Use of Caenorhabditis elegans to study the potential bioactivity of

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Perspective

Use of Caenorhabditis elegans to study the potential bioactivity of natural compounds Vivian Hsiu-Chuan Liao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05700 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 6, 2018

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Journal of Agricultural and Food Chemistry

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Manuscript to: Journal of Agricultural and Food Chemistry

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Perspectives / Viewpoints 2017

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Manuscript ID: jf-2017-05700p-R2

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Use of Caenorhabditis elegans to study the potential bioactivity of natural compounds

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Vivian Hsiu-Chuan Liao*

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Department of Bioenvironmental Systems Engineering, National Taiwan University, No. 1 Roosevelt Road, Sec. 4, Taipei 106, Taiwan

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* Correspondence: Vivian Hsiu-Chuan Liao, Tel: +886-2-33665239; Fax: +886-2-33663462; E-mail:

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[email protected]

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ABSTRACT

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There is growing need and interest in finding specific compounds in natural products that have

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health benefits. Despite ongoing efforts to discover such compounds, the scientific evidence lags behind

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the vision, and it is important to find an effective paradigm for discovering such compounds. The model

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organism Caenorhabditis elegans offers a promising solution for studying the potential bioactivity and

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molecular mechanisms of natural compounds in vivo. This article discusses its use to study potential

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human health benefits with focus on anti-oxidative, anti-aging, anti-metabolic disorders (diabetes and

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obesity), and anti-neurodegenerative activities (Alzheimer’s disease and Parkinson’s disease) with

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practical examples. Finally, future directions in using C. elegans-based model for discovering bioactive

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compounds for health promotion are discussed.

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KEYWORDS: Caenorhabditis elegans; natural compounds; bioactivity; health benefits; in vivo

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1. Introduction

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Natural chemical substances produced by living organisms or found in nature (plants, animals,

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microflora, minerals) encompass an extremely wide and diverse range of chemical compounds. Due to

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the huge diversity in chemical structures, natural products have been rich sources and inspiration for a

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substantial fraction of human therapeutics and have played a significant role in drug discovery. For

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example, some widely used drugs are derived from natural products, such as metformin, vincristine,

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acetyldigoxin, and atropine. Hence, the search for bioactive compounds from natural sources to improve

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health and prevent diseases continues to play an important role in new medicinal therapies.

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Whereas pharmaceutical drugs are designed to cure or treat a specific disease, natural bioactive

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compounds that are used to promote health are found in agricultural products and food.1 There is

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increasing evidence that such bioactive natural compounds may help to promote health or reduce the risk

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of chronic lifestyle diseases.1 For example, several bioactive plant-derived compounds have been

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intensively investigated for their potential human health benefits, such as tea phenolics, ascorbic acid,

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epigallocatechin gallate (EGCG), and curcumin. Their potential for anti-oxidative stress, anticancer, and

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anti-inflammatory activities have been explored. The desire to improve health and prevent diseases

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continues to drive the search for efficacious bioactive agricultural and food compounds.

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Efforts to discover such compounds have been deeply engaged in investigating the detailed

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chemical and biological properties, yet the scientific evidence lags behind the vision to exploit the

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potential health benefits.1 Challenges lie in the detailed chemical characterization of the compounds’

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molecular structures, unraveling the bioavailability and bioefficacy of bioactive molecules, and 3 ACS Paragon Plus Environment


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understanding how they promote health.1 Therefore, it is important to find an effective and reliable in

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vivo paradigm for discovering such compounds.

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The nematode Caenorhabditis elegans offers a promising solution for studying the potential

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bioactivity and molecular mechanisms of natural compounds in vivo. This perspective article discusses

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the use of C. elegans as a model organism in this capacity. The focus is on using C. elegans to study

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potential human health benefits related to anti-oxidative activity, anti-aging activity, anti-metabolic

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disorders (diabetes and obesity), and anti-neurodegenerative disorders (Alzheimer’s and Parkinson’s

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diseases), with practical examples.

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2. The nematode C. elegans as a model organism

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C. elegans is a small, transparent nematode that lives in soil. It is a genetically tractable multicellular

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organism that has been a popular model for biological and basic medical research for several decades. It

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has been successfully used as a model system to address fundamental questions in many aspects of

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biology, such as development, cell fate specification, neurobiology, tumorigenesis, RNA-mediated

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interference (RNAi) of gene expression, and aging.

