RESUMEN
Reactive washing (RW) is a key process for disinfecting, purifying, and bleaching of cork stoppers to seal bottles with alcoholic beverages. Excessively severe treatment conditions deteriorate the surface properties of cork stoppers and must be strictly controlled. In this study, the conventional RW of natural cork stoppers was optimized employing a fractional factorial design. The RW variables (H2O2 and NaOH concentrations, oxidation time, and washing water volume) were correlated with the final ISO brightness of the stoppers. A three-level and four-factor fractional factorial design within the response surface methodology approach allowed a quadratic model to predict the process response, where the H2O2 concentration is the variable with the highest response (ISO brightness), followed by the NaOH concentration. The model obtained was validated, allowing the optimization of the process with savings of 37% in the concentration of H2O2 and 33% in the concentration of NaOH and volume of washing water, without deteriorating the final appearance of the stoppers. In addition, the less severe treatment of stoppers under optimized conditions led to less degradation of their surface, thus favoring the receptivity to functional coatings.
RESUMEN
The dioxane lignin was isolated from extractives- and suberin-free cork (Quercus suber L.) by a modified acidolytic procedure and submitted to structural analysis by permanganate oxidation, analytical pyrolysis coupled with gas chromatography and mass spectrometry (Py-GC-MS), liquid- and solid-state nuclear magnetic resonance (NMR) spectroscopy, and Fourier transform infrared (FTIR) spectroscopy. The molecular weight (Mw = 2500 Da) was assessed by size exclusion chromatography (SEC). The results obtained show that the cork lignin is of syringyl (S)/guaiacyl (G) type with a small proportion of p-hydroxyphenyl (H) units (S:G:H molar ratio of 23:72:5). Among a dozen detected lignin structures, those linked by ether bonds, such as ß-O-4' (38 mol %) and 4-O-5' (5 mol %), were the most abundant. The frequency of occurrence of ß-5', ß-ß', 5-5', tetrahydrofuran type, and structures arising from the condensation with concomitant procyanidins was assessed. Ferulates were the only cinnamic structure detected in the cork dioxane lignin.
Asunto(s)
Lignina , Quercus , Dioxanos , Peso Molecular , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Trehalose-6-phosphate (T6P) is an important signaling metabolite involved in plant growth control that inhibits the sucrose nonfermenting-1-related protein kinase 1 (SnRK1), a key regulator of energy and carbon metabolism in plants. The quantification of T6P in plant tissues is fundamental to improve our understanding of sugar signaling and the links between plant growth and development in response to stress conditions. However, the almost undetectable levels of T6P together with the complex plant matrix and the presence of T6P isomers such as sucrose-6-phosphate (S6P), makes the detection of this metabolite challenging. This work describes the development and validation of a hydrophilic interaction chromatography (HILIC) method for the on-line coupling with negative ion electrospray (ESI) triple quadrupole tandem mass spectrometry (MS/MS) in the highly sensitive and selective multiple reaction monitoring (MRM) mode for the target analysis of metabolic intermediates of the biosynthesis of trehalose, including glucose-6-phosphate (G6P), uridine 5-diphospho-glucose (UDPG), T6P (and its isomer S6P). Enhanced signal in the MRM mode and improved chromatographic separation for each compound were obtained using piperidine and methylphosphonic acid as additives in the HILIC mobile phase. The optimized HILIC-ESI-QqQ-MS/MS method increases the range of sensitive analytical methodologies for the quantification of key low-abundant metabolites, and was applied to quantify the fluctuations of S6P, T6P and G6P in Medicago truncatula plants in response to environmental stress. The levels of S6P, T6P, and G6P in M. truncatula plant tissues (roots and leaves) exposed to a water deficit and recovery treatment, ranged from 30 to 150pmolg-1 FW, 16-120pmolg-1 FW, and 330-1690pmolg-1 FW, respectively.
