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This research work investigates the development of alginate-based films incorporating phenolic compounds extracted from Amaranthus cruentus grain using different solvents. Alginate, glycerol, and amaranth grain phenolic compounds at various concentrations were used to produce the films. An experimental Central Composite Rotatable Design (CCRD) was used to evaluate the effect of these variables on different film's properties, i.e., water vapor permeability, hydrophobicity, moisture content, solubility, thermal, mechanical, and optical properties. This study demonstrated that high phenolic compound content and antioxidant capacity were obtained from amaranth grain using ethanol as the extraction solvent. Alginate films incorporating amaranth phenolic compounds were successfully manufactured, and this study can be used to tailor the formulation of alginate films containing amaranth phenolic compounds, depending on their final food application. For example, less flexible but more resistant and water-soluble films can be produced by increasing the alginate concentration, which was confirmed by a Principal Component Analysis (PCA) and Partial Least Squares (PLS) analysis. This study showed that active alginate films with amaranth phenolic compounds can be tailored to be used as food packaging material with potential antioxidant activity.
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Amaranthus , Alginatos , Antioxidantes/análisis , Antioxidantes/farmacología , Grano Comestible/química , Etanol/análisis , Glicerol/análisis , Fenoles/análisis , Extractos Vegetales , Solventes/análisis , Vapor/análisisRESUMEN
OBJECTIVE: To investigate whether milk polar lipids (PL) impact human intestinal lipid absorption, metabolism, microbiota and associated markers of cardiometabolic health. DESIGN: A double-blind, randomised controlled 4-week study involving 58 postmenopausal women was used to assess the chronic effects of milk PL consumption (0, 3 or 5 g-PL/day) on lipid metabolism and gut microbiota. The acute effects of milk PL on intestinal absorption and metabolism of cholesterol were assessed in a randomised controlled crossover study using tracers in ileostomy patients. RESULTS: Over 4 weeks, milk PL significantly reduced fasting and postprandial plasma concentrations of cholesterol and surrogate lipid markers of cardiovascular disease risk, including total/high-density lipoprotein-cholesterol and apolipoprotein (Apo)B/ApoA1 ratios. The highest PL dose preferentially induced a decreased number of intestine-derived chylomicron particles. Also, milk PL increased faecal loss of coprostanol, a gut-derived metabolite of cholesterol, but major bacterial populations and faecal short-chain fatty acids were not affected by milk PL, regardless of the dose. Acute ingestion of milk PL by ileostomy patients shows that milk PL decreased cholesterol absorption and increased cholesterol-ileal efflux, which can be explained by the observed co-excretion with milk sphingomyelin in the gut. CONCLUSION: The present data demonstrate for the first time in humans that milk PL can improve the cardiometabolic health by decreasing several lipid cardiovascular markers, notably through a reduced intestinal cholesterol absorption involving specific interactions in the gut, without disturbing the major bacterial phyla of gut microbiota. TRIAL REGISTRATION NUMBER: NCT02099032 and NCT02146339; Results.
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Enfermedades Cardiovasculares/sangre , Metabolismo de los Lípidos/efectos de los fármacos , Lípidos/farmacología , Sobrepeso/metabolismo , Esfingomielinas/metabolismo , Animales , Apolipoproteína A-I/sangre , Apolipoproteína B-100/sangre , Colestanol/metabolismo , Colesterol/metabolismo , HDL-Colesterol/sangre , Estudios Cruzados , Suplementos Dietéticos , Método Doble Ciego , Emulsionantes/farmacología , Heces/química , Femenino , Microbioma Gastrointestinal/efectos de los fármacos , Humanos , Ileostomía , Absorción Intestinal/efectos de los fármacos , Lípidos/administración & dosificación , Lípidos/análisis , Persona de Mediana Edad , Leche/química , Posmenopausia , Factores de RiesgoRESUMEN
This paper has been prepared by the Symphony collaboration (University of Warsaw, Uniwersytet Jagiellonski, DESY/CNR and ICFO) on the occasion of the 25th anniversary of the 'simple man's models' which underlie most of the phenomena that occur when intense ultrashort laser pulses interact with matter. The phenomena in question include high-harmonic generation (HHG), above-threshold ionization (ATI), and non-sequential multielectron ionization (NSMI). 'Simple man's models' provide both an intuitive basis for understanding the numerical solutions of the time-dependent Schrödinger equation and the motivation for the powerful analytic approximations generally known as the strong field approximation (SFA). In this paper we first review the SFA in the form developed by us in the last 25 years. In this approach the SFA is a method to solve the TDSE, in which the non-perturbative interactions are described by including continuum-continuum interactions in a systematic perturbation-like theory. In this review we focus on recent applications of the SFA to HHG, ATI and NSMI from multi-electron atoms and from multi-atom molecules. The main novel part of the presented theory concerns generalizations of the SFA to: (i) time-dependent treatment of two-electron atoms, allowing for studies of an interplay between electron impact ionization and resonant excitation with subsequent ionization; (ii) time-dependent treatment in the single active electron approximation of 'large' molecules and targets which are themselves undergoing dynamics during the HHG or ATI processes. In particular, we formulate the general expressions for the case of arbitrary molecules, combining input from quantum chemistry and quantum dynamics. We formulate also theory of time-dependent separable molecular potentials to model analytically the dynamics of realistic electronic wave packets for molecules in strong laser fields. We dedicate this work to the memory of Bertrand Carré, who passed away in March 2018 at the age of 60.
