Zhao J et al. (2022) Nat Commun "Bond-selective intensity diffraction tomography."

Zhan Y, Chen Z, Wang B, Matlock A, Chen F, Song Z, Xu Y, Zhao J, Cheng JX, Lin X, Zhu J, Zhu H, Tian L

[Nat Commun, 2022]

Recovering molecular information remains a grand challenge in the widely used holographic and computational imaging technologies. To address this challenge, we developed a computational mid-infrared photothermal microscope, termed Bond-selective Intensity Diffraction Tomography (BS-IDT). Based on a low-cost brightfield microscope with an add-on pulsed light source, BS-IDT recovers both infrared spectra and bond-selective 3D refractive index maps from intensity-only measurements. High-fidelity infrared fingerprint spectra extraction is validated. Volumetric chemical imaging of biological cells is demonstrated at a speed of ~20 s per volume, with a lateral and axial resolution of ~350 nm and ~1.1 µm, respectively. BS-IDT's application potential is investigated by chemically quantifying lipids stored in cancer cells and volumetric chemical imaging on Caenorhabditis elegans with a large field of view (~100 µm x 100 µm).

Schwartz S et al. (2008) Genome Res "Large-scale comparative analysis of splicing signals and their corresponding splicing factors in eukaryotes."

Burstein D, Silva J, Eyras E, Pupko T, Schwartz S, Ast G

[Genome Res, 2008]

Introns are among the hallmarks of eukaryotic genes. Splicing of introns is directed by three main splicing signals: the 5'' splice site (5''ss), the branch site (BS), and the polypyrimdine tract/3''splice site (PPT-3''ss). To study the evolution of these splicing signals, we have conducted a systematic comparative analysis of these signals in over 1.2 million introns from 22 eukaryotes. Our analyses suggest that all these signals have dramatically evolved: The PPT is weak among most fungi, intermediate in plants and protozoans, and strongest in metazoans. Within metazoans it shows a gradual strengthening from Caenorhabditis elegans to human. The 5''ss and the BS were found to be degenerate among most organisms, but highly conserved among some fungi. A maximum parsimony-based algorithm for reconstructing ancestral position-specific scoring matrices suggested that the ancestral 5''ss and BS were degenerate, as in metazoans. To shed light on the evolutionary variation in splicing signals, we have analyzed the evolutionary changes in the factors that bind these signals. Our analysis reveals coevolution of splicing signals and their corresponding splicing factors: The strength of the PPT is correlated to changes in key residues in its corresponding splicing factor U2AF2; limited correlation was found between changes in the 5''ss and U1 snRNA that binds it; but not between the BS and U2 snRNA. Thus, although the basic ability of eukaryotes to splice introns has remained conserved throughout evolution, the splicing signals and their corresponding splicing factors have considerably evolved, uniquely shaping the splicing mechanisms of different organisms.

Wicky C et al. (2004) Mol Cell Biol "Multiple genetic pathways involving the Caenorhabditis elegans Bloom's syndrome genes him-6, rad-51, and top-3 are needed to maintain genome stability in the germ line."

Passannante M, Alpi A, Wicky C, Rose A, Gartner A, Muller F

[Mol Cell Biol, 2004]

Bloom's syndrome (BS) is an autosomal-recessive human disorder caused by mutations in the BS RecQ helicase and is associated with loss of genomic integrity and an increased incidence of cancer. We analyzed the mitotic and the meiotic roles of Caenorhabditis elegans him-6, which we show to encode the ortholog of the human BS gene. Mutations in him-6 result in an enhanced irradiation sensitivity, a partially defective S-phase checkpoint, and in reduced levels of DNA-damage induced apoptosis. Furthermore, him-6 mutants exhibit a decreased frequency of meiotic recombination that is probably due to a defect in the progression of crossover recombination. In mitotically proliferating germ cells, our genetic interaction studies, as well as the assessment of the number of double-strand breaks via RAD-51 foci, reveal a complex regulatory network that is different from the situation in yeast. Although the number of double-strand breaks in him-6 and top-3 single mutants is elevated, the combined depletion of him-6 and top-3 leads to mitotic catastrophe concomitant with a massive increase in the level of double-strand breaks, a

Zheng J et al. (2016) Front Nutr "Dietary Plant Lectins Appear to Be Transported from the Gut to Gain Access to and Alter Dopaminergic Neurons of Caenorhabditis elegans, a Potential Etiology of Parkinson's Disease."

