Turner LM et al. (1987) International C. elegans Meeting "gamma-irradiation in C elegans."

Turner LM, Baillie DL, Rosenbluth RE

[International C. elegans Meeting, 1987]

Turner LM et al. (1986) Worm Breeder's Gazette "differential gamete sensitivity to gamma-irradiation in C elegans."

Turner LM, Baillie DL, Rosenbluth RE

[Worm Breeder's Gazette, 1986]

Genetic analysis in the nematode Caenorhabditis pends on the ability to induce mutations using gamma-radiation. Previously a gamma-ray dose-response curve was obtained for self-fertilizing hermaphrodites (Rosenbluth et al. Mutat. Res. 110:39-48, 1985). It was found that 1500R induced recessive lethals in the region balanced by eT1 at a rate of 4.5%. This rate did not distinguish oocyte sensitivity from that of sperm. We have now obtained 1500R rates for hermaphrodite oocytes alone and for male sperm. {Figure 1} As can be seen, in general male sperm were more sensitive than hermaphrodite oocytes. Furthermore the gametes appeared to be more sensitive in the mature rather than the immature stages. These results are not surprising since it was known that in Drosophila sperm are more sensitive than oocytes to gamma-radiation. Therefore consideration should be given to the gamete type and/or stage mutagenized when designing screening protocols. Genetic analysis of the mutations recovered in our studies will determine if there are qualitative differences between mutations in the different gametes and stages. Experiments are in progress which will: l) determine more precisely, the rates in the different oocyte stages and 2) compare the rate in mature sperm of hermaphrodites with that in mature sperm of males.

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.

Rosenbluth RE et al. (1985) International C. elegans Meeting "deficiency mapping and crossover suppression on LGV (left) of C elegans."

Rosenbluth RE, Baillie DL, Turner LM

[International C. elegans Meeting, 1985]

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.

McKim KS et al. (1986) Worm Breeder's Gazette "fine structure analysis of unc-60 V."

McKim KS, Turner LM, Baillie DL

[Worm Breeder's Gazette, 1986]

Thin filament assembly of C. elegans muscle is severely disorganized in strains homozygous for the unc-60 mutation (Waterston et al. 1980). unc-60 is located near the left end of LGV, a region of interest to our lab. We have undertaken a fine structure analysis of unc-60 mutations. Available for study were the alleles e677, e890, e723 (Brenner 1974), m35 (D. Riddle) and r398. All are paralyzed except r398 which moves well as an adult but exhibits a paralyzed phenotype when linked to unc- 34. This allele was isolated as a unc-105 suppressor by P. Anderson. Three new alleles were induced in this lab in an F1 screen utilizing a let-x(s704) 310) eT1 balancer chromosome (see McKim et al. this issue). The induction frequency of unc-60 alleles was 1/4000 using 0.012M EMS. Heterozygotes of the genotype unc-34(e556) +/+ unc-60(y) 24) were selfed and the F2's screened for mobile animals. The map shown in Figure 1 has been constructed. Points to note are: 1) The recombinational size (0.01-0.017 mu.) of unc-60 is larger than unc-15 or unc-13 and equivalent to unc-22 and unc-54. unc-54 makes a better comparison because like unc-60, it is found outside a gene cluster. 2) Except for r398, all 6 mapped alleles are clustered at two points. This may reflect a limited number of sites available for mutational alteration to the severely paralyzed phenotype. s1307 has not been mapped because like r398, it is a weak allele. e677 and e890 are lethal over sDf28. We thus consider unc-60 as an essential gene. For example it may have function in the pharyngeal musculature. sup-7 (Waterston 1981) does not suppress e677, e890, e723 or m35. The rest are being tested. All alleles are hypomorphs by deficiency tests. [See Figure 1]

Rosenbluth RE et al. (1984) Worm Breeder's Gazette "analysis of the left half of linkage group V in C elegans."

Baillie DL, Rosenbluth RE, Turner LM

[Worm Breeder's Gazette, 1984]

We have begun an analysis of the left half of LGV using recessive lethals, balanced over eT1, which were generated with low doses of gamma radiation or EMS (Rosenbluth, Cuddeford and Baillie, Mutation Research 110: 39-48,1983). At present, the breakpoints of five 1500 R induced deficiencies divide the region balanced by eT1 into 9 zones. [See Figure 1] To date the distribution of lethal mutations relative to these zones is as follows. (Inter-se complementation tests for mutations in the same zone have not yet been carried out;) [See Figure 2] These preliminary results indicate that the distribution of lethal mutations on LGV(left) follows that of the 'visible' genes. Over half the mutations lie within zones 6 to 9,-i.e. in the 'visible' gene cluster. An additional 1500R induced lethal is being analyzed. The mutation s741 is to the left of unc-46. When no longer balanced over eT1, it recombines with unc-46, giving an apparent map distance of 10 m.u., which would place it between unc-60 and dpy-11. However, s741 is complemented by sDf's 28, 27 and 26. Furthermore, the apparent recombination distance between unc-60 and dpy-11 in s741/unc-60 dpy-11 heterozygotes is reduced from the normal 18 m.u. to 10 m.u. Therefore s741 is associated with a dominant crossover suppressor that affects the unc-60 to dpy-11 region. The recessive lethality may be due to a mutation that has a hypermorphic effect, or one that lies to the left of sDf28.Cloning of the unc-60 s underway, employing BO/N2 Tc1 polymorphisms. We hope to correlate soon the developing lethal map with the recombinant DNA map.

Rosenbluth RE et al. (1985) Worm Breeder's Gazette "update on linkage group V (left)."

Johnsen RC, McKim KS, Rosenbluth RE, Baillie DL, Turner LM

[Worm Breeder's Gazette, 1985]

Our analysis of LGV(left) is continuing. At present 10 deletions, providing 13 different breakpoints, divide the region into 14 zones ( figure). The table shows recessive lethal mutations that have been mapped to the zones. [See Figure 1] At Cold Spring Harbor (1985) we noted that deletion mapping placed unc-34 to the left of unc-60. This has been additionally confirmed by 3-factor mapping. The two genes are 0.8 - 3.4 m.u. apart. Another gene, let-336 has now been deletion-mapped to the left of unc-34 and therefore becomes the current left-end gene of LGV. One of us (Kim McKim) has begun an intragenic mapping study of unc- 60. We are in possession of the alleles, e677, e723, e890 and m35 and would greatly appreciate receiving others. We have noticed that at least e677 and m-35 differ greatly regarding their viability as heterozygotes over sDf28: e677/sDf28 barely survives while m35/sDf28 survives well. This relationship correlates with their respective homozygous phenotypes, - m35 being more mobile than e677.