Application of 3Rs in Caenorhabditis elegans Research for the Identification of Health-Promoting Natural Products.

The average age of the population is increasing worldwide, which has a profound impact on our society. This leads to an increasing demand for medicines and requires the development of new strategies to promote health during the additional years. In the search for resources and therapeutics for improved health during an extended life span, attention has to be paid to environmental exposure and ecosystem burdens that inevitably emerge with the extended consumption of medicines and drug development, even in the preclinical stage. The hereby introduced sustainable strategy for drug discovery is built on 3Rs, "R: obustness, R: eliability, and saving R: esources", inspired by both the 3Rs used in animal experiments and environmental protection, and centers on the usefulness and the variety of the small model organism Caenorhabditis elegans for detecting health-promoting natural products. A workflow encompassing a multilevel screening approach is presented to maximize the amount of information on health-promoting samples, while considering the 3Rs. A detailed, methodology- and praxis-oriented compilation and discussion of proposed C. elegans health span assays and more disease-specific assays are presented to offer guidance for scientists intending to work with C. elegans, thus facilitating the initial steps towards the integration of C. elegans assays in their laboratories.

Brominated flame retardants in surface sediment from Western Guangdong, South China: Occurrence, distribution and toxicity in Caenorhabditis elegans.

Sediment is the ultimate sink of environmental pollutants. A total of 128 surface sediment samples were collected from 8 rivers and 3 reservoirs in Maoming City, Guangdong Province. This study assessed the content and distribution of brominated flame retardants in sediments. The acute toxicity effects of tetrabromobisphenol A (TBBPA) and hexabromocyclododecane (HBCDs) in sediments were evaluated using Caenorhabditis elegans as model organisms. The concentration of TBBPA in sediments ranged from not detected (ND) to 12.59 µg/kg and was mainly distributed in the central area, which was affected by the emission of TBBPA from residential and factory. The concentration of HBCDs ranged from ND to 6.31 µg/kg, and the diastereoisomer distribution was consistent, showing a trend close to the South China Sea. The composition pattern of HBCDs in the surface sediments from rivers were 41.73%-62.33%, 7.89%-25.54%, and 18.76%-40.65% for α-, ß-, and γ-HBCD, respectively, and in the sediments from reservoirs were 26.15%-45.52%, 7.44%-19.23%, and 47.04%-61.89% for α-, ß-, and γ-HBCD, respectively. When the sum of concentrations of TBBPA and HBCD in sediments were above high levels, reactive oxygen species in nematodes significantly increased, resulting in an oxidative stress response. Intestinal permeability was also enhanced, causing intestinal damage. In addition, in terms of this study, TBBPA had a greater impact on biotoxicity compared to HBCDs, and more attention should be paid to the toxic effects of the river ecosystem organisms in Maoming City, Guangdong Province. This study can complement the pollution database in the study area and provide basic data for pollution control.

Time-course swRNA-seq uncovers a hierarchical gene regulatory network in controlling the response-repair-remodeling after wounding.

Wounding initiates intricate responses crucial for tissue repair and regeneration. Yet, the gene regulatory networks governing wound healing remain poorly understood. Here, employing single-worm RNA sequencing (swRNA-seq) across 12 time-points, we delineated a three-stage wound repair process in C. elegans: response, repair, and remodeling. Integrating diverse datasets, we constructed a dynamic regulatory network comprising 241 transcription regulators and their inferred targets. We identified potentially seven autoregulatory TFs and five cross-autoregulatory loops involving pqm-1 and jun-1. We revealed that TFs might interact with chromatin factors and form TF-TF combinatory modules via intrinsically disordered regions to enhance response robustness. We experimentally validated six regulators functioning in transcriptional and translocation-dependent manners. Notably, nhr-76, daf-16, nhr-84, and oef-1 are potentially required for efficient repair, while elt-2 may act as an inhibitor. These findings elucidate transcriptional responses and hierarchical regulatory networks during C. elegans wound repair, shedding light on mechanisms underlying tissue repair and regeneration.

Clock gene homologs lin-42 and kin-20 regulate circadian rhythms in C. elegans.

