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WormBook,
2014]
Mass spectrometry (MS)-based shotgun proteomics is an enabling technology for the study of C. elegans proteins. When coupled with co-immunoprecipitation (CoIP), new interactions and functions among proteins can be discovered. We provide a general background on protein complexes and methods for their analysis, along with the lifecycle and interaction types of proteins that ultimately define the identifiable components of protein complexes. We highlight traditional biochemical methods to evaluate whether the complexes are sufficiently pure and abundant for analysis with shotgun proteomics. We present two CoIP-MS case studies of protein complexes from C. elegans, using both endogenous and fusion protein antibodies to illustrate the important aspects of their analyses. We discuss results from mass spectrometers with differences in mass accuracy and resolution, along with the relevant information that can be extracted from the data generated, such as protein relative abundance, post-translational modifications, and identification confidence. Finally, we illustrate how comparative analysis can reveal candidate binding partners for biological follow-up and validation. This chapter should act as a complement and extension to the WormBook chapter Biochemistry and molecular biology, which describes tandem affinity purification (TAP) of protein complexes for analysis by mass spectrometry.
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J Proteomics,
2010]
Mass spectrometry-based proteomics is rapidly becoming an essential tool for biologists. One of the most common applications is identifying the components of protein complexes isolated by co-immunoprecipitation. In this review, we discuss the co-immunoprecipitation, mass spectrometry and data analysis techniques that have been used successfully to define protein complexes in C. elegans research. In this discussion, two strategies emerged. One approach is to use stringent biochemical purification methods and attempt to identify a small number of complex components with a high degree of certainty based on MS data. A second approach is to use less stringent purification and identification parameters, and ultimately test a longer list of potential binding partners in biological validation assays. This should provide a useful guide for biologists planning proteomic experiments.
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Interdiscip Top Gerontol,
2010]
Dramatic changes in body composition accompany aging in humans, particularly with respect to adiposity and the musculature. People accumulate fat as they age and lose muscle mass and strength. Caenorhabditis elegans nematodes are small, hermaphroditic soil nematodes that offer a flexible model for studying genetic pathways regulating body composition in humans. While there are significant physiological differences between worms and people, many of the genetic pathways relevant to human lipid and muscle homeostasis are present in worms. Initial studies indicate that adiposity increases in C. elegans during aging, as occurs in humans. Furthermore, substantial evidence demonstrates age-related loss of muscle mass in worms. Possible mechanisms for these changes in C. elegans are presented. Recent studies have highlighted neuroendocrine and environmental signals regulating C. elegans fat metabolism. Potential dysfunction of these pathways during aging could affect overall fat accumulation. By contrast, muscle decline in aging worms results from accumulated damage and 'wear-and-tear' over life span. However, neuroendocrine pathways also regulate muscle mass in response to food availability. Such pathways might provide useful therapeutic approaches for combating muscle loss during aging. From this chapter, readers will develop a deeper understanding of the ways that C.elegans can be used for mechanistic gerontological studies.
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Metabolites,
2021]
Metabolomics and lipidomics recently gained interest in the model organism <i>Caenorhabditis elegans</i> (<i>C. elegans</i>). The fast development, easy cultivation and existing forward and reverse genetic tools make the small nematode an ideal organism for metabolic investigations in development, aging, different disease models, infection, or toxicology research. The conducted type of analysis is strongly depending on the biological question and requires different analytical approaches. Metabolomic analyses in <i>C. elegans</i> have been performed using nuclear magnetic resonance (NMR) spectroscopy, direct infusion mass spectrometry (DI-MS), gas-chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) or combinations of them. In this review we provide general information on the employed techniques and their advantages and disadvantages in regard to <i>C. elegans</i> metabolomics. Additionally, we reviewed different fields of application, e.g., longevity, starvation, aging, development or metabolism of secondary metabolites such as ascarosides or maradolipids. We also summarised applied bioinformatic tools that recently have been used for the evaluation of metabolomics or lipidomics data from <i>C. elegans</i>. Lastly, we curated metabolites and lipids from the reviewed literature, enabling a prototypic collection which serves as basis for a future <i>C. elegans</i> specific metabolome database.
