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Olive pomace is an agro-industrial waste product generated from the olive oil industry and constituted by bioactive compounds with potential applications in several industrial sectors. The purpose of this work was to evaluate the effects of electron beam (e-beam) radiation on olive pomace, specifically on phenolic compounds (by HPLC-DAD-ESI/MS) and the bioactive properties (antioxidant, antiproliferative, and antimicrobial activities) of crude olive pomace (COP) and extracted olive pomace (EOP) extracts. The amount of total flavonoid content and the reducing power of COP extracts were higher than those obtained for EOP extracts. The results suggested that e-beam radiation at 6 kGy increased both total phenolic and total flavonoid contents as well as the reducing power of COP extracts, due to the higher extractability (>2.5-fold) of phenolic compounds from these samples, while decreasing the scavenging activity of extracts. The extracts of both olive pomaces showed antibacterial potential, and COP extracts at 400 µg/mL also presented antiproliferative activity against A549, Caco-2, 293T, and RAW264.7 cell lines, with both properties preserved with the e-beam treatment. All in all, e-beam radiation at 6 kGy appears to be a promising technology to valorize the pollutant wastes of the olive oil industry through enhancing phenolic extractability and bioactive properties, and, furthermore, to contribute to the environmental and economical sustainability of the olive oil industry.
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It has increasingly been recognized that electrical currents play a pivotal role in cell migration and tissue repair, in a process named "galvanotaxis". In this review, we summarize the current evidence supporting the potential benefits of electric stimulation (ES) in the physiology of peripheral nerve repair (PNR). Moreover, we discuss the potential of piezoelectric materials in this context. The use of these materials has deserved great attention, as the movement of the body or of the external environment can be used to power internally the electrical properties of devices used for providing ES or acting as sensory receptors in artificial skin (e-skin). The fact that organic materials sustain spontaneous degradation inside the body means their piezoelectric effect is limited in duration. In the case of PNR, this is not necessarily problematic, as ES is only required during the regeneration period. Arguably, piezoelectric materials have the potential to revolutionize PNR with new biomedical devices that range from scaffolds and nerve-guiding conduits to sensory or efferent components of e-skin. However, much remains to be learned regarding piezoelectric materials, their use in manufacturing of biomedical devices, and their sterilization process, to fine-tune their safe, effective, and predictable in vivo application.
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The biocatalysis of ß-myrcene into value-added compounds, with enhanced organoleptic/therapeutic properties, may be performed by resorting to specialized enzymatic machinery of ß-myrcene-biotransforming bacteria. Few ß-myrcene-biotransforming bacteria have been studied, limiting the diversity of genetic modules/catabolic pathways available for biotechnological research. In our model Pseudomonas sp. strain M1, the ß-myrcene catabolic core-code was identified in a 28-kb genomic island (GI). The lack of close homologs of this ß-myrcene-associated genetic code prompted a bioprospection of cork oak and eucalyptus rhizospheres, from 4 geographic locations in Portugal, to evaluate the environmental diversity and dissemination of the ß-myrcene-biotransforming genetic trait (Myr+). Soil microbiomes were enriched in ß-myrcene-supplemented cultures, from which ß-myrcene-biotransforming bacteria were isolated, belonging to Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, and Sphingobacteriia classes. From a panel of representative Myr+ isolates that included 7 bacterial genera, the production of ß-myrcene derivatives previously reported in strain M1 was detected in Pseudomonas spp., Cupriavidus sp., Sphingobacterium sp., and Variovorax sp. A comparative genomics analysis against the genome of strain M1 found the M1-GI code in 11 new Pseudomonas genomes. Full nucleotide conservation of the ß-myrcene core-code was observed throughout a 76-kb locus in strain M1 and all 11 Pseudomonas spp., resembling the structure of an integrative and conjugative element (ICE), despite being isolated from different niches. Furthermore, the characterization of isolates not harboring the Myr+-related 76-kb locus suggested that they may biotransform ß-myrcene via alternative catabolic loci, being thereby a novel source of enzymes and biomolecule catalogue for biotechnological exploitation. KEY POINTS: ⢠The isolation of 150 Myr+ bacteria hints the ubiquity of such trait in the rhizosphere. ⢠The Myr+ trait is spread across different bacterial taxonomic classes. ⢠The core-code for the Myr+ trait was detected in a novel ICE, only found in Pseudomonas spp.