Circadian rhythms are endogenous oscillations in nearly all organisms, from prokaryotes to humans, allowing them to adapt to cyclical environments for close to 24 h. Circadian rhythms are regulated by a central clock, based on a transcription-translation feedback loop. One important protein in the central loop in metazoan clocks is PERIOD, which is regulated in part by Casein kinase 1ε/δ (CK1ε/δ) phosphorylation. In the nematode Caenorhabditis elegans, period and casein kinase 1ε/δ are conserved as lin-42 and kin-20, respectively. Here, we studied the involvement of lin-42 and kin-20 in the circadian rhythms of the adult nematode using a bioluminescence-based circadian transcriptional reporter. We show that mutations of lin-42 and kin-20 generate a significantly longer endogenous period, suggesting a role for both genes in the nematode circadian clock, as in other organisms. These phenotypes can be partially rescued by overexpression of either gene under their native promoter. Both proteins are expressed in neurons and epidermal seam cells, as well as in other cells. Depletion of LIN-42 and KIN-20, specifically in neuronal cells after development, was sufficient to lengthen the period of oscillating sur-5 expression. Therefore, we conclude that LIN-42 and KIN-20 are critical regulators of the adult nematode circadian clock through neuronal cells.

Network pharmacology and experimental verification to explore the effect of Hedyotis diffusa on Alzheimer's disease.

This study aimed to explore the active components and the effect of Hedyotis diffusa (HD) against Alzheimer's disease (AD) via network pharmacology, molecular docking, and experimental evaluations. We conducted a comprehensive screening process using the TCMSP, Swiss Target Prediction, and PharmMapper databases to identify the active components and their related targets in HD. In addition, we collected potential therapeutic targets of AD from the Gene Cards, Drugbank, and OMIM databases. Afterward, we utilized Cytoscape to establish both protein-protein interaction (PPI) networks and compound-target (C-T) networks. To gain further insights into the functional aspect, we performed GO and KEGG pathway analyses using the David database. Next, we employed Autodock vina to estimate the binding force between the components and the hub genes. To validate our network pharmacology findings, we conducted relevant experiments on Caenorhabditis elegans, further confirming the reliability of our results. Then a total of six active compounds and 149 therapeutic targets were detected. Through the analysis of the association between active compounds, therapeutic targets, and signaling pathways, it was observed that the therapeutic effect of HD primarily encompassed the inhibition of Aß, suppression of AChE activity, and mitigating oxidative stress. Additionally, our investigation revealed that the key active compounds in HD primarily consisted of iridoids, which exhibited resistance against AD by acting on the Alzheimer's disease pathway and the AGE-RAGE signaling pathway in diabetic complications.

Use of Viscous medium to study anthelmintic drug action in Caenorhabditis elegans.

Caenorhabditis elegans is an appealing tool for experimental evolution and for working with antiparasitic drugs, from understanding the molecular mechanisms of drug action and resistance to uncover new drug targets. We present a new methodology for studying the impact of antiparasitic drugs in C. elegans. Viscous medium was initially designed for C. elegans maintenance during long-term evolution experiments. Viscous medium provides a less structured environment than the standard nematode growth media agar, yet the bacteria food source remains suspended. Further, the Viscous medium offers the worm population enough support to move freely, mate, and reproduce at a rate comparable to standard agar cultures. Here, the Viscous medium was adapted for use in antiparasitic research. We observed a similar sensitivity of C. elegans to anthelmintic drugs as in standard liquid media and statistical difference to the standard agar media through a larval development assay. Using Viscous medium in C. elegans studies will considerably improve antiparasitic resistance research, and this medium could be used in studies aimed at understanding long-term multigenerational drug activity.

Zebrafish and nematodes as whole organism models to measure developmental neurotoxicity.

