MS Shah et al. (1999) International C. elegans Meeting "Genetic Analysis of Signaling Pathways that Determine the Deaths of Sex-Specific Neurons"

MS Shah, J Nyugen, D Xue

[International C. elegans Meeting, 1999]

In C. elegans 131 of the 1090 cells generated during development of the hermaphrodite adult animal undergo programmed cell death. We are interested in identifying new genes involved in the life vs. death decisions of normally dying cells. Specifically, we are interested in studying the signaling pathways that regulate the deaths of hermaphrodite specific neurons (HSNs) and male specific chemosensory neurons (CEMs) in different sexes. HSNs in C. elegans control egg-laying in hermaphrodite animals and normally undergo programmed cell death in males in which they are not needed. In contrast, CEM neurons mediate male chemotaxis towards the hermaphrodite during mating but are programmed to die in hermaphrodites where their function is not needed. The life vs. death decisions of the HSN and CEM neurons present an interesting paradigm for studying sex-specific cell death specification. Mutations in several genes ( her-1 , tra-2 , and egl-1 ) have been identified that cause inappropriate death of HSNs in hermaphrodites. her-1 encodes a novel secreted molecule whose activity is high in males to promote male somatic cell fate, while tra-2 encodes a putative transmembrane receptor for the HER-I protein. The egl-1 gene is generally required for the cell-killing in nematodes but is not involved in sex determination. Thus her-1 and tra-2 mediate a novel signal transduction pathway that integrates into the cell-death pathway through the egl-1 gene to control HSN cell death. In order to identify new components in this signaling pathway, we have performed several genetic screens to isolate suppressors of inappropriate HSN cell death caused by loss-of-function mutations in the tra-2 gene. We expect to isolate mutations in genes that control general sex determination, genes that control general programmed cell death, and genes that are involved in specifying the death of HSN neurons in males. So far, we have isolated more than forty mutations. One of them is an allele of the ced-4 gene, a general cell-death gene. Eleven mutations result in fem phenotypes and are likely to be alleles of fem genes. Several mutations seem to affect only HSN cell death and are likely to affect genes that are involved in HSN death specification. We are focusing on studying these HSN-specific mutations.

Wielsch N et al. (2006) J Proteome Res "Rapid validation of protein identifications with the borderline statistical confidence via de novo sequencing and MS BLAST searches."

Surendranath V, Pevzner P, Wielsch N, Frank A, Waridel P, Thomas H, Shevchenko A

[J Proteome Res, 2006]

Protein identifications with the borderline statistical confidence are typically produced by matching a few marginal quality MS/MS spectra to database peptide sequences and represent a significant bottleneck in the reliable and reproducible characterization of proteomes. Here, we present a method for rapid validation of borderline hits that circumvents the need in, often biased, manual inspection of raw MS/MS spectra. The approach takes advantage of the independent interpretation of corresponding MS/MS spectra by PepNovo de novo sequencing software followed by mass spectrometry-driven BLAST (MS BLAST) sequence-similarity database searches that utilize all partially inaccurate, degenerate and redundant candidate peptide sequences. In a case study involving the identification of more than 180 Caenorhabditis elegans proteins by nanoLC -MS/MS analysis on a linear ion trap LTQ mass spectrometer, the approach enabled rapid assignment (confirmation or rejection) of more than 70% of Mascot hits of borderline statistical confidence.

Soto MC (2017) Dev Cell "Sequential Rosettes Drive C.elegans Ventral Nerve Cord Assembly."

Soto MC

[Dev Cell, 2017]

Planar cell polarity (PCP) signaling orients developmental events in vertebrates and invertebrates, including convergent extension (CE). In this issue of Development Cell, Shah and Tanner etal. (2017) report that ROBO/SAX-3 signaling acts in parallel with PCP signaling to drive the CE required for ventral nerve cord assembly in C.elegans.

Morris F Maduro et al. (2002) West Coast Worm Meeting "Identification of Embryonic Mesoderm Genes Using Microarrays"

Joel H Rothman, Gina Broitman-Maduro, Morris F Maduro

[West Coast Worm Meeting, 2002]

At the 8-cell stage of C. elegans embryogenesis, the MS and E blastomeres are born. E will give rise to the entire intestine, while MS makes many mesodermal cell types, including body wall muscle and the posterior half of the pharynx. The fates of MS and E are specified by the MED-1,2 GATA transcription factors. While many of the regulators that function in E lineage development have been identified, there is comparatively little known about how MS development proceeds downstream of the MEDs. As a first approximation toward identifying MED target genes in MS, and identifying regulators that might function in the elaboration of the MS lineage, we are using microarray analysis.

Masselon C et al. (2003) Proteomics "Identification of tryptic peptides from large databases using multiplexed tandem mass spectrometry: simulations and experimental results."

