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[
Int J Food Microbiol,
2016]
The current study explores the in vitro and in vivo antibiofilm efficacy of morin against a leading foodborne pathogen-Listeria monocytogenes (LM). Minimum inhibitory concentration (MIC) of morin against LM strains was found to be 100g/ml. The non-antibacterial effect of morin at its sub-MICs (6.25, 12.5 and 25g/ml) was determined through growth curve and XTT assay. Morin at its sub-MICs demonstrated a significant dose dependent inhibitory efficacy against LM biofilm formation which was also evidenced through light, confocal and scanning electron microscopic analyses. However, morin failed to disperse the mature biofilm of LM even at its MIC. Our data also revealed the anti-virulence efficacy of morin, as it significantly inhibited the production of hemolysin and motility of LM. Concentration-dependent susceptibility of morin treated LM cells to normal human serum was observed. In vivo studies revealed that morin extended the lifespan of LM infected Caenorhabditis elegans by about 85%. Furthermore, the non-toxic nature and in vivo anti-adherence efficacy of morin were also ascertained through C. elegans-LM infection model. Overall, the data of the current study identifies morin as a promising antibiofilm agent and its suitability to formulate protective strategies against biofilm associated infections caused by LM.
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[
Pathog Dis,
2016]
The current study was intentionally focused on cyclo(L-leucyl- L-prolyl) (CLP)-a cyclic dipeptide with myriad pharmaceutical significance, to explore its antivirulence efficacy against the predominant food-borne pathogen-Listeria monocytogenes (LM). Minimum inhibitory concentration (MIC) of CLP against LM ATCC 19111 was found to be 512 g mL(-1). CLP at sub-MICs (64,128, 256 g mL(-1)) demonstrated a profound non-bactericidal dose-dependent antibiofilm efficacy (on polystyrene and glass) against LM, which was further confirmed through confocal and scanning electron microscopic analysis (on stainless steel surface). In vitro bioassays divulged the phenomenal inhibitory efficacy of CLP towards various virulence traits of LM, specifically its overwhelming suppression of swimming and swarming motility. Data of in vivo assay using Caenorhabditis elegans signified that the plausible mechanism of CLP could be by impeding the pathogen's initial adhesion and thereby attenuating the biofilm assemblage and its associated virulence. This was further confirmed by significant decrease in exopolymeric substance, auto-aggregation, hydrophobicity index and extracellular DNA (eDNA) of the CLP treated-LM cells. Collectively, the current study unveils the antivirulence efficacy of CLP against the Gram-positive food borne-pathogen and the strain Bacillus amyloliquifaciens augurs well to be a promising probiotic in controlling infections associated with LM.
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[
Nat Methods,
2023]
Our understanding of nerve regeneration can be enhanced by delineating its underlying molecular activities at single-neuron resolution in model organisms such as Caenorhabditis elegans. Existing cell isolation techniques cannot isolate neurons with specific regeneration phenotypes from C. elegans. We present femtosecond laser microdissection (fs-LM), a single-cell isolation method that dissects specific cells directly from living tissue by leveraging the micrometer-scale precision of fs-laser ablation. We show that fs-LM facilitates sensitive and specific gene expression profiling by single-cell RNA sequencing (scRNA-seq), while mitigating the stress-related transcriptional artifacts induced by tissue dissociation. scRNA-seq of fs-LM isolated regenerating neurons revealed transcriptional programs that are correlated with either successful or failed regeneration in wild-type and
dlk-1 (0) animals, respectively. This method also allowed studying heterogeneity displayed by the same type of neuron and found gene modules with expression patterns correlated with axon regrowth rate. Our results establish fs-LM as a spatially resolved single-cell isolation method for phenotype-to-genotype mapping.
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Ma, K., Maiya, R., Jiang, N., Kreeger, L., Martin, C., Zemelman, B., Arur, S., Ben-Yakar, A., ZHAO, P., Trimmer, K., Messing, R.
