Elegans Worms Research Articles

Investigating the Roles of RNA Binding Protein Combinations in Neuronal Function and Organismal Behavior

The Norris lab recently identified two RNA binding proteins required for proper neuron-specific splicing. The lab conducted touch- response behavioral assays to assess the function of these proteins in touch-sensing neurons. After isolating C. elegans worms with specific phenotypes, the lab used automated computer tracking and video analysis to record the worms’ behavior. The behavior of mutant worms differed from that of wild-type worms. The Norris lab also discovered two possible RNA binding protein sites in SAD-1, a neuronal gene implicated in the neuronal development of C. elegans1. These two binding sites may control the splicing of SAD-1. The lab transferred mutated DNA into the genome of wild-type worms by injecting a mutated plasmid. The newly transformed worms fluoresced green, indicating that the two binding sites control SAD-1 splicing.

Label-free molecular mapping and assessment of glycogen in C. elegans.

Caenorhabditis elegans is an animal model frequently used in research on the effects of metabolism on organismal aging. This comes with a requirement for methods to investigate metabolite content, turnover, and distribution. The aim of our study was to assess the use of a label-free approach to determine both content and distribution of glycogen, the storage form of glucose, in C. elegans. To this end, we grew C. elegans worms under three different dietary conditions for 24-48 h, representing starvation, regular diet and a high glucose diet, followed by analysis of glycogen content. Glycogen analysis was performed on fixed individual whole worms using Raman micro-spectroscopy (RMS). Results were confirmed by comparison with two conventional assays, i.e. iodine staining of worms and enzymatic determination of glycogen. RMS was further used to assess overall lipid and protein content and distribution in the same samples used for glycogen analysis. Expectedly, both glycogen and lipid content were highest in worms grown on a high glucose diet, lower in regularly fed, and lowest in starved nematodes. In summary, RMS is a method suitable for analysis of glycogen content in C. elegans that has the advantage over established methods that (i) individual worms (rather than hundreds per sample) can be analyzed, (ii) glycogen distribution can be assessed at subcellular resolution and (iii) the distribution patterns of other macromolecules can be assessed from the same worms. Thus, RMS has the potential to be used as a sensitive, accurate, cost-effective and high throughput method to evaluate glycogen stores in C. elegans.

A-to-I RNA Editing Affects lncRNAs Expression after Heat Shock.

Adenosine to inosine (A-to-I) RNA editing is a highly conserved regulatory process carried out by adenosine-deaminases (ADARs) on double-stranded RNA (dsRNAs). Although a considerable fraction of the transcriptome is edited, the function of most editing sites is unknown. Previous studies indicate changes in A-to-I RNA editing frequencies following exposure to several stress types. However, the overall effect of stress on the expression of ADAR targets is not fully understood. Here, we performed high-throughput RNA sequencing of wild-type and ADAR mutant Caenorhabditis elegans worms after heat-shock to analyze the effect of heat-shock stress on the expression pattern of genes. We found that ADAR regulation following heat-shock does not directly involve heat-shock related genes. Our analysis also revealed that long non-coding RNAs (lncRNAs) and pseudogenes, which have a tendency for secondary RNA structures, are enriched among upregulated genes following heat-shock in ADAR mutant worms. The same group of genes is downregulated in ADAR mutant worms under permissive conditions, which is likely, considering that A-to-I editing protects endogenous dsRNA from RNA-interference (RNAi). Therefore, temperature increases may destabilize dsRNA structures and protect them from RNAi degradation, despite the lack of ADAR function. These findings shed new light on the dynamics of gene expression under heat-shock in relation to ADAR function.

The Nif3-Family Protein YqfO03 from Pseudomonas syringae MB03 Has Multiple Nematicidal Activities against Caenorhabditis elegans and Meloidogyne incognita.

