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.

Bellver-Sanchis, Aina et al. (2021) International Worm Meeting "Discovery of new small molecule inhibitors of methyltransferase G9a for Alzheimer's disease treatment"

Pallas, Merce, Malik, Ruchi, Bellver-Sanchis, Aina, Singh Choudhary, Bhanwar, Grinan-Ferre, Christian

[International Worm Meeting, 2021]

Alzheimer's disease (AD) is the most important neurodegenerative disorder, with no effective cure. The epigenetic control of gene expression might be affected by the addition and/or removal of post-translational changes. Alterations of these modifications are linked with altered gene expression, resulting in cognitive decline. G9a is a lysine methyltransferase that forms a heterodimeric complex able to mono- and di-methylate lysine 9 of histone H3 (H3K9me1 and H3K9me2) of the N-terminal tail. Those epigenetic modifications lead to transcription repression, synaptic plasticity reduction, autophagy dysfunction, increased OS, and neuroinflammation. Hence, we hypothesize that overexpression of the G9a and its repressive mark serves as a driver of the repression of learning memory genes. Novel strategies are based on the synthesis of epigenetic drugs, regulating gene expression and translation modifications. While several small-molecules have been published to inhibit G9a, one of the most common problems of the firsts inhibitors, as BIX01294, is that the concentration values of the toxicity range and the last activity were akin one to the other, strongly limiting its uses. Moreover, these inhibitors were not or low effective in in vivo studies. In our study, several compounds were identified as a potential inhibitor through structure-based virtual screening of the Cambridge CNS MPO library of more than 4,50,000 compounds against G9a (PDB id: 5TTF) Maestro software (Schrodinger), and further in vivo screenings. Firstly, we assessed the IC50 of eleven compounds using AlphaLISA technology. To identify the best new candidates G9a inhibitors for AD, a compound screening method using transgenic Caenorhabditis elegans (C. elegans) was used to characterize and select the most promising results for further studies. Among all the compounds, four were confirmed as the best candidates through different in vivo assays, such as food clearance and motility assays, which revealed that toxicity/function ranges were safe in C. elegans. The treatment with all G9a inhibitors recovered the age-dependent paralysis presented by the CL2006 strain, like the well-established G9a inhibitor, UNC0638. Thus, this work presents 4 candidates (called "CAB1, CAB2, CAB3 and CAB4") as inhibitors of G9a with novel structures, providing both leads in G9a inhibitors design and demonstrating the participation in AD pathology.

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.

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.

Pir GJ et al. (2017) FASEB J "Caenorhabditis elegans models of tauopathy."

Pir GJ, Choudhary B, Mandelkow E

[FASEB J, 2017]

One of the hallmarks of the tauopathies, which include the neurodegenerative disorders, such as Alzheimer disease (AD), corticobasal degeneration, frontotemporal dementia, and progressive supranuclear palsy (PSP), is the abnormal accumulation of post-translationally modified, insoluble tau. The result is a loss of neurons, decreased mental function, and complete dependence of patients on others. Aggregation of tau, which under physiologic conditions is a highly soluble protein, is thought to be central to the pathogenesis of these diseases. Indeed one of the strongest lines of evidence is the MAPT gene polymorphisms that lead to the familial forms of tauopathy. Extensive research in animal models over the years has contributed some of the most important findings regarding the pathogenesis of these diseases. Despite this, the precise molecular mechanisms that lead to abnormal tau folding, accumulation, and spreading remain unknown. Owing to the fact that most of the biochemical pathways are conserved, Caenorhabditis elegans provides an alternative approach to identify cellular mechanisms and druggable genes that operate in such disorders. Many human genes implicated in neurodegenerative diseases have counterparts in C. elegans, making it an excellent model in which to study their pathogenesis. In this article, we review some of the important findings gained from C. elegans tauopathy models.-Pir, G. J., Choudhary, B., Mandelkow, E. Caenorhabditiselegans models of tauopathy.

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.