Driesschaert, Brecht et al. (2022) MicroPubl Biol

Driesschaert, Brecht, Temmerman, Liesbet

[MicroPubl Biol, 2022]

The Q system is a genetic tool developed to deliver spatiotemporal control over gene expression (Giles et al. 1991; Potter et al. 2010; Wei et al. 2012). Although it has already been adapted for use in C. elegans by Wei et al. in 2012, to date, the Q system has not been applied extensively in this nematode. In the relatively few available reports, it is mainly used to constitutively restrict gene expression in a spatial manner (e.g. Schild et al. 2014; Schild and Glauser 2015; Jee et al. 2016; Tolstenkov et al. 2018; Chiyoda et al. 2021), while but a handful of studies also explore the temporal aspect of the system (Matus et al. 2015; Yuan et al. 2016; Cottee et al. 2017; Hoang and Miller 2017). We aimed to apply this tool in the C. elegans nervous system to gain both spatial and temporal control over expression of a gene encoding a reporter protein that is targeted to the secretory pathway. Despite our efforts, we here report that in our hands, the Q system is not suitable for application in the neurons due to a lack of dynamic range.

Luck AN et al. (2016) FASEB J "Heme acquisition in the parasitic filarial nematode Brugia malayi."

Hamza I, Voronin D, Slatko BE, Yuan X, Foster JM, Luck AN

[FASEB J, 2016]

Nematodes lack a heme biosynthetic pathway and must acquire heme from exogenous sources. Given the indispensable role of heme, this auxotrophy may be exploited to develop drugs that interfere with heme uptake in parasites. Although multiple heme-responsive genes (HRGs) have been characterized within the free-living nematode Caenorhabditis elegans, we have undertaken the first study of heme transport in Brugia malayi, a causative agent of lymphatic filariasis. Through functional assays in yeast, as well as heme analog, RNAi, and transcriptomic experiments, we have shown that the heme transporter HRG-1 (BmHRG-1) is indeed functional in B. malayi In addition, BmHRG-1 localizes both to the endocytic compartments and cell membrane when expressed in yeast cells. Transcriptomic sequencing revealed that BmHRG-1, BmHRG-2, and BmMRP-5 (all orthologs of HRGs in C. elegans) are down-regulated in heme-treated B. malayi, as compared to non-heme-treated control worms. Likely because of short gene lengths, multiple exons, other HRGs in B. malayi (BmHRG-3, -4, -5, and -6) remain unidentified. Although the precise mechanisms of heme homeostasis in a nematode with the ability to acquire heme remains unknown, this study clearly demonstrates that the filarial nematode B. malayi is capable of transporting exogenous heme.-Luck, A. N., Yuan, X., Voronin, D., Slatko, B. E., Hamza, I., Foster, J. M. Heme acquisition in the parasitic filarial nematode Brugia malayi.

Yue-E H et al. (2016) Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi "[Meta regression analysis of five heavy metal biotoxicity effects onCaenorhabditis elegans]."

Yue-E H, Chao-Pin L, Nan Z

[Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi, 2016]

OBJECTIVE: To evaluate five heavy metal biotoxicity effects on Caenorhabditis elegans with Meta regression analysis. METHODS: The data were collected from the following electronic databases：PubMed, Web of Science, EBSCO, the Chinese Biomedical Database, Chinese National Knowledge Infrastructure, Wan Fang (Chinese) Databases and so on, and all updated through January 2004 to December 2014. A literature review was made on articles about Cd, Hg, Pb, As and Cr toxicity effects on C. elegans, and the data was extracted from figure information by Scan It software. A Meta regression model was built by stata12.0 software, using toxicity indices such as the dependent variables, and different metals, concentrations, exposure time, and development stages of C. elegans as independent variables. RESULTS:The study articles asociated with heavy metal biotoxicity effects on C. elegans were selected out, 10 studies of Cu, 12 studies of Cr, 12 studies of Pb, 12 studies of Cd, 3 studies of As and 14 studies of Hg. The mortality of toxicities of 5 heavy metals on C. elegans ranked Hg > Pb > Cr> Cd>As; the biotoxicity of the indexes of body size based on development, generational time based on reproductive toxicity, body bend frequencies, and head thrash frequencies based on neurotoxicity ranked Hg > Pb > Cr > Cd; the biotoxicity ranked Hg > Pb > Cr> Cd>As by LC50 indexes of toxicities of 5 heavy metals on C. elegans. The sensitivity analysis proved that the Meta regression model was reliable. CONCLUSIONS: The biotoxicities on C. elegans of Hg and Pb are stronger than Cr, Cd and As.

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.

Williams W et al. (2011) J Vis Exp "Constructing a low-budget laser axotomy system to study axon regeneration in C. elegans."

