Quantitative proteomics reveals new insights into calcium-mediated resistance mechanisms in Aspergillus flavus against the antifungal protein PgAFP in cheese

The ability of Aspergillus flavus to produce aflatoxins in dairy products presents a potential hazard. The antifungal protein PgAFP from Penicillium chrysogenum inhibits various foodborne toxigenic fungi, including Aspergillus flavus. However, PgAFP did not inhibit A. flavus growth in cheese, which was related to the associated cation content. CaCl2 increased A. flavus permeability and prevented PgAFP-mediated inhibition in potato dextrose broth (PDB). PgAFP did not elicit any additional increase in permeability of CaCl2-incubated A. flavus. Furthermore, PgAFP did not alter metabolic capability, chitin deposition, or hyphal viability of A. flavus grown with CaCl2. Comparative proteomic analysis after PgAFP treatment of A. flavus in calcium-enriched PDB revealed increased abundance of 125 proteins, including oxidative stress-related proteins, as determined by label-free mass spectrometry (MS)-based proteomics. Seventy proteins were found at lower abundance, with most involved in metabolic pathways and biosynthesis of secondary metabolites. These changes do not support the blockage of potential PgAFP receptors in A. flavus by calcium as the main cause of the protective role. A. flavus resistance appears to be mediated by calcineurin, G-protein, and γ-glutamyltranspeptidase that combat oxidative stress and impede apoptosis. These findings could serve to design strategies to improve PgAFP activity against aflatoxigenic moulds in dairy products

Quantitative proteomic profiling of ochratoxin A repression in Penicillium nordicum by protective cultures

Dry-cured meat products are usually contaminated with moulds during ripening. Although fungal development contributes to the desired sensory characteristics, some moulds, such as Penicillium nordicum are able to produce ochratoxin A (OTA) on meat products. Therefore, strategies to prevent OTA contamination in ripened meat products are required. Microorganisms isolated from these meat products can be adequate as biocontrol agents, given that no negative sensory impact is expected. The PgAFP antifungal protein-producer Penicillium chrysogenum (Pc) and Debaryomyces hansenii (Dh) have been shown to successfully inhibit toxigenic moulds. However, scarce information about the mechanism of action of these biocontrol agents on toxigenic mould inhibition is available. Comparative proteomic analysis is a powerful tool to investigate the physiological response of microorganisms to stimuli. Proteomic analysis was carried out on P. nordicum co-cultured with Pc, Dh, PgAFP, and their combinations on a dry-cured ham-based medium. Additionally, OTA production by P. nordicum in the different cultures was measured. The individual inoculation of Pc or Dh repressed OTA production by P. nordicum by 5 and 3.15 fold, respectively. A total of 2844 unique P. nordicum proteins were identified by proteomic analysis. The impact of the biocontrol agents on the proteome of P. nordicum was higher for Pc-containing cultures, followed by Dh-containing treatments. PgAFP alone had minimal impact on the proteome of P. nordicum. Proteomic analyses indicated Pc repressed P. nordicum OTA production through nutrient competition, potentially reducing glucose availability. Data also suggest that Dh and Pc inhibited P. nordicum through cell wall integrity impairment. Both Pc and Dh seem to hamper P. nordicum secondary metabolism (SM) as indicated by lower levels of MAP kinases and SM-associated proteins found in the co-inoculated P. nordicum. This work paves the way to use antifungal agents in the most efficient way to prevent OTA formation in meat products

Analytical derivation of the probability for the escape of stars from colliding galaxies

We use the impulse approximation to derive analytical formulae for the escape probability from a simple binary system that interacts with a third body. The binary system is made up of a mass-less body in circular orbit around a massive object, and we assume that the two massive bodies follow a Schuster (or Plummer) distribution. Within the ranges imposed by the impulsive approximation to the parameters of the encounter, we find good agreement between our results and those obtained from numerical experiments.Facultad de Ciencias Astronómicas y Geofísica

Revista de educación

Resumen tomado del autor. Este artículo se incluye en el monográfico ïLa Ley de CalidadïIntenta identificar los factores que subyacen tras las altas tasas de abandono y fracaso que se producen en los primeros cursos de las carreras científicas y de las Ingenierías Técnicas Superiores. Los objetivos específicos de esta primera parte de la investigación son : averiguar cuál es la formación inicial en física de los alumnos que ingresan en la universidad, y estudiar la validez tanto de la prueba inicial diseñada en esta investigación, como de la prueba de selectividad para predecir el rendimiento en asignaturas de física del primer curso universitario. Las muestras están constituídas por un total de 663 estudiantes de las universidades de Alcalá y Burgos, y los resultados señalan la existencia de deficiencias importantes en destrezas científicas y ponen de manifiesto la escasa validez de ambas medidas de formación inicial para predecir el rendimiento posterior. En consecuencia, la explicación del éxito o fracaso debe buscarse en otros factores distintos al conocimiento inicial de los alumnos.MadridBiblioteca de Educación del Ministerio de Educación, Cultura y Deporte; Calle San Agustín, 5 - 3 Planta; 28014 Madrid; Tel. +34917748000; [email protected]

