Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Evalución de la inclusión de polialcoholes, fructooligosacáridos y péptidos antihipertensivos de origen lácteo en la formulación de un masmelo depositado [recurso electrónico]

Se desarrollaron prototipos que incluyen proporciones fijas de agua, citrato de sodio, y saborizante de vainilla, con la inclusión de edulcorantes a 3 niveles; Nutraflora (FOS), maltitol (M) y jarabe de sorbitol (JS) en las siguientes proporciones: FOS-M-JS (15-30-35)%w/w; FOS-M-JS (10-40-30) %w/w y FOS-M-JS (5-50-25) %w/w; dos niveles de gelatina tipo B de 270 Bloom al 3,5 y 4%w/w , y dos niveles de péptidos antihipertensivos al 1,5 y 3 %w/w. Se incluyó sucralosa en los prototipos para ajustar el dulzor debido al bajo aporte de los polialcoholes. Para determinar los módulos elásticos (ME) se realizaron pruebas de compresión uniaxial. Los datos experimentales F(N) Vs distancia (mm) se utilizaron para determinar el módulo de elasticidad (ME). El esfuerzo lineal (σL), límite de fluencia (γL), esfuerzo de fractura (σF) y deformación de fractura (γF) no fueron determinados debido a que los masmelos no presentaron punto de fractura. Los ensayos de análisis de perfil de textura se desarrollaron a doble compresión. Para el análisis estadístico de los datos se desarrolló un arreglo factorial (AF) 3x2², con doce (12) tratamientos, tres réplicas y diez mediciones, para evaluar los efectos de las proporciones de polialcoholes, fructooligosacáridos, gelatina y péptidos antihipertensivos, sobre el ME de los masmelos desarrollados; posterior a ello se desarrolló un DCA con cuatro (4) tratamientos, tres (3) de ellos con 12 mediciones y un (1) tratamiento control con 10 mediciones para establecer comparaciones con el masmelo control. Una vez identificado el prototipo con las propiedades mecánicas cercanas al control, se procedió a optimizar la formulación mediante un diseño de mezclas con vértices extremos; por último, se determinó el TPA del masmelo desarrollado. Los resultados fueron analizados en Minitab 17. Las proporciones de polialcoholes en el AF causaron efecto estadístico significativo sobre los ME de los prototipos desarrollados (p<0.05), los niveles de gelatina y péptido no causaron efecto sobre el ME, las interacciones dobles de los factores no presentaron diferencia estadística. El factor proporción de polialcoholes en el DCA presentó efecto estadístico significativo sobre el ME (p<0.05), el masmelo control presentó la mayor media