Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria
Revista de los másteres de Biotecnología de la UPO
Study of the role of importin alpha Imp1 in the heat shock response.
Motivation: Protein homeostasis or proteostasis is essential for cellular survival and tissue homeostasis. Cellular proteostasis is maintained by a tight balance between protein synthesis, folding and degradation. Various stress conditions, mutations, translation defects, etc. can disrupt this balance, leading to the accumulation of misfolded proteins prone to aggregation, a hallmark of several human diseases [1]. In this situation, cells activate the heat shock response (HSR), a transcriptional response driven by the universal transcription factor Hsf1, which upregulates molecular chaperones to counteract folding stress [2]. Among these chaperones, Hsp104 plays a key role in disaggregating misfolded proteins, promoting their refolding or degradation. However, the mechanisms regulating Hsp104 subcellular localization remain poorly understood. Previous results from the laboratory have shown that in the absence of Importin alpha Imp1, which functions in nucleocytoplasmic transport, the cellular distribution of the chaperone disaggregase Hsp104 is altered [3]. Methods: In this work, we used a combination of live-cell fluorescence microscopy and genetic analysis to characterize the role of Imp1 in the regulation of Hsp104 nucleocytoplasmic transport and its contribution to cellular proteostasis under folding stress conditions. Results: Our results show that, in the absence of Imp1, nuclear levels of Hsp104-GFP are significantly reduced compared to wild-type cells and it is accumulated in nuclear foci or aggregates under non-stressed conditions. Upon heat stress, Hsp104 nuclear levels further decrease in imp1Δ cells, correlating with an impaired stress response and reduced cellular fitness. Currently we are testing whether Imp1 is required for the nuclear localization of other HSR chaperones as part of a general mechanism to maintain nuclear proteostasis in unperturbed conditions and upon folding stress. Conclusions: Our findings demonstrate that Importin-α Imp1 is required to maintain cellular fitness upon folding stress through the regulation of Hsp104 nucleocytoplasmic transport.
Derechos de autor 2025 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria https://creativecommons.org/licenses/by-nc-sa/4.0
Serial repitching: its effect on lager yeast fermentation properties.
Motivation: The term serial repitching is given to the act of harvesting yeast at the end of beer fermentation with the aim of using it in the brewing of subsequent batches. It has been shown that serial repitching causes progressive deterioration of the physiological state of the yeast cells due to damage accumulation as a result of exposure to stress, and subsequently, an alteration in the brewing product. Nevertheless, the magnitude of this physiological deterioration varies according to the yeast strain [1]. The aim of the research is to evaluate the effect of serial repitching of the lager yeast used by an industrial brewery under its regular production conditions for eight consecutive generations. Methods: For this purpose, the lager beer data collected during production by the brewery’s quality control laboratory on a routine basis during the years 2023 and 2024 was analyzed. The parameters considered were total fermentation time, final attenuation, pH and viability, vitality and size of the lager yeast cells. In addition, the tasting notes made by the brewers from the brewery's tasting panel were also taken into account. Results: Our analysis shows that most of the parameters do not show any significant difference. However, a slight difference was observed for the fermentation time, which could be caused due to the extended lag phase which is usually seen in freshly propagated yeast [2]. Conclusions: The results suggest that serial repitching at the level studied in the research has a minimal effect on the physiological state of the lager yeast strain used by the brewery and, therefore, on the quality of the resulting beer. Although this result was expected [3], the possibility that this lack of significance in the results is caused due to a low precision in the analysis cannot be ruled out, and further research with more homogeneous data would be necessary.
Modulation of the tumor microenvironment in pancreatic cancer.
Motivation: Pancreatic cancer is one of the most aggressive malignancies, with a five-year survival rate below 5%. Tumor microenvironment (TME) plays a key role in tumor progression, promoting cell proliferation, angiogenesis, and immune system evasion. Pancreatic stellate cells (PCSs) play a major role in this TME. Normally, PSCs are quiescent and store vitamin A in lipid droplets and regulate extracellular matrix (ECM) homeostasis. Upon activation by tumor-derived signals, they lose lipid droplets, gain a myofibroblast-like phenotype, overexpress α-smooth muscle actin (α-SMA) and increase production of ECM (collagens, laminin, etc.). Activated PSCs can facilitate cancer progression by interacting with pancreatic cancer cells and other cells in the TME, which in turn leads to excessive fibrosis, promotes tumor metastasis, induces chemo-radio resistance, and contributes to immunosuppression [1]. The inactivation of PSCs is a promising approach to target pancreatic cancer. Pancreatic and hepatic stellate cells share many features. Previous studies from our lab have shown that the transcription factor GATA4 induces the inactivation of hepatic stellate cells (HSC) [2]. In this work we show that another member of the GATA transcription factor family, GATA6, is a regulator of PSCs phenotype, inducing the reversion towards an inactive state. Methods: GATA6 was overexpressed in PSCs via adenoviral infection to evaluate its role in reversing activation. Protein expression was assessed by Western blot, and immunocytochemistry analysis were used to analyze α-SMA, laminin and collagen expression. RT-qPCR was performed to quantify gene expression changes, and Oil Red staining was used to evaluate lipid droplet accumulation. Results: Western blot and immunocytochemistry showed that GATA6 overexpression reduced α-SMA levels, indicating the inactivation of PSC. Immunocytochemistry analyses also confirmed the inactivation by GATA6, displaying a decrease in laminin expression. RT-qPCR and Oil Red staining have not yet provided conclusive data. Conclusions: These preliminary results suggest that GATA6 may play an important role in the regulation of PSC activation, as the reduction of key activation markers such as α-SMA indicates a potential for GATA6 to revert PSCs phenotype. This could provide new insights into therapeutic strategies for modulating the fibrotic TME and improving treatment responses in pancreatic cancer.
Impact of expression of the carbon flow regulation on sucrose production in Synechocystis sp. PCC 6803.
In response to the increasing demand for sustainable production solutions [1], cyanobacteria have emerged as a promising alternative. Synechocystis sp. PCC 6803 is a model organism commonly used to study carbon metabolic fluxes. This moderately halotolerant cyanobacterium accumulates sucrose and glucosylglycerol as compatible solutes under salt stress. Adaptation of carbon flux during nitrogen deficiency promotes the accumulation of glycogen as a carbon pool not combinable with nitrogen [2]. In this context, the potential biotechnological interest of modulating carbon flux to redirect it towards sucrose production was explored [3]. Strains overexpressing regulatory proteins involved in carbon flux and carrying a plasmid inducibly expressing sucrose-phosphate synthase (SPS) and the heterologous permease CscB were phenotypically characterized. Under salt stress, the expression of these regulators led to a 40% increase in sucrose production compared to non-induced levels, reaching a maximum productivity of 48.16 mg L⁻¹ h⁻¹. Under these conditions, a partial redistribution of fixed carbon toward sucrose production occurred, at the expense of glycogen accumulation and biomass generation. Additionally, an improvement in the photosynthetic activity of these strains was observed due to the presence of this carbon sink. The effect of eliminating glucosylglycerol synthesis on sucrose production was also analyzed. Under high salinity conditions (400 mM NaCl), this mutation contributed to an additional 10% increase in sucrose production, reaching a maximum of 2.72 g/L. These results suggest that modulation of carbon flux can significantly increase sucrose production, making it a valuable exogenous source of carbon or in co-cultures with heterotrophic organisms producing compounds of interest.
Studing the potential of complex sugars as new proteasome inhibitors
The proteasome is the main cellular proteolytic complex responsible for degrading short-lived and damaged proteins. The proteasome is composed of a catalytic complex named 20S proteasome and a regulatory particle named 19S that provides substrate specificity. Proteasome function is critical for many different cellular processes such as cell-cycle regulation, apoptosis, immune response etc. (Adams 2003) and consequently proteasome dysfunction is associated to several human diseases such as cancer or neurodegenerative diseases. Furthermore, during aging, there is a progressive decline in the activity of the proteasome which contributes to the accumulation of protein aggregates resulting in loss of cell and tissue fitness (Hegde 2023). Thus, the search for proteasome modulators is one of the current challenges in cancer, aging, and neurodegeneration therapies. Several proteasome inhibitors such as Bortezomib or Carfilzomib…, are currently used for the treatment of certain types of cancer However, these inhibitors frequently lead to drug resistance, which highlight the necessity for a continuous search for new proteasome inhibitors (Nunes 2017). In this context, the main motivation of this work is the search for new structure-based proteasome inhibitors. Complex sugars are macrocyclic oligosaccharides with truncated cone geometry (Díaz-Moscoso 2010) which show structural similarity with the catalytic 20S proteasome. In this work, we are testing the effect of several complex sugars, specifically designed to match proteasome structure as potential inhibitors of the proteasome. By using live cell microscopy, cell fitness assays and a combination of in vivo and in vitro techniques to measure proteasome activity we show that several variants of these complex sugars have inhibitory activity on the proteasome in vitro and in vivo. In view of the results, we are currently characterizing the cellular effects of this complex sugars that have shown inhibitory activity on the proteasome and also, we are testing the effect on proteasome activity of new and improved variants of these complex sugars.
