Xu S et al. (2014) J Vis Exp "Methods for skin wounding and assays for wound responses in C. elegans."

Xu S, Chisholm AD

[J Vis Exp, 2014]

The C. elegans epidermis and cuticle form a simple yet sophisticated skin layer that can repair localized damage resulting from wounding. Studies of wound responses and repair in this model have illuminated our understanding of the cytoskeletal and genomic responses to tissue damage. The two most commonly used methods to wound the C. elegans adult skin are pricks with microinjection needles, and local laser irradiation. Needle wounding locally disrupts the cuticle, epidermis, and associated extracellular matrix, and may also damage internal tissues. Laser irradiation results in more localized damage. Wounding triggers a succession of readily assayed responses including elevated epidermal Ca(2+) (seconds-minutes), formation and closure of an actin-containing ring at the wound site (1-2 hr), elevated transcription of antimicrobial peptide genes (2-24 hr), and scar formation. Essentially all wild type adult animals survive wounding, whereas mutants defective in wound repair or other responses show decreased survival. Detailed protocols for needle and laser wounding, and assays for quantitation and visualization of wound responses and repair processes (Ca dynamics, actin dynamics, antimicrobial peptide induction, and survival) are presented.

Xu S et al. (2012) Worm "The wounded worm: Using C. elegans to understand the molecular basis of skin wound healing."

Hsiao TI, Xu S, Chisholm AD

[Worm, 2012]

The ability to heal wounds is an ancient and conserved function of epidermal epithelial layers. The importance of skin wound healing to human life and biology has long been evident, however many of the molecular mechanisms underlying wound repair remain little understood. In the past several years, analysis of the C. elegans innate immune response to fungal infection of the epidermis has led to investigations of the ability of the C. elegans skin to respond to damage. In a recent paper we used live imaging to investigate the cell biological basis of wound repair in the adult C. elegans epidermis. We found that needle or laser injury of the skin triggers a large and sustained increase in epidermal calcium. Epidermal calcium signals appear to specifically promote actin-dependent processes of wound closure. The innate immune and wound closure responses act in parallel to promote survival after injury. Our findings indicate that wounding triggers multiple signals in the C. elegans skin. C. elegans offers a tractable model to dissect how epidermal epithelia activate coordinated responses to repair damage.

Xu S et al. (2016) Food Funct "Antioxidant activity in vitro and in vivo of the polysaccharides from different varieties of Auricularia auricula."

Jiang K, Zhang Y, Xu S

[Food Funct, 2016]

In this study, five different kinds of polysaccharides (AAP1, AAP2, AAP3, AAP4, and AAP5) were extracted from different varieties of Auricularia auricula through an alkali extraction process. Furthermore, the crude polysaccharides were deproteinized by the Sevag method. Auricularia auricula produced in the Shanxi province had the highest content of polysaccharide, 53.02%. The monosaccharide composition was determined by the GC method. Their antioxidant capacities in vitro were assessed by radical-scavenging capacity (DPPH, superoxide, and hydroxyl radicals), metal chelating ability and reducing-power methods. In addition, the evaluation of their antioxidant effects in vivo was performed using the C. elegans model. The yield of crude polysaccharides, monosaccharide composition and antioxidant activity of Auricularia auricula polysaccharides (AAPs) were different among samples from various sources. Among them, the strongest antioxidant activity was shown for AAP1, consisting of arabinose, xylose, 2-deoxy-d-glucose, mannose, glucose, and N-acetyl-d-glucosamine with the molar ratio of 1:0.44:0.33:1.67:1:0.17. It could scavenge free radicals, up-regulate stress-resistance-related enzymes including superoxide dismutase (SOD) by 70.04 8.75% and CAT by 117.32 8.06% and reduce the level of reactive oxygen species (ROS) in C. elegans under oxidative stress. The present results suggested that variety was an important factor that affects the antioxidant activity of A. auricula polysaccharides.

Xu S et al. (2016) Sci Rep "Highly efficient optogenetic cell ablation in C. elegans using membrane-targeted miniSOG."

