Araujo Agarrayua de Souza, Danielle et al. (2021) International Worm Meeting "Safaty evaluation of nanoparticles prepared with differents polymers in Caenorhabditis elegans"

Araujo Agarrayua de Souza, Danielle, Carvalho, Felipe, Avila , Daiana, Elisa Haas, Sandra

[International Worm Meeting, 2021]

Nano-sized drug delivery systems have been the subject of intense research in recent years because polymeric materials allow the absorption and release of active substances in a controlled manner. The use of polymeric nanoparticles as a drug carrier has emerged as an alternative. Despite the benefits, the safety of nanoparticulate systems is an aspect to be understood, particularly in in vivo systems. Caenorhabditis elegans is a very useful alternative model for nanotoxicology and has been recently applied in this field. The aim of this study was to evaluate toxicological endpoints in worms exposed to nanomaterials prepared with different polymers: polyethylene glycol (PEG), chitosan (CH), eudragit (EU) and polysorbate 80 (P80) in C. elegans. First larval staged worms were obtained by a synchronization process and treated with nanomaterials at concentrations of 0.015, 0.225 and 0.45 mg/mL for 30 minutes in liquid medium (acute exposure). Soon after, they were washed to remove the treatments and transferred to Petri dishes containing NGM and E. coli OP50 for 48 h. Survival rate, brood size and worms length were determined. Data were expressed as mean ± standard error, and statistical analysis were done by one-way ANOVA followed by Tukey post-hoc test. We observed that the EU nanoparticles did not cause any significant change in toxicological endpoints when compared to control. On the other hand, exposure to CH, PEG and P80 nanoparticles decreased worms survival, reduced their progeny and significantly altered worms size. This work demonstrates the toxicological differences between polymers and a potential for EU to be used in new formulations for future drug vectoring and targeting systems.

Masler EP et al. (1998) East Coast Worm Meeting "FMRFamide-Like Immunoreactivity in Heterodera glycines: Changes During Development and Comparison with Caenorhabditis elegans and Panagrellus redivivus."

Sardanelli S, Masler EP, Kovaleva ES

[East Coast Worm Meeting, 1998]

A peptide family with a characteristic arginine-phenylalanine-amide C-terminal motif ("FMRFamide") and myoactive properties has been described for both free-living and animal parasitic nematodes. Primary structures reported to date probably represent only a fraction of those present in these animals, and their neuromodulatory nature suggests a potentially extensive involvement of this peptide family in the behavior, development and reproduction of nematodes. Immunocytochemical studies (Atkinson et al. 1988) detected the presence of FMRFamide-like peptides in the nervous system of the plant parasitic nematode Heterodera glycines, but no analyses of these peptides from plant parasitic nematodes have been available. We have developed methods for the collection and extraction of H. glycines and the detection of FMRFamide-like peptides using a competitive ELISA. HPLC-ELISA procedures reveal species-specific differences in the quality and quantity of FMRFamide-like peptides from H. glycines and the free-living nematodes Caenorhabditis elegans and Panagrellus redivivus. In addition, changes in FMRFamide-like peptide immunoreactivity were observed during H. glycines development and maturation. The significance of these developmental changes and species differences is discussed, and arguments for the involvement of FMRFamides in parasitic nematode growth and development are presented.

Wang, Juan et al. (2017) International Worm Meeting "Biogenesis and Function of Social Extracellular Vesicles (EVs)."

Barr, Maureen, Wang, Juan, Silva, Malan

[International Worm Meeting, 2017]

