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Joseph, B. et al. (2017) International Worm Meeting "Sibling Subtraction Method: A novel approach to identify causal mutations by WGS."

Fay, D., Joseph, B., Blouin, N.

[

International Worm Meeting,

2017]

Sequencing technologies have made it possible to carry out whole genome sequencing (WGS) to identify the genetic variants present in mutant organisms. In addition, methods have been developed that simultaneously facilitate mapping of the causal mutation. Currently, two principal strategies for "mapping by sequencing" are used by C elegans researchers. In one approach, mutants are crossed to the polymorphic HA strain, which provides extensive SNP mapping information and localization of the causal mutation. This approach, however, is not highly practical for complex genetic backgrounds, such as the case with genetic suppressors, and the numerous polymorphisms present in the HA strain can alter phenotypes in ways that are unpredictable. A second method, EMS density mapping, exploits of the higher concentration of signature-EMS alterations in regions surrounding causal mutations following multiple backcrosses. The resolution of EMS density mapping, however, can be quite low and this method is not applicable for alleles that arise spontaneously or through other methods. We recently obtained ~30 strong suppressors nekl-2; nekl-3 double-mutant larval lethality. Our screens included a novel strategy using counter-selection that was enabled by the peel-1 toxin. Notably, neither the nekl-2 or nekl-3 single mutants used for this screen display phenotypes on their own, nor did the majority of obtained suppressors. In order to identify the causal mutations that suppress nekl-2; nekl-3 lethality, we designed and implemented a novel refinement strategy we call the Sibling Subtraction Method (SSM). For our approach, suppressed strains were backcrossed several times to ensure Mendelian inheritance and to partially remove background mutations. While performing the final backcross, we isolated strains that were homozygous for the suppressor mutation as well as strains that were non-suppressed (i.e, homozygous wild type at the suppressor loci). These isolates were combined to create a Suppressed DNA pool and a Non-suppressed Comparitor DNA pool, respectively. Following WGS, alignment, and filtering steps, common variants present in both the Suppressed and Comparitor pools were subtracted. This resulted in a very small number of candidate variants (~5) that caused non-synonymous coding changes in each of the suppressor strains. Notably, we were able to subtract more than 95% of background variants using this method. The bioinformatics analysis was performed using Cloudmap workflows in the Galaxy web platform, which are freely available and widely used by the C elegans field. Our strategy was validated on five suppressor strains and, most importantly, is generally applicable for all varieties of mutations and genetic backgrounds. As such, SSM largely obviates the need to use the HA strain and provides better candidate refinement than EMS density mapping methods.

Arena JP (1994) Parasitol Today "Expression of Caenorahbditis elegans messenger RNA in Xenopus oocytes: A model systems to study the mechanism of action of avermectins."

Arena JP

[

Parasitol Today,

1994]

It has recently been shown that Xenopus oocytes injected with mRNA from the free-living nematode Caenorhabditis elegans express avermectin-sensitive chloride channels(1). Joseph Arena here reviews whet is known about the mechanism of action of avermectin and how these recent results relate to the mechanism in nematodes.

Neupane RP et al. (2019) Mar Drugs "Cytotoxic Sesquiterpenoid Quinones and Quinols, and an 11-Membered Heterocycle, Kauamide, from the Hawaiian Marine Sponge Dactylospongia elegans."

Williams PG, Neupane JB, Yip MLR, Turkson J, Parrish SM, Yoshida WY, Head JD, Harper MK, Neupane RP

[

Mar Drugs,

2019]

Several known sesquiterpenoid quinones and quinols (1-9), and kauamide (10), a new polyketide-peptide containing an 11-membered heterocycle, were isolated from the extracts of the Hawaiian marine sponge Dactylospongiaelegans. The planar structure of 10 was determined from spectroscopic analyses, and its relative and absolute configurations were established from density functional theory (DFT) calculations of the GIAO NMR shielding tensors, and advanced Marfey's analysis of the N-MeLeu residue, respectively. Compounds 1 and 3 showed moderate inhibition of -secretase 1 (BACE1), whereas 1-9 exhibited moderate to potent inhibition of growth of human glioma (U251) cells. Compounds 1-2 and 4-7 were also active against human pancreatic carcinoma (Panc-1) cells.