Xu S, Chisholm AD

[Sci Rep, 2016]

The genetically encoded photosensitizer miniSOG (mini Singlet Oxygen Generator) can be used to kill cells in C. elegans. miniSOG generates the reactive oxygen species (ROS) singlet oxygen after illumination with blue light. Illumination of neurons expressing miniSOG targeted to the outer mitochondrial membrane (mito-miniSOG) causes neuronal death. To enhance miniSOG's efficiency as an ablation tool in multiple cell types we tested alternative targeting signals. We find that membrane targeted miniSOG allows highly efficient cell killing. When combined with a point mutation that increases miniSOG's ROS generation, membrane targeted miniSOG can ablate neurons in less than one tenth the time of mito-miniSOG. We extend the miniSOG ablation technique to non-neuronal tissues, revealing an essential role for the epidermis in locomotion. These improvements expand the utility and throughput of optogenetic cell ablation in C. elegans.

Xu S et al. (2011) Int J Parasitol "In vivo transfection of developmentally competent Brugia malayi infective larvae."

Tzertzinis G, Liu C, Evans CC, Unnasch TR, Xu S, Kaplan R, Ghedin E

[Int J Parasitol, 2011]

Transient transfection of isolated Brugia malayi embryos by biolistics has proven to be useful in defining promoter structure and function in this parasite. However, isolated transfected embryos are developmentally incompetent. A method of producing developmentally competent transfected parasites is therefore needed. We report that L3 parasites can be chemically transfected in situ in the peritoneal cavity of a gerbil with a construct consisting of a secreted luciferase reporter gene containing a promoter, the 3' untranslated region and first intron derived from the B. malayi 70 kDa heat shock protein gene. The in situ chemically transfected parasites are developmentally competent, producing adult parasites with an efficiency similar to that obtained from implanted untreated L3s. Cultured adult parasites and progeny microfilariae (mf) derived from L3s transfected with this construct secreted luciferase into the culture medium. When the transfected mf were fed to mosquitoes and the resulting L3s collected, the L3s also secreted luciferase into the culture medium. Progeny mf from transgenic adult parasites contained transgenic DNA, and the transgenic mRNA produced in these parasites was found to be correctly cis- and trans-spliced. In situ chemical transformation thus results in developmentally competent transfected B. malayi in which the transgenic sequences remain transcriptionally active in all life cycle stages and are present in the subsequent generation.

Xu S et al. (2014) Dev Cell "C. elegans epidermal wounding induces a mitochondrial ROS burst that promotes wound repair."

Chisholm AD, Xu S

[Dev Cell, 2014]

Reactive oxygen species (ROS) such as hydrogen peroxide are generated at wound sites and act as long-range signals in wound healing. The roles of other ROS in wound repair are little explored. Here, we reveal a cytoprotective role for mitochondrial ROS (mtROS) in Caenorhabditis elegans skin wound healing. We show that skin wounding causes local production of mtROS superoxide at the wound site. Inhibition of mtROS levels by mitochondrial superoxide-specific antioxidants blocks actin-based wound closure, whereas elevation of mtROS promotes wound closure and enhances survival of mutant animals defective in wound healing. mtROS act downstream of wound-triggered Ca(2+) influx. We find that the mitochondrial calcium uniporter MCU-1 is essential for rapid mitochondrial Ca(2+) uptake and mtROS production after wounding. mtROS can promote wound closure by local inhibition of Rho GTPase activity via a redox-sensitive motif. These findings delineate a pathway acting via mtROS that promotes cytoskeletal responses in wound healing.

Xu S et al. (2011) Curr Biol "A Gq-Ca signaling pathway promotes actin-mediated epidermal wound closure in C. elegans."

