Dhillon HS et al. (1994) Worm Breeder's Gazette "More Degenerins in the Worm?"

Dhillon HS, Driscoll MA

[Worm Breeder's Gazette, 1994]

More degenerins in the worm? Harbinder Singh Dhillon and Monica Driscoll. Department of Molecular Biology and Biochemistry, Rutgers University, Center for Advanced Biotechnology and Medicine, 679 Hoes lane, Piscataway, N.J. 08855

Pinheiro D et al. (2014) Dev Cell "Making the most of the midbody remnant: specification of the dorsal-ventral axis."

Bellaiche Y, Pinheiro D

[Dev Cell, 2014]

In this issue of Developmental Cell, Singh and Pohl (2014) report that myosin II cortical flow and the midbody remnant participate in the specification of the C.elegans embryo dorsal-ventral axis.

Lee YT et al. (2019) Dev Cell "The Bacterivore's Solution: Fight and Flight to Promote Survival."

Lee YT, Wang MC

[Dev Cell, 2019]

Bacterial avoidance and innate immune response are two ways by which C.elegans respond to pathogenic bacteria. In this issue of Developmental Cell, Kumar etal. (2019) and Singh and Aballay (2019) demonstrate that bacterial colonization is essential to induce both responses, which may be associated with somatic and reproductive longevity.

Huang H et al. (2017) Bio Protoc "Measuring Caenorhabditis elegans Sleep During the Transition to Adulthood Using a Microfluidics-based System."

Hart AC, Singh K, Huang H

[Bio Protoc, 2017]

C. elegans sleep during development is regulated by genes and cellular mechanisms that are conserved across the animal kingdom (Singh et al., 2014; Trojanowski & Raizen, 2016). C. elegans developmental sleep is usually assessed during the transition to adulthood, a 2.6 h time interval called lethargus (Raizen et al., 2008; Singh et al., 2011). During lethargus, animals cycle between periods of immobility (sleep bouts) and periods of active locomotion (motion bouts). Sleep bouts resemble sleep in other species based on behavioral criteria, including cessation of feeding and locomotion, increased arousal threshold for response to sensory stimulation, rapid reversibility, and homeostatic response to sleep loss. Several assays have been developed to study sleep in C. elegans (Belfer et al., 2013; Bringmann, 2011; Nelson et al., 2013; Raizen et al., 2008). Here, we contribute a detailed protocol for assessment of C. elegans sleep during lethargus, which has been used successfully by many research groups, incorporating simple microfluidic chambers, a low cost camera with lighting system, and computational analysis based on image subtraction. We note that this system could be easily adapted to assess sleep in any small animal.

Iyer, Sangeetha et al. (2015) International Worm Meeting "A high-throughput screening approach using model organisms for Niemann-Pick Type C disease."

Hartl, Tom, Singh, Kiran, Ludin, Alec, DiPrimio, Nina, Perlstein, Ethan, Iyer, Sangeetha, Portillo, Tamy

[International Worm Meeting, 2015]

Perlstein Lab, PBC is a San Francisco based startup using model organisms to find small molecule therapies for rare genetic disorders. In this abstract, we describe a parallel model organism platform encompassing yeast, C.elegans, drosophila and mammalian cells for Niemann-Pick type C (NPC) disease. NPC, an autosomal recessive disorder caused by mutations in the NPC1 gene, afflicts 1 in 1,50,000 people. Clinically, this disease manifests during childhood in the form of progressive ataxia, dystonia, liver disease in addition to progressive cognitive decline. Biochemically, this disease is characterized by accumulation of free or unesterified cholesterol in the lysosomes. The NPC1 gene is remarkably conserved across species, with an ortholog present in yeast, nematode and fruit flies. In C.elegans, deletion of the homologous ncr-1 gene leads to mutant animals that develop slowly, have smaller brood sizes and importantly, inappropriately form dauer larvae under favorable conditions. These animals are also hypersensitive to the absence of cholesterol. Building on this, Perlstein Lab generated new lines of ncr-1 null mutants using CRISPR technology. After genotyping and validating that two newly generated strains phenocopied similar to their predecessors, one strain was selected for a rapid drug screening assay. Using slowed growth in cholesterol-deprived animals as an endpoint, a high throughput chemical modifier screen was designed to identify compounds that rescued the effects of cholesterol deprivation to near wildtype levels. Hits arising from this primary screen were compared for duplication and structural similarity against hits obtained from parallel screens in yeast and drosophila model organisms. Multi-organism hits were then tested in mammalian cell models of NPC using a filipin stain assay. Using this approach, we demonstrate that, for genetically conserved diseases, a parallel organism screening platform can be used to rapidly screen through tens of thousands of compounds thus paving the way for translational therapeutics.