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Bacterias , Rizosfera , Monoterpenos Acíclicos , Bacterias/genética , Pseudomonas/genética , Pseudomonas/metabolismoRESUMEN
Lanthanides are critical elements, and their recovery from wastewater increases the availability of these elements and reduces their impacts on the environment. In this study, tentative approaches to extract lanthanides from low-concentration aqueous solutions were investigated. PVDF membranes soaked with different active compounds or synthesized chitosan-based membranes containing these active compounds were used. The membranes were immersed in 10-4 M of aqueous solutions of selected lanthanides, and their extraction efficiency was assessed using ICP-MS. The PVDF membranes showed quite poor results, with only the membrane with oxamate ionic liquid giving some positive results (0.75 mg of Yb, 3 mg of lanthanides per gram of membrane). However, the chitosan-based membranes led to very interesting results, with the maximum concentration factor for the final solution relative to the initial solution being 13 times higher for Yb, which was obtained with the chitosan-sucrose-citric acid membrane. Several of the chitosan membranes, namely the one with 1-Butyl-3-methylimidazolium-di-(2-ethylhexyl)-oxamate, could extract around 10 mg of lanthanides per gram of membrane, with the better one being the membrane with sucrose/citric acid that achieved more than 18 mg/g of membrane. The use of chitosan for this purpose is a novelty. Since these membranes are easily prepared and have a very low cost, practical applications can be envisaged after further studies to better understand the underlying mechanism.
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In 2017, aquaculture producers of the Albufeira lagoon, Portugal, reported an invasion of tunicates that was disrupting mussel production, particularly the tunicate Styela plicata (Lesueur, 1823). A totally effective eradication method still does not exist, particularly for S. plicata, and the effects of the eradication treatments on bivalves' performance are also poorly understood. Our study examined the effectiveness of eradication treatments using three laboratory trials and five treatments (air exposure, freshwater immersion, sodium hypochlorite, hypersaline solution and acetic acid) for S. plicata, as well as their effects on survival and growth of blue mussel Mytilus edulis Linnaeus, 1758. While air exposure and freshwater immersion caused a 27% mortality rate in S. plicata, the acetic acid treatment was the most effective in eliminating this species (>90% mortality). However, a 33-40% mortality rate was registered in mussels. Both species were not affected by the hypersaline treatment in the last trial, but the sodium hypochlorite treatment led to a 57% mortality rate in mussels. Differences in mussels' growth rates were not detected. These trials represent a step forward in responding to the needs of aquaculture producers. However, further studies are needed to investigate the susceptibility of tunicates to treatments according to sexual maturation, as well as to ensure minimum mussel mortality in the most effective treatments, and to better understand the effects on mussel physiological performance in the long-term.
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INTRODUCTION: Technological applications are an innovative way of providing reminiscence therapy and must meet the users' needs. Intangible cultural heritage as a basis for such therapy has not been explored yet. We evaluated the applicability of a new technological application supported by artificial intelligence for reminiscence therapy based on intangible cultural heritage aimed at older people. MATERIAL AND METHODS: A prospective observational study was carried out with people aged 65 or over, without cognitive impairment and with mild and moderate cognitive impairment who attended six centers for older people in Spain and Portugal. Participants tested the first prototype of the individualized LONG-REMI program in four consecutive weekly sessions. The usability and satisfaction of the experience were evaluated using the VAS scale at the end of the intervention. Emotions were evaluated using the PANAS scale before and at the end of the intervention. RESULTS: Data from 56 participants were analysed. For all participants, usability and satisfaction were highly perceived, with scores of 7.75±1.88 and 8.38±1.57, respectively. The positive affect subscale PANAS showed significant changes (28.86±8.88 before the intervention versus 36.70±9.43 post intervention, Z = -4.18, P = 0.000). There were no significant changes in the PANAS negative affect subscale. CONCLUSIONS: The first prototype of the LONG-REMI technological application can be used by older people both with and without cognitive impairment. This has the potential to be an instrument for future cognitive therapies with stimulating activities and benefits for emotions.