Chen CH et al. (2024) J Agric Food Chem "Novel Flavonol Alkaloids in Green Tea: Synthesis, Detection, and Anti-Alzheimer's Disease Effect in a Transgenic Caenorhabditis elegans CL4176 Model."

Ke JP, Liu Z, Yang Z, Liu G, Yang Y, Chen CH, Bao GH, Li JY, Yao G, Zhang YX

[

J Agric Food Chem,

2024]

Novel N-ethy-2-pyrrolidinone-substituted flavonols, myricetin alkaloids A-C (1-3), quercetin alkaloids A-C (4a, 4b, and 5), and kaempferol alkaloids A and B (6 and 7), were prepared from thermal reaction products of myricetin, quercetin, kaempferol─l-theanine, respectively. We used HPLC-ESI-HRMS/MS to detect 1-7 in 14 cultivars of green tea and found that they were all present in "Shuchazao," "Longjing 43", "Fudingdabai", and "Zhongcha 108" green teas. The structures of 1-4 and 6 were determined by extensive 1D and 2D NMR spectroscopies. These flavonol alkaloids along with their skeletal flavonols were assessed for anti-Alzheimer's disease effect based on molecular docking, acetylcholinesterase inhibition, and the transgenic Caenorhabditis elegans CL4176 model. Compound 7 strongly binds to the protein amyloid β (Aβ1-42) through hydrogen bonds (BE: -9.5 kcal/mol, Ki: 114.3 nM). Compound 3 (100 μM) is the strongest one in significantly extending the mean lifespan (13.4 ± 0.5 d, 43.0% promotion), delaying the Aβ1-42-induced paralysis (PT50: 40.7 ± 1.9 h, 17.1% promotion), enhancing the locomotion (140.0% promotion at 48 h), and alleviating glutamic acid (Glu)-induced neurotoxicity (153.5% promotion at 48 h) of CL4176 worms (p < 0.0001).

Phukhamsakda C et al. (2019) J Nat Prod "Sparticolins A-G, Biologically Active Oxidized Spirodioxynaphthalene Derivatives from the Ascomycete Sparticola junci."

Hyde KD, Phukhamsakda C, Cheng T, Macabeo APG, Stadler M, Huch V

[

J Nat Prod,

2019]

To explore the chemical diversity of metabolites from new species of Dothideomycetes, the ex-type strain of Sparticola junci was investigated. Seven highly oxygenated and functionalized spirodioxynaphthalene natural products incorporating carboxyalkylidene-cyclopentanoid (1-4), carboxyl-functionalized oxabicyclo[3.3.0]octane (5-6), and annelated 2-cyclopentenone/-lactone (7) units, sparticolins A-G, were isolated from submerged cultures of the fungus. Their chemical structures including their relative (and absolute) configurations were established through spectroscopic and X-ray crystallographic analyses. Sparticolin B (2) exhibited inhibitory activity against the Gram-positive bacteria Bacillus subtilis, Micrococcus luteus, and Staphylococcus aureus, while sparticolin G (7) showed antifungal activities against Schizosaccharomyces pombe and Mucor hiemalis. All other sparticolins were only weakly active against S. aureus and also showed weak activities against the nematode Caenorhabditis elegans. Compounds 2 and 7 also showed moderate cytotoxic activities against seven mammalian cell lines.

Sakoguchi H et al. (2019) Biosci Biotechnol Biochem "Nematocidal activity of 6-O-octanoyl- and 6-O-octyl-d-allose against larvae of Caenorhabditis elegans."

