1,952 research outputs found

    Discovery of selective saccharide receptors via Dynamic Combinatorial Chemistry

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    The diagnosis of various diseases and pathological conditions can be accomplished by screening and detecting glycans in cells. Certain glycans serve as excellent biomarkers, being related to cell malfunctioning, while other structurally similar glycans perform completely different functions and are naturally present in healthy cells. Despite the theoretical feasibility of using glycans as biomarkers for early disease detection, our current inability to discriminate between them limits their use. One promising approach to distinguishing between glycans is targeting their dissimilarities in saccharide chains. However, designing selective receptors for saccharides is challenging due to the complexity of these molecules. Their vast diversity, the fact that they exist in many interconvertible forms, their lack of recognisable functional groups, or the fact that they are normally heavily solvated in aqueous environments have made the design of receptors for saccharides a challenge that has kept the scientific community busy for the last 35 years. Although there have been ground-breaking discoveries in the field, improvements are needed to enhance our disease detection and risk stratification tools. To address this challenge, we employed a technique known as Dynamic Combinatorial Chemistry (DCC). DCC enables the self-formation and self-selection of the best possible receptor for a given target from a pool or library of potentially good ligands. DCC has been effective for creating receptors for biomolecules such as DNA, RNA, and proteins, but its use for discovering sugar receptors is less explored. In this work, we filled this gap by implementing DCC for screening common saccharides (glucose, galactose, mannose, and fructose) using small, simple, and inexpensive building blocks. Our results indicated that molecule 2DD, which consists of a benzene ring with 2 units of amino acid aspartic acid derivatives connected in positions 1 and 3, is the best receptor in a library of very similar structures for the saccharides glucose, galactose, and mannose. For fructose, molecule 1P, a benzene ring linked to just one unit of the amino acid phenylaldehyde, was appointed as the best receptor. The differential behaviour of fructose can provide insight into the relatively unknown processes behind molecular recognition of sugars. Molecules 2DD and 1P, as well as some other library members as negative controls, were then synthesised for further testing and DCC results were then validated by Isothermal Titration Calorimetry (ITC) and NMR techniques, proving the effectiveness of DCC as a molecular recognition tool for the creation of receptors for saccharides. Moreover, molecule 1P was found to have a high binding constant (Ka_{a} = 1762 M1^{-1}) and selectivity (50-100 times over other sugars) for fructose, which is surprisingly good considering the simplicity of the receptor. A much more challenging approach was attempted by employing short peptides as scaffolds in DCC experiments. The benefits of using peptides are numerous but can be summarised in three bullet points: customisability, flexibility, and easiness in their synthesis. Unfortunately, we encountered many difficulties for the complete functionalisation of the peptides within the Dynamic Combinatorial Library (DCL) and this approach did not yield the desired results before the research project came to an end. However, we believe in its potential and the knowledge that we gained on the topic helped to stablish the foundations on which new research will be carried out in the near future within the research group. In summary, this thesis reports the development of a rapid methodology for the discovery of selective receptors for monosaccharides, employing a library of simple and inexpensive starting building blocks. While this was a proof-of-concept study, it can be scalable to larger library sizes and to target more complex biomolecules, becoming a useful tool that could accelerate the discovery of new molecules with biomedical applications

    Genomic architecture of selection for adaptation to challenging environments in aquaculture

