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."

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

[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."

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

[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.

Zhao, P. et al. (2019) International Worm Meeting "Femtosecond laser microdissection isolates single C. elegans neurons for nerve regeneration RNA-seq studies."

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.

Krakow D et al. (2001) Gene "Identification of human FEM1A, the ortholog of a C. elegans sex-differentiation gene."

Sebald E, Krakow D, King LM, Cohn DH

[Gene, 2001]

We report the isolation, genomic structure, chromosomal location, and expression pattern of the FEM1A gene, the human ortholog of the Caenorhabditis elegans fem-1 and mouse Fem1a genes. The coding sequence is 1851 bp and encodes a 617 amino acid protein. The human FEM1A protein has 65% identity with the mouse Fem1a protein and 34% identity with the C. elegans fem-1 protein, indicating conservation of this protein. The N-terminal region of the encoded protein contains six ankyrin repeat elements, a motif found in signaling and transcriptional regulatory molecules such as Notch and glp1. The gene was highly expressed in human kidney and cardiac tissue, and was expressed at lower levels in multiple tissues, including cartilage. FEM1A was localized to chromosome 5q23.1, a region of conserved synteny with a portion of mouse chromosome 17 that contains Fem1a. In C. elegans, fem-1 is involved in a pathway necessary for sex determination. The identification of a human homolog of this conserved gene suggests a potential role for this sex-determining molecule in humans.

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

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

[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."

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

[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.

Vajo Z et al. (1999) Mamm Genome "Conservation of the Caenorhabditis elegans timing gene clk-1 from yeast to human: a gene required for ubiquinone biosynthesis with potential implications for aging."

Francomano CA, Wilkin DJ, King LM, Jonassen T, Ho N, Vajo Z, Munnich A, Clarke CF

[Mamm Genome, 1999]

Mutations in the Caenorhabditis elegans gene clk-1 have a major effect on slowing development and increasing life span. The Saccharomyces cerevisiae homolog COQ7 encodes a mitochondrial protein involved in ubiquinone biosynthesis and, hence, is required for respiration and gluconeogenesis. In this study, RT-PCR and 5' RACE were used to isolate both human and mouse clk-1/COQ7 homologs. Human CLK-1 was mapped to Chr 16(p12-13.1) by Radiation Hybrid (RH) and fluorescence in situ hybridization (FISH) methods. The number and location of human CLK1 introns were determined, and the location of introns II and IV are the same as in C. elegans. Northern blot analysis showed that three different isoforms of CLK-1 mRNA are present in several tissues and that the isoforms differ in the amount of expression. The functional equivalence of human CLK-1 to the yeast COQ7 homolog was tested by introducing either a single or multicopy plasmid containing human CLK-1 cDNA into yeast coq7 deletion strains and assaying for growth on a nonfermentable carbon source. The human CLK-1 gene was able to functionally complement yeast coq7 deletion mutants. The protein similarities and the conservation of function of the CLK-1/clk-1/COQ7 gene products suggest a potential link between the production of ubiquinone and aging.

Ehtesham N et al. (2002) Am J Hum Genet "Evidence that Smith-McCort dysplasia and Dyggve-Melchior-Clausen dysplasia are allelic disorders that result from mutations in a gene on chromosome 18q12."

Shanske A, King LM, Reinker K, Powell BR, Cantor RM, Rimoin DL, Cohn DH, Unger S, Ehtesham N

[Am J Hum Genet, 2002]

Smith-McCort dysplasia is a rare autosomal recessive osteochondrodysplasia characterized by short limbs and a short trunk with a barrel-shaped chest. The radiographic phenotype includes platyspondyly, generalized abnormalities of the epiphyses and metaphyses, and a distinctive lacy appearance of the iliac crest. We performed a genomewide scan in a consanguineous family from Guam and found evidence of linkage to loci on chromosome 18q12. Analysis of a second, smaller family was also consistent with linkage to this region, producing a maximum combined two-point LOD score of 3.04 at a recombination fraction of 0 for the marker at locus D18S450. A 10.7-cM region containing the disease gene was defined by recombination events in two affected individuals in the larger family. Furthermore, all affected children in the larger family were homozygous for a subset of marker loci within this region, defining a 1.5-cM interval likely to contain the defective gene. Analysis of three small, unrelated families with Dyggve-Melchior-Clausen syndrome, a radiographically identical disorder with the additional clinical finding of mental retardation, provided evidence of linkage to the same region, a result consistent with the hypothesis that the two disorders are allelic.

King KV et al. (1998) Midwest Worm Meeting "DAF-8 is a SMAD protein required in amphid chemosensory neurons to promote growth."

Riddle DL, King KV, Estevez AOZ

[Midwest Worm Meeting, 1998]

Non-dauer development in C. elegans involves activation of a signal transduction pathway analogous to Transforming Growth Factor-beta (TGF-beta) signaling pathways in other organisms. Previous genetic analysis indicates that daf-8 and daf-14 are partially redundant positive regulators of non-dauer development, whereas daf-3 promotes dauer formation. The daf-8 (King et al., unpublished), daf-3 (Patterson et al., 1997) and daf-14 (J. Thomas, personal communication) genes each encode SMAD proteins. SMAD proteins mediate transcriptional regulation of TGF-beta-responsive genes in response to phosphorylation by TGF-beta receptors. Mutations in daf-8 result in constitutive dauer larva formation, and some embryonic and larval lethality. One mutation truncates the protein in the putative DNA binding domain and eight other daf-8 mutations change residues in the putative transcriptional activation domain. Expression of a daf-8::GFP fusion gene is restricted to one pair of amphid chemosensory neurons in the head and is down-regulated in dauer larvae. This implies that dauer formation is sensitive to the levels of daf-8. This limited expression pattern suggests that DAF-8s function in regulating dauer formation may be different from DAF-14.