Highlights • Zebrafish have advanced genetic and functional imaging toolkits to study sleep. • Zebrafish have conserved sleep genes and neurons. • Recent zebrafish studies have identified novel ...regulators of sleep and arousal. • Behavioral drug screens can identify links between disease and sleep disruption.
Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral ...insertion sleep screen in larval zebrafish, we identified a novel gene,
(
), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expressed
gene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na
,K
-ATPase regulator, FXYD1/Phospholemman. Disruption of either
or
, a Na
,K
-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Since
and
mutants have elevated intracellular Na
levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na
pump function modulates neuronal excitability to maintain normal sleep behaviour.
Novel invertebrate‐killing compounds are required in agriculture and medicine to overcome resistance to existing treatments. Because insecticides and anthelmintics are discovered in phenotypic ...screens, a crucial step in the discovery process is determining the mode of action of hits. Visible whole‐organism symptoms are combined with molecular and physiological data to determine mode of action. However, manual symptomology is laborious and requires symptoms that are strong enough to see by eye. Here, we use high‐throughput imaging and quantitative phenotyping to measure Caenorhabditis elegans behavioral responses to compounds and train a classifier that predicts mode of action with an accuracy of 88% for a set of ten common modes of action. We also classify compounds within each mode of action to discover substructure that is not captured in broad mode‐of‐action labels. High‐throughput imaging and automated phenotyping could therefore accelerate mode‐of‐action discovery in invertebrate‐targeting compound development and help to refine mode‐of‐action categories.
Synopsis
A combination of imaging and machine learning is used to predict compound mode of action using the unique behavioural responses of the roundworm C. elegans to different pesticides and anthelmintics.
Insecticides affect phenotypes in multiple behavioural dimensions.
Compounds with the same mode of action have similar effects on behaviour.
Combining classifiers by voting enables mode of action prediction.
The approach allows mode of action deconvolution within classes.
A combination of imaging and machine learning is used to predict compound mode of action using the unique behavioural responses of the roundworm C. elegans to different pesticides and anthelmintics.
Tracking small laboratory animals such as flies, fish, and worms is used for phenotyping in neuroscience, genetics, disease modelling, and drug discovery. An imaging system with sufficient throughput ...and spatiotemporal resolution would be capable of imaging a large number of animals, estimating their pose, and quantifying detailed behavioural differences at a scale where hundreds of treatments could be tested simultaneously. Here we report an array of six 12-megapixel cameras that record all the wells of a 96-well plate with sufficient resolution to estimate the pose of C. elegans worms and to extract high-dimensional phenotypic fingerprints. We use the system to study behavioural variability across wild isolates, the sensitisation of worms to repeated blue light stimulation, the phenotypes of worm disease models, and worms' behavioural responses to drug treatment. Because the system is compatible with standard multiwell plates, it makes computational ethological approaches accessible in existing high-throughput pipelines.
Localizing messenger RNAs at specific subcellular sites is a conserved mechanism for targeting the synthesis of cytoplasmic proteins to distinct subcellular domains, thereby generating the asymmetric ...protein distributions necessary for cellular and developmental polarity. However, the full range of transcripts that are asymmetrically distributed in specialized cell types, and the significance of their localization, especially in the nervous system, are not known. We used the EP-MS2 method, which combines EP transposon insertion with the MS2/MCP in vivo fluorescent labeling system, to screen for novel localized transcripts in polarized cells, focusing on the highly branched Drosophila class IV dendritic arborization neurons. Of a total of 541 lines screened, we identified 55 EP-MS2 insertions producing transcripts that were enriched in neuronal processes, particularly in dendrites. The 47 genes identified by these insertions encode molecularly diverse proteins, and are enriched for genes that function in neuronal development and physiology. RNAi-mediated knockdown confirmed roles for many of the candidate genes in dendrite morphogenesis. We propose that the transport of mRNAs encoded by these genes into the dendrites allows their expression to be regulated on a local scale during the dynamic developmental processes of dendrite outgrowth, branching, and/or remodeling.