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Terapia Cognitivo-Conductual , Disfunción Cognitiva , Anciano , Humanos , Proyectos Piloto , Inteligencia Artificial , Disfunción Cognitiva/psicología , Estudios ProspectivosRESUMEN
Sol-gel production of hybrid materials has, to some extent, revolutionised materials' engineering and the way science and technology perceive the creation of new materials. Despite that, the method presents some limitations that are circumvented by radiation processing. Electron beam irradiation was used to promote synthesis of hybrid structures while using silanol-terminated PDMS, TEOS and TPOZ as precursors. Evaluation of the method's performance was executed by gel fraction determination, WDXRF and FTIR-ATR. Results showed that, although there is some pre-irradiation reactivity between precursors, radiolysis induces scission on multiple sites of precursor's structures, which induces hybrid network formation to a greater extent. Characterisation allowed determining electron beam irradiation to be effective in the creation of Si-O-Zr bonds, resulting in the production of a Class II hybrid material.
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Frequently, deep partial and full-thickness skin wounds do not spontaneously regenerate. To restore the normal function of skin, epidermal and dermal components have to be supplied to the wound bed by grafting various substrates. Available options are limited and frequently costly. Herein, authors present a possible approach using 3D skin scaffolds capable of mimicking structure and biological functions of the extracellular matrix, providing, in parallel, a good environment for cell attachment, proliferation and differentiation. Low-molecular weight chitosan-based membranes were prepared by freeze-drying and ionizing radiation techniques to be used as skin scaffolds. Poly (vinyl alcohol), PVA, vinyl pyrrolidone, VP, and gelatin from cold water fish were incorporated. Information regarding membranes' physical-chemical properties from SEM analysis, swelling and weight loss, together with biological response through in vitro assays (using Human Caucasian Fetal Foreskin Fibroblast) allowed the selection of an optimized batch of membranes that was used as skin scaffold in a dorsal rat model wound. The in vivo implantation assays (in Wistar rats) resulted in very promising results: (i) healing process faster than control; (ii) good vascularization; (iii) viable new tissues morphologically functional.
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Reminiscence therapy (RT) consists of thinking about one's own experiences through the presentation of memory-facilitating stimuli, and it has as its fundamental axis the activation of emotions. An innovative way of offering RT involves the use of technology-assisted applications, which must also satisfy the needs of the user. This study aimed to develop an AI-based computer application that recreates RT in a personalized way, meeting the characteristics of RT guided by a therapist or a caregiver. The material guiding RT focuses on intangible cultural heritage. The application incorporates facial expression analysis and reinforcement learning techniques, with the aim of identifying the user's emotions and, with them, guiding the computer system that emulates RT dynamically and in real time. A pilot study was carried out at five senior centers in Barcelona and Portugal. The results obtained are very positive, showing high user satisfaction. Moreover, the results indicate that the high frequency of positive emotions increased in the participants at the end of the intervention, while the low frequencies of negative emotions were maintained at the end of the intervention.
