Duangjan C et al. (2019) Oxid Med Cell Longev "Lifespan Extending and Oxidative Stress Resistance Properties of a Leaf Extracts from Anacardium occidentale L. in Caenorhabditis elegans."

Duangjan C, Gu X, Rangsinth P, Wink M, Tencomnao T

[Oxid Med Cell Longev, 2019]

<i>Anacardium occidentale</i> (AO) contains a number of polyphenolic secondary metabolites with antioxidant activity. The objectives of this study were aimed at investigating the roles of AO leaf extracts in antioxidative stress and longevity, as well as their underlying mechanisms, in the <i>Caenorhabditis elegans</i> (<i>C. elegans</i>) model. AO extracts mediated the survival rate of nematodes under oxidative stress by attenuating intracellular reactive oxygen species (ROS) via the DAF-16/FoxO and SKN-1/Nrf-2 signaling pathways. AO extracts stimulated the expression of stress response genes including SOD-3 and GST-4. Moreover, AO extracts exhibited antiaging activities and enhanced longevity. We observed improved pharyngeal pumping function, attenuation of pigment accumulation (lipofuscin), and an increased lifespan of the worms. Collectively, our results demonstrated that AO extracts exerted both oxidative stress resistance and antiaging properties in the <i>C. elegans</i> model and may lead to new agents to benefit humans in the near future.

Thomas B et al. (2015) J Biomed Opt "Enhanced resolution through thick tissue with structured illumination and adaptive optics."

Wolstenholme A, Kner P, Kipreos ET, Thomas B, Chaudhari SN

[J Biomed Opt, 2015]

Structured illumination microscopy provides twice the linear resolution of conventional fluorescence microscopy, but in thick samples, aberrations degrade the performance and limit the resolution. Here, we demonstrate structured illumination microscopy through 35 m of tissue using adaptive optics (AO) to correct aberrations resulting in images with a resolution of 140 nm. We report a 60% minimum improvement in the signal-to-noise ratio of the structured illumination reconstruction through thick tissue by correction with AO.

Jeong J et al. (2019) Nanotoxicology "Developing adverse outcome pathways on silver nanoparticle-induced reproductive toxicity via oxidative stress in the nematode Caenorhabditis elegans using a Bayesian network model."

Choi I, Song T, Choi J, Chatterjee N, Jeong J, Cha YK

[Nanotoxicology, 2019]

An adverse outcome pathway (AOP) is a framework that organizes the mechanistic or predictive relationships between molecular initiating events (MIEs), key events (KEs), and adverse outcomes (AOs). Previously, we intensively investigated the molecular mechanism that underlies toxicity caused by AgNPs in the nematode Caenorhabditis elegans. Using transcriptomics, functional genetics, and various molecular/biochemical tools, we identified oxidative stress as the major mechanism underlying toxicity and reproduction failure as the outcome. With this information, here we conducted a case study of building an AOP to link oxidative stress with reproductive toxicity. To validate this AOP, we filled the gaps by conducting further experiments on its elements, such as NADPH oxidase, ROS formation, PMK-1 P38 MAPK activation, HIF-1 activation, mitochondrial damage, DNA damage, and apoptosis. The establishment of a causal link between the MIE and AO is critical for the construction of an AOP. Therefore, causal relationships between each KE and AO were verified by using functional genetic mutants of each KE. By combining these experimental data with our previously published results, we established causal relationships between the MIE, KEs, and AO using a Bayesian network (BN) model, culminating in an AOP entitled 'NADPH oxidase and P38 MAPK activation leading to reproductive failure in C. elegans ( https://aopwiki.org/aops/207)' . Overall, our approach shows that an AOP can be developed using existing data and further experiments can be conducted to fill the gaps between the MIE, KEs, and the AO. This study also shows that BN modeling has the potential to identify causal relationships in an AOP.

Yassin L et al. (2002) Biochemistry "Mutations in the extracellular domain and in the membrane-spanning domains interfere with nicotinic acetylcholine receptor maturation."

Halevi S, Yassin L, Treinin M, Eshel M, Samson AO

[Biochemistry, 2002]

The deg-3 (u662) mutation is a degeneration-causing mutation in a Caenorhabditis elegans nicotinic acetylcholine receptor. In a large screen for mutations that suppress the deleterious effects this mutation we identified 32 mutations in the deg-3 gene. Among these, 11 are missense mutations, affecting seven residues within the extracellular domain or the membrane-spanning domains. All of these mutations greatly reduce the degeneration-causing activity of deg-3 (u662). All but one of these mutations cause defective localization of the DEG-3 protein, as seen in immunohistochemical analysis. Thus our screen identifies multiple residues within the nicotinic acetylcholine receptor needed for normal folding, assembly, or trafficking of this receptor. Interestingly, these mutations lead to distinct localization defects suggesting differences in their effect on DEG-3's maturation process. Specifically, mutation sin the extracellular domain lead to a phenotype more severe than mutations in the membrane-spanning domains. Differences in the effects of the mutations are also predicted by homology-based modeling, showing that some mutations in the extracellular domain are likely to disrupt the native fold of the protein, while other are likely to disrupt trafficking.