Manuscript title: antifungal proteins from moulds: analytical tools and potential application to dry-ripened foods

Moulds growing on the surface of dry-ripened foods contribute to their sensory qualities, but some of them are able to produce mycotoxins that pose a hazard to consumers. Small cysteine-rich antifungal proteins (AFPs) from moulds are highly stable to pH and proteolysis and exhibit a broad inhibition spectrum against filamentous fungi, providing new chances to control hazardous moulds in fermented foods. The analytical tools for characterizing the cellular targets and affected pathways are reviewed. Strategies currently employed to study these mechanisms of action include ‘omics’ approaches that have come to the forefront in recent years, developing in tandem with genome sequencing of relevant organisms. These techniques contribute to a better understanding of the response of moulds against AFPs, allowing the design of complementary strategies to maximize or overcome the limitations of using AFPs on foods. AFPs alter chitin biosynthesis, and some fungi react inducing cell wall integrity (CWI) pathway. However, moulds able to increase chitin content at the cell wall by increasing proteins in either CWI or calmodulin-calcineurin signalling pathways will resist AFPs. Similarly, AFPs increase the intracellular levels of reactive oxygen species (ROS), and moulds increasing G-protein complex β subunit CpcB and/or enzymes to efficiently produce glutathione may evade apoptosis. Unknown aspects that need to be addressed include the interaction with mycotoxin production by less sensitive toxigenic moulds. However, significant steps have been taken to encourage the use of AFPs in intermediate-moisture foods, particularly for mould-ripened cheese and meat products

Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus

Antifungal proteins produced by molds are generally small, highly basic, and cysteine-rich. The best known effects of these proteins include morphological changes, metabolic inactivation, and membrane perturbation on sensitive fungi. Reactive oxygen species (ROS) generation leads to apoptosis, with G -protein playing a key role in transduction of cell death signals. The antifungal protein PgAFP from Penicillium chrysogenum inhibits growth of some toxigenic molds. Here we analyzed the effect of the antifungal protein PgAFP on the growth of Aspergillus flavus. For this, comparative proteomic analysis was used to identify the whole protein profile and protein change in abundance after PgAFP treatment. PgAFP provoked metabolic changes related to reduced energy metabolism, cell wall integrity alteration, and increased stress response due to higher levels of ROS. The observed changes in protein abundance, favoring a higher glutathione concentration as well as the increased abundance in heat shock proteins, do not seem to be enough to avoid necrosis. The decreased chitin deposition observed in PgAFP-treated A. flavus is attributed to a lower relative quantity of Rho1. The reduced relative abundance of a β subunit of G -protein seems to be the underlying reason for modulation of apoptosis in PgAFP-treated A. flavus hyphae. We propose Rho1 and G -protein subunit β CpcB to be the main factors in the mode of action of PgAFP in A. flavus. Additionally, enzymes essential for the biosynthesis of aflatoxin were no longer detectable in A. flavus hyphae at 24 h, following treatment with PgAFP. This presents a promising effect of PgAFP, which may prevent A. flavus from producing mycotoxins. However, the impact of PgAFP on actual aflatoxin production requires further study

Inhibitory Effect of PgAFP and Protective Cultures on Aspergillus parasiticus Growth and Aflatoxins Production on Dry-Fermented Sausage and Cheese

Aflatoxigenic molds can grow and produce aflatoxins on dry-fermented meat and cheese. The small, basic, cysteine-rich antifungal protein PgAFP displays a time-limited inhibitory ability against unwanted molds by increasing reactive oxygen species (ROS), which can lead to increased aflatoxin production. However, calcium abolishes the inhibitory effect of PgAFP on certain Aspergillus spp. To maximize the antifungal effect, this protein may be combined with protective cultures. Yeasts and lactic acid bacteria may counteract the impact of calcium on PgAFP fungal inhibition. The objective of this work was to study the effect of PgAFP and different combined treatments with Debaryomyces hansenii and/or Pediococcus acidilactici against growth of and aflatoxin production by an aflatoxigenic strain of Aspergillus parasiticus in both culture media and dry-fermented foods with low or high calcium levels. Aflatoxins production was increased by PgAFP but dramatically reduced by P. acidilactici in low calcium culture medium, whereas in the Ca-enriched culture medium, all treatments tested led to low aflatoxins levels. To study whether PgAFP and the protective microorganisms interfere with ROS and aflatoxin production, the relative expression of genes foxA, which is involved in peroxisomal β-oxidation, and aflP, which is required for aflatoxin biosynthesis, were evaluated. The aflatoxin overproduction induced by PgAFP seems not to be linked to peroxisomal β-oxidation. The combination of PgAFP and D. hansenii provided a successful inhibitory effect on A. parasiticus growth as well as on aflatoxin production on sliced dry-fermented sausage and cheese ripened up to 15 days, whereas P. acidilactici did not further enhance the protective effect of the two former agents. Therefore, the combined treatment of PgAFP and D. hansenii seems to provide a promising protective mean against aflatoxin-producing A. parasiticus on dry-fermented foods