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PURPOSE OF REVIEW: We review recent findings on the involvement of vitamin D in skeletal muscle trophicity. RECENT FINDINGS: Vitamin D deficiencies are associated with reduced muscle mass and strength, and its supplementation seems effective to improve these parameters in vitamin D-deficient study participants. Latest investigations have also evidenced that vitamin D is essential in muscle development and repair. In particular, it modulates skeletal muscle cell proliferation and differentiation. However, discrepancies still exist about an enhancement or a decrease of muscle proliferation and differentiation by the vitamin D. Recently, it has been demonstrated that vitamin D influences skeletal muscle cell metabolism as it seems to regulate protein synthesis and mitochondrial function. Finally, apart from its genomic and nongenomic effects, recent investigations have demonstrated a genetic contribution of vitamin D to muscle functioning. SUMMARY: Recent studies support the importance of vitamin D in muscle health, and the impact of its deficiency in regard to muscle mass and function. These 'trophic' properties are of particular importance for some specific populations such as elderly persons and athletes, and in situations of loss of muscle mass or function, particularly in the context of chronic diseases.
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Músculo Esquelético/metabolismo , Enfermedades Musculares/metabolismo , Deficiencia de Vitamina D/metabolismo , Vitamina D/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Suplementos Dietéticos , Humanos , Enfermedades Musculares/etiología , Vitamina D/uso terapéutico , Deficiencia de Vitamina D/complicaciones , Vitaminas/uso terapéuticoRESUMEN
Filamentous fungi produce a great variety of enzymes, and research on their biotechnological potential has recently intensified. The objective of this work was to identify, at the species level, using DNA barcoding, 46 fungal isolates obtained from maize grains with rot symptoms. We also analyzed the production of extracellular amylases, cellulases, proteases and lipases of 33 of those fungal isolates. The enzymatic activities were evaluated by the formation of a clear halo or a white precipitate around the colonies in defined substrate media. The found fungi belong to the genera Talaromyces, Stenocarpella, Penicillium, Phlebiopsis, Cladosporium, Hyphopichia, Epicoccum, Trichoderma, Aspergillus, Irpex, Fusarium, Microdochium, Mucor and Sarocladium. In the genus Fusarium, the species Fusarium verticillioides was predominant and this genus presented the highest diversity, followed by the genera Aspergillus. The best genera for lipase production were Cladosporium and Penicillium; while Cladosporium, Aspergillus and Penicillium were best for cellulase activity; Hyphopichia, Aspergillus and Irpex for amylase activity; and Cladosporium and Sarocladium for proteases activity. In conclusion, a collection of fungi from maize seeds presenting rotten symptoms were obtained, among which exist important producers of hydrolases.