The nematicidal activity of the common plant-pathogenic bacterium Pseudomonas syringae against certain nematodes has been recently identified, but little is known about its virulence factors. In the current study, predictive analysis of nematode-virulent factors in the genome of a P. syringae wild-type strain MB03 revealed a variety of factors with the potential to be pathogenic against nematodes. One of these virulence factors that was predicted with a high score, namely, YqfO03, was a protein with structural domains that are similar to the Nif3 superfamily. This protein was expressed and purified in Escherichia coli, and was investigated for nematicidal properties against the model nematode Caenorhabditis elegans and an agriculturally important pest Meloidogyne incognita. Our results showed that YqfO03 exhibits lethal activity toward C. elegans and M. incognita worms, and it also caused detrimental effects on the growth, brood size, and motility of C. elegans worms. However, C. elegans worms were able to defend themselves against YqfO03 via a physical defense response by avoiding contact with the protein. Discovery of the diverse nematicidal activities of YqfO03 provides new knowledge on the biological function of a bacterial Nif3-family protein and insight into the potential of this protein as a specific means of controlling agricultural nematode pests.

High-Throughput Analysis of Behavior Under the Control of Optogenetics in Caenorhabditis elegans.

In this unit, we describe an inexpensive and versatile method for optogenetic stimulation of a large population of genetically engineered Caenorhabditis elegans worms while quantitatively analyzing behavior. A custom light-emitting diode light source is used to deliver blue-light stimuli, causing direct depolarization of neurons expressing the light-gated cation channel Channelrhodopsin-2, which in turn evokes behavioral responses. The behavioral responses are recorded by a high-throughput machine vision-based tracking system, the Multi-Worm Tracker, for detailed analysis. This approach allows researchers to bypass technical obstacles to simultaneously deliver uniform stimuli to a large number of freely behaving animals and investigate the neural underpinnings of behavior. © 2018 by John Wiley & Sons, Inc.

ALA-mediated biphasic downregulation of α-7nAchR/HIF-1α along with mitochondrial stress modulation strategy in mammary gland chemoprevention.

The study elucidates the effect of ɑ-linolenic acid (ALA) on mitochondrial stress, hypoxic cancer microenvironment, and intervention of cholinergic anti-inflammatory pathway using N-methyl-N-nitrosourea (MNU) induced estrogen receptor (ER+) mammary gland carcinoma and Caenorhabditis elegans model, respectively. The efficacy of ALA was scrutinized in vivo and in vitro using various experiments like hemodynamic studies, morphological analysis, antioxidants parameters, immunoblotting, and quantitative reverse transcription polymerase chain reaction. The effect of ALA was also validated using C. elegans worms. ALA administration had a positive effect on tissue architecture of the malignancy when scrutinized through the whole mount carmine staining, hematoxylin and eosin staining, and scanning electron microscopy. The proteomic and genomic checkpoint revealed the participation of mitochondrial dysfunction, alteration of hypoxic microenvironment, and involvement of cholinergic anti-inflammatory response after treatment with ALA. ALA treatment has also increased the level of synaptic acetylcholine and acetylcholine esterase with a significant decrease in lipid content. It was concluded that ALA persuaded the mitochondrial stress, activation of downstream cholinergic anti-inflammatory markers, and favorable regulation of hypoxia microenvironment through inhibition of fatty acid synthase and sterol regulatory element-binding protein.

Using microfluidic impedance cytometry to measure C. elegans worms and identify their developmental stages

Microfluidic impedance cytometry allows for label-free detection of cells. But it has not yet been used to detect large organisms, such as Caenorhabditis elegans (C. elegans) worms, due to their variable morphology and strong motility. Here, we report on a C. elegans microfluidic impedance cytometry (CeMIC), which enables the electrical impedance measurement of worms when flowing through a straight microchannel and the identification of their developmental stages based on impedance signals. With the optimized configuration of impedance-sensing structures, namely the microchannel and integrated microelectrodes, the influence of undulation and position variation of worms upon the measured impedance can be eliminated. In signal processing, a kernel density estimation method is employed to extract worm-length-related values that can be directly assigned to the developmental stages of worms. The accuracy for impedance-based identification of worm stages can reach 90%. Additionally, the CeMIC device is developed into a simple and automated system for size-based enrichment of worms. Large and small worms from a mixed population are successfully separated to different outlets after identifying their sizes with impedance measurement. Therefore, our CeMIC system provides a promising platform to measure the worm size, identify developmental stages, and prepare size/stage-homogenized populations for C. elegans experiments.