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Longevidad , Psicoterapia , Inteligencia Artificial , Humanos , Proyectos Piloto , TecnologíaRESUMEN
Pseudomonas aeruginosa can alter its lifestyle in response to changes in environmental conditions. The switch to a pathogenic host-associated lifestyle can be triggered by the luminosity settings, resorting to at least one photoreceptor which senses light and regulates cellular processes. This study aimed to address how light exposure affects the dynamic and adaptability of two P. aeruginosa pneumonia-associated isolates, HB13 and HB15. A phenotypic characterization of two opposing growth conditions, constant illumination and intensity of full-spectrum light and total absence of light, was performed. Given the nature of P. aeruginosa pathogenicity, distinct fractions were characterized, and its inherent pathogenic potential screened by comparing induced morphological alterations and cytotoxicity against human pulmonary epithelial cells (A549 cell line). Growth in the dark promoted some virulence-associated traits (e.g., pigment production, LasA proteolytic activity), which, together with higher cytotoxicity of secreted fractions, supported an increased pathogenic potential in conditions that better mimic the lung microenvironment of P. aeruginosa. These preliminary findings evidenced that light exposure settings may influence the P. aeruginosa pathogenic potential, likely owing to differential production of virulence factors. Thus, this study raised awareness towards the importance in controlling light conditions during bacterial pathogenicity evaluation approaches, to more accurately interpret bacterial responses.
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Portobello variety of Agaricus bisporus mushrooms, appreciated for its taste, makes it desirable to be eaten fresh and also as flour in soups and gravies. Gamma and electron-beam radiation at four doses (1, 2, 5, and 10 kGy) were used to analyze its preservation effect on Portobello mushroom flour. A proximate analysis, as well as the impact on fatty acids, tocopherols, soluble sugars, organic acids, and ergosterol profiles, were performed every 3 months, during a storage period of 1 year. Gamma rays preserved mannitol (most abundant soluble sugar) over the 12 months, while electron beam radiation preserved organic acids. No significant changes were sought for any radiation type, and the slight changes extracted from the estimated marginal means reveal a tendency for irradiation as having preserving effects of nutrients and other important molecules. Thus, both irradiation types, up to 10 kGy are suitable for preservation of A. bisporus Portobello flour.
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Agaricus/química , Irradiación de Alimentos , Valor Nutritivo , Gusto , Rayos gamma , Factores de TiempoRESUMEN
A wide array of fungal endophytes is known to inhabit plant tissues and were recently recognized as essential for plant health. A better description of the scarcely known endophyte microbiota in olive tree phyllosphere is the first step for elucidating the microbial interactions that lead to olive disease establishment. In this work, the fungal endophytic community of the phyllosphere of different olive tree cultivars (Cobrançosa, Galega vulgar, Madural, Picual, Verdeal Transmontana) is revealed by using a metabarcoding strategy targeting ITS1 barcode. A total of 460 OTUs were obtained, increasing the broad view of fungal endophytes inhabiting the olive tree phyllosphere, in particular yeast endophytes. New endophytes were persistently found in all cultivar tissues. Different olive tree cultivars depicted distinct endophyte communities. Olive cultivars exhibited dissimilar amounts of fungi with distinct ecological functions, which could explain at least in part their differential susceptibility/tolerance to olive diseases.
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Endófitos/clasificación , Endófitos/genética , Hongos/clasificación , Hongos/fisiología , Micobioma , Olea/microbiología , Código de Barras del ADN Taxonómico , Endófitos/aislamiento & purificación , Hongos/aislamiento & purificaciónRESUMEN
Monoterpenes are plant secondary metabolites, widely used in industrial processes as precursors of important aroma compounds, such as vanillin and (-)-menthol. However, the physicochemical properties of monoterpenes make difficult their conventional conversion into value-added aromas. Biocatalysis, either by using whole cells or enzymes, may overcome such drawbacks in terms of purity of the final product, ecological and economic constraints of the current catalysis processes or extraction from plant material. In particular, the ability of oxidative enzymes (e.g., oxygenases) to modify the monoterpene backbone, with high regio- and stereo-selectivity, is attractive for the production of "natural" aromas for the flavor and fragrances industries. We review the research efforts carried out in the molecular analysis of bacterial monoterpene catabolic pathways and biochemical characterization of the respective key oxidative enzymes, with particular focus on the most relevant precursors, ß-pinene, limonene and ß-myrcene. The presented overview of the current state of art demonstrates that the specialized enzymatic repertoires of monoterpene-catabolizing bacteria are expanding the toolbox towards the tailored and sustainable biotechnological production of values-added aroma compounds (e.g., isonovalal, α-terpineol, and carvone isomers) whose implementation must be supported by the current advances in systems biology and metabolic engineering approaches.
