoma formation in Pten-deficient mice.
https://www.upo.es/revistas/index.php/biosaia/article/view/8402
<p>Pituitary tumors are abnormal masses developed in the pituitary gland. Although they are generally benign, between 40-50% of pituitary adenomas cannot be removed by surgery alone due to local invasion. Moreover, they are associated with hormonal dysregulation. Prolactinoma is the most common type (50-60%), followed by somatotropic cell adenoma (10-15%), corticotropic cell adenoma (5-10%) and finally thyrotropinoma (less than 1%) (Cano González et al., 2015).<br>Previous descriptive studies have suggested a possible role for the PI3K/AKT/mTOR signaling pathway in the formation of pituitary adenomas. In this study, we used genetic mouse models to assess the oncogenic capacity of this signaling pathway in the pituitary. For this purpose, conditional knockout mice have been generated in which the Pten gene is inactivated specially in the pituitary, indirectly causing the AKT overexpression. To accomplish this, a HesX1-Cre mouse line, whose expression is controlled by a pituitary-specific promoter and which is present in very early stages of embryonic development (Rizzoti, 2015) were crossed with mouse lines in which the Pten gene is floxed by two LoxP sequences.<br>We have analyzed the pituitary in Pten-deficient mice at three different ages: 12, 6 and 1 month of age, comparing genotype and sex. At young ages, Pten-deficient mice show pituitary hyperplasia. After 12 months of age, Pten-deficient mice develop pituitary tumors. However, this is only observed in mutant female mice, whereas male mice simply display pituitary hyperplasia. Data from immunohistochesmistry, immunofluorescence, and blood hormones show that Pten-deficient mice developed prolactinomas. These tumors show high rates of cell proliferation as well as alterations in the expression levels of several cell cycle inhibitors.</p>Alicia Moreno PantojaÁlvaro Flores MartínezAlexia Barroso Romero,Eva María Venegas MorenoElena Dios FuentesAlfonso Manuel Soto MorenoDavid Antonio Cano González
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Uso de reactivos colorimétricos como marcadores químicos con importante aplicación industrial.
https://www.upo.es/revistas/index.php/biosaia/article/view/8374
<p>Colorimetry is a technique used to measure and analyse colours, mainly to determine the concentration of colored compounds in solutions by the application of the Beer–Lambert law. It is applied in a variety of fields, such as chemistry, medicine, biology, and in the pharmaceutical and food industries. The basis of this technique is often the use of colorimetric reagents that provide an easily identifiable colour that depends on factors both internal and external to the samples. These reagents are usually pigments that vary in colour depending on the conditions to which they are subjected. In addition, they can interact with the packaging or the environment in which they are placed. The stimulated colour response becomes a practical tool to assess in real time the status of products in the distribution chain. This is why pigments modulated by other reagents have been applied to measure the colour change of samples applicable to products as quality markers.<br>Pigments and the modulator have been prepared in a hand-made device of plastic nature. The colour change reaction of the pigment has been characterised at different temperatures using water baths and the influence of movement on the sample has been measured by means of orbital shaking. The colour change of the pigment has been measured by means of an RGB device and UV-vis spectrophotometry. Subsequently, real trials will be carried out with volunteers to test the effectiveness of the colourimetric device attached to a product.<br>It has been observed that the reaction kinetics is temperature-dependent. The higher the temperature the shorter the reaction time. The reaction time ranged from 3 to 14 hours at temperatures of 40°C down to 5°C. Stirring does not influence the reaction time.<br>Compounds such as pigments, once modulated, can be an effective tool for monitoring product quality quite accurately. Once the reactants ratios are adjusted to the shelf life of the products, their reliability increases considerably, bringing benefits to the final consumer.<br>NOTE: This work is bound by confidentiality in the framework of a research contract.</p>Miguel Ángel López FuentesJavier Roales BataneroJosé María Pedrosa Poyato
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312The mitochondrial phosphatase PPTC7, ¿a biomarker of coenzyme Q10 deficiency or a therapeutic target?
https://www.upo.es/revistas/index.php/biosaia/article/view/8400
<p>PPTC7 is a PP2C-type cation divalent phosphatase of the mitochondrial matrix that catalyzes the dephosphorylation of phosphoserine and phosphothreonine residues. This enzyme is involved in post-translational modifications to regulate mitochondrial biogenesis and mammalian development through coenzyme Q10 biosynthesis (CoQ10) and the mitochondrial import proteins. Several models have demonstrated the importance of PPTC7 as a regulator of mitochondrial proteins. For instance, Ptc7 is an orthologue of PPTC7 in S. cerevisiae that regulates mitochondrial function by activating citrate synthase, a crucial enzyme in the Krebs cycle. Furthermore, PPTC7 expression can be modulated according to mouse liver metabolic changes, and low PPTC7 expression has been associated with altered oxidative phosphorylation profiles and mitochondrial dysfunction in obese C57BL/6 mice (ob/ob). These studies have proven its role as a regulator of mitochondrial metabolism, in particular, PPTC7 activates the COQ7 hydroxylase encoded by the COQ7 gene, which is one of the 11 genes involved in the CoQ10 biosynthetic pathway. Some COQ proteins facilitate the complex assembly involved in the synthesis process and others act enzymatically in the synthesis, such as COQ7, which is critical in the conversion of the molecule demethoxyubiquinone (DMQ) to 5-hydroxyubiquinone. Mutations in COQ genes cause primary coenzyme Q10 deficiency, a rare disease characterized by ataxia, myalgia and renal dysfunction, among others. Preliminary transcriptomic analysis by this group has revealed low levels of PPTC7 expression in fibroblast samples from patients with primary and secondary coenzyme Q10 deficiency. Its role in coenzyme Q10 synthesis and the relationship between CoQ deficiencies in humans has not been fully elucidated, so this study aims to examine the role of the PPTC7 as a biomarker of coenzyme Q10 deficiency. To identify a cause-effect relationship, we performed PPTC7 gene and protein expression, cellular respiration, and the variation of coenzyme Q10 concentration in two different conditions: a) in control fibroblasts after the inhibition of CoQ10 synthesis generated by para-aminobenzoic acid (pABA), and b) in patients fibroblasts cultured in presence of exogenous CoQ10. Moreover, this study explores the PPTC7 overexpression in HEK293 cells as a potential therapeutic approach for mitochondrial diseases.</p> Elena García DíazCarlos Santos OcañaMaría Victoria Cascajo Almenara
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Validation of new matrices to detect gluten using sandwich ELISA.
https://www.upo.es/revistas/index.php/biosaia/article/view/8367
<p>Celiac disease is a chronic inflammatory bowel disease that has an important genetic component. It is induced by wheat gluten and rye and barley prolamines, which generate an abnormal immune response that causes inflammation and damage to the lining of the small intestine and reduces the absorption of nutrients such as iron. calcium, vitamins A , D, E, K and folic acid [1].</p> <p>Celiac disease affects about 1% of people in the world and a strict gluten-free diet for life is the only treatment available [1].</p> <p>Nonceliac gluten sensitivity syndrome (NCGS) is gluten intolerance in people who do not have any wheat allergy or celiac disease [2].</p> <p>Gluten is a mixture of proteins present in cereals. Alpha-gliadin stands out, a multimeric protein that presents a peptide toxic to celiac people. This peptide is called 33-mer and triggers the activation of the immune response, generating antigliadine antibodies (AGA) [3].</p> <p>Our project has the objective of validating aromas, colorings and preservatives as matrices for the detection of gluten. To achieve this validation, a series of tests regulated by the ISO 17025 law must be carried out and will be supervised by the National Accreditation Entity through an audit. The objective of the validation activity is to confirm the reliability of the test information. One of the purposes of validation is to provide confidence to the recipient of information, such as administrations, consumers, businesses, etc.</p> <p>The validation process consists in repeatability and reproducibility.</p> <p>Repeatability consists of analyzing for each matrix three replicates of each fortification range: low (5ppm), medium (10-20 ppm) and high (50 ppm). Each replicate shall be analysed in the ELISA sandwich R5 test in triplicate.</p> <p>On the other hand, reproducibility consists in analyzing for each matrix a replica of each fortification range in two different days. Also, each replicate will be analyzed in triplicate in ELISA sandwich R5.</p> <p>Fortification consists of doping, for a certain concentration, a white matrix with an analyte of interest that we want to quantify. This allows us to obtain reliability in the quantification made during a test.</p> <p>The ELISA sandwich R5 involves two antibodies with epitopes for the same antigen. R5 is fixed to the microplate and binds by complementarity to the 33-mer peptide of gliadin (antigen). The conjugate antibody has a HRP substrate binding region, which by oxidation causes color change to be quantified [4].</p>Miguel Ángel Siglez GranadoJesús González ColcheroTania Isabel Lopes Da Costa
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Development of a tool for the study of the reproductive microbiome and its relationship with endometrial receptivity and functionality in infertility patients.
