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.

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.

Kiontke K et al. (2000) European Worm Meeting ""Saltational evolution" of phasmid position in nematodes"

Sudhaus W, Kiontke K, Fitch DHA

[European Worm Meeting, 2000]

Phasmids are structures in the tail region of secernentean nematodes. The two large groups within Nematoda, Secernentea and "Adenophorea", are distinguished by this character: "Adenophorea" lack phasmids. Phasmids are similarly built in C. elegans(Rhabditidae) Tylenchidae and Filariidae. They consist of 1-2 socket cells that contact the body epidermis, a glandular sheath cell, and one or two sensory processes projecting into a receptor cavity within the sheath cell. Through a pore in the socket cell, these recessed processes are exposed to the exterior. Phasmids are generally described for females of most secernentean species. However, especially in Rhabditidae, phasmids in males have rarely been reported. This is due to the fact that in males phasmids are easily confused with rays if they are integrated into the velum (Fitch & Emmons, 1995). With SEM and interference contrast LM, the pore in the phasmid socket cell is clearly visible, whereas in rays either one sensory process protrudes through an opening in the structural cell or this opening is very small. We studied 53 species of Rhabditidae including Heterorhabditis as well as Diplogastrina, Panagrolaimidae, Cephalobidae, Brevibuccidae, Myolaimus, Steinernema, and Strongylida with LM and SEM, and scanned the literature on animal parasitic Secernentea. Phasmids are present in males of all species. The rhabditid ancestor had 9 pairs of rays and one pair of phasmids instead of 10 pairs as rays as was previously assumed. Two alternative positions of the phasmids relative to the rays could be distinguished: an anterior position with 3-4 rays posterior to the phasmid, and a posterior position with all rays anterior to the phasmid as in C. elegans. There are never more than 4 rays posterior to the phasmid. Phasmids are anterior in Cephalobidae and Diplogastrina and posterior in Panagrolaimidae, in Steinernema, and in Strongylidae. Within Rhabditidae both character states occur. We mapped the phasmid position on a cladogram based on small subunit rDNA (Fitch et al. unpublished) and found that multiple changes between anterior and posterior phasmid position must have occurred during evolution. This could be explained in terms of the development of phasmid socket cells and the posterior three rays, which are all derived from the same blast cell (T) in the L1 larva. In C. elegans, the phasmid socket cells are descendants of the posterior daughter of the T cell. The polarity of the first division of the T cell might be reversed in species with anterior phasmids, such that the phasmid socket cells are now descendants of its anterior daughter (Fitch, 1997; Kiontke & Sudhaus in press). We have begun to test this hypothesis. References: Fitch, D.H.A. & Emmons S. (1995) Dev. Biol. 170: 564-582 Fitch, D.H.A. (1997) Syst. Biol. 56: 145-179. Kiontke, K. & Sudhaus, W. (in Press) J. Nemat. Morph. Syst.

Zhao Y et al. (2007) J Biol Chem "The notch regulator MAML1 interacts with p53 and functions as a coactivator."

Zeng MS, Dimri G, Kung A, Delmolino LM, Ma H, Zhao Y, Griffin JD, Wazer DE, Band H, Katzman RB, Solomon A, Reddi HV, Gao Q, Miele L, Germaniuk-Kurowska A, Zhang Y, Bhat I, Stanculescu A, Wu L

[J Biol Chem, 2007]

Members of the evolutionarily-conserved Mastermind (MAM) protein family, including the three related mammalian Mastermind-like (MAML) proteins, MAML1-3, function as crucial coactivators of Notch-mediated transcriptional activation. Given the recent evidence of crosstalk between the p53 and Notch signal transduction pathways and the increasing evidence that coactivator proteins can function in multiple transcriptional pathways, we have investigated if MAML1 may also be a transcriptional coactivator of p53. Indeed, we show here that MAML1 is able to interact with p53 both in vitro and in vivo. We show that MAML1-p53 interaction involves the N-terminus region of MAML1 and the DNA-binding domain of p53 and we use a ChIP assay to show that MAML1 is part of the activator complex that binds to native p53 response elements within the promoter of the p53 target genes. Overexpression of wild-type MAML1 as well as a mutant, defective in Notch signaling enhanced the p53-dependent gene induction in mammalian cells whereas MAML1 knockdown reduced the p53-dependent gene expression. MAML1 increases half-life of p53 protein and enhances its phosphorylation/ acetylation upon DNA damage of cells. Finally, RNAi-mediated knockdown of the single C. elegans MAML homolog, Lag-3, led to substantial abrogation of p53-mediated germ-cell apoptotic response to DNA damage and markedly reduced the expression of Ced-13 and egl-1, downstream pro-apoptotic targets of C. elegans p53 homolog, Cep-1. Thus, we present evidence for a novel coactivator function of MAML1 for p53, independent of its function as a coactivator of Notch signaling pathway.

Cha, S. et al. (2015) International Worm Meeting "An annotated genome of the root-knot nematode, Meloidogyne chitwoodi."

Cha, S., Bird, D.

