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

Generation, analysis and functional annotation of expressed sequence tags from the ectoparasitic mite <it>Psoroptes ovis</it>

<p>Abstract</p> <p>Background</p> <p>Sheep scab is caused by <it>Psoroptes ovis </it>and is arguably the most important ectoparasitic disease affecting sheep in the UK. The disease is highly contagious and causes and considerable pruritis and irritation and is therefore a major welfare concern. Current methods of treatment are unsustainable and in order to elucidate novel methods of disease control a more comprehensive understanding of the parasite is required. To date, no full genomic DNA sequence or large scale transcript datasets are available and prior to this study only 484 <it>P. ovis </it>expressed sequence tags (ESTs) were accessible in public databases.</p> <p>Results</p> <p>In order to further expand upon the transcriptomic coverage of <it>P. ovis </it>thus facilitating novel insights into the mite biology we undertook a larger scale EST approach, incorporating newly generated and previously described <it>P. ovis </it>transcript data and representing the largest collection of <it>P. ovis </it>ESTs to date. We sequenced 1,574 ESTs and assembled these along with 484 previously generated <it>P. ovis </it>ESTs, which resulted in the identification of 1,545 unique <it>P. ovis </it>sequences. BLASTX searches identified 961 ESTs with significant hits (E-value < 1E-04) and 584 novel <it>P. ovis </it>ESTs. Gene Ontology (GO) analysis allowed the functional annotation of 880 ESTs and included predictions of signal peptide and transmembrane domains; allowing the identification of potential <it>P. ovis </it>excreted/secreted factors, and mapping of metabolic pathways.</p> <p>Conclusions</p> <p>This dataset currently represents the largest collection of <it>P. ovis </it>ESTs, all of which are publicly available in the GenBank EST database (dbEST) (accession numbers <ext-link ext-link-id="FR748230" ext-link-type="gen">FR748230</ext-link> - <ext-link ext-link-id="FR749648" ext-link-type="gen">FR749648</ext-link>). Functional analysis of this dataset identified important homologues, including house dust mite allergens and tick salivary factors. These findings offer new insights into the underlying biology of <it>P. ovis</it>, facilitating further investigations into mite biology and the identification of novel methods of intervention.</p

Echinodermata: The Complex Immune System in Echinoderms

View references (418) The Echinodermata are an ancient phylum of benthic marine invertebrates with a dispersal-stage planktonic larva. These animals have innate immune systems characterized initially by clearance of foreign particles, including microbes, from the body cavity of both larvae and adults, and allograft tissue rejection in adults. Immune responsiveness is mediated by a variety of adult coelomocytes and larval mesenchyme cells. Echinoderm diseases from a range of pathogens can lead to mass die-offs and impact aquaculture, but some individuals can recover. Genome sequences of several echinoderms have identified genes with immune function, including expanded families of Toll-like receptors, NOD-like receptors, and scavenger receptors with cysteine-rich domains, plus signaling pathways and cytokines. The set of transcription factors that regulate proliferation and differentiation of the cellular immune system are conserved and indicate the ancestral origins of hematopoiesis. Both larval and adult echinoderms are in constant contact with potential pathogens in seawater, and they respond to infection by phagocytosis and encapsulation, and employ proteins that function in immune detection and response. Antipathogen responses include activation of the SpTransformer genes, a complement system, and the production of many types of antimicrobial peptides. Echinoderms have homologues of the recombinase activating genes plus all associated genes that function in vertebrates for immunoglobulin gene family rearrangement, although their gene targets are unknown. The echinoderm immune system has been characterized as unexpectedly complex, robust, and flexible. Many echinoderms have very long life-spans that correlate with an excellent capacity for cell damage repair. In many marine ecosystems, echinoderms are keystone predators and herbivores, and therefore are species that can serve as optimal sentinels of environmental health. Coelomocytes can be employed in sensor systems to test for the presence of marine pollutants. When Elie Metchnikoff inserted a rose prickle into a larval sea star and observed chemotaxis, phagocytosis, and encapsulation by the mesenchyme cells, he initiated not only the field of immunology but also that of comparative immunology, of which the echinoderms have been an important part