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Bacterias/metabolismo , Monoterpenos/metabolismo , Monoterpenos Acíclicos/metabolismo , Animales , Monoterpenos Bicíclicos/metabolismo , Biotecnología/métodos , Humanos , Limoneno/metabolismo , OdorantesRESUMEN
The drilling, processing and transportation of oil are the main sources of pollution in water and soil. The current work analyzes the microbial diversity and aromatic compounds degradation potential in the metagenomes of communities in the wastewater treatment plant (WWTP) of a crude oil refinery. By focusing on the degradation of phenol, we observed the involvement of diverse indigenous microbial communities at different steps of the WWTP. The anaerobic bacterial and archaeal genera were replaced by aerobic and facultative anaerobic bacteria through the biological treatment processes. The phyla Proteobacteria, Bacteroidetes and Planctomycetes were dominating at different stages of the treatment. Most of the established protein sequences of the phenol degradation key enzymes belonged to bacteria from the class Alphaproteobacteria. From 35 isolated strains, 14 were able to grow on aromatic compounds, whereas several phenolic compound-degrading strains also degraded aliphatic hydrocarbons. Two strains, Acinetobacter venetianus ICP1 and Pseudomonas oleovorans ICTN13, were able to degrade various aromatic and aliphatic pollutants and were further characterized by whole genome sequencing and cultivation experiments in the presence of phenol to ascertain their metabolic capacity in phenol degradation. When grown alone, the intermediates of catechol degradation, the meta or ortho pathways, accumulated into the growth environment of these strains. In the mixed cultures of the strains ICP1 and ICTN13, phenol was degraded via cooperation, in which the strain ICP1 was responsible for the adherence of cells and ICTN13 diminished the accumulation of toxic intermediates.
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Bacteria of the Burkholderia cepacia complex (Bcc) are ubiquitous multidrug resistant organisms and opportunistic pathogens capable of causing life threatening lung infections among cystic fibrosis (CF) patients. No effective therapies are currently available to eradicate Bcc bacteria from CF patients, as these organisms are inherently resistant to the majority of clinically available antimicrobials. An immunoproteomics approach was used to identify Bcc proteins that stimulate the humoral immune response of the CF host, using bacterial cells grown under conditions mimicking the CF lung environment and serum samples from CF patients with a clinical record of Bcc infection. 24 proteins of the Bcc strain B. cenocepacia J2315 were identified as immunoreactive, 19 here reported as immunogenic for the first time. Ten proteins were predicted as extracytoplasmic, 9 of them being conserved in Bcc genomes. The immunogenic Bcc extracytoplasmic proteins are potential targets for development of novel therapeutic strategies and diagnostic tools to protect patients against the onset of chronic Bcc lung infections.
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Burkholderia cenocepacia/inmunología , Fibrosis Quística/sangre , Fibrosis Quística/inmunología , Proteoma/inmunología , Niño , Biología Computacional , Electroforesis en Gel Bidimensional , Humanos , Masculino , Proteoma/análisisRESUMEN
The biological treatment of oil refinery effluents in wastewater treatment plants (WWTPs) relies on specialized bacteria contributing to remove organic load, nitrogen, sulfur, and phosphorus compounds. Knowledge about bacterial dynamics in WWTPs and how they affect the performance of the wastewater treatment is limited, particularly in tropical countries. The bacterial communities from three compartments of an oil refinery WWTP in Uran, India, were assessed using 16S-metabarcoding, in winter and monsoon seasons, upstream (from the surge pond) and downstream the biotower (clarifier and guard pond), to understand the effects of seasonal variations in WWTP's efficiency. The organic load and ammonia levels of the treated wastewater increased by 3- and 9-fold in the monsoon time-point. A decreased abundance and diversity of 47 genera (325 OTUs) comprising ammonia and nitrite oxidizing bacteria (AOB, NOB, denitrifiers) was observed in the monsoon season downstream the biotower, whereas 23 OTUs of Sulfurospirillum, Desulfovibrio, and Bacillus, putatively performing dissimilatory nitrate reduction to ammonia (DNRA), were 3-fold more abundant in the same compartments (DNRA/denitrifiers winter ratio < 0.5 vs. monsoon ratio around 3). The total abundance of reported sulfate- and sulfite-reducing bacteria also increased 250- and 500-fold downstream the biotower, in the monsoon time-point. Bacteria performing DNRA may thus outcompete denitrification in this WWTP, limiting the biodegradation process. The alterations detected in bacterial populations involved in the removal of nitrogen and sulfur species evidenced a reduced quality of the released wastewater and may be good candidates for the following monitoring strategies and optimization of the wastewater treatment.
