Harder A et al. (2001) Parasitol Res "Activity of the cyclic depsipeptide emodepside (BAY 44-4400) against larval and adult stages of nematodes in rodents and the influence on worm survival."

von Samson-Himmelstjerna G, Harder A

[Parasitol Res, 2001]

The present investigations deal with the activity of the cyclic depsipeptide emodepside (BAY 44-4400) against larval and adult stages of three rodent nematodes. While emodepside acts strongly against the adult stages of the rat nematodes Nippostrongylus brasiliensis and Strongyloides ratti, as well as against the mouse nematode Heligmosomoides polygyrus, its actions against the larval stages of these nematodes vary according to the species. Thus, emodepside is highly effective against the lung and intestine larval stages of N. brasiliensis and S. ratti. By contrast. the larval stages of H. polygyrus in the intestine are only partly affected by higher emodepside dosages.

Harders RH et al. (2018) Cell Death Dis "Dynein links engulfment and execution of apoptosis via CED-4/Apaf1 in C. elegans."

Lande AD, Olsen A, Hesselager MO, Mandrup OA, Stensballe A, Morthorst TH, Harders RH, Bendixen E

[Cell Death Dis, 2018]

Apoptosis ensures removal of damaged cells and helps shape organs during development by removing excessive cells. To prevent the intracellular content of the apoptotic cells causing damage to surrounding cells, apoptotic cells are quickly cleared by engulfment. Tight regulation of apoptosis and engulfment is needed to prevent several pathologies such as cancer, neurodegenerative and autoimmune diseases. There is increasing evidence that the engulfment machinery can regulate the execution of apoptosis. However, the underlying molecular mechanisms are poorly understood. We show that dynein mediates cell non-autonomous cross-talk between the engulfment and apoptotic programs in the Caenorhabditis elegans germline. Dynein is an ATP-powered microtubule-based molecular motor, built from several subunits. Dynein has many diverse functions including transport of cargo around the cell. We show that both dynein light chain 1 (DLC-1) and dynein heavy chain 1 (DHC-1) localize to the nuclear membrane inside apoptotic germ cells in C. elegans. Strikingly, lack of either DLC-1 or DHC-1 at the nuclear membrane inhibits physiological apoptosis specifically in mutants defective in engulfment. This suggests that a cell fate determining dialogue takes place between engulfing somatic sheath cells and apoptotic germ cells. The underlying mechanism involves the core apoptotic protein CED-4/Apaf1, as we find that DLC-1 and the engulfment protein CED-6/GULP are required for the localization of CED-4 to the nuclear membrane of germ cells. A better understanding of the communication between the engulfment machinery and the apoptotic program is essential for identifying novel therapeutic targets in diseases caused by inappropriate engulfment or apoptosis.

Harders RH et al. (2024) G3 (Bethesda) "CED-6/GULP and Components of the Clathrin-mediated Endocytosis Machinery Act Redundantly to Correctly Display CED-1 on the Cell Membrane in C. elegans."

Feteira-Montero V, Morthorst TH, Landgrebe LE, Mortensen SB, Olsen A, Jensen HH, Lande AD, Wesseltoft JB, Fuglsang MS, Harders RH

[G3 (Bethesda), 2024]

CED-1 is a transmembrane receptor involved in the recognition of "eat-me" signals displayed on the surface of apoptotic cells and thus central for the subsequent engulfment of the cell corpse in C. elegans. The roles of CED-1 in engulfment are well established, as are its downstream effectors. The latter includes the adapter protein CED-6/GULP and the ABC family homolog CED-7. However, how CED-1 is maintained on the plasma membrane in the absence of engulfment is currently unknown. Here, we show that CED-6 and CED-7 have a novel role in maintaining CED-1 correctly on the plasma membrane. We propose that the underlying mechanism is via endocytosis as CED-6 and CED-7 act redundantly with clathrin and its adaptor, the AP2 complex, in ensuring correct CED-1 localization. In conclusion, CED-6 and CED-7 impact other cellular processes than engulfment of apoptotic cells.

Angier N (1991) New York Times "Life's machinery, seen in a transparent worm."

