The SNARE complex formed by RIC-4/SEC-22/SYX-2 promotes C. elegans epidermal wound healing.

Maintaining cellular viability relies on the integrity of the plasma membrane, which must be repaired upon damage. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion is a crucial mechanism involved in membrane repair. In C. elegans epidermal cell hyp 7, syntaxin-2 (SYX-2) facilitates large membrane wound repair; however, the underlying molecular mechanism remains unclear. Here, we found that SNAP-25 protein RIC-4 and synaptobrevin protein SEC-22 are required for SYX-2 recruitment at the wound site. They interact to form a SNARE complex to promote membrane repair in vivo and fusion in vitro. Moreover, we found that SEC-22 localized in multiple intracellular compartments, including endosomes and the trans-Golgi network, which recruited to the wounds. Furthermore, inhibition of RAB-5 disrupted SEC-22 localization and prevented its interaction with SYX-2. Our findings suggest that RAB-5 facilitates the formation of the RIC-4/SEC-22/SYX-2 SNARE complex and provides valuable insights into the molecular mechanism of how cells repair large membrane wounds.

High zoonotic potential and heavy environmental burden of Cryptosporidium spp. and Enterocytozoon bieneusi in farmed and pet African pygmy hedgehogs (Atelerix albiventris).

African pygmy hedgehogs (Atelerix albiventris) are widely farmed in southern China and Japan for medicinal materials and as pets. However, little is known about the prevalence, zoonotic potential, and environmental burden of Cryptosporidium spp., Enterocytozoon bieneusi and Giardia duodenalis in these animals. In this study, 380 fecal samples were collected from farmed and pet African pygmy hedgehogs in Guangdong of China, and analyzed for these pathogens by PCR and DNA sequencing. Overall, the detection rates of Cryptosporidium spp., E. bieneusi and G. duodenalis were 35.5%, 70.0% and 0, respectively. By living condition, the highest detection rates of Cryptosporidium spp. (61.5%) and E. bieneusi (100.0%) were both obtained from animals kept in the cave, which could be due to the overcrowding and poor hygiene conditions. Two Cryptosporidium species were identified, including C. erinacei (n = 22) and Cryptosporidium horse genotype (n = 113). The C. erinacei isolates belonged to a new subtype family (XIIIb), which has been identified in a patient with cryptosporidiosis recently. The horse genotype isolates are of a known subtype VIbA13, which was previously identified in a pet store employee in care of hedgehogs with diarrhea. Eleven genotypes of the zoonotic Group 1 were identified in E. bieneusi, with the known genotype SCR05 previously detected in pet rabbits being dominant (235/266, 88.3%). In longitudinal monitoring of Cryptosporidium infection in 11 naturally infected African pygmy hedgehogs, the oocyst shedding intensity decreased gradually from the mean oocysts per gram of feces of ∼6 logs to ∼2 logs over 42 days. The high intensity and long duration of oocyst shedding could lead to heavy environmental contamination and increase the potential for zoonotic transmission of the pathogens. Results of the study suggest that zoonotic Cryptosporidium spp. and E. bieneusi are common in farmed and pet African pygmy hedgehogs. Hygiene and One Health measures should be implemented by pet owners and farmers to prevent zoonotic transmission and environmental contamination of Cryptosporidium spp. and E. bieneusi.

Triggered Golgi membrane enrichment promotes PtdIns(4,5)P2 generation for plasma membrane repair.

The maintenance of plasma membrane integrity and a capacity for efficiently repairing damaged membranes are essential for cell survival. Large-scale wounding depletes various membrane components at the wound sites, including phosphatidylinositols, yet little is known about how phosphatidylinositols are generated after depletion. Here, working with our in vivo C. elegans epidermal cell wounding model, we discovered phosphatidylinositol 4-phosphate (PtdIns4P) accumulation and local phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] generation at the wound site. We found that PtdIns(4,5)P2 generation depends on the delivery of PtdIns4P, PI4K, and PI4P 5-kinase PPK-1. In addition, we show that wounding triggers enrichment of the Golgi membrane to the wound site, and that is required for membrane repair. Moreover, genetic and pharmacological inhibitor experiments support that the Golgi membrane provides the PtdIns4P for PtdIns(4,5)P2 generation at the wounds. Our findings demonstrate how the Golgi apparatus facilitates membrane repair in response to wounding and offers a valuable perspective on cellular survival mechanisms upon mechanical stress in a physiological context.