In vitro modelization of totipotency to study DNA damage response.
Embryonic development begins with the fusion of gametes that form a zygote, capable of differentiating into all cell types in the organism, in a state known as totipotency. This process involves intense epigenetic and metabolic reprogramming that are likely to generate high levels of stress in embryonic cells, which, paradoxically, are not only tolerable but may be necessary for the induction of totipotency (Xu & Liang, 2022). The mechanisms behind this response and their relationship to cell differentiation are still not fully understood. Furthermore, obtaining totipotent cells in the lab is a complex process, so alternative models are being developed to study these changes in vitro. Our study aims to investigate different responses to DNA damage in an in vitro model of totipotency. To achieve this, we first generated a mouse embryonic stem cell (mESC) line that allows to detect totipotent cell by GFP expression. We transfected mESC with a plasmid that includes the GFP gene under the control of the MERVL retrotransposon promoter, whose expression is linked to the totipotent state (Ishiuchi et al., 2015). After transfection, the cells were selected and genotyped to confirm the correct insertion of the construct, thereby generating the MERVL-GFP cell line. We then performed reprogramming assays through splicing inhibition, a recently discovered method that induces a state of totipotent-like cells (Shen et al., 2021). For this, pladienolide B was used as indicated in the previous study. After a temporal study, we identified the optimal time point for reprogramming with the highest proportion of GFP-positive cells, confirmed by qPCR, sorting, and confocal microscopy. Additionally, we performed differentiation assays and alkaline phosphatase staining, as well as embryoid body studies. The results showed that the MERVL-GFP cell line exhibits behaviour similar to non-transfected mESCs, indicating that our new cell line retains its pluripotency and differentiation capacity. With this cell line, we aim to enrich the population of reprogrammed cells to study the effects of DNA damage in the context of the totipotent state. This model could be key to solving problems related to fertility and in vitro fertilization, opening new avenues in the study of developmental biology.
Mechanisms Regulating the Morphogenesis and Homeostasis of Epithelia
Motivation: Epithelia are robust tissues that support the structure of embryos and organs. These tissues have evolved diverse cellular architectures, which are acquired through a complex combination of cellular processes, including cell shape changes and cell migration. The control of these processes is fundamental for proper epithelia morphogenesis, and to maintain epithelial integrity in homeostasis and regeneration. Furthermore, disruption of these processes can lead to diseases, such as cancer. Thus, it's imperative to understand the molecular and cellular mechanisms by which precise multicellular structures arise during development and become disrupted during disease. The follicular epithelium (FE) of the Drosophila melanogaster ovary provides us with a monolayered epithelium model, which, as part of its normal development, suffers a big event of morphogenesis, involving coordinated changes in cell shape and collective cell migration, which ultimately leads to elongation of the tissue and egg formation. This process is a potent model to perform functional and dynamic assays of epithelia morphogenesis in vivo. In the search for factors that could participate in this processes, we came across with capicua (cic). Cic is a transcriptional repressor involved in many types of cancer, and has been identified as mediator of EGFR in control of cell proliferation and cell differentiation. In this work, we focused on uncovering the role of cic in regulating cell shape changes and cell migration. Methods: We analysed morphogenetic events in Drosophila ovary in both wild-type and cic mutant conditions using different genetic approaches, such as Gal4/UAS and FRT/FLP systems. We are using in vivo confocal microscopy and biophisycal tools, such as laser ablation, to characterize the effects of modifying cic expression in cell and tissue shape. Results and Conclusions: We have shown that cic expression pattern changes during FE development. Functionally, down regulation of cic blocks changes in cell shape and cell migration leading to defects at a tissue level. Furthermore, cic loss of function induces alterations in the expression of two key regulators of cell shape changes, the actin cytoskeleton and the integrin adhesion proteins. These results lead us to propose a model in which cic might regulate the cell shape changes underlying epithelia morphogenesis acting as a repressor to modulate cytoskeletal and adhesion proteins levels.
Molecular analysis of the symbiosis between Nostoc punctiforme and Oryza sativa.
Nostoc punctiforme is a filamentous, diazotrophic cyanobacteria capable of forming symbiotic relationships with diverse plant species (Álvarez et al., 2020; Álvarez et al., 2023). Given the widespread use of synthetic nitrogen fertilizers in rice cultivation and their associated environmental issues, biological nitrogen fixation (BNF) by cyanobacteria presents a promising alternative (Álvarez et al., 2023). However, the molecular mechanisms underpinning the symbiotic association between N. punctiforme and rice remain poorly understood. In well-characterized symbiotic interactions, such as those occurring between legumes and nitrogen-fixing rhizobial bacteria, the common symbiotic signalling pathway (CSSP) is activated. This process begins with Rhizobium secreting Nod factors in response to flavonoids released by compatible plants. Álvarez et al. (2022) demonstrated for the first time the expression of Nod-like proteins in N. punctiforme and the participation of the CSSP pathway in rice during symbiosis. Based on these previous results, this study aims to elucidate the genetic basis of this interaction by characterizing orthologous genes in Nostoc related to the nodD regulatory gene in Rhizobium leguminosarum, which play a pivotal role in symbiosis by regulating the expresion of the genes involved in the generation of nod factors. Bioinformatic analyses were conducted to identify homologous sequences of nodD from R. leguminosarum in N. punctiforme, and structural modelling compared their potential functional roles. Three key nodD homologs with conserved DNA-binding and inducer recognition domains were found in N. punctiforme, called nodD1, nodD2 and nodD3. Using homologous recombination, loss-of-function nodD gene mutants were generated in N. punctiforme and characterized. Genetic characterization revealed the maintenance of wild type copies of genes nodD1 and nodD3 in their respective mutants, suggesting that those genes may be essential. Preliminary symbiosis analysis results with different symbiotic plant species seem to indicate reduced root adherence, highlighting their possible involvement in stablishing symbiosis. Further symbiosis assays with rice are needed to evaluate endophytic colonization efficiency. These insights contribute to understanding microbial symbiosis and support the development of cyanobacteria-based biofertilizers for sustainable agriculture.
Patient-derived brain organoids: A new approach to studying brain tumors.
Motivation: Brain tumors are the most common type of solid tumors in children and the leading cause of cancer-related deaths in this age group [1]. Although advances in diagnosis and treatment have significantly improved survival rates, patients are still dying due to this disease, so further research is still crucial. Current models used to study these tumors are cell cultures and animal models, which do not fully replicate the tumor biological complexity and the dynamic environment of the human brain. To address these limitations, we are developing patient-derived brain organoids—miniature brain models that closely mimic the human brain— which incorporate immune cells and tumor cells. These advanced organoids will provide an invaluable tool for studying current therapies and exploring potential future treatments. Methods: Urine samples were collected from patients and processed until colonies of urine-derived epithelial cells appeared. These colonies were reprogrammed into iPSCs using the CytoTune™-iPS 2.0 Sendai Reprogramming Kit [2]. Once iPSC lines were established, patient-derived organoids (PDOs) were generated following Lancaster’s protocol [3]. Some organoids were then co-cultured with microglia to promote its development, while others were injected with CFSE-labeled DAOY medulloblastoma cells on day 40 and allowed to grow. PDOs were subsequently analyzed using immunohistochemistry techniques to assess cell composition, as well as the presence of microglia and tumor cells. Results: PDOs exhibited markers of neural progenitors (Nestin) which were actively proliferating (Ki67 positive), and immature neurons (Doublecortin), indicating that the organoids accurately mimic the structure of the brain. Additionally, microglia successfully populated the brain organoid, rendering the model immunocompetent. We also assessed engraftment of CFSE-labeled tumor cells within the PDOs, which survived and proliferated, further supporting the relevance of the model. The next step will be integrating these insights into a single organoid to create a comprehensive model and to test how the PDOs respond to cancer treatments, such as radiotherapy. Conclusions: These findings demonstrate that our model will serve as a valuable and reliable tool for investigating therapeutic strategies for brain tumors. Moreover, since it is patient-derived, the model offers great potential for advancing personalized medicine by enabling more customized and precise treatment approaches.