Huang, Huiyan et al. (2013) International Worm Meeting "A genetic screen for Notch downstream targets regulating C. elegans sleep."

Zhu, Chen-Tsen, Hart, Anne, Huang, Huiyan

[International Worm Meeting, 2013]

The conserved Notch signaling pathway plays well-defined roles in cell fate determination during development. Recently, Notch has also been implicated in non-developmental neuronal processes across species, including invertebrate sleep. In C. elegans, the level of Notch activity affects both quality and quantity of lethargus quiescence, a sleep-like behavior coinciding with larval molts. Also, transiently overexpressing the Notch co-ligand, OSM-11, is sufficient to drive inappropriate quiescence in adult animals, which can be suppressed by loss of either Notch receptor or downstream players in Notch signaling (Singh et al., 2011). To find pertinent transcriptional targets of the Notch pathway and to gain insight into regulation of sleep, we undertook a classical suppressor screen to identify suppressors of inappropriate quiescence in adult hsp::osm-11 animals. In this screen, 2122 mutant lines were assessed; 79 independent isolates suppressed the OSM-11-induced inappropriate quiescence. To exclude mutations with pervasive effects on locomotion, we examined endogenous L4/adult lethargus quiescence in these 79 strains using both an adaption of the Multi-Worm Tracker (Swierczek et al., 2011) and the microfluidic chamber-based assay (Singh et al., 2011). Twenty-seven strains had defects in endogenous lethargus quiescence. We are in the process of identifying the corresponding genes by whole genome sequencing. As Notch signaling also modulates sleep behavior in Drosophila (Seugnet et al., 2011), we are confident that the genes identified in this screen will be conserved regulators of arousal and sleep across species.

Fischer, Christian et al. (2021) International Worm Meeting "MitoSegNet: Easy-to-use Deep Learning Segmentation for Analysing Mitochondrial Morphology"

Fischer, Christian, Haeussler, Simon, Marr, Carsten, Duchen, Michael, Conradt, Barbara, Rolland, Stephane, Singh, Kritarth, Besora-Casals, Laura

[International Worm Meeting, 2021]

While the analysis of mitochondrial morphology has emerged as an important tool in the study of mitochondrial function, efficient quantification of mitochondrial microscopy images presents a difficult task and bottleneck for statistically robust conclusions. Here, we present the Mitochondrial Segmentation Network (MitoSegNet), a pretrained deep learning segmentation model that enables researchers to easily exploit the power of deep learning for the quantification of mitochondrial morphology (Fischer, Besora-Casals et al. 2020). The MitoSegNet was generated by training a modified fully convolutional neural network with fluorescent microscopy, maximum-intensity projection images, depicting mitochondria in body wall muscle cells of adult C. elegans worms. We tested the performance of MitoSegNet against three feature-based segmentation algorithms and the machine-learning segmentation tool Ilastik. MitoSegNet outperformed all other methods in both pixelwise and morphological segmentation accuracy. We successfully applied MitoSegNet to unseen fluorescence microscopy images of mitoGFP expressing mitochondria in wild-type and catp-6ATP13A2 mutant C. elegans adults. Additionally, MitoSegNet was capable of accurately segmenting mitochondria in HeLa cells treated with fragmentation inducing reagents. We provide MitoSegNet for all operating systems as an easy-to-use graphical user interface tool that combines segmentation with morphological analysis. Reference Fischer, C. A., L. Besora-Casals, S. G. Rolland, S. Haeussler, K. Singh, M. Duchen, B. Conradt and C. Marr (2020). "MitoSegNet: Easy-to-use Deep Learning Segmentation for Analyzing Mitochondrial Morphology." iScience 23(10).

Bennett, Heather L. et al. (2013) International Worm Meeting "Notch DSL ligand lag-2 is required for C. elegans lethargus quiescence."