https://www.upo.es/revistas/index.php/biosaia/article/view/8396
<p>Motivation: The uterine cavity has been considered a sterile niche until few years ago; however, recent studies have shown a characteristic and functional microbiota resides over the endometrial tissue [1]. Thanks to the next generation sequencing (NGS) of the gene encoding the rRNA 16S of prokaryotic ribosomes, microorganisms present in the endometrium were identified; among those predominantly species of the genus Lactobacillus [2]. Bacteria of this genus are classified as acid-lactic bacteria, so they are able to secrete lactic acid, hydrogen peroxide and bacteriocins in order to generate a suitable environment with other microorganisms and avoid the proliferation of potential pathogens [3]. Despite the high percentage of lactobacilli in the endometrium of most women, the microbial composition of each one is highly individualized and the microbial profile can change due to different causes appearing episodes of dysbiosis, that can affect reproductive health, causing embryonic implantation failures, spontaneous miscarriage, premature deliveries and infectious diseases such as chronic endometritis.<br>The main objective of this project is to develop a new tool to analyze the composition of endometrial microbiota of women with infertility problems using microfluidic techniques and studying its relationship with endometrial receptivity and functionality.<br>Methods: In order to analyze the endometrial microbiota, a bibliographical research of the main microorganisms that reside in this tissue, as well as differential genes of these species was carried out. With these sequences, we designed specific primers for the genes of the microorganisms we identify, and they have will be associated with TaqMan probes. After a correct verification of primers and probes, we perform a DNA extraction from endometrial biopsies using a commercial extraction kit and the DNA obtained was pre-amplified by PCR. Finally, thanks to current microfluidic techniques, it was possible to make a single analysis with 96 samples of patients and the primers previously synthesized, to study the microbiological profile of each woman by qPCR.</p> Rosa Gómez MoranoClaudia Díaz LópezAntonio Martínez LaraMarta Pérez Sánchez David Cotán
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Study of scale-up and productivity of an acidotolerant and halotolerant microalgae (Coccomyxa onubensis) as a function of light exposure.
https://www.upo.es/revistas/index.php/biosaia/article/view/8365
<p>The inherent need to feed a growing population that is more aware of the importance of the nutritional quality of food in health has led nutraceutical products to an increasingly relevant position in the food industry. In this context microalgae acquire an essential role, due to their high nutrient content, being rich in various biomolecules, minerals, vitamins and antioxidant compounds1 . The main challenge in large-scale microalgae production is to achieve robust and economically viable growth, even under non-laboratory conditions such as those found outdoors. One promising alternative is extremophilic microalgae that have developed mechanisms that allow them to withstand these conditions2. Among them, Coccomyxa onubensis ACCV1, isolated from the acidic pH water of Río Tinto (Huelva), stands out. It is a halotolerant and acid-tolerant microalgae capable of growing in a wide range of pH values (2.5-9)3 , which potentially allows production while avoiding contamination by other microorganisms. This, together with its ability to accumulate antioxidant compounds such as carotenoids, polyphenols and polyunsaturated fatty acids make it an interesting candidate to study its scale-up for production. Thus, this project focuses on one of scale up stages by studying the variation of the productivity of this microalgae as a function of the different light exposure that occurs as a result of the different optical density of the cultures in 10 L bags. Thus, the trial consists of 3 bags maintained at different optical density (2-4, 6-8 and 10-12, respectively) by means of a repeated bath culture, with constant gassing and illumination. The incident radiation is progressively increased. In this way, the aim is to observe how productivity varies as a function of the different light exposure of the cells in each of the bags, as well as to observe their response to the increase in incident light, in terms of growth, photosynthetic efficiency and accumulation of bioactive compounds. To this end, growth is being evaluated through the quantification of parameters such as dry weight and optical density, while the measurement of quantum yield informs us of their photosynthetic state. The cell content in lipids, chlorophylls, carotenoids, flavonoids and polyphenols is being quantified.</p>Laureano Calviño OlivaresAlejandro Cuetos MenéndezCarlos Vílchez Lobato
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Using Drosophila to analyse organogenetic gene network evolution.
https://www.upo.es/revistas/index.php/biosaia/article/view/8414
<p>Motivation: Cell migration during development is key to the correct development and morphogenesis of animals. This process normally starts with an Epithelial to Mesenchymal Transition (EMT) allowing the normally cohesive and static epithelial cells to migrate. In Drosophila melanogaster the ring gland is formed by three endocrine organs that are specified in different locations and then migrate together: the corpora cardiaca (CC), the corpora allata (CA) and the prothoracic glands (PG). Previous investigations found that CA, PG and trachea can be homeotically transformed into each other, suggesting they all evolved from a metamerically repeated organ. To investigate the lesser known migration of both CA and PG, a reporter gene named sna-rg-eGFP was made using a specific enhancer that activates the expression of the EMT inducer snail (sna) gene in the CA and the PG.This reporter allows us to track the movement of the CA and the PG during their migration. In this way it was possible to identify deletions affecting the migration process. We will present the defects caused by some of these deletions that were found to have a phenotype of apoptosis. Also, we are studying variants altering the sna-rg enhancer as they can provide information on the up-stream regulators of sna activation and CA/PG specification. These showed that, although STAT sites and a fragment called A1 are required for its expression, in the CA the abscence of STAT sites only delays sna-rg expression. This could mean that after its initial activation, the Sna protein could be self regulating or activating other genes to maintain its expression.<br>Methods: For the purpose of finding which gene in the deletion causes the apoptosis phenotype we study with fluorescent microscopy different overlapping deletions to narrow down the suspects. After that we check if the mutation of just one gene causes the phenotype and use UAS/SpaltG4 constructs to find if introducing the gene rescues the phenotype. To study the regulation of the sna-rg enhancer and check if sna is maintaining its own expression we are generating a series of progressively shorter versions of the enhancer regulating GFP and performing RNA in situ hybridization to check the expression of the resulting reporters. This way we study directly the expression directed by the enhancer instead of the protein that is more stable and can last longer.</p>Álvaro Marrufo MenaMar García-Ferres James Castelli-Gair Hombría
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Healthy gummies based on the microalgae Spirulina as functional ingredient.