Kawanami Y, Sakoguchi H, Ishiyama H, Yanagita RC, Sato M, Shintani T

[

Biosci Biotechnol Biochem,

2019]

The nematocidal activities of the fatty acid esters of d-allose were examined using the larvae of C. elegans. Among the fatty acid esters, 6-O-octanoyl-d-allose (3) showed significant activity. 6-O-octanoyl-d-glucose (5) showed no activity, indicating that the D-allose moiety is essential for the nematocidal activity of 3. A nonhydrolyzable alkoxy analog 6-O-octyl-d-allose (6) also showed activity equivalent to that of 3.

Lazetic, V. et al. (2017) International Worm Meeting "A NimA-kinase pathway controls CDC-42 activity and actin organization in the epidermis."

Joseph, B., Fay, D., Lazetic, V.

[

International Worm Meeting,

2017]

During molting the epidermal apical extracellular matrix (cuticle) is extensively remodeled. We have shown that the NimA-related kinases, NEKL-2/NEK8 and NEKL-3/NEK6/NEK7, as well as their conserved ankyrin-repeat partners, MLT-2/ANKS6, MLT-3/ANKS3 and MLT-4/INVS, are essential for molting. Both NEKLs and MLTs are expressed in the epidermal syncytia and are specifically required in hyp7 for normal molting. Our studies indicate that NEKLs and MLTs act primarily within two complexes composed of NEKL-2-MLT-2-MLT-4 and NEKL-3-MLT-3, and that the MLTs function as scaffolds to ensure proper subcellular localization of the NEKLs. To understand how the NEKL-MLT network controls molting, we identified suppressors of nekl mutant molting defects. Our screens identified CDC-42, a highly conserved Rho-family GTPase, and its upstream regulator, KIN-25/SID-3. CDC-42 is important for the establishment of cell polarity and for organization of the actin cytoskeleton. KIN-25 is the ortholog of Activated CDC42 Kinase 1, which binds to CDC42 and inhibits its GTPase activity, thereby maintaining CDC42 in an active state. Notably, molting has been proposed to require extensive reorganization of actin within the epidermis. Specifically, actin is reorganized at each molt to form a series of circumferential bundles along the apical surface of hyp7, however, the mechanisms underlying this process are unknown. We found that inhibition of NEKL-MLT activities leads to defects in the pattern of apical actin and failure to form molting-specific actin bundles. Furthermore, expression studies indicate that components of the NEKL-MLT network colocalize with actin in specific regions of the epidermis. In addition, normal localization of CDC-42 in the epidermis depends on presence of NEKL-MLT proteins, and CDC-42 partially colocalizes with MLT-2. Our findings suggest that the NEKL-MLT network may negatively regulate CDC-42 activity and that partial inhibition of CDC-42 may thus reduce defects in nekl-mlt mutants. Interestingly, we also observed that downregulation of cdc-42 on its own can lead to molting defects, suggesting that there is a tight balance between NEKL-MLT and CDC-42 activities in the epidermis. Notably, studies in mammalian cells have implicated the MLT-4 ortholog, Inversin, in the regulation of CDC42 activity and apical actin organization. In addition, it was independently shown that the mammalian orthologs of NEKL-3, NEK6 and NEK7, physically associate with CDC42, however no functional connection between these proteins has been described. For the first time, our data provide in vivo evidence for a functional link between the NEKL-MLT network and regulation of the actin cytoskeleton through the CDC42 pathway.

Teixeira-Castro A et al. (2015) Brain "Serotonergic signalling suppresses ataxin 3 aggregation and neurotoxicity in animal models of Machado-Joseph disease."