Computational analysis of an integrative and conjugative element in a biodegrader bacterium.
In this study, we explore a shared sequence of an integrative and conjugative element (ICE) in Sphingopyxis granuli strain TFA. ICEs are significant mobile genetic elements, which are integrated into the bacterial chromosome and encode a fully functional conjugation machinery, making them self-transmissible between bacterial cells. ICE cargo genes can confer host selective advantages such as antibiotic resistance and virulence factors (Delavat et al., 2017). S. granuli strain TFA is a member of the Sphingomonadaceae family, has garnered attention for its metabolic capabilities, particularly in the degradation of aromatic compounds such as tetralin. This strain has been characterized through genomic analyses that reveal its potential for environmental adaptability and resistance mechanisms (García-Romero et al., 2020). For these reasons, the presence of ICEs in the S. granuli genome has been identified and confirmed using the ICEfinder tool (Liu et al., 2019). A unique sequence has been selected that does not occur in all other S. granuli strains but in other bacteria in which the conserved sequence is found with more than 99% identity and 80-96% coverage using skani, which is a program for calculating average nucleotide identity (ANI). Each of the genes present in the shared ICE was annotated using BLAST (Basic Local Alignment Search Tool) from NCBI. In addition, structural annotation of all genomes and shared ICE was performed using Bakta (Schwengers et al., 2021). Raw sequencing data have been used to confirm the integration site of the shared ICE. The ends of the ICE are currently being investigated with an evolutionary approach, a set of genetic elements around the ICE known as genetic archipelago is conserved among bacteria
From Yeast to Fly: A Study of the Evolutionary Conservation of Cohesin in Schizosaccharomyces pombe and Drosophila melanogaster.
Motivation: Cohesin is a crucial protein complex composed of Psm1, Psm3 and Rad21, arranged in a like-ring conformation. This complex plays a key role in maintaining chromosome structure, facilitating accurate chromosome segregation, regulating DNA repair, and controlling gene expression by influencing chromatin architecture and transcriptional processes. Defects in cohesin can lead to severe cellular consequences, including chromosome missegregation, genomic instability, and developmental disorders. Schizosaccharomyces pombe is an wide-spread eukaryotic model organism for genetic studies due to its simplicity, ease of genetic manipulation, and well-characterized genetic structure, making it an ideal tool for research in fundamental cellular processes. The evolutionary gap between Schizosaccaromyces pombe and Drosophila melanogaster (approximately 1.2 billion years) and between D. melanogaster and humans (around 600 million years) provides a unique opportunity to investigate the conservation and divergence [3] of cohesin function across a broad evolutionary spectrum. This study aims to explore the conservation and functional roles of cohesin in these species, providing valuable insights into its molecular mechanisms and the potential impact of its dysfunction across different eukaryotic organisms. Methods: Each of the three S. pombe cohesin subunits, Psm1, Psm3 and Rad21, were replaced by their D. melanogaster orthologues. This was achieved by transforming a diploid strain with a linear fragment containing the Drosophila gene, a selection marker and homology sequences with the upstream and downstream regions of the gene to be replaced. The correct insertion of the fragment was verified by PCR. Viability of the transgenic yeast in haploidy was studied by sporulating the heterozygous mutants and segregating their four spores using a micromanipulator. Results: Psm1 and Psm3 genes were replaced by their fly orthologues, SMC1 and SMC3, in a diploid strain resulting in a double heterozygous transgenic mutant. Upon tetrad segregation, it was observed that drosophila´s version can not substitute yeast´s natives genes.
Identification and characterization of last-resort antibiotic resistance genes and new antimicrobial compounds in environmental samples.
Motivation: The misuse of antibiotics has led to a global health crisis, with antimicrobial resistance projected to surpass cancer-related deaths by 2050 and potentially reaching ten million deaths annually. This resistance originates in environmental bacteria, which transmit resistance genes to pathogens via horizontal gene transfer. Additionally, most antimicrobials are natural products, primarily produced by bacteria. However, studying these mechanisms in environmental bacteria is limited, as fewer than 1% of them can be cultured. To overcome this, functional metagenomics and massive sequencing are key tools for identifying these genes in microbial communities(1). Previously, in our laboratory, metagenomic libraries from Los Alcornocales Natural Park soils were constructed and screened, isolating clones conferring resistance to last-resort antibiotics or producing novel antimicrobials. Methods: This work has two objectives: 1️.Evaluating MPO223 for NRPS (non-ribosomic peptide synthetase) expression: This new strain produces a PPTase (phosphopantethenil transferase) required for NRP synthesis(2). Its ability to generate NRP compounds is being compared with the control strain MPO554. Resistance to last-resort antibiotics: On the one hand, the capacity of the fosmids that confer resistance to ciprofloxacin in Pseudomonas to confer resistance in Escherichia coli was evaluated by conjugation and determination of the minimum inhibitory concentration (MIC). On the other hand, we have checked whether a specific region of the MerA25 fosmid is associated with resistance to meropenem, through its deletion. Results: Pseudomonas fosmids confer ciprofloxacin resistance in Escherichia coli, suggesting that resistance genes can be transferred between different bacterial genera. MIC analysis with and without inducers revealed that one of the fosmids carries a salicylate-independent promoter, indicating that heterologous expression is not required for resistance gene expression(3). For the MerA25 fosmid, deletion of a specific region resulted in loss of resistance, indicating that this fragment is essential for that function. For the evaluation of strain MPO223, we are currently constructing plasmids with the bpsA reporter gene to measure the PPTase activity.
Valorization of Seaweed and Seagrass Waste in Flocculant Synthesis.
Motivation: The accumulation of tide waste generates serious environmental and economic impacts on coastlines around the world. Among the most negative effects are the emission of greenhouse gases and the death of various marine species, caused by the creation of anoxic habitats due to eutrophication. In this context, there is growing interest in exploring the potential of tide waste as a source of cellulose materials for the production of flocculants, which would be used in wastewater treatment, aligning with the principles of the circular economy. [1] This approach also enables the production of new functionalized products, including flocculants synthesized from the extracted cellulose, in order to reduce dependence on petrochemical flocculants such as polyacrylamide.[2] Methods: The collected tide waste was subjected to a water washing process, followed by detailed chemical characterization. Experiments were then designed to investigate the morphological properties of the tide waste, specifically the ones derived from Halopteris scoparia, using various cooking conditions and the soda-anthraquinone process to extract cellulose. Lignin removal was carried out using hydrogen peroxide, and the morphological characterization of the product was performed with specialized equipment, the Morfi Lab (Techpap).[3] Results: The results showed key properties in the extracted cellulose fibers, through statistical analysis that helped identify the most suitable samples for achieving efficient flocculation. These findings highlight the use of tide waste as a sustainable alternative to traditional chemical flocculants, suggesting a promising path towards reducing the environmental impact derived from the use of chemicals in water treatment. Additionally, the utilization of these waste materials as resources in the circular economy helps mitigate the accumulation of marine debris, transforming it into a valuable material for industrial applications.
Increasing the vitamin E content of virgin olive oil: cloning and molecular characterization of genes involved in the biosynthesis of phytyl-diphosphate in olive fruit.