Extracellular vesicles are emerging as an important aspect of intercellular communication by delivering a parcel of proteins, lipids even nucleic acids to specific target cells over short or long distances (Maas 2017). A subset of C. elegans ciliated neurons release EVs to the environment and elicit changes in male behaviors in a cargo-dependent manner (Wang 2014, Silva 2017). Our studies raise many questions regarding these social communicating EV devices. Why is the cilium the donor site? What mechanisms control ciliary EV biogenesis? How are bioactive functions encoded within EVs? EV detection is a challenge and obstacle because of their small size (100nm). However, we possess the first and only system to visualize and monitor GFP-tagged EVs in living animals in real time. We are using several approaches to define the properties of an EV-releasing neuron (EVN) and to decipher the biology of ciliary-released EVs. To identify mechanisms regulating biogenesis, release, and function of ciliary EVs we took an unbiased transcriptome approach by isolating EVNs from adult worms and performing RNA-seq. We identified 335 significantly upregulated genes, of which 61 were validated by GFP reporters as expressed in EVNs (Wang 2015). By characterizing components of this EVN parts list, we discovered new components and pathways controlling EV biogenesis, EV shedding and retention in the cephalic lumen, and EV environmental release. We also identified cell-specific regulators of EVN ciliogenesis and are currently exploring mechanisms regulating EV cargo sorting. Our genetically tractable model can make inroads where other systems have not, and advance frontiers of EV knowledge where little is known. Maas, S. L. N., Breakefield, X. O., & Weaver, A. M. (2017). Trends in Cell Biology. Silva, M., Morsci, N., Nguyen, K. C. Q., Rizvi, A., Rongo, C., Hall, D. H., & Barr, M. M. (2017). Current Biology. Wang, J., Kaletsky, R., Silva, M., Williams, A., Haas, L. A., Androwski, R. J., Landis JN, Patrick C, Rashid A, Santiago-Martinez D, Gravato-Nobre M, Hodgkin J, Hall DH, Murphy CT, Barr, M. M. (2015).Current Biology. Wang, J., Silva, M., Haas, L. A., Morsci, N. S., Nguyen, K. C. Q., Hall, D. H., & Barr, M. M. (2014). Current Biology.

Chi-Hao Luan et al. (2003) International Worm Meeting "High Throughput Recombinant Protein Expression for C. elegans"

Chi-Hao Luan, Rita J Gray, Lawrence J DeLucas, Ming Luo, Wenying Huang, James B Finley, Shihong Qiu

[International Worm Meeting, 2003]

C. elegans is one of the most investigated multicellular organisms. The complete sequencing of the worm's genome and the cloning of its protein encoding open reading frames (ORFs) using the Gateway system (Invitrogen) greatly facilitates the genomic and proteomic study of the model Eukaryotic system. Instead of traditional approach whereby genes and gene products are studied a few at a time, the entirety of 19,099 predicted C. elegans genes are selected by Southeast Collaboratory for Structural Genomics as targets in a genomic-scale structural biology initiative. To support this effort, we are establishing a state-of-the-art facility for high throughput recombinant protein expression and refolding. We developed an automated recombinant protein expression system using the Gateway cloning and expression technology. The system, based on a strategy of step-wise automation on an integrated robotic platform with in-house technology development, successfully automated nearly all aspects of recombinant protein expression. To achieve total automation, we investigated and optimized conditions for miniaturization of bacterial growth, DNA plasmid propagation, protein expression and purification in 96-well plate of 0.6 ml. We developed a novel transformation platform including a robotic device for heat-shock in 96-well plate, as well as an ElectroTip for automated electroporation. We developed fully automated dynamic ELISA for profiling protein expression level and solubility, which increased the dynamic range of 96-well plate ELISA up to two orders of magnitude. We also introduced sigma, a new parameter defined as the ratio of ELISA signal in soluble fraction to that of insoluble fraction, to predict a proteins solubility and how to improve it in scale up production, and to predict the easiness of refolding if the protein is not readily soluble. The solubility profiling is almost 100% accurate as confirmed by 1 L scale up expressions. Currently, 384 unique C. elegans ORFs in Gateway entry vector are processed in 3-weeks, processing four 96-well plates in parallel. This throughput can be further increased in an assembly line fashion by staggering new plates in the process each day as demand dictates. To increase the number of soluble proteins, we developed new Gateway compatible destination vectors, utilized multiple E. coli strains, and are exploring alternative expression systems, for instance, the Baculovirus system. The automation developed using the E. coli expression system can be readily adapted to other expression systems.

Sithigorngul P et al. (1995) International C. elegans Meeting "AF1-like and other -RFamide like immunoreactive substances in Caenorhabditis elegans."

Stretton AOW, Sithigorngul P

[International C. elegans Meeting, 1995]

A high sensitivity dot-ELISA technique has been used to detect -RFamide like peptides in an extract of C. elegans fractionated by HPLC. Monoclonal antibodies AF1-243 (highly specific to AF1), AF2-003 (detects AF2), AF3-442 (recognizes the -VLRFamide family of peptides) and AF1-62 (recognizes -RFamide peptides) were used to monitor the fractionation. AF1-like immunoreactivity was detected in the fractions with an elution time similar to AF1 in Ascaris. Neither AF2 nor -VLRFamide like immunoreactive substances were found, however numerous -RFamide like immunoreactive peptides were revealed with antibody AF1-62. This indicates that additional of-RFamide like peptides, besides those encoded by the flp-1 gene (Rosoff et al., 1992) are present in C. elegans. Purification of -RFamide like peptides from C. elegans is in progress.

Link CD (1996) West Coast Worm Meeting "MAKING AMYLOID WORMS MORE SENILE?"

