Anthony A Ferrante et al. (2001) International C. elegans Meeting "Automated Sorting of C. elegans Muv Mutants According to Pseudovulva Number"

Russell Gershman, Anthony A Ferrante, W Peter Hansen, Steve Alam, Peter Kalutkiewicz

[International C. elegans Meeting, 2001]

The UBI COPAS BIOSORT system disperses and maintains nematodes in liquid suspension, straightens them, and flows them one-by-one through a laser beam. The system analyzes and sorts each animal according to both size and fluorescence without harm. For this study the system was modified such that positional information was retained as the nematodes were scanned along their longitudinal axis. Rather than averaging the fluorescence signal over the length of the animal, the signal was measured at approximately 100 points along the length of adult C. elegans . Profiling algorithms were developed that determined the location and the number of fluorescent features of the animals. The prototype was used to characterize and sort multivulva (Muv) C. elegans according to the number of vulvae. The Muv strains MT309 lin-15 and MT388 lin-12 (both obtained from the C. elegans Genetics Center, University of Minnesota, St. Paul), as well as wild-type strain N2, were stained using NEMASELECT Male/Herma stain (Union Biometrica, Inc.) to mark the location of their vulvae. Electronic profiles were collected with the new signal processing electronics and single nematodes were deposited in individual wells of a 96-well plate. Nematodes and their associated profiles were visually compared. Close correlation between the number of stained vulva and the number of fluorescence peaks in the profiles was observed. The prototype software accurately scored and sorted the animals according to vulva number.

Anthony A Ferrante et al. (2002) East Coast Worm Meeting "Check out my Profile! Isolation of mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence profiling"

Britta Moellers, Bruce Holcombe, Steve Alam, Vance Chang, Anthony A Ferrante

[East Coast Worm Meeting, 2002]

Autofluorescence background, the natural fluorescence from biological materials, reduces sensitivity of detection of localized fluorescence in multicellular organisms. For example in the case of a transgenic C. elegans that expresses GFP in two cells, that fluorescence can be masked by the autofluorescence of the remaining 1000 somatic cells. We have solved this problem with an add-on to our COPAS analysis and sorter technology that allows the instrument to measure and store the pattern of fluorescence along the length of the animal. That information was used to restrict fluorescence analysis to the region of the animal that contained the fluorescent cells thus improving the signal to autofluorescence ratio more than 20-fold. We used the system to perform a genetic screen in which we isolated mutants of PY1089 (kindly provided by Piali Sengupta), a transgenic C. elegans that expresses GFP in the AWB sensory neurons under control of the str-1 promoter. Without the Profiler technology the COPAS system was unable to distinguish PY1089 from N2 wild type animals, and it could not be used in a genetic screen for mutants with reduced GFP expression. With the addition of Profiler we were able to clearly distinguish PY1089 from N2 and were further able to sort animals that had either reduced or increased GFP expression in the sensory neurons. F2 progeny of mutagenized PY1089 were analyzed and sorted using the COPAS BIOSORT with prototype Profiler hardware. First, 100 animals were run and their peak fluorescence intensities were determined. That information was used to set a range of normal peak heights in the Profiler. The Profiler was instructed to dispense those animals whose peak heights were outside of the normal range, either higher or lower, one per well into 96-well plates. Mutants were confirmed both by regrowing the sorted animals and running them through the Profiler a second time and visually by microscopy. Approximately 50,000 animals from 8 separate pools were screened and 1,000 were dispensed to wells in the first pass. We have retested 96 lines from the sorted animals to date. We have isolated 4 independent mutant lines with decreased GFP expression, 3 independent lines with increased GFP expression and 2 lines with a mix of increased, wild-type, and decreased GFP expression. We are in the process of further characterizing the mutants by fluorescence microscopy and we will further characterize the mutants for their chemotactic response to 2-nonanone, which is dependent on proper AWB neuronal function.