Virgin olive oil (VOO) is characterized by its distinctive organoleptic characteristics and exceptional nutritional properties due to its well-balanced fatty acid composition, as well as the presence of minor components such as natural antioxidants. Among them, alpha-, beta-, and gamma-tocopherols have been detected, with alpha-tocopherol representing more than 95% of the total tocopherol content, which ranges between 50 and 300 mg/kg. VOO with improved functional quality, such as the increase in vitamin E (alpha-tocopherol) content, is considered an important target for generating new cultivars in olive breeding programs. With this purpose, the identification of molecular markers associated with high vitamin E content is needed. Therefore, the general objective of this study is to improve the VOO nutritional quality by increasing the alpha-tocopherol content of olive fruit. To achieve this, we focus on the isolation and characterization of genes involved in alpha-tocopherol biosynthesis in olive fruit. Tocopherols are generated in photosynthetic organisms from the condensation of phytyl-diphosphate (PDP) and homogentisic acid, followed by cyclization and methylation reactions. The precursor PDP can be synthesized from phytol by the successive action of two kinase enzymes, phytol kinase (PK) and phytyl-phosphate kinase (PPK)(1). In the present work, the identification of olive genes with a high degree of similarity with Arabidopsis PK(2) and PPK(3) genes was carried out using the olive transcriptome and genome. From the identified sequences, a pair of specific oligonucleotides for each sequence were designed and the full-length cDNA clones encoding for olive PK and PPK were obtained using a PCR approach. Alignment of the sequences and phylogenetic analysis indicated that they code for PK and PPK enzymes. In addition, expression analysis by qRT-PCR was performed using different olive tissues, particularly in mesocarp and seeds from Picual and Arbequina at distinct developmental stages, as well as in mesocarp from cultivars with low and high tocopherol content. Furthermore, PK and PPK transcript levels were determined in olive mesocarp from fruits subjected to different abiotic stresses, including water deficit, low and high temperature, darkness, and wounding. Overall, this study constitutes a significant advance in elucidating the factors that regulate the tocopherol biosynthesis in olive fruit to obtain VOO with enhanced alfa-tocopherol content.
Cell death by ferroptosis in Caenorhabditis elegans models of neurodegenerative diseases. Implications of the mitochondria.
Ferroptosis is an iron-dependent cell death driven by the peroxidation of membrane phospholipids. To protect themselves from this harm, cells possess several defense systems among which we find peroxiredoxins (Prdxs). This protein family is characterized by the ability to scavenge peroxides and is composed of six members in mammals and three in Caenorhabditis elegans, PRDX-2, -3 and -6 (Ismail et al, 2019). C.elegans is a nematode that is an ideal model for the study of neurodegenerative diseases as it has conserved neurological pathways and simple organ and tissue organization (Caldero-Escudero et al, 2024). Among the three Prdx members in C.elegans, PRDX-2 and PRDX-6 are cytoplasmatic proteins, while PRDX-3 is located in the mitochondria. In this study, we have focused on the effect of these proteins in C. elegans models of polyglutamine-repeats (polyQ) protein aggregation and the possible implication in ferroptosis protection. Bearing this in mind, we have performed genetic crossing strategies to generate all double and triple mutant combinations of prdx-2, prdx-3 and prdx-6 genes. These strains have been used to determine the role of peroxiredoxins in development, progeny generation and effect on PolyQ aggregation in muscle cells using fluorescence. We demonstrate that mutations of peroxiredoxin genes, especially prdx-2, slow down the development of the nematode and reduce egg laying. Combinations of mutations in the other prdx genes enhance this phenotype. Moreover, prdx-2;prdx-3 mutants are letal, being arrested at the L3 state of the development, which might show a possible mitochondrial implication. prdx-6 simple mutants are barely affected when compared with wild type (N2) worms. However, prdx-2; prdx-6 mutants are greatly affected, having a huge decrease in lifespan and egg laying. Furthermore, there is also a reduction in the size of the worms. Finally, microscopy analysis on the integrity of the mitocondrial network are yet to be done in these mutants. In conclusion, the results obtained so far suggest that peroxiredoxins have influence on worms´ developement, maybe protecting them from ferroptosis. Also, mitochondria seem to play its own role during this process. Studies on the implication of ferroptosis and mitochondria, as well as on protein aggregation, are being carried out and need to be completed.
The iINS-4F mouse: A novel tool to explore the regenerative effect of partial reprogramming in the pancreas.
Type 1 diabetes mellitus is a metabolic disease caused by autoinmmune destruction of pancreatic β-cells. Although current therapies allow glycemic control, they do not restore the function of these cells, underscoring the need for innovative therapeutic approaches. In this context, partial reprogramming by transient overexpression of Yamanaka factors (OSKM) has emerged as a promising strategy in regenerative medicine (Chondronasiou et al., 2022). In this study, we introduce the iINS-4F murine model, designed to induce partial reprogramming specifically in pancreatic β-cells. This model combines the TetOn system with Cre recombinase, allowing controlled activation of OSKM exclusively in β-cells following doxycycline administration. To evaluate its potential, we implemented a partial reprogramming protocol based on short induction cycles in both young and old animals. We have performed a comprehensive characterization of the iINS-4F model, analyzing the specificity and expression level of OSKM as well as its impact on key β-cell identity markers. Our results reveal an initial phase of transient dedifferentiation, marked by the reversible loss of insulin and Glut2. The ability to recover after a rest period indicates a remarkable flexibility of the system. Furthermore, our data suggest that although age does not subtantially limit reprogramming and recovery, sex could influence the process. These findings position the iINS-4F model as a valuable tool for the study of partial reprogramming in pancreatic β-cells and open new possibilities for its application in innovative diabetes therapies.
Regulatory elements of VANGL2 expression – Implication in Neural Tube Defects.
Motivation: Neurulation is a complex process aimed at closing neural tube (NT) that occurs during embryogenesis. It is a highly regulated process which combines molecular and cellular events; if any disturbance happens and the closure is not complete, neuroepithelium remains open and neural tube defects (NTD) appear, which are among the commonest of birth defects. A breakthrough in understanding the basis of neurulation, and thus NTDs, was the discovery of the role of the non-canonical Wnt signalling pathway in this process, which is involved in coordinating cell polarity [1]. Mutations in Vangl2, a member of this pathway, have been linked to human NTDs. While studying Vangl2 regulatory regions in NTDs patients, it was found an alteration in CTCF binding sites. CTCF is a conserved transcription factor expressed ubiquitously which plays a key role during development and in the 3D organisation of the genome. Recently, it has been described that curaxin, a DNA-binding compound with epigenetic abilities, interferes with CTCF expression [2]. The purpose of this work is to elucidate the implication of CTCF on the regulation of Vangl2 expression and consequently on neurulation and the development of the disease. Methods: The approach was carried out by testing different concentrations of curaxin in vitro (human neuroblastoma cell lines) and in vivo (NTD-mouse model loop-tail, Lp). Lp mice carry a mutation in Vangl2 that causes craneorachischisis in homozygosity and spina bifida in heterocygosity, two types of NTDs [1]. In the in vivo model, the effect of curaxin will be tested in embryo culture in stages previous to the initial NT closure. Analysis studies include gene expression by qPCR, histology and image analysis (Qupath, Cell profiler). Results: The exposition to curaxin seem to have a negative impact in CTCF expression, afecting indirectly Vangl2 expression. This effect is reflected in vitro, causing an increase in neurites number, in line with previous results [3]; an in vivo, leading to impairments on NT closure. Conclusions: In conclusion, the results obtained suggest that the diminution in Vangl2 expression that causes neural tube defects could be related to an alteration in one of its transcription factors, CTCF. Nevertheless, the current experiments and more exhaustive studies need to be completed.
Bioreactor’s optimization through an innovative agitation-aeration system.
Motivation: Bioreactor systems are essential for optimizing biotechnological processes in industries like pharmaceuticals and food. Traditional systems, such as Rushton impellers, face challenges with energy inefficiency and poor oxygen transfer. The BioMixing’s aeration and agitation system, based on CFD simulations, offers a patented solution to improve oxygen transfer, mixing efficiency, and overall productivity (Ashok et al., 2017). Methods: The system’s performance was evaluated in two main experiments: 1) kLa (volumetric mass transfer coefficient) measurement via oxygen saturation and nitrogen purging (Vanags & Suleiko, 2022), and 2) mixing time measurement with the iodine-starch method.The experimental procedures were carried out following the guidelines outlined in the Dechema protocol. Both experiments were conducted to compare Rushton's aeration-agitation system with our innovative system, aiming to identify significant differences between them. Results:The BioMixing system achieved a remarkable 170% improvement in kLa at 250 rpm, and up to 60% at 400 rpm, compared to the conventional Rushton system. This enhanced oxygen transfer, especially at lower revolutions, is crucial for optimizing production processes in industries such as pharmaceuticals. Furthermore, the mixing time experiment demonstrated significant improvements in homogenization and medium distribution. At 700 rpm, the BioMixing system delivered performance enhancements of up to 29.4%, reinforcing its potential for more efficient bioprocessing (Nienow, 1998; Kelly, 2008). Conclusion:The BioMixing system outperforms traditional bioreactors in key parameters such as oxygen transfer and mixing efficiency, offering a promising solution for enhancing biotechnological productivity and sustainability.y. Future work will refine its application for broader microbial cultivation (Wang et al., 2020).
