Toxicological response of the model fungus Saccharomyces cerevisiae to different concentrations of commercial graphene nanoplatelets

Graphene nanomaterials have attracted a great interest during the last years for different applications, but their possible impact on different biological systems remains unclear. Here, an assessment to understand the toxicity of commercial polycarboxylate functionalized graphene nanoplatelets (GN) on the unicellular fungal model Saccharomyces cerevisiae was performed. While cell proliferation was not negatively affected even in the presence of 800 mg L−1 of the nanomaterial for 24 hours, oxidative stress was induced at a lower concentration (160 mg L−1), after short exposure periods (2 and 4 hours). No DNA damage was observed under a comet assay analysis under the studied conditions. In addition, to pinpoint the molecular mechanisms behind the early oxidative damage induced by GN and to identify possible toxicity pathways, the transcriptome of S. cerevisiae exposed to 160 and 800 mg L−1 of GN was studied. Both GN concentrations induced expression changes in a common group of genes (337), many of them related to the fungal response to reduce the nanoparticles toxicity and to maintain cell homeostasis. Also, a high number of genes were only differentially expressed in the GN800 condition (3254), indicating that high GN concentrations can induce severe changes in the physiological state of the yeast.European Union’s H2020 research and innovation programme under the Marie Skłodowska-Curie grant agreements N° 691095 and N° 734873; and Junta de Castilla y Leon-FEDER under grants N° BU079U16, and N° UBU-16-B

La evaluación de la calidad de enseñanza universitaria mediante la percepción del alumno de una actividad práctica: propuesta de escalas de medida

El panorama universitario actual, centrado en la valoración y juicio de los distintos agentes del proceso de enseñanza-aprendizaje, supone un campo adecuado para el desarrollo de investigaciones que pretendan alcanzar indicadores fiables del desempeño de las labores docentes. La futura implantación del Espacio Europeo de Educación Superior (EEES), acrecienta el interés por este tipo de investigaciones.Este trabajo, centrado en este contexto, pretende evaluar y valorar, por parte del discente, una actividad práctica constitutiva del programa de dos asignaturas universitarias impartidas en dos licenciaturas oficiales. Para su evaluación desarrollaremos una escala de medida que permita conocer la percepción de calidad de dicha actividad por parte de los alumnos y las posibles fuentes de mejora. Con los resultados obtenidos podremos conocer que aportación tiene esta práctica en el proceso de aprendizaje de esta materia y para la formación general del alumno

Colonization of electrospun polycaprolactone fibers by relevant pathogenic bacterial strains

Electrospun biodegradable polymers have emerged as promising materials for their applications in several fields, including biomedicine and food industry. For this reason, the susceptibility of these materials to be colonized by different pathogens is a critical issue for public health, and their study can provide future knowledge to develop new strategies against bacterial infections. In this work, the ability of three pathogenic bacterial species (Pseudomonas aeruginosa, Acinetobacter baumannii, and Listeria monocytogenes) to adhere and form biofilm in electrospun polycaprolactone (PCL) microfibrous meshes was investigated. Bacterial attachment was analyzed in meshes with different microstructure, and comparisons with other materials (borosilicate glass and electrospun polylactic acid (PLA)) fibers were assessed. Analysis included colony forming unit (CFU) counts, scanning electron microscopy (SEM), and crystal violet (CV) staining. All the obtained data suggest that PCL meshes, regardless of their microstructure, are highly susceptible to be colonized by the pathogenic relevant bacteria used in this study, so a pretreatment or a functionalization with compounds that present some antimicrobial activity or antibiofilm properties is highly recommended before their application. Moreover, an experiment designed to simulate a chronic wound environment was used to demonstrate the ability of these meshes to detach biofilms from the substratum where they have developed, thus making them promising candidates to be used in wound cleaning and disinfection.European Union’s H2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 691095 and Junta de Castilla y Leon-FEDER (projects BU079U16 and BU092U16)

Global transcriptional response of aspergillus niger to blocked active citrate export through deletion of the exporter gene