https://www.upo.es/revistas/index.php/biosaia/article/view/8394
<p>The growing obesity epidemic could be combated with new generations of less caloric but nutritious foods at the same time. Microalgae are considered as the functional ingredients of these new foods due to their nutritional properties . The company G2GAlgaesolutions develops a line of research on healthy gummies with high nutritional value thanks to the aggregation of microalgae of the Spirulina genus. In this work, research is focus on optimizing the recipe for healthy gummies from an initial recipe. Specifically, different concentrations of microalgae and methods to improve the consistency of the jelly based on different types of gelatin were tested. The major objective is to optimize the nutritional properties, prioritizing low sugar levels and an adequate amount of nutrients from the microalgae. The ingredient mixtures were tested iteratively to avoid a too intense flavor derived from the microalgae although preserving sweetness in the gummy. A mixture of strawberry gelatin marketed by ROYAL with neutral ROYAL gelatin was finally chosen as the base of a consistent and sweet-tasting gummy. The maximum threshold concentration of microalgae was determined to avoid unwanted taste and accumulation problems at the bottom of the gelatin. One important finding regarding the manufacturing protocol was that, for an adequate integration, the microalgae must be added once the water is at the boiling point and before adding the gelatin mixture.<br>In conclusion, the research support microalgae as functional ingredients for a healthier nutrition in our society, therefore helping to remedy a variety of diseases associated with obesity and metabolic syndrome.</p>Cheyenne Lorena Pulgar Hilmer
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Metagenomic and metabarcoding analysis of microbial populations in agricultural soils using 16S ribosomal sequencing and database development
https://www.upo.es/revistas/index.php/biosaia/article/view/8359
<p>Motivation: Metagenomic and metabarcoding analyses of microbial populations present in agricultural soils using 16S ribosomal region sequencing and processing technology are performed to understand the microbial diversity and their role in crop productivity, as well as to identify possible environmental impacts.<br>Methods: A bioinformatic analysis is carried out involving taxonomic assignment of the sequences to different microbial groups. This analysis is performed by comparing the obtained sequences with public databases containing information on the diversity of 16S gene sequences. A data filtering and purification process is carried out to eliminate low-quality sequences. The methodology used involves the use of three taxonomic assignment methods: 1. a classical method of taxonomical assignment using a generalist database of public use, 2. a single assigment step using a specific database with a predictive algorithm , and 3. two-step method of taxonomical assigment involving different algorithms using a specific database. A comparison is made between the three methods to determine which is the most effective in taxonomic identification. Additionally, statistical tools can be used to determine the abundance of different microbial taxa present in soils. Finally, work is done on improving the specific database by expanding its information to obtain more accurate results.<br>Results:The results obtained show that the use of a specific database for agricultural soils produces more accurate results in taxonomic identification.This is because the databases contain general information on a wide variety of samples, which can introduce noise and make it difficult to accurately identify the microbes present. By expanding the specific database, an improvement in the accuracy of the results is observed.<br>Conclusions:In conclusion, metabarcoding is an important tool for understanding microbial diversity. The characteristics of the database used in taxonomic identification have a great impact on the taxonomic results. It can be affirmed that the use of different databases has a significant impact on the identification of the most abundant taxa. Additionally, the improvement and maintenance of databases are essential to obtain more accurate results and should be continued to be researched and updated to maintain the effectiveness of the technology used.</p>Sonia Arrojo Viejo
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Effect of priming on the immunomodulatory response of mesenchymal stem cells (adMSC) with immunogenic synthetic peptides from Leishmania braziliensis.
https://www.upo.es/revistas/index.php/biosaia/article/view/8412
<p>Leishmaniasis is a zoonotic disease that causes major public health problems, mainly in tropical or subtropical countries,<br>causing approximately 70,000 deaths per year and currently there is no approved vaccine. Therefore, research into the<br>development of effective therapies that help both the elimination of the parasite and the aesthetic recovery of wounds, through<br>the search for synthetic antigenic systems with immunogenic activity, is nowadays a necessity. Recently, the<br>immunotherapeutic potential of human mesenchymal stem cells (hMSCs) and the secretome and exocytic vesicles they<br>produce when cultured in specific media have been described [1]. It has also been described that they have regenerative and<br>antimicrobial potential and that these effects are enhanced when stimulated with different antigens. The present study aimed<br>to characterize the secretome of hMSCs, treated with synthetic immunogenic peptides from Leishmania braziliensis, and to<br>evaluate their immunomodulatory potential. Adipose tissue-derived hMSCs, which were characterized according to<br>international standards [2], were used. For this work, cells at passage eight were exposed to two synthetic immunogenic<br>peptides MSD1 and CBP, designed in silico, from L. braziliensis and lipopolysaccharide (LPS) as a positive control. The<br>secretome was collected at 48 and 96h and characterized firstly by measuring the expression of Interleukin 6 (IL-6) by an<br>ELISA method and the expression of cytokines and other proteins using an 80- antibodies targets in a chemiluminescence<br>panel. The results have shown a proinflammatory profile at the level of cytokine expression for the treatments with MSD1, CPB<br>and LPS [3,4]. These were characterized by an increase in the expression of proinflammatory cytokines such as INF-γ, IL-6,<br>IL-16, TNF-α and TNF-β. In summary, the secretome of adipose tissue hMSC pre-treated with synthetic immunogenic<br>peptides from L. braziliensis presents differences in the level of cytokine expression with a tendency towards a<br>proinflammatory profile.</p>Ana Paula Flores ThomasJuan Rigoberto Tejedo HuamanGladys Margot Cahuana Macedo
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Modified natural zeolites for water disinfection using heterogeneous photo-Fenton at near neutral pH.
https://www.upo.es/revistas/index.php/biosaia/article/view/8390
<p>According to the World Health Organisation (WHO) [1], microbiologically contaminated water for for human consumption can transmit diarrheal diseases or acute respiratory infections, among others, causing 485,000 deaths from diarrhea each year. Therefore, water contaminated with pathogenic organisms (bacteria resistant to antibiotics, viruses, etc.) must be adequately treated for its safe use.<br>Currently there are several types of treatments for water disinfection, and in recent years the interest of the scientific community in Advanced Oxidation Processes (POAs) has increased significantly [2]. Among all types of POAs, the heterogeneous photo-Fenton process has been used for the degradation of a large number of contaminants in water. Likewise, there are studies that demonstrate its efficacy for the inactivation of pathogens. Its effectiveness lies in the generation of hydroxyl radicals through the oxidation-reduction reactions that occur on the surface of a photocatalyst due to the action of UV-Vis light and the presence of an oxidising agent (hydrogen peroxide) capable of producing profound changes in the chemical structure of contaminants and irreversible damage to microorganisms.<br>In this work, modified natural zeolites (NZ) were used as an efficient photocatalytic and low-cost support [3] for the inactivation of a model microorganism (E. coli). To do this, first, natural zeolite clinoptilolite from the Tasajeras site (Villa Clara) was was subjected to ion exchange with Fe2+, following an usual procedure [4]. Once the material was obtained, it was characterised by DRX, SEM and IR, to ensure that the synthesis had been carried out properly. Next, its efficiency was evaluated in an 850 mL jacketed glass reactor with constant agitation and illuminated with UV or visible light at a constant temperature of 25ºC and initial pH of 6.5. The tests were performed with an initial concentration of E. coli of 106 CFU/mL and adding H2O2 (10 mM) and modified zeolite<br>2<br>(0.85 gr) at the beginning. The reduction of bacterial concentration was then evaluated every 30 minutes through serial dilutions. Likewise, in addition to the photo-Fenton process, to understand the isolated effect of the different parameters of the process, radiation disinfection; radiation - H2O2; H2O2; modified zeolite; modified radiation-zeolite; H2O2 - modified zeolite (Fenton) and the viability of the cells and their recreation were evaluated. Finally, the ability of the used catalyst to be recovered and cyclically reused without losing its photocatalytic activity was evaluated.</p>Carlos Salameh BorreroTania FaríasSurey RamirezInes CanosaAmando FloresA. Rabdel Ruiz-SalvadorMenta Ballesteros
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Evaluation of the efficiency of CRISPR-Cas systems in Gram-negative bacteria
https://www.upo.es/revistas/index.php/biosaia/article/view/8335
<p>CRISPR-Cas is a genetic tool based on a prokaryotic defense system. This is a technology that involves the simple design of an RNA molecule (crRNA) and its binding to a Cas nuclease, forming a complex capable of breaking DNA. This system, in the presence of a repair system, also allows the introduction of mutations in a targeted manner. CRISPR-Cas has been widely used in eukaryotic cell genome editing, but not so much in prokaryotes [1]. The absence of efficient repair systems in bacteria could be the main reason, but this could be supplemented by the exogenous introduction of a bacterial non-homologous repair system, or NHEJ. Most bacterial NHEJs have only two proteins: Ku, which binds to and protects the broken ends; and LigD, which ligates them [2]. In this work a CRISPR-Cas system has been implemented and studied in Pseudomonas putida, a Gram-negative bacterium of great biotechnological interest, which lacks efficient NHEJ repair systems [3]. For this, two plasmids for the expression of Cas12a and NHEJ from Sphingopyxis granuli TFA have been introduced, in addition to a reporter plasmid that includes the necessary spacer and the target. The efficacy of Cas12 and the NHEJ in double-strand break repair in P. putida has been verified by fluorescence, PCR, and sequencing. However, when the repaired plasmids were analyzed, an unexpected homologous recombination repair (HDR) mechanism was found. The characterization of this phenomenon is currently being carried out.</p>Alba González BarneoGovantes Romero Fernando
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Generation and characterization of mecp2 mutant.