Asunto(s)
Cromatografía Liquida/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Medicago truncatula/metabolismo , Metaboloma , Fosfatos de Azúcar/análisis , Espectrometría de Masas en Tándem/métodos , Trehalosa/análogos & derivados , Vías Biosintéticas , Límite de Detección , Hojas de la Planta/metabolismo , Estándares de Referencia , Soluciones , Sacarosa/análogos & derivados , Sacarosa/metabolismo , Fosfatos de Azúcar/metabolismo , Trehalosa/análisisRESUMEN
The mitochondrion maintains and regulates its proteome with chaperones primarily inherited from its bacterial endosymbiont ancestor. Among these chaperones is the AAA+ unfoldase ClpX, an important regulator of prokaryotic physiology with poorly defined function in the eukaryotic mitochondrion. We observed phenotypic similarity in S. cerevisiae genetic interaction data between mitochondrial ClpX (mtClpX) and genes contributing to heme biosynthesis, an essential mitochondrial function. Metabolomic analysis revealed that 5-aminolevulinic acid (ALA), the first heme precursor, is 5-fold reduced in yeast lacking mtClpX activity and that total heme is reduced by half. mtClpX directly stimulates ALA synthase in vitro by catalyzing incorporation of its cofactor, pyridoxal phosphate. This activity is conserved in mammalian homologs; additionally, mtClpX depletion impairs vertebrate erythropoiesis, which requires massive upregulation of heme biosynthesis to supply hemoglobin. mtClpX, therefore, is a widely conserved stimulator of an essential biosynthetic pathway and uses a previously unrecognized mechanism for AAA+ unfoldases.
Asunto(s)
Endopeptidasa Clp/metabolismo , Eritropoyesis , Eucariontes/metabolismo , Hemo/biosíntesis , 5-Aminolevulinato Sintetasa/metabolismo , Secuencia de Aminoácidos , Ácido Aminolevulínico/metabolismo , Animales , Evolución Biológica , Endopeptidasa Clp/química , Endopeptidasa Clp/genética , Eucariontes/genética , Humanos , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Pez Cebra/metabolismoRESUMEN
We used exome sequencing to identify mutations in sideroflexin 4 (SFXN4) in two children with mitochondrial disease (the more severe case also presented with macrocytic anemia). SFXN4 is an uncharacterized mitochondrial protein that localizes to the mitochondrial inner membrane. sfxn4 knockdown in zebrafish recapitulated the mitochondrial respiratory defect observed in both individuals and the macrocytic anemia with megaloblastic features of the more severe case. In vitro and in vivo complementation studies with fibroblasts from the affected individuals and zebrafish demonstrated the requirement of SFXN4 for mitochondrial respiratory homeostasis and erythropoiesis. Our findings establish mutations in SFXN4 as a cause of mitochondriopathy and macrocytic anemia.
Asunto(s)
Anemia Macrocítica/genética , Proteínas de la Membrana/genética , Enfermedades Mitocondriales/genética , Adolescente , Animales , Niño , Eritropoyesis/genética , Exoma , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Mitocondriales/genética , Mutación , Pez Cebra/genéticaRESUMEN
Growth Factor Independence (Gfi) transcription factors play essential roles in hematopoiesis, differentially activating and repressing transcriptional programs required for hematopoietic stem/progenitor cell (HSPC) development and lineage specification. In mammals, Gfi1a regulates hematopoietic stem cells (HSC), myeloid and lymphoid populations, while its paralog, Gfi1b, regulates HSC, megakaryocyte and erythroid development. In zebrafish, gfi1aa is essential for primitive hematopoiesis; however, little is known about the role of gfi1aa in definitive hematopoiesis or about additional gfi factors in zebrafish. Here, we report the isolation and characterization of an additional hematopoietic gfi factor, gfi1b. We show that gfi1aa and gfi1b are expressed in the primitive and definitive sites of hematopoiesis in zebrafish. Our functional analyses demonstrate that gfi1aa and gfi1b have distinct roles in regulating primitive and definitive hematopoietic progenitors, respectively. Loss of gfi1aa silences markers of early primitive progenitors, scl and gata1. Conversely, loss of gfi1b silences runx-1, c-myb, ikaros and cd41, indicating that gfi1b is required for definitive hematopoiesis. We determine the epistatic relationships between the gfi factors and key hematopoietic transcription factors, demonstrating that gfi1aa and gfi1b join lmo2, scl, runx-1 and c-myb as critical regulators of teleost HSPC. Our studies establish a comparative paradigm for the regulation of hematopoietic lineages by gfi transcription factors.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Hematopoyesis/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero/metabolismo , Epistasis Genética , Eritropoyesis/genética , Evolución Molecular , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Sistema Hematopoyético/embriología , Sistema Hematopoyético/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Pez Cebra/embriología , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/metabolismoRESUMEN
