The cys-loop ligand-gated ion channel gene superfamily of the nematode, Caenorhabditis elegans

The nematode, Caenorhabditis elegans, possesses the most extensive known superfamily of cys-loop ligand-gated ion channels (cys-loop LGICs) consisting of 102 subunit-encoding genes. Less than half of these genes have been functionally characterised which include cation-permeable channels gated by acetylcholine (ACh) and γ-aminobutyric acid (GABA) as well as anion-selective channels gated by ACh, GABA, glutamate and serotonin. Following the guidelines set for genetic nomenclature for C. elegans, we have designated unnamed subunits as lgc genes (ligand-gated ion channels of the cys-loop superfamily). Phylogenetic analysis shows that several of these lgc subunits form distinct groups which may represent novel cys-loop LGIC subtypes

The Glucuronyltransferase GlcAT-P Is Required for Stretch Growth of Peripheral Nerves in Drosophila

During development, the growth of the animal body is accompanied by a concomitant elongation of the peripheral nerves, which requires the elongation of integrated nerve fibers and the axons projecting therein. Although this process is of fundamental importance to almost all organisms of the animal kingdom, very little is known about the mechanisms regulating this process. Here, we describe the identification and characterization of novel mutant alleles of GlcAT-P, the Drosophila ortholog of the mammalian glucuronyltransferase b3gat1. GlcAT-P mutants reveal shorter larval peripheral nerves and an elongated ventral nerve cord (VNC). We show that GlcAT-P is expressed in a subset of neurons in the central brain hemispheres, in some motoneurons of the ventral nerve cord as well as in central and peripheral nerve glia. We demonstrate that in GlcAT-P mutants the VNC is under tension of shorter peripheral nerves suggesting that the VNC elongates as a consequence of tension imparted by retarded peripheral nerve growth during larval development. We also provide evidence that for growth of peripheral nerve fibers GlcAT-P is critically required in hemocytes; however, glial cells are also important in this process. The glial specific repo gene acts as a modifier of GlcAT-P and loss or reduction of repo function in a GlcAT-P mutant background enhances VNC elongation. We propose a model in which hemocytes are required for aspects of glial cell biology which in turn affects the elongation of peripheral nerves during larval development. Our data also identifies GlcAT-P as a first candidate gene involved in growth of integrated peripheral nerves and therefore establishes Drosophila as an amenable in-vivo model system to study this process at the cellular and molecular level in more detail

Mining Predicted Essential Genes of Brugia malayi for Nematode Drug Targets

We report results from the first genome-wide application of a rational drug target selection methodology to a metazoan pathogen genome, the completed draft sequence of Brugia malayi, a parasitic nematode responsible for human lymphatic filariasis. More than 1.5 billion people worldwide are at risk of contracting lymphatic filariasis and onchocerciasis, a related filarial disease. Drug treatments for filariasis have not changed significantly in over 20 years, and with the risk of resistance rising, there is an urgent need for the development of new anti-filarial drug therapies. The recent publication of the draft genomic sequence for B. malayi enables a genome-wide search for new drug targets. However, there is no functional genomics data in B. malayi to guide the selection of potential drug targets. To circumvent this problem, we have utilized the free-living model nematode Caenorhabditis elegans as a surrogate for B. malayi. Sequence comparisons between the two genomes allow us to map C. elegans orthologs to B. malayi genes. Using these orthology mappings and by incorporating the extensive genomic and functional genomic data, including genome-wide RNAi screens, that already exist for C. elegans, we identify potentially essential genes in B. malayi. Further incorporation of human host genome sequence data and a custom algorithm for prioritization enables us to collect and rank nearly 600 drug target candidates. Previously identified potential drug targets cluster near the top of our prioritized list, lending credibility to our methodology. Over-represented Gene Ontology terms, predicted InterPro domains, and RNAi phenotypes of C. elegans orthologs associated with the potential target pool are identified. By virtue of the selection procedure, the potential B. malayi drug targets highlight components of key processes in nematode biology such as central metabolism, molting and regulation of gene expression

Iron Behaving Badly: Inappropriate Iron Chelation as a Major Contributor to the Aetiology of Vascular and Other Progressive Inflammatory and Degenerative Diseases

The production of peroxide and superoxide is an inevitable consequence of aerobic metabolism, and while these particular "reactive oxygen species" (ROSs) can exhibit a number of biological effects, they are not of themselves excessively reactive and thus they are not especially damaging at physiological concentrations. However, their reactions with poorly liganded iron species can lead to the catalytic production of the very reactive and dangerous hydroxyl radical, which is exceptionally damaging, and a major cause of chronic inflammation. We review the considerable and wide-ranging evidence for the involvement of this combination of (su)peroxide and poorly liganded iron in a large number of physiological and indeed pathological processes and inflammatory disorders, especially those involving the progressive degradation of cellular and organismal performance. These diseases share a great many similarities and thus might be considered to have a common cause (i.e. iron-catalysed free radical and especially hydroxyl radical generation). The studies reviewed include those focused on a series of cardiovascular, metabolic and neurological diseases, where iron can be found at the sites of plaques and lesions, as well as studies showing the significance of iron to aging and longevity. The effective chelation of iron by natural or synthetic ligands is thus of major physiological (and potentially therapeutic) importance. As systems properties, we need to recognise that physiological observables have multiple molecular causes, and studying them in isolation leads to inconsistent patterns of apparent causality when it is the simultaneous combination of multiple factors that is responsible. This explains, for instance, the decidedly mixed effects of antioxidants that have been observed, etc...Comment: 159 pages, including 9 Figs and 2184 reference

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

Instantaneous inactivation of cofilin reveals its function of F-actin disassembly in lamellipodia

Cofilin is a key regulator of the actin cytoskeleton. It can sever actin filaments, accelerate filament disassembly, act as a nucleation factor, recruit or antagonize other actin regulators, and control the pool of polymerization-competent actin monomers. In cells these actions have complex functional outputs. The timing and localization of cofilin activity are carefully regulated, and thus global, long-term perturbations may not be sufficient to probe its precise function. To better understand cofilin's spatiotemporal action in cells, we implemented chromophore-assisted laser inactivation (CALI) to instantly and specifically inactivate it. In addition to globally inhibiting actin turnover, CALI of cofilin generated several profound effects on the lamellipodia, including an increase of F-actin, a rearward expansion of the actin network, and a reduction in retrograde flow speed. These results support the hypothesis that the principal role of cofilin in lamellipodia at steady state is to break down F-actin, control filament turnover, and regulate the rate of retrograde flow