Ben-Yakar A (2019) Assay Drug Dev Technol "High-Content and High-Throughput In Vivo Drug Screening Platforms Using Microfluidics."

Ben-Yakar A

[Assay Drug Dev Technol, 2019]

The drug-discovery process is expensive and lengthy, and has been causing a rapid increase in the global health care cost. Despite extensive efforts, many human diseases still lack a cure. To improve the outcomes, there is a growing need to implement novel approaches into the early stages of the drug-discovery pipeline. A specific such effort has focused on the development of novel disease models such as cellular models (genetically modified cell lines, spheroids, and organoids) and whole-animal models (small animal models and genetically modified large animal models). The whole-animal screens are advantageous as they can provide system-level information, off-target effects, complete absorption, distribution, metabolism, excretion, and toxicity architectures, and early in vivo toxicity, which help to prioritize compounds before using them for human trials. Such multivariate analysis helps to improve the translational potential of drug compounds. Drug testing in large animals is expensive and time consuming. A solution is small animal models that have simplified biological system with intact physiology and sufficient homology with human genes. In recent times, many such models have constantly been developed and tested to identify new disease mechanisms. Caenorhabditis elegans is one such small animal model that has been considered for large-scale drug testing. In this review, we will discuss the current state-of-the-art technologies, including two platforms developed in my group that have enabled high-throughput and high-content screening using C. elegans disease models.

Ben-Yakar A et al. (2009) Curr Opin Biotechnol "Ultrafast laser nanosurgery in microfluidics for genome-wide screenings."

Ben-Yakar A, Bourgeois F

[Curr Opin Biotechnol, 2009]

The use of ultrafast laser pulses in surgery has allowed for unprecedented precision with minimal collateral damage to surrounding tissues. For these reasons, ultrafast laser nanosurgery, as an injury model, has gained tremendous momentum in experimental biology ranging from in vitro manipulations of subcellular structures to in vivo studies in whole living organisms. For example, femtosecond laser nanosurgery on such model organism as the nematode Caenorhabditis elegans has opened new opportunities for in vivo nerve regeneration studies. Meanwhile, the development of novel microfluidic devices has brought the control in experimental environment to the level required for precise nanosurgery in various animal models. Merging microfluidics and laser nanosurgery has recently improved the specificities and increased the speed of laser surgeries enabling fast genome-wide screenings that can more readily decode the genetic map of various biological processes.

Ben-Yakar A et al. (2009) Curr Opin Neurobiol "Microfluidics for the analysis of behavior, nerve regeneration, and neural cell biology in C. elegans."

Lu H, Chronis N, Ben-Yakar A

[Curr Opin Neurobiol, 2009]

The nematode Caenorhabditis elegans is a widely adopted model organism for studying various neurobiological processes at the molecular and cellular level in vivo. With a small, flexible, and continuously moving body, the manipulation of C. elegans becomes a challenging task. In this review, we highlight recent advances in microfluidic technologies for the manipulation of C. elegans. These new family of microfluidic chips are capable of handling single or populations of worms in a high-throughput fashion and accurately controlling their microenvironment. So far, they have been successfully used to study neural circuits and behavior, to perform large-scale phetotyping and morphology-based screens as well as to understand axon regeneration after injury. We envision that microfluidic chips can further be used to study different aspects of the C. elegans nervous system, extending from fundamental understanding of behavioral dynamics to more complicated biological processes such as neural aging and learning and memory.

Driscoll MA et al. (1989) J Cell Biol "Genetic and molecular analysis of a Caenorhabditis elegans beta-tubulin that conveys benzimidazole sensitivity."