https://www.upo.es/revistas/index.php/biosaia/article/view/8409
<p>Motivation: Mecp2 gene encodes the Methyl CpG binding protein (MeCP2). This protein is known for being an important regulator of gene expression that interacts with methylated and unmethylated genomic DNA regions, and enhances or silences transcriptional processes. Different mutations of the MeCP2 protein in humans can lead to a variety of symptoms in Rett syndrome (RTT), a rare disease which causes abnormalities during female brain development as well as acute mental and physical disability. These misfunctions are caused by mutations in one or two of the domains that can be found in the MeCP2 protein: the methyl binding domain (MBD) and the transcriptional repression domain (TRD). The aim of this study is the generation of a knock-out (KO) mutant of the mecp2 gene in zebrafish by employing the CRISPR/Cas9 technique. Once we obtain the homozygous mutant, we will elucidate the significance of this mutation by deep RNA sequencing (RNA-Seq), focusing on the differences between transcripts present in wild type (WT) and homozygous mutant fish. These results will give us an indication of which genes are potentially regulated by the MeCP2 protein, which could render zebrafish as a useful model system aimed at understanding RTT etiology in vivo.<br>Methods: To create the mecp2 KO, we designed sgRNA guides targeting the mecp2 exon 2, using the CRISPRscan software. We then tested the sgRNAs in F0 by microinjection of single-cell stage zebra-fish embryos. The embryos were allowed to develop to 24-48 hours post fertilization (hpf), after which they were genotyped. Amplification of regions of interest by PCR followed by electrophoresis in agarose gel, showed us that the sgRNAs worked, as we could see a band different to that of the WT (471bp). After the raising of the F0, we genotyped the mutant individually to identify founders. The founders of interest were named as follows: (i) mecp2 26⚦, with a deletion of 181bp, (ii) mecp2 27, ⚦with deletion of 15bp and (iii) mecp2 13,⚦ with a deletion of 8bp. These founders were out-crossed with WT zebra-fish to stabilize the mutation (F1), and the offspring was genotyped and raised. We then genotyped the F1 generation by extracting the DNA from fin tissue (fin-clip); it is expected that 25% of this generation should be heterozygous. Once we have sequenced the heterozygous candidates for the mecp2 gene, we will in-cross a male and a female with the same mutation, in order to have a homozygous F2. Finally, we will genotype the F2 individually at 72h, extracting the DNA and RNA at the same time, using the qiagen DNAeasy blood and tissue kit for DNA and Trizol for RNA. The RNA of the confirmed homozygous mutants will be sent for RNA-Seq analysis.<br>Results and conclusions: We have generated heterozygous mecp2 mutants, that we have to genotype, from the founders mentioned above (F1). The deletions sequenced cause the generation of a stop codon, so the MeCP2 protein should not be expressed in homozygous fish. The next step will be generating the homozygous generation in order to study the phenotype and perform the RNA-Seq at 72h of embryo development. Once we obtain the results of the RNA-seq analysis, we will be able to explain which are the genes whose expression is modulated by MeCP2 and that are potentially implicated in RTT.</p>Juan Aperador RedondoAna María Fernández Miñán Ozren Bogdanovic
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Development of a novel and rapid molecular diagnostic system for Hepatitis C virus (HCV) detection.
https://www.upo.es/revistas/index.php/biosaia/article/view/8388
<p>Hepatitis C virus (HCV) is an enveloped, single-stranded positive sense RNA [ssRNA(+)] virus which causes cirrhosis, hepatocellular carcinoma and liver failure in infected patients [1]. The Global Hepatitis Report posted in 2017, indicates that around 71 million people were living with HCV in 2015 [2]. Moreover, the most recent data collected in 2019 by WHO was able to estimate that 58 million people live with chronic hepatitis C infection and about 1.5 million new infections occur per year. Due to the increasing number of infections, one of the recommendations postulated in the 2022 guidelines ("Updated recommendations on HCV simplified service delivery and HCV diagnostics: policy brief") was to achieve more efficient and simplified hepatitis diagnosis [3]. The diagnosis of an active HCV infection requieres the detection of HCV RNA, and nowadays it is performed by the gold standard real-time reverse transcription polymerase chain reaction (RT-qPCR). Nevertheless, it involves specific laboratory facilities, well-trained personnel and high-cost equipment, maintenance and reagents [4]. Consequently, this project was born on the need of developing a new diagnostic system for direct HCV molecular detection based on the RT-LAMP (reverse transcription loop-mediated isothermal amplification) technique that could be implemented in resource-limited settings. This strategy is focused on the nucleic acid-based amplification method using from four to six primers under isothermal conditions to amplify specifically and effectively the target sequence [4]. The aim of the project is to validate the system and integrate it in a future point-of-care (POC) diagnostic device.</p>Cristina Yépez NotarioSonia Arca de LafuenteFernando Govantes RomeroRicardo Madrid González
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Genomic selection of new low-gliadin wheat lines.
https://www.upo.es/revistas/index.php/biosaia/article/view/8327
<p>Gluten proteins are responsible for the quality and properties of processed wheat products. However, consuming gluten proteins (gliadins and glutenins) from wheat, barley, and rye can cause celiac disease (CD) or non-celiac wheat sensitivity (NCWS) in genetically predisposed individuals. The immunogenic epitopes that trigger celiac disease are mainly present in the α-, β-, and γ-Gliadins. The only alternative currently available for this type of diseases is a strict, lifelong gluten-free diet.</p> <p>The use of genetic engineering techniques such as RNA interference (RNAi) for post-transcriptional regulation of α-gliadins [1] and gene editing using CRISPR/Cas for silencing the α-gliadins , has resulted in wheat lines (T. aestivum ssp. aestivum,) with reduced α-gliadin content. [2]. These methods produce offspring with silenced, deleted and/or edited gliadins, which can reduce patients' exposure to CD epitopes.</p> <p>To produce new lines with silenced α-gliadins, Francisco Barro's group at the Institute of Sustainable Agriculture (IASCSIC), crossed lines treated with CRISPR/Cas and RNAi and, using anther culture for the production of double haploid (DH) plants, they fixed the mutations of the offspring of these crosses into homozygous lines. (Unpublished).</p> <p>In this Master's thesis, a bioinformatics pipeline based on Next Generation Sequencing (NGS) amplicon sequencing (previously designed by Francisco Barro's group at the Institute for Sustainable Agriculture IAS-CSIC [3]) will be used to analyze the insertion and deletions (InDels) in target genes of lines treated by DH process. In addition, A-PAGE gels will be used to examine the protein profiles of these lines with the main objective of selecting those lines containing lower quantities of gliadins.</p>Jose Antonio Berlanga TorresFrancisco Barro Losada
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Evaluation of the antiviral-HAdV activity of venom extracts obtained from arachnids and snakes.
