scholarly journals A high-throughput device for size based separation of C. elegans developmental stages

Lab on a Chip ◽  
2014 ◽  
Vol 14 (10) ◽  
pp. 1746-1752 ◽  
Author(s):  
Xiaoni Ai ◽  
Weipeng Zhuo ◽  
Qionglin Liang ◽  
Patrick T. McGrath ◽  
Hang Lu
Keyword(s):  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Developmental Stages ◽  
C Elegans ◽  
Size Based Separation

We present an efficient and fast device for size-based separation of Caenorhabditis elegans.

Broad oxygen tolerance in the nematode Caenorhabditis elegans

2000 ◽  
Vol 203 (16) ◽  
pp. 2467-2478 ◽  
Author(s):  
W.A. Van Voorhies ◽  
S. Ward
Keyword(s):  
Caenorhabditis Elegans ◽  
Metabolic Rate ◽  
Developmental Stages ◽  
Small Body ◽  
Metabolic Cost ◽  
Soil Nematode ◽  
Nematode Caenorhabditis Elegans ◽  
Oxygen Levels ◽  
C Elegans ◽  
Short Period

This study examined the effects of oxygen tensions ranging from 0 to 90 kPa on the metabolic rate (rate of carbon dioxide production), movement and survivorship of the free-living soil nematode Caenorhabditis elegans. C. elegans requires oxygen to develop and survive. However, it can maintain a normal metabolic rate at oxygen levels of 3.6 kPa and has near-normal metabolic rates at oxygen levels as low as 2 kPa. The ability to withstand low ambient oxygen levels appears to be a consequence of the small body size of C. elegans, which allows diffusion to supply oxygen readily to the cells without requiring any specialized respiratory or metabolic adaptations. Thus, the small size of this organism pre-adapts C. elegans to living in soil environments that commonly become hypoxic. Movement in C. elegans appears to have a relatively minor metabolic cost. Several developmental stages of C. elegans were able to withstand up to 24 h of anoxia without major mortality. Longer periods of anoxia significantly increased mortality, particularly for eggs. Remarkably, long-term exposure to 100 % oxygen had no effect on the metabolic rate of C. elegans, and populations were able to survive for a least 50 generations in 100 % (90 kPa) oxygen. Such hyperoxic conditions are fatal to most organisms within a short period.

scholarly journals CAENORHABDITIS ELEGANS AS A MODEL OF AIR POLLUTION TOXICITY DURING DEVELOPMENT AND LIFESPAN

2019 ◽  
Vol 3 (Supplement_1) ◽  
pp. S97-S97
Author(s):  
Amin Haghani ◽  
Hans M Dalton ◽  
Nikoo Safi ◽  
Farimah Shirmohammadi ◽  
Constantinos Sioutas ◽  
...  
Keyword(s):  
Air Pollution ◽  
Caenorhabditis Elegans ◽  
Mouse Models ◽  
High Throughput ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Exposure Model ◽  
C Elegans ◽  
Biological Responses ◽  
Short Term Exposure

Abstract Air pollution (AirPoll) is among the leading human mortality risk factors and yet little is known about the molecular mechanisms of this global environmental toxin. Our recent studies using mouse models even showed genetic variation and sex can alter biological responses to air pollution. To expand genetic studies of AirPoll toxicity throughout the lifespan, we introduced Caenorhabditis elegans as a new AirPoll exposure model which has a short lifespan, high throughput capabilities and shared longevity pathways with mammals. Acute exposure of C. elegans to airborne nanosized AirPoll matter (nPM) caused similar gene expression changes to our prior findings in cell culture and mouse models. Initial C. elegans responses to nPM included antioxidant, inflammatory and Alzheimer homolog genes. The magnitude of changes was dependent on the developmental stage of the worms. Even short term exposure of C. elegans to nPM altered developmental and lifespan hormetic effects, with pathways that included skn-1/Nrf family antioxidant responses. We propose C. elegans as a new and complementary model for mouse and cultured cells to study AirPoll across the lifespan. Future chronic nPM exposure and high throughput genetic screening of C. elegans can identify other major regulators of the developmental and lifespan effects of air pollution. This work was supported by grants R01AG051521 (CEF); R21AG05020 (CEF); Cure Alzheimer’s Fund (CEF); R01GM109028 (SPC), F31AG051382 (HMD) and T32AG000037 (HMD), T32AG052374 (AH).

scholarly journals A scalable method for automatically measuring pharyngeal pumping in C. elegans

2016 ◽  
Author(s):  
Monika Scholz ◽  
Dylan J. Lynch ◽  
Kyung Suk Lee ◽  
Erel Levine ◽  
David Biron
Keyword(s):  
Caenorhabditis Elegans ◽  
Food Availability ◽  
High Throughput ◽  
Visual Inspection ◽  
Automated Analysis ◽  
C Elegans ◽  
Automated Method ◽  
Controlled Environments ◽  
Pharyngeal Pumping ◽  
Feeding Environments

We describe a scalable automated method for measuring the pharyngeal pumping of Caenorhabditis elegans in controlled environments. Our approach enables unbiased measurements for prolonged periods, a high throughput, and measurements in controlled yet dynamically changing feeding environments. The automated analysis compares well with scoring pumping by visual inspection, a common practice in the field. In addition, we observed overall low rates of pharyngeal pumping and long correlation times when food availability was oscillated.