Anthony A Ferrante et al. (2002) Mid-west Worm Meeting "Check out my Profile! Isolation of chemotaxis defective mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence profiling"

Bruce Holcombe, Peter Van Osta, Vance Chang, Jennifer Kean, Britta Moellers, Anthony A Ferrante, Steve Alam, Gregory O'Connor

[Mid-west Worm Meeting, 2002]

Autofluorescence background, the natural fluorescence from biological materials, reduces sensitivity of detection of localized fluorescence in multicellular organisms. For example in the case of a transgenic C. elegans that expresses GFP in two cells, that fluorescence can be masked by the autofluorescence of the remaining 1000 somatic cells. We have solved this problem with an add-on to our COPAS analysis and sorter technology that allows the instrument to measure and store the pattern of fluorescence along the length of the animal. That information was used to restrict fluorescence analysis to the region of the animal that contained the fluorescent cells thus improving the signal to autofluorescence ratio more than 20-fold. We used the Profiler system to perform a genetic screen in which we isolated mutants of PY1089 (kindly provided by Piali Sengupta), a transgenic C. elegans that expresses GFP in the AWB sensory neurons under control of the str-1 promoter. Proper expression of the odorant receptor, STR-1, in the AWB sensory neurons is required for appropriate chemotactic response to the repulsive odorant 2-nonanone. Without the Profiler technology the COPAS system was unable to distinguish PY1089 from N2 wild type animals, and it could not be used in a genetic screen for mutants with reduced GFP expression. With the addition of Profiler we were able to clearly distinguish PY1089 from N2 and were further able to sort animals that had either reduced or increased GFP expression in the sensory neurons. F2 progeny of mutagenized PY1089 were analyzed and sorted using the COPAS BIOSORT with prototype Profiler hardware. First, 100 animals were run and their peak fluorescence intensities were determined. That information was used to set a range of normal peak heights in the Profiler. The Profiler was instructed to dispense those animals whose peak heights were outside of the normal range, either higher or lower, one per well into 96-well plates. Mutants were confirmed by regrowing the sorted animals and running them through the Profiler a second time. Approximately 50,000 animals from 8 separate pools were screened and 1,000 were dispensed to wells in the first pass. We have retested 109 lines from the sorted animals to date. We have isolated 5 independent mutant lines with decreased GFP expression, 5 independent lines with increased GFP expression and 2 lines with a mix of increased, wild-type, and decreased GFP expression. So far, we have tested 5 mutant lines for their chemotactic response to 2-nonanone. Of these one line has demonstrated defective chemotaxis

Anthony A Ferrante et al. (2002) West Coast Worm Meeting "Check out my Profile! Isolation of chemotaxis defective mutants with altered str-1 expression levels using automated, high-sensitivity fluorescence profiling."

Gregory O'Connor, Steven Alam, Bruce Holcombe, Anthony A Ferrante, Jennifer Kean, Peter Van Osta, Vance Chang, Britta Moellers

[West Coast Worm Meeting, 2002]

Autofluorescence background, the natural fluorescence from biological materials, reduces sensitivity of detection of localized fluorescence in multicellular organisms. For example in the case of a transgenic C. elegans that expresses GFP in two cells, that fluorescence can be masked by the autofluorescence of the remaining 1000 somatic cells. We have solved this problem with an add-on to our COPAS analysis and sorter technology that allows the instrument to measure and store the pattern of fluorescence along the length of the animal. That information was used to restrict fluorescence analysis to the region of the animal that contained the fluorescent cells thus improving the signal to autofluorescence ratio more than 20-fold. We used the Profiler system to perform a genetic screen in which we isolated mutants of PY1089 (kindly provided by Piali Sengupta), a transgenic C. elegans that expresses GFP in the AWB sensory neurons under control of the str-1 promoter. Proper expression of the odorant receptor, STR-1, in the AWB sensory neurons is required for appropriate chemotactic response to the repulsive odorant 2-nonanone. Without the Profiler technology the COPAS system was unable to distinguish PY1089 from N2 wild type animals, and it could not be used in a genetic screen for mutants with reduced GFP expression. With the addition of Profiler we were able to clearly distinguish PY1089 from N2 and were further able to sort animals that had either reduced or increased GFP expression in the sensory neurons.