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    Aquaculture, including freshwater and marine farming, has been important for global fish production during the past few decades. However, climate change presents a major risk threatening both quality and quantity of aquaculture production. The environmental stressors in aquaculture resulting from climate change, are temperature rise, salinity changes, sea level rise, acidification and changes of other chemical properties and changes of oxygen levels. Although a reasonable genetic gain can be achieved by selective breeding, this genetic response may not be enough to adapt fish species to the effects of climate change. Marker assisted selection focusing on specific genes or alleles that allow fish to cope with these changes would allow more rapid adaptation of fish to these new environments. In this thesis, I focused on three essential environmental stressors - dissolved oxygen, salinity and temperature as primarily determined in aquaculture production. The main objective is to provide insight in the genomic architecture underlying the mechanism of adaptation to challenging environments of aquaculture species under farming conditions. First, I determined candidate QTL associated with phenotypic variation during adaptation to hypoxia or normoxia. I identified overrepresented pathways that could explain the genetic regulation of hypoxia on growth. To identify fish with better hypoxia tolerance and growth under a hypoxic environment, I quantified the genetic correlations between an indicator trait for hypoxia tolerance (critical swimming performance) and growth. Moreover, the genomic architecture associated with swimming performance was demonstrated, while the effect of significant QTLs on growth was estimated. Beyond applying genome-wide association studies, I used selection signatures to identify QTLs and genes contributing to salinity tolerance. In addition, I also compared the genome of the saline-tolerant and highly productive tilapia “Sukamandi”, that was developed by the aquaculture research institute in Indonesia, to that of blue tilapia and Nile tilapia, to identify the QTLs contributing to salinity tolerance. Finally, I investigated QTLs associated with growth-related traits and organ weights at two distinct commercial Mediterranean product sites differing in temperature (farms in Spain and Greece). Overall, this thesis considerably adds to insight into how fish adapt to challenging environments, which will aid marker-assisted selection for improved resilience of aquaculture species under climate change

    Effects of municipal smoke-free ordinances on secondhand smoke exposure in the Republic of Korea

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    ObjectiveTo reduce premature deaths due to secondhand smoke (SHS) exposure among non-smokers, the Republic of Korea (ROK) adopted changes to the National Health Promotion Act, which allowed local governments to enact municipal ordinances to strengthen their authority to designate smoke-free areas and levy penalty fines. In this study, we examined national trends in SHS exposure after the introduction of these municipal ordinances at the city level in 2010.MethodsWe used interrupted time series analysis to assess whether the trends of SHS exposure in the workplace and at home, and the primary cigarette smoking rate changed following the policy adjustment in the national legislation in ROK. Population-standardized data for selected variables were retrieved from a nationally representative survey dataset and used to study the policy action’s effectiveness.ResultsFollowing the change in the legislation, SHS exposure in the workplace reversed course from an increasing (18% per year) trend prior to the introduction of these smoke-free ordinances to a decreasing (−10% per year) trend after adoption and enforcement of these laws (β2 = 0.18, p-value = 0.07; β3 = −0.10, p-value = 0.02). SHS exposure at home (β2 = 0.10, p-value = 0.09; β3 = −0.03, p-value = 0.14) and the primary cigarette smoking rate (β2 = 0.03, p-value = 0.10; β3 = 0.008, p-value = 0.15) showed no significant changes in the sampled period. Although analyses stratified by sex showed that the allowance of municipal ordinances resulted in reduced SHS exposure in the workplace for both males and females, they did not affect the primary cigarette smoking rate as much, especially among females.ConclusionStrengthening the role of local governments by giving them the authority to enact and enforce penalties on SHS exposure violation helped ROK to reduce SHS exposure in the workplace. However, smoking behaviors and related activities seemed to shift to less restrictive areas such as on the streets and in apartment hallways, negating some of the effects due to these ordinances. Future studies should investigate how smoke-free policies beyond public places can further reduce the SHS exposure in ROK

    Impacts of marine heat extremes on bivalves

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    As the global ocean continues to experience the consequences of an increase in the frequency and intensity of heat waves, the trend is expected to persist into the 21st century, with a projected tripling of heat waves by 2040. This phenomenon poses a significant threat to marine ecosystems and the survival of marine organisms, including the ecologically and economically vital bivalves. Bivalves are vulnerable to harm from heat stress at various levels of biological organization, and their growth can be negatively impacted by high temperatures, potentially leading to mass mortalities and posing a threat to ecosystem quality and food security. In light of these concerns, this review aims to provide a comprehensive examination of the effects of heat stress on bivalves. It summarizes the physiological and biochemical changes that bivalves undergo in response to extreme heat events and offers an overview of the strategies they employ to mitigate their impacts. A better understanding of the underlying mechanisms of bivalve responses to heat stress is crucial in order to fully appreciate the impact of these events on these organisms. This review synthesizes the current knowledge on heat stress in bivalves and highlights the importance of further research in this area. By providing a comprehensive overview of the physiological and biochemical changes that bivalves experience during heat stress and the strategies they use to mitigate its impact, this review aims to support the development of more effective approaches to minimize heat stress in bivalves