Several researchers investigated the induction of metallothioneins (MTs) in the presence of metals, namely Cadmium (Cd). Fewer studies observed the induction of MTs due to oxidizing agents, and literature comparing the sensitivity of MTs to different stressors is even more scarce or even nonexistent. The role of MTs in metal and oxidative stress and thus their use as a stress biomarker, remains to be clearly elucidated. To better understand the role of MTs as a biomarker in Cerastoderma edule, a bivalve widely used as bioindicator, a laboratory assay was conducted aiming to assess the sensitivity of MTs to metal and oxidative stressors. For this purpose, Cd was used to induce metal stress, whereas hydrogen peroxide (H2O2), being an oxidizing compound, was used to impose oxidative stress. Results showed that induction of MTs occurred at very different levels in metal and oxidative stress. In the presence of the oxidizing agent (H2O2), MTs only increased significantly when the degree of oxidative stress was very high, and mortality rates were higher than 50 percent. On the contrary, C. edule survived to all Cd concentrations used and significant MTs increases, compared to the control, were observed in all Cd exposures. The present work also revealed that the number of ions and the metal bound to MTs varied with the exposure conditions. In the absence of disturbance, MTs bound most (60-70 percent) of the essential metals (Zn and Cu) in solution. In stressful situations, such as the exposure to Cd and H2O2, MTs did not bind to Cu and bound less to Zn. When organisms were exposed to Cd, the total number of ions bound per MT molecule did not change, compared to control. However the sort of ions bound per MT molecule differed; part of the Zn and all Cu ions where displaced by Cd ions. For organisms exposed to H2O2, each MT molecule bound less than half of the ions compared to control and Cd conditions, which indicates a partial oxidation of thiol groups in the cysteine residues through ROS scavenging. The present results suggest that MTs are excellent markers of metal stress, but not of oxidative stress.
Asunto(s)
Biomarcadores/análisis , Cadmio/toxicidad , Cardiidae/efectos de los fármacos , Metalotioneína/análisis , Estrés Oxidativo/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Animales , Cardiidae/química , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Oxidantes/toxicidad , Oxidación-ReducciónRESUMEN
Commercial and recreational harvesting of shellfish within the coastal systems is usually very extensive. Since these ecosystems are frequently subjected to contamination, namely from agricultural, urban and industrial activities, and shellfish generally display a high capacity to bioaccumulate metals, populations may be at risk in terms of toxic metal exposure as a consequence of the harvesting and ingestion of near shore coastal marine organisms. Shellfish is regularly tested for concentrations of metals and other contaminants by legal authorities for commercial purposes, but although health officials use total metal as standards of food safety, only a part of the metal accumulated in shellfish is available to be assimilated and to cause toxic effect. In order to elucidate these issues an investigation on cockles inhabiting the Aveiro estuary was conducted. Element levels in sediments and wild Cerastoderma edule from sampling areas with different levels of contamination were measured; total element burden of cockles was related to accessible fraction for assimilation (TAM); element concentrations in wild C. edule were compared to EFSA (European Food Safe Authorities), USFDA (United States Food and Drug Administration) and FSANZ (Food Standards Australia and New Zealand) maximum levels (MLs); and the amount of cockle flesh needed to be consumed to exceed provisional tolerable weekly intake (PTWI) was determined. The present work showed that although sediment metal and metalloid contamination in Aveiro estuary is low the concentration of elements in C. edule does not reflect the contamination of the sediment. Aluminium (Al) and mercury (Hg) were the less and nickel (Ni), lead (Pb), zinc (Zn) and cadmium (Cd) were the most bioaccumulated metals by cockles. Comparison of MLs from international organisations with the concentration of elements in C. edule showed that arsenic (As) and Pb exceeded standard levels. The ingestion of less than 1 kg for As and 1.5 kg for Pb of cockles would result in exceeding the PTWI threshold (0.015 and 0.025 mg kg⻹ week⻹ respectively) in any of the areas considered in the study. Cd and Al also appear to be limiting elements for human consumption. Indeed, consumption of more than 3.1 kg and 2.1 kg of whole cockle soft part from one of the study areas during a single week would lead to exceedance of the recommended PTWI value for Cd (0.007 mg kg⻹ week⻹) and Al (7 mg kg⻹ week⻹) respectively. The health concerns to humans from cockle consumption from Aveiro estuary are discussed.