Sleep is a nearly universal feature of animal behaviour, yet many of the molecular, genetic, and neuronal substrates that orchestrate sleep/wake transitions lie undiscovered. Employing a viral ...insertion sleep screen in larval zebrafish, we identified a novel gene,
dreammist
(
dmist
), whose loss results in behavioural hyperactivity and reduced sleep at night. The neuronally expressed
dmist
gene is conserved across vertebrates and encodes a small single-pass transmembrane protein that is structurally similar to the Na
+
,K
+
-ATPase regulator, FXYD1/Phospholemman. Disruption of either
fxyd1
or
atp1a3a
, a Na
+
,K
+
-ATPase alpha-3 subunit associated with several heritable movement disorders in humans, led to decreased night-time sleep. Since
atpa1a3a
and
dmist
mutants have elevated intracellular Na
+
levels and non-additive effects on sleep amount at night, we propose that Dmist-dependent enhancement of Na
+
pump function modulates neuronal excitability to maintain normal sleep behaviour.
Data collected for the eLife OpenAccess paper: Systematic creation and phenotyping of Mendelian disease models in C. elegans: towards large-scale drug repurposing. (doi: 10.7554/eLife.92491.1)
...Contains: extracted features, calculated stats, normalised z-scores and timerseries data of all the disease model mutants generated. In addition, there is a static .html file that allows for mousing over the clustermaps to easily view differences in strains compared to the N2 wild-type.
Staphylococcus aureus
is one of the most prevalent organisms responsible for nosocomial infections, and cases of community-acquired
S. aureus
infection have continued to increase despite widespread ...preventative measures. Pathologies attributed to
S. aureus
infection are diverse; ranging from dermal lesions to bacteremia, abscesses, and endocarditis. Reported cases of
S. aureus
-associated meningitis and brain abscesses have also increased in recent years, however, the precise mechanism whereby
S. aureus
leave the bloodstream and gain access to the central nervous system (CNS) are not known. Here we demonstrate for the first time that
S. aureus
efficiently adheres to and invades human brain microvascular endothelial cells (hBMEC), the single-cell layer which constitutes the blood–brain barrier (BBB). The addition of cytochalasin D, an actin microfilament aggregation inhibitor, strongly reduced bacterial invasion, suggesting an active hBMEC process is required for efficient staphylococcal uptake. Furthermore, mice injected with
S. aureus
exhibited significant levels of brain bacterial counts and histopathologic evidence of meningeal inflammation and brain abscess formation, indicating that
S. aureus
was able to breech the BBB in an experimental model of hematogenous meningitis. We found that a YpfP-deficient mutant, defective in lipoteichoic acid (LTA) membrane anchoring, exhibited a decreased ability to invade hBMEC and correlated to a reduced risk for the development of meningitis in vivo. Our results demonstrate that LTA-mediated penetration of the BBB may be a primary step in the pathogenesis of staphylococcal CNS disease.
► A general framework for integrating biodiversity concerns into REDD+ programmes. ► A tiered approach to assessing biodiversity safeguards to help guide implementation. ► Progress can be achieved in ...respecting safeguards using available data.
The UNFCCC mechanism for Reducing Emissions from Deforestation and Degradation in developing countries (REDD+) represents an unprecedented opportunity for the conservation of forest biodiversity. Nevertheless, there are widespread concerns surrounding the possibility of negative environmental outcomes if biodiversity is not given adequate consideration throughout the REDD+ process. We propose a general framework for incorporating biodiversity concerns into national REDD+ programmes based on well-established ecological principles and experiences. First, we identify how biodiversity distribution and threat data, together with data on biodiversity responses to forest change and management, can be readily incorporated into the strategic planning process for REDD+ in order to identify priority areas and activities for investment that will deliver returns for both carbon and biodiversity. Second, we propose that assessments of changes in biodiversity following REDD+ implementation could be greatly facilitated by paralleling, where possible, the existing IPCC architecture for assessing carbon emissions. A three-tiered approach is proposed for biodiversity assessment, where lower tiers can provide a realistic starting point for countries with fewer data and lower technical capacities. Planning and assessment of biodiversity safeguards for REDD+ need not overburden an already encumbered UNFCCC process. Immediate progress is already possible for a large number of developing countries, and a gradual, phased approach to implementation would minimise risks and facilitate the protection of additional biodiversity benefits from REDD+ activities. Greater levels of coordination between the UNFCCC and CBD, as well as other agencies and stakeholder groups interested in forest conservation are needed if biodiversity safeguards are to be fully adopted and implemented.
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