Synthesis and characterization of cationic cellulose-based flocculants from tidal waste.
Motivation: Synthetic flocculants such as polyacrylamide are widely used in industry, but they present several environmental problems due to their toxicity and dependence on non-renewable resources [1]. In this context, cellulose-based flocculants have emerged as a biodegradable and sustainable alternative capable of replacing synthetic polymers. Methods: This study aims to obtain and characterize cationized cellulose from algae and phanerogams of tidal waste in order to evaluate its potential application as a flocculant in wastewater treatment and other industrial processes. To this end, the waste was subjected to several process of conditioning, pulping and bleaching for the extraction of cellulose, followed by cationization by chemical treatment with CHPTAC [1]. In order to optimize cationization, preliminary tests were carried out using pure cellulose as reference material, varying the concentration of reactants and the liquid-to-solid ratio [2]. Structural and chemical characterization of the cationized samples was performed by X-ray diffraction (XRD) to evaluate the changes in the crystal structure of the cellulose after chemical modification [3], determination of the nitrogen content by the Kjeldahl method to quantify the degree of functionalization and analysis of intrinsic viscosity according to TAPPI T230. These analyses will allow correlating the reaction parameters with the level of cationization achieved, establishing the optimal conditions for the production of cellulosic flocculants. Results and conclusions: The results obtained in this study will contribute to the development of new bio-based materials that may reduce the dependence on synthetic polymers in flocculation, promoting sustainable solutions in the context of circular economy and management of renewable resources.
Search for biomarkers in an experimental model of hypoxic-ischaemic brain damage
Motivation: Hypoxic-ischaemic encephalopathy (HIE) results from sudden impaired brain blood flow, leading to oxygen deprivation and potential brain damage. Its incidence is neonates is 1 to 8 per 1,000 births, and despite being a rare disease, it ranks among the top 10 causes of childhood mortality in developed countries and contributes to life-long disabilities. The extent of motor, cognitive, and behavioural impairments depends on factors such as the affected brain region, duration of oxygen deprivation, and severity of the event. Therapeutic hypothermia is the primary intervention but early diagnostics are lacking, which highlights the need for advanced detection techniques. Our group’s single-cell transcriptomics (scRNA-seq) studies have analysed blood and vascular precursor responses in neonatal hypoxic-ischaemic injury. This study aims to validate scRNA-seq findings via flow cytometry, RT-qPCR and immunofluorescence, seeking for blood biomarkers. Methods: A neonatal hypoxia-ischaemia (HI) experimental model in mice was developed based on the Rice-Vannucci’s protocol. Postnatal day 7 (P7) mice underwent unilateral ligation of the left common carotid artery followed by 90 minutes hypoxia with 9% O2. 48 hours post-insult, a multiparametric analysis was conducted assessing peripheral blood via flow cytometry and brain tissue via immunofluorescence to characterise cellular and molecular responses. Results: Neonatal HI triggers neuroinflammatory and vascular alterations. Increased microglial activation (IBA1+) and leukocyte infiltration (CD45+) are expected in brain tissue, potentially modulated by CD31+ endothelial cells, which facilitate immune cell recruitment. Preliminary findings indicate increased Ki67+ proliferating cells in infarcted areas. Optimization of leukocyte (CD45, CD11b) and endothelial (CD31) staining in SHAM and HI samples is ongoing. Flow cytometry has been initiated, focusing on monocyte subsets due to previous scRNA-seq assays. An 8-colour panel has been optimized in control samples using markers: DAPI, CD45, Ly6C, Lyve1, CD172, CD36, and Siglec-F. While quantitative significance is pending, preliminary results align with previous scRNA-seq data. Conclusions: Expected findings suggest immune activation in brain tissue and peripheral blood, indicating the neuroinflammatory response after an HI episode. Aligning these results with previous transcriptomic studies could aid in developing rapid diagnostic strategies for HIE in newborns.
Use of brain organoids for the evaluation of toxicity and biodistribution of nanomedicines.
The evaluation of safety and efficacy is a crucial requirement in the development of new drugs. Traditionally, animal models, especially murine, have been the standard approach. However, growing evidence suggests that these models often fail to accurately replicate human physiology, a limitation that is particularly pronounced in the study of drugs targeting the central nervous system (CNS). This is due to significant molecular and cellular differences between the human brain and rodent models. Furthermore, animal experimentation entails high costs, requires specialized personnel, and is increasingly restricted by regulatory and funding agencies, driving the need for more predictive human-based models(García-Delgado et al., 2022). Human 2D cell cultures offer an alternative, yet they present limitations, such as the inability to replicate the three-dimensional architecture of tissues and the complex cell-cell interactions found in vivo. In contrast, 3D in vitro models, like brain organoids, have emerged as powerful tools to better simulate human brain tissue and to explore novel therapies for neurological diseases. These multicellular models, typically derived from pluripotent stem cells, mimic the structural and functional complexity of the human brain more closely than traditional 2D cultures(Lancaster et al., 2013). Nanoparticles (NPs) represent a promising approach in both therapeutic and diagnostic fields. Functionalized NPs loaded with bioactive compounds, such as cannabinoids, have shown anti-inflammatory, antioxidant, and antinociceptive effects without psychoactive properties, offering alternative mechanisms to currently available CNS drugs (Álvarez-Fuentes et al., 2012). This study aims to develop cerebral organoids to assess the biodistribution and toxicity of PLGA-PEG polymeric nanoparticles loaded with cannabidiol (CBD), synthesized via nanoprecipitation (NPP). The evaluation of CBD-NP behavior within the CNS, using cerebral organoids, will provide insight into their potential therapeutic application. For biodistribution analysis, NPs were labeled with the fluorophore Nile Red for visualization under fluorescence microscopy. Immunohistochemical staining was also performed on organoid sections to evaluate the biodistributión of NPs and assess potential cytotoxic effects.This approach aims to advance CNS drug delivery by combining cerebral organoids with nanoparticles, promoting safer and more effective therapies.
Isolation and characterization of natural yeasts for beer brewing
In the last two decades, there has been a rise in the craft brewery sector in our country. Although this industry operates on a small scale, craft brewers are willing to experiment with new flavors, produce native beer varieties, or develop healthier options in response to consumer demand, aiming to challenge the long-established and consolidated beer market [1]. One of the approaches to achieving these novel beer varieties is through the use of new yeast strains. Traditionally, brewing yeasts belong to a small group within the genus Saccharomyces. However, many brewers are inclined to explore alternative yeasts to introduce new characteristics into their beers. The main objective of this study is the identification and characterization of wild yeasts to assess their potential for beer production. The isolated yeast strains were classified through restriction fragment analysis of the ITS region. This method yielded distinct banding patterns corresponding to each isolated strain. Sequencing of the ITS fragment will allow for precise species identification [2]. To characterize the yeasts phenotypically, their fermentative capacity was evaluated. Sugar consumption was measured through growth curves in wort, and CO₂ production was analyzed by monitoring weight variation over ten days. Additionally, the ability to ferment maltose, the most abundant sugar in wort, was assessed using Durham tube assays in a minimal medium containing maltose as the sole carbon source [3]. Finally, we examined the killer activity of the analyzed strains to determine their ability to dominate fermentation over other brewing yeast strains. Commercial brewing yeast strains were used as controls in all experiments. Currently, we are analyzing the growth capacity of selected strains under different carbon sources, temperature conditions, and hop concentrations, as well as assessing their potential for producing undesirable phenolic compounds in beer. Finally, we will brew beer with selected strains to evaluate their brewing potential. Based on our findings, we conclude that we have identified new yeast strains with potential applications in beer production, which require further characterization.
Assessment of the Subchronic Toxicity of Salicornia ramosissima Extract in Rats Over a 90-Day Period.