Link CD

[West Coast Worm Meeting, 1996]

Transgenic worms containing a signal peptide/ beta peptide 1-42 minigene (derived from the human Amyloid Precursor Protein, APP) driven by the unc-54 promoter/enhancer produce intracellular immunoreactive beta peptide deposits that react with the amyloid specific dye thioflavin S. Animals containing an identical construct expressing the 1-40 version of the beta peptide (lacking two C-terminal amino acids) also produce immunoreactive beta peptide deposits, but these deposits do not react with thioflavin S. Recent experiments have been directed towards: 1) determining if the difference in thioflavin reactivity between transgenic lines expressing the 1-42 and 1-40 is due to quantitative or qualitative differences in the expressed peptide, 2) determining what the engineered signal peptide is (or isn't) doing in these constructs, and 3) investigating whether formation of thioflavin-reactive (amyloid) deposits can be enhanced by co-expression of human apolipoprotein E 4, which is strongly associated with increased Alzheimer's Disease susceptibility and has been postulated to directly interact with beta peptide to form amyloid. Quantitative ELISA assays done in collaboration with the lab of S. Younkin (Mayo Clinic) indicate that the failure of beta 1-40 peptide to make amyloid deposits is due to qualitative differences in the expressed peptide (not due to lower overall levels of peptide expression). However, both peptide versions also show evidence of N-terminal modification, raising questions about the role of the signal peptide in beta peptide expression, and complicating interpretation of the ELISA data. I have therefore generated a new series of transgenic constructs with the signal peptide replaced with an initiator methionine, employing unc-119, mec-7, and unc-54 promoters. For currently unknown reasons, none of these lines express immunohistochemically detectable beta peptide. Extrachromosomal transgenic lines have also been constructed that coexpress an unc-54/Apo E4 construct and either of the original unc-54/signal peptide/beta peptide constructs. Although immunohistochemistry clearly demonstrates that these lines coexpress both proteins in muscle cells, significant enhancement of thioflavin S-reactive deposits has not been observed in the extrachromosomal lines examined so far. Integrated lines are currently being generated to more carefully examine the interaction of Apo E and beta peptide in this model system.

KA Swan et al. (1999) International C. elegans Meeting "or191ts and or182ts are Embryonic Lethal, Temperature-Sensitive Mutations that Disrupt Spindle Rotation and Cell Cycle Timing, Respectively, in Two-Cell Stage C. elegans Embryos."

DR Hamill, BA Bowerman, KA Swan

[International C. elegans Meeting, 1999]

In order to understand the processes governing the establishment of asymmetry within the early C. elegans embryo, our lab has begun an investigation of several conditional mutants isolated in a screen for temperature sensetive, embryonic lethal mutations (see abstract by D. Hamill). Two of these mutations, or191ts and or182ts disrupt different aspects of asymmetry normally observed at the two-cell stage. The or191ts mutation results in random spindle orientation within the two-cell stage blastomeres AB and P 1 , which normally divide transversely and longitudinally, respectively. In addition to dividing along different axes, the cell cycle times of AB and P 1 are unequal in wild-type embryos, with AB dividing slightly ahead of P 1 . A second mutation we have identified increases this asymmetry. The mutation or182ts , delays the cell cycle timing of both AB and P 1 . The AB cell cycle time, however, is increased only slightly, while the time of the P 1 cell cycle is doubled. or191ts maps to the center of chromosome V while or182ts maps to the far left arm of chomosome III. Phenotypic analysis of these mutants and progress on identifying the affected genes will be presented. For information concerning the analysis of other mutants affecting similar processes see posters presented by Danielle Hamill and Sandra Encalada in our laboratory.

Brad D Barrows et al. (2005) International Worm Meeting "Analysis of a gene associated with resistance to a specific crystal protein produced by Bacillus thuringiensis"

Raffi V Aroian, Brad D Barrows

[International Worm Meeting, 2005]

The crystal proteins made by Bacillus thuringiensis (Bt toxins) are important, naturally occurring agents for the control of insects that eat crops and carry disease. Mutant animals (C. elegans) resistant to the nematode-active crystal toxin (Cry 5B) can be isolated, providing the first genetic system for the study of these important toxins. Resistant animals are called bre mutants. All of the four bre mutants cloned to date (bre-2 bre-5) encode putative glycosyltransferases. The bre-1 gene has been found to be an exception to the pattern established by other bre genes. Instead of functioning as a glycosyltransferase involved in a carbohydrate biosynthetic pathway, bre-1 appears to be involved in a monosaccharide biosynthetic pathway. Collected data suggests that bre-1, like the other bre genes, is involved in the biosynthesis of a glycolipid receptor, but unlike the other bre genes, the involvement of bre-1 is less direct. bre-1 is also intriguing in other aspects. Unlike the other bre mutants, bre-1 has an overall reduced brood size that is more severe than that of other resistance mutants in addition to also having a weak resistance phenotype. bre-1 mutant animals have been characterized by examining pharyngeal pumping in response to toxin, temperature sensitivity, intestinal injection of specific sugars, glycolipid analysis by thin layer chromatography, and ELISA-based receptor binding studies. These results will be presented.