Inhibition of Sarco-Endoplasmic Reticulum Ca2+ ATPase Extends the Lifespan in C. elegans Worms

The sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) refills the endoplasmic reticulum (ER) with Ca2+ up to the millimolar range and is therefore the main controller of the ER [Ca2+] level ([Ca2+]ER), which has a key role in the modulation of cytosolic Ca2+ signaling and ER-mitochondria Ca2+ transfer. Given that both cytosolic and mitochondrial Ca2+ dynamics strongly interplay with energy metabolism and nutrient-sensitive pathways, both of them involved in the aging process, we have studied the effect of SERCA inhibitors on lifespan in C. elegans. We have used thapsigargin and 2,5-Di-tert-butylhydroquinone (2,5-BHQ) as SERCA inhibitors, and the inactive analog 2,6-Di-tert-butylhydroquinone (2,6-BHQ) as a control for 2,5-BHQ. Every drug was administered to the worms either directly in the agar or via an inclusion compound with γ-cyclodextrin. The results show that 2,6-BHQ produced a small but significant increase in survival, perhaps because of its antioxidant properties. However, 2,5-BHQ produced in all the conditions a much higher increase in lifespan, and the potent and specific SERCA inhibitor thapsigargin also extended the lifespan. The effects of 2,5-BHQ and thapsigargin had a bell-shaped concentration dependence, with a maximum effect at a certain dose and smaller or even toxic effects at higher concentrations. Our data show therefore that submaximal inhibition of SERCA pumps has a pro-longevity effect, suggesting that Ca2+ signaling plays an important role in the aging process and that it could be a promising novel target pathway to act on aging.

Microfluidics-enabled phenotyping of a whole population of C. elegans worms over their embryonic and post-embryonic development at single-organism resolution

The organism Caenorhabditis elegans is a performant model system for studying human biological processes and diseases, but until now all phenome data are produced as population-averaged read-outs. Monitoring of individual responses to drug treatments would however be more informative. Here, a new strategy to track different phenotypic traits of individual C. elegans nematodes throughout their full life-cycle—i.e., embryonic and post-embryonic development, until adulthood onset, differently from life-span—is presented. In an automated fashion, single worms were synchronized, isolated, and cultured from egg to adulthood in a microfluidic device, where their identity was preserved during their whole development. Several phenotypes were monitored and quantified for each animal, resulting in high-content phenome data. Specifically, the method was validated by analyzing the response of C. elegans to doxycycline, an antibiotic fairly well-known to prolong the development and activate mitochondrial stress-response pathways in different species. Interestingly, the obtained extensive single-worm phenome not only confirmed the dramatic doxycycline effect on the worm developmental delay, but more importantly revealed subtle yet severe treatment-dependent phenotypes that are representative of minority subgroups and would have otherwise stayed hidden in an averaged dataset. Such heterogeneous response started during the embryonic development, which makes essential having a dedicated chip that allows including this early developmental stage in the drug assay. Our approach would therefore allow elucidating pharmaceutical or therapeutic responses that so far were still being overlooked.