Despite the growing epidemiological evidence of an association between toxin exposure and developmental neurotoxicity (DNT), systematic testing of DNT is not mandatory in international regulations for admission of pharmaceuticals or industrial chemicals. However, to date around 200 compounds, ranging from pesticides, pharmaceuticals and industrial chemicals, have been tested for DNT in the current OECD test guidelines (TG-443 or TG-426). There are calls for the development of new approach methodologies (NAMs) for DNT, which has resulted in a DNT testing battery using in vitro human cell-based assays. These assays provide a means to elucidate the molecular mechanisms of toxicity in humans which is lacking in animal-based toxicity tests. However, cell-based assays do not represent all steps of the complex process leading to DNT. Validated models with a multi-organ network of pathways that interact at the molecular, cellular and tissue level at very specific timepoints in a life cycle are currently missing. Consequently, whole model organisms are being developed to screen for, and causally link, new molecular targets of DNT compounds and how they affect whole brain development and neurobehavioral endpoints. Given the practical and ethical restraints associated with vertebrate testing, lower animal models that qualify as 3 R (reduce, refine and replace) models, including the nematode (Caenorhabditis elegans) and the zebrafish (Danio rerio) will prove particularly valuable for unravelling toxicity pathways leading to DNT. Although not as complex as the human brain, these 3 R-models develop a complete functioning brain with numerous neurodevelopmental processes overlapping with human brain development. Importantly, the main signalling pathways relating to (neuro)development, metabolism and growth are highly conserved in these models. We propose the use of whole model organisms specifically zebrafish and C. elegans for DNT relevant endpoints.

Identification of a family of species-selective complex I inhibitors as potential anthelmintics.

Soil-transmitted helminths (STHs) are major pathogens infecting over a billion people. There are few classes of anthelmintics and there is an urgent need for new drugs. Many STHs use an unusual form of anaerobic metabolism to survive the hypoxic conditions of the host gut. This requires rhodoquinone (RQ), a quinone electron carrier. RQ is not made or used by vertebrate hosts making it an excellent therapeutic target. Here we screen 480 structural families of natural products to find compounds that kill Caenorhabditis elegans specifically when they require RQ-dependent metabolism. We identify several classes of compounds including a family of species-selective inhibitors of mitochondrial respiratory complex I. These identified complex I inhibitors have a benzimidazole core and we determine key structural requirements for activity by screening 1,280 related compounds. Finally, we show several of these compounds kill adult STHs. We suggest these species-selective complex I inhibitors are potential anthelmintics.

Barley Protein LFBEP-C1 from Lactiplantibacillus plantarum dy-1 Fermented Barley Extracts by Inhibiting Lipid Accumulation in a Caenorhabditis elegans Model.

Objective: This study aimed to investigate the lipid-lowering activity of LFBEP-C1 in high glucose-fed Caenorhabditis elegans (C. elegans). Methods: In this study, the fermented barley protein LFBEP-C1 was prepared and tested for its potential anti-obesity effects on C. elegans. The worms were fed Escherichia coli OP50 ( E. coli OP50), glucose, and different concentrations of LFBEP-C1. Body size, lifespan, movement, triglyceride content, and gene expression were analyzed. The results were analyzed using ANOVA and Tukey's multiple comparison test. Results: Compared with the model group, the head-swing frequency of C. elegans in the group of LFBEP-C1 at 20 µg/mL increased by 33.88%, and the body-bending frequency increased by 27.09%. This indicated that LFBEP-C1 improved the locomotive ability of C. elegans. The average lifespan of C. elegans reached 13.55 days, and the body length and width of the C. elegans decreased after LFBEP-C1 intake. Additionally, LFBEP-C1 reduced the content of lipid accumulation and triglyceride levels. The expression levels of sbp-1, daf-2, and mdt-15 significantly decreased, while those of daf-16, tph-1, mod-1, and ser-4 significantly increased after LFBEP-C1 intake. Changes in these genes explain the signaling pathways that regulate lipid metabolism. Conclusion: LFBEP-C1 significantly reduced lipid deposition in C. elegans fed a high-glucose diet and alleviated the adverse effects of a high-glucose diet on the development, lifespan, and exercise behavior of C. elegans. In addition, LFBEP-C1 regulated lipid metabolism mainly by mediating the expression of genes in the sterol regulatory element-binding protein, insulin, and 5-hydroxytryptamine signaling pathways.

Isolation and Identification of Nematode-Infecting Microsporidia.