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Bacterias/aislamiento & purificación , Microbiota , Aguas Residuales/microbiología , Amoníaco/metabolismo , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Desnitrificación , India , Nitratos/metabolismo , Nitritos/metabolismo , Petróleo/metabolismo , Filogenia , Estaciones del AñoRESUMEN
Pseudomonas sp. strain M1 is able to mineralize highly hydrophobic and recalcitrant compounds, such as benzene, phenol, and their methylated/halogenated derivatives, as well as the backbone of several monoterpenes. The ability to use such a spectrum of compounds as the sole carbon source is, most probably, associated with a genetic background evolved under different environmental constraints. The outstanding performance of strain M1 regarding ß-myrcene catabolism was elucidated in this work, with a focus on the biocatalytical potential of the ß-myrcene-associated core code, comprised in a 28-kb genomic island (GI), predicted to be organized in 8 transcriptional units. Functional characterization of this locus with promoter probes and analytical approaches validated the genetic organization predicted in silico and associated the ß-myrcene-induced promoter activity to the production of ß-myrcene derivatives. Notably, by using a whole-genome mutagenesis strategy, different genotypes of the 28-kb GI were generated, resulting in the identification of a novel putative ß-myrcene hydroxylase, responsible for the initial oxidation of ß-myrcene into myrcen-8-ol, and a sensor-like regulatory protein, whose inactivation abolished the myr+ trait of M1 cells. Moreover, it was demonstrated that the range of monoterpene substrates of the M1 enzymatic repertoire, besides ß-myrcene, also includes other acyclic (e.g., ß-linalool) and cyclic [e.g., R-(+)-limonene and (-)-ß-pinene] molecules. Our findings are the cornerstone for following metabolic engineering approaches and hint at a major role of the 28-kb GI in the biotransformation of a broad monoterpene backbone spectrum for its future biotechnological applications.IMPORTANCE Information regarding microbial systems able to biotransform monoterpenes, especially ß-myrcene, is limited and focused mainly on nonsystematic metabolite identification. Complete and detailed knowledge at the genetic, protein, metabolite, and regulatory levels is essential in order to set a model organism or a catabolic system as a biotechnology tool. Moreover, molecular characterization of reported systems is scarce, almost nonexistent, limiting advances in the development of optimized cell factories with strategies based on the new generation of metabolic engineering platforms. This study provides new insights into the intricate molecular functionalities associated with ß-myrcene catabolism in Pseudomonas, envisaging the production of a molecular knowledge base about the underlying catalytic and regulatory mechanisms of plant-derived volatile catabolic pathways.