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Hongos/enzimología , Hongos/aislamiento & purificación , Semillas/microbiología , Zea mays/microbiología , Amilosa/metabolismo , Secuencia de Bases , Celulosa/metabolismo , ADN Ribosómico/genética , Evolución Molecular , Hongos/genética , Hidrólisis , Lipólisis , Proteolisis , Factores de TiempoRESUMEN
The aging of parents results in changes in the filial relationship. The increasing vulnerability of parents leads adult children to realize that they have individual needs and cannot fully function as sources of security and protection, as they did before. Simultaneously, the evidence of losses and disability imposes the need for care, which tends to be assumed by adult children. Therefore, there is a progressive change in the volume of support exchanges between parents and children, with more support from adult children to parents. The way adult children adapt to these transitions is influenced by several internal and relational factors. Filial maturity has been associated with filial caregiving towards aging parents. The concept of filial maturity describes a developmental stage in which the adult child overcomes the filial crisis, realizing and accepting that the parent also needs support and comfort and starting to relate to him/her beyond the strictly parental role. Thus, this study aims to explore the role of attachment and mental representation of caregiving in filial maturity. A total of 304 children aged between 35 and 64 years old participated in this study, with at least one of the living parents aged 65 years or older, not institutionalized. Attachment was assessed with the Adult Attachment Scale, mental representation of caregiving with the Mental Representations of Caregiving Scale and filial maturity with the Filial Maturity Measure. The results suggest that attachment, mental representation of caregiving and the interaction between the two explain 24.5% (p < 0.01) of variability in Comprehending and 11.1% (p < 0.05) of variability in Distance, two dimensions of filial maturity. These findings suggest that it is important to consider mental representation of caregiving and attachment when adult children must adapt to changes in the filial relationship and to the need to care for parents.
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The globalization in plant material trading has caused the emergence of invasive pests in many ecosystems, such as the alder pathogen Phytophthora ×alni in European riparian forests. Due to the ecological importance of alder to the functioning of rivers and the increasing incidence of P. ×alni-induced alder decline, effective and accessible decision tools are required to help managers and stakeholders control the disease. This study proposes a Bayesian belief network methodology to integrate diverse information on the factors affecting the survival and infection ability of P. ×alni in riparian habitats to help predict and manage disease incidence. The resulting Alder Decline Network (ADnet) management tool integrates information about alder decline from scientific literature, expert knowledge and empirical data. Expert knowledge was gathered through elicitation techniques that included 19 experts from 12 institutions and 8 countries. An original dataset was created covering 1189 European locations, from which P. ×alni occurrence was modeled based on bioclimatic variables. ADnet uncertainty was evaluated through its sensitivity to changes in states and three scenario analyses. The ADnet tool indicated that mild temperatures and high precipitation are key factors favoring pathogen survival. Flood timing, water velocity, and soil type have the strongest influence on disease incidence. ADnet can support ecosystem management decisions and knowledge transfer to address P. ×alni-induced alder decline at local or regional levels across Europe. Management actions such as avoiding the planting of potentially infected trees or removing man-made structures that increase the flooding period in disease-affected sites could decrease the incidence of alder disease in riparian forests and limit its spread. The coverage of the ADnet tool can be expanded by updating data on the pathogen's occurrence, particularly from its distributional limits. Research on the role of genetic variability in alder susceptibility and pathogen virulence may also help improve future ADnet versions.
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Alnus , Teorema de Bayes , Enfermedades de las Plantas/microbiología , Enfermedades de las Plantas/estadística & datos numéricos , Phytophthora , Ecosistema , Europa (Continente)/epidemiología , Bosques , Conservación de los Recursos NaturalesRESUMEN
Follicular lymphoma (FL), the most common indolent non-Hodgkin lymphoma, constitutes a paradigm of immune tumor microenvironment (TME) contribution to disease onset, progression, and heterogenous clinical outcome. Here we present the first FL-Patient Derived Lymphoma Spheroid (FL-PDLS), including fundamental immune actors and features of TME in FL lymph nodes (LNs). FL-PDLS is organized in disc-shaped 3D structures composed of proliferating B and T cells, together with macrophages with an intermediate M1/M2 phenotype. FL-PDLS recapitulates the most relevant B-cell transcriptional pathways present in FL-LN (proliferation, epigenetic regulation, mTOR, adaptive immune system, among others). The T cell compartment in the FL-PDLS preserves CD4 subsets (follicular helper, regulatory, and follicular regulatory), also encompassing the spectrum of activation/exhaustion phenotypes in CD4 and CD8 populations. Moreover, this system is suitable for chemo and immunotherapy testing, recapitulating results obtained in the clinic. FL-PDLS allowed uncovering that soluble galectin-9 limits rituximab, rituximab, plus nivolumab/TIM-3 antitumoral activities. Blocking galectin-9 improves rituximab efficacy, highlighting galectin-9 as a novel immunotherapeutic target in FL. In conclusion, FL-PDLS maintains the crosstalk between malignant B cells and the immune LN-TME and constitutes a robust and multiplexed pre-clinical tool to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.