    Studies on the host-pathogen interactions for Rhizoctonia solani AG2-1 causing damping-off disease of Brassica napus (Oilseed Rape)

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    Rhizoctonia solani is a necrotrophic soil-borne plant pathogen species complex, of which anastomosis group (AG) 2-1 causes devastating disease on oilseed rape (OSR, Brassica napus). It is frequently isolated from arable crop fields where it affects establishment and yield via pre- and post-emergence damping off, hypocotyl and root rot. Genetic resistance to R. solani AG2-1 has not been observed and long-lived sclerotia, plus a broad host range allow the pathogen to survive in the soil for many years. Here, the interactions between Rhizoctonia solani AG2-1, its crop host OSR and the model organism Arabidopsis thaliana were explored. Variation in responses to R. solani were observed between commercial OSR varieties and gene expression data showed that susceptibility was associated with auxin and abscisic acid signalling, and the MYC2 branch of jasmonate signalling, while reactive oxygen species, ethylene signalling and the ERF/PDF branch of jasmonate signalling were associated with increased tolerance. This was supported by inoculation of A. thaliana defence mutants and microscopy using Jas9:VENUS and IAA2pro:GUS lines. Further investigations into the role of auxins in R. solani AG2-1 – A. thaliana interactions showed that R. solani was able to differentially affect the root architecture of WT and aux1 transport mutants. Experiments showing the effects of 2,4-D, PAA and NAA demonstrated that PAA was able to restore gravitropism in aux1. R. solani produced both IAA and PAA when grown in broth culture and growth stimulation was observed when R. solani was grown in broth with low concentrations of exogenous PAA. Analysis of gene expression markers (GEMS) from a previous genome wide association study (GWAS) provided further evidence for the involvement of auxins, jasmonates and ethylene in the defence responses of OSR to R. solani AG2-1. Corresponding A. thaliana candidate gene mutants were inoculated with R. solani AG2-1 under experimental conditions to identify potential susceptibility genes. Two of these were taken further and B. rapa TILLING line resources were developed. This thesis increases understanding of the defence pathways involved in resistance and susceptibility to R. solani AG2-1, examines the influence that R. solani has on the root architecture of auxin mutants, and provides candidate gene TILLING line resources for future work

    Green revolution to genome revolution: driving better resilient crops against environmental instability

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    Crop improvement programmes began with traditional breeding practices since the inception of agriculture. Farmers and plant breeders continue to use these strategies for crop improvement due to their broad application in modifying crop genetic compositions. Nonetheless, conventional breeding has significant downsides in regard to effort and time. Crop productivity seems to be hitting a plateau as a consequence of environmental issues and the scarcity of agricultural land. Therefore, continuous pursuit of advancement in crop improvement is essential. Recent technical innovations have resulted in a revolutionary shift in the pattern of breeding methods, leaning further towards molecular approaches. Among the promising approaches, marker-assisted selection, QTL mapping, omics-assisted breeding, genome-wide association studies and genome editing have lately gained prominence. Several governments have progressively relaxed their restrictions relating to genome editing. The present review highlights the evolutionary and revolutionary approaches that have been utilized for crop improvement in a bid to produce climate-resilient crops observing the consequence of climate change. Additionally, it will contribute to the comprehension of plant breeding succession so far. Investing in advanced sequencing technologies and bioinformatics will deepen our understanding of genetic variations and their functional implications, contributing to breakthroughs in crop improvement and biodiversity conservation