https://www.upo.es/revistas/index.php/biosaia/article/view/8407
<p>Motivation: Human adenoviruses (HAdVs) can cause life-threatening infections and disseminated disease in hematopoietic stem cell transplant (HSCT) recipients, especially in paediatric patients, and is also a well-known cause of community-acquired pneumonia in the general population. Despite this significant impact on public health, there are no specific antiviral therapies approved by the health authorities to treat HAdV infections. Thus, it is necessary to identify and characterize molecules that exert their antiviral action against HAdVs infection as the first step in the clinical development of an specific antiviral drug. In this sense, we have selected a collection of crude extracts of arachnid and snake venoms to evaluate their antiviral activity against HAdV.<br>Methods: A total of 67 extracts from different arachnid species were subjected to a first screening at a concentration of 50 μg/ml in A549 cells infected with HAdVs-RFP at a MOI of 2000 vp/cell for 48 h. Those that showed an inhibition of the viral infection higher than 60% were tested through the same procedure using HAdV-GFP to determine if the extract exerts its antiviral action in early or late stages of the HAdV replicative cycle. In parallel, the selected compounds were subjected to a methabolic assay to determine their cytotoxicity (CC50). Finally, the selected compounds were tested at a low MOI (0.06 vp/cell) with HAdV-GFP at concentrations ranging from 10 μg/ml to 0,625 μg/ml. For the extracts that their inhibitory concentration 50% (IC50) could be calculated, the selectivity index (SI) was determined, as the coefficient between the CC50 and the IC50.<br>Results: Sixteen out of the 67 extracts analyzed with HAdVs-RFP and HAdV-GFP, showed an inhibition of the infection >60%. The CC50 values obtained ranged from 87.43 ± 27.86 to 1.24 ± 0.21 μg/ml. So far, most of the extracts evaluated present a IC50 value above 10 μg/ml, with the exception of Bothrops atrox and Bothrops pictus, with an IC50 value of 0.75 ± 0.047 μg/ml (SI = 24,26) and 1.3 ± 0.00565 μg/ml (SI = 23,20), respectively.<br>Conclusions: Sixteen extracts have been identified with the ability to inhibit HAdV infection by more than 60%, and presenting CC50 above their IC50. Thus, in this situation, the next step will be to carry out the deconvolution of these 16 extracts, to identify the molecule/s responsible for the observed activity in order to accurately characterize their antiviral activity and mechanism of action.</p>Jaime Rampérez Barrajón María Balsera ManzaneroAlexander Vassilevski Javier Sánchez Céspedes
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
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2023-09-132023-09-1312Generation and characterization of mecp2 mutant.
https://www.upo.es/revistas/index.php/biosaia/article/view/8381
<p><span id="page1R_mcid18" class="markedContent"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*280.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.10123);" role="presentation">Motivation:</span></span><span id="page1R_mcid19" class="markedContent"></span><span id="page1R_mcid20" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*105.90px); top: calc(var(--scale-factor)*280.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.999445);" role="presentation">Mecp2</span></span><span id="page1R_mcid21" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*136.80px); top: calc(var(--scale-factor)*280.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.04759);" role="presentation">gene encodes the Methyl CpG binding protein (MeCP2). This protein is known for being an important</span></span><span id="page1R_mcid22" class="markedContent"></span><span id="page1R_mcid23" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*292.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.07266);" role="presentation">regulator of gene expression that interacts with methylated and unmethylated genomic DNA regions, and enhances or</span></span><span id="page1R_mcid24" class="markedContent"></span><span id="page1R_mcid25" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*304.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02119);" role="presentation">silences transcriptional processes. Different mutations of the MeCP2 protein in humans can lead to a variety of symptoms in</span></span><span id="page1R_mcid26" class="markedContent"></span><span id="page1R_mcid27" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*316.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02882);" role="presentation">Rett syndrome (RTT), a rare disease which causes abnormalities during female brain development as well as acute mental</span></span><span id="page1R_mcid28" class="markedContent"></span><span id="page1R_mcid29" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*328.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.04367);" role="presentation">and physical disability. These misfunctions are caused by mutations in one or two of the domains that can be found in the</span></span><span id="page1R_mcid30" class="markedContent"></span><span id="page1R_mcid31" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*340.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02657);" role="presentation">MeCP2 protein: the methyl binding domain (MBD) and the transcriptional repression domain (TRD). The aim of this study is</span></span><span id="page1R_mcid32" class="markedContent"></span><span id="page1R_mcid33" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*352.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02342);" role="presentation">the generation of a knock-out (KO) mutant of the</span></span><span id="page1R_mcid34" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*255.60px); top: calc(var(--scale-factor)*352.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.999445);" role="presentation">mecp2</span></span><span id="page1R_mcid35" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*285.70px); top: calc(var(--scale-factor)*352.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.01708);" role="presentation">gene in zebrafish by employing the CRISPR/Cas9 technique. Once</span></span><span id="page1R_mcid36" class="markedContent"></span><span id="page1R_mcid37" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*364.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02841);" role="presentation">we obtain the homozygous mutant, we will elucidate the significance of this mutation by deep RNA sequencing (RNA-Seq),</span></span><span id="page1R_mcid38" class="markedContent"></span><span id="page1R_mcid39" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*376.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.0049);" role="presentation">focusing on the differences between transcripts present in wild type (WT) and homozygous mutant fish. These results will give</span></span><span id="page1R_mcid40" class="markedContent"></span><span id="page1R_mcid41" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*388.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.05044);" role="presentation">us an indication of which genes are potentially regulated by the MeCP2 protein, which could render zebrafish as a useful</span></span><span id="page1R_mcid42" class="markedContent"></span><span id="page1R_mcid43" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*400.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.00052);" role="presentation">model system aimed at understanding RTT etiology</span></span><span id="page1R_mcid44" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*262.30px); top: calc(var(--scale-factor)*400.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.998502);" role="presentation">in vivo.</span></span><span id="page1R_mcid45" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*424.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.07908);" role="presentation">Methods:</span></span><span id="page1R_mcid46" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*96.59px); top: calc(var(--scale-factor)*424.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.0175);" role="presentation">To create the</span></span><span id="page1R_mcid47" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*152.20px); top: calc(var(--scale-factor)*424.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.999445);" role="presentation">mecp2</span></span><span id="page1R_mcid48" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*181.70px); top: calc(var(--scale-factor)*424.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.999283);" role="presentation">KO, we designed sgRNA guides targeting the</span></span><span id="page1R_mcid49" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*365.40px); top: calc(var(--scale-factor)*424.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.999445);" role="presentation">mecp2</span></span><span id="page1R_mcid50" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*394.89px); top: calc(var(--scale-factor)*424.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.998528);" role="presentation">exon 2, using the</span></span><span id="page1R_mcid51" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*466.00px); top: calc(var(--scale-factor)*424.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.999378);" role="presentation">CRISPRscan</span></span><span id="page1R_mcid52" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*521.49px); top: calc(var(--scale-factor)*424.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.998632);" role="presentation">software.</span></span><span id="page1R_mcid53" class="markedContent"></span><span id="page1R_mcid54" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*436.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.04379);" role="presentation">We then tested the sgRNAs in F0 by microinjection of single-cell stage zebra-fish embryos. The embryos were allowed to</span></span><span id="page1R_mcid55" class="markedContent"></span><span id="page1R_mcid56" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*448.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.03394);" role="presentation">develop to 24-48 hours post fertilization (hpf), after which they were genotyped. Amplification of regions of interest by PCR</span></span><span id="page1R_mcid57" class="markedContent"></span><span id="page1R_mcid58" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*460.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.00184);" role="presentation">followed by electrophoresis in agarose gel, showed us that the sgRNAs worked, as we could see a band different to that of the</span></span><span id="page1R_mcid59" class="markedContent"></span><span id="page1R_mcid60" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*472.