Discovery of Natural Small Molecules Promoting Collagen Secretion by High-Throughput Screening in Caenorhabditis elegans.

Advancing approaches for drug screening are in great demand to explore natural small molecules that may play important roles in collagen biogenesis, secretion, and assembly, which may find novel lead compounds for treating collagen-related diseases or preventing skin aging. In this study, we generated a single copy insertion transgenic Pcol-19- COL-12::GFP Caenorhabditis elegans (C. elegans) strain to label epidermis collagen XII (COL-12), a cuticle structure component, and established an efficient high-content screening techniques to discover bioactive natural products in this worm strain through quantification of fluorescence imaging. We performed a preliminary screening of 614 compounds from the laboratory's library of natural small molecule compounds on the COL-12 labeling worm model, which was tested once at a single concentration of 100 µM to screen for compounds that promoted COL-12 protein amount. Besides col-12, the transcriptional levels of worm-associated collagen coding genes col-19 and sqt-3 were also examined, and none of the compounds affected their transcriptional levels. Meanwhile, the protein levels of COL-12 were significantly upregulated after treating with Danshensu, Lawsone, and Sanguinarine. The effects of these drugs on COL-12 overexpressing worms occur mainly after collagen transcription. Through various validation methods, Danshensu, Lawsone, and Sanguinarine were more effective in promoting the synthesis or secretion of COL-12.

Identification and Characterization of Three Spore Wall Proteins of Enterocytozoon Bieneusi.

Enterocytozoon bieneusi is the most common microsporidian pathogen in farm animals and humans. Although several spore wall proteins (SWPs) of other human-pathogenic microsporidia have been identified, SWPs of E. bieneusi remain poorly characterized. In the present study, we identified the sequences of three E. bieneusi SWPs from whole genome sequence data, expressed them in Escherichia coli, generated a monoclonal antibody (mAb) against one of them (EbSWP1), and used the mAb in direct immunofluorescence detection of E. bieneusi spores in fecal samples. The amino acid sequence of EbSWP1 shares some identity to EbSWP2 with a BAR2 domain, while the sequence of EbSWP3 contains a MICSWaP domain. No cross-reactivity among the EbSWPs was demonstrated using the polyclonal antibodies generated against them. The mAb against EbSWP1 was shown to react with E. bieneusi spores in fecal samples. Using chromotrope 2R staining-based microscopy as the gold standard, the sensitivity and specificity of the direct immunofluorescence for the detection of E. bieneusi were 91.4 and 73.7%. Data generated from the study could be useful in the characterization of E. bieneusi and immunological detection of the pathogen.

Recruitment of tetraspanin TSP-15 to epidermal wounds promotes plasma membrane repair in C. elegans.

Maintaining the integrity of the plasma membrane after cellular damage is essential for cell survival. However, it is unclear how cells repair large membrane injuries in vivo. Here, we report that the tetraspanin protein, TSP-15, is recruited to large membrane wounds and forms a ring-like structure in C. elegans epidermis and promotes membrane repair after an injury. TSP-15 recruits from the adjacent region underneath the plasma membrane to the wound site in a RAB-5-dependent manner upon membrane damage. Genetic and live-imaging analysis suggested that the endosomal sorting complex required for transport III (ESCRT III) is necessary for recruiting TSP-15 from the early endosome to the damaged membrane. Moreover, TSP-15 interacts with and is required for the accumulation of t-SNARE protein Syntaxin-2, which facilitates membrane repair. These findings provide valuable insights into the role of the conserved tetraspanin TSP-15 in the cellular repair of large wounds resulting from environmental insults.

Cryptosporidiosis outbreak caused by Cryptosporidium parvum subtype IIdA20G1 in neonatal calves.

