http://www.maths.qmul.ac.uk/%7Elatora/report_06.pdf

Complex networks: Structure and dynamics

Autophagy in the Pathogenesis of Disease

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 flux 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, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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 target by gene knockout or RNA interference more than one autophagy-related protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that 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 assays, we hope to encourage technical innovation in the field.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-ijf2t30pbq.pdf

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. 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 vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most 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 field 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. 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. 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.

/pdf/guidelines-for-the-use-and-interpretation-of-assays-for-28d1pi1jvo.pdf

Guidelines for the use and interpretation of assays for monitoring autophagy

N G van Kampen 1981 Amsterdam: North-Holland xiv + 419 pp price Dfl 180 This is a book which, at a lower price, could be expected to become an essential part of the library of every physical scientist concerned with problems involving fluctuations and stochastic processes, as well as those who just enjoy a beautifully written book. It provides an extensive graduate-level introduction which is clear, cautious, interesting and readable.

https://iopscience.iop.org/article/10.1088/0031-9112/34/4/040/pdf

Stochastic Processes in Physics and Chemistry

The pharynx of Caenorhabditis elegans is a nearly self-contained neuromuscular organ responsible for feeding. To identify genes involved in the development or function of the excitable cells of the pharynx, I screened for worms with visible defects in pharyngeal feeding behavior. Fifty-two mutations identified 35 genes, at least 22 previously unknown. The genes broke down into three broad classes: 2 pha genes, mutations in which caused defects in the shape of the pharynx, 7 phm genes, mutations in which caused defects in the contractile structures of the pharyngeal muscle, and 26 eat genes, mutants in which had abnormal pharyngeal muscle motions, but had normally shaped and normally birefringent pharynxes capable of vigorous contraction. Although the Eat phenotypes were diverse, most resembled those caused by defects in the pharyngeal nervous system. For some of the eat genes there is direct evidence from previous genetic mosaic and pharmacological studies that they do in fact affect nervous system. In eat-5 mutants the motions of the different parts of the pharynx were poorly synchronized. eat-6 and eat-12 mutants failed to relax their pharyngeal muscles properly. These pharyngeal motion defects are most easily explained as resulting from abnormal electrical excitability of the pharyngeal muscle membrane.

https://www.genetics.org/content/genetics/133/4/897.full.pdf

The Genetics of Feeding in Caenorhabditis elegans

The ability of organisms to evolve resistance threatens the effectiveness of every antibiotic drug. We show that in the nematode Caenorhabditis elegans, simultaneous mutation of three genes, avr-14, avr-15, and glc-1, encoding glutamate-gated chloride channel (GluCl) α-type subunits confers high-level resistance to the antiparasitic drug ivermectin. In contrast, mutating any two channel genes confers modest or no resistance. We propose a model in which ivermectin sensitivity in C. elegans is mediated by genes affecting parallel genetic pathways defined by the family of GluCl genes. The sensitivity of these pathways is further modulated by unc-7, unc-9, and the Dyf (dye filling defective) genes, which alter the structure of the nervous system. Our results suggest that the evolution of drug resistance can be slowed by targeting antibiotic drugs to several members of a multigene family.

https://www.pnas.org/content/pnas/97/6/2674.full.pdf

The genetics of ivermectin resistance in Caenorhabditis elegans

A guanylyl cyclase (GC-D) was recently shown to be expressed in a subclass of neurons within the neuroepithelim of the rat, but given that only a single cyclase was discovered, whether it represents an odorant/pheromone receptor as has been suggested for the large family of seven-transmembrane receptors remains unclear Through cloning and expression of cDNA we now demonstrate that at least 29 genomic or cDNA sequences found in Caenorhabditis elegans represent guanylyl cyclases Many of the membrane forms retain cysteine residues conserved within the extracellular, ligand-binding domain of known cyclase receptors Of eight orphan cyclase receptor::GFP (green fluroescence protein) fusion constructs for which signals were obtained, all were expressed in specific sensory neurons Furthermore, a cyclase/GFP fusion protein (GCY-10/GFP) was principally expressed in the sensory cilium, suggesting these cyclases function as primary chemosensory receptors For the first time, we also found that chemosensory neurons (ASE), known to be bilaterally symmetric, demonstrate absolute right or left sidedness with respect to the expression of three different cyclases Thus, the guanylyl cyclases represent an unexpectedly large and new family of sensory neuron receptors that may complement the 7-transmembrane family of odorant/pheromone receptors

https://www.pnas.org/content/pnas/94/7/3384.full.pdf

Guanylyl cyclase expression in specific sensory neurons: A new family of chemosensory receptors

Animals have evolved diverse behaviors that serve the purpose of finding food in the environment. We investigated the food seeking strategy of the soil bacteria-eating nematode Caenorhabditis elegans. C. elegans bacterial food varies in quality: some species are easy to eat and support worm growth well, while others do not. We show that worms exhibit dietary choice: they hunt for high quality food and leave hard-to-eat bacteria. This food seeking behavior is enhanced in animals that have already experienced good food. When hunting for good food, worms alternate between two modes of locomotion, known as dwelling: movement with frequent stops and reversals; and roaming: straight rapid movement. On good food, roaming is very rare, while on bad food it is common. Using laser ablations and mutant analysis, we show that the AIY neurons serve to extend roaming periods, and are essential for efficient food seeking.

Leon Avery

Papers

The Genetics of Feeding in Caenorhabditis elegans

The genetics of ivermectin resistance in Caenorhabditis elegans

Guanylyl cyclase expression in specific sensory neurons: A new family of chemosensory receptors

Dietary choice behavior in Caenorhabditis elegans

Active Currents Regulate Sensitivity and Dynamic Range in C. elegans Neurons