Bessa C, Duarte-Silva S, Maciel P, Bessa J, Silverman RB, Miranda A, Kang S, Summavielle T, Oliveira S, da Silva Santos L, Neto MF, Esteves S, Brielmann RM, Neves-Carvalho A, Teixeira-Castro A, Oliveira P, Morimoto RI, Silva-Fernandes A, Jalles A

[

Brain,

2015]

Polyglutamine diseases are a class of dominantly inherited neurodegenerative disorders for which there is no effective treatment. Here we provide evidence that activation of serotonergic signalling is beneficial in animal models of Machado-Joseph disease. We identified citalopram, a selective serotonin reuptake inhibitor, in a small molecule screen of FDA-approved drugs that rescued neuronal dysfunction and reduced aggregation using a Caenorhabditis elegans model of mutant ataxin 3-induced neurotoxicity. MOD-5, the C. elegans orthologue of the serotonin transporter and cellular target of citalopram, and the serotonin receptors SER-1 and SER-4 were strong genetic modifiers of ataxin 3 neurotoxicity and necessary for therapeutic efficacy. Moreover, chronic treatment of CMVMJD135 mice with citalopram significantly reduced ataxin 3 neuronal inclusions and astrogliosis, rescued diminished body weight and strikingly ameliorated motor symptoms. These results suggest that small molecule modulation of serotonergic signalling represents a promising therapeutic target for Machado-Joseph disease.

Dube M et al. (2021) Biomolecules "Anthelmintic Activity and Cytotoxic Effects of Compounds Isolated from the Fruits of Ozoroa insignis Del. (Anacardiaceae)."

Haberli C, Fotso GW, Arnold N, Andrae-Marobela K, Saoud M, Rennert R, Dube M, Imming P, Keiser J

[

Biomolecules,

2021]

Ozoroa insignis Del. is an ethnobotanical plant widely used in traditional medicine for various ailments, including schistosomiasis, tapeworm, and hookworm infections. From the so far not investigated fruits of Ozoroa insignis, the anthelmintic principles could be isolated through bioassay-guided isolation using Caenorhabditis elegans and identified by NMR spectroscopic analysis and mass spectrometric studies. Isolated 6-[8(Z)-pentadecenyl] anacardic (1), 6-[10(Z)-heptadecenyl] anacardic acid (2), and 3-[7(Z)-pentadecenyl] phenol (3) were evaluated against the 5 parasitic organisms Schistosoma mansoni (adult and newly transformed schistosomula), Strongyloides ratti, Heligmosomoides polygyrus, Necator americanus, and Ancylostoma ceylanicum, which mainly infect humans and other mammals. Compounds 1-3 showed good activity against Schistosoma mansoni, with compound 1 showing the best activity against newly transformed schistosomula with 50% activity at 1M. The isolated compounds were also evaluated for their cytotoxic properties against PC-3 (human prostate adenocarcinoma) and HT-29 (human colorectal adenocarcinoma) cell lines, whereby compounds 2 and 3 showed antiproliferative activity in both cancer cell lines, while compound 1 exhibited antiproliferative activity only on PC-3 cells. With an IC50 value of 43.2 M, compound 3 was found to be the most active of the 3 investigated compounds.

Goncalves IL et al. (2020) Future Med Chem "New pharmacological findings linked to biphenyl DHPMs, kinesin Eg5 ligands: anticancer and antioxidant effects."

Figueiro F, Kagami LP, das Neves GM, Goethel G, Garcia SC, Munhoz T, Eifler-Lima VL, Sauer E, Rockenbach L, Goncalves IL, Oliveira Batasttini AM

[

Future Med Chem,

2020]

Background: Dihydropyrimidin-2-thiones (DHPMs) are a class of heterocyclic compound which have been intensively investigated mainly due to their anticancer activity as kinesin Eg5 inhibitors. Materials & methods: A library of N1 aryl substituted DHPMs were tested against glioma and bladder cancer cell lines. Quantitative structure-activity relationship (QSAR) investigation was performed in order to identify key elements of DHPMs linked with their antiproliferative effect. The toxicity of most active compounds was investigated using Caenorhabditis elegans as the model. Results & conclusion: DHPMs 9, 13 and 17 have been identified as having improved activity against glioma and bladder cell lines as compared with monastrol. Flow cytometry investigations showed that the new compounds induce cell cycle arrest in phase G2/M and cell death by apoptosis. In addition, compound 13was able to modulate the reactive oxygen species production in vivo in C. elegans. The biphenyl dihydropyrimidinthiones provided a safety profile in C. elegans.

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