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C. elegans can be either self-fertilizing hermaphrodites or males, but males account for only about

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0.1% of the population. An adult hermaphrodite consists of 959 somatic cells with a complete cell lineage

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map, all of which are visible with a microscope throughout the life of the organism. C. elegans has a short

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life cycle of ~3 days to develop into fertile adults (Figure 1), a lifespan of ~3 weeks, and an ability to

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produce ~300 genetically identical progeny. It has a nervous system containing 302 neurons with a

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complete connectome. In addition, C. elegans has many different organs and tissues, including muscle, a 4 ACS Paragon Plus Environment

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hypoderm, an intestine, a reproductive system, a secretory-excretory system, and glands.

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In the laboratory, C. elegans is usually grown on small Petri agar plates or in liquid media with

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auxotrophic Escherichia coli OP50 as a food source. This makes it very easy and cost-effective to grow.

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Other advantages of C. elegans include mutants’ ability to be frozen indefinitely and revived easily, easy

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delivery of RNAi, the ability to readily create transgenic strains, free online resources such as WormBook

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(http://www.wormbook.org/), and databases such as WormBase (http://www.wormbase.org/).

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An important feature for the usefulness of C. elegans as a model organism in vivo is its relevance to

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human disease. It is estimated that over 83% of the C. elegans proteome has human homologues, as well

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as counterparts for an estimated ~65% of human disease genes.2 Therefore, C. elegans has been used

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extensively as a key model for investigating molecular and cellular aspects of a growing number of

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complex human diseases, such as Alzheimer’s disease, Parkinson’s disease, diabetes, and cancer.3

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To translate the experimental results to humans, research on mammals has some advantages, but

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there are limitations in mammalian animals such as ethical constraints, methodological difficulties, long

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life cycle, small brood size, large genome size, large number of neurons in adult, and difficulties in

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genetic screens. Therefore, in both biological and biomedical studies, C. elegans provides several

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advantages over vertebrate models such as mice (Table 1). Table 1 compares model organisms that are

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commonly used in biomedical research.

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3. Use of C. elegans to study antioxidative activity

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Oxidative stress is characterized as an imbalance between the production of intracellular reactive

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oxygen/nitrogen species (ROS/RNS) and antioxidant defense activity in an organism, as well as a 5 ACS Paragon Plus Environment


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disturbance in the cell redox balance. ROS/RNS include superoxide anion radicals, singlet oxygen,

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hydrogen peroxide, hydroxyl, alkoxyl and lipid peroxyl radicals, nitric oxide, and peroxynitrite. Excessive

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free radicals are associated with damage to many biomolecules, including lipids, proteins, and nucleic

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acids. Free-radical-induced damage in oxidative stress has been linked to a number of chronic health

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problems, such as cancer, diabetes, neurodegenerative diseases, cardiovascular diseases, and

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inflammatory diseases.4 Increasing evidence suggests that the consumption of antioxidant-rich foods or

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medicinal plants can retard or help to avoid the incidence of some diseases.5 Therefore, there is growing

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effort and great interest in the search for effective, nontoxic natural compounds with antioxidative activity

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with associated health benefits.

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The antioxidant properties of natural compounds are investigated through either chemical-based or

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cell-based in vitro or in vivo methods.6 There are various in vitro antioxidant activity assays, and each one

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has a specific target within the matrix with advantages and disadvantages.6 Although in vitro chemical

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methods are fairly straight forward, they lack information about the bioavailability of the test compounds.

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For most in vivo models, the tested samples are usually administered to test animals such as mice or rats,

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which is usually followed by the sacrifice of the animals and the use of blood or tissues for antioxidative

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activity assay.6 In C. elegans, the signal transduction pathways for oxidative stress are highly conserved,

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including the insulin signaling pathway, TOR signaling pathway, and autophagy pathway, as are the

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mechanisms that involve the detoxification of ROS, such as superoxide dismutase and catalase.7 C.

