Assays to monitor aggrephagy in Drosophila brain

Accumulation of ubiquitinated protein aggregates is a hallmark of most ageingrelated neurodegenerative disorders. Autophagy has been found to be involved in the selective clearance of these protein aggregates, and this process is called aggrephagy. Here we provide two protocols for the investigation of protein aggregation and their removal by autophagy using western blotting and immunofluorescence techniques in Drosophila brain. Investigating the role of aggrephagy at the cellular and organismal level is important for the development of therapeutic interventions against ageing-related diseases

Health promotion through self-care and community participation: Elements of a proposed programme in the developing countries

BACKGROUND: The concepts of health promotion, self-care and community participation emerged during 1970s, primarily out of concerns about the limitation of professional health system. Since then there have been rapid growth in these areas in the developed world, and there is evidence of effectiveness of such interventions. These areas are still in infancy in the developing countries. There is a window of opportunity for promoting self care and community participation for health promotion. DISCUSSION: A broad outline is proposed for designing a health promotion programme in developing countries, following key strategies of the Ottawa Charter for health promotion and principles of self care and community participation. Supportive policies may be framed. Self care clearinghouses may be set up at provincial level to co-ordinate the programme activities in consultation with district and national teams. Self care may be promoted in the schools and workplaces. For developing personal skills of individuals, self care information, generated through a participatory process, may be disseminated using a wide range of print and audio-visual tools and information technology based tools. One such potential tool may be a personally held self care manual and health record, to be designed jointly by the community and professionals. Its first part may contain basic self care information and the second part may contain outlines of different personally-held health records to be used to record important health and disease related events of an individual. Periodic monitoring and evaluation of the programme may be done. Studies from different parts of the world indicate the effectiveness and cost-effectiveness of self care interventions. The proposed outline has potential for health promotion and cost reduction of health services in the developing countries, and may be adapted in different situations. SUMMARY: Self care, community participation and health promotion are emerging but dominant areas in the developed countries. Elements of a programme for health promotion in the developing countries following key principles of self care and community participation are proposed. Demonstration programmes may be initiated to assess the feasibility and effectiveness of this programme before large scale implementation

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Use of Peptide Arrays for Identification and Characterization of LIR Motifs

The mammalian ATG8 proteins (LC3A-C/GABARAP, GABARAPL1, and GABARAPL2) are small ubiquitin-like proteins critically involved in macroautophagy. Their processed C-termini are posttranslationally conjugated to a phosphatidylethanolamine moiety, enabling their insertion into the lipid bilayers of both the inner and outer membranes of the forming autophagosomes. The ATG8s bind a diverse selection of proteins including cargo receptors for selective autophagy, members of the core autophagy machinery, and other proteins involved in formation, transport, and maturation (fusion to lysosomes) of autophagosomes. Protein binding to the ATG8s is in most cases mediated by short, conserved sequence motifs known as LC3-interacting regions (LIRs). Here, we present a protocol for identifying putative LIR motifs in a whole protein sequence using peptide arrays generated by SPOT synthesis on nitrocellulose membranes. The use of two-dimensional peptide arrays allows for further identification of specific residues critical for LIR binding