Shaw M et al. (2013) Opt Express "Investigation of the confocal wavefront sensor and its application to biological microscopy."

O'Holleran K, Shaw M, Paterson C

[Opt Express, 2013]

Wavefront sensing in the presence of background light sources is complicated by the need to restrict the effective depth of field of the wavefront sensor. This problem is particularly significant in direct wavefront sensing adaptive optic (AO) schemes for correcting imaging aberrations in biological microscopy. In this paper we investigate how a confocal pinhole can be used to reject out of focus light whilst still allowing effective wavefront sensing. Using a scaled set of phase screens with statistical properties derived from measurements of wavefront aberrations induced by C. elegans specimens, we investigate and quantify how the size of the pinhole and the aberration amplitude affect the transmitted wavefront. We suggest a lower bound for the pinhole size for a given aberration strength and quantify the optical sectioning provided by the system. For our measured aberration data we find that a pinhole of size approximately 3 Airy units represents a good compromise, allowing effective transmission of the wavefront and thin optical sections. Finally, we discuss some of the practical implications of confocal wavefront sensing for AO systems in microscopy.

Agnes K.Y. HUI et al. (2008) Development & Evolution Meeting "ER Stress induced Autophagy-regulated Cell Death in C. elegans"

Agnes K.Y. HUI, King L. Chow

[Development & Evolution Meeting, 2008]

Misfolded protein accumulated in the endoplasmic reticulum(ER) compartment generates subcellular stress. Three ER localized sensor proteins, IRE-1, ATF-6 and PEK-1, monitor the ER lumen environment and collectively forms the unfolded protein response (UPR) pathways to cope with the stress. ER expansion, enhanced protein degradation, reduced protein synthesis are strategies to restore the function of the overloaded ER. Different degrees of responses being employed for increasing severity of stress ranging from autophagy for bulk degradation of stressed organelles to cell death to eliminate the stressed cell. The connection among UPR pathways, autophagy and cell death are largely unknown. Here, we utilize a dominant negative mutation of a male tail-specific collagen, ram-2(bx32) as a model to evaluate this mechanistic coordination. We envision that G to E substitution in RAM-2(bx32) results in an aberrant protein to generate ER stress at the male tail. Organelle volume of mutants was found to be increased 8-fold as visualized with aid of an ER marker. We asked whether autophagy is involved for the clearance of this expanded ER. dsRED::LGG-1 marker confirmed the presence of autophagosome. The elevation of both number and size of autophagosomes suggested that bulk degradation is activated. As autophagy can be pro-survival or pro-apoptotic, we differentiated these possibilities by nuclear membrane marker to visualize the nuclear morphology and acridine orange(AO) to monitor the presence of cell corpse DNA. Punctuated nuclear membrane signal and positive AO staining confirmed cell death is initiated. To delineate the cellular pathway leading to cell death, double mutants of ram-2(bx32) with UPR pathways and autophagy mutations were constructed. ram-2(bx32);unc-51(e369) double mutant showed enhanced Ram phenotype and AO staining. Loss of the autophagy inducing unc-51 function exacerbated cell death, which suggested a protective role of autophagy in this ER stress induced cell death. Further investigation on the molecular mechanism of ER stress induced cell death will be discussed. (This study is funded by Research Grants Council, Hong Kong.)

Guillaume Lettre et al. (2003) International Worm Meeting "A genetic suppressor screen for genes that trigger germ cell apoptosis in C. elegans"

Michael O Hengartner, Guillaume Lettre

[International Worm Meeting, 2003]

Apoptosis is part of the developmental program of the nematode C. elegans: out of the 1090 somatic cells that are born in a hermaphrodite, exactly 131 will die by apoptosis. These deaths require the activity of four genes, all of them being functionally conserved in human. The BH3-only protein EGL-1 binds the anti-apoptotic BCL-2-like protein CED-9; this interaction releases the CED-4/CED-3 complex. ced-4 and ced-3 encode an Apaf-1 homologue and a caspase, respectively. Upon release, the caspase CED-3 becomes activated and triggers apoptosis. Although we understand relatively well the molecular events that lead to apoptosis in the worm, other important questions remain to be answered. Among them, why are some cells born to inevitably die, and what are the developmental cues that cause cell death? Apoptosis also occurs in the germ line of C. elegans hermaphrodites, and the core apoptotic machinery (ced-3, -4, -9) is required for these deaths. However, three features distinguish physiological germ cell death from somatic cell death. First, contrary to somatic cell death, physiological germ cell death is unpredictable; this is also a feature of mammalian apoptosis. Second, the regulation of apoptosis in the germ line is genetically different than in the soma since loss of egl-1 function or gain of ced-9 function do not block germ line apoptosis, but prevent somatic cell death. Third, we also know that genotoxic treatment can activate germ cell death but not somatic cell death. All these lines of evidence suggest that distinct pathways regulate physiological germ line apoptosis and somatic apoptosis in C. elegans. In order to identify novel genes that specifically regulate physiological germ cell death, we performed a genetic suppressor screen for mutants with less germline apoptosis. We mutagenized gla-1(op234) worms (a strain with a 10-fold increase in the number of apoptotic corpses in the germ line) and screened the F2s for an absence of acridine orange (AO) staining under epifluorescence. AO is a vital dye that distinctively stains corpses in the germ line. We screened 21,500 haploid genomes and found 16 mutants. As expected, we identified new alleles of ced-3 and ced-4, as well as new alleles of engulfment genes (un-engulfed corpses do not stain with AO). Here, we will present epistatic and mapping data regarding the two strongest suppressors identified in our screen.