Lifestyle behaviours have been shown to influence the risk of stroke. Polyphenol-rich diet or the use of polyphenol-containing supplements are suggested as a potential strategy for stroke prevention, by improving lipid profiles, blood pressure, insulin resistance and systemic inflammation. In this context, Salicornia ramosissima, or glasswort, is a halophyte found along the Mediterranean and Atlantic coast that have been reported to exert a benefit in flies and mice animal models of stroke. Moreover, Salicornia extracts have been shown to be safe in healthy volunteers at short term, and to modulate several vascular parameters in patients after suffering a transient ischaemic attack. These positive results encourage further research to develop effective food supplements for the prevention or treatment of neurovascular disorders, particularly stroke. However, its use cannot be fully recognized without comprehensive biosafety studies. Therefore, a subchronic 90-days toxicity study was conducted following EFSA guidelines. A total of 80 Wistar rats (40 males and 40 females) were randomly assigned to three different doses of Salicornia extract or placebo. Animals were monitored for clinical symptoms as well as for food and water consumption. At the end of the study, the animals were sacrificed and blood samples were collected for haematological analysis. In addition, the organs were dissected and processed for histological analysis. The results obtained showed no signs of toxicity at the clinical, biochemical or histological levels at any dose, supporting the safety of this extract for future clinical studies.
Development of a semicontinuous system to evaluate the effects of K2 toxins on wine yeasts.
Some yeast species can be infected by viral sequences from the Totiviridae family, which encode toxins (1). These toxins provide a competitive advantage to the yeast carriers by inducing the death of sensitive cells (2). Additionally, "killer" yeasts must possess another auxiliary virus, called LA, responsible for providing the capsid and the replication functions for the killer virus. This study focuses on wine yeasts infected with the M2 killer virus, which encodes the K2 toxin, maintained by the auxiliary virus, L2. The main objective of this work is to develop and assay a semicontinuous culture system that allows maintaining the culture in its exponential growth phase for as long as possible. This will enable the evaluation of competition between infected and non- carrier yeasts, as well as the effects of these toxins on wild wine yeasts. Since toxins are only active in a narrow range of pH the variation in the pH of the culture, caused by yeast metabolism, must be monitored and controlled using a pH -coupled peristaltic bomb that adds fresh medium to keep cells in proliferative state while keeping pH in the toxin active range. To assess competitiveness between two yeast strains and observe the behavior and evolution in co-culture, strains must be genetically labeled to distinguish between them, to differentiate between yeasts that produce K2 and those that do not. Our last goal is to select yeast strains suitable for their use in wineries, so we have chosen to label the strains with spontaneous resistance mutations. This strategy allows the marked strain to be used in the food industry without restrictions. The spontaneous generation of mutants is carried out using different protocols, in which various toxins are employed, such as sulfometuron (3), copper sulfate (4), or 2-deoxyglucose (5). In this poster we present preliminary results on the setup of the pH controlled bioreactor and the analysis of different strategies to isolate yeast spontaneous mutants.
Revaluation of tidal waste in wastewater treatment plants.
Motivation: The latest European legislation on the use of chemical products in wastewater treatment requires a reduction in their usage. Consequently, essential operations in this process, such as contaminant removal and sludge flocculation, require alternative solutions [1]. Although replacing the efficiency and cost-effectiveness of chemical flocculants is challenging, natural flocculants derived from plant residues and algae are proposed as a promising solution to reduce these polluting products while also valorizing waste, thus promoting a circular economy [2]. Methods: This study focuses on two approaches: phosphorus removal from wastewater and improved sludge flocculation. The optimal flocculant dose required for phosphorus reduction was determined using a jar test. Specifically, 500 mL of wastewater was used for each sample, and soluble phosphorus levels were measured before and after the procedure. The testing protocol for each chemical substitute was as follows [3]: 1. One minute of stirring at 80 rpm (revolutions per minute). 2. Fifteen minutes of stirring at 30 rpm. 3. Twenty minutes of settling. For the sludge tests, the experiment was conducted manually using 100 mL of sludge. Results and conclusions: The tests confirmed that natural flocculants effectively reduce contaminant levels. Similarly, they were observed to aid in sludge flocculation and decrease the amount of chemical flocculant required. Additionally, while the cationization of flocculants did not significantly affect sludge flocculation, it did enhance the removal of contaminants such as phosphorus. However, further research in this area is necessary, as these natural alternatives are still far from achieving the cost-effectiveness and performance levels of traditional chemical flocculants.
Characterising the role of palmitoylation in metastatic breast cancer.
Motivation: Metastasis is the final stage and main cause of cancer-related deaths. In a study by Altea Manzano et al., they observed that increased dietary fat uptake can facilitate metastasis of the primary breast tumour in the lung. Primary breast cancer tumour cells can promote increased palmitate production in the lungs, creating a premetastatic niche that allows the colonisation and growth of tumour cells in the lung. This excess of palmitate can be used by tumour cells as an energy source or to activate oncogenic pathways. In this context, this study aims to characterise the role of palmitoylation in metastatic breast cancer. Protein S-palmitoylation is a reversible posttranslational modification where the fatty acid palmitate is attached to a specific cysteine residue in the target protein. Palmitoylation affects the localisation, trafficking, stability and activity of hundreds of proteins and is mediated by the ZDHHC family of proteins, while the depalmitoylation process is carried out by depalmitoylases. Proteins that are encoded by oncogenes and tumour suppressor genes are palmitoylated and dysregulation of protein palmitoylation could be related to metastatic invasion. Methods: We performed 3D in vitro cultures of mouse (4T1 and D2.A1) and human (HCC1428 and MCF10A-HRas) breast cancer cell lines to determine the role of palmitate in tumour spheroid growth. We also generated knockdown cell lines for a specific depalmitoylase to study how this modification affects the growth of 3D spheroids. We also tested a chemical inhibitor for the depalmitoylase enzyme to determine if the effect of the inhibition in the 3D spheroids growth is similar to the effect of the knockdown. Results: Cell lines were cultured with different concentrations of palmitate and we observed that breast cancer spheroids showed an increase in 3D growth in the presence of 75 µM of palmitate. We also found that higher concentrations than 75 µM are no longer beneficial for 3D growth. We also tested D2.A1 3D growth upon depalmitoylation inhibition (knockdown and chemical inhibition). Notably, we found that inhibiting depalmitoylation prevents the growth advantage conferred by palmitate supplementation. Conclusions: Palmitate promotes the growth of 3D tumour spheroids, and this effect is dependent on depalmitoylase enzymes. When depalmitoylases are inhibited, 3D spheroids are not able to grow using palmitate, which may be related to a reduced ability to form metastases.
Use of brain organoids for the development of new treatments for Amyotrophic Lateral Sclerosis (ALS).
Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease with no cure, leading to patient death within 2-5 years after symptom onset. As a result, there is a loss of muscle strength that spreads, affecting mobility, speech, swallowing, and breathing, while the senses, intellectual capacity, and eye muscles remain unaffected. Many drugs showing promising results in animal models failed in clinical trials, likely due to poor predictability of human brain responses. Developing more predictive models based on human cells is crucial to advance our understanding of ALS pathophysiology and promoting the development of more effective and safer therapies. Animal experimentation, while widely used, has significant limitationsfor the development of medicines targeting central nervous system and is increasingly questioned for ethical and scientific reasons. In response, brain organoids have emerged as a promising alternative (García-Delgado et al; 2022). Here we show a way to generate a robust ALS model using cortical organoids derived from induced pluripotent stem cells (iPSCs). To enhance model reliability, we have applied a small-molecule-guided protocol that reduces heterogeneity and improve cortical specificity. We applied dual SMAD inhibition using SB431542 and Dosomorphin to reduce non-ectodermal contamination and improve cortical specification. Additionally, we incorporated decellularized extracellular matrix (cECM) (Cho et al; 2021)derived from ALS patients and healthy controls. Cortical-derived cECM, rich in proteoglycans, laminins, and integrins, better mimics the neural niche, enhancing neural differentiation and maturation compared to conventional matrigel (García-Delgado et al; 2024). These advancements establish a highly predictive ALS model for evaluating drug efficacy and safety, driving the development of novel therapies and optimized drug delivery strategies.
Decoding the Role of a Microtubule-related protein in Meiosis.