Aspergillus niger is the major industrial citrate producer worldwide. Export as well as uptake of citric acid are believed to occur by active, proton-dependent, symport systems. Both are major bottlenecks for industrial citrate production. Therefore, we assessed the consequences of deleting the citT gene encoding the A. niger citrate exporter, effectively blocking active citrate export. We followed the consumption of glucose and citrate as carbon sources, monitored the secretion of organic acids and carried out a thorough transcriptome pathway enrichment analysis. Under controlled cultivation conditions that normally promote citrate secretion, the knock-out strain secreted negligible amounts of citrate. Blocking active citrate export in this way led to a reduced glucose uptake and a reduced expression of high-affinity glucose transporter genes, mstG and mstH. The glyoxylate shunt was strongly activated and an increased expression of the OAH gene was observed, resulting in a more than two-fold higher concentration of oxalate in the medium. Deletion of citT did not affect citrate uptake suggesting that citrate export and citrate uptake are uncoupled from the system

Forward genetics by genome sequencing uncovers the central role of the aspergillus niger goxB locus in hydrogen peroxide induced glucose oxidase expression

Aspergillus niger is an industrially important source for gluconic acid and glucose oxidase (GOx), a secreted commercially important flavoprotein which catalyses the oxidation of b-D-glucose by molecular oxygen to D-glucolactone and hydrogen peroxide. Expression of goxC, the GOx encoding gene and the concomitant two step conversion of glucose to gluconic acid requires oxygen and the presence of significant amounts of glucose in the medium and is optimally induced at pH 5.5. The molecular mechanisms underlying regulation of goxC expression are, however, still enigmatic. Genetic studies aimed at understanding GOx induction have indicated the involvement of at least seven complementation groups, for none of which the molecular basis has been resolved. In this study, a mapping-by-sequencing forward genetics approach was used to uncover the molecular role of the goxB locus in goxC expression. Using the Illumina and PacBio sequencing platforms a hybrid high quality draft genome assembly of laboratory strain N402 was obtained and used as a reference for mapping of genomic reads obtained from the derivative NW103:goxB mutant strain. The goxB locus encodes a thioredoxin reductase. A deletion of the encoding gene in the N402 parent strain led to a high constitutive expression level of the GOx and the lactonase encoding genes required for the two-step conversion of glucose in gluconic acid and of the catR gene encoding catalase R. This high constitutive level of expression was observed to be irrespective of the carbon source and oxidative stress applied. A model clarifying the role of GoxB in the regulation of the expression of goxC involving hydrogen peroxide as second messenger is presented

Mechano-Optical Analysis of Single Cells with Transparent Microcapillary Resonators

The study of biophysical properties of single cells is becoming increasingly relevant in cell biology and pathology. The measurement and tracking of magnitudes such as cell stiffness, morphology, and mass or refractive index have brought otherwise inaccessible knowledge about cell physiology, as well as innovative methods for high-throughput label-free cell classification. In this work, we present hollow resonator devices based on suspended glass microcapillaries for the simultaneous measurement of single-cell buoyant mass and reflectivity with a throughput of 300 cells/minute. In the experimental methodology presented here, both magnitudes are extracted from the devices' response to a single probe, a focused laser beam that enables simultaneous readout of changes in resonance frequency and reflected optical power of the devices as cells flow within them. Through its application to MCF-7 human breast adenocarcinoma cells and MCF-10A nontumorigenic cells, we demonstrate that this mechano-optical technique can successfully discriminate pathological from healthy cells of the same tissue type

Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus niger and Trichoderma reesei

Background: Global climate change and fossil fuels limitations have boosted the demand for robust and efficient microbial factories for the manufacturing of bio-based products from renewable feedstocks. In this regard, efforts have been done to enhance the enzyme-secreting ability of lignocellulose-degrading fungi, aiming to improve protein yields while taking advantage of their ability to use lignocellulosic feedstocks. Access to sugars in complex polysaccharides depends not only on their release by specific hydrolytic enzymes, but also on the presence of transporters capable of effectively transporting the constituent sugars into the cell. This study aims to identify and characterize xylose transporters from Aspergillus Niger and Trichoderma reesei, two fungi that have been industrially exploited for decades for the production of lignocellulose-degrading hydrolytic enzymes. Results: A hidden Markov model for the identification of xylose transporters was developed and used to analyze the A. Niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. Niger (XltA, XltB and XltC) and three T. reesei (Str1, Str2 and Str3) transporters were selected, functionally validated and biochemically characterized through their expression in a Saccharomyces cerevisiae hexose transport null mutant, engineered to be able to metabolize xylose but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs recently associated to xylose transporters. Transcriptional analysis of A. Niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, signifying a biological role for these transporters in xylose utilization. Conclusions: This study revealed the existence of a variety of xylose transporters in the cell factories A. Niger and T. reesei. The particular substrate specificity and biochemical properties displayed by A. Niger XltA and XltB suggested a possible biological role for these transporters in xylose uptake. New insights were also gained into the molecular mechanisms regulating the pentose utilization, at inducer uptake level, in these fungi. Analysis of the A. Niger and T. reesei predicted transportome with the newly developed hidden Markov model showed to be an efficient approach for the identification of new xylose transporting proteins.</p