Angier N

[New York Times, 1991]

Through a microscope, they look like tiny crystal serpents, curving and slithering across the dish with an almost opiated languor, doubling back on themselves as though discovering their tails for the first time, or bumping up against a neighbor clumsily and then slowly recoiling. Beneath their translucent skin the pulsing muscle cells and nerve fibers are clearly visible, a sight so strange and so exquisite that it is hard to believe these creatures are common roundworms, found in gardens and compost heaps everywhere. And it is harder still to believe that such slippery squiggles of life are fast changing the face of fundamental biology.

Boris Shtonda et al. (2002) Worm Breeder's Gazette "Use of eat-5 as a selection marker for transgenics"

Leon Avery, Boris Shtonda

[Worm Breeder's Gazette, 2002]

eat-5 encodes a Caenorhabditis elegans innexin, a component of gap junctions between the muscle cells in the pharynx. The eat-5 null worms grow normally on an easy-to-eat strain of E. coli, HB101, but arrest as L1s on a harder-to-eat strain, DA837, a derivative of OP50. This difference in growth on two E. coli strains makes eat-5 a convenient co-transformation marker. This is particularly useful for situations when rol-6 is not good because the movement phenotype is studied. The gene of interest is co-injected into the eat-5(ad1402) null strain DA1402 with the genomic fragment containing the eat-5 gene, and the injected worms are transferred on the DA837 E. coli. Only transgenic F1s will grow. (It is important to use the null allele ad1402--mutants carrying the weak allele ad464 can grow on DA837.) Once the transgenic strains are isolated, they can be maintained on DA837, so a population of adult transgenic worms is easily obtained. If non-transgenic sibs are needed for controls, a transgenic worm can be transferred on HB101, so that non-transgenics can grow. In this case, however, a second marker, such as GFP, is needed to identify transgenics.

K Bull et al. (2004) European Worm Meeting "FURTHER INVESTIGATIONS TO CHARACTERISE THE PHYSIOLOGICAL EFFECTS OF THE NOVEL ANTHELMINTIC EMODEPSIDE ON C. ELEGANS"

A Harder, K Bull, R Walker, L Holden-Dye

[European Worm Meeting, 2004]

The cyclodepsipeptide, emodepside, is a novel broad spectrum anthelmintic with a distinct mode of action. Previous research in our laboratory, using the model nematode Caenorhabditis elegans, has demonstrated that emodepside potently paralyses pharyngeal and body wall muscle by binding to a latrophilin-like receptor at the neuromuscular junction, leading to activation of a G protein signalling pathway and the exocytosis of, possibly, an inhibitory neuropeptide(s). The present study reports on the effects of emodepside on C. elegans growth and development to obtain a better understanding of the physiological effects of this novel anthelmintic. The action of emodepside on the growth and locomotion of a synchronized population of C. elegans (N2 Bristol strain) was determined. Eggs were isolated from gravid adult hermaphrodite worms by the alkaline hypochlorite method (Lewis & Fleming, 1995) and were resuspended in M9 buffer (composition in g/litre: 6g Na2HPO4, 3g KH2PO4, 5g NaCl, 0.25g MgSO4.H2O). Approximately 100 eggs were placed on agar plates containing OP50 E. coli and either emodepside (10nM to 500nM) or vehicle control (1% ethanol). The plates were incubated for 5 days at 20C and the developmental progress of the worms, from hatching to fertile adulthood, was monitored. Locomotion was quantified by counting body bends/minute. Emodepside possesses a clear effect on the locomotion of C. elegans larval stage four (L4), with a concentration-dependent effect on normal sinusoidal movements. The IC50 for this effect on L4 worms was ~10nM. At an emodepside concentration of 50nM or more, the majority of L4 worms appeared unable to produce body bends in their anterior region, although they were still capable of forward locomotion via shallow and irregular sinusoidal movement in their posterior region. It was also observed that at 10nM or higher, emodepside retarded C. elegans growth to adulthood. Worm development in the presence of emodepside (10nM to 500nM) was measured quantitatively 43 and 64 hours after transfer of synchronised egg population onto the agar plates. Emodepside also appears to have a significant concentration-dependent impact on C. elegans egg laying. At 100nM or more, hermaphrodite adult worms became bloated with unlaid eggs, and at 500nM adult C. elegans appeared completely incapable of egg laying, eventually rupturing due to hatching of progeny within the parent worm.