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elegans is thus an attractive in vivo model where the whole organism can be used to evaluate the

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antioxidative activity of natural compounds. 6 ACS Paragon Plus Environment

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In recent years, an increasing number of studies have used C. elegans to explore the antioxidative

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activity of natural compounds, many of which have previously been shown antioxidative activity in other

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in vitro or in vivo models. Examples include curcumin, monascin, selenium, epigallocatechin gallate

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(EGCG) and alpha-lipoic acid, quercetin, etc. This demonstrates the usefulness of C. elegans for studying

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the antioxidative activity of natural compounds. The antioxidative activity of natural compound in C.

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elegans can be evaluated by assays such as performing oxidative stress resistance assay, measuring

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intracellular ROS level, analyzing the responses of transgenic strains expressing antioxidant genes such as

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superoxide dismutase (SOD-3) and glutathione S-transferase (GST-4). Recently, Possik and Pause8

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developed a protocol to measure oxidative stress resistance of C. elegans in liquid in a 96-well microtiter

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plate which might facilitate the investigation of potential antioxdative activity of natural compounds

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while a large number of samples screening is needed.

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4. Use of C. elegans to study anti-aging activity

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Aging is an inevitable process characterized by accumulating functional declines of physiological

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integrity that lead to impaired function and ultimately result in death. Aging has been linked to several

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chronic human diseases, including various cancers, type 2 diabetes (T2DM), and cardiovascular and

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neurodegenerative diseases. Therefore, there is great interest and urgency in studying how to delay the

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process of aging and eliminate or prevent age-related diseases.

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Many mutations have been identified to prolong lifespan in model organisms ranging from yeast to

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mammals.9 The rate of aging is regulated at least in part by genetic pathways and biochemical processes

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that are evolutionarily conserved.9 For example, the signaling pathways of aging including 7 ACS Paragon Plus Environment


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insulin/insulin-like growth factor (IGF) (IIS) pathway, germline signaling pathway, and target of

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rapamycin (TOR) pathway are evolutionarily conserved in metazoan model organisms, such as C. elegans,

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Drosophila, and mice. Thus, compounds with anti-aging activity may be useful in treating or delaying

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age-related human diseases. Natural compounds have a special advantage as resource with highly diverse

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structural scaffolds that might offer promising candidate chemical constituents for aging research. Many

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natural compounds (either pure forms or extracts) have been reported to have anti-aging activity, such as

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slowing cellular senescence or aging and extending lifespan.10 Some natural compounds such as curcumin,

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resveratrol, and α-lipoic acid have received great interest for their various anti-aging activities in different

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models, including C. elegans.10

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The study of C. elegans has provided a wealth of information for understanding the role of genetics

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in modulating aging. In addition to the advantages mentioned, there are other several unique features that

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make C. elegans an ideal model organism for aging research. For example, the organism has a relatively

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short lifespan (~3 weeks), which is largely invariant. This allows for identifying mutants with shorter or

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longer average lifespans. Second, the somatic cells are postmitotic in adult animals, making them useful

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for studying chronological aging. Furthermore, several important signaling pathways involved in aging

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and longevity have been studied extensively, such as insulin/IGF-1 and dietary restriction (DR), which

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allows for the analysis of molecular mechanisms involved in aging.

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Various assays have been developed to study aging in C. elegans. These include lifespan analyses in

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solid and liquid media and assays for measuring age-related changes.11 C. elegans shows certain

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phenotypes that are correlated with aging, such as muscle decline, which is usually analyzed with 8 ACS Paragon Plus Environment

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locomotory behaviors and pharyngeal pumping assays; various types of stress, which are analyzed using

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oxidative stress, UV stress, and heat stress assays; proteostasis, which can be analyzed with a paralysis

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assay; and lipofuscin accumulation, which is measured with lipofuscin autofluorescence in the intestine.11

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Therefore, to evaluate the potential anti-aging activity of natural compounds in C. elegans, it is important

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to measure both the lifespan and age-related changes, which might suggest potential mechanisms for the

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influence on longevity. However, it is noted that compounds with antioxidant activity are not necessary to

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extend the lifespan of C. elegans, for which organic selenium Glu-SeMet have been previously reported.12

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Interestingly, Glu-SeMet shows an ability to improve aging indicators that is mediated by the

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selenoprotein TRXR-1,12 suggesting the potential of natural compounds to improve “healthy aging.”