Aspergillus niger citrate exporter revealed by comparison of two alternative citrate producing conditions

Currently, there is no consensus regarding the mechanism underlying Aspergillus niger citrate biosynthesis and secretion. We hypothesise that depending on the experimental setup, extracellular citrate accumulation can have fundamentally different underlying transcriptomic landscapes. We show that varying the amount and type of supplement of an arginine auxotrophic A. niger strain results in transcriptional down-regulation of citrate metabolising enzymes in the condition in which more citrate is accumulated extracellularly. This contrasts with the transcriptional adaptations when increased citrate production is triggered by iron limitation. By combining gene expression data obtained from these two very distinct experimental setups with hidden Markov models and transporter homology approaches, we were able to compile a shortlist of the most likely citrate transporter candidates. Two candidates (An17g01710 and An09g06720m.01) were heterologously expressed in the yeast Saccharomyces cerevisiae, and one of the resultant mutants showed the ability to secrete citrate. Our findings provide steps in untangling the complex interplay of different mechanisms underlying A. niger citrate accumulation, and we demonstrate how a comparative transcriptomics approach complemented with further bioinformatics analyses can be used to pinpoint a fungal citrate exporter

Uso potencial de subproductos biorrefinados de Moringa oleifera en la alimentación de codornices japonesas

&nbsp; With the aim of evaluating the degreased paste from seed pressing (SMOD) and a residue flour from leaves fermented by Mucor circinelloides-Saccharomyces cerevisiae (HMHF), biorefining byproducts from the processes of biodiesel and bioethanol production from Moringa oleifera (MO), the effect of their inclusion was tested in fattening Japanese quails, measuring productive response parameters and carcass yields. The results were compared to diets containing unfermented MO leaf (HMNT) and a conventional one (TEST). For this purpose, 180 quails were fed during 35 days with the formulated diets. The diet with HMNT showed the lowest weight gain and the highest mortality. SMOD obtained the highest average of final live weight, weight gain and the lowest food conversion. SMOD presented the highest weights and HMNT the highest percentages of warm and cold carcass. It is concluded that the degreased pastes from seed pressing and the flour from fermented MO can be used at the inclusion levels tested in diets to feed Japanese quails, without negative effects on their growth.&nbsp; Con el fin de evaluar la pasta desgrasada por prensado de semilla (SMOD) y una harina de residuo de hojas fermentadas por Mucor circinelloides-Saccharomyces cerevisiae (HMHF), subproductos biorrefinados de los procesos de producción de biodiesel y bioetanol a partir de Moringa oleifera (MO), se probó el efecto de su inclusión en dietas de iniciación y finalización para codornices japonesas, midiéndose parámetros de desarrollo y rendimientos de canal. Los resultados se compararon con dietas conteniendo hoja de MO no fermentada (HMNT) y una convencional (TEST). Para ello, se alimentaron 180 codornices durante 35 días con las dietas formuladas. Durante la etapa de iniciación la dieta con HMNT mostró la menor ganancia de peso y la mayor mortalidad. SMOD obtuvo el mayor promedio de peso vivo final, ganancia de peso y menor conversión alimenticia. SMOD presentó los pesos más altos y HMNT los mayores porcentajes de canal caliente y fría. Se concluye que las pastas de semilla desgrasada por prensado y la harina de hoja fermentada de MO pueden usarse a los niveles de inclusión probados, en dietas para alimentar codornices japonesas sin efectos negativos en su crecimiento