Reilly E, Dean E, Bergholz E, Driscoll MA, Chalfie M

[J Cell Biol, 1989]

Benzimidazole anti-microtubule drugs, such as benomyl, induce paralysis and slow the growth of the nematode Caenorhabditis elegans. We have identified 28 mutations in C. elegans that confer resistance to benzimidazoles. All resistant mutations map to a single locus, ben-1. Virtually all these mutations are genetically dominant. Molecular cloning and DNA sequence analysis established that ben-1 encodes a beta-tubulin. Some resistant mutants are completely deleted for the ben-1 gene. Since the deletion strains appear to be fully resistant to the drugs, the ben-1 product appears to be the only benzimidazole-sensitive beta-tubulin in C. elegans. Furthermore, since animals lacking ben-1 are viable and coordinated, the ben-1 beta-tubulin appears to be nonessential for growth and movement. The ben-1 function is likely to be redundant in the nematode genome.

Bourgeois F et al. (2008) Opt Express "Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans."

Ben-Yakar A, Bourgeois F

[Opt Express, 2008]

We present a study characterizing the properties of femtosecond laser nanosurgery applied to individual axons in live Caenorhabditis elegans (C. elegans) using nano-Joule laser pulses at 1 kHz repetition rate. Emphasis is placed on the characterization of the damage threshold, the extent of damage, and the statistical rates of axonal recovery as a function of laser parameters. The ablation threshold decreases with increasing number of pulses applied during nanoaxotomy. This dependency suggests the existence of an incubation effect. In terms of extent of damage, the energy per pulse is found to be a more critical parameter than the number of pulses. Axonal recovery improves when surgery is performed using a large number of low energy pulses.

Bourgeois F et al. (2007) Opt Express "Femtosecond laser nanoaxotomy properties and their effect on axonal recovery in C. elegans."

Bourgeois F, Ben-Yakar A

[Opt Express, 2007]

We present a study characterizing the properties of femtosecond laser nanosurgery applied to individual axons in live Caenorhabditis elegans (C. elegans) using nano-Joule laser pulses at 1 kHz repetition rate. Emphasis is placed on the characterization of the damage threshold, the extent of damage, and the statistical rates of axonal recovery as a function of laser parameters. The ablation threshold decreases with increasing number of pulses applied during nanoaxotomy. This dependency suggests the existence of an incubation effect. In terms of extent of damage, the energy per pulse is found to be a more critical parameter than the number of pulses. Axonal recovery improves when surgery is performed using a large number of low energy pulses.

Driscoll MA et al. (1988) Worm Breeder's Gazette "notes on the DNA sequence of most of the ben-1 gene."

Bergholz E, Driscoll MA, Chalfie M

[Worm Breeder's Gazette, 1988]

The ben-1 gene encodes one of the four (or so) C. elegans - tubulins. All mutations that confer resistance to the antimicrotubule drug benomyl map to this locus. The ben-1-encoded -tubulin is not essential for C. elegans coordination or viability since animals harboring deletions of the ben-1 gene appear wild-type in growth and behavior. We have sequenced all but the very 3' end of the ben-1 gene. 408 of approximately 450 amino acids are represented. (The sequence data is available upon request). In Genbank, the deduced protein sequence is most homologous to a human -tubulin (showing 93% identity, with all amino acid changes conservative). Comparison to other C. elegans - tubulins indicates that ben-1 is 96% identical to the tub-1 gene sequenced by L. Gremke and J. Culotti. ben-1 shares 87% identity with the mec-7-encoded -tubulin. The C. elegans -tubulin gene organization can be summarized as follows: [See Figure 1] ben-1 and tub-1 are similar in organization of coding regions, whereas mec-7 differs markedly in the relative positioning of exons and introns. The ben-1 gene contains two relatively long introns, of 973 and 809 bp. The putative splice sites in ben-1 conform to consensus C. elegans splice sequences. Interestingly, there is a potential splice acceptor site upstream of the AUG initiator codon. We are concerned that the sequenced 350bp 5' to the translational start may not include the promoter for this gene. Possibly, the message may acquire a trans- spliced leader. The DNA sequence of the 5' region in a ben-1 cDNA should clarify the transcript structure.