[International Worm Meeting, 2015]

Plant-parasitic nematodes are responsible for annual crop losses in excess of USD123 billion worldwide. Most important are the root-knot nematodes (RKN: Meloidogyne spp.), which establish an intimate association with their host. As a genus, Meloidogyne has a host-range that spans the tracheophyta, although individual isolates are more restricted. In cool climates, M. hapla and M. chitwoodi predominate and are a significant problem on potato in Europe and the US. Because of its genetic tractability, we selected M. hapla for sequencing, and an annotated public release (HapPep1) was made in 2008 (Opperman et al., 2008). Since then we have undertaken on-going curation, and the current release (HapPep5) includes ~2xE9 RNA-Seq reads (Guo et al., 2014). We have also sequenced two additional strains of M. hapla (VW8 and LM) but they have not yet been released. Because M. hapla and M. chitwoodi are sympatric, they presumably have similar gene compliments. To test this, we sequenced the M. chitwoodi genome.Genomic DNA was isolated from nematodes collected in a potato field in Washington State, and confirmed by Axel Elling to be M. chitwoodi. Using an Illumina MiSeq II we obtained 20,079,197 short (~300 bp) paired-end reads. The assembly parameters were empirically optimized, and 4,735 contigs assembled; N50 is 82 kbp. The longest is ~758 kbp with coverage of 33 reads per bp. Average coverage genome-wide is 289 reads. At the protein level, CEGMA identified 245 (98.79%) of the core proteins, pointing to near 100% genome coverage. When CEGMA proteins as a query were blasted against the assembled contigs as a database, it was observed that one protein had hits with more than two contigs. Using CEGMA (and other) proteins, as well as M. chitwoodi ESTs, we trained AUGUSTUS for gene prediction. GO categorization was performed using InterProScan. Analysis of these data is in progress, with an emphasis on genes encoding replicas of plant peptide hormones (xenomones). Because of a potential role in speciation of the closely-related parasites, we have, in collaboration with David Lunt (University of Hull), examined the numbers and distribution of transposable elements: not surprising, M. chitwoodi is much more similar to M. hapla than it is to M. incognita.Operman et al., 2008. Proc Nat Acad Sci 105: 14802 -14807.Guo et al. 2014. Worm 3:e29158.

(1982) Worm Breeder's Gazette "Goettingen worm group activities 1981 and prospects 1982"

[Worm Breeder's Gazette, 1982]

After Gunter von Ehrenstein's death of a heart attack December 26, 1980, the 6 directors of the Max-Planck-Institute for Experimental Medicine decided to close the Department of Molecular Biology as of the end of 1982. We are continuing work to finish up and publish work in progress with gradual attrition as people find new positions elsewhere and with correspondingly diminishing budget. A list of our recent papers and manuscripts is given below. Eddi Isnenghi completed his paper on the phenotypes of the Gottingen emb mutants (including TSP's and maternal tests) and is trying to microinject cloned DNA to rescue worm mutants. He is also trying to transform yeast mutants with worm DNA. Randy Cassada is still trying to map embs on LG III with deficiencies and duplications and is looking for new strains of C. elegans in the wild. Eddi has received a 6-months' extension of his DFG (= NSF) fellowship until July. Ken Denich is writing up his work on emb mutant lineages in Gottingen for a paper and as PhD Thesis for the Department of Zoology, University of Manitoba and will be returning there this summer to finish up PhD requirements. Einhard Schierenberg has continued with laser-induced cell fusion experiments. If the membrane between 2 cells is disrupted at the right time, the division of both nuclei results in 4 more or less normal cells. Development continues to a twitching lima bean monster. Fusing cells in later stages results in uncoordinated animals. All treated embryos show timing defects in the E-cell line similar to emb mutants, perhaps due to remixing of cytoplasm. A copy of Einhard's film on embryogenesis is available (with commentary) from Scott Emmons for those who want to show it to students. Einhard is going to Bill Wood's lab this fall. Khosro Radnia has left for a job in industry. Ulli Certa and Franz Scharfenberg are characterizing histone H2A variants in emb mutants. They have also identified several histone H4 DNA clones in a C. elegans genomic library using a sea urchin probe obtained from Mike Grunstein. Ulli has finished his PhD and plans to go to Grunstein's lab at UCLA this spring for a post- doc on yeast histones. Ulli and the other gene cloners have been working successfully with similar minded members of the Institute's Department of Chemistry. Franz hopes to do his PhD in that department. Shahid Siddiqui has just joined Joe Culotti at Northwestern. Tom Cole is very happy with his new Zeiss EM 109 electron microscope. Our computer-aided reconstruction program with color display now works for LM and EM input. We are trying to transfer the programs and data to the MRC, so they will still be available after our department has closed. Chris Carlson has gone to the EMBL in Heidelberg as of Jan 1 to work on computer graphics of molecules. Ursula Reuter also has a position at EMBL as of Jan 1 as an EM technician. Ed Hedgecock visited for 3 weeks this fall to show us microinjection, antibody staining (with anti-actin from Mary Osborne and Klaus Weber), post- embryonic lineage techniques and the like. It was a very useful and pleasant visit. We hope to have some of you as visitors in 1982!