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Galectinas , Ganglios Linfáticos , Linfoma Folicular , Microambiente Tumoral , Humanos , Linfoma Folicular/inmunología , Linfoma Folicular/patología , Linfoma Folicular/terapia , Ganglios Linfáticos/patología , Ganglios Linfáticos/inmunología , Microambiente Tumoral/inmunología , Esferoides Celulares , Inmunoterapia/métodos , Transducción de Señal , Células Tumorales CultivadasRESUMEN
OBJECTIVES: Human aging is a multidirectional, multidimensional, and multicausal process that reflects biological, psychological, and sociocultural influences, which act in distinct combinations throughout the life-span. Proactivity towards avoiding the usual aging process is needed. This study analyses the long-term effects of participation in Community-Based Programs on psychological well-being. METHOD: A sample of 150 community-dwelling participants enrolled in Community-Based Programs, aged 55 to 84 years and living in three Portuguese localities were matched by age (55-64, 65-74, 75-84 years), gender, and locality with a comparison group of non-participants. We administered a multidimensional gerontological protocol which included socio-demographic information, measures of health/disease, functional ability, social network, cognitive performance and psychological well-being. Hierarchical regression models were used to test the effects of Community-Based Programs on psychological well-being adjusting for remaining variables. RESULTS: Overall, psychological well-being is positively associated with household income and satisfaction with health. Nevertheless, in participants, psychological well-being builds predominantly upon social network and is not associated with a moderate inability or cognitive deficits, contrasting with psychological well-being in non-participants. After adjusting for background variables, psychological well-being was positively associated with health satisfaction and social network and negatively related to moderate inability. Further, a significant interaction of participation in Community-Based Programs with age, points out higher levels of psychological well-being in participants contrasting with a downward trend in non-participants. After stratification by age, psychological well-being increases with time attending Community-Based Programs in the oldest (75-84 years) contrasting with the remainder. CONCLUSIONS: Participation in Community-Based Programs may improve the negative effects of the aging process on psychological well-being. This positive effect as age increases may be linked to a reinforcement of social network, valued more by participants in Community-Based Programs. Furthermore, the programs may act as a healing/maintenance strategy in persons with moderate inability and/or cognitive deficits.
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Actividades Cotidianas , Bienestar Psicológico , Humanos , Adulto , Anciano , Anciano de 80 o más Años , Portugal , Envejecimiento/psicología , Vida IndependienteRESUMEN
Despite the advancements in therapy for B cell malignancies and the increase in long-term survival of patients, almost half of them lead to relapse. Combinations of chemotherapy and monoclonal antibodies such as anti-CD20 leads to mixed outcomes. Recent developments in immune cell-based therapies are showing many encouraging results. γδ T cells, with their potential of functional plasticity and their anti-tumoral properties, emerged as good candidates for cancer immunotherapies. The representation and the diversity of γδ T cells in tissues and in the blood, in physiological conditions or in B-cell malignancies such as B cell lymphoma, chronic lymphoblastic leukemia or multiple myeloma, provides the possibility to manipulate them with immunotherapeutic approaches for these patients. In this review, we summarized several strategies based on the activation and tumor-targeting of γδ T cells, optimization of expansion protocols, and development of gene-modified γδ T cells, using combinations of antibodies and therapeutic drugs and adoptive cell therapy with autologous or allogenic γδ T cells following potential genetic modifications.