Bastiani M, Williams W, Nix P

[J Vis Exp, 2011]

Laser axotomy followed by time-lapse microscopy is a sensitive assay for axon regeneration phenotypes in C. elegans(1). The main difficulty of this assay is the perceived cost ($25-100K) and technical expertise required for implementing a laser ablation system(2,3). However, solid-state pulse lasers of modest costs (<$10K) can provide robust performance for laser ablation in transparent preparations where target axons are "close" to the tissue surface. Construction and alignment of a system can be accomplished in a day. The optical path provided by light from the focused condenser to the ablation laser provides a convenient alignment guide. An intermediate module with all optics removed can be dedicated to the ablation laser and assures that no optical elements need be moved during a laser ablation session. A dichroic in the intermediate module allows simultaneous imaging and laser ablation. Centering the laser beam to the outgoing beam from the focused microscope condenser lens guides the initial alignment of the system. A variety of lenses are used to condition and expand the laser beam to fill the back aperture of the chosen objective lens. Final alignment and testing is performed with a front surface mirrored glass slide target. Laser power is adjusted to give a minimum size ablation spot (<1 um). The ablation spot is centered with fine adjustments of the last kinematically mounted mirror to cross hairs fixed in the imaging window. Laser power for axotomy will be approximately 10X higher than needed for the minimum ablation spot on the target slide (this may vary with the target you use). Worms can be immobilized for laser axotomy and time-lapse imaging by mounting on agarose pads (or in microfluidic chambers(4)). Agarose pads are easily made with 10% agarose in balanced saline melted in a microwave. A drop of molten agarose is placed on a glass slide and flattened with another glass slide into a pad approximately 200 um thick (a single layer of time tape on adjacent slides is used as a spacer). A "Sharpie" cap is used to cut out a uniformed diameter circular pad of 13 mm. Anesthetic (1 ul Muscimol 20mM) and Microspheres (Chris Fang-Yen personal communication) (1 ul 2.65% Polystyrene 0.1 um in water) are added to the center of the pad followed by 3-5 worms oriented so they are lying on their left sides. A glass coverslip is applied and then Vaseline is used to seal the coverslip and prevent evaporation of the sample.

Dhillon I et al. (2018) MicroPubl Biol "GABA Receptor mutant gbb-1 accelerates morphological aging of GABA neurons in Caenorhabditis elegans"

Dhillon I, Chin-Sang I, Momin A

[MicroPubl Biol, 2018]

The aging of an organism is heterogeneous, that is to say, tissues and organs may age at unique rates compared to overall organism longevity. Inhibition of the insulin/insulin-like growth factor signaling (IIS), such as the daf-2 IIS receptor increases Caenorhabditis elegans longevity. The daf-2 pathway plays a key role in the regulation of metabolism and stress responses and as such, impacts the longevity of the organism (Uno and Nishida 2016). Evidence suggests that an increase in insulin signaling results in a reduction in overall lifespan and a decrease in neuronal longevity (Kenyon 2010). Recently, the gbb-1 GABA signaling pathway has also been implicated in the regulation of adult longevity (Chun et al. 2015). The GABAergic motor neurons innervate the dorsal and ventral body muscles controlling locomotion, foraging and defecation of the animal. GABA, an inhibitory neurotransmitter, has been found to decrease the lifespan of the nematodes (Chun et al. 2015), however, its role in neuronal aging has yet to be determined. The formation of ectopic neurite branches and production of axonal beads are two known phenotypes of neuronal aging in C. elegans. In this study, we examined the aging of the GABAergic motor neuron nerve cords and dorsal commissures. Fifty age-synchronized worms were imaged every three days (days 3, 6, 9, 12, 15, 18). Worms were plated on Fluorodeoxyuridine (FUdR) treated agar plates in order to maintain only adult worms throughout the imaging process (Mitchell et al. 1979). Neurons were visible using the GABAergic specific unc-25::GFP marker strain. To keep the data consistent, images were taken of the GABAergic axons and dorsal commissures between the VD5 and DD5 cell bodies (Panel A, scale bar 10 microns). Fluorescent images were taken using the Zeiss Axioplan fluorescent microscope under 40x objective. To prepare slides for fluorescence microscopy, an agarose immobilization protocol was followed (Fang-Yen et al. 2009). Aging of the neurons was quantified by counting the number of axonal beads and branches present on the neural processes. Branched processes emanating from the cell body, neural axon or dorsal commissures were scored as aging phenotypes. As predicted, GABAergic neurons displayed phenotypic evidence of aging. The C. elegans genome encodes two metabotropic GABAB receptor genes, gbb-1 and gbb-2, which are homologous to their mammalian counterparts (Chun et al. 2015). GABA binds to the gbb-1 receptor initiating a signaling cascade that ultimately inhibits the daf-16 transcription factor. A reduction of function mutation in the gbb-1 encoded GABA receptor subunit increases worm lifespan; however, it seems to be specific to the GBB-1 subunit as no effect was observed in a loss of function gbb-2 mutation. When overexpressed, gbb-1 mutants were short-lived which is consistent with GABA signaling regulating lifespan (Chun et al. 2015). To determine the effect of GABA signaling on neuronal aging, we observed the effect of a loss-of-function gbb-1 mutant on the aging of the GABAergic motor neurons. Unexpectedly, the GABAergic dorsal commissures and axons in the gbb-1 mutants displayed a significant increase in aging morphology compared to the wild type (Panel A). gbb-1 mutants displayed a significant increase in beading (Panel B, Table 1) and branching (Panel C, Table 2) in the GABAergic motor neurons compared to the wild type from day 9 onwards (p<0.05, independent t-test, n=50). Thus gbb-1(tm1406) mutants although long lived, have neurons that appear to age faster. This work was done with a single gbb-1 allele, in the future it will be important to verify our findings with additional gbb-1 alleles or show that a transgene with the wildtype gbb-1 can suppress the observed aging phenotypes.