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.03787);" role="presentation">WT (471bp). After the raising of the F0, we genotyped the mutant individually to identify founders. The founders of interest</span></span><span id="page1R_mcid61" class="markedContent"></span><span id="page1R_mcid62" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*484.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02028);" role="presentation">were named as follows: (i)</span></span><span id="page1R_mcid63" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*164.10px); top: calc(var(--scale-factor)*484.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.01393);" role="presentation">mecp2 26</span><span dir="ltr" style="left: calc(var(--scale-factor)*204.20px); top: calc(var(--scale-factor)*484.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif;" role="presentation">⚦</span></span><span id="page1R_mcid64" class="markedContent"><span dir="ltr" style="left: calc(var(--scale-factor)*211.70px); top: calc(var(--scale-factor)*484.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.0273);" role="presentation">, with a deletion of 181bp, (ii)</span></span><span id="page1R_mcid65" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*333.30px); top: calc(var(--scale-factor)*484.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.01393);" role="presentation">mecp2 27</span> <span dir="ltr" style="left: calc(var(--scale-factor)*380.88px); top: calc(var(--scale-factor)*484.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif;" role="presentation">,</span><span dir="ltr" style="left: calc(var(--scale-factor)*373.40px); top: calc(var(--scale-factor)*484.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif;" role="presentation">⚦</span></span><span id="page1R_mcid66" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*386.50px); top: calc(var(--scale-factor)*484.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02239);" role="presentation">with deletion of 15bp and (iii)</span></span><span id="page1R_mcid67" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*507.00px); top: calc(var(--scale-factor)*484.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.01393);" role="presentation">mecp2 13</span> <span dir="ltr" style="left: calc(var(--scale-factor)*554.58px); top: calc(var(--scale-factor)*484.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif;" role="presentation">,</span><span dir="ltr" style="left: calc(var(--scale-factor)*547.10px); top: calc(var(--scale-factor)*484.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif;" role="presentation">⚦</span></span><span id="page1R_mcid68" class="markedContent"></span><span id="page1R_mcid69" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*496.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02808);" role="presentation">with a deletion of 8bp. These founders were out-crossed with WT zebra-fish to stabilize the mutation (F1), and the offspring</span></span><span id="page1R_mcid70" class="markedContent"></span><span id="page1R_mcid71" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*508.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.00355);" role="presentation">was genotyped and raised. We then genotyped the F1 generation by extracting the DNA from fin tissue (fin-clip); i</span></span><span id="page1R_mcid72" class="markedContent"><span dir="ltr" style="left: calc(var(--scale-factor)*507.40px); top: calc(var(--scale-factor)*508.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.00237);" role="presentation">t is expected</span></span><span id="page1R_mcid73" class="markedContent"></span><span id="page1R_mcid74" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*520.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02675);" role="presentation">that 25% of this generation should be heterozygous. Once we have sequenced the heterozygous candidates for the</span></span><span id="page1R_mcid75" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*530.20px); top: calc(var(--scale-factor)*520.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.999445);" role="presentation">mecp2</span></span><span id="page1R_mcid76" class="markedContent"></span><span id="page1R_mcid77" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*532.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.07328);" role="presentation">gene, we will in-cross a male and a female with the same mutation, in order to have a homozygous F2. Finally, we will</span></span><span id="page1R_mcid78" class="markedContent"></span><span id="page1R_mcid79" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*544.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.04416);" role="presentation">genotype the F2 individually at 72h, extracting the DNA and RNA at the same time, using the</span></span><span id="page1R_mcid80" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*444.40px); top: calc(var(--scale-factor)*544.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02771);" role="presentation">qiagen DNAeasy blood and</span></span><span id="page1R_mcid81" class="markedContent"></span><span id="page1R_mcid82" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*556.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.998767);" role="presentation">tissue</span></span><span id="page1R_mcid83" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*80.09px); top: calc(var(--scale-factor)*556.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.998657);" role="presentation">kit for DNA and</span></span><span id="page1R_mcid84" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*143.70px); top: calc(var(--scale-factor)*556.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.01453);" role="presentation">Trizol</span></span><span id="page1R_mcid85" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*168.20px); top: calc(var(--scale-factor)*556.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.999257);" role="presentation">for RNA. The RNA of the confirmed homozygous mutants will be sent for RNA-Seq analysis.</span></span><span id="page1R_mcid86" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*580.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.10811);" role="presentation">Results and conclusions:</span></span><span id="page1R_mcid87" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*167.89px); top: calc(var(--scale-factor)*580.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.01741);" role="presentation">We have generated heterozygous</span></span><span id="page1R_mcid88" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*308.60px); top: calc(var(--scale-factor)*580.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(0.999445);" role="presentation">mecp2</span></span><span id="page1R_mcid89" class="markedContent"> <span dir="ltr" style="left: calc(var(--scale-factor)*338.90px); top: calc(var(--scale-factor)*580.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02701);" role="presentation">mutants, that we have to genotype, from the founders</span></span><span id="page1R_mcid90" class="markedContent"></span><span id="page1R_mcid91" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*592.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.02024);" role="presentation">mentioned above (F1). The deletions sequenced cause the generation of a stop codon, so the MeCP2 protein should not be</span></span><span id="page1R_mcid92" class="markedContent"></span><span id="page1R_mcid93" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*604.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.01918);" role="presentation">expressed in homozygous fish. The next step will be generating the homozygous generation in order to study the phenotype</span></span><span id="page1R_mcid94" class="markedContent"></span><span id="page1R_mcid95" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*616.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.00139);" role="presentation">and perform the RNA-Seq at 72h of embryo development. Once we obtain the results of the RNA-seq analysis, we will be able</span></span><span id="page1R_mcid96" class="markedContent"></span><span id="page1R_mcid97" class="markedContent"><br role="presentation"><span dir="ltr" style="left: calc(var(--scale-factor)*54.10px); top: calc(var(--scale-factor)*628.67px); font-size: calc(var(--scale-factor)*9.00px); font-family: sans-serif; transform: scaleX(1.00202);" role="presentation">to explain which are the genes whose expression is modulated by MeCP2 and that are potentially implicated in RTT</span></span></p>Juan Aperador RedondoAna María Fernández MiñánOzren Bogdanovic
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
https://creativecommons.org/licenses/by-nc-sa/4.0
2023-09-132023-09-1312Study of the secondary metabolism of phytopathogenic fungi as a source of compounds with antibiotic activity.
https://www.upo.es/revistas/index.php/biosaia/article/view/8405
<p>Eutypa lata is a phytopathogenic fungus responsible for a "Grapevine Trunk Disease" commonly called "eutypiosis" or "eutypa dieback" which affects several agricultural crops. Its secondary metabolism has been studied in order to find the main toxins that cause the symptoms of the disease, and to learn how to control its production. One of the most phytophatogenic toxins is Eutypine, a hydroxybenzaldehyde, among several acetylenic derivatives related with it. Syccaine is one of them and exhibits antimicrobial activity against Aerobacter aerogenes and a variety of Gram-positive bacteria, and few fungi. That is why, in this study, we enviosioned the study of the secondary metabolism of E. lata as a producer of natural acetylenic compounds with antimicrobial activity to give response to the worlwide problem posed by the shortage of new antibiotics development.<br>Throught OSMAC (One Strain Many Compounds) approach, two strains of the fungus, E. lata 355 y E. lata 311, were grown using different cultivation conditions like culture media composition, aeration, type of culture flask or incubation time. The extract obtained from the extraction with ethyl acetate of the culture media, was tested against Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae using the broth dilution method. Then, the extracts with antibiotical activity were purified in order to obtain pure compounds to find which compound are responsible for the antibiotical activity.<br>The preliminary results of the research have indicated that OSMAC approach estimulates the production of compounds with antibiotic activity in some of the culture conditions tested.</p>Ana Cotán-GarcíaInmaculada Izquierdo-Bueno RCristina Pinedo-Rivilla
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
https://creativecommons.org/licenses/by-nc-sa/4.0
2023-09-132023-09-1312Chromatin regulation of virulence gene clusters in Ustilago maydis.