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Mieloma Múltiple , Receptores de Antígenos de Linfocitos T gamma-delta , Humanos , Inmunoterapia , Anticuerpos Monoclonales/uso terapéutico , Linfocitos TRESUMEN
Mantle cell lymphoma (MCL), a rare and aggressive B-cell non-Hodgkin lymphoma, mainly develops in the lymph node (LN) and creates a protective and immunosuppressive niche that facilitates tumor survival, proliferation and chemoresistance. To capture disease heterogeneity and tumor microenvironment (TME) cues, we have developed the first patient-derived MCL spheroids (MCL-PDLS) that recapitulate tumor oncogenic pathways and immune microenvironment in a multiplexed system that allows easy drug screening, including immunotherapies. MCL spheroids, integrated by tumor B cells, monocytes and autologous T-cells self-organize in disc-shaped structures, where B and T-cells maintain viability and proliferate, and monocytes differentiate into M2-like macrophages. RNA-seq analysis demonstrated that tumor cells recapitulate hallmarks of MCL-LN (proliferation, NF-kB and BCR), with T cells exhibiting an exhaustion profile (PD1, TIM-3 and TIGIT). MCL-PDLS reproduces in vivo responses to ibrutinib and demonstrates that combination of ibrutinib with nivolumab (anti-PD1) may be effective in ibrutinib-resistant cases by engaging an immune response with increased interferon gamma and granzyme B release. In conclusion, MCL-PDLS recapitulates specific MCL-LN features and in vivo responses to ibrutinib, representing a robust tool to study MCL interaction with the immune TME and to perform drug screening in a patient-derived system, advancing toward personalized therapeutic approaches.
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Linfoma de Células del Manto , Humanos , Adulto , Línea Celular Tumoral , Linfoma de Células del Manto/patología , Resistencia a Antineoplásicos , Adenina/uso terapéutico , Microambiente TumoralRESUMEN
BACKGROUND: Follicular lymphoma (FL), the most common indolent non-Hodgkin's Lymphoma, is a heterogeneous disease and a paradigm of the contribution of immune tumor microenvironment to disease onset, progression, and therapy resistance. Patient-derived models are scarce and fail to reproduce immune phenotypes and therapeutic responses. METHODS: To capture disease heterogeneity and microenvironment cues, we developed a patient-derived lymphoma spheroid (FL-PDLS) model culturing FL cells from lymph nodes (LN) with an optimized cytokine cocktail that mimics LN stimuli and maintains tumor cell viability. RESULTS: FL-PDLS, mainly composed of tumor B cells (60% on average) and autologous T cells (13% CD4 and 3% CD8 on average, respectively), rapidly organizes into patient-specific three-dimensional (3D) structures of three different morphotypes according to 3D imaging analysis. RNAseq analysis indicates that FL-PDLS reproduces FL hallmarks with the overexpression of cell cycle, BCR, or mTOR signaling related gene sets. FL-PDLS also recapitulates the exhausted immune phenotype typical of FL-LN, including expression of BTLA, TIGIT, PD-1, TIM-3, CD39 and CD73 on CD3+ T cells. These features render FL-PDLS an amenable system for immunotherapy testing. With this aim, we demonstrate that the combination of obinutuzumab (anti-CD20) and nivolumab (anti-PD1) reduces tumor load in a significant proportion of FL-PDLS. Interestingly, B cell depletion inversely correlates with the percentage of CD8+ cells positive for PD-1 and TIM-3. CONCLUSIONS: In summary, FL-PDLS is a robust patient-derived 3D system that can be used as a tool to mimic FL pathology and to test novel immunotherapeutic approaches in a context of personalized medicine.
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Linfoma Folicular , Humanos , Linfoma Folicular/tratamiento farmacológico , Linfoma Folicular/genética , Receptor 2 Celular del Virus de la Hepatitis A , Receptor de Muerte Celular Programada 1/metabolismo , Microambiente Tumoral , Medicina de PrecisiónRESUMEN
Fusarium verticillioides and Fusarium subglutinans are important fungal pathogens of maize and other cereals worldwide. In this study, we developed PCR-based protocols for the identification of these pathogens targeting the gaoB gene, which codes for galactose oxidase. The designed primers recognized isolates of F. verticillioides and F. subglutinans that were obtained from maize seeds from several producing regions of Brazil but did not recognize other Fusarium spp. or other fungal genera that were either obtained from fungal collections or isolated from maize seeds. A multiplex PCR protocol was established to simultaneously detect the genomic DNA from F. verticillioides and F. subglutinans. This protocol could detect the DNA from these fungi growing in artificially or naturally infected maize seeds. Another multiplex reaction with a pair of primers developed in this work combined with a pre-existing pair of primers has allowed identifying F. subglutinans, F. konzum, and F. thapsinum. In addition, the identification of F. nygamai was also possible using a combination of two PCR reactions described in this work, and another described in the literature.