    Comparative genomics of recent adaptation in Candida pathogens

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    [eng] Fungal infections pose a serious health threat, affecting >1,000 million people and causing ~1.5 million deaths each year. The problem is growing due to insufficient diagnostic and therapeutic options, increased number of susceptible patients, expansion of pathogens partly linked to climate change and the rise of antifungal drug resistance. Among other fungal pathogens, Candida species are a major cause of severe hospital-acquired infections, with high mortality in immunocompromised patients. Various Candida pathogens constitute a public health issue, which require further efforts to develop new drugs, optimize currently available treatments and improve diagnostics. Given the high dynamism of Candida genomes, a promising strategy to improve current therapies and diagnostics is to understand the evolutionary mechanisms of adaptation to antifungal drugs and to the human host. Previous work using in vitro evolution, population genomics, selection inferences and Genome Wide Association Studies (GWAS) have partially clarified such recent adaptation, but various open questions remain. In the three research articles that conform this PhD thesis we addressed some of these gaps from the perspective of comparative genomics. First, we addressed methodological issues regarding the analysis of Candida genomes. Studying recent adaptation in these pathogens requires adequate bioinformatic tools for variant calling, filtering and functional annotation. Among other reasons, current methods are suboptimal due to limited accuracy to identify structural variants from short read sequencing data. In addition, there is a need for easy-to-use, reproducible variant calling pipelines. To address these gaps we developed the “personalized Structural Variation detection” pipeline (perSVade), a framework to call, filter and annotate several variant types, including structural variants, directly from reads. PerSVade enables accurate identification of structural variants in any species of interest, such as Candida pathogens. In addition, our tool automatically predicts the structural variant calling accuracy on simulated genomes, which informs about the reliability of the calling process. Furthermore, perSVade can be used to analyze single nucleotide polymorphisms and copy number-variants, so that it facilitates multi-variant, reproducible genomic studies. This tool will likely boost variant analyses in Candida pathogens and beyond. Second, we addressed open questions about recent adaptation in Candida, using perSVade for variant identification. On the one hand, we investigated the evolutionary mechanisms of drug resistance in Candida glabrata. For this, we used a large-scale in vitro evolution experiment to study adaptation to two commonly-used antifungals: fluconazole and anidulafungin. Our results show rapid adaptation to one or both drugs, with moderate fitness costs and through few mutations in a narrow set of genes. In addition, we characterize a novel role of ERG3 mutations in cross-resistance towards fluconazole in anidulafungin-adapted strains. These findings illuminate the mutational paths leading to drug resistance and cross-resistance in Candida pathogens. On the other hand, we reanalyzed ~2,000 public genomes and phenotypes to understand the signs of recent selection and drug resistance in six major Candida species: C. auris, C. glabrata, C. albicans, C. tropicalis, C. parapsilosis and C. orthopsilosis. We found hundreds of genes under recent selection, suggesting that clinical adaptation is diverse and complex. These involve species-specific but also convergently affected processes, such as cell adhesion, which could underlie conserved adaptive mechanisms. In addition, using GWAS we predicted known drivers of antifungal resistance alongside potentially novel players. Furthermore, our analyses reveal an important role of generally-overlooked structural variants, and suggest an unexpected involvement of (para)sexual recombination in the spread of resistance. Taken together, our findings provide novel insights on how Candida pathogens adapt to human-related environments and suggest candidate genes that deserve future attention. In summary, the results of this thesis improve our knowledge about the mechanisms of recent adaptation in Candida pathogens, which may enable improved therapeutic and diagnostic applications.