P-18 - Iron-homeostasis regulation by neuronal oxygen sensors in the nematode Caenorhabditis elegans

Iron presents cells with a double-edged sword. On the one hand, iron is crucial for fundamental physiological processes such as DNA synthesis, oxygen (O2) transport, and mitochondrial respiration. On the other hand, iron can catalyze the formation of toxic oxygen radicals that can destroy any biological molecule. Therefore, it is crucial to understand how the concentration of labile iron is regulated at the whole animal level. And, in particular, how it is controlled in stress conditions in which the level of reactive oxygen species (ROS) is high. Here, we characterized the changes in intestinal gene expression occurring during the adaptation of C. elegans worms to hypoxia and reoxygenation stress. Among the 770 genes that were differentially expressed, the iron storage ferritin 1 (ftn-1) gene was significantly upregulated in hypoxia. Intriguingly, we show that this upregulation is controlled by neuronal oxygen-sensors in a HIF-1-dependent manner. Moreover, our data suggest that FTN-1 plays an important role in innate immunity in hypoxia. These data may provide novel insights about the way iron level is regulated in tumor development where cells may experience mild to severe hypoxia conditions.

20. Discovery of Novel Proteins Involved the Insulin/IGF-1 Signalling Pathway

The insulin/insulin growth factor-1 (IGF-1) signalling (IIS) pathway plays a key role in metabolism, growth and development. Though research has elucidated aspects of this pathway, it is not fully characterized or understood. A better understanding of the pathway will give insight into related diseases such as cancer. To discover novel proteins involved in the IIS pathway, the C. elegans worm was used due to the homology its insulin/IGF-1 receptor shares with that of humans. 
 To identify novel protein interactions with the insulin/IGF-1 receptor, we performed a yeast two-hybrid screen using a library of worm proteins. We found several separate interactions with the worm homolog of the HSP90 protein.
 To support the involvement of HSP90 in the IIS pathway, we studied the phenotypes of worm strains with a mutant form of HSP90. They showed a similar phenotype to those that have a mutant form of the insulin/IGF-1 receptor, inappropriately entering a developmental stage known as dauer. This strongly suggests the involvement of HSP90 in the IIS pathway.
 Based on previous research, we hypothesized the interaction between HSP90 and the insulin/IGF-1 receptor may allow it to bind other proteins. Thus, we performed a modified yeast two-hybrid screen to identify proteins which interact with the receptor in the presence of HSP90. The screen identified 15 interactions, many more than with the insulin/IGF-1 receptor alone, supporting this hypothesis.
 Overall, we provide evidence of a novel interaction with insulin/IGF-1 receptor, suggesting HSP90 may be a potential target for developing therapies for IIS pathway related diseases.

Lactobacillus paracasei 28.4 reduces in vitro hyphae formation of Candida albicans and prevents the filamentation in an experimental model of Caenorhabditis elegans

The objective of this study was to evaluate the influence of microbe-microbe interactions to identify a strain of Lactobacillus that could reduce the filamentation of Candida albicans ATCC 18804 using in vitro and in vivo models. Thus presenting a probiotic effect against the fungal pathogen. First, we analyzed the ability of 25 clinical isolates of Lactobacillus to reduce filamentation in C. albicans in vitro. We found that L. paracasei isolate 28.4 exhibited the greatest reduction of C. albicans hyphae (p = 0.0109). This reduction was confirmed by scanning electron microscopy analysis. The influence of C. albicans filamentation was found to be contributed through reduced gene expression of filament associated genes (TEC1 and UME6). In an in vivo study, prophylactic provisions with L. paracasei increased the survival of Caenorhabditis elegans worms infected with C. albicans (p = 0.0001) by 29%. Prolonged survival was accompanied by the prevention of cuticle rupture of 27% of the worms by filamentation of C. albicans, a phenotype that is characteristic of C. albicans killing of nematodes, compared to the control group. Lactobacillus paracasei isolate 28.4 reduced the filamentation of C. albicans in vitro by negatively regulating the TEC1 and UME6 genes that are essential for the production of hyphae. Prophylactic provision of Lactobacillus paracasei 28.4 protected C. elegans against candidiasis in vivo. L. paracasei 28.4 has the potential to be employed as an alternative method to control candidiasis.