https://www.upo.es/revistas/index.php/biosaia/article/view/8376
<p>Pathogenic fungi possess chromatin modification mechanisms that are involved in their virulence and interaction with the host 1. This modification usually occurs as a consequence of the presence or absence of certain molecular marks on histones, such as methylations or acetylations. It allows the transition between their two conformational states, euchromatin and heterochromatin, associated with gene transcriptional activation and gene silencing, respectively 2,3. The maize fungus Ustilago maydis has become a model organism for the study of plant pathogenesis due to its ease of handling and the multiple available genetic and cellular tools 4. This pathogen uses a large number of factors and pathways that regulate gene expression at each stage of the infective process, but little is known about the role of chromatin in that 1. Many genes involved in that process are clustered in gene regions and are activated at specific times 4,5. Therefore, we should think of an active role of chromatin in this regulation. However, the absence of typical heterochromatin-associated marks and regulators in U. maydis has so far been observed 1. In this laboratory, we have sought other likely regulators of this process and found that the histone methyltransferase Ash1 affects the virulence activity through the silencing of gene clusters involved in this process. In this project we try to find out if this is the only regulator of heterochromatin in U. maydis. For this purpose, different strains with antibiotic resistance markers in silenced regions controlled by Ash1 were developed, allowing antibiotic resistance when these regions are derepressed. Spontaneous resistant mutants were obtained from these strains and Ash1 was consequently sequenced to determine if the silencing was affected by a mutation in Ash1. Surprisingly, the methyltransferase gene was not mutated, suggesting the presence of other regulators. In addition, we performed a ChIP-qPCR to see if the epigenetic mark associated with Ash1 was still present. We observed a decrease in this mark in one of the spontaneous mutants in different virulence regions, which also suggests the presence of other regulators, such as some demethylases. Subsequently, in order to discover whether the decrease in this mark is maintained over time, we have studied its stability in successive generations. We noticed antibiotic-resistant colonies trended towards a decrease which might imply a reacquisition of the epigenetic mark.</p>Martín Fernández SalvadorSanz Martí EstelaJosé Ignacio IbeasRamón Ramos Barrales
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
https://creativecommons.org/licenses/by-nc-sa/4.0
2023-09-132023-09-1312Synthesis of sustainable flocculants for phosphorus removal in urban wastewater from algae, marine and terrestrial plant wastes.
https://www.upo.es/revistas/index.php/biosaia/article/view/8486
<p>Motivation:<br>Wastewater treatment is a process that is constantly evolving in order to reduce both economic costs and the environmental impact. Since the Urban Waste Water Treatment Directive (UWWTD) was approved in 1991 [1], the minimum requirements for wastewater treatment have been increasing, and there are now plans until 2040 that introduce new limit values for certain elements such as Nitrogen and Phosphorus [2].<br>For this reason, there are several lines of research focused on finding new, less polluting alternatives for this process. In our study, we seek to reuse organic wastes from algae, marine and terrestrial plants, from which we can extract cellulose [3], subject it to a cationization process [4] and use it as a flocculant to clarify the water and remove a portion of the phosphorus.<br>The objective of this study is not only to try to substitute the chemical flocculants that are normally used, but also testing wether our cellulose can be combined with this flocculant (Ferrous Chloride) in order to achieve the same performance while decreasing economic costs and environmental impact.<br>Methods:<br>The most important method we are going to use is the jar test. This test allows us to analyze at the same time several 500 ml samples with different amounts of coagulant to evaluate the optimal dose and its efficacy.<br>Different methods will also be used to measure pH (Hach pH meter), conductivity (Hach conductivity meter) and concentrations of different elements such as P or N (Merck kits). A study of the microfauna will also be carried out by microscopic observation.<br>These tests are performed to ensure that the coagulants used do not modify the characteristics (physical, chemical and biological) in a negative way.<br>Results:<br>After preliminary tests with different cellulose samples, positive results were obtained in 3 different samples of cationized cellulose from terrestrial plants. In these tests, a reduction of soluble phosphorus by 14 to 20% was achieved.<br>Conclusions:<br>The results obtained so far are promising, as other types of cellulose are still to be tested and a higher P removal is expected when combining this flocculant with the Ferrous Chloride.</p>José Miguel Pérez LombardoAna Moral RamaEva González Rodríguez
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
https://creativecommons.org/licenses/by-nc-sa/4.0
2023-09-132023-09-1312Transformation of process water in the agri-food industry for its use in the agriculture in order to improve the agricultural processes.
https://www.upo.es/revistas/index.php/biosaia/article/view/8395
<p>The agricultural sector is facing a challenge because they need to increase the productivity of the agricultural crops due to the growing global population. Moreover, another problem is the negative impact that the chemical fertilizers<br />and pesticides have in the ecosystems and human health. For this reason, investigation is needed to find other type of fertilizers to avoid this detrimental impact. Nowadays, biostimulants are playing an important role, they are considered as plant growth regulators because they enhance flowering, germination, plant growth and crop productivity and are<br />environmental-friendly [1].<br />In this study, the efficiency of the microalgaes and their use as biostimulants have been put to test. In order to prove the effects in the plant growth and crop productivity using lettuce as test crop, seven solutions have been prepared with different concentrations of natural fertilizer, microorganisms and microalgae. After that, these solutions were<br />tested to prove if microalgae promoted seed germination and plant development in radishes' seed. Finally, cut flowers (daisies) were used to prove the efficiency of microalgaes in the maintenance of post-harvey, so four solutions which had water, aspirin, microalgae culture (with the microalgae) and seaweed liquid extract (without the microalgae) were<br />prepared.<br />The results showed improve in the test crops as well as in the cut flowers. This can be explained because microalgae produce phytohormones like auxins, gibberelins, cytokinins and abscisc acid and other exogenous molecules as lipids, aminoacids, polysaccharide, etc. That can cause this beneficial effect [2].<br />In conclusion, the microalgae seems to be a perfect substitute for the chemical fertilizers because of its benefits and non-harmful effects.</p>Verónica Castillo RuizAroa Sánchez LópezManuel Del Valle González
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
https://creativecommons.org/licenses/by-nc-sa/4.0
2023-09-132023-09-1312Characterization of the antimicrobial activity of a clone with metagenomic DNA from a refinery.
https://www.upo.es/revistas/index.php/biosaia/article/view/8411
<p>Motivation: The increase in the number of antibiotic resistance mechanisms in microorganisms added to the low rate of new<br>antimicrobial (AM) development supposes a threat for public health that should be urgently approached. Bacteria are one of<br>the biggest sources of AM but only a small fraction of them can be cultured in vitro. To overcome this limitation our lab uses a<br>functional metagenomic approach and had previously developed a heterologous expression system that allows the study of all<br>the gene pool from a specific environmen1,2. Using this strategy we found different clones whose metagenomic DNA expresssion produces AM activity against Micrococcus luteus and also against the Methicillin-resistant Staphylococcus aureus (MRSA), one of the World Health Organization’s main priorities regarding resistant bacteria. In this work we study the properties and the activity of the AM produced by three different subclones with homologous genes related to phenol metabolism that were found in two different metagenomic libraries and we further characterize one of these subclones, pMPO1718, which proceeds from a refinery metagenomic library.<br>Methods: The AM is produced in liquid cultures, the antimicrobial production is increased with arabinose for 6-7h and then<br>the supernatant is filtered and lyophilized. The AM activity is tested on LB soft agar plates inoculated with the target strains.<br>The AM is separated in different fractions by chromatography to study in which of them the activity is present.<br>Results: We have defined a protocol to produce the AM in minimum media complemented with tryptophan and we have<br>observed AM activity against M.luteus in the filtered culture and even higher activity in the total culture extracted with acetone<br>50%. The last one was analyzed by high performance liquid chromatography and mass spectrometry by Fundación MEDINA<br>and the comparison with databases showed that it may be an AM not previously described. Some AM properties are<br>characterized in this work such as the minimum inhibitory concentration, the minimum bactericidal concentration, its<br>thermostability, its activity in different solvents or against different bacteria.<br>Conclusions: The filtered supernatant of the three different subclones has AM activity against M.luteus and MRSA produced<br>by phenol hydroxylase related genes. This AM is thermostable until more than 100ºC, has bactericidal activity and can be<br>produced either in LB medium or in M9 medium complemented with tryptophan.</p>Luis Andreo-Andreu Amando Flores DíazCynthia Alias-VillegasSebastián Acosta-Jurado Alejandro Díaz-Moscoso Eva María Camacho Fernández
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
https://creativecommons.org/licenses/by-nc-sa/4.0
2023-09-132023-09-1312Analysis of the association between bacteriophages and related strains Pseudomonas aeruginosa bacteria.
