Liang J et al. (2017) PLoS One "Chloride intracellular channel proteins respond to heat stress in Caenorhabditis elegans."

Boissinot S, Shaulov Y, Hoque T, Savage-Dunn C, Liang J

[PLoS One, 2017]

Chloride intracellular channel proteins (CLICs) are multi-functional proteins that are expressed in various cell types and differ in their subcellular location. Two CLIC homologs, EXL-1 (excretory canal abnormal like-1) and EXC-4 (excretory canal abnormal- 4), are encoded in the Caenorhabditis elegans genome, providing an excellent model to study the functional diversification of CLIC proteins. EXC-4 functions in excretory canal formation during normal animal development. However, to date, the physiological function of EXL-1 remains largely unknown. In this study, we demonstrate that EXL-1 responds specifically to heat stress and translocates from the cytoplasm to the nucleus in intestinal cells and body wall muscle cells under heat shock. In contrast, we do not observe EXC-4 nuclear translocation under heat shock. Full protein sequence analysis shows that EXL-1 bears a non-classic nuclear localization signal (NLS) that EXC-4 is lacking. All mammalian CLIC members have a nuclear localization signal, with the exception of CLIC3. Our phylogenetic analysis of the CLIC gene families across various animal species demonstrates that the duplication of CLICs in protostomes and deuterostomes occurred independently and that the NLS was subsequently lost in amniotes and nematodes, suggesting convergent evolution. We also observe that EXL-1 nuclear translocation occurs in a timely ordered manner in the intestine, from posterior to anterior regions. Finally, we find that exl-1 loss of function mutants are more susceptible to heat stress than wild-type animals, demonstrating functional relevance of the nuclear translocation. This research provides the first link between CLICs and environmental heat stress. We propose that C. elegans CLICs evolved to achieve different physiological functions through subcellular localization change and spatial separation in response to external or internal signals.

Liang J et al. (2007) Dev Biol "Transcriptional repressor and activator activities of SMA-9 contribute differentially to BMP-related signaling outputs."

Yu L, Liang J, Yin J, Savage-Dunn C

[Dev Biol, 2007]

In the nematode Caenorhabditis elegans, the BMP-related growth factor DBL-1 regulates body size and male tail morphogenesis via a conserved receptor/Smad signaling pathway. Smads are transcription factors, but rely on transcription cofactors for appropriate regulation of target genes in response to TGF-beta- and BMP-related signals. In the DBL-1 pathway, sma-9 encodes multiple zinc finger transcription factors homologous to Drosophila Schnurri, which functions in Dpp/BMP signaling. We have studied the molecular functions of SMA-9 as a model for transcription cofactor-dependent regulation of gene expression. Using SMA-9 fusions to known transcriptional activators and repressors, we demonstrate that SMA-9 acts primarily as a transcriptional repressor in body size regulation in vivo. In contrast, both activator and repressor functions contribute to male tail patterning. We further show that different SMA-9 regions have intrinsic repressor and activator activities using a yeast transcription assay. We use microarray analysis to identify transcriptional target genes in body size regulation. Consistent with the importance of repression in mediating body size regulation, we find more repressed genes than activated genes in this pool. Finally, we identify five transcriptional targets with body size and/or male tail patterning phenotypes, including transcription factors related to Runx and fos and signaling molecules related to hedgehog and patched. Our results thus suggest that SMA-9 products function differentially as transcriptional repressors and activators in DBL-1/BMP pathway regulated body size and male tail morphogenesis.

Liang J et al. (2018) J Vis Exp "Detecting Protein Subcellular Localization by Green Fluorescence Protein Tagging and 4',6-Diamidino-2-phenylindole Staining in Caenorhabditis elegans."

