Automated, high-throughput, motility analysis in Caenorhabditis elegans and parasitic nematodes: Applications in the search for new anthelmintics

The scale of the damage worldwide to human health, animal health and agricultural crops resulting from parasitic nematodes, together with the paucity of treatments and the threat of developing resistance to the limited set of widely-deployed chemical tools, underlines the urgent need to develop novel drugs and chemicals to control nematode parasites. Robust chemical screens which can be automated are a key part of that discovery process. Hitherto, the successful automation of nematode behaviours has been a bottleneck in the chemical discovery process. As the measurement of nematode motility can provide a direct scalar readout of the activity of the neuromuscular system and an indirect measure of the health of the animal, this omission is acute. Motility offers a useful assay for high-throughput, phenotypic drug/chemical screening and several recent developments have helped realise, at least in part, the potential of nematode-based drug screening. Here we review the challenges encountered in automating nematode motility and some important developments in the application of machine vision, statistical imaging and tracking approaches which enable the automated characterisation of nematode movement. Such developments facilitate automated screening for new drugs and chemicals aimed at controlling human and animal nematode parasites (anthelmintics) and plant nematode parasites (nematicides)

Phenotypic screening in C. elegansas as a tool for the discovery of new geroprotective drugs

Population aging is one of the largest challenges of the 21st century. As more people live to advanced ages, the prevalence of age-related diseases and disabilities will increase placing an ever larger burden on our healthcare system. A potential solution to this conundrum is to develop treatments that prevent, delay or reduce the severity of age-related diseases by decreasing the rate of the aging process. This ambition has been accomplished in model organisms through dietary, genetic and pharmacological interventions. The pharmacological approaches hold the greatest opportunity for successful translation to the clinic. The discovery of such pharmacological interventions in aging requires high-throughput screening strategies. However, the majority of screens performed for geroprotective drugs inC. elegansso far are rather low throughput. Therefore, the development of high-throughput screening strategies is of utmost importance

Massively parallel C. elegans tracking provides multi-dimensional fingerprints for phenotypic discovery.

BACKGROUND: The nematode worm C. elegans is a model organism widely used for studies of genetics and of human disease. The health and fitness of the worms can be quantified in different ways, such as by measuring their bending frequency, speed or lifespan. Manual assays, however, are time consuming and limited in their scope providing a strong motivation for automation. NEW METHOD: We describe the development and application of an advanced machine vision system for characterising the behaviour of C. elegans, the Wide Field-of-View Nematode Tracking Platform (WF-NTP), which enables massively parallel data acquisition and automated multi-parameter behavioural profiling of thousands of worms simultaneously. RESULTS: We screened more than a million worms from several established models of neurodegenerative disorders and characterised the effects of potential therapeutic molecules for Alzheimer's and Parkinson's diseases. By using very large numbers of animals we show that the sensitivity and reproducibility of behavioural assays is very greatly increased. The results reveal the ability of this platform to detect even subtle phenotypes. COMPARISON WITH EXISTING METHODS: The WF-NTP method has substantially greater capacity compared to current automated platforms that typically either focus on characterising single worms at high resolution or tracking the properties of populations of less than 50 animals. CONCLUSIONS: The WF-NTP extends significantly the power of existing automated platforms by combining enhanced optical imaging techniques with an advanced software platform. We anticipate that this approach will further extend the scope and utility of C. elegans as a model organism

High-throughput phenotyping of multicellular organisms: finding the link between genotype and phenotype

High-throughput phenotyping approaches (phenomics) are being combined with genome-wide genetic screens to identify alterations in phenotype that result from gene inactivation. Here we highlight promising technologies for 'phenome-scale' analyses in multicellular organisms

Identification Of Hydralazine As An Anti-Infective Compound In Pseudomonas Aeruginosa-Infected C. Elegans