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5. Use of C. elegans to study anti-metabolic disorder activity: diabetes and obesity

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In recent decades, there has been increasing prevalence of metabolic disorders such as obesity and

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T2DM, which affect millions of people worldwide. In fact, there is increasing evidence to support the

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relationships between T2DM, obesity, Alzheimer’s disease, and cancer. Diabetes mellitus is characterized

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by poor control of glucose homeostasis, including insufficient or inefficient insulin secretary response and

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hyperglycemia. Diabetes is commonly divided into type 1 diabetes mellitus (T1DM), which is caused by

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insufficient insulin secretion, and T2DM, which is a consequence of insulin resistance and hyperglycemia.

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Clinically, diabetic patients with T2DM are more common (90–95%).13 The pathogenic mechanisms of

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diabetes are complicated and involve several distinct signaling pathways, including the insulin signaling

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pathway, carbohydrate metabolism pathway, endoplasmic reticulum (ER) stress pathways, and

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inflammation related pathways. Recently, an increasing number of active components from natural 9 ACS Paragon Plus Environment


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products have been reported to exhibit anti-diabetic activity and regulate pathophysiological signaling

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pathways involved in diabetes. Examples include monascin, quercetin, and resveratrol. The activity has

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been reported in various model organisms, and some of these products have gone through clinical trials.14

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The insulin/IGF-1 signaling (IIS) pathway and the effect of lower levels of its activity in increasing

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lifespan are conserved across diverse metazoa.15 In C. elegans, the pathway regulates fat storage,

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reproduction, and lifespan. DAF-2 is the single ortholog of the human insulin and IGF-1 receptor.15

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Growing evidence suggests that impaired insulin signaling plays a crucial role in the pathogenesis of

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obesity and T2DM.16 C. elegans thus provides a promising model to examine the molecular mechanisms

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of glucose toxicity that lead to diabetic complications.

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Enhanced blood glucose levels are generally observed in diabetes and are recognized as the major

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cause of diabetic complications. Several natural compounds or extracts are reported to prevent high

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glucose-induced toxicity in C. elegans, such as quercetin.17 Quercetin is also reported to have a protective

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effect on hyperglycemia in diabetic mice.18 This suggests the usefulness of C. elegans for investigating

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the potential anti-diabetic activity of natural compounds.

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Another prevalent metabolic disorder is obesity, which is a significant risk for various chronic

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diseases, such as T2DM, heart disease, hyperlipidemia, and certain cancers.19 The causes of obesity are

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complicated and include genetic susceptibility, excessive caloric intake, and sedentary life style.

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Currently, there are only a few FDA-approved medications for obesity, and most have undesired side

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effects.13 Natural compounds might be good candidates for anti-obesity treatments due to their fewer side

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effects compared to synthetic drugs.13 Several natural compounds or extracts have been reported to have 10 ACS Paragon Plus Environment

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anti-obesity activity, and some of them have gone through clinical trials. Examples include Yerba mate,

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Euiiyin-tang, red wine polyphenol supplement, quercetin, resveratrol.14

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Factors controlling energy metabolism and fat regulatory pathways are evolutionarily conserved

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between mammals and C. elegans, which has thus emerged in the last decade as a genetically and

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metabolically tractable model to decipher the homeostatic mechanisms of lipid regulation that lead to

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obesity. Several methods have been employed to examine lipid storage in C. elegans. Fixed staining

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methods use colorimetric dyes or fluorescent dyes followed by quantification of the amount of bound dye

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to reflect fat content. Biochemical methods use lipid extracts in C. elegans and thin layer chromatography

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(TLC) or gas chromatography/mass spectrometry (GC/MS).19 Recently, several natural compounds or

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extracts have been reported to reduce fat accumulation in C. elegans, such as proanthocyanidin trimer

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gallate.20 This suggests that C. elegans is useful for studying the potential anti-obesity activity of natural

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compounds.

