Donahue LM et al. (1987) Proc Natl Acad Sci U S A "Molecular analysis of X chromosome dosage compensation in Caenorhabditis elegans."

Wood WB, Donahue LM, Quarantillo BA

[Proc Natl Acad Sci U S A, 1987]

We used a convenient quantitative dot blot assay to measure transcript levels for two X chromosome-linked genes, myo-2 and act-4, in the nematode Caenorhabditis elegans. We show that there is dosage compensation of transcript levels for these two genes between XX hermaphrodites and X0 males and that a mutation in the dpy-21 gene, postulated from genetic analysis to be involved in control of X chromosome expression, can affect these transcript levels in the manner predicted. However, we observe the dpy-21 effects only at some stages of the life cycle and not at others. These results are generally consistent with earlier genetic and molecular evidence.

Sivaranjani M et al. (2016) Int J Food Microbiol "Morin inhibits biofilm production and reduces the virulence of Listeria monocytogenes - An in vitro and in vivo approach."

Ravi AV, Balamurugan K, Kamaladevi A, Sivaranjani M, Pandian SK, Gowrishankar S

[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.

Gowrishankar S et al. (2016) Pathog Dis "Cyclic dipeptide-cyclo(L-leucyl-L-prolyl) from marine Bacillus amyloliquefaciens mitigates biofilm formation and virulence in Listeria monocytogenes."

Pandian SK, Kamaladevi A, Ravi AV, Gowrishankar S, Balamurugan K, Sivaranjani M

[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.

Zhao P et al. (2023) Nat Methods "Femtosecond laser microdissection for isolation of regenerating C. elegans neurons for single-cell RNA sequencing."

Jiang N, Ma KY, Chizari S, Ben-Yakar A, DuPlissis A, Martin C, Mondal S, Messing RO, Maiya R, Zhao P

[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.

Wood WB et al. (1985) Cold Spring Harb Symp Quant Biol "Aspects of dosage compensation and sex determination in Caenorhabditis elegans."

Schedin P, Wood WB, Donahue LM, Meneely PM

[Cold Spring Harb Symp Quant Biol, 1985]

The nematode Caenorhabditis elegans has two sexes, hermaphrodites and males. Hermaphrodites normally have two X chromosomes (XX or 2X) and males only one (XO or 1X). There is no Y chromosome, and sex is determined by the X/A ratio, i.e., the ratio of X chromosomes to sets of autosomes. The X/A ratio acts to determine sex through a set of at least seven interacting autosomal genes, which have been defined, characterized, and shown to act as a regulatory pathway, primarily by Hodgkin and co-workers. The first gene in the pathway, her-1, acts through five intervening genes to regulate the major switch gene tra-1, whose activity determines somatic sexual development. At an X/A ratio of 1.0, her-1 activity is low and tra-1 acivity is high, leading to hermaphrodite development. At an X/A ratio of 0.5, her-1 activity is high and tra-1 activity is low, leading to male development. Loss-of-function mutations in the her-1 gene transform 1X animals into fertile hermaphrodites and have no effect on 2X hermaphrodites. Loss-of-function mutations in the tra-1 gene transform 2X animals into fertile males and have no effect on 1X males....

Schieber NL et al. (2017) Methods Cell Biol "Minimal resin embedding of multicellular specimens for targeted FIB-SEM imaging."

Markert SM, Schieber NL, Stigloher C, Schwab Y, Steyer AM, Machado P

[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.

Lucas MS et al. (2012) Methods Cell Biol "Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures."

Gasser P, Guenthert M, Lucas F, Lucas MS, Wepf R

[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.