Ratiometric Calcium Imaging of Individual Neurons in Behaving <em>Caenorhabditis Elegans</em>

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified. Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 µm and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

Ratiometric Calcium Imaging of Individual Neurons in Behaving <em>Caenorhabditis Elegans</em>

It has become increasingly clear that neural circuit activity in behaving animals differs substantially from that seen in anesthetized or immobilized animals. Highly sensitive, genetically encoded fluorescent reporters of Ca2+ have revolutionized the recording of cell and synaptic activity using non-invasive optical approaches in behaving animals. When combined with genetic and optogenetic techniques, the molecular mechanisms that modulate cell and circuit activity during different behavior states can be identified.Here we describe methods for ratiometric Ca2+ imaging of single neurons in freely behaving Caenorhabditis elegans worms. We demonstrate a simple mounting technique that gently overlays worms growing on a standard Nematode Growth Media (NGM) agar block with a glass coverslip, permitting animals to be recorded at high-resolution during unrestricted movement and behavior. With this technique, we use the sensitive Ca2+ reporter GCaMP5 to record changes in intracellular Ca2+ in the serotonergic Hermaphrodite Specific Neurons (HSNs) as they drive egg-laying behavior. By co-expressing mCherry, a Ca2+-insensitive fluorescent protein, we can track the position of the HSN within ~ 1 µm and correct for fluctuations in fluorescence caused by changes in focus or movement. Simultaneous, infrared brightfield imaging allows for behavior recording and animal tracking using a motorized stage. By integrating these microscopic techniques and data streams, we can record Ca2+ activity in the C. elegans egg-laying circuit as it progresses between inactive and active behavior states over tens of minutes.

WorMachine: machine learning-based phenotypic analysis tool for worms

BackgroundCaenorhabditis elegans nematodes are powerful model organisms, yet quantification of visible phenotypes is still often labor-intensive, biased, and error-prone. We developed WorMachine, a three-step MATLAB-based image analysis software that allows (1) automated identification of C. elegans worms, (2) extraction of morphological features and quantification of fluorescent signals, and (3) machine learning techniques for high-level analysis.ResultsWe examined the power of WorMachine using five separate representative assays: supervised classification of binary-sex phenotype, scoring continuous-sexual phenotypes, quantifying the effects of two different RNA interference treatments, and measuring intracellular protein aggregation.ConclusionsWorMachine is suitable for analysis of a variety of biological questions and provides an accurate and reproducible analysis tool for measuring diverse phenotypes. It serves as a “quick and easy,” convenient, high-throughput, and automated solution for nematode research.

Live imaging of C. elegans oocytes and early embryos.

Caenorhabditis elegans is a self-fertilizing hermaphroditic worm. A single C. elegans worm therefore produces both male and female gametes that fuse to generate embryos. While sperm production stops at the end of the C. elegans larval development, oocytes are continuously generated and fertilized during the entire reproductive life of the adult worm. The molecular and cellular mechanisms involved in gametogenesis and the early embryonic divisions are highly conserved between worms and humans; thus C. elegans is a powerful model to study meiotic and mitotic cell divisions in a metazoan system. Additionally, the optical transparency of the worm combined with the ease of the genome-editing methods can be used to easily follow the subcellular behavior of any fluorescently tagged protein of interest using light microscopy approaches. Here we describe two methods for preparing live samples to study oocyte meiotic and early embryonic mitotic divisions by confocal microscopy in C. elegans.

A Marine Actinomycete Rescues Caenorhabditis elegans from Pseudomonas aeruginosa Infection through Restitution of Lysozyme 7.