Mounsey A et al. (2002) Genome Res "Evidence suggesting that a fifth of annotated Caenorhabditis elegans genes may be pseudogenes."

Mounsey A, Bauer P, Hope IA

[Genome Res, 2002]

Only a minority of the genes, identified in the Caenorhabditis elegans genome sequence data by computer analysis, have been characterized experimentally. We attempted to determine the expression patterns for a random sample of the annotated genes using reporter gene fusions. A low success rate was obtained for evolutionarily recently duplicated genes. Analysis of the data suggests that this is not due to conditional or low-level expression. The remaining explanation is that most of the annotated genes in the recently duplicated category are pseudogenes, a proportion corresponding to 20% of all of the annotated C elegans genes. Further Support for this Surprisingly high figure was sought by comparing sequences for families of recently duplicated C elegans genes. Although only a preliminary analysis, clear evidence for a gene having been recently inactivated by genetic drift was found for many genes in the recently duplicated category. At least 4% of the annotated C elegans genes call be recognized as pseudogenes simply from closer inspection of the sequence data. Lessons learned in identifying pseudogenes in C elegans could be of value in the annotation of the genomes of other species where, although there may be fewer pseudogenes, they may be harder to detect.

Janin L et al. (2014) Bioinformatics "Adaptive reference-free compression of sequence quality scores."

Janin L, Cox AJ, Rosone G

[Bioinformatics, 2014]

MOTIVATION: Rapid technological progress in DNA sequencing has stimulated interest in compressing the vast datasets that are now routinely produced. Relatively little attention has been paid to compressing the quality scores that are assigned to each sequence, even though these scores may be harder to compress than the sequences themselves. By aggregating a set of reads into a compressed index, we find that the majority of bases can be predicted from the sequence of bases that are adjacent to them and, hence, are likely to be less informative for variant calling or other applications. The quality scores for such bases are aggressively compressed, leaving a relatively small number at full resolution. As our approach relies directly on redundancy present in the reads, it does not need a reference sequence and is, therefore, applicable to data from metagenomics and de novo experiments as well as to re-sequencing data. RESULTS: We show that a conservative smoothing strategy affecting 75% of the quality scores above Q2 leads to an overall quality score compression of 1 bit per value with a negligible effect on variant calling. A compression of 0.68 bit per quality value is achieved using a more aggressive smoothing strategy, again with a very small effect on variant calling. AVAILABILITY: Code to construct the BWT and LCP-array on large genomic data sets is part of the BEETL library, available as a github repository at git@github.com:BEETL/BEETL.git.

K Amliwala et al. (2002) European Worm Meeting "A comparison of the action of ivermectin and emodepside on C.elegans"

K Amliwala, R J Walker, A Harder, J Willson, L Holden-Dye

[European Worm Meeting, 2002]