Asunto(s)
Cardiidae/metabolismo , Inocuidad de los Alimentos , Metales/metabolismo , Contaminantes Químicos del Agua/metabolismo , Animales , Arsénico/análisis , Arsénico/metabolismo , Arsénico/toxicidad , Carga Corporal (Radioterapia) , Cadmio/análisis , Cadmio/metabolismo , Cadmio/toxicidad , Ecosistema , Exposición a Riesgos Ambientales/análisis , Contaminación de Alimentos/estadística & datos numéricos , Sedimentos Geológicos/química , Humanos , Mercurio/análisis , Mercurio/metabolismo , Mercurio/toxicidad , Metales/análisis , Metales/toxicidad , Medición de Riesgo , Agua de Mar/química , Contaminantes Químicos del Agua/análisis , Contaminantes Químicos del Agua/toxicidad , Zinc/análisis , Zinc/metabolismo , Zinc/toxicidadRESUMEN
Human activities have been increasing the cadmium levels in soils and waters, disturbing many organisms in the primary trophic levels such as microalgae. Toxic metal pollution is a focus point of serious concern and the examination and monitoring water quality are becoming essential procedures. Diatoms are important bioindicators to monitor the metal concentrations in diverse habitats. The present study was planned to determine the biochemical mechanisms used by freshwater diatoms to cope with cadmium stress and to identify biomarkers of metal stress. For this, Nitzschia palea (Kützing) W. Smith was grown under different concentrations of Cd (0.01-0.1 mg l(-1)) and the IC(50) determined. Three concentrations (0.1, 0.2 and 0.3 mg Cd l(-1)) and a control (no cadmium) were used to undergo the experimental assays which allowed the determination of cadmium accumulation and several biochemical markers currently used to assess metal stress. N. palea was sensitive to cadmium, as the IC(50) calculated was 0.0276 mg Cd l(-1). Cadmium accumulation increased sharply and was mainly associated to the frustule. Total protein content increased with cadmium exposure, inducing increases and decreases in polypeptide expression, indicating an attempt of N. palea cells to adjust to the new prevailing conditions induced by metal stress. In order to cope with cadmium stress, cells induced the synthesis of chelating molecules such as phytochelatins (PCs). The enzymatic (SOD and CAT) and non-enzymatic (glutathione and proline) ROS scavenging mechanisms were also induced. Our results indicate the existence of diverse metal stress-mediated mechanisms in order to lessen metal damages to the cell. PCs showed to be a suitable biomarker of metal stress; besides being metal specific and concentration respondent it also allows to infer about the level of stress imposed to cells, constituting a useful tool to complement the evaluation of diatom communities when accessing aquatic metal toxicity.
Asunto(s)
Biomarcadores/análisis , Cadmio/toxicidad , Diatomeas/química , Estrés Fisiológico/efectos de los fármacos , Contaminantes Químicos del Agua/toxicidad , Regulación de la Expresión Génica/efectos de los fármacos , Concentración 50 Inhibidora , Fitoquelatinas/metabolismo , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: Sucrose content is a highly desirable trait in sugarcane as the worldwide demand for cost-effective biofuels surges. Sugarcane cultivars differ in their capacity to accumulate sucrose and breeding programs routinely perform crosses to identify genotypes able to produce more sucrose. Sucrose content in the mature internodes reach around 20% of the culms dry weight. Genotypes in the populations reflect their genetic program and may display contrasting growth, development, and physiology, all of which affect carbohydrate metabolism. Few studies have profiled gene expression related to sugarcane's sugar content. The identification of signal transduction components and transcription factors that might regulate sugar accumulation is highly desirable if we are to improve this characteristic of sugarcane plants. RESULTS: We have evaluated thirty genotypes that have different Brix (sugar) levels and identified genes differentially expressed in internodes using cDNA microarrays. These genes were compared to existing gene expression data for sugarcane plants subjected to diverse stress and hormone treatments. The comparisons revealed a strong overlap between the drought and sucrose-content datasets and a limited overlap with ABA signaling. Genes associated with sucrose content were extensively validated by qRT-PCR, which highlighted several protein kinases and transcription factors that are likely to be regulators of sucrose accumulation. The data also indicate that aquaporins, as well as lignin biosynthesis and cell wall metabolism genes, are strongly related to sucrose accumulation. Moreover, sucrose-associated genes were shown to be directly responsive to short term sucrose stimuli, confirming their role in sugar-related pathways. CONCLUSION: Gene expression analysis of sugarcane populations contrasting for sucrose content indicated a possible overlap with drought and cell wall metabolism processes and suggested signaling and transcriptional regulators to be used as molecular markers in breeding programs. Transgenic research is necessary to further clarify the role of the genes and define targets useful for sugarcane improvement programs based on transgenic plants.