Lee C et al. (2017) Bio Protoc "Single-molecule RNA Fluorescence in situ Hybridization (smFISH) in Caenorhabditis elegans."

Sorensen EB, Lee C, Seidel HS, Lynch TR, Crittenden SL, Kimble J

[Bio Protoc, 2017]

Single-molecule RNA fluorescence <i>in situ</i> hybridization (smFISH) is a technique to visualize individual RNA molecules using multiple fluorescently-labeled oligonucleotide probes specific to the target RNA ( Raj <i>et al.</i>, 2008 ; Lee <i>et al.</i>, 2016a ). We adapted this technique to visualize RNAs in the <i>C. elegans</i> whole adult worm or its germline, which enabled simultaneous recording of nascent transcripts at active transcription sites and mature mRNAs in the cytoplasm ( Lee <i>et al.</i>, 2013 and 2016b). Here we describe each step of the smFISH procedure, reagents, and microscope settings optimized for <i>C. elegans</i> extruded gonads.

Markowicz J et al. (2019) Eur J Pharmacol "Antitumor and anti-nematode activities of alpha-mangostin."

Mangiardi L, Markowicz J, Maj P, Uram L, Rode W, Sobich J

[Eur J Pharmacol, 2019]

alpha-Mangostin, one of the major xanthones isolated from pericarp of mangosteen (Garcinia mangostana Linn), exhibits a wide range of pharmacological activities, including antioxidant, anti-inflammatory, antimicrobial as well as anticancer, both in in vitro and in vivo studies. In the present study, alpha-mangostin's anti-cancer and anti-parasitic properties were tested in vitro against three human cell lines, including squamous carcinoma (SCC-15) and glioblastoma multiforme (U-118 MG), compared to normal skin fibroblasts (BJ), and in vivo against Caenorhabditis elegans. The drug showed cytotoxic activity, manifested by decrease of cell viability, inhibition of proliferation, induction of apoptosis and reduction of adhesion at concentrations lower than 10M (the IC₅₀ values were 6.43, 9.59 and 8.97M for SCC-15, U-118 MG and BJ, respectively). The toxicity, causing cell membrane disruption and mitochondria impairment, was selective against squamous carcinoma with regard to normal cells. Moreover, for the first time anti-nematode activity of alpha-mangostin toward C. elegans was described (the LC₅₀=3.8+/-0.5M), with similar effect exerted by mebendazole, a well-known anthelmintic drug.

Dawes-Hoang RE et al. (2003) Dev Cell "Bringing classical embryology to C elegans gastrulation."

Zallen JA, Wieschaus EF, Dawes-Hoang RE

[Dev Cell, 2003]

In a recent paper, Lee and Goldstein develop an explant assay that recapitulates key aspects of gastrulation in C. elegans and permits classical embryological manipulations. The resulting detailed analysis of cell behavior will ultimately extend to broader issues, such as, whether morphogenesis can be described as the sum of single-cell events or if unique phenomena emerge at the multicellular level.

Bauer, Jack et al. (2021) MicroPubl Biol

Labb, Jean-Claude, Lacroix, La, Bauer, Jack

[MicroPubl Biol, 2021]

To perturb actomyosin function in the primordial germ line, we first monitored germ line organization in L1-stage animals bearing temperature-sensitive (ts) alleles in genes encoding actomyosin regulators and that were reported to interfere with cytokinesis during embryogenesis (Davies et al. 2014). Previous work demonstrated that the initial stages of germline expansion occur normally in cyk-4(ts) and zen-4(ts) animals raised at restrictive temperature from the L1 stage (Lee et al. 2018). We found that primordial germ line organization in cyk-1(ts), nmy-2(ts), cyk-4(ts) or zen-4(ts) L1 larvae maintained at restrictive temperature for 12h was no different than control (Figure 1A-B). Furthermore, the first primordial germ cell (PGC) division occurred normally upon feeding these animals at restrictive temperature with typical bacterial food (E. coli OP50). As noted previously (Lee et al. 2018), germ line disorganization and sterility were observed in all cases when animals reached adulthood (Figure 1B).

Kwon JY et al. (2001) FEBS Lett "The MED-7 transcriptional mediator encoded by let-49 is required for gonad and germ cell development in Caenorhabditis elegans."