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Integr Comp Biol,
2015]
This review provides an overview of two complementary approaches to identify biologically active compounds for studies in chemical ecology. The first is activity-guided fractionation and the second is metabolomics, particularly focusing on a new liquid chromatography-mass spectrometry-based method called isotopic ratio outlier analysis. To illustrate examples using these approaches, we review recent experiments using Caenorhabditis elegans and related free-living nematodes.
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[
1982]
The small soil nematode Caenorhabditis elegans is an attractive organism for the molecular study of muscle function and development because of its anatomical simplicity and suitability for genetic and biochemical analysis (Brenner 1974; Sulston and Horvitz 1977). The body-wall musculature of C. elegans is composed of 95 cell disposed in four quadrants, which run the length of the animal beneath the cuticle. The musculature is obliquely striated, and the sarcomeres are oriented parallel to the long axis of the animal. Since these cells represent a large reaction of the animal mass, isolation of contractile proteins is comparatively simple (Epstein et al. 1974; Waterston et al. 1974, 1977a; Harris and Epstein 1977; Mackenzie and Epstein 1980). Mutants affecting the characteristic pattern of motility of C. elegans can be easily identified, and microscopic examination of these "uncoordinated," or unc strains, in the living animal by polarized light microscopy or, more carefully, by electron microscopy has led to the identification of 22 genes that produce altered muscle phenotypes (Waterston et al. 1980; Zengel and Epstein 1980). Of these, two are known to code for major structural proteins of muscle: The
unc-54 gene codes for the major heavy chain of myosin (Epstein et al. 1974; MacLeod et al. 1977b), whereas the un-15 gene codes for paramyosin, the core protein of the thick filaments (Waterston et al. 1974; MacLeod et al. 1977a; Harris and Epstein 1977).
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Crit Rev Biochem Mol Biol,
2012]
The CCAAT box promoter element and NF-Y, the transcription factor (TF) that binds to it, were among the first cis-elements and trans-acting factors identified; their interplay is required for transcriptional activation of a sizeable number of eukaryotic genes. NF-Y consists of three evolutionarily conserved subunits: a dimer of NF-YB and NF-YC which closely resembles a histone, and the "innovative" NF-YA. In this review, we will provide an update on the functional and biological features that make NF-Y a fundamental link between chromatin and transcription. The last 25 years have witnessed a spectacular increase in our knowledge of how genes are regulated: from the identification of cis-acting sequences in promoters and enhancers, and the biochemical characterization of the corresponding TFs, to the merging of chromatin studies with the investigation of enzymatic machines that regulate epigenetic states. Originally identified and studied in yeast and mammals, NF-Y - also termed CBF and CP1 - is composed of three subunits, NF-YA, NF-YB and NF-YC. The complex recognizes the CCAAT pentanucleotide and specific flanking nucleotides with high specificity (Dorn et al., 1997; Hatamochi et al., 1988; Hooft van Huijsduijnen et al, 1987; Kim & Sheffery, 1990). A compelling set of bioinformatics studies clarified that the NF-Y preferred binding site is one of the most frequent promoter elements (Suzuki et al., 2001, 2004; Elkon et al., 2003; Marino-Ramirez et al., 2004; FitzGerald et al., 2004; Linhart et al., 2005; Zhu et al., 2005; Lee et al., 2007; Abnizova et al., 2007; Grskovic et al., 2007; Halperin et al., 2009; Hakkinen et al., 2011). The same consensus, as determined by mutagenesis and SELEX studies (Bi et al., 1997), was also retrieved in ChIP-on-chip analysis (Testa et al., 2005; Ceribelli et al., 2006; Ceribelli et al., 2008; Reed et al., 2008). Additional structural features of the CCAAT box - position, orientation, presence of multiple Transcriptional Start Sites - were previously reviewed (Dolfini et al., 2009) and will not be considered in detail here.