Flores L, Liang J, De Castro A

[J Vis Exp, 2018]

In this protocol, a green fluorescence protein (GFP) fusion protein and 4',6-diamidino-2-phenylindole (DAPI) staining are used to track protein subcellular localization changes; in particular, a nuclear translocation under a heat stress condition. Proteins react correspondingly to external and internal signals. A common mechanism is to change its subcellular localization. This article describes a protocol to track protein localization that does not require an antibody, radioactive labeling, or a confocal microscope. In this article, GFP is used to tag the target protein EXL-1 in C. elegans, a member of the chloride intracellular channel proteins (CLICs) family, including mammalian CLIC4. An integrated translational exl-1::gfp transgenic line (with a promoter and a full gene sequence) was created by transformation and -radiation, and stably expresses the gene and gfp. Recent research showed that upon heat stress, not oxidative stress, EXL-1::GFP accumulates in the nucleus. Overlapping the GFP signal with both the nuclei structure and the DAPI signals confirms the EXL-1 subcellular localization changes under stress. This protocol presents two different fixation methods for DAPI staining: ethanol fixation and acetone fixation. The DAPI staining protocol presented in this article is fast and efficient and preserves both the GFP signal and the protein subcellular localization changes. This method only requires a fluorescence microscope with Nomarski, a FITC filter, and a DAPI filter. It is suitable for a small laboratory setting, undergraduate student research, high school student research, and biotechnology classrooms.

Liang J et al. (2013) J Vis Exp "Using RNA-mediated interference feeding strategy to screen for genes involved in body size regulation in the nematode C. elegans."

Liang J, Savage-Dunn C, Xiong S

[J Vis Exp, 2013]

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- -llike ligand DBL-1, so this assay is appropriate for identification of TGF- signaling components. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Liang J et al. (2003) Development "The Caenorhabditis elegans schnurri homolog sma-9 mediates stage- and cell type-specific responses to DBL-1 BMP-related signaling."

Foehr ML, Savage-Dunn C, Tokarz R, Emmons SW, Lints R, Liang J, Liu J, Yu L

[Development, 2003]

In Caenorhabditis elegans, the DBL-1 pathway, a BMP/TGFbeta-related signaling cascade, regulates body size and male tail development. We have cloned a new gene, sma-9, that encodes the C elegans homolog of Schnurri, a large zinc finger transcription factor that regulates dpp target genes in Drosophila. Genetic interactions, the sma-9 loss-of-function phenotype, and the expression pattern suggest that sma-9 acts as a downstream component and is required in the DBL-1 signaling pathway, and thus provide the first evidence of a conserved role for Schnurri proteins in BMP signaling. Analysis of sma-9 mutant phenotypes demonstrates that SMA-9 activity is temporally and spatially restricted relative to known DBL-1 pathway components. In contrast with Drosophila schnurri, the presence of multiple alternatively spliced sma-9 transcripts suggests protein isoforms with potentially different cell sublocalization and molecular functions. We propose that SMA-9 isoforms function as transcriptional cofactors that confer specific responses to DBL-1 pathway

Liang JJH et al. (2020) J Neurogenet "The contribution of C. elegans neurogenetics to understanding neurodegenerative diseases."

McKinnon IA, Rankin CH, Liang JJH

[J Neurogenet, 2020]

Since <i>Caenorhabditis elegans</i> was first introduced as a genetic model organism by Sydney Brenner, researchers studying it have made significant contributions in numerous fields including investigations of the pathophysiology of neurodegenerative diseases. The simple anatomy, optical transparency, and short life-span of this small nematode together with the development and curation of many openly shared resources (including the entire genome, cell lineage and the neural map of the animal) allow researchers using <i>C. elegans</i> to move their research forward rapidly in an immensely collaborative community. These resources have allowed researchers to use <i>C. elegans</i> to study the cellular processes that may underlie human diseases. Indeed, many disease-associated genes have orthologs in <i>C. elegans,</i> allowing the effects of mutations in these genes to be studied in relevant and reproducible neuronal cell-types at single-cell resolution <i>invivo.</i> Here we review studies that have attempted to establish genetic models of specific human neurodegenerative diseases (ALS, Alzheimer's Disease, Parkinson's Disease, Huntington's Disease) in <i>C. elegans</i> and what they have contributed to understanding the molecular and genetic underpinnings of each disease. With continuous advances in genome engineering, research conducted using this small organism first established by Brenner, Sulston and their contemporaries will continue to contribute to the understanding of human nervous diseases.