Cryptosporidium parvum is a major zoonotic pathogen responsible for outbreaks of severe diarrhoea in humans and calves. Almost all investigations of cryptosporidiosis outbreaks caused by C. parvum have focused on its IIa subtype family in industrialized nations. From December 2018 to April 2019, approximately 200 neonatal calves on a large cattle farm in Hebei Province, China, were diagnosed with watery diarrhoea and over 40 died. To investigate the cause of the outbreak, faecal samples were taken during and after the outbreak from neonatal calves of ≤4 weeks of age (n = 40 and n = 56) and older calves of 4-24 weeks of age (n = 79 and n = 38). A total of 18 faecal samples collected from ill calves at the peak of the outbreak were analysed for four common enteric pathogens using an enzymatic immunoassay (EIA). In addition, 75 samples from neonatal calves were tested for rotavirus by EIA. All samples were analysed for Cryptosporidium spp. using PCR and sequencing techniques. Of the initial 18 samples from sick calves, ten were positive for C. parvum, five for rotavirus, and one for coronavirus. The overall prevalence of rotavirus in neonatal calves was 20.0% (15/75), with no significant differences during and after the outbreak. In contrast, Cryptosporidium parvum infections were significantly higher during the outbreak (60.0%, 24/40) than after the outbreak (30.4%, 17/56; p = .004). Cryptosporidium parvum infection was associated with the presence of watery diarrhoea in neonatal calves (OR = 11.19), while no association was observed between C. bovis infection and diarrhoea. All C. parvum isolates were identified as subtype IIdA20G1. This is one of the few reports of outbreaks of severe diarrhoea caused by C. parvum IId subtypes in calves. More attention should be directed towards the dissemination of C. parvum in China.

Redox-sensitive CDC-42 clustering promotes wound closure in C. elegans.

Tissue damage induces immediate-early signals, activating Rho small GTPases to trigger actin polymerization essential for later wound repair. However, how tissue damage is sensed to activate Rho small GTPases locally remains elusive. Here, we found that wounding the C. elegans epidermis induces rapid relocalization of CDC-42 into plasma membrane-associated clusters, which subsequently recruits WASP/WSP-1 to trigger actin polymerization to close the wound. In addition, wounding induces a local transient increase and subsequent reduction of H2O2, which negatively regulates the clustering of CDC-42 and wound closure. CDC-42 CAAX motif-mediated prenylation and polybasic region-mediated cation-phospholipid interaction are both required for its clustering. Cysteine residues participate in intermolecular disulfide bonds to reduce membrane association and are required for negative regulation of CDC-42 clustering by H2O2. Collectively, our findings suggest that H2O2-regulated fine-tuning of CDC-42 localization can create a distinct biomolecular cluster that facilitates rapid epithelial wound repair after injury.

Protocol to Induce Wounding and Measure Membrane Repair in Caenorhabditis elegans Epidermis.

Efficient membrane repair after injury is essential for cell and animal survival. Caenorhabditis elegans epidermal cell hpy7 has emerged as a powerful genetic system to investigate the molecular mechanism of membrane repair in vivo. This protocol describes detailed approaches for how to perform wounding on the epidermis and how to examine membrane repair by trypan blue staining, confocal imaging, and data analysis. For details on the use and execution of this protocol, please refer to Meng et al. (2020).

Actin Polymerization and ESCRT Trigger Recruitment of the Fusogens Syntaxin-2 and EFF-1 to Promote Membrane Repair in C. elegans.

Membrane repair is essential for cell and organism survival. Exocytosis and endocytosis facilitate membrane repair in small wounds within a single cell; however, it remains unclear how large wounds in the plasma membrane are repaired in metazoans. Here, we show that wounding triggers rapid transcriptional upregulation and dynamic recruitment of the fusogen EFF-1 to the wound site in C. elegans epidermal cells. EFF-1 recruitment at the wounded membrane depends on the actin cytoskeleton and is important for membrane repair. We identified syntaxin-2 (SYX-2) as an essential regulator of EFF-1 recruitment. SYX-2 interacts with the C terminus of EFF-1 to promote its recruitment, facilitating both endoplasmic and exoplasmic membrane repair. Furthermore, we show that SYX-2-EFF-1 repair machinery acts downstream of the ESCRT III signal. Together, our findings identify a key pathway underlying membrane repair and provide insights into tissue repair and regenerative medicine after injury.

Wounding triggers MIRO-1 dependent mitochondrial fragmentation that accelerates epidermal wound closure through oxidative signaling.