Lisozim adalah model enzim yang penting dalam bidang penyelidikan bioperubatan. Sehubungan dengan taburan meluas dalam pelbagai tisu dan rembesan, lisozim dikenali dengan fungsi sebagai enzim pertahanan anti-bakteria manakala fungsi sampingan sebagai enzim pencernaan juga dilaporkan dalam segelintiran taksa Lysozymes are important enzyme models with biomedical importance. In accordance with its widespread distribution in various biological tissues and secretions, lysozyme is implicated primarily in the antibacterial defense, although an additional function as digestive enzyme has been described as well for several tax

Caenorhabditis elegans Semi-Automated Liquid Screen Reveals a Specialized Role for the Chemotaxis Gene cheB2 in Pseudomonas aeruginosa Virulence

Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity

Automated parallel immobilization microfluidic platforms for high-throughput neuronal degeneration studies with C. elegans

C. elegans has emerged as an invaluable model organism for in vivo neurobiology research to understand disease mechanisms and pathology relevant in humans. Simple anatomy, short lifecycle, fully characterized genome, and miniature body scale make these nematodes an ideal model organism for phenotyping and bio-molecular studies using microfluidic platforms. Advancements in soft-lithography have improved the functionality of microfluidic technology for C. elegans, leading to whole organism studies in high-throughput manner that were not otherwise possible. In order to study phenomena that require large amounts of data such as drug screens for neurological disorders and phenotyping, high-throughput imaging platforms with high-speed, high-resolution image acquisition become essential. With this in mind, we have developed and tested microfluidic immobilization devices to enable high-throughput optical interrogation of C. elegans for neurodegenerative diseases and large scale drug screens. Initially, we designed, developed, and tested single-layer and double-layer SU8 mold PDMS chips with parallel tapered channels to immobilize 40 adult C. elegans for high-resolution fluorescence imaging of their neurons in a parallel manner. vi We achieved over 90% immobilization efficiency using these initial devices, but could achieve only ~50% of the trapped worms with proper orientation to allow scoring of the VC neurons of interest. To improve worm orientation, we developed a three-layer microfluidic chip that can immobilize and orient the adult worms for optical interrogation of these VC neurons with 90% efficiency. Finally, we scaled the platform to accommodate a large scale platform with standard multi-well format on-chip wells where each well leads to the optimized trapping channels. The final optimized multi-well platform provides comprehensive easy to use 96-well microfluidic system to orient, immobilize, and image adult C. elegans in high-throughput manner. The novel gasket system can pressurize the multi-well device pre-loaded with 96 individual worm populations. Using a sequence of on-off applied gasket pressure, we can orient and immobilize worms in all 96 devices simultaneously in less than 5 minutes. Custom designed software can capture 12 z-stack images per worm from all 96-well in less than 12 minutes. With 95% trapping efficiency, approximately 90% of the worms can be scored successfully for neuronal phenotyping of VC neurons. This 96-well platform and the automated imaging system enable high-throughput optical interrogation of adult C. elegans for large-scale drug screens relating to ageing and various neurodegenerative diseases.Mechanical Engineerin

Identification of Antifungal Compounds Active against Candida albicans Using an Improved High-Throughput Caenorhabditis elegans Assay

Candida albicans, the most common human pathogenic fungus, can establish a persistent lethal infection in the intestine of the microscopic nematode Caenorhabditis elegans. The C. elegans–C. albicans infection model was previously adapted to screen for antifungal compounds. Modifications to this screen have been made to facilitate a high-throughput assay including co-inoculation of nematodes with C. albicans and instrumentation allowing precise dispensing of worms into assay wells, eliminating two labor-intensive steps. This high-throughput method was utilized to screen a library of 3,228 compounds represented by 1,948 bioactive compounds and 1,280 small molecules derived via diversity-oriented synthesis. Nineteen compounds were identified that conferred an increase in C. elegans survival, including most known antifungal compounds within the chemical library. In addition to seven clinically used antifungal compounds, twelve compounds were identified which are not primarily used as antifungal agents, including three immunosuppressive drugs. This assay also allowed the assessment of the relative minimal inhibitory concentration, the effective concentration in vivo, and the toxicity of the compound in a single assay