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Aging Cell,
2020]
Sarcopenia is the age-related decline in muscle mass and function without any underlying disease. The exact molecular mechanisms responsible for this pathology remain unknown. The use of model organisms, such as mice, rats, flies, and worms, has advanced the field of sarcopenia research by identifying therapeutic strategies and genetic mutations that result in improved muscle mass and function of elderly animals. This review discusses molecular and therapeutic discoveries made using these model organisms and how these animals can be further utilized to better understand sarcopenia pathogenesis. In rodents, flies, and worms, dietary restriction improves muscle performance in old animals. In rodents and worms, exercise and a number of naturally occurring compounds alleviate sarcopenia. Reduction in the insulin/IGF1 receptor pathway, well known to promote longevity, improves sarcopenia in worms and flies. Mitochondrial dysfunction may contribute to the pathogenesis of sarcopenia: In rodents, there is age-dependent reduction in mitochondrial mass and a change in morphology; in nematodes, there is age-dependent fragmentation of mitochondria that precedes sarcomeric disorganization. In Drosophila and rats, components of the 26S proteasome are elevated in aged muscle. An advantage of the worm and fly models is that these organisms lack muscle stem cells, and thus processes that promote the maintenance of already assembled muscle, can be identified without the confounding influence of muscle regeneration. Zebrafish are an up and coming model of sarcopenia for future consideration. A better understanding of the molecular changes behind sarcopenia will help researchers develop better therapies to improve the muscle health of elderly individuals.
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Clin Microbiol Infect,
2011]
Lymphatic filariasis (LF) and onchocerciasis are parasitic nematode infections that are responsible for a major disease burden in the African continent. Disease symptoms are induced by the immune reactions of the host, with lymphoedema and hydrocoele in LF, and dermatitis and ocular inflammation in onchocerciasis. Wuchereria bancrofti and Onchocerca volvulus, the species causing LF and onchocerciasis in Africa, live in mutual symbiosis with Wolbachia endobacteria, which cause a major part of the inflammation leading to symptoms and are antibiotic targets for treatment. The standard microfilaricidal drugs ivermectin and albendazole are used in mass drug administration programmes, with the aim of interrupting transmission, with a consequent reduction in the burden of infection and, in some situations, leading to regional elimination of LF and onchocerciasis. Co-endemicity of Loa loa with W. bancrofti or O. volvulus is an impediment to mass drug administration with ivermectin and albendazole, owing to the risk of encephalopathy being encountered upon administration of ivermectin. Research into new treatment options is exploring several improved delivery strategies for the classic drugs or new antibiotic treatment regimens for anti-wolbachial chemotherapy.
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Front Genet,
2019]
Onchocerciasis and lymphatic filariasis are targeted for elimination, primarily using mass drug administration at the country and community levels. Elimination of transmission is the onchocerciasis target and global elimination as a public health problem is the end point for lymphatic filariasis. Where program duration, treatment coverage, and compliance are sufficiently high, elimination is achievable for both parasites within defined geographic areas. However, transmission has re-emerged after apparent elimination in some areas, and in others has continued despite years of mass drug treatment. A critical question is whether this re-emergence and/or persistence of transmission is due to persistence of local parasites-i.e., the result of insufficient duration or drug coverage, poor parasite response to the drugs, or inadequate methods of assessment and/or criteria for determining when to stop treatment-or due to re-introduction of parasites <i>via</i> human or vector movement from another endemic area. We review recent genetics-based research exploring these questions in <i>Onchocerca volvulus</i>, the filarial nematode that causes onchocerciasis, and <i>Wuchereria bancrofti</i>, the major pathogen for lymphatic filariasis. We focus in particular on the combination of genomic epidemiology and genome-wide associations to delineate transmission zones and distinguish between local and introduced parasites as the source of resurgence or continuing transmission, and to identify genetic markers associated with parasite response to chemotherapy. Our ultimate goal is to assist elimination efforts by developing easy-to-use tools that incorporate genetic information about transmission and drug response for more effective mass drug distribution, surveillance strategies, and decisions on when to stop interventions to improve sustainability of elimination.