[cat] Les infeccions fúngiques representen una greu amenaça per a la salut, afectant a més de 1.000 milions de persones i causant aproximadament 1,5 milions de morts cada any. El problema està augmentant a causa d’unes opcions terapèutiques i diagnòstiques insuficients, l'increment del nombre de pacients susceptibles, l'expansió dels patògens parcialment vinculada al canvi climàtic i l'augment de la resistència als fàrmacs antifúngics. D’entre diversos fongs patògens, els llevats del gènere Candida són una causa important d'infeccions nosocomials, amb una alta mortalitat en pacients immunodeprimits. Diverses espècies de Candida constitueixen un problema de salut pública, cosa que requereix més esforços per a desenvolupar nous medicaments, optimitzar els tractaments disponibles i millorar els diagnòstics. Tenint en compte el dinamisme genòmic d’aquests patògens, una estratègia prometedora per millorar les teràpies i diagnòstics actuals és comprendre els mecanismes evolutius d'adaptació als fàrmacs antifúngics i a l’hoste humà. Treballs anteriors utilitzant l'evolució in vitro, la genòmica de poblacions, les inferències de selecció i els estudis d'associació de genoma complet (GWAS, per les sigles en anglès) han aclarit parcialment aquesta adaptació recent, però encara hi ha diverses preguntes obertes. En els tres articles que conformen aquesta tesi doctoral, hem abordat algunes d'aquestes preguntes des de la perspectiva de la genòmica comparativa. En primer lloc, hem abordat qüestions metodològiques relatives a l'anàlisi dels genomes de les espècies Candida. L'estudi de l'adaptació recent en aquests patògens requereix eines bioinformàtiques adequades per a la detecció, filtratge i anotació funcional de variants genètiques. Entre altres raons, els mètodes actuals són subòptims a causa de la limitada precisió per identificar variants estructurals a partir de dades de seqüenciació amb lectures curtes. A més, hi ha una necessitat d’eines computacionals per a la detecció de variants que siguin senzilles d'utilitzar i reproduibles. Per abordar aquestes mancances, hem desenvolupat el mètode bioinformàtic "personalized Structural Variation detection" (perSVade), una eina que permet la detecció, filtratge i anotació de diversos tipus de variants, incloent-hi les variants estructurals, directament des de les lectures. PerSVade permet la identificació precisa de les variants estructurals en qualsevol espècie d'interès, com ara els patògens Candida. A més, la nostra eina prediu automàticament la precisió de la detecció d’aquestes variants en genomes simulats, la qual cosa informa sobre la fiabilitat del procés. Finalment, perSVade es pot utilitzar per analitzar altres tipus de variants, com els polimorfismes de nucleòtid únic o els canvis en el nombre de còpies, facilitant així estudis genòmics integrals i reproduibles. Aquesta eina probablement impulsarà les anàlisis genòmiques en els patògens Candida i també en altres espècies. En segon lloc, hem abordat algunes de les preguntes obertes sobre l'adaptació recent en els llevats Candida, utilitzant perSVade per a la identificació de variants. D'una banda, hem investigat els mecanismes evolutius de resistència als fàrmacs antifúngics en Candida glabrata. Per a això, hem utilitzat un experiment d'evolució in vitro a gran escala per estudiar l'adaptació a dos antifúngics comuns: el fluconazol i l’anidulafungina. Els nostres resultats mostren una adaptació ràpida a un o ambdós fàrmacs, amb un cost per al creixement moderat i a través de poques mutacions en un nombre reduït de gens. A més, hem caracteritzat un paper nou de les mutacions en ERG3 en la resistència creuada al fluconazol en soques adaptades a anidulafungina. Aquests descobriments aclareixen els processos mutacionals que condueixen a la resistència als fàrmacs i a la resistència creuada en els patògens Candida. D'altra banda, hem re-analitzat aproximadament 2.000 genomes i fenotips disponibles en repositoris públics per a comprendre els senyals genòmics de selecció recent i de resistència a fàrmacs antifúngics, en sis espècies rellevants de Candida: C. auris, C. glabrata, C. albicans, C. tropicalis, C. parapsilosis i C. orthopsilosis. Hem trobat centenars de gens sota selecció recent, suggerint que l'adaptació clínica és diversa i complexa. Aquests gens estan relacionats amb funcions específiques de cada espècie, però també trobem processos alterats de manera similar en diferents patògens, com per exemple l’adhesió cel·lular, cosa que indica fenòmens d’adaptació conservats. A part, utilitzant GWAS hem predit mecanismes esperats de resistència a antifúngics i també possibles nous factors. A més, les nostres anàlisis revelen un paper important de les variants estructurals, generalment poc estudiades, i suggereixen una implicació inesperada de la recombinació (para)sexual en la propagació de la resistència. En conjunt, els nostres descobriments proporcionen noves perspectives sobre com els patògens Candida s'adapten als entorns humans, i suggereixen gens candidats que mereixen investigacions futures. En resum, els resultats d’aquesta tesi milloren el nostre coneixement sobre els mecanismes d'adaptació recent en els patògens Candida, cosa que pot permetre el disseny de noves teràpies i diagnòstics