Differential assembly of alpha- and gamma-filagenins into thick filaments in Caenorhabditis elegans

2000 ◽  
Vol 113 (22) ◽  
pp. 4001-4012 ◽  
Author(s):  
F. Liu ◽  
I. Ortiz ◽  
A. Hutagalung ◽  
C.C. Bauer ◽  
R.G. Cook ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Immunoelectron Microscopy ◽  
Body Wall ◽  
Developmental Stages ◽  
Body Wall Muscle ◽  
Developmentally Regulated ◽  
C Elegans ◽  
Novel Proteins ◽  
Thick Filaments ◽  
Associated Proteins

Muscle thick filaments are highly organized supramolecular assemblies of myosin and associated proteins with lengths, diameters and flexural rigidities characteristic of their source. The cores of body wall muscle thick filaments of the nematode Caenorhabditis elegans are tubular structures of paramyosin sub-filaments coupled by filagenins and have been proposed to serve as templates for the assembly of native thick filaments. We have characterized alpha- and gamma-filagenins, two novel proteins of the cores with calculated molecular masses of 30,043 and 19,601 and isoelectric points of 10.52 and 11.49, respectively. Western blot and immunoelectron microscopy using affinity-purified antibodies confirmed that the two proteins are core components. Immunoelectron microscopy of the cores revealed that they assemble with different periodicities. Immunofluorescence microscopy showed that alpha-filagenin is localized in the medial regions of the A-bands of body wall muscle cells whereas gamma-filagenin is localized in the flanking regions, and that alpha-filagenin is expressed in 1.5-twofold embryos while gamma-filagenin becomes detectable only in late vermiform embryos. The expression of both proteins continues throughout later stages of development. C. elegans body wall muscle thick filaments of these developmental stages have distinct lengths. Our results suggest that the differential assembly of alpha- and gamma-filagenins into thick filaments of distinct lengths may be developmentally regulated.

scholarly journals Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans

Genetics ◽  
2019 ◽  
Vol 213 (4) ◽  
pp. 1197-1207 ◽  
Author(s):  
Ivana Sfarcic ◽  
Theresa Bui ◽  
Erin C. Daniels ◽  
Emily R. Troemel
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Fluorescent Reporters ◽  
C Elegans ◽  
Link Type ◽  
Highly Sensitive ◽  
Green Fluorescent

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Caenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in C. elegans. We optimize detection of bioluminescent signals from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult “hidden” in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the pals-5 stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in C. elegans.

scholarly journals Staphylococcus aureus Virulence Factors Identified by Using a High-Throughput Caenorhabditis elegans-Killing Model

2005 ◽  
Vol 73 (2) ◽  
pp. 872-877 ◽  
Author(s):  
Jakob Begun ◽  
Costi D. Sifri ◽  
Samuel Goldman ◽  
Stephen B. Calderwood ◽  
Frederick M. Ausubel
Keyword(s):  
Staphylococcus Aureus ◽  
Caenorhabditis Elegans ◽  
Dna Replication ◽  
Virulence Factors ◽  
High Throughput ◽  
Human Pathogen ◽  
Renal Abscess ◽  
C Elegans ◽  
Transposon Insertion ◽  
Strain Nctc

ABSTRACT Staphylococcus aureus is an important human pathogen that is also able to kill the model nematode Caenorhabditis elegans. We constructed a 2,950-member Tn917 transposon insertion library in S. aureus strain NCTC 8325. Twenty-one of these insertions exhibited attenuated C. elegans killing, and of these, 12 contained insertions in different genes or chromosomal locations. Ten of these 12 insertions showed attenuated killing phenotypes when transduced into two different S. aureus strains, and 5 of the 10 mutants correspond to genes that have not been previously identified in signature-tagged mutagenesis studies. These latter five mutants were tested in a murine renal abscess model, and one mutant harboring an insertion in nagD exhibited attenuated virulence. Interestingly, Tn917 was shown to have a very strong bias for insertions near the terminus of DNA replication.

scholarly journals Insect-Derived Cecropins Display Activity against Acinetobacter baumannii in a Whole-Animal High-Throughput Caenorhabditis elegans Model

2015 ◽  
Vol 59 (3) ◽  
pp. 1728-1737 ◽  
Author(s):  
Elamparithi Jayamani ◽  
Rajmohan Rajamuthiah ◽  
Jonah Larkins-Ford ◽  
Beth Burgwyn Fuchs ◽  
Annie L. Conery ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Acinetobacter Baumannii ◽  
High Throughput ◽  
Sequence Data ◽  
Treatment Options ◽  
Automated Image Analysis ◽  
Screening Assay ◽  
Content Type ◽  
C Elegans ◽  
Cecropin A