Several meiosis-specific proteins of Schizosaccharomyces pombe play essential roles in meiotic progression. In this study we report that a conserved microtubule-related protein is required for proper spore formation. Sporulation in fission yeast represents a unique model to study gametogenesis in sexual reproducing organisms. This event is accompanied by formation of the forespore membrane (FSM), which becomes the plasma membrane of spores [1]. After the two sequential meiotic divisions, FSMs expand and encapsulate the haploid nuclei, producing the membrane-surrounded spore precursors [2]. During anaphase II, two FSM sacs expand from the poles encasing the nuclei, and these elongates with the intranuclear spindle to divide into two. Finally, these sacs enclose four newborn nuclei individually. The leading edge of the elongating FSM forms a ring and comprises some associated proteins [3]. The leading edge ring is a crucial structure in meiosis that remains poorly understood. Our research investigates its role and dynamics, focusing on the localization and function of this microtubule-related protein during spore formation in fission yeast. Transgenic S. pombe strains were generated by tagging the gene of interest with GFP along with either mCherry-labeled microtubules or mCherry-Psy1 to observe simultaneous dynamics of these markers throughout meiosis in vivo. Fluorescence microscopy was used to capture images at different Z-plane levels for 3D reconstruction. These experiments reveal that this protein is localizes in the leading-edge ring. In addition, we studied the lack-of-function by deletion of this gene, revealing a phenotype characterized by malformed spindles and delayed assembly/disassembly during meiosis II. This could lead to issues in chromosome segregation and result in mutants with altered chromosomal content.
Pharmaceutical contaminated water treatment with inmobilized bacteria in zeolite.
Pollution is one of the top three global challenges, along with biodiversity loss and climate change. Among the pollutants, emerging contaminants are attracting more attention. These are natural or synthetic products that have either recently appeared in the environment and there are concerns about their effects. Pharmaceuticals can be found in these contaminants because they have increased in use over the last few decades, with more than 3000 active ingredients. These products can reach receiving waters and contaminate them, causing adverse effects in aquatic ecosystems. The low efficiency of their treatment in wastewater treatment plants leads to the investigation of new methods to deal with this pollutant. One of them is absorption by absorbents such as zeolite and another is biodegradation by living organisms such as bacteria. In this study, we aimed to combine both approaches. To do this, we extracted silicate from rice husk ash under different conditions to find the best one. With this silicate, zeolite is synthesised under different parameters to determine the most suitable ones. This is verified by X-ray difraction to see if the synthesised zeolite contains only zeolite X. Three different treatments are then used to attach the zeolite to a polycarbonate substrate so that bacteria can form a biofilm on it. The successful attachment of the zeolite to the polycarbonate is expected to promote bacterial growth and biodegradation studies will then be carried out under these conditions.
Isolation and characterization of thermophilic, sulfur-oxidyzing bacteria from agriculture soils.
In the soil, sulfur undergoes constant transformation through biochemical reactions resulting from interactions with organisms. This element is essential for all living beings, from prokaryotes to multicellular organisms (Paniagua & Garcia, 2022). Thermophilic bacteria play a crucial role in the mineralization of organic sulfur compounds, actively breaking down organic matter and generating inorganic nutrients of interest to microorganisms and plants, such as inorganic sulfur compounds. Additionally, they are of great importance due to their high persistence and enzymatic activity in warm environments (Gonzalez et al., 2023). Bacterial isolates were obtained from soil samples from different regions of Spain. In this process, two culture media with different characteristics and pH levels were used: one nutrient-rich medium C (nutrient broth/agar) and two sulfur-oxidyzing bacteria selective media. The samples were incubated at 50ºC and 65ºC, resulting in a total collection of 294 thermophilic strains. To assess their sulfur-oxidizing capacity, sulfate determination was performed on all strains (Amala et al., 2020). The ten thermophilic strains that achieved the best results, with a sulfate concentration of approximately 300 mg/L, were selected and their 16S rRNA genes were PCR amplified, purified and sequenced to obtain their taxanomic identification. In high-temperature agricultural regions, solarization is carried out to maximize soil temperature using solar energy. This is a common practice for pathogen control. This process can influence soil microbial dynamics, particularly thermophilic bacteria with sulfur-oxidizing capabilities. As the next phase of the project, the inhibitory capacity of the thermophilic isolates will be assayed against relevant phytopathogens will be evaluated. The identification of strains with high sulfur-oxidizing activity opens the possibility of their application in sustainable agricultural practices, improving nutrient availability and potentially contributing to the biological control of phytopathogens. This study highlights the importance of exploring microbial diversity in soils managed with techniques such as solarization to develop integrated strategies that promote soil health and agricultural productivity.
Impact of a ferrotherapy intervention on the immune system.
Motivation: Absolute iron deficiency is a prevalent condition among women, characterized by low iron levels that can precede the onset of iron-deficiency anemia. Research utilizing animal models indicates a potential influence on the immune system (IS), although evidence from human studies remains limited. We hypothesized that the restoration of normal iron levels could favorably modified the immunological profile of iron-deficient patients. Methods: This study included 42 non-obese, menstruating adult women devoid of comorbidities, all presenting with ferritin levels below 30 ng/mL, who received supplementation with ferric carboxymaltose. Out of the initial cohort, 32 patients completed the treatment protocol. Blood samples were collected prior to and following the therapy. Additionally, a control group consisting of 3 individuals with ferritin levels exceeding 65 ng/mL was recruited. We evaluated parameters related to iron metabolism, complete blood counts, lymphocyte subpopulations, immunoglobulin levels, inflammatory markers (including DD, B2M, hsCRP), and complement components (C3, C4). Statistical analyses employed Wilcoxon tests for longitudinal data and Mann-Whitney U tests for comparisons against the control group. Results: The patient cohort had a median age of 43 years and a body mass index (BMI) of 24.8 kg/m². Clinical symptoms were reported by 90% of participants, with 44% meeting the diagnostic criteria for anemia. Menorrhagia was identified as the primary etiology of iron deficiency. Treatment resulted in the correction of iron metabolism parameters and a reduction in leukocyte, neutrophil, monocyte, and platelet counts. Percentages of %B and %T lymphocytes increased, concomitant with a reduction in the CD4/CD8 T-cell ratio, while the percentage of %NK decreased. IgM levels increased, whereas IgG, C3, and DD levels demonstrated a decrease; C4 and other inflammatory markers showed no significant changes. Importantly, iron-deficient patients did not exhibit pathological alterations in immune parameters, since both, pre- and post-treatment values remained within the normal range. Conclusions: Ferric carboxymaltose treatment was effective and revealed effects on the IS, including changes in leukocyte subpopulations, a potential benefit in humoral response, and activation of the alternative complement pathway.
Tumor microenvironment modulation in hepatocarcinoma.
Motivation: Hepatocellular carcinoma, one of the most aggressive malignancies with the lowest survival rates, has a complex tumor microenvironment containing immune cells, tumor cells, HSCs, and other cancer-associated cells. The tumor microenvironment signals tumoral cells to become more tumorigenic. In liver fibrosis and HCC, HSCs transdifferentiate and become active, expressing a-SMA and producing ECM proteins. Energy-intensive HSC activation needs glycolysis, mitochondrial respiration, and glutaminolysis. Targeting HSC activation in HCC is a possible therapy for this prevalent disease. Previous research from our group has demonstrated that GATA4 modulates HSC, leading it to switch from active to inactive state. This work investigates the effect of GATA4 on the metabolism of active HSCS and the efficacy of pharmacological treatments to increase endogenous GATA4 expression in active HSCs to inactivate them. Methods: Activated human-derived activated hepatic stellate cell line, LX2, was used to determine the effect of GATA4 overexpression in cellular respiration. Previously identified putative activators of GATA4 endogenous expression were interrogated for their potential to inactivate HSCs. Results: GATA4 overexpression in activated HSCs induces decreased in mitochondrial respiration and metabolomic analysis showed a decreased in a-cetoglutarate. The addition of Dimetil a-cetoglutarate, DMKG, did not restored the inactivation of HSCs promoted by GATA4. Two different molecules with potential of activated the endogenous expression of GATA4 were not able to revert the active phenotype of HSCs. Conclusions: The decrease of mitochondrial respiration induced by GATA4 to revert the active phenotype of HSCs is independent of a-cetoglutarate. The potential activators of GATA4 expression identified in a previous screening from a repurposing library were not able to induce the reversion of the activate state, as measured by a-SMA expression and ECM proteins.
