We report a suite of key microfluidic devices for complex highthroughput whole-animal genetic and drug screens. We demonstrate a high-speed microfluidic sorter that can isolate and immobilize Caenorhabditis elegans in a well defined geometry for screening phenotypic features at subcellular resolution in physiologically active animals. We show an integrated chip containing individually addressable screening-chamber devices for incubation and exposure of individual animals to biochemical compounds and high-resolution time-lapse imaging of many animals on a single chip without the need for anesthesia. We describe a design for delivery of compound libraries in standard multiwell plates to microfluidic devices and also for rapid dispensing of screened animals into multiwell plates. When used in various combinations, these devices will facilitate a variety of high-throughput assays using whole animals, including mutagenesis and RNAi and drug screens at subcellular resolution, as well as high-throughput high-precision manipulations such as femtosecond laser microsurgery for large-scale in vivo neural degeneration and regeneration studies.Caenorhabditis elegans ͉ femtosecond laser microsurgery ͉ immobilization and time-lapse imaging ͉ mutagenesis ͉ RNAi and drug screening E xisting large vertebrate animal models currently cannot be used in high-throughput assays for rapid identification of new genes and drug targets because of the size and complexity of the instrumentation with which these models are studied. In recent years, the advantages of using small invertebrate animals as model systems for human disease have become increasingly apparent and have resulted in two Nobel Prizes in physiology and medicine during the last five years for studies conducted on the nematode Caenorhabditis elegans. The availability of a wide array of species-specific genetic techniques, along with the transparency of the worm and its ability to grow in minute volumes make C. elegans an extremely powerful model organism.However, since the first studies on C. elegans in the early 1960s, little has changed in how scientists manipulate this tiny organism by manually picking, sorting, and transferring individual animals. As a result, large-scale assays such as mutagenesis and RNAi screens (1-3) can take months or even years to complete manually. Currently, high-throughput C. elegans assays are performed by adapting techniques developed for screening cell lines, such as flow-through sorters and microplate readers (4-6). Because of the significant limitations of these methods, highthroughput small-animal studies either have to be dramatically simplified before they can be automated or cannot be conducted at all.Here, we report key components of an integrated, wholeanimal, high-throughput sorting and large-scale screening platform for drug and genetic assays with subcellular resolution using microfluidic devices. Although microfluidics have previously been used to perform novel assays on C. elegans, so far research has been limited to specific appl...
This international guideline proposes improving clozapine package inserts worldwide by using ancestry-based dosing and titration. Adverse drug reaction (ADR) databases suggest that clozapine is the third most toxic drug in the United States (US), and it produces four times higher worldwide pneumonia mortality than that by agranulocytosis or myocarditis. For trough steady-state clozapine serum concentrations, the therapeutic reference range is narrow, from 350 to 600 ng/mL with the potential for toxicity and ADRs as concentrations increase. Clozapine is mainly metabolized by CYP1A2 (female non-smokers, the lowest dose; male smokers, the highest dose). Poor metabolizer status through phenotypic conversion is associated with co-prescription of inhibitors (including oral contraceptives and valproate), obesity, or inflammation with C-reactive protein (CRP) elevations. The Asian population (Pakistan to Japan) or the Americas’ original inhabitants have lower CYP1A2 activity and require lower clozapine doses to reach concentrations of 350 ng/mL. In the US, daily doses of 300–600 mg/day are recommended. Slow personalized titration may prevent early ADRs (including syncope, myocarditis, and pneumonia). This guideline defines six personalized titration schedules for inpatients: 1) ancestry from Asia or the original people from the Americas with lower metabolism (obesity or valproate) needing minimum therapeutic dosages of 75–150 mg/day, 2) ancestry from Asia or the original people from the Americas with average metabolism needing 175–300 mg/day, 3) European/Western Asian ancestry with lower metabolism (obesity or valproate) needing 100–200 mg/day, 4) European/Western Asian ancestry with average metabolism needing 250–400 mg/day, 5) in the US with ancestries other than from Asia or the original people from the Americas with lower clozapine metabolism (obesity or valproate) needing 150–300 mg/day, and 6) in the US with ancestries other than from Asia or the original people from the Americas with average clozapine metabolism needing 300–600 mg/day. Baseline and weekly CRP monitoring for at least four weeks is required to identify any inflammation, including inflammation secondary to clozapine rapid titration.
Techniques for stable, rapid and repeatable small-animal immobilization are necessary for high-throughput in vivo genetic/drug screens using cellular and sub-cellular features in multi-cellular organisms. We demonstrate a method for non-invasive and high-throughput on-chip immobilization of physiologically active C. elegans without the use of anesthesia or cooling, but with comparable stability even for the most demanding purposes. We show observation and manipulation of sub-cellular features in immobilized animals using two-photon microscopy and femtosecond-laser microsurgery.
Discovery of molecular mechanisms and chemical compounds that enhance neuronal regeneration can lead to development of therapeutics to combat nervous system injuries and neurodegenerative diseases. By combining high-throughput microfluidics and femtosecond laser microsurgery, we demonstrate for the first time largescale in vivo screens for identification of compounds that affect neurite regeneration. We performed thousands of microsurgeries at single-axon precision in the nematode Caenorhabditis elegans at a rate of 20 seconds per animal. Following surgeries, we exposed the animals to a hand-curated library of approximately one hundred small molecules and identified chemicals that significantly alter neurite regeneration. In particular, we found that the PKC kinase inhibitor staurosporine strongly modulates regeneration in a concentration-and neuronal type-specific manner. Two structurally unrelated PKC inhibitors produce similar effects. We further show that regeneration is significantly enhanced by the PKC activator prostratin. C. elegans | chemical screen | microfluidicsT he ability of neurons in the adult mammalian central nervous system to regenerate their axons after injury is extremely limited, which has been attributed to both extrinsic signals of the inhibitory glial environment (1) as well as intrinsic neuronal factors (2-4). The discovery of cell-permeable small molecules that modulate axon regrowth can potentiate the development of efficient therapeutic treatments for spinal cord injuries, brain trauma, stroke, and neurodegenerative diseases. Identification of such molecules can also provide valuable tools for fundamental investigations of the mechanisms involved in the regeneration process. Currently, small-molecule screens for neuronal regeneration are performed in simple in vitro cell culture systems. Such screens have already revealed large numbers of chemicals that enhance regeneration and/or affect cellular morphogenesis, yet many of these hits still remain untested in vivo. In addition, most in vitro studies do not translate to animal models and also fail to reveal off-target, toxic, or lethal effects. Thus, a thorough investigation of neuronal regeneration mechanisms requires in vivo neuronal injury models.In vivo investigation of neuronal regeneration has been performed mainly in mice and rats. However, their long developmental periods, complicated genetics and biology, and expensive maintenance prevent large-scale studies on these animals. The nematode Caenorhabditis elegans is a simple, well-studied, invertebrate model-organism with a fully mapped neuronal network comprising 302 neurons. Its short developmental cycle, simple and low-cost laboratory maintenance, and genetic amenability make it an ideal model for large-scale screens, rapid identification of the molecular targets of screened compounds, and discovery of novel signaling pathways implicated in regeneration.Until recently however, the small size of C. elegans (∼50 μm in diameter) prevented its use for investigation of neuronal re...
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