Chisholm AD, Xu S

[Curr Biol, 2011]

BACKGROUND: Repair of skin wounds is essential for animals to survive in a harsh environment, yet the signaling pathways initiating wound repair in vivo remain little understood. In Caenorhabditis elegans, a p38 mitogen-activated protein kinase (MAPK) cascade promotes innate immune responses to wounding but is not required for other aspects of wound healing. We therefore set out to identify additional wound response pathways in C. elegans epidermis. RESULTS: We show here that wounding the adult C. elegans skin triggers a rapid and sustained rise in epidermal Ca(2+) that is critical for survival after wounding. The wound-triggered rise in Ca(2+) requires the epidermal transient receptor potential channel, melastatin family (TRPM) channel GTL-2 and IP(3)R-stimulated release from internal stores. We identify an epidermal signal transduction pathway that includes the G(q) EGL-30 and its effector PLC EGL-8. Loss of function in this pathway impairs survival after wounding. The G(q)-Ca(2+) pathway is not required for known innate immune responses to wounding but instead promotes actin-dependent wound closure. Wound closure requires the Cdc42 small GTPase and Arp2/3-dependent actin polymerization and is negatively regulated by Rho and nonmuscle myosin. Finally, we show that the death-associated protein kinase DAPK-1 acts as a negative regulator of wound closure. CONCLUSIONS: Skin wounding in C. elegans triggers a Ca(2+)-dependent signaling cascade that promotes wound closure, in parallel to the innate immune response to damage. Wound closure requires actin polymerization and is negatively regulated by nonmuscle myosin.

Vinci CR et al. (2007) J Biol Chem "Recognition of age-damaged (R,S)-adenosyl-L-methionine by two methyltransferases in the yeast Saccharomyces cerevisiae."

Vinci CR, Clarke SG

[J Biol Chem, 2007]

The biological methyl donor, S adenosylmethionine (AdoMet), can exist in two diastereoisomeric states with respect to its sulfonium ion. The "S" configuration, (S,S)AdoMet, is the only form that is produced enzymatically as well as the only form used in almost all biological methylation reactions. Under physiological conditions, however, the sulfonium ion can spontaneously racemize to the "R" form, producing (R,S)AdoMet. As of yet, (R,S)AdoMet has no known physiological function and may inhibit cellular reactions. In this study, two enzymes have been found in Saccharomyces cerevisiae that are capable of recognizing (R,S)AdoMet and using it to methylate homocysteine to form methionine. These enzymes are the products of the SAM4 and MHT1 genes, previously identified as homocysteine methyltransferases dependent upon AdoMet and S-methylmethionine respectively. We find here that Sam4 recognizes both (S,S) and (R,S)AdoMet, but its activity is much higher with the R,S form. Mht1 reacts with only the R,S form of AdoMet while no activity is seen with the S,S form. R,S-specific homocysteine methyltransferase activity is also shown here to occur in extracts of Arabidopsis thaliana, Drosophila melanogaster, and Caenorhabditis elegans, but has not been detected in several tissue extracts of Mus musculus. Such activity may function to prevent the accumulation of (R,S)AdoMet in these organisms.

Bonini NM (2001) Dev Cell "Stores to die for."

Bonini NM

[Dev Cell, 2001]

Genes that regulate apoptosis are well defined. In contrast, it has not been clear what genes are central to necrotic cell loss. In the September 27th issue of Neuron, Xu et al. (2001) report a critical role for genes that regulate storage and release of Ca2+ from the endoplasmic reticulum as important to necrotic-like cellular degeneration in Caenorhabditis elegans.

Qi Y et al. (2013) Bio Protoc "MiniSOG-mediated Photoablation in Caenorhabdtis elegans."

Qi Y, Xu S

[Bio Protoc, 2013]

This protocol describes a method for light-inducible cell ablation in live worms. miniSOG (mini Singlet Oxygen Generator) generates singlet oxygen upon blue light illumination (Shu et al., 2011). Mitochondrially membrane targeted miniSOG (the first 55 a. a. of C. e. tomm-20 fused at the N'-terminus of miniSOG, termed as mito-miniSOG in the following) is transgenically expressed in specific cells/tissues (Qi et al., 2012). Groups of transgenic animals are illuminated under open field fluorescence light on a compound microscope or LED light setup for photo-ablation.