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ADN de Hongos/análisis , Proteínas Fúngicas/genética , Fusarium/enzimología , Galactosa Oxidasa/genética , Gibberella/enzimología , Reacción en Cadena de la Polimerasa Multiplex/métodos , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , ADN de Hongos/aislamiento & purificación , Fusarium/genética , Genoma Fúngico , Gibberella/genética , Zea mays/microbiologíaRESUMEN
ABSTRACT: We investigate twisted electrons with a well-defined orbital angular momentum, which have been ionised via a strong laser field. By formulating a new variant of the well-known strong field approximation, we are able to derive conservation laws for the angular momenta of twisted electrons in the cases of linear and circularly polarised fields. In the case of linear fields, we demonstrate that the orbital angular momentum of the twisted electron is determined by the magnetic quantum number of the initial bound state. The condition for the circular field can be related to the famous ATI peaks, and provides a new interpretation for this fundamental feature of photoelectron spectra. We find the length of the circular pulse to be a vital factor in this selection rule and, employing an effective frequency, we show that the photoelectron OAM emission spectra are sensitive to the parity of the number of laser cycles. This work provides the basic theoretical framework with which to understand the OAM of a photoelectron undergoing strong field ionisation.
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Follicular lymphoma (FL) is an indolent B cell lymphoproliferative disorder of transformed follicular center B cells, which accounts for 20-30 percent of all non-Hodgkin lymphoma (NHL) cases. Great advances have been made to identify the most relevant targets for precision therapy. However, no relevant models for in vitro studies have been developed or characterized in depth. To this purpose, we generated a 3D cell model from t(14;18)-positive B-NHL cell lines cultured in ultra-low attachment 96-well plates. Morphological features and cell growth behavior were evaluated by classical microscopy (2D imaging) and response to treatment with different drugs was evaluated by a high-content analysis system to determine the robustness of the model. We show that the ultra-low attachment (ULA) method allows the development of regular, spherical and viable ULA-multicellular aggregates of lymphoma cells (MALC). However, discrepancies in the results obtained after 2D imaging analyses on drug-treated ULA-MALC prompted us to develop 3D imaging and specific analyses. We show by using light sheet microscopy and specifically developed 3D imaging algorithms that 3D imaging and dedicated analyses are necessary to characterize morphological properties of 3D models and drug effects. This study proposes a new method, but also imaging tools and informatic solutions, developed for FL necessary for future preclinical studies.
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Galactose oxidase (GO) converts galactose to an aldehyde and has several biotechnological applications, including cancer diagnosis. It is mainly produced by Fusarium austroamericanum but is also produced by Fusarium acuminatum and by isolates of the Fusarium graminearum and Gibberella fujikuroi complexes. The F. austroamericanum GO gaoA gene has been cloned, but the GO genes from other secreting species have not been characterized. Problems associated with the F. austroamericanum GO such as high pI and low catalytic efficiency and thermostability, and the difficult purification process makes the search for homologous genes attractive. In this work, the GO genes from Fusarium verticillioides and Fusarium subglutinans, two species of the G. fujikuroi complex, were cloned, sequenced, and analyzed. New GO genes were found in databases and were used to construct a phylogenetic tree, which revealed the existence of three orthologous lineages of GO genes in Fusarium spp. In addition, RT-PCR analyses revealed that the new GO cloned gene may be endogenously expressed in F. subglutinans but not in F. verticillioides, in the used culture conditions.
