Silencing of the Laccase (lacc2) Gene from Pleurotus ostreatus Causes Important Effects on the Formation of Toxocyst-like Structures and Fruiting Body.

A wide variety of biological functions, including those involved in the morphogenesis process of basidiomycete fungi, have been attributed to laccase enzymes. In this work, RNA interference (RNAi) was used to evaluate the role of the laccase (lacc2) gene of Pleurotus ostreatus PoB. Previously, transformant strains of P. ostreatus were obtained and according to their level of silencing they were classified as light (T7), medium (T21) or severe (T26 and T27). The attenuation of the lacc2 gene in these transformants was determined by RT-PCR. Silencing of lacc2 resulted in a decrease in laccase activity between 30 and 55%, which depended on the level of laccase expression achieved. The silenced strains (T21, T26, and T27) displayed a delay in the development of mycelium on potato dextrose agar (PDA) medium, whereas in the cultures grown on wheat straw, we found that these strains were incapable of producing aerial mycelium, primordia, and fruiting bodies. Scanning electron microscopy (SEM) showed the presence of toxocyst-like structures. The highest abundance of these structures was observed in the wild-type (PoB) and T7 strains. However, the abundance of toxocysts decreased in the T21 and T26 strains, and in T27 they were not detected. These results suggest that the presence and abundance of toxocyst-like structures are directly related to the development of fruiting bodies. Furthermore, our data confirm that lacc2 is involved in the morphogenesis process of P. ostreatus.

Laccase Production from Agrocybe pediades: Purification and Functional Characterization of a Consistent Laccase Isoenzyme in Liquid Culture.

Laccases are valuable enzymes as an excellent ecological alternative for bioremediation issues because they can oxidize persistent xenobiotic compounds. The production and characterization of extracellular laccases from saprotrophic fungi from disturbed environments have been scarcely explored, even though this could diversify their functional characteristics and expand the conditions in which they carry out their catalysis. Agrocybe pediades, isolated from a disturbed forest, produces an extracellular laccase in liquid culture. The enzyme was purified, identified and characterized. Copper and hexachlorobenzene do not function as inducers for the laccase produced. Partial amino acid sequences were obtained by LC-MS/MS that share similarity with laccases from other fungi. Purified laccase is a monomer with a molecular mass between 55-60 kDa and had an optimum activity at pH 5.0 and the optimum temperature at 45 °C using 2,6-dimethoxyphenol (2,6-DMP) as substrate. The Km and Vmax also determined with 2,6-DMP were 100 µM and 285 µmol∙min-1∙mg-1, respectively, showing that the laccase of A. pediades has a higher affinity for this substrate than that of other Agaricales. These features could provide a potential catalyst for different toxic substrates and in the future laccase could be used in environmental recovery processes.

Análisis microbiológico a corto y largo plazo del material usado para esterilizar instrumental odontológico / Short and long-term microbiological analysis of the material for sterilize dental instruments

Introducción: el material para empaquetar el instrumental odontológico, como pueden ser bolsas de tela, papel o plástico, es usado por profesionales de la salud; sin embargo, es necesario esclarecer la efectividad de cada uno y determinar el tiempo que permanece estéril luego del procedimiento. Objetivo: identificar la eficacia de tela, plástico y papel como materiales para esterilizar instrumental a corto y largo plazo. Material y métodos: se realizaron cultivos sólidos y líquidos de instrumental esterilizado en tres materiales y con diferentes tiempos de postesterilización. Se incubaron a 36 oC por 72 horas en condiciones aerobias y anaerobias. Los resultados se analizaron usando una prueba de Kruskal-Wallis, seguida de una prueba de Dunn. Resultados: los resultados mostraron que inmediatamente después del proceso de esterilización, los tres materiales son efectivos (Kruskal-Wallis test, p = 0.2752), 24 horas (p = 0.2492), siete (p = 0.0509) y 14 días (p = 0.0006). Veinticuatro horas posterior a la esterilización la tela no es efectiva, el plástico disminuye su efectividad y el papel sigue siendo efectivo. Conclusión: en nuestros resultados, el papel es la mejor opción para esterilizar instrumental (AU)

Introduction: material such as cloth, paper or plastic bags to wrap dental instruments is used by health professionals, however, it is necessary to clarify the effectiveness of each one and determine if it remains sterile after the procedure. Objective: to determine the effectiveness of cloth, plastic and paper as materials to sterilize dental instruments in the short and long term. Material and methods: we carry out solid and liquid cultures of sterilized instruments in three materials, at different post-sterilization times, incubated at 36 oC for 72 hours under aerobic and anaerobic conditions, and the results were analyzed using a Kruskal-Wallis test, followed by from a Dunn's test. Results: our results showed that immediately after the sterilization process the three materials are effective (Kruskal-Wallis; p = 0.2752), 24 hours (p = 0.2492), 7 (p = 0.0509) and 14 (p = 0.0006) days. Twenty-four hours after the cloth is not effective, plastic decreases its effectiveness and paper remain effective. Conclusion: in our results, paper is the best option to sterilize dental instruments (AU)