https://www.upo.es/revistas/index.php/biosaia/article/view/8488
<p>Motivation: Bacteriaphages can stay as prophages inside host genome when bacteria are infected by them. Bacteria use numerous defense systems against them, some of them were known years ago. CRISPR-Cas systems are an acquired inmunity systems in bacteria that recognize and degrade phages specifically through the spacers. Using numerous genomes of different strains from Pseudomonas aeruginosa, a ESKAPE group bacteria, prophages can be associated with certain strains by some of their features, as the isolation source, the geographic location or the CRISPR-Cas systems which they have, in order to know the which features must have a bacteria to be infected by a particular bacteriophage.<br>Methods: It was used some bioinformatic tools to predict multiple prophages in P. aeruginosa genomes and a few scripts written in some programation languages such as Python or Bash. In order to analyze the relationship between strains features and prophages, it was built some graphical plots by using RStudio, such as heatmaps and barplots. Also, it was performed some statistical tests to determinate the significancy of the results.<br>Results: Some of most present phages in P. aeruginosa are correlated with specific strains and some of their feature, specially the CRISPR-Cas systems, as the CRISPR type E or strains without CRISPR-Cas systems. In addition, some of these phages are present in strains with only one particular CRISPR-Cas system or isolated from only a certain source. Finally, phages were associated to the presence of particular transmembrane protein, to discover what protein use the phages to infect host cells.<br>Conclusions: The results suggest that bacteria have a set of certain genes, such as transmembrane proteins that allow the adhesion and infection of particular phages in the host cell, so bacteria have to obtain specific CRISPR-Cas systems against these bacteriphages to recognize and degrade them, in order to avoid the infection.</p>Antonio Moreno RodríguezAntonio Jesús Pérez Pulido
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
https://creativecommons.org/licenses/by-nc-sa/4.0
2023-09-132023-09-1312Generation of new tools to fight/counteract protein aggregation.
https://www.upo.es/revistas/index.php/biosaia/article/view/8416
<p>Cells need to eliminate unfolded, damaged, or aged protein in order to maintain protein homeostasis, a mechanism known as proteostasis. Whereas molecular chaperones contribute to proteostasis by promoting correct protein folding, the proteasome, and autophagy are the main proteolytic machinery for the selective elimination of these proteins.¹ Mutation in components of these systems or stress situations that overwhelm these proteostasis mechanisms results in the accumulation of damaging protein aggregates that in humans cause neurodegenerative diseases. Furthermore, during aging, there is an overall decrease in the activity of these proteins’ clearance machinery which generates a state of chronic proteostasis stress that eventually leads to cell death.²<br>In this project, we aim to build different genetic tools to assay proteolytic activities in S. pombe cells and determine the relative level of protein aggregation. These tools will allow us to characterize in more detail both, the stress-induced protein aggregations and the activity and relative level of aggregation in different proteasome mutants. In addition, using different molecular biology techniques, we will develop genetic tools to induce proteotoxicity through the expression of human proteins prone to aggregation, and hallmarks of neurodegenerative diseases.<br>Finally, it is known that molecular chaperones prevent protein aggregation by interacting and blocking aggregation-prone domains. In this context, using both, a mutant of the proteasome which accumulates protein aggregates and mimics an aged cell, and some of the tools generated.., we will screen compounds that function as chemical chaperones and decrease proteotoxicity. These chemical chaperones might help to develop new treatments for neurodegenerative diseases and aging.³</p>María Dolores Berdún-Reina Rafael Rodríguez DagaSilvia Salas-Pino
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
https://creativecommons.org/licenses/by-nc-sa/4.0
2023-09-132023-09-1312¿Qué puede hacer la bioinformática para responder preguntas microbiológicas?
https://www.upo.es/revistas/index.php/biosaia/article/view/8494
<p>Motivation: Bioinformatics now makes it possible to analyze thousands of bacterial genomes and answer biological questions such as how many different genes we can associate with a species and how to discover gene markers linked to resistance and other bacterial defense systems.<br>Methods: In this talk I showed how to perform bacterial pangenomes using several public and home-made bioinformatics tools, including the Sma3s gene annotator, or the Roary pangenome software.<br>Results: With this methodology, we have analyzed groups of genomes of human pathogenic bacteria with CRISPR-Cas-acquired immunity systems and have discovered that they are highly specialized in phages, especially in variants called phagoplasmids. In addition, we have found that some of the genomes carrying these systems may have been forced to acquire them because they have genes that encode possible receptors for entry of specific phages.</p>Antonio Jesús Pérez-Pulido
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
https://creativecommons.org/licenses/by-nc-sa/4.0
2023-09-132023-09-1312Heterologous models for the detection of subcellular targets of rhizobacterial effector proteins
https://www.upo.es/revistas/index.php/biosaia/article/view/8497
<p>Many soil bacteria have evolved to interact with root plants in a special environment called rhizosphere. This interaction is mediated by different bacterial molecules as surface polysaccharides or proteins that bacteria secrete through distinct mechanisms. Among them, secretion system types III, IV and VI are specialized in inject proteins named effectors directly in the cytoplasm of the eukaryotic host cell, whose role is involved in the interference of different plant cell molecular pathways. The compatibility of plant-bacteria interaction is mainly determined by the plant recognition of molecular signals of a friendly or a phytopathogen bacteria, being the effectors one of the most determinant bacterial signals. Some effectors can be recognized by plant defenses what induces a blocking in the bacterial invasion, while others have evolved to counteract the immune response of the plant promoting therefore the plant invasion.</p> <p>Rhizobia, as many other plant interacting bacteria, use distinct protein secretion systems to translocate proteins into legumes root cells, what contribute to the invasion of vegetal tissues to establish a mutual beneficial interaction known as nodulation. In this mutualism while the plant provides bacteria with nutrients and a safe non-competitive environment, bacteria promote the fixation of atmospheric nitrogen that is directly transferred to the legume. Rhizobial effectors secreted by type three secretion system has two possible effects in the establishment of the legume symbiosis. They can be recognized by plant receptors that block the nodulation process, but in other cases they are necessary to induce the nodulation. This duality is strictly related to the specificity of plant-bacteria relationship, arguing some clues in the co-evolution of rhizobia and legumes, and explaining the nodulation range of bacteria.</p> <p>At molecular level, the study of the rhizobial effectors inside root cells is difficult to face, and therefore the use of heterologous models to contribute to its study is desirable. In this work, we use a rhizobial effector (NopL) to illustrate by a multidisciplinary approach where does it localizes in heterologous hosts as tobacco leave cells, and even in transfected and/or Salmonella-infected animal cells. The consistency of these results serves as example to study the location inside eukaryotic cells of effectors in distinct hosts with different handling technics that can be used in almost every research laboratory.</p>Irene Jimenez-GuerreroFco Javier Lopez-BaenaEva Mª CamachoCarlos Medina
Derechos de autor 2023 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
https://creativecommons.org/licenses/by-nc-sa/4.0
2023-09-132023-09-1312