Singh, Akanksha (2021) International Worm Meeting "CRISPR- Nanobodies from C. elegans as an therapeutic approach for Erythroblastosis Fetalis"

Singh, Akanksha

[International Worm Meeting, 2021]

Erythroblastosis fetalis is a consequence of incompatibility amongst Rh-negative antigen from mother and Rh-positive antigen of foetus thus resulting in hemolysis. The hemolysis usually results in respiratory problems, neurological disorders or even heart failure. C.elegans is known to be homologous to some extent with human genome and produces monoclonal antibodies. The nanobodies from C. elegans are engineered with CRISPR Cas 9 technology where Cas 9 protein has been incorporated into nanobodies that will accelerate its efficiency. The engineered nanobodies will target genes like RHD and RhCE that are prominent in RBC membranous environment. The nanobodies with Cas-9 might block the interaction of Rh-negative antibodies with Rh-positive antibodies of foetus as by refusing the recruitment of Rh-negative antibodies in placenta. Antibodies from the C. elegans are extracted and engineered to produce nanobodies with encoded Cas 9 protein. The haemoglobin obtained from foetus are mixed with haemoglobin from mother with Rh-antigens in vitro. After a period of 24-48 hours, when engineered nanobodies are treated with blood by ELISA technique indicated reduced count of Rh-positive antibodies thus blocking RHD gene not allowing the interaction with Rh-negative antibodies. The aim of the study was to study molecular interaction occurring after exposure of nanobodies to Rh- antigens present in foetal blood. Keywords: Erythroblastosis fetalis, Rh-negative, hemolysis, C. elegans, CRISPR Cas 9, RHD , RHCE, haemoglobin, nanobodies.

Gill, R. et al. (2019) International Worm Meeting "Identifying biogenesis mechanisms for discrete subpopulations of extracellular vesicles."

Gill, R., Touroutine, D., Clupper, M., Tanis, J.

[International Worm Meeting, 2019]

Extracellular vesicles (EVs) are membrane wrapped structures containing proteins, RNAs, lipids, and metabolites that are released from most if not all cell types to mediate intercellular communication in physiological and pathological conditions. EVs fall into subclasses based on their mode of biogenesis. Exosomes are released following fusion of multivesicular bodies (MVBs) with the plasma membrane, while microvesicles form directly from plasma membrane budding. Our goal is to use C. elegans to identify proteins required for EV biogenesis in vivo. EVs are released from the cilia of the male specific cephalic male (CEM) neurons in the head and hook B type (HOB) and bilateral ray B type neurons (RnB) in the tail. These EVs are likely microvesicles as there is no evidence of MVBs in these neurons1. We discovered that the ion channel CLHM-1 is cargo in EVs released from C. elegans ciliated male sensory neurons by performing high resolution imaging of animals expressing functional GFP-tagged CLHM-1 at endogenous levels. Remarkably, when we co-expressed tdTomato-tagged CLHM-1 with GFP-tagged PKD-2, a known EV cargo protein expressed in the same neurons1, we rarely observed colocalization of the fluorescent proteins in vesicles, suggesting that CLHM-1 and PKD-2 are in discrete EV subpopulations. We are using the power of genetics to manipulate candidate EV biogenesis pathways to identify factors required for release of CLHM-1 containing EVs. Lipid asymmetry between the inner and outer leaflets of the plasma membrane induces curvature which could drive microvesicle release. Type IV-ATPase flippases translocate phospholipids from the outer to the inner leaflet to maintain bilayer asymmetry, while scramblases disrupt membrane asymmetry. We are determining if the flippases TAT-1, TAT-3, and TAT-6 as well as the scramblases ANOH-1 and SCRM-4 play a role in the biogenesis of one or both fluorescently marked EV subpopulations derived from male ciliated neurons. 1. Wang J, Silva M, Haas LA, Morsci NS, Nguyen KC, Hall DH, Barr MM. (2014) C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication. Curr Biol. 24(5):519-25.