R Bongaarts et al. (2002) European Worm Meeting "Multi-parametric & Dual Color Fluorescent Analysis and Flow Sorting of C. elegans"

A Ferrante, B Dell'Orfano, S Zusman, Johan Geysen, R Bongaarts, L Bols

[European Worm Meeting, 2002]

The COPAS BIOSORT is a high speed flow sorter that analyzes and sorts C. elegans. The system is equipped with two lasers. A 633 nm excitation diode laser analyzes two physical parameters of the organism: time of flight (TOF, a measure of the length of an organism) and extinction (EXT, a measurement of body opacity). TOF and EXT allow sorting of pure embryos, a larval stage or adults from a wild type population as well as a number of mutants with affected body length or opacity of internal structures. The 488/514 nm multi-line argon laser is used to measure the expression levels of fluorescent proteins like GFP, YFP, DsRed, ZsGreen, ZsYellow or the binding of fluorophores like SytoxTM- green, propidium iodide (PI) or phycoerythrin (PE)-tagged lectins to C. elegans (sub-) populations. Single color applications such as live-from-dead (PI, Sytox green) or male-from- hermaphrodites animals (wheat germ agglutinin-PE) have been developed as well as protocols to isolate animals with predefined levels of expression of a fluorescent protein for e.g. high speed genetic/enhancer/suppressor screens or bulk isolates for micro-array experiments. Here we exemplify how the three photon multiplyer tubes (PMT's) of the COPAS BIOSORT allow multi parametric plus dual color sort setting of gatings for TOF/EXT plus two fluorescent signals. Isolation of viable-ZsGreen positive animals.

Cindy Voisine et al. (2005) International Worm Meeting "Assessing drug efficacy in a C. elegans model of polyglutamine toxicity"

Nicola Walker, Brent R Stockwell, Anne C Hart, Cindy Voisine, Hemant Varma

[International Worm Meeting, 2005]

Huntington's Disease (HD) is one of nine neurodegenerative disorders caused by polyglutamine (polyQ) domain expansion. There is currently no cure nor effective treatment available for HD. In our C. elegans model of polyQ (CGC # 3360), the N-terminus of the toxic expanded human huntingtin protein causes age dependent degeneration of ASH neurons. We are developing drug efficacy assays to test possible therapeutic agents and assess their impact on polyQ mediated neurodegeneration in C. elegans. Drug studies should also provide insight into the contributions of different cellular pathways to the disease process. Our first assay format used animals that also carry a mutation in pqe-1, a previously identified, genetic enhancer of polyQ toxicity (CGC # 5585). In pqe-1 mutant animals, the vast majority of ASH neurons undergo cell death before the animals reach adulthood. L1s were incubated with drugs and food for two or three days before the death of ASH neurons was evaluated. We identified several compounds, but some therapeutic doses also slowed growth. To assess the impact of slower growth on polyQ toxicity, we developed a second assay that was independent of growth. ASH cell death increases over time in arrested L1 pqe-1 animals. L1s were incubated with drugs in the absence of food for one or two days before the death of ASH neurons was evaluated. We tested several compounds in this new assay format and found that LiCl and mithramycin suppressed neurodegeneration. Both compounds have similar effects in other HD models. LiCl is a GSK-3 inhibitor that has been shown to reduce polyQ toxicity in cell culture (Carmichael J et al, 2002). Mithramycin binds to GC rich regions of DNA and has been shown to reduce polyQ toxicity in a mouse HD model (Ferrante et al, 2004). Currently, we are developing an additional assay utilizing aged animals with normal pqe-1 function to further evaluate what effect these compounds have on the age dependent degeneration associated with expression of the toxic expanded human huntingtin protein. We believe that our drug assays provide an efficient means of identifying therapeutic agents for further testing in vertebrate models of polyQ diseases.

Quartey MO et al. (2021) Sci Rep "The A(1-38) peptide is a negative regulator of the A(1-42) peptide implicated in Alzheimer disease progression."