Masselon C, Harkewicz R, Li L, Pasa-Tolic L, Anderson GA, Lee SW, Smith RD

[Proteomics, 2003]

Multiplexed tandem mass spectrometry (MS/MS) has recently been demonstrated as a means to increase the throughput of peptide identification in liquid chromatography (LC) MS/MS experiments. In this approach, a set of parent species is dissociated simultaneously and measured in a single spectrum (in the same manner that a single parent ion is conventionally studied), providing a gain in sensitivity and throughput proportional to the number of species that can be simultaneously addressed. In the present work, simulations performed using the Caenorhabditis elegans predicted proteins database show that multiplexed MS/MS data allow the identification of tryptic peptides from mixtures of up to ten peptides from a single dataset with only three "y" or "b" fragments per peptide and a mass accuracy of 2.5 to 5 ppm. At this level of database and data complexity, 98% of the 500 peptides considered in the simulation were correctly identified. This compares favorably with the rates obtained for classical MS/MS at more modest mass measurement accuracy. LC multiplexed Fourier transform-ion cyclotron resonance MS/MS data obtained from a 66 kDa protein (bovine serum albumin) tryptic digest sample are presented to illustrate the approach, and confirm that peptides can be effectively identified from the C. elegans database to which the

Salzer L et al. (2023) Metabolomics "Capillary electrophoresis-mass spectrometry as a tool for Caenorhabditis elegans metabolomics research."

Witting M, Salzer L, Schmitt-Kopplin P

[Metabolomics, 2023]

INTRODUCTION: Polar metabolites in Caenorhabditis elegans (C. elegans) have predominantly been analyzed using hydrophilic interaction liquid chromatography coupled to mass spectrometry (HILIC-MS). Capillary electrophoresis coupled to mass spectrometry (CE-MS) represents another complementary analytical platform suitable for polar and charged analytes. OBJECTIVE: We compared CE-MS and HILIC-MS for the analysis of a set of 60 reference standards relevant for C. elegans and specifically investigated the strengths of CE separation. Furthermore, we employed CE-MS as a complementary analytical approach to study polar metabolites in C. elegans samples, particularly in the context of longevity, in order to address a different part of its metabolome. METHOD: We analyzed 60 reference standards as well as metabolite extracts from C. elegans daf-2 loss-of-function mutants and wild-type (WT) samples using HILIC-MS and CE-MS employing a Q-ToF-MS instrument. RESULTS: CE separations showed narrower peak widths and a better linearity of the estimated response function across different concentrations which is linked to less saturation of the MS signals. Additionally, CE exhibited a distinct selectivity in the separation of compounds compared to HILIC-MS, providing complementary information for the analysis of the target compounds. Analysis of C. elegans metabolites of daf-2 mutants and WT samples revealed significant alterations in shared metabolites identified through HILIC-MS, as well as the presence of distinct metabolites. CONCLUSION: CE-MS was successfully applied in C. elegans metabolomics, being able to recover known as well as identify novel putative biomarkers of longevity.

Asef CK et al. (2023) Anal Chem "Unknown Metabolite Identification Using Machine Learning Collision Cross-Section Prediction and Tandem Mass Spectrometry."

Rainey MA, Fernandez FM, McIntyre LM, Garcia BM, Gouveia GJ, Asef CK, Edison AS, Shaver AO, Leach FE, Morse AM

[Anal Chem, 2023]

Ion mobility (IM) spectrometry provides semiorthogonal data to mass spectrometry (MS), showing promise for identifying unknown metabolites in complex non-targeted metabolomics data sets. While current literature has showcased IM-MS for identifying unknowns under near ideal circumstances, less work has been conducted to evaluate the performance of this approach in metabolomics studies involving highly complex samples with difficult matrices. Here, we present a workflow incorporating de novo molecular formula annotation and MS/MS structure elucidation using SIRIUS 4 with experimental IM collision cross-section (CCS) measurements and machine learning CCS predictions to identify differential unknown metabolites in mutant strains of Caenorhabditis elegans. For many of those ion features, this workflow enabled the successful filtering of candidate structures generated by in silico MS/MS predictions, though in some cases, annotations were challenged by significant hurdles in instrumentation performance and data analysis. While for 37% of differential features we were able to successfully collect both MS/MS and CCS data, fewer than half of these features benefited from a reduction in the number of possible candidate structures using CCS filtering due to poor matching of the machine learning training sets, limited accuracy of experimental and predicted CCS values, and lack of candidate structures resulting from the MS/MS data. When using a CCS error cutoff of +/-3%, on average, 28% of candidate structures could be successfully filtered. Herein, we identify and describe the bottlenecks and limitations associated with the identification of unknowns in non-targeted metabolomics using IM-MS to focus and provide insights into areas requiring further improvement.

Mello CC et al. (1992) Cell "The pie-1 and mex-1 genes and maternal control of blastomere identity in early C. elegans embryos."