Lant B et al. (2014) Cold Spring Harb Protoc "Fluorescent visualization of germline apoptosis in living Caenorhabditis elegans."

Derry WB, Lant B

[Cold Spring Harb Protoc, 2014]

Visualization of apoptosis using fluorescent tools is quite straightforward in living Caenorhabditis elegans. Several transgenic lines are available that mark dying cells with fluorescent proteins, making it possible to quantify kinetics at various stages of the apoptotic process. Proteins required for the engulfment of cell corpses are particularly useful for detecting early apoptotic stages using this approach. For example, expression of the engulfment protein CED-1 fused to green fluorescent protein (CED-1::GFP) creates a halo around cells during early apoptosis, before their refractile morphology can be detected by differential interference contrast (DIC) optics. In addition, vital dyes such as acridine orange (AO) and SYTO-12 are selectively retained in apoptotic cells and can be used to visualize apoptosis in the germlines of living animals. It is also possible to use vital dyes in combination with transgenic strains expressing fluorescent markers of cell corpses to examine, in detail, multiple stages of apoptosis in vivo. Because of the high optical contrast of fluorescent reagents, apoptosis can be visualized even at low magnification, facilitating the use of screening platforms to identify apoptosis regulators. This protocol describes multiple uses of fluorescent reagents for visualization of germline apoptosis in living C. elegans, including AO staining, time-course studies using fluorescent proteins, and low-throughput screening of cell death genes using RNA interference (RNAi).

Sergio M Pinto et al. (2007) International Worm Meeting "A candidate based reverse genetic screen for negative regulators of corpse engulfment in Caenorhabditis elegans ."

Sergio M Pinto, Michael O Hengartner, Johann Almendinger

[International Worm Meeting, 2007]

During C. elegans hermaphrodite development, 1090 cells are formed, of which 131 die by apoptosis. In adult hermaphrodites, programmed cell death also occurs in the germ line. Phagocytosis of apoptotic cells in C. elegans is performed by the neighbouring cells. At least two partially redundant pathways contribute to apoptotic cell corpse removal; the first pathway comprises CED-1/MEGF10, which signals through the adaptor protein CED-6/GULP, and CED-7/ABCA1, which is required for proper localization of CED-1. In the second pathway, CED-2/CrkII recruits the GEF complex formed by CED-5/DOCK180 and CED-12/ELMO to the membrane. These pathways promote actin remodelling through regulated activation of CED-10/Rac1. Interestingly, all these genes have functional homologs in higher vertebrates, suggesting that the mechanisms mediating cell corpse removal are evolutionary conserved. Despite the intensive studies that this pathway has been subjected to, only very few negative regulators of engulfment have been found to date. In order to identify additional negative regulators, we screened by RNAi a set of potential candidate genes, chosen based on their predicted domains and cellular function. To measure apoptotic cell engulfment, we used an Acridine Orange (AO) based assay, as AO is known to accumulate in acidic compartments such as engulfed corpses upon fusion with the acidic lysosomes. We screened our set of candidate in several engulfment-defective backgrounds, in order to identify both general and pathway-specific negative regulators of engulfment. Here we will present an overview of the screens that we performed to date, and the results obtained from these screens.

Lant B et al. (2014) Cold Spring Harb Protoc "High-throughput RNAi screening for germline apoptosis genes in Caenorhabditis elegans."

Lant B, Derry WB

[Cold Spring Harb Protoc, 2014]

Among the greatest tools that Caenorhabditis elegans can provide researchers are the capabilities to perform high-throughput, genome-wide screens. Using bacterial RNAi libraries, which cover the majority (&gt;85%) of the worm genome, genes can be rapidly and systematically evaluated for apoptosis phenotypes in the germline. Screens can be designed to directly assess the levels of apoptotic corpses under normal physiological conditions using transgenic strains expressing fluorescent reporters that mark apoptotic bodies. Vital dyes that are selectively retained in apoptotic cells, such as acridine orange (AO), can also be used to screen for genes that regulate germline apoptosis. Using these reagents, screens can be performed in wild-type worms or mutant backgrounds that suppress or enhance apoptosis phenotypes. This protocol describes methods for designing and carrying out high-throughput or targeted RNAi screens for germline apoptosis regulators.