Kwon JY, Kim-Ha J, Lee J, Lee BJ

[FEBS Lett, 2001]

Transcription mediators are evolutionarily conserved from yeast to human. We previously reported the specific in vivo roles of mediators during development. Transcriptional mediators including med-6, med-7, and med-10 were shown to be involved in the regulated transcription of specific genes., but not in the transcription of ubiquitous genes. In this report we have identified and characterized the Caenorhabditis elegans med-7 gene. A genetic mutation in the med-7 gene was identified by comparing genetic and physical maps and determining the molecular lesion. let-49 was found to have a nonsense mutation in the coding region of the med-17 gene. The identification of let-49 as the med-7 gene was confirmed by rescue experiments. The phenotype of the let-49 mutation indicated that the med-7 gene is required for normal postembryonic development. RNAi experiments showed that med-7 is also involved in embryogenesis and the gonad and germ cell development.

Miranda MC et al. (2024) MicroPubl Biol "Investigating the effects of antipsychotic drugs as a treatment for improving the activity of the unc-33 /Dpysl2 gene in C. elegans."

Miranda MC, Hyde J, Holgado A, Salazar K, Bell B

[MicroPubl Biol, 2024]

Prenatal stress is hypothesized to contribute to the development of schizophrenia. Lee and colleagues determined that prenatal stress in rats decreases levels of Dpysl2, which is found to be inactivated in schizophrenic patients. UNC-33 , the homolog to Dpysl2 in <i>C. elegans</i> , is important for axonal outgrowth and synapse formation. Herein, we study the effects of antipsychotic drugs on developing <i>C.elegans</i> exposed to stress through high temperatures. Results indicate that the <i>unc-33</i> promoter was not impacted by antipsychotic drug treatment, but the lifespan was decreased.

Neuhaus K et al. (2016) Arch Microbiol "Probiotic Enterococcus faecalis Symbioflor() down regulates virulence genes of EHEC in vitro and decrease pathogenicity in a Caenorhabditis elegans model."

Spanier B, Zolch B, Vogel RF, Ehrmann MA, Simon S, Lamparter MC, Neuhaus K, Landstorfer R

[Arch Microbiol, 2016]

Enterohemorrhagic E. coli O157:H7 (EHEC) shorten the lifespan of Caenorhabditis elegans compared to avirulent bacteria. Co-feeding EHEC with Enterococcus faecalis Symbioflor() significantly increased the worms' lifespan. The transcriptome of EHEC grown in vitro with or without Symbioflor() was analyzed using RNA-seq. The analysis revealed downregulation of several virulence-associated genes in the presence of Symbioflor(), including virulence key genes (e.g., LEE, flagellum, quorum-sensing). The downregulation of the LEE genes was corroborated by lux-transposon mutants. Upregulated genes included acid response genes, due to a decrease in pH exerted by Symbioflor(). Further genes indicate cellular stress in EHEC (e.g. prophage/mobile elements involved in excision, cell lysis, and cell division inhibition). Thus, the observed protection of C. elegans during an EHEC infection by the probiotic Symbioflor() is suggested to be caused by triggering concomitant transcriptomic changes. To verify the biological relevance of this modulation, exemplary genes found to be influenced by Symbioflor() were knocked out (fliD, espB, Z3136, Z3917, and L7052). The lifespan of nematodes changed when using knock-outs as food source and the effect could be complemented in trans. In summary, Symbioflor() appears to be a protective probiotic in the nematode model.

Wang ST et al. (2021) Mol Microbiol "OmpR coordinates the expression of virulence factors of enterohemorrhagic Escherichia coli in the alimentary tract of Caenorhabditis elegans."