Zheng J, Adhikari B, Keller JN, Peng N, King JF, Wang M, King ML, Laine RA, Wei W

[Front Nutr, 2016]

Lectins from dietary plants have been shown to enhance drug absorption in the gastrointestinal tract of rats, be transported trans-synaptically as shown by tracing of axonal and dendritic paths, and enhance gene delivery. Other carbohydrate-binding protein toxins are known to traverse the gut intact in dogs. Post-feeding rhodamine- or TRITC-tagged dietary lectins, the lectins were tracked from gut to dopaminergic neurons (DAergic-N) in transgenic Caenorhabditis elegans (C. elegans) [egIs1(Pdat-1:GFP)] where the mutant has the green fluorescent protein (GFP) gene fused to a dopamine transport protein gene labeling DAergic-N. The lectins were supplemented along with the food organism Escherichia coli (OP50). Among nine tested rhodamine/TRITC-tagged lectins, four, including Phaseolus vulgaris erythroagglutinin (PHA-E), Bandeiraea simplicifolia (BS-I), Dolichos biflorus agglutinin (DBA), and Arachis hypogaea agglutinin (PNA), appeared to be transported from gut to the GFP-DAergic-N. Griffonia Simplicifolia and PHA-E, reduced the number of GFP-DAergic-N, suggesting a toxic activity. PHA-E, BS-I, Pisum sativum (PSA), and Triticum vulgaris agglutinin (Succinylated) reduced fluorescent intensity of GFP-DAergic-N. PHA-E, PSA, Concanavalin A, and Triticum vulgaris agglutinin decreased the size of GFP-DAergic-N, while BS-I increased neuron size. These observations suggest that dietary plant lectins are transported to and affect DAergic-N in C. elegans, which support Braak and Hawkes' hypothesis, suggesting one alternate potential dietary etiology of Parkinson's disease (PD). A recent Danish study showed that vagotomy resulted in 40% lower incidence of PD over 20years. Differences in inherited sugar structures of gut and neuronal cell surfaces may make some individuals more susceptible in this conceptual disease etiology model.

Choi, S. et al. (2017) International Worm Meeting "Real time observation of DNA unwinding mode of Caenorhabditis elegans HIM-6 helicase."

Cho, H., Kim, H., Ahn, B., Choi, S.

[International Worm Meeting, 2017]

Bloom's syndrome(BS) is caused by mutations in the BLM gene encoding BLM helicase. BLM helicase contains conserved RecQ helicase domain which includes 3'-5' helicase activity. BLM unwinds various DNA helicase substrates including replication forks. Cells from BS patients, that lack the BLM, show defects in the response to replicative stress and contain a multitude of chromosomal aberrations. A BLM homolog, him-6, has been identified in C. elegans based on its close homology with human BLM. Here, we used single-molecule FRET to measure DNA unwinding in real time. Our results showed that the HIM-6 repetitively unwound a long forkd DNA depending on ATP concentrations and resolved flap and D-loop DNA, all of which are intermediates in DNA repair. Also, we found that HIM-6 unwound at most 25 base pairs of duplex DNA in a speed of 28 nucleotides per second before moving back. Besides, repetitive unwinding mode of HIM-6 was changed to unidirectional unwinding in the presence of RPA, indicating that its unwinding mode can be modulated by proteins in vivo. This study can be used to search proteins which would modulated HIM-6 unwinding mode on different DNA structures, such as Holliday junction and G4-quadruplex.

Wadsworth WG et al. (2000) East Coast Worm Meeting "Elongation factor-2 kinase affects Caenorhabditis elegans life span by regulating protein turnover"

Tavernarakis NN, Wadsworth WG, Mendola CE, Ryazanov AG, Altun-Gultekin ZF, Pavur KS, Driscoll MA, Nefsky BS

[East Coast Worm Meeting, 2000]

Accumulation of damaged proteins is postulated to be a major contributor to senescent decline. Decreases in both protein synthesis and degradation rates may result in the persistence of defective or modified proteins and thus the overall rate of protein turnover could affect the rate of aging. We characterized a Caenorhabditis elegans mutation disrupting the efk-1 gene which encodes elongation factor-2 (eEF-2) kinase, an enzyme that specifically phosphorylates eEF-2, inhibiting the elongation step of protein synthesis. We find that loss of eEF-2 kinase activity increases protein synthesis and degradation rates and extends lifespan. Conversely, over-expression of eEF-2 kinase in transgenic nematodes shortens lifespan. efk-1 mutants appear nearly normal in morphology, co-ordination and fertility but exhibit slowed development and altered rates of several rhythmic behaviors, reminiscent of the clk lifespan extension mutants. Interestingly, double mutants of efk-1 and age-1 or clk-1 mutants live no longer than either single mutant. This suggests both age-1 and clk-1 mutants may affect lifespan, at least in part, by modulating protein turnover. Strikingly, eEF-2 kinase activity is also decreased in starved nematodes, suggesting that up-regulation of protein synthesis via decreased eEF-2 kinase activity could contribute to the extension of lifespan by caloric restriction. Our data provide the first direct evidence that an increase in protein turnover can extend lifespan.