The cyclodepsipeptide, emodepside, is a novel broad spectrum anthelmintic with a distinct mode of action. Here we report preliminary studies comparing the commercially available broad spectrum anthelmintic ivermectin with emodepside. The free-living nematode Caenorhabditis elegans was used to identify the effective concentration window of emodepside inhibitory action on the growth and development of C.elegans. This information will subsequently be utilized in forward genetic strategies to identify emodepside resistant phenotypes, with the aim of identifying genetic determinants of emodepside action. The action of ivermectin and emodepside on the growth and locomotion of a synchronized population of C.elegans (N2 Bristol strain) was determined. Eggs were isolated from gravid adult hermaphrodite worms by the alkaline hypochlorite method (Lewis, Fleming, 1995) resuspended in M9 buffer (composition in g/liter Na2HPO4 6, KH2PO4 3, NaCl 5, MgSO4.7H2O 0.25). Approximately 100 eggs were placed on agar plates containing OP50-E.coli and either emodepside (100pM 950nM), ivermectin (100pm-9.5nM) or vehicle control (1% ethanol). The plates were incubated for 5 days at 20C and the developmental progress of the worms, through larval stages, to fertile adulthood, monitored. Locomotion was quantified by counting body bends/min. Ivermectin inhibits the development of larval stages to fertile adults at concentrations as low as 2.25nM. There exists a concentration dependent inhibition of larval development to adulthood up to 9.5nM, at which point 90% of juvenile worms are arrested at L1 stage (n=2). Emodepside has no observable effect on larval stages of C.elegans at any of the concentrations tested (n=4). However, in mature fertile adults there was a concentration-dependent effect on locomotion. The threshold for this effect was observed at 4.5nM (13 2 compared to control 25 1, body bends/min, n=4), and at 90nM locomotion was dramatically decreased to 1.3 0.3 body bends/min. It was observed that above 180nM emodepside growth to adulthood was retarded, on average by 24 hrs, when compared with worms on lower emodepside concentration and control plates. Furthermore, at 950nM hermaphrodite adults were arrested before egg laying could occur (n=2). In conclusion, these data suggest that emodepside has a distinctly different anthelmintic mechanism to ivermectin, in that it interacts with a target that is expressed, or functionally important, in the adult stage only. Furthermore, this target is involved in motor control.

J Willson et al. (2002) European Worm Meeting "An investigation into the mechanism of action of the novel anthelmintic emodepside using A.suum"

J Willson, A Harder, L Holden-Dye, R J Walker, K Amliwala

[European Worm Meeting, 2002]

Resistance of parasitic nematodes to existing anthelmintics has encouraged the search for novel compounds. A new compound, emodepside, a 24 membered cyclic depsipeptide, acts as a potent, broad-spectrum anthelmintic. It causes fast onset paralysis of nematodes, favouring the view that it is neuropharmalogically active. The aim of this project is to define and characterise the site of action of emodepside. The action of emodepside on the body wall muscle of the nematode Ascaris suum was investigated. It was shown that emodepside relaxes the muscle in a concentration-dependent and irreversible manner. Dorsal muscle strips (DMS) were bathed in artificial perienteric fluid (APF). The DMS was contracted using acetylcholine (ACh; 30M, 30secs). The amplitude of this contraction after pretreatment of the DMS with emodepside (10M) was expressed as a percentage of the control contraction (i.e. without emodepside pretreatment). Emodepside reduced the contraction by 39% 4 (n=9). Emodepside was also shown to relax the DMS following prolonged contraction with ACh. The DMS was contracted with ACh (30M, 10mins). A slow relaxation of the DMS was then observed over a period of 10mins. Addition of 10M emodepside at the point of maximum contraction, caused a significantly (P<0.005) faster relaxation of the DMS, 9.0% min-1 0.3 (n=5), compared to control (6.1% min-1 0.6, n=5). The action of emodepside was similar to that of an inhibitory neuropeptide PF2 (SADPNFLRFamide), and differed from that of the inhibitory neurotransmitter GABA. Application of PF2 (1M) at the maximum point of ACh contraction caused a faster relaxation of the DMS (9.2%min-1 0.4 n=6) compared to ACh control (7.4% min-1 0.3, n=6), which was similar to emodepside (9% min-1 0.4, n=6). In contrast to the slow relaxation of the muscle observed with emodepside and PF2, the response to GABA was rapid and returned to base line tension. To elucidate whether emodepside acts pre-or post-synaptically at the neuromuscular junction, the effect of emodepside was investigated on a denervated muscle strip preparation. Application of emodepside (10M) to the denervated muscle strip caused no reduction in ACh induced contraction (n=7). These data suggest that emodepside may inhibit nematode motility by triggering the release of an inhibitory neuropeptide at the neuromuscular junction. Emodepside therefore acts pre-synaptically to cause a relaxation of worm muscle. Emodepside action is similar to that of the inhibitory peptide PF2 and differs from that of GABA. Currently I am determining how emodepside acts pre-synaptically to cause release of an inhibitory peptide. To this end the effect of emodepside on c.elegans will be investigated, to give an insight into the molecular mechanisms of emodepside action.