ABSTRACTThe rise of multidrug-resistantAcinetobacter baumanniiand a concomitant decrease in antibiotic treatment options warrants a search for new classes of antibacterial agents. We have found thatA. baumanniiis pathogenic and lethal to the model host organismCaenorhabditis elegansand have exploited this phenomenon to develop an automated, high-throughput, high-content screening assay in liquid culture that can be used to identify novel antibiotics effective againstA. baumannii. The screening assay involves coincubatingC. eleganswithA. baumanniiin 384-well plates containing potential antibacterial compounds. At the end of the incubation period, worms are stained with a dye that stains only dead animals, and images are acquired using automated microscopy and then analyzed using an automated image analysis program. This robust assay yields a Z′ factor consistently greater than 0.7. In a pilot experiment to test the efficacy of the assay, we screened a small custom library of synthetic antimicrobial peptides (AMPs) that were synthesized using publicly available sequence data and/or transcriptomic data from immune-challenged insects. We identified cecropin A and 14 other cecropin or cecropin-like peptides that were able to enhanceC. eleganssurvival in the presence ofA. baumannii. Interestingly, one particular hit, BR003-cecropin A, a cationic peptide synthesized by the mosquitoAedes aegypti, showed antibiotic activity against a panel of Gram-negative bacteria and exhibited a low MIC (5 μg/ml) againstA. baumannii. BR003-cecropin A causes membrane permeability inA. baumannii, which could be the underlying mechanism of its lethality.

scholarly journals Combined flow cytometry and high throughput image analysis for the study of essential genes in Caenorhabditis elegans

2017 ◽  
Author(s):  
Blanca Hernando-Rodríguez ◽  
Annmary Paul Erinjeri ◽  
María Jesús Rodríguez-Palero ◽  
Val Millar ◽  
Sara González-Hernández ◽  
...  
Keyword(s):  
Image Analysis ◽  
Caenorhabditis Elegans ◽  
High Throughput ◽  
Rnai Screen ◽  
Essential Genes ◽  
Genetic Interactions ◽  
Automated Image Analysis ◽  
C Elegans ◽  
Larval Stages ◽  
Lethal Mutations

ABSTRACTBackgroundThe advancement in automated image based microscopy platforms coupled with high throughput liquid workflows has facilitated the design of large scale screens utilizing multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high throughput approaches.ResultsIn C. elegans, non-conditional lethal mutations can be maintained in heterozygosis using chromosome balancers, commonly labelled with GFP in the pharynx. Moreover gene-expression is typically monitored by the use of fluorescent reporters marked with the same fluorophore. Therefore, the separation of the different populations of animals at early larval stages represents a challenge. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at early larval stages. Because sorting is not completely error-free, we develop an automated high-throughput image-analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image-analysis in a functional genomic RNAi screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while, homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both, known and new PHB genetic interactors affecting the UPRmt and growth.ConclusionsA systematic way to analyse genetic interactions of essential genes in multicellular organisms is lacking. The method presented here allows the study of balanced lethal mutations in a high-throughput manner and can be easily adapted depending on the user’s requirements. Therefore, it will serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.

DevStaR: High-Throughput Quantification of C. elegans Developmental Stages

2013 ◽  
Vol 32 (10) ◽  
pp. 1791-1803 ◽  
Author(s):  
Amelia G. White ◽  
Brandon Lees ◽  
Huey-Ling Kao ◽  
P. Giselle Cipriani ◽  
Eliana Munarriz ◽  
...  
Keyword(s):  
High Throughput ◽  
Developmental Stages ◽  
C Elegans

scholarly journals GENETIC AND PHENOTYPIC CHARACTERIZATION OF ROLLER MUTANTS OF CAENORHABDITIS ELEGANS

Genetics ◽  
1980 ◽  
Vol 95 (2) ◽  
pp. 317-339
Author(s):  
George N Cox ◽  
John S Laufer ◽  
Meredith Kusch ◽  
Robert S Edgar
Keyword(s):  
Caenorhabditis Elegans ◽  
Linkage Group ◽  
Developmental Stages ◽  
Developmental Process ◽  
Phenotypic Characterization ◽  
Gene Interactions ◽  
C Elegans ◽  
Nematode Cuticle ◽  
Six Genes

ABSTRACT Eighty-eight mutants of C. elegans that display a roller phenotype (a helically twisted body) have been isolated and characterized genetically and phenotypically. The mutations are located in 14 different genes. Most genes contain a number of alleles. Their distribution among the chromosomes appears nonrandom, with seven of the genes being located on linkage group 11, some very closely linked. The phenotypes of the mutants suggest that there are five different classes of genes, each class representing a set of similar phenotypic effects: Left Roller (four genes), Right Roller (one gene), Left Squat (one gene), Right Squat (two genes) and Left Dumpy Roller (six genes). The classes of mutants differ with respect to a number of characteristics that include the developmental stages affected and the types of aberrations observed in cuticle structure. A variety of gene interactions were found, arguing that these genes are involved in a common developmental process. The presence of alterations in cuticle morphology strongly suggests that these genes are active in the formation of the nematode cuticle.

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