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Genes Bacterianos , Islas Genómicas , Redes y Vías Metabólicas/genética , Monoterpenos/metabolismo , Pseudomonas/genética , Pseudomonas/metabolismo , Monoterpenos Acíclicos , Biotransformación , Biología Computacional , Análisis Mutacional de ADN , Perfilación de la Expresión Génica , Orden Génico , Genotipo , Fitoquímicos/metabolismo , Transcripción Genética , Compuestos Orgánicos Volátiles/metabolismoRESUMEN
A comprehensive assessment of the toxicity and antioxidant activity of cork boiling wastewater and the effects of gamma radiation on these parameters was performed. Antioxidant activity was evaluated using different methodologies as DPPH radical scavenging activity, reducing power and inhibition of ß-carotene bleaching. The results have shown that gamma radiation can induce an increase on the antioxidant activity of cork boiling wastewater. Toxicity tests were performed to access the potential added value of the irradiated wastewaters and/or minimization of the impact for discharge in the environment. Two different methods for toxicity evaluation were followed, bacterial growth inhibition test and cytotoxicity assay, in order to predict the behavior of different cells (prokaryotic and eukaryotic) in the presence of cork wastewater. Non-treated cork boiling wastewater seemed to be non-toxic for prokaryotic cells (Pseudomonas fluorescens and Bacillus subtilis) but toxic for eukaryotic cells (A549 human cells and RAW264.7 mouse cells). The gamma radiation treatment at doses of 100 kGy appeared to increase the toxicity of cork compounds for all tested cells, which could be related to a toxic effect of radiolytic products of cork compounds in the wastewaters.
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Bacterias/efectos de la radiación , Rayos gamma , Eliminación de Residuos Líquidos/métodos , Aguas Residuales/química , Contaminantes Radiactivos del Agua/toxicidad , Animales , Línea Celular , Residuos Industriales/análisis , Ratones , Oxidación-Reducción , Pruebas de Toxicidad , beta CarotenoRESUMEN
Burkholderia multivorans is an opportunistic pathogen capable of causing severe disease in patients with cystic fibrosis (CF). Patients may be chronically infected for years, during which the bacterial population evolves in response to unknown forces. Here we analyze the genomic and functional evolution of a B. multivorans infection that was sequentially sampled from a CF patient over 20 years. The population diversified into at least four primary, coexisting clades with distinct evolutionary dynamics. The average substitution rate was only 2.4 mutations/year, but notably, some lineages evolved more slowly, whereas one diversified more rapidly by mostly nonsynonymous mutations. Ten loci, mostly involved in gene expression regulation and lipid metabolism, acquired three or more independent mutations and define likely targets of selection. Further, a broad range of phenotypes changed in association with the evolved mutations; they included antimicrobial resistance, biofilm regulation, and the presentation of lipopolysaccharide O-antigen repeats, which was directly caused by evolved mutations. Additionally, early isolates acquired mutations in genes involved in cyclic di-GMP (c-di-GMP) metabolism that associated with increased c-di-GMP intracellular levels. Accordingly, these isolates showed lower motility and increased biofilm formation and adhesion to CFBE41o- epithelial cells than the initial isolate, and each of these phenotypes is an important trait for bacterial persistence. The timing of the emergence of this clade of more adherent genotypes correlated with the period of greatest decline in the patient's lung function. All together, our observations suggest that selection on B. multivorans populations during long-term colonization of CF patient lungs either directly or indirectly targets adherence, metabolism, and changes in the cell envelope related to adaptation to the biofilm lifestyle. IMPORTANCE Bacteria may become genetically and phenotypically diverse during long-term colonization of cystic fibrosis (CF) patient lungs, yet our understanding of within-host evolutionary processes during these infections is lacking. Here we combined current genome sequencing technologies and detailed phenotypic profiling of the opportunistic pathogen Burkholderia multivorans using sequential isolates sampled from a CF patient over 20 years. The evolutionary history of these isolates highlighted bacterial genes and pathways that were likely subject to strong selection within the host and were associated with altered phenotypes, such as biofilm production, motility, and antimicrobial resistance. Importantly, multiple lineages coexisted for years or even decades within the infection, and the period of diversification within the dominant lineage was associated with deterioration of the patient's lung function. Identifying traits under strong selection during chronic infection not only sheds new light onto Burkholderia evolution but also sets the stage for tailored therapeutics targeting the prevailing lineages associated with disease progression.
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The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care.