Organisms respond to tissue damage through the upregulation of protective responses which restore tissue structure and metabolic function. Mitochondria are key sources of intracellular oxidative metabolic signals that maintain cellular homeostasis. Here we report that tissue and cellular wounding triggers rapid and reversible mitochondrial fragmentation. Elevated mitochondrial fragmentation either in fzo-1 fusion-defective mutants or after acute drug treatment accelerates actin-based wound closure. Wounding triggered mitochondrial fragmentation is independent of the GTPase DRP-1 but acts via the mitochondrial Rho GTPase MIRO-1 and cytosolic Ca2+. The fragmented mitochondria and accelerated wound closure of fzo-1 mutants are dependent on MIRO-1 function. Genetic and transcriptomic analyzes show that enhanced mitochondrial fragmentation accelerates wound closure via the upregulation of mtROS and Cytochrome P450. Our results reveal how mitochondrial dynamics respond to cellular and tissue injury and promote tissue repair.

[CRISPR-Cas9 mediated genome editing in Caenorhabditis elegans].

The development of genome editing techniques based on CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas9 system has revolutionized biomedical researches. It can be utilized to edit genome sequence in almost any organisms including Caenorhabditis elegans, one of the most convenient and classic genetic model animals. The application of CRISPR-Cas9 mediated genome editing in C. elegans promotes the functional analysis of gene and proteins under many physiological conditions. In this mini-review, we summarized the development of CRISPR-Cas9-based genome editing in C. elegans.

Effect of total flavonoids of Spatholobus suberectus Dunn on PCV2 induced oxidative stress in RAW264.7 cells.

BACKGROUND: This study was carried out to investigate the effect of total flavonoids of Spatholobus suberectus Dunn (TFSD) on PCV2 induced oxidative stress in RAW264.7 cells. METHODS: Oxidative stress model was established in RAW264.7 cells by infecting with PCV2. Virus infected cells were then treated with various concentrations (25 mg/ml, 50 mg/ml and 100 mg/ml) of TFSD. The levels of oxidative stress related molecules (NO, ROS, GSH and GSSG) and activities of associated enzymes (SOD, MPO and XOD were analyzed using ultraviolet spectrophotometry, fluorescence method and commercialized detection kits. RESULTS: PCV2 infection induced significant increase of NO secretion, ROS generation, GSSG content, activities of both XOD and MPO, and dramatically decrease of GSH content and SOD activity in RAW264.7 cells (P < 0.05). After treating with TFSD, PCV2 induced alteration of oxidative stress related molecule levels and enzyme activities were recovered to a level similar to control. CONCLUSION: Our findings indicated that TFSD was able to regulate oxidative stress induced by PCV2 infection in RAW264.7 cells, which supports the ethnomedicinal use of this herb as an alternative or complementary therapeutic drug for reactive oxygen-associated pathologies.

Quantitative live cell fluorescence-microscopy analysis of fission yeast.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP). In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

Influences of acid-treated multiwalled carbon nanotubes on fibroblasts: proliferation, adhesion, migration, and wound healing.

With the increasing applications of carbon nanotubes (CNTs) in fields of biomedical engineering and medical chemistry, it is important to understand the response of mammalian cells to the CNTs exposure and treatment. In this study, the influences of multiwalled carbon nanotubes (MWCNTs) on cellular behavior of human dermal fibroblasts and NIH 3T3 murine fibroblasts were investigated. Results showed that the MWCNTs treatment induced dose-dependent cytotoxicity and arrested the cell cycle in the G1 phase, indicating inhibition of DNA synthesis. The presence of MWCNTs also down regulated the expression level of adhesion-related genes, and simultaneously caused cytoskeleton damage and disturbance of actin stress fibers, thereby inducing dramatically adverse effects on the cell physiological functions such as cell spreading, adhesion, migration, and wound healing ability.

Fabrication of cellular polycaprolactone films for cell culture.

Hierarchical cellular structures consisting of multilayers of ordered pores were created on a poly-epsilon-caprolactone (PCL) film by introducing a colloidal crystal mask/template in thermomechanical lithography. The surface characteristics were measured by scanning electron microscopy, atomic force microscopy, water contact angle and analyzed by Fourier Transform (FT). The resultant PCL films present a metastable superhydrophobicity. Their water contact angles were initially 150 degrees and then declined with time eventually to 115-120 degrees . In vitro culture of human fibroblasts found that the cells could spread on the cellular PCL surface as normal, but showed higher viability compared with the control cells on a flat substrate.