Wang ST, Kuo CJ, Chen JW, Chen CS, Lee TM, Huang CW

[Mol Microbiol, 2021]

Enterohemorrhagic Escherichia coli (EHEC), an enteropathogen that colonizes in the intestine, causes severe diarrhea and hemorrhagic colitis in humans by the expression of the type III secretion system (T3SS) and Shiga-like toxins (Stxs). However, how EHEC can sense and respond to the changes in the alimentary tract and coordinate the expression of these virulence genes remains elusive. The T3SS-related genes are known to be regulated by the locus of enterocyte effacement (LEE)-encoded regulators, such as Ler, as well as non-LEE-encoded regulators in response to different environmental cues. Herein, we report that OmpR, which participates in the adaptation of E. coli to osmolarity and pH alterations, is required for EHEC infection in Caenorhabditis elegans. OmpR protein was able to directly bind to the promoters of ler and stx1 (Shiga-like toxin 1) and regulate the expression of T3SS and Stx1, respectively, at the transcriptional level. Moreover, we demonstrated that the expression of ler in EHEC is in response to the intestinal environment and is regulated by OmpR in C. elegans. Taken together, we reveal that OmpR is an important regulator of EHEC which coordinates the expression of virulence factors during gastrointestinal infection in vivo.

Miller MA et al. (2001) Science "A sperm cytoskeletal protein that signals oocyte meiotic maturation and ovulation."

Schedl TB, Kosinski ME, Miller MA, Lee MH, Greenstein D, Caprioli RM, Nguyen VQ

[Science, 2001]

Caenorhabditis elegans oocytes, like those of most animals, arrest during meiotic prophase. Sperm promote the resumption of meiosis (maturation) and contraction of smooth muscle-like gonadal sheath cells, which are required for ovulation. We show that the major sperm cytoskeletal protein (MSP) is a bipartite signal for oocyte maturation and sheath contraction. MSP also functions in sperm locomotion, playing a role analogous to actin. Thus, during evolution, MSP has acquired extracellular signaling and intracellular cytoskeletal functions for reproduction. Proteins with MSP-like domains are found in plants, fungi, and other animals, suggesting that related signaling functions may exist in other phyla.AD - Department of Cell Biology, Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.FAU - Miller, M AAU - Miller MAFAU - Nguyen, V QAU - Nguyen VQFAU - Lee, M HAU - Lee MHFAU - Kosinski, MAU - Kosinski MFAU - Schedl, TAU - Schedl TFAU - Caprioli, R MAU - Caprioli RMFAU - Greenstein, DAU - Greenstein DLA - engID - CA09592/CA/NCIID - GM57173/GM/NIGMSID - GM58008/GM/NIGMSID - HD07043/HD/NICHDID - HD25614/HD/NICHDPT - Journal ArticleCY - United StatesTA - ScienceJID - 0404511RN - 0 (Carrier Proteins)RN - 0 (Helminth Proteins)RN - 0 (MAP Kinase Signaling System)RN - 0 (Membrane Proteins)RN - 0 (Recombinant Proteins)RN - 0 (VAP-33 protein)RN - 0 (major sperm protein, nematode)RN - EC 2.7.1.- (Mitogen-Activated Protein Kinases)SB - IM

Robins H et al. (2005) Proc Natl Acad Sci U S A. "Incorporating structure to predict microRNA targets."

Li Y, Robins H, Padgett RW

[Proc Natl Acad Sci U S A., 2005]

MicroRNAs (miRNAs) are a recently discovered set of regulatory genes that constitute up to an estimated 1% of the total number of genes in animal genomes, including Caenorhabditis elegans, Drosophila, mouse, and humans [Lagos-Quintana, M., Rauhut, R., Lendeckel, W. M Tuschl, T. (2001) Science 294, 853-858; Lai, E. C., Tomancak, P., Williams, R. W. M Rubin, G.M. (2003) Genome Biol. 4, R42; Lau, N. C., Lim, L. P., Weinstein, E. G. M Bartel, D. P. (2001) Science 294, 858-862; Lee, R. C. M Ambros, V. (2001) Science 294, 862-8644; and Lee, R. C., Feinbaum, R. L. M Ambros, V. (1993) Cell 115, 787-798]. In animals, miRNAs regulate genes by attenuating protein translation through imperfect base pair binding to 3' UTR sequences of target genes. A major challenge in understanding the regulatory role of miRNAs is to accurately predict regulated targets. We have developed an algorithm for predicting targets that does not rely on evolutionary conservation. As one of the features of this algorithm, we incorporate the folded structure of mRNA. By using Drosophila miRNAs as a test case, we have validated our predictions in 10 of 15 genes tested. One of these validated genes is mad as a target for bantam. Furthermore, our computational and experimental data suggest that miRNAs have fewer targets than previously reported.