Donati LM et al. (1984) Worm Breeder's Gazette "a hybrid dysgenesis-like phenomenon in C elegans."

Baillie DL, Donati LM

[Worm Breeder's Gazette, 1984]

We are studying the unc-22 f linkage group IV . Our experiments make use of R. Horvitz's balancer nT1. In one experiment devised to study the effect of heat shock mutagenesis in C. elegans , we noticed that when Bergerac (BO) hermaphrodites were mated to Bristol (N2) males, a high rate of mutation was detected both in our control and in our experimental. unc-22 BO hermaphrodites (EMS induced by Teresa Rogalski) were mated to unc-22 1 N2 males. Single wild-type F1's were set and their progeny analyzed for the absence of Unc-22 individuals. Of 537 chromosomes screened, 24 lethal bearing chromosomes were recovered (4-5%). This is an increase of approximately 50 fold over the spontaneous rate of lethal induction in the Bristol strain (Rosenbluth et. al., Mutation Research 110:39-48). Several of these mutations have been mapped relative to unc-22 . One maps 8.5 map units from unc-22 , another maps 4 map units away. Of interest are three independently derived mutations which fail to complement one another and let-52 and appear tightly linked to unc-22 . These mutations may either be point mutations of let-52 or may be chromosomal rearrangements in this region. We are able to detect lethal bearing chromosomes in the progeny of the F1 suggesting that the mutations are fixed in the F1. Therefore, two possibilities exist as to the nature of these mutations. The unc-22 BO strain may have an unusually high spontaneous mutation rate. Alternatively, the high mutation rate may be due to a hybrid dysgenesis-like phenomenon, occurring sometime after fertilization and prior to the development of the germline of the F1. If the unc-22 BO strain had an endogenous high spontaneous mutation rate, one would expect a large number of clustered mutational events. We have evidence of one such event. We are in the process of establishing a N2/BO let-52 strain which has been repetitively backcrossed to Bergerac. Examination of this strain may allow us to detect reversion. Also of interest is the distribution of these lethals. EMS induced lethals seem to be primarily to the left of unc-22 . There is only one EMS induced allele of let-52 , while there are three N2/BO interstrain cross induced alleles. The molecular basis of these mutations is being investigated. If these mutations are due to transposable element insertions, either Tc1 or some other element, it will be possible to isolate insertional mutations in genes of interest. These molecularly marked strains will be invaluable for the cloning and molecular identification of specific genes.

Donati LM et al. (1985) International C. elegans Meeting "genetic analysis of the unc-22 to dpy-4 region in C elegans."

Donati LM, Wild GC, Baillie DL

[International C. elegans Meeting, 1985]

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.

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

Kreeger, L., Arur, S., ZHAO, P., Ben-Yakar, A., Trimmer, K., Messing, R., Ma, K., Martin, C., Zemelman, B., Jiang, N., Maiya, 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.

Clark DV et al. (1987) International C. elegans Meeting "organization of essential genes on LGIV(right)."

Clark DV, Donati LM, Baillie DL, Rogalski TM

[International C. elegans Meeting, 1987]

Clark DV et al. (1988) Genetics "The unc-22(IV) region of Caenorhabditis elegans: genetic analysis of lethal mutations."

Rogalski TM, Baillie DL, Donati LM, Clark DV

[Genetics, 1988]

The organization of essential genes in the unc-22 region, defined by the deficiency sDf2 on linkage group IV, has been studied. Using the balancer nT1 (IV;V), which suppresses recombination over 49 map units, 294 lethal mutations on LGIV(right) and LGV(left) were recovered using EMS mutagenesis. Twenty-six of these mutations fell into the unc-22 region. Together with previously isolated lethal mutations, there is now a total of 63 lethal mutations which fall into 31 complementation groups. Mutations were positioned on the map using eight overlapping deficiencies in addition to sDf2. The lethal alleles and deficiencies in the unc-22 region were characterized with respect to their terminal phenotypes. Mapping of these lethal mutations shows that sDf2 deletes a minimum of 1.8 map units and a maximum of 2.5 map units. A minimum estimate of essential gene number for the region using a truncated Poisson calculation is 48. The data indicate a minimum estimate of approximately 3500 essential genes in the Caenorhabditis elegans genome.

Rose AM et al. (1983) International C. elegans Meeting "molecular and genetic analysis of the dpy-14 unc-13 region of chromosome I."

Donati LM, Rose AM, Harris LJ, Mawji NR

[International C. elegans Meeting, 1983]

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.