Shekhar Saxena A et al. (2018) Mol Biol Evol "Evolution of the mutational process under relaxed selection in Caenorhabditis elegans."

Salomon MP, Baer CF, Shekhar Saxena A, Matsuba C, Yeh SD

[Mol Biol Evol, 2018]

The mutational process varies at many levels, from within genomes to among taxa. Many mechanisms have been linked to variation in mutation, but understanding of the evolution of the mutational process is rudimentary. Physiological condition is often implicated as a source of variation in microbial mutation rate and may contribute to mutation rate variation in multicellular organisms.Deleterious mutations are a ubiquitous source of variation in condition. We test the hypothesis that the mutational process depends on the underlying mutation load in two groups of Caenorhabditis elegans mutation accumulation (MA) lines that differ in their starting mutation loads. "First-Order MA" (O1MA) lines maintained under minimal selection for 250 generations were divided into high-fitness and low-fitness groups and sets of "second-order MA" (O2MA) lines derived from each O1MA line were maintained for 150 additional generations. Genomes of 48 O2MA lines and their progenitors were sequenced. There is significant variation among O2MA lines in base-substitution rate (bs), but no effect of initial fitness; the indel rate is greater in high-fitness O2MA lines. Overall, bs is positively correlated with recombination and proximity to short tandem repeats and negatively correlated with 10bp and 1Kb GC content. However, probability of mutation is sufficiently predicted by the three-nucleotide motif alone. 90% of the variance in standing nucleotide variation is explained by mutability. Total mutation rate increased in the O2MA lines, as predicted by the "drift barrier" model of mutation rate evolution. These data, combined with experimental estimates of fitness, suggest that epistasis is synergistic.

Jung H et al. (2014) PLoS One "Characterization of the Caenorhabditis elegans HIM-6/BLM helicase: unwinding recombination intermediates."

Jung H, Lee JA, Lee H, Ahn B, Choi S

[PLoS One, 2014]

Mutations in three human RecQ genes are implicated in heritable human syndromes. Mutations in BLM, a RecQ gene, cause Bloom syndrome (BS), which is characterized by short stature, cancer predisposition, and sensitivity to sunlight. BLM is a RecQ DNA helicase that, with interacting proteins, is able to dissolve various DNA structures including double Holliday junctions. A BLM ortholog, him-6, has been identified in Caenorhabditis elegans, but little is known about its enzymatic activities or its in vivo roles. By purifying recombinant HIM-6 and performing biochemical assays, we determined that the HIM-6 has DNA-dependent ATPase activity HIM-6 and helicase activity that proceeds in the 3'-5' direction and needs at least five 3' overhanging nucleotides. HIM-6 is also able to unwind DNA structures including D-loops and Holliday junctions. Worms with him-6 mutations were defective in recovering the cell cycle arrest after HU treatment. These activities strongly support in vivo roles for HIM-6 in processing recombination intermediates.

Melissa M Grabowski et al. (2005) International Worm Meeting "Mutation in the C. elegans him-6 Blooms Ortholog Causes a Mutator Phenotype"

Nenad Svrzikapa, Melissa M Grabowski, Heidi A Tissenbaum

[International Worm Meeting, 2005]

Individuals with Bloom's Syndrome display a high incidence of cancer caused by genomic instability. Bloom Syndrome (BS) is caused by mutation in a member of the RecQ helicase family, the Bloom helicase (BLM). We use C. elegans to elucidate the in vivo functions of BLM in maintaining genomic stability. We find him-6 mutants show a mutator phenotype that specifically results in an increased amount of insertions and deletions. This leads to the appearance of random visible phenotypes in a mutant him-6 population. We also observe additional germ line phenotypes in him-6 mutants including an increased level of germ line apoptosis and a mortal germ line. Finally, the increased genomic instability in the him-6 mutants leads to a shortened life span. Our data suggests a model where loss of HIM-6 causes genomic instability either due to an increased number of DNA lesions, an inability to repair these lesions, or low fidelity recombination events.

Glenwinkel, Lori et al. (2013) International Worm Meeting "CisOrtho V 2.0: a comparative genomic approach to genome wide identification of transcription factor target genes."

Glenwinkel, Lori, Hobert, Oliver

[International Worm Meeting, 2013]

CisOrtho V 2.0 is able to predict novel regulatory target genes using an experimentally derived PSSM and species conservation among five nematode genomes. CisOrtho ranks transcription factor (TF) binding sites (BS) by the PSSM log-odds score, BS enrichment, and inter-species preservation of BSs in orthologous regions. I have validated CisOrtho's predictive power using a bioinformatics approach and in vivo validation of novel regulatory targets. Three sets of regulatory target genes for the cholinergic, AIY, and ASE neuron terminal selector TFs (UNC-3,TTX-3/CEH-10,CHE