Bennett, Heather L., Hart, Anne C., Singh, Komudi, Huang, Huiyan

[International Worm Meeting, 2013]

Sleep is an evolutionarily conserved behavior defined as a rapidly reversible period of inactivity with decreased sensory responsiveness. Despite this knowledge, the genetic underpinnings, mechanisms of sleep regulation, and the specific tissues involved in this behavior remain elusive. C. elegans undergoes sleep-like quiescence during lethargus in coordination with each larval cuticle molt (Raizen et al., 2008). Our laboratory has shown that Notch signaling regulates quiescence, as perturbations in the Notch pathway change the arousal thresholds and amount of quiescence during the last larval lethargus during transition from L4 stage to adult (Singh et al., 2011). We find that decreased function of lag-2, which encodes a Notch DSL ligand, results in increased quiescence and reduced arousal thresholds during the L4 to adult lethargus. However, it remains unclear how specific changes in lag-2 gene function disrupt aspects of this behavior. We are now characterizing the role of lag-2 in regulating behavioral quiescence. The specific cells where LAG-2 functions in this behavior and when lag-2 is required to regulate quiescence is being determined. The knowledge gained from addressing these aims will help us understand the role of Notch signaling, particularly the role of lag-2 in regulating C. elegans quiescence and may illuminate evolutionary conserved mechanisms required to regulate sleep across species.

Jennifer L. Tenor et al. (2007) International Worm Meeting "A conserved Toll-like receptor is required for Caenorhabditis elegans innate immunity."

Jennifer L. Tenor, Alejandro Aballay

[International Worm Meeting, 2007]

Pathogen recognition through Toll-like receptors (TLRs) is the first step required to mount an appropriate immune response against invading pathogens. The only toll-like receptor, tol-1, in Caenorhadbitis elegans was shown to be required for behavioral avoidance to a particular Gram-negative bacterium, Serratia marcescens (1). Although we did not observe significant differences in behavioral avoidance between wild type and the tol-1(nr2033) mutant to various other Gram-negative pathogens, the tol-1 mutant was hypersusceptible to these pathogens suggesting that TOL-1 may provide protection through other mechanisms. Based on survival assays and microscopy, we found that TOL-1 is essential for protecting the pharynx against the enteropathogenic bacterium, Salmonella enterica serovar Typhimurium. Loss of tol-1 not only led to rapid death of C. elegans but also resulted in rapid accumulation of S. enterica in the terminal bulb of the pharynx. We showed that TOL-1 is required for the proper expression of ABF-2 which is a defensin-like molecule expressed in the pharynx (2) and HSP-16.41 which is also expressed in the pharynx and is part of a family of heat shock proteins required for C. elegans immunity (3). These results indicate that TOL-1 plays a direct role in innate immunity. (1) Pujol et al., 2001; (2) Kato, 2002; (3) Singh and Aballay, 2006.

Chamoli, Manish et al. (2020) MicroPubl Biol

Chinta, Shankar J, Andersen, Julie K, Lithgow, Gordon J, Chamoli, Manish

[MicroPubl Biol, 2020]

Kavain belongs to a group of lactone-based compounds collectively known as kavalactones, present in the pepper plant kava (P. methysticum). Kavalactones have been shown to possess diverse biological activities including sedation and anxiolysis (Ooi et al., 2018). Kavain in particular has been demonstrated to show potent anti-inflammatory properties in various in vitro and animal models (Guo et al., 2018; Singh et al., 2018; Tang and Amar, 2016; Yuan et al., 2011). A study in C. elegans reported that kavain increases lifespan by inhibiting advance glycation end-products (AGEs), which are known to suppress lifespan (Chaudhuri et al., 2016; Upadhyay et al., 2014). Another study reported that kavain increases acetylcholine (ACh) transmission at the neuromuscular junction (Kautu et al., 2017). Since loss in ACh transmission and increased formation of AGEs are closely linked to A-pathology, we hypothesized that kavain may protect against A-induced toxicity (Kar et al., 2004; Li et al., 2013). We tested kavain in the C. elegans GMC101 strain that over-expresses human Ain body wall muscle cells (McColl et al., 2012). Kavain at a concentration of 40 and 80 M was shown to increase lifespan, thus we decided to use a dose between these ranges (Upadhyay et al., 2014). We observed GMC101 animals fed 50 M kavain showed significantly less paralysis when shifted to the higher permissive temperature (25o C). The result shows that kavain suppresses A-induced proteotoxicity.