    Dietary Debaryomyces hansenii promotes skin and skin mucus defensive capacities in a marine fish model

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    The present study explores the effects of two supplementation levels of Debaryomyces hansenii (1.1% and 2.2%) as a probiotic in a reference low fish meal-based diet on the skin mucosal tissue in Sparus aurata. This study includes the evaluation of fish performance coupled with a holistic study of the skin mucosa: i) a transcriptomic study of the skin tissue, and ii) the evaluation of its secreted mucus both in terms of skin mucosal-associated biomarkers and its defensive capacity by means of co-culture analysis with two pathogenic bacteria. Results showed that after 70 days of diet administration, fish fed the diet supplemented with D. hansenii at 1.1% presented increased somatic growth and a better feed conversion ratio, compared to fish fed the control diet. In contrast, fish fed the diet including 2.2% of the probiotic presented intermediate values. Regarding gene regulation, the probiotic administration at 1.1% resulted in 712 differentially expressed genes (DEGs), among which 53.4% and 46.6% were up- and down-regulated, respectively. In particular, D. hansenii modulated some skin biological processes related to immunity and metabolism. Specifically, D. hansenii administration induced a strong modulation of some immune biological-related processes (61 DEGs), mainly involved in B- and T-cell regulatory pathways. Furthermore, dietary D. hansenii promoted the skin barrier function by the upregulation of anchoring junction genes (23 DEGs), which reinforces the physical defense against potential skin damage. In contrast, the skin showed modulated genes related to extracellular exosome and membrane organization (50 DEGs). This modulated functioning is of great interest, particularly in relation to the increased skin mucus defensive capacity observed in the bacterial co-culture in vitro trials, which could be related to the increased modulation and exudation of the innate immune components from the skin cells into the mucus. In summary, the modulation of innate immune parameters coupled with increased skin barrier function and cell trafficking potentiates the skin's physical barrier and mucus defensive capacity, while maintaining the skin mucosa's homeostatic immune and metabolic status. These findings confirmed the advantages of D. hansenii supplementation in low fish meal-based diets, demonstrating the probiotic benefits on cultured marine species

    Data management challenges for artificial intelligence in plant and agricultural research [version 2; peer review: 2 approved]

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    Artificial Intelligence (AI) is increasingly used within plant science, yet it is far from being routinely and effectively implemented in this domain. Particularly relevant to the development of novel food and agricultural technologies is the development of validated, meaningful and usable ways to integrate, compare and visualise large, multi-dimensional datasets from different sources and scientific approaches. After a brief summary of the reasons for the interest in data science and AI within plant science, the paper identifies and discusses eight key challenges in data management that must be addressed to further unlock the potential of AI in crop and agronomic research, and particularly the application of Machine Learning (AI) which holds much promise for this domain

    Industrial chicory genome gives insights into the molecular timetable of anther development and male sterility

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    Industrial chicory (Cichorium intybus var. sativum) is a biannual crop mostly cultivated for extraction of inulin, a fructose polymer used as a dietary fiber. F1 hybrid breeding is a promising breeding strategy in chicory but relies on stable male sterile lines to prevent self-pollination. Here, we report the assembly and annotation of a new industrial chicory reference genome. Additionally, we performed RNA-Seq on subsequent stages of flower bud development of a fertile line and two cytoplasmic male sterile (CMS) clones. Comparison of fertile and CMS flower bud transcriptomes combined with morphological microscopic analysis of anthers, provided a molecular understanding of anther development and identified key genes in a range of underlying processes, including tapetum development, sink establishment, pollen wall development and anther dehiscence. We also described the role of phytohormones in the regulation of these processes under normal fertile flower bud development. In parallel, we evaluated which processes are disturbed in CMS clones and could contribute to the male sterile phenotype. Taken together, this study provides a state-of-the-art industrial chicory reference genome, an annotated and curated candidate gene set related to anther development and male sterility as well as a detailed molecular timetable of flower bud development in fertile and CMS lines
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