Cho, Jaehyoung et al. MicroPubl Biol

Cho, Jaehyoung, Sternberg, Paul W., Chan, Juancarlos, Gao, Sibyl, Grove, Christian, Van Auken, Kimberly

[MicroPubl Biol, 2018]

Protein interaction is an important data type to understand the biological function of proteins involved in the interaction, and helps researchers to deduce the biological nature of unknown proteins from the well-characterized functions of their interaction partners. High-throughput studies, coupled with the aggregation of individual experiments, provides a global 'snapshot' of the protein interactions occurring at all levels of biological processes or circumstances. This snapshot of the interaction network, the interactome, is important to understand the overall events up to the level of comparison between species or pathway simulation, or to find new factors yet undefined in the processes, or to add details to the biological processes and pathways. As of September 2018, WormBase (www.wormbase.org) (Lee et al. 2018) contains 28,279 physical protein-protein interactions for the roundworm Caenorhabditis elegans. Among these, 1500 protein-protein interactions have been curated by BioGRID as a collaboration with WormBase. Within the data set, 17,990 protein-protein interactions are unique, and 6,079 unique genes are involved in these interactions. In order to visualize the overall interaction map, a network diagram for all the unique interactions was generated by using the Cytoscape program, version 3.6.1 (Shannon et al. 2003) (Figure 1A). These numbers represent a 108% increase in the number of interaction annotations since last year, 2017. These interaction data were curated from 1,251 peer-reviewed papers, which were selected from the literature by Textpresso Central using automatic SVM (Support Vector Machine)-based text mining approaches (Fang et al. 2012; Mller et al. 2018) and manual verification. Compared to other databases providing C. elegans protein-protein interaction, WormBase now presents the largest data set, which has 1.72-fold more interaction annotations than IMEx (Orchard et al. 2012) and 4.51-fold more than BioGRID (Chatr-Aryamontri et al. 2017) (Figure 1B). Most significantly, WormBase now houses the complete protein interaction data from almost all of the C. elegans literature published from 1993 to 2018. The data sets presented at IMEx and BioGRID are annotated from 253 and 174 papers, respectively. All the physical interaction data in WormBase are supported by experimental evidence from original research papers. The statistics of the detection methods used as experimental evidence are shown in Figure 1C. The majority of the interaction data came from high throughput analysis such as large-scale yeast two-hybrid assays or mass-spectrometry, however, a significant portion of the data (13.1%) are supported by more direct detection methods using small-scale, low throughput methods such as co-immunoprecipitation or co-crystallography (Figure 1C). In WormBase, protein-protein interaction data can be found as a subclass of physical interaction data in the Interactions widget on the gene report page. The Interactions widget provides all types of interaction data related to the gene of interest, such as physical, genetic, regulatory, and predicted interactions. All the interaction data are represented together in a graph created with Cytoscape.js and a table. In the table, the gene names of interaction partners (bait-target) in the interaction are displayed along with the publication. The interaction details including the detection method are also captured in the summary and the remark field in the Interactions page. Users can query the data by using the search bar on the WormBase front page or download all the available data files from the WormBase FTP site (ftp://ftp.wormbase.org/pub/wormbase/releases/current-production-release/species/c_elegans/PRJNA13758 /annotation/c_elegans.PRJNA13758.WSXXX.interactions.txt.gz, where WSXXX is the database version release, like WS267). All the interaction data in WormBase will be available soon at the new information resource for multiple model organisms, the Alliance of Genome Resources (https://www.alliancegenome.org/). This site will integrate all the interaction data from human and from model organisms C. elegans, budding yeast (Saccharomyces cerevisiae), fruit fly (Drosophila melanogaster), zebrafish (Danio rerio), mouse (Mus musculus) and rat (Rattus norvegicus). Integrated views of interaction data from diverse model organisms will be extremely helpful to build interaction databases for species-to-species comparison, and to establish a disease model quickly based on the database. For the most efficient analysis of the interaction data in WormBase, we are now working on developing a new Venn diagram tool and integrating the Gene Set Enrichment Analysis tool (https://wormbase.org/tools/enrichment/tea/tea.cgi) into the Interactions widget. We will continue to curate other types of macro-molecular interactions including protein-DNA, protein-RNA and RNA-RNA interactions, as well as newly reported protein-protein interaction data to serve our research community.