Energy potential, nutrients, and contaminants of olive mill solid waste.
Motivation: Olive oil production is a key activity in the Mediterranean, with Spain as the world's leading producer. The twophase system generates approximately 4 tons of olive mill solid waste (alpeorujo) for every ton of oil produced. Due to its high organic matter concentration and pollutant potential, its management represents an environmental challenge (Doula et al., 2021). This study aims to characterize different alpeorujo samples, determine their nutrient and toxic content, and evaluate their biochemical methane potential (BMP). Methods: Six alpeorujo samples were collected from three olive mills in Andalusia, including fresh and stored samples. Chemical Oxygen Demand (COD), total and volatile solids, N-NH₃, C/N ratio, protein, and fat content were analyzed. Elemental composition was determined using ICP, while polyphenols were analyzed by HPLC-DAD. BMP tests were conducted in Erlenmeyer flasks (240 mL) with an inoculum/substrate ratio of 2:1 under anaerobic conditions at 37°C for 20 days. Methane production was measured under standard conditions. Results: Total COD ranged between 300–400 mg O₂/L, with higher solid concentrations in fresh samples due to sedimentation. Alpeorujo exhibited a high C/N ratio (~30) and low protein content. BMP values ranged from 150 to 350 mL CH₄·g VS⁻¹, with higher methane production in stored samples, indicating that prolonged storage improves biodegradability. It was also observed that alpeorujo samples containing high-molecular-weight phenols exhibited greater microbial inhibition (El Gnaoui et al., 2020), resulting in lower methane production.
Computational analysis of a bacteriophage pangenome with multiple defense systems.
Motivation: Antimicrobial resistance has been declared as one of the biggest threats to human health. The World Health Organization (WHO) has described a list of pathogens for which the development of new treatments is essential. One of these pathogens is Acinetobacter baumannii, which is classified with "Priority 1" [1]. A. baumannii carries different antiviral defense systems. In our group, nearly 10,000 genomes of A. baumannii were analyzed and in half of them, a bacteriophage, DgiS1, appeared integrated downstream of the ssrA gene. In most cases, the phage presents defense systems which seems to antagonyze with the CRISPR-Cas system from another phage [2]. For this reason, we are characterizing the DgiS1 genome, with particular focus on its defense systems and genes, which will help to shed light on the constant warfare between bacteria and phages. Methods: The pangenome of DgiS1 was created using Roary Version 3.13.0 [3], with protein-coding genes previously predicted by Prokka Version 1.14.6. Proteins were functionally annotated using Sma3s v2. To identify defense systems, DefenseFinder v2.0.0 software was used [4]. In order to represent the strains features of DgiS1, heatmaps were built with R programming language. AlphaFold3 and USAlign Version 20240510 were used to analyze the structural similarity. Results: 2,199 genomes of DgiS1 were analyzed and they presented an average of 93 genes. The core genome was composed of 77 cluster genes while its accessory genome had 223 cluster genes. Due to differences in its accessory genome, strains were classified in six groups, based on the presence/absence of those genes. The annotation of the genome showed that core genes were phage genes and accessory genes were mostly defense genes. Furthermore, DefenseFinder identified an antitoxin belonging to a toxin-antitoxin system in five of these groups, while in the remaining one, another type of defense system was identified in the same region. The analysis of the closest proteins to the antitoxin and its sequential and structural comparison with other reference proteins suggested the presence of a potential toxin. Conclusions: DgiS1 presents a core genome composed mainly of phage-annotated genes, while its accessory genome includes a diverse range of defense systems. Strains with different types of defense systems suggest an evolutionary pressure, likely resulting from the fight that happens against other bacteriophages and to guarantee its permanence in the host bacteria.
Characterization of an innovative agitation and aeration system for the optimization of industrial bioprocesses.
Motivation: In the biotechnology industry, optimizing bioprocesses is crucial for improving efficiency, sustainability, and competitiveness. Stirred-tank bioreactors are widely used, but traditional aeration and agitation systems face challenges like inefficient oxygen transfer and high energy consumption, which drive the need for more effective, sustainable solutions (Shukla et al., 2001). Biomixing S.L. addresses this gap by developing innovative agitation and aeration systems that improve oxygen transfer, nutrient distribution, and reduce shear stress on microorganisms. The company uses advanced tools like computational fluid dynamics (CFD) and mathematical modeling to optimize system performance (Doran, 2013). Methods: This study characterizes and validates the Biomixing system using methods based on the DECHEMA protocol, focusing on mixing time and volumetric oxygen transfer coefficient (kLa).To achieve this objective, we compared the performance of bioreactor from a golden brand using two different agitation/aeration systems: (1) the Biomixing system and (2) the widely used Rushton system. These methods allow for a thorough evaluation of system performance, providing insights into mixing efficiency, oxygen transfer, and biological impact in industrial bioprocesses (Meusel et al., 2016). Results: The results showed improvements in mixing time and kLa values compared to the traditional Rushton turbine, confirming better oxygen transfer and mixing efficiency. These improvements were mainly observed at low rotation speeds, such as 400 and 250 rpm, where kLa increased by up to 150%. However, at speeds above 1000 rpm, the improvement were not so significant. Regarding mixing time, a 24% improvement was achieved at 700 rpm. Conclusions: In conclusion, the optimized agitation and aeration system can lead to significant energy and resource savings by enhancing mixing efficiency and oxygen transfer. The system we have characterized is particularly well-suited for bioprocesses that demand rapid and uniform distribution of gases, nutrients and additives, ensuring optimal conditions for microbial or cellular growth. Additionally, its ability to improve oxygen transfer efficiency makes it especially beneficial for processes with high oxygen requirements, where maintaining adequate aeration is crucial for productivity and overall process performance.
Identification of defense genes targeted by CRISPR-Cas systems in Acinetobacter baumannii.
Motivation: CRISPR-Cas systems are an acquired immunity system in bacteria and archaea, which allows them to recognise and degrade foreign DNA sequences. They do this thanks to short sequences they store in their genome, called spacers. Understanding which defense genes are recognised by these spacers may be of interest for understanding the phage fighting that often occurs in bacteria [1]. Building on a previous study in which the Acinetobacter baumannii pangenome was constructed and its spacers identified [2], this work aims to analyse the genes of other defense systems, out of the more than one hundred that the bacterium presents, which can be recognised by the CRISPR-Cas system. Methods: The A. baumannii pangenome was used and analysed with DefenseFinder v1.3.0 [3] to identify its defense genes. Subsequently, a similarity search was performed using the BLAST tool [4], configured to search for short fragments, between the identified genes and the previously obtained spacers. After analysing the results, the most abundant defense gene typologies were identified and a new BLAST analysis [4] focused on these genes was carried out to evaluate their recognition by CRISPR-Cas systems. Results: . Multiple genes from different defense systems recognized by CRISPR-Cas spacers were identified in A. baumannii. Among them, bacteriophage restriction-modification systems were prominent. Further analysis of these genes revealed a large sequence diversity among genes with the same annotation, not all of which were recognised as defense systems. However, some of these genes, belonging to phage sequences, showed a high level of recognition by spacers, suggesting a selective pressure on them. Conclusions:The results indicate that A. baumannii CRISPR-Cas systems are in part directed against phage defense genes, including restriction-modification systems. The large variability in the sequences of these genes suggests a continuous evolutionary race between phages and bacteria.
A journey from academia to industry through the language of the genome.
From a very young age, I have been deeply fascinated by the simplicity and elegance of the genetic code—just four letters, A, G, C, and T, encoding the immense complexity of nature’s biodiversity. This fascination has driven my passion for understanding how decoding this language can lead to endless biotechnological and medical advancements, with the potential to address some of society’s most pressing challenges. For over fourteen years, I dedicated my career to academic research, focusing on the application of functional genomics to explore various biological processes, such as evolution, embryonic development, and the molecular foundations of human diseases. However, some time ago, my professional path took a new direction, and I am currently part of a growing pharmacogenomics company, Duponte Research and Development, whose vision is to integrate pharmacogenomic analysis into routine clinical practice to ensure that every patient receives the most effective treatment, at the optimal dose, and at the right time. I was also a student in these master’s programs many years ago, and this talk aims to offer a personal perspective on the opportunities that both academia and industry can provide, as well as the importance of education, passion, and curiosity in professional development.