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Besides age and genetic predisposition, obesity has been suggested as a significant risk factor for

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developing insulin resistance, which is a key feature of T2DM. Therefore, compounds that

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simultaneously address obesity and diabetes are highly desirable and anticipated. Such candidates include

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red wine polyphenol supplements, quercetin, resveratrol, and cinnamon, and some of them have gone

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through clinical trials.13,14

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6. Use of C. elegans to study anti-neurodegenerative disorder activity: Alzheimer’s disease and Parkinson’s disease

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Neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease seriously affect

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millions of people worldwide. These age-associated disorders lead to a progressive loss of neurons and

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neuronal dysfunction. The pathophysiology involves a combination of genetic and environmental factors.

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So far, the medications to completely cure these diseases are unavailable or ineffective.21 Effective

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compounds and a practical experimental model are needed to decipher the molecular determinants of

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these disorders. There is molecular conservation in neuronal signaling pathways such as dopamine (DA)

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signaling between invertebrates and vertebrates,21 as well as a diverse range of chemical entities of natural

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compounds. Thus, the use of C. elegans to study the beneficial effects of natural compounds on

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neurodegenerative disorders might provide a promising paradigm.

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Alzheimer’s disease is the most common neurodegenerative disorder and is characterized by the

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loss of memory and cognitive impairments. The histopathological hallmarks of Alzheimer’s patients

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include deposition of β-amyloid (Aβ) plaques and neurofibrillary tangles of tau microtubule protein.22 Aβ

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peptides derive from the sequential proteolytic cleavage of amyloid precursor protein (APP).22 The

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oligomers Aβ 1-42 are toxic species are thus a biomarker for Alzheimer’s disease progression.22 In addition,

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many factors are associated with Alzheimer’s disease, such as oxidative stress, inflammation, metabolic

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disturbances, and reduction of cholinergic neuron activity.23 Several natural compounds have been

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reported to have protective effects against Aβ toxicity in various experimental models. Examples are

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quercetin, EGCG, curcumin, resveratrol, and some of them are in clinical trials.23

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Although it is unlikely that C. elegans can completely capture the pathology of Alzheimer’s disease,

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it has several models that can be used to assess Aβ and tau induced toxicity, which have two crucial

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hallmarks.21,22 Transgenic C. elegans strains expressing human Aβ or human tau are used to assess the 12 ACS Paragon Plus Environment

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toxicity.21,22 These models have led to the discovery of a number of candidate compounds for modulating

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the disease. Natural compounds or extracts such as curcumin and resveratrol have been reported to reduce

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Aβ or tau toxicity. These compounds have been shown to have protective effects against Aβ toxicity in

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mammalian models, and in particular, compounds such curcumin and resveratrol have gone through

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clinical trials.23 Thus, C. elegans is useful for studying the potential bioactivity of compounds against

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Alzheimer’s disease.

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Parkinson’s disease is the second most common neurodegenerative disorder after Alzheimer’s

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disease, and it is mainly characterized by motor impairment, the progressive loss of dopaminergic

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neurons, and the accumulation of Lewy bodies in the brain.21 The cause and pathogenic mechanisms of

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Parkinson’s disease are not well understood, and so far, there is no effective treatment. Several factors

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have been linked to its pathogenesis, such as oxidative stress, neuroinflammation, impaired function in

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the ubiquitin-proteasome system, and mitochondrial impairment.24 The presence of Lewy bodies in

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neurons is an important neurohistological characteristic of the disease and is considered as a preclinical or

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presymptomatic marker.25 Self-assembling α-synuclein (α-syn) is the most abundant protein in Lewy

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bodies and is closely associated with Parkinson’s disease.21 A growing number of studies have indicated

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that several natural compounds protect against the neurotoxins 6-hydroxydopamine (6-OHDA) or

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1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in animal models. Examples are green tea

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polyphenols, EGCG, curcumin, and resveratrol.26

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Several unique features make C. elegans a valuable model for investigating Parkinson’s disease.