Efecto de los surfactantes sobre la absorción de agroquímicos en plantas

Introduction. The uptake and translocation of molecules and chemicals compounds applied to biological systems is a function of several interactions between biological and abiotics factors. In plant systems, these factors include physical-chemical composition and concentration of molecule, physiological stage of the target plant organ and management of chemical compound (doses and form of application. Technologies dealing with tensoactives or surfactants enhance the uptake of chemical compounds applied to crops. The objective of this study was to evaluate the effect of surfactant INEX-A (COSMOCELTM) on the uptake of agrochemical compounds using isotopic techniques under greenhouse conditions. Methods. Several assays using agrochemical compounds isotopic labeled were conducted: 1) herbicide 14C-Glyphosate applied to foliage of Johnson grass (Sorghum halepense), 2) insecticide 14C-Imidacloprid applied to aerial part, 3) insecticide 14C-Imidacloprid applied to soil, and 4) fertilizer 32P-Phosphate of ammonium monobasic applied to aerial part of tomato plant (Lycopersicum esculentum). Results. The use of INEX-A increased the absorption of the molecules under study (herbicide 14C-Glyphosate, insecticide 14C-Imidacloprid applied to foliage and soil, and fertilizer 32PPhosphate of ammonium monobasic). The uptake of the molecules increased in a range from 22 to 100 % when compared to the control treatments (without INEX-A). The effect of the INEX-A showed a direct relation with the types of compounds tested and the effect decreased in the following order: 14C-Imidacloprid (aerial part)>14C-Glyphosate (aerial part)>14C-Imidacloprid (soil)>32P-Phosphate of ammonium monobasic (aerial part). Conclusions. The results showed a high tensoactive capability of INEX-A (COSMOCELMR) increasing the uptake of molecules with systemic activity by the crops.Introducción. La eficiencia de absorción y removilización de una molécula o compuesto químico aplicado a un sistema biológico depende de interacciones entre factores bióticos y abióticos. Para el caso de vegetales, destaca la composición físico-química y concentración de la molécula, el estado fisiológico del órgano donde se aplica y el manejo del producto (dosis y forma de aplicación). Existen diferentes tecnologías a base de compuestos químicos tensoactivos que permiten aumentar la absorción del compuesto aplicado en plantas de interés agrícola. El objetivo de este trabajo fue evaluar el efecto del surfactante INEX-A (COSMOCELMR) sobre la absorción de compuestos con actividad sistémica comúnmente aplicados en la agricultura a través del marcaje isotópico bajo condiciones de invernadero. Métodos. Se desarrollaron los ensayos siguientes: 1) herbicida (14C-Glifosato) aplicado vía foliar en zacate Johnson (Sorghum halepense), 2) insecticida (14C-Imidacloprid) aplicado vía foliar, 3) insecticida (14C-Imidacloprid) aplicado vía suelo y 4) nutrimento (32P-Fosfato de amonio monobásico) aplicado vía foliar en tomate (Lycopersicum esculentum). Resultados. El efecto del surfactante INEX-A aumentó la absorción de las moléculas en estudio en comparación con los tratamientos donde no se aplicó éste surfactante, esto generó una mayor removilización de las moléculas y probablemente una mayor producción de metabolitos secundarios que tienen como origen o intermediario a dichas moléculas. El efecto del surfactante INEX-A osciló en el rango 22-100 % en relación al incremento de la absorción de las moléculas en estudio. Cuando se aplicó el surfactante INEX-A se incrementó 12 veces la absorción del insecticida 14C-Imidacloprid aplicado vía foliar. Asimismo, la removilización hacia el grupo de hojas superiores vs. hojas inferiores fue mayor. Además, el efecto del INEX-A presentó una relación directa en función al tipo de molécula y la forma de aplicación, disminuyendo en el orden siguiente: 14C-Imidacloprid (foliar)>14C-Glifosato (foliar)>14C-Imidacloprid (suelo)>32PFosfato de amonio monobásico (foliar). Conclusiones. El surfactante INEX-A (COSMOCELMR) incrementa significativamente la absorción de diferentes compuestos en plantas de importancia económica