Krause MW, Mello CC, Draper BW, Weintraub H, Priess JR

[Cell, 1992]

During C. elegans embryogenesis an 8-cell stage blastomere, called MS, undergoes a reproducible cleavage pattern, producing pharyngeal cells, body wall muscles, and cell deaths. We show here that maternal-effect mutations in the pie-1 and mex-1 genes cause additional 8-cell stage blastomeres to adopt a fate very similar to that of the wild-type MS blastomere. In pie-1 mutants one additional posterior blastomere adopts an MS-like fate, and in mex-1 mutants four additional anterior blastomeres adopt an MS-like fate. We propose that maternally provided pie-1(+) and mex-1(+) gene products may function in the early embryo to localize or regulate factors that determine the fate of the MS blastomere.

Owraghi M et al. (2010) Dev Biol "Roles of the Wnt effector POP-1/TCF in the C. elegans endomesoderm specification gene network."

Luu T, Owraghi M, Roberson H, Maduro MF, Broitman-Maduro G

[Dev Biol, 2010]

In C. elegans the 4-cell stage blastomere EMS is an endomesodermal precursor. Its anterior daughter, MS, makes primarily mesodermal cells, while its posterior daughter E generates the entire intestine. The gene regulatory network underlying specification of MS and E has been the subject of study for more than 15 years. A key component of the specification of the two cells is the involvement of the Wnt/beta-catenin asymmetry pathway, which through its nuclear effector POP-1, specifies MS and E as different from each other. Loss of pop-1 function results in the mis-specification of MS as an E-like cell, because POP-1 directly represses the end-1 and end-3 genes in MS, which would otherwise promote an endoderm fate. A long-standing question has been whether POP-1 plays a role in specifying MS fate beyond repression of endoderm fate. This question has been difficult to ask because the only chromosomal lesions that remove both end-1 and end-3 are large deletions removing hundreds of genes. Here, we report the construction of bona fide end-1 end-3 double mutants. In embryos lacking activity of end-1, end-3 and pop-1 together, we find that MS fate is partially restored, while E expresses early markers of MS fate and adopts characteristics of both MS and C. Our results suggest that POP-1 is not critical for MS specification beyond repression of endoderm specification, and reveal that Wnt-modified POP-1 and END-1/3 further reinforce E specification by repressing MS fate in E. By comparison, a previous work suggested that in the related nematode C. briggsae, Cb-POP-1 is not required to repress endoderm specification in MS, in direct contrast with Ce-POP-1, but is critical for repression of MS fate in E. The findings reported here shed new light on the flexibility of combinatorial control mechanisms in endomesoderm specification in Caenorhabditis.

Saini V et al. (2013) Vaccine "Poly(d,l)-lactide-co-glycolide (PLGA) microspheres as immunoadjuvant for Brugia malayi antigens."

Verma SK, Murthy PK, Saini V, Kohli D

[Vaccine, 2013]

Recently we identified in Brugia malayi adult worm extract (BmA) a pro-inflammatory 54-68kDa SDS-PAGE resolved fraction F6 that protects the host from the parasite via Th1/Th2 type responses. We are currently investigating F6 as a potential source of vaccine candidate(s) and the present study is aimed at investigating the suitability of poly(d,l)-lactide-co-glycolide microspheres (PLGA-Ms) as immunoadjuvant for the antigen administration in a single dose. PLGA-Ms were prepared aseptically by a modified double emulsion (w/o/w) solvent evaporation technique and their size, shape, antigen adsorption efficiency, in-process stability, and antigen release were characterized. Swiss mice were immunized by a single subcutaneous administration of BmA and F6 adsorbed on PLGA-Ms (lactide:glycolide ratios 50:50 and 75:25) and the immune responses were compared with administration of 1 or 2 doses of plain BmA and F6. Specific IgG, IgG1, IgG2a, IgG2b, IgE levels in serum, cellular-proliferative response and release of IFN-, TNF- and nitric oxide from the cells of immunized host in response to the antigens/LPS/Con A challenge and antibody-dependant cellular cytotoxicity (ADCC) to parasite life stages were determined. The average size of PLGA-Ms 50:50 was smaller than the size of PLGA-Ms 75:25 and the % antigen adsorption efficiency of PLGA-Ms 50:50 was greater than PLGA-Ms 75:25. Single shot injection of PLGA-Ms 50:50/75:25-BmA/F6 produced better and stronger IgG, IgG1/IgG2a and cell-mediated immune responses than even two injections of plain BmA or F6. Further, PLGA-Ms 50:50-F6 produced stronger responses than PLGA-Ms 50:50-BmA. Anti-PLGA-Ms 50:50-F6 antibodies elicited higher ADCC response to infective larval and microfilarial stages of the parasite than anti-PLGA-Ms 75:25-F6 antibodies. The findings demonstrate that PLGA-Ms 50:50 is an excellent adjuvant for use with F6 in a single administration. This is the first ever report on PLGA as immunoadjuvant for filarial antigens.