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With only 302 neurons, including 8 dopaminergic neurons, C. elegans is quite simple compared with 13 ACS Paragon Plus Environment


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billions of neurons in the brains of mammals, or even fruit flies (Drosophila), which have ~10,000

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neurons (Table 1). The pathways involved in dopamine neurons are evolutionally conserved. Due to the

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transparency of C. elegans, neuronal cell death can be readily observed within living organisms. Several

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transgenic strains have been generated to examine α-syn aggregation and dopaminergic neuron

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degeneration, which are two pathological hallmarks of the disease.27 These transgenic strains include a

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strain expressing human α-syn and a strain expressing green fluorescent protein (GFP) specifically in the

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dopaminergic neurons.27 Recently, a few studies have used C. elegans models of Parkinson’s disease to

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examine the potential activity of natural compounds against Parkinson’s disease, such as β-amyrin.28

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7. Concluding remarks and future directions

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This perspective article has highlighted the advantages of using C. elegans to study the potential

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bioactivity of natural compounds. The article has also described how researchers have used this versatile

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model organism to investigate several aspects of human health benefits, as well as how these natural

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compounds have contributed to our understanding in promoting health. Mammalian models remain

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invaluable experimental tools for the discovery of new compounds, especially considering the wide range

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of clinical features and many analogues to the organs and circulatory system in humans. However,

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mammalian models are usually time-consuming, expensive, and complex, thereby hindering the

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efficiency of discovering compounds, especially for screening a large numbers of candidates. Cell-based

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in vitro assay is another common research tool that is used to observe bioactivity in cell-based in vitro

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assays, but the results might not translate to in vivo health effects.1 To address the limitations of

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mammalian models and cell cultures, C. elegans seems to be a practical, promising, versatile, and 14 ACS Paragon Plus Environment

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relevant model for providing multifaceted aspects to study the potential bioactivity of natural compounds,

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as well as the underlying molecular determinants of the associated health effects.

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In the future, in addition to the human health benefits aforementioned in the article, C. elegans can

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be further explored to study other potential bioactivities of natural compounds, such as circadian rhythms,

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anti-cancer, anti-microbial, polyglutamine-expansion disorders, e.g., Huntington’s disease. Moreover, C.

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elegans can be explored as a model for high throughput in the discovery of natural compounds to promote

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health benefits. Therefore, future large-scale of screening bioactive compounds for candidate leads to

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potential bioactivity is possible. Natural compounds that can simultaneously promote multiple health

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benefits are highly desirable and anticipated. Therefore, future studies using C. elegans-based model to

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simultaneously investigate multiple bioactivities of a specific natural compound are desirable. In the

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future, C. elegans-based model can serve as the first pass screen and an effective paradigm for identifying

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genes and bioactive compounds before the studies in mammalian models or clinical trials that might

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facilitate the development for successful health promotion.

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Conflict of interest statement

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The author declares that no competing interests exist.

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(17) Fitzenberger, E.; Deusing, D. J.; Marx, C.; Boll, M.; Lüersen, K.; Wenzel, U., The polyphenol

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357 358

Table 1. Comparison of commonly used model organisms in biomedical research. Organism

C. elegans

Drosophila

Mouse

Life cycle

3 - 4 days

11 - 12 days

50 - 60 days

Adult size

1 - 1.3 mm

3 - 4 mm

6 - 10 cm

Brood size

~140 eggs per day

~120 eggs per day

6 - 12 pups per month

97 Mb

180 Mb

3,000 Mb

Fully annotated genome

○

○

No ethical constraints

○

○

routine

routine

difficult

plates, liquid

vials

cages

weeks

weeks

months

65%

77%

> 90%

302

> 100,000

> 70,000,000

Distinct tissues and cell diversity

○

○

○

Amenable to drug testing

○

○

○

High throughput drug screening

○

Genome size

Genetic screens Growth conditions Transgenic organisms generation Mutants can be frozen and revived easily Gene homology for human

○

diseases Number of neurons in adult

359 360 361 20 ACS Paragon Plus Environment

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Figure Captions

363

Figure 1. C. elegans hermaphrodite life cycle at 20 ºC. The reproductive life cycle of hermaphrodite

364

includes 4 larval stages (L1 through L4), each ending in a molt. The dauer larva is a diapause stage

365

representing an alternative L3 stage, which is entered when unfavorable conditions such as crowding or

366

low food availability occur.

367 368



369 370

Figure 1

371 372

373 374 375 376 377


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Table of contents (TOC)

379 380 381 382 383 384

385 386 387 388 389


Use of Caenorhabditis elegans to study the potential bioactivity of

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