Liang W et al. (2019) J Comput Biol "Bayesian Detection of Abnormal Asynchrony of Division Between Sister Cells in Mutant Caenorhabditis elegans Embryos."

Yang Y, Zhao Z, Hu J, Liang W, Fang Y

[J Comput Biol, 2019]

Cell division timing is critical for cell fate specification and morphogenesis during embryogenesis, but how division timings are regulated among cells during development is poorly understood. In this article, we focus on the comparison of asynchrony of division, that is, difference of lifetime, between sister cells (ADS) among wild-type and mutant individuals of Caenorhabditis elegans. On the one hand, due to extreme imbalance between wild-type individuals and mutant-type samples, direct comparison of two distributions of ADS between wild type and mutant type is not feasible. On the other hand, we originally found that the ADS is correlated with the lifespan of the corresponding mother cell in wild type. Hence, a semiparametric Bayesian quantile regression method where lifetime of the mother cell is taken as covariate is developed to estimate the 95% confidence curve of ADS in wild type and then ADS of mutant type is classified as abnormal if outside the corresponding confidence interval. A high accuracy of our method is demonstrated by a large-scale simulation study. Real data analysis shows that ADS is related to gene function and expression quantitatively.

Liang S et al. (2020) Int J Nanomedicine "Comprehensive Analysis of SiNPs on the Genome-Wide Transcriptional Changes in Caenorhabditis elegans."

Hu H, Sun Z, Duan J, Liang S, Li G, Jing H, Gao S, Zhang J

[Int J Nanomedicine, 2020]

Background: remain largely unclear. Purpose: . Methods and Results: . Conclusion: .

Liang L et al. (2023) Int J Biol Macromol "Anti-aging activities of Rehmannia glutinosa Libosch. crude polysaccharide in Caenorhabditis elegans based on gut microbiota and metabonomic analysis."

Liang L, Shen X, Wang Y, Liang Y, Luo R, Zhong L, Shi R, Yue Y, Zhao M, Du J, Shu Z, Cao X, Yang M

[Int J Biol Macromol, 2023]

Aging is a degenerative progress, accompanied by oxidative damage, metabolic disorders and intestinal flora imbalance. Natural macromolecular polysaccharides have shown excellent anti-aging and antioxidant properties, while maintaining metabolic and intestinal homeostasis. The molecular weight, monosaccharide composition, infrared spectrum and other chemical structure information of four Rehmannia glutinosa polysaccharides (RG50, RG70, RG90, RGB) were determined, and their free radical scavenging ability was assessed. Molecular weight and monosaccharide composition analysis exhibited that RG50 (2-72&#x202f;kDa), RG70 (3.2-37&#x202f;kDa), RG70 (3-42&#x202f;kDa), and RGB (3.1-180&#x202f;kDa) were heteropolysaccharide with significant different monosaccharide species and molar ratios. We found that RG70 had the best antioxidant activity in vitro and RG70 could enhance the antioxidant enzyme system of Caenorhabditis elegans, diminished lipofuscin and reactive oxygen species levels, up-regulate the expression of daf-16, skn-1 and their downstream genes, and down-regulate the expression of age-1. Metabolomics results showed that RG70 mainly influenced glycine, serine and threonine metabolism and citric acid cycle. 16S rRNA sequencing showed that RG70 significantly up-regulated the abundance of Lachnospiraceae_NK4B4_group, which were positively correlated with amino acid metabolism and energy cycling. These results suggest that RG70 may delay aging by enhancing antioxidant effects, affecting probiotics and regulating key metabolic pathways.

McGhee JD (1987) Biochemistry "Purification and characterization of a carboxylesterase from the intestine of the nematode Caenorhabditis elegans."

McGhee JD

[Biochemistry, 1987]

The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].