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Fusarium/enzimología , Fusarium/genética , Galactosa Oxidasa/genética , Secuencia de Aminoácidos , Clonación Molecular , Análisis por Conglomerados , ADN de Hongos/química , ADN de Hongos/genética , Perfilación de la Expresión Génica , Genes Fúngicos , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Galactose oxidase catalyzes a two-electron oxidation, mainly from the C6 hydroxyl group of D-galactose, with the concomitant reduction of water to hydrogen peroxide. This enzyme is secreted by Fusarium species and has several biotechnological applications. In this study, a screening of galactose oxidase production among species of the Fusarium fujikuroi species complex demonstrated Fusarium subglutinans to be the main producer. The truncated F. subglutinans gaoA gene coding for the mature galactose oxidase was expressed from the prokaryotic vector pTrcHis2B in the E. coli Rosetta™ (DE3) strain. The purified recombinant enzyme presented temperature and pH optima of 30 °C and 7.0, respectively, KM of 132.6 ± 18.18 mM, Vmax of 3.2 ± 0.18 µmol of H2O2/min, kcat of 12,243 s-1, and a catalytic efficiency (kcat/KM) of 9.2 × 104 M-1 s-1. In the presence of 50% glycerol, the enzyme showed a T50 of 59.77 °C and was stable for several hours at pH 8.0 and 4 °C. Besides D-(+)-galactose, the purified enzyme also acted against D-(+)-raffinose, α-D-(+)-melibiose, and methyl-α-D-galactopyranoside, and was strongly inhibited by SDS. Although the F. subglutinans gaoA gene was successfully expressed in E. coli, its endogenous transcription was not confirmed by RT-PCR.
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Fusarium/enzimología , Galactosa Oxidasa/metabolismo , Galactosa/química , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fusarium/química , Galactosa/metabolismo , Galactosa Oxidasa/química , Galactosa Oxidasa/genética , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Concentración de Iones de Hidrógeno , Melibiosa/química , Melibiosa/metabolismo , Metilgalactósidos/química , Metilgalactósidos/metabolismo , Modelos Moleculares , Oxidación-Reducción , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Rafinosa/química , Rafinosa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TemperaturaRESUMEN
Quantifying the genetic diversity of riparian trees is essential to understand their chances to survive hydroclimatic alterations and to maintain their role as foundation species modulating fluvial ecosystem processes. However, the application of suitable models that account for the specific dendritic structure of hydrographic networks is still incipient in the literature. We investigate the roles of ecological and spatial factors in driving the genetic diversity of Salix salviifolia, an Iberian endemic riparian tree, across the species latitudinal range. We applied spatial stream-network models that aptly integrate dendritic features (topology, directionality) to quantify the impacts of multiple scale factors in determining genetic diversity. Based on the drift hypothesis, we expect that genetic diversity accumulates downstream in riparian ecosystems, but life history traits (e.g. dispersal patterns) and abiotic or anthropogenic factors (e.g. drought events or hydrological alteration) might alter expected patterns. Hydrological factors explained the downstream accumulation of genetic diversity at the intermediate scale that was likely mediated by hydrochory. The models also suggested upstream gene flow within basins that likely occurred through anemophilous and entomophilous pollen and seed dispersal. Higher thermicity and summer drought were related to higher population inbreeding and individual homozygosity, respectively, suggesting that increased aridity might disrupt the connectivity and mating patterns among and within riparian populations.
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Bosques , Variación Genética , Genética de Población , Árboles/clasificación , Árboles/genética , Ecología , Modelos TeóricosRESUMEN
SCOPE: In recent years, several studies reported the role of eIF4E-binding proteins (4E-BPs) on the development of diet-induced obesity and insulin resistance. Our aim was to investigate the effect of 4E-BP protein deletion on lipid accumulation and metabolism in skeletal muscle in response to a high-fat diet induced obesity in 4E-BP1/2 DKO mice. METHODS AND RESULTS: Diet-induced obesity engendered increased ectopic accumulation of lipotoxic species in skeletal muscle of 4E-BP1 and 4E-BP2 double knockout mice (4E-BP1/2 DKO), namely diacylglycerols and ceramides. Increased lipid accumulation was associated with alterations in the expression of genes involved in fatty acid transport (FATP, CD36), diacylglycerol/triacylglycerol biosynthesis (GPAT1, AGPAT1, DGAT1), and ß-oxidation (CPT1b, MCAD). Diet-induced obesity resulted in increased lean mass and muscle in 4E-BP1/2 DKO mice despite the development of a more severe systemic insulin resistance. Since increased expression of genes of several proteolytic systems (MuRF1, atrogin/MAFbx, and cathepsin-l) in 4EBP1/2 DKO skeletal muscle was reported, the increase of skeletal muscle mass in 4E-BP1/2 DKO mice suggests that ablation of 4E-BPs compensate with activation of muscle anabolism. CONCLUSIONS: These findings indicate that 4E-BP proteins may prevent excess lipid accumulation in skeletal muscle and suggest that 4E-BPs are key regulators of muscle homeostasis regardless of insulin sensitivity.