Pennington PR, Heistad RM, Nyarko JNK, Barnes JR, Bolanos MAC, Parsons MP, Knudsen KJ, De Carvalho CE, Leary SC, Mousseau DD, Buttigieg J, Maley JM, Quartey MO

[Sci Rep, 2021]

The pool of -Amyloid (A) length variants detected in preclinical and clinical Alzheimer disease (AD) samples suggests a diversity of roles for A peptides. We examined how a naturally occurring variant, e.g. A(1-38), interacts with the AD-related variant, A(1-42), and the predominant physiological variant, A(1-40). Atomic force microscopy, Thioflavin T fluorescence, circular dichroism, dynamic light scattering, and surface plasmon resonance reveal that A(1-38) interacts differently with A(1-40) and A(1-42) and, in general, A(1-38) interferes with the conversion of A(1-42) to a -sheet-rich aggregate. Functionally, A(1-38) reverses the negative impact of A(1-42) on long-term potentiation in acute hippocampal slices and on membrane conductance in primary neurons, and mitigates an A(1-42) phenotype in Caenorhabditis elegans. A(1-38) also reverses any loss of MTT conversion induced by A(1-40) and A(1-42) in HT-22 hippocampal neurons and APOE 4-positive human fibroblasts, although the combination of A(1-38) and A(1-42) inhibits MTT conversion in APOE 4-negative fibroblasts. A greater ratio of soluble A(1-42)/A(1-38) [and A(1-42)/A(1-40)] in autopsied brain extracts correlates with an earlier age-at-death in males (but not females) with a diagnosis of AD. These results suggest that A(1-38) is capable of physically counteracting, potentially in a sex-dependent manner, the neuropathological effects of the AD-relevant A(1-42).

Danielle Thierry-Mieg et al. (2003) Worm Breeder's Gazette "www.wormgenes.org , a new gene centered database"

Vahan Simonyan, Michel Potdevin, Mark Sienkiewicz, Yuji KOHARA, Jean Thierry-Mieg, Danielle Thierry-Mieg, Tadasu SHIN-I

[Worm Breeder's Gazette, 2003]

Wormgenes is a new resource for C.elegans offering a detailed summary about each gene and a powerful query system.

Tangrodchanapong T et al. (2020) Front Pharmacol "Frondoside A Attenuates Amyloid- Proteotoxicity in Transgenic Caenorhabditis elegans by Suppressing Its Formation."

Tangrodchanapong T, Sobhon P, Meemon K

[Front Pharmacol, 2020]

Oligomeric assembly of Amyloid- (A) is the main toxic species that contribute to early cognitive impairment in Alzheimer's patients. Therefore, drugs that reduce the formation of A oligomers could halt the disease progression. In this study, by using transgenic <i>Caenorhabditis elegans</i> model of Alzheimer's disease, we investigated the effects of frondoside A, a well-known sea cucumber <i>Cucumaria frondosa</i> saponin with anti-cancer activity, on A aggregation and proteotoxicity. The results showed that frondoside A at a low concentration of 1 M significantly delayed the worm paralysis caused by A aggregation as compared with control group. In addition, the number of A plaque deposits in transgenic worm tissues was significantly decreased. Frondoside A was more effective in these activities than ginsenoside-Rg3, a comparable ginseng saponin. Immunoblot analysis revealed that the level of small oligomers as well as various high molecular weights of A species in the transgenic <i>C. elegans</i> were significantly reduced upon treatment with frondoside A, whereas the level of A monomers was not altered. This suggested that frondoside A may primarily reduce the level of small oligomeric forms, the most toxic species of A. Frondoside A also protected the worms from oxidative stress and rescued chemotaxis dysfunction in a transgenic strain whose neurons express A. Taken together, these data suggested that low dose of frondoside A could protect against A-induced toxicity by primarily suppressing the formation of A oligomers. Thus, the molecular mechanism of how frondoside A exerts its anti-A aggregation should be studied and elucidated in the future.

Hodgkin JA (1998) International Journal of Developmental Biology "Seven types of pleiotropy."

Hodgkin JA

[International Journal of Developmental Biology, 1998]

Pleiotropy , a situation in which a single gene influences multiple phenotypic tra its, can arise in a variety of ways. This paper discusses possible underlying mechanisms and proposes a classification of the various phenomena involved.