[
International Worm Meeting,
2019]
C. elegans has become a versatile system for studying in vivo nerve regeneration since the advent of precise laser axotomy method for severing specific axons. Through mutant and RNAi screening, a number of regeneration regulator genes have been identified. Nevertheless, their downstream effectors remain elusive. As a complementary approach, we propose to perform single-cell RNA-sequencing on regrowing neurons to capture the genome-wide dynamics underlying nerve regeneration. However, it has been technically unfeasible to isolate regrowing neurons from living C. elegans. The prevalent isolation method uses FACS to sort neurons of interest from chemo-mechanically dissociated animals, thus requires thousands of animals with synchronized nerve injury, which cannot be obtained even with state-of-the-art automated microfluidic systems. We developed a new femtosecond laser microdissection (fs-LM) method to rapidly and precisely isolate single cells directly from living tissue or organisms by leveraging femtosecond laser ablation as a high-precision cutting tool. Compared to traditional laser capture microdissection, our method provides a few crucial advantages. 1) fs-LM yields intact single cells without sample sectioning, freezing, or fixing, thus preventing sample degradation or contamination. 2) compared to the dissociation and sorting method, fs-LM induces less stress response in isolated cells. 3) fs-LM preserves the spatial and phenotypic information of the collected neurons. In addition, by correlating gene expression to the context-dependent regeneration phenotypes, it is possible to further dissect the genetic activities encoding nerve regeneration. 4) fs-LM does can isolate unlabeled cells. We isolated regrowing posterior lateral microtubule (PLM) neurons from larval 4 stage animals. Single cell RNA-sequencing on the isolated neurons identified gene expression patterns underlying axon regeneration. To demonstrate the versatility of our method, we have also dissected and sequenced single C. elegans oocytes and mammalian brain neurons.
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[
Nematologica,
1977]
SEM observations of adult Caenorhabditis briggsae females showed differences between young and old nematodes. In young nematodes the cuticle was generally smooth, whereas in old ones it was wrinkled. Deirids were located at the level of the excretory pore in the lateral field. They were distinct in young nematodes but indistinct in old ones. The oral opening was formed by six lips, which were closed in old nematodes and open in young ones. The vulva possessed two semi-circular lips and was bordered by two lateral flaps. These lips were smooth in young specimens and wrinkled in old ones. Cryofractures of old nematodes showed cavities in the intestinal epithelium corresponding to areas in which age pigment granules normally occur. No such cavities were seen in young nematodes.
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[
Elife,
2019]
Young <i>Caenorhabditis elegans</i> hermaphrodites use their own sperm to protect against the negative consequences of mating.
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[
Methods Cell Biol,
2017]
Correlative light and electron microscopy (CLEM) is a powerful tool to perform ultrastructural analysis of targeted tissues or cells. The large field of view of the light microscope (LM) enables quick and efficient surveys of the whole specimen. It is also compatible with live imaging, giving access to functional assays. CLEM protocols take advantage of the features to efficiently retrace the position of targeted sites when switching from one modality to the other. They more often rely on anatomical cues that are visible both by light and electron microscopy. We present here a simple workflow where multicellular specimens are embedded in minimal amounts of resin, exposing their surface topology that can be imaged by scanning electron microscopy (SEM). LM and SEM both benefit from a large field of view that can cover whole model organisms. As a result, targeting specific anatomic locations by focused ion beam-SEM (FIB-SEM) tomography becomes straightforward. We illustrate this application on three different model organisms, used in our laboratory: the zebrafish embryo Danio rerio, the marine worm Platynereis dumerilii, and the dauer larva of the nematode Caenorhabditis elegans. Here we focus on the experimental steps to reduce the amount of resin covering the samples and to image the specimens inside an FIB-SEM. We expect this approach to have widespread applications for volume electron microscopy on multiple model organisms.
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[
Journal of Nematology,
1972]
The specific gravity of old Caenorhabditis briggsae was shown to be greater than that of young nematodes. The possible explanations for this age-associated change are discussed.
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[
Sci Rep,
2020]
Maternal behaviors benefit the survival of young, contributing directly to the mother's reproductive fitness. An extreme form of this is seen in matriphagy, when a mother performs the ultimate sacrifice and offers her body as a meal for her young. Whether matriphagy offers only a single energy-rich meal or another possible benefit to the young is unknown. Here, we characterized the toxicity of a bacterial secondary metabolite, namely, violacein, in Caenorhabditis elegans and found it is not only toxic towards adults, but also arrests growth and development of C. elegans larvae. To counteract this, C. elegans resorted to matriphagy, with the mothers holding their eggs within their bodies and hatching the young larvae internally, which eventually led to the mothers' death. This violacein-induced matriphagy alleviated some of the toxic effects of violacein, allowing a portion of the internally-hatched young to bypass developmental arrest. Using genetic and pharmacological experiments, we found the consumption of oleate, a monounsaturated fatty acid produced by the mother, during matriphagy is partially responsible. As such, our study provides experimental evidence of why such a drastic and peculiar maternal behavior may have arisen in nematode natural habitats.
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[
Methods Cell Biol,
2012]
The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.