Nematodes are naturally infected by the fungal-related pathogen microsporidia. These ubiquitous eukaryotic parasites are poorly understood, despite infecting most types of animals. Identifying novel species of microsporidia and studying them in an animal model can expedite our understanding of their infection biology and evolution. Nematodes present an excellent avenue for pursuing such work, as they are abundant in the environment and many species are easily culturable in the laboratory. The protocols presented here describe how to isolate bacterivorous nematodes from rotting substrates, screen them for microsporidia infection, and molecularly identify the nematode and microsporidia species. Additionally, we detail how to remove environmental contaminants and generate a spore preparation of microsporidia from infected samples. We also discuss potential pitfalls and provide suggestions on how to mitigate them. These protocols allow for the identification of novel microsporidia species, which can serve as an excellent starting point for genomic analysis, determination of host specificity, and infection characterization. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Gathering samples Support Protocol 1: Generating 10× and 40× Escherichia coli OP50 and seeding NGM plates Basic Protocol 2: Microsporidia screening, testing for Caenorhabditis elegans susceptibility, and sample freezing Basic Protocol 3: DNA extraction, PCR amplification, and sequencing to identify nematode and microsporidia species Basic Protocol 4: Removal of contaminating microbes and preparation of microsporidia spores Support Protocol 2: Bleach-synchronizing nematodes.

Biclustering analysis on tree-shaped time-series single cell gene expression data of Caenorhabditis elegans.

BACKGROUND: In recent years, gene clustering analysis has become a widely used tool for studying gene functions, efficiently categorizing genes with similar expression patterns to aid in identifying gene functions. Caenorhabditis elegans is commonly used in embryonic research due to its consistent cell lineage from fertilized egg to adulthood. Biologists use 4D confocal imaging to observe gene expression dynamics at the single-cell level. However, on one hand, the observed tree-shaped time-series datasets have characteristics such as non-pairwise data points between different individuals. On the other hand, the influence of cell type heterogeneity should also be considered during clustering, aiming to obtain more biologically significant clustering results. RESULTS: A biclustering model is proposed for tree-shaped single-cell gene expression data of Caenorhabditis elegans. Detailedly, a tree-shaped piecewise polynomial function is first employed to fit non-pairwise gene expression time series data. Then, four factors are considered in the objective function, including Pearson correlation coefficients capturing gene correlations, p-values from the Kolmogorov-Smirnov test measuring the similarity between cells, as well as gene expression size and bicluster overlapping size. After that, Genetic Algorithm is utilized to optimize the function. CONCLUSION: The results on the small-scale dataset analysis validate the feasibility and effectiveness of our model and are superior to existing classical biclustering models. Besides, gene enrichment analysis is employed to assess the results on the complete real dataset analysis, confirming that the discovered biclustering results hold significant biological relevance.

Nutritional, molecular, and functional properties of a novel enzymatically hydrolyzed porcine plasma product.

In the present study, an enzymatically hydrolyzed porcine plasma (EHPP) was nutritionally and molecularly characterized. EHPP molecular characterization showed, in contrast to spray-dried plasma (SDP), many peptides with relative molecular masses (Mr) below 8,000, constituting 73% of the protein relative abundance. IIAPPER, a well-known bioactive peptide with anti-inflammatory and antioxidant properties, was identified. In vivo functionality of EHPP was tested in C. elegans and two different mouse models of intestinal inflammation. In C. elegans subjected to lipopolysaccharide exposure, EHPP displayed a substantial anti-inflammatory effect, enhancing survival and motility by 40% and 21.5%, respectively. Similarly, in mice challenged with Staphylococcus aureus enterotoxin B or Escherichia coli O42, EHPP and SDP supplementation (8%) increased body weight and average daily gain while reducing the percentage of regulatory Th lymphocytes. Furthermore, both products mitigated the increase of pro-inflammatory cytokines expression associated with these challenged mouse models. In contrast, some significant differences were observed in markers such as Il-6 and Tnf-α, suggesting that the products may present different action mechanisms. In conclusion, EHPP demonstrated similar beneficial health effects to SDP, potentially attributable to the immunomodulatory and antioxidant activity of its characteristic low Mr bioactive peptides.

Lactobacillus paracasei subsp. paracasei 2004 improves health and lifespan in Caenorhabditis elegans.

Recent research has highlighted the importance of the gut microbiome in regulating aging, and probiotics are interventions that can promote gut health. In this study, we surveyed several novel lactic acid bacteria to examine their beneficial effect on organismal health and lifespan in C. elegans. We found that animals fed some lactic acid bacteria, including L. acidophilus 1244 and L. paracasei subsp. paracasei 2004, grew healthy. Supplementation with the lactic acid bacterial strains L. acidophilus 1244 or L. paracasei subsp. paracasei 2004 significantly improved health, including food consumption, motility, and resistance to oxidative stressor, hydrogen peroxide. Our RNA-seq analysis showed that supplementation with L. paracasei subsp. paracasei 2004 significantly increased the expression of daf-16, a C. elegans FoxO homolog, as well as genes related to the stress response. Furthermore, daf-16 deletion inhibited the longevity effect of L. paracasei subsp. paracasei 2004 supplementation. Our results suggest that L. paracasei subsp. paracasei 2004 improves health and lifespan in a DAF-16-dependent manner.

Functional implications of NHR-210 enrichment in C. elegans cephalic sheath glia: insights into metabolic and mitochondrial disruptions in Parkinson's disease models.

Glial cells constitute nearly half of the mammalian nervous system's cellular composition. The glia in C. elegans perform majority of tasks comparable to those conducted by their mammalian equivalents. The cephalic sheath (CEPsh) glia, which are known to be the counterparts of mammalian astrocytes, are enriched with two nuclear hormone receptors (NHRs)-NHR-210 and NHR-231. This unique enrichment makes the CEPsh glia and these NHRs intriguing subjects of study concerning neuronal health. We endeavored to assess the role of these NHRs in neurodegenerative diseases and related functional processes, using transgenic C. elegans expressing human alpha-synuclein. We employed RNAi-mediated silencing, followed by behavioural, functional, and metabolic profiling in relation to suppression of NHR-210 and 231. Our findings revealed that depleting nhr-210 changes dopamine-associated behaviour and mitochondrial function in human alpha synuclein-expressing strains NL5901 and UA44, through a putative target, pgp-9, a transmembrane transporter. Considering the alteration in mitochondrial function and the involvement of a transmembrane transporter, we performed metabolomics study via HR-MAS NMR spectroscopy. Remarkably, substantial modifications in ATP, betaine, lactate, and glycine levels were seen upon the absence of nhr-210. We also detected considerable changes in metabolic pathways such as phenylalanine, tyrosine, and tryptophan biosynthesis metabolism; glycine, serine, and threonine metabolism; as well as glyoxalate and dicarboxylate metabolism. In conclusion, the deficiency of the nuclear hormone receptor nhr-210 in alpha-synuclein expressing strain of C. elegans, results in altered mitochondrial function, coupled with alterations in vital metabolite levels. These findings underline the functional and physiological importance of nhr-210 enrichment in CEPsh glia.

Actin-nucleation promoting factor N-WASP influences alpha-synuclein condensates and pathology.

Abnormal intraneuronal accumulation of soluble and insoluble α-synuclein (α-Syn) is one of the main pathological hallmarks of synucleinopathies, such as Parkinson's disease (PD). It has been well documented that the reversible liquid-liquid phase separation of α-Syn can modulate synaptic vesicle condensates at the presynaptic terminals. However, α-Syn can also form liquid-like droplets that may convert into amyloid-enriched hydrogels or fibrillar polymorphs under stressful conditions. To advance our understanding on the mechanisms underlying α-Syn phase transition, we employed a series of unbiased proteomic analyses and found that actin and actin regulators are part of the α-Syn interactome. We focused on Neural Wiskott-Aldrich syndrome protein (N-WASP) because of its association with a rare early-onset familial form of PD. In cultured cells, we demonstrate that N-WASP undergoes phase separation and can be recruited to synapsin 1 liquid-like droplets, whereas it is excluded from α-Syn/synapsin 1 condensates. Consistently, we provide evidence that wsp-1/WASL loss of function alters the number and dynamics of α-Syn inclusions in the nematode Caenorhabditis elegans. Together, our findings indicate that N-WASP expression may create permissive conditions that promote α-Syn condensates and their potentially deleterious conversion into toxic species.

Neuronal basis and diverse mechanisms of pathogen avoidance in Caenorhabditis elegans.

Pathogen avoidance behaviour has been observed across animal taxa as a vital host-microbe interaction mechanism. The nematode Caenorhabditis elegans has evolved multiple diverse mechanisms for pathogen avoidance under natural selection pressure. We summarise the current knowledge of the stimuli that trigger pathogen avoidance, including alterations in aerotaxis, intestinal bloating, and metabolites. We then survey the neural circuits involved in pathogen avoidance, transgenerational epigenetic inheritance of pathogen avoidance, signalling crosstalk between pathogen avoidance and innate immunity, and C. elegans avoidance of non-Pseudomonas bacteria. In this review, we highlight the latest advances in understanding host-microbe interactions and the gut-brain axis.

Gelsenicine disrupted the intestinal barrier of Caenorhabditis elegans.

Gelsemium elegans Benth. (G. elegans) is a traditional medicinal herb that has anti-inflammatory, analgesic, sedative, and detumescence effects. However, it can also cause intestinal side effects such as abdominal pain and diarrhea. The toxicological mechanisms of gelsenicine are still unclear. The objective of this study was to assess enterotoxicity induced by gelsenicine in the nematodes Caenorhabditis elegans (C. elegans). The nematodes were treated with gelsenicine, and subsequently their growth, development, and locomotion behavior were evaluated. The targets of gelsenicine were predicted using PharmMapper. mRNA-seq was performed to verify the predicted targets. Intestinal permeability, ROS generation, and lipofuscin accumulation were measured. Additionally, the fluorescence intensities of GFP-labeled proteins involved in oxidative stress and unfolded protein response in endoplasmic reticulum (UPRER) were quantified. As a result, the treatment of gelsenicine resulted in the inhibition of nematode lifespan, as well as reductions in body length, width, and locomotion behavior. A total of 221 targets were predicted by PharmMapper, and 731 differentially expressed genes were screened out by mRNA-seq. GO and KEGG enrichment analysis revealed involvement in redox process and transmembrane transport. The permeability assay showed leakage of blue dye from the intestinal lumen into the body cavity. Abnormal mRNAs expression of gem-4, hmp-1, fil-2, and pho-1, which regulated intestinal development, absorption and catabolism, transmembrane transport, and apical junctions, was observed. Intestinal lipofuscin and ROS were increased, while sod-2 and isp-1 expressions were decreased. Multiple proteins in SKN-1/DAF-16 pathway were found to bind stably with gelsenicine in a predictive model. There was an up-regulation in the expression of SKN-1:GFP, while the nuclear translocation of DAF-16:GFP exhibited abnormality. The UPRER biomarker HSP-4:GFP was down-regulated. In conclusion, the treatment of gelsenicine resulted in the increase of nematode intestinal permeability. The toxicological mechanisms underlying this effect involved the disruption of intestinal barrier integrity, an imbalance between oxidative and antioxidant processes mediated by the SKN-1/DAF-16 pathway, and abnormal unfolded protein reaction.

Identification of a novel oligopeptide from defatted walnut meal hydrolysate as a potential neuroprotective agent.

Free radical damage and oxidative stress are thought to play a crucial role in the development of neurodegenerative diseases. Walnut peptides, especially walnut oligopeptides, have been shown to protect nerve cells from oxidative stress and inflammatory damage, as well as improve memory function. In this study, walnut peptides were obtained from walnut meal through enzymatic hydrolysis, ultrafiltration, and gel filtration chromatography. A novel oligopeptide called AQ was successfully isolated and its chemical structure was identified as AASCDQ using ESI-MS/MS. AQ demonstrated remarkable scavenging activity against O2- free radicals (81.00%), DPPH free radicals (79.40%), and ABTS free radicals (67.09%) at a concentration of 1 mg mL-1. Furthermore, AQ exhibited strong neuroprotective effects against hydrogen peroxide-induced damage in SH-SY5Y cells, reducing cell injury and apoptosis. AQ also effectively inhibited the secretion of pro-inflammatory factors NO (IC50 = 46.03 ± 0.32 µM) and suppressed the expression of IL-6 and TNF-α in RAW264.7 cells stimulated by LPS. In vivo experiments demonstrated that AQ promoted angiogenesis in the quail chick chorioallantoic membrane assay and reduced ROS accumulation in Caenorhabditis elegans, thereby extending its lifespan. The anti-inflammatory mechanism of AQ was further confirmed by western blotting. In summary, the novel oligopeptide AQ possesses potential neuroprotective effects, including antioxidant, anti-inflammatory, angiogenic, and anti-aging properties, making it a promising candidate for the development of functional foods and pharmaceutical products.