Chokshi TV et al. (2009) Lab Chip "CO2 and compressive immobilization of C. elegans on-chip."

Chokshi TV, Ben-Yakar A, Chronis N

[Lab Chip, 2009]

We present two microfluidic approaches for immobilizing the roundworm C. elegans on-chip. The first approach creates a CO(2) micro-environment while the second one utilizes a deformable PDMS membrane to mechanically restrict the worm's movement. An on-chip 'behavior' module was used to characterize the effect of these methods on the worm's locomotion pattern. Our results indicate that both methods are appropriate for the short-term (minutes) worm immobilization. The CO(2) method offers the additional advantages of long-term immobilization (1-2 hours) and reduced photobleaching, if fluorescent imaging during immobilization is required. We envision the use of these methods in a wide variety of biological studies in C. elegans, including cell developmental and neuronal regeneration studies.

Gibson SB et al. (2022) Int J Parasitol Drugs Drug Resist "Benzimidazoles cause lethality by inhibiting the function of Caenorhabditis elegans neuronal beta-tubulin."

Andersen EC, Gibson SB, Ness-Cohn E

[Int J Parasitol Drugs Drug Resist, 2022]

Parasitic nematode infections cause an enormous global burden to both humans and livestock. Resistance to the limited arsenal of anthelmintic drugs used to combat these infections is widespread, including benzimidazole (BZ) compounds. Previous studies using the free-living nematode Caenorhabditis elegans to model parasitic nematode resistance have shown that loss-of-function mutations in the beta-tubulin gene ben-1 confer resistance to BZ drugs. However, the mechanism of resistance and the tissue-specific susceptibility are not well known in any nematode species. To identify in which tissue(s) ben-1 function underlies BZ susceptibility, transgenic strains that express ben-1 in different tissues, including hypodermis, muscles, neurons, intestine, and ubiquitous expression were generated. High-throughput fitness assays were performed to measure and compare the quantitative responses to BZ compounds among different transgenic lines. Significant BZ susceptibility was observed in animals expressing ben-1 in neurons, comparable to expression using the ben-1 promoter. This result suggests that ben-1 function in neurons underlies susceptibility to BZ. Subsetting neuronal expression of ben-1 based on the neurotransmitter system further restricted ben-1 function in cholinergic neurons to cause BZ susceptibility. These results better inform our current understanding of the cellular mode of action of BZs and also suggest additional treatments that might potentiate the effects of BZs in neurons.

Ghorashian N et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "A Parallelized Microfluidic Chamber Device for High-Throughput Nerve Regeneration Studies in C. elegans"

Ghorashian N, Hilliard M A, Ben-Yakar A

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

We fabricated and tested a high-throughput microfluidic platform to study nerve regeneration in C. elegans. The device consists of an array of small chambers in a parallel fluidic circuit allowing for simultaneous trapping of dozens of C. elegans worms in individual visualization chambers for in-vivo imaging and laser ablation of fluorescently labeled axons. With proper liquid nutrients, the animals can easily survive in the microfluidic chambers for three days or more for monitoring nerve regeneration. This device could serve as the optical and fluidic interface for automated genome-wide nerve regeneration studies using femtosecond laser nano-axotomy and fluorescence microscopy. Using our device and conventional methods, we investigated the regenerative capacity of the oxygen sensory neuron, PQR. This neuron is located in the left lumbar ganglion on the posterior-lateral side of the worm's body, and has only two processes emerging from the cell body – a dendrite extending posterior toward the tip of the tail and an axon extending anterior joining the ventral nerve cord. We looked at regeneration rates in animals in which either only one or both neurites were severed. We observed that the dendrite process regenerated with a higher frequency when the axon was simultaneously severed. This result suggests that the molecular machinery responsible for regeneration is more efficiently recruited in a given process when there is additional damage to other parts of the neuron.