The resistance of Pseudomonas aeruginosa to conventional antimicrobial treatment is a major scourge in healthcare. Therefore, it is crucial that novel potent anti-infectives are discovered. The aim of the present study is to screen marine actinomycetes for chemical entities capable of overcoming P. aeruginosa infection through mechanisms involving anti-virulence or host immunity activities. A total of 18 actinomycetes isolates were sampled from marine sediment of Songsong Island, Kedah, Malaysia. Upon confirming that the methanolic crude extract of these isolates do not display direct bactericidal activities, they were tested for capacity to rescue Caenorhabditis elegans infected with P. aeruginosa strain PA14. A hexane partition of the extract from one isolate, designated as Streptomyces sp. CCB-PSK207, could promote the survival of PA14 infected worms by more than 60%. Partial 16S sequence analysis on this isolate showed identity of 99.79% with Streptomyces sundarbansensis. This partition did not impair feeding behavior of C. elegans worms. Tested on PA14, the partition also did not affect bacterial growth or its ability to colonize host gut. The production of biofilm, protease, and pyocyanin in PA14 were uninterrupted, although there was an increase in elastase production. In lys-7::GFP worms, this partition was shown to induce the expression of lysozyme 7, an important innate immunity defense molecule that was repressed during PA14 infection. GC-MS analysis of the bioactive fraction of Streptomyces sp. CCB-PSK207 revealed the presence of methyl esters of branched saturated fatty acids. In conclusion, this is the first report of a marine actinomycete producing metabolites capable of rescuing C. elegans from PA14 through a lys-7 mediated activity.

Worm optimization for the multiple level warehouse layout problem

In this paper, the NP-complete multiple level warehouse layout problem is addressed. The problem consists of assigning items to cells and levels with the objective of minimizing transportation costs. A worm optimization algorithm (WO) is introduced, based on the foraging behaviors of Caenorhabditis elegans (Worms), and its performance was assessed by comparing with a genetic algorithm (GA), ant colony optimization (ACO), and an exact solution (B&B) for small problems. The computational results reflected the superiority of WO in large problems, with a marginally better performance than ACO and GA in smaller ones, while solving the tested problems within a reasonable computational time. Furthermore, WO was able to attain most of the known optimal solutions.

Effective drug combination for Caenorhabditis elegans nematodes discovered by output-driven feedback system control technique.

Infections from parasitic nematodes (or roundworms) contribute to a significant disease burden and productivity losses for humans and livestock. The limited number of anthelmintics (or antinematode drugs) available today to treat these infections are rapidly losing their efficacy as multidrug resistance in parasites becomes a global health challenge. We propose an engineering approach to discover an anthelmintic drug combination that is more potent at killing wild-type Caenorhabditis elegans worms than four individual drugs. In the experiment, freely swimming single worms are enclosed in microfluidic drug environments to assess the centroid velocity and track curvature of worm movements. After analyzing the behavioral data in every iteration, the feedback system control (FSC) scheme is used to predict new drug combinations to test. Through a differential evolutionary search, the winning drug combination is reached that produces minimal centroid velocity and high track curvature, while requiring each drug in less than their EC50 concentrations. The FSC approach is model-less and does not need any information on the drug pharmacology, signaling pathways, or animal biology. Toward combating multidrug resistance, the method presented here is applicable to the discovery of new potent combinations of available anthelmintics on C. elegans, parasitic nematodes, and other small model organisms.

Optimization and evaluation of zein nanoparticles to improve the oral delivery of glibenclamide. In vivo study using C. elegans

The aim of this work was to evaluate the capability of zein nanoparticles as oral carriers for glibenclamide (GB). Nanoparticles were prepared by a desolvation procedure in the presence of lysine as stabilizer. A central composite design was used to optimize this preparative process. Under the selected conditions, nanoparticles displayed a size of about 190 nm, a surface charge of −37mV and a payload of 45µg GB/mg. Small-angle neutron scattering and X-ray diffraction techniques suggested an internal fractal-like structure, based on the repetition of spherical blocks of zein units (about 20nm) grouped to form the nanoparticles. This structure, stabilized by lysine molecules located at the surface, would determine the release of GB (molecularly trapped into the nanoparticles) by a pure diffusion mechanism. Moreover, GB-loaded nanoparticles induced a significant hypolipidemic effect with a reduction of about 15% in the fat content of C. elegans worms. In addition, did not induce any significant modification in the lifespan of worms. In summary, the employment of zein nanoparticles as delivery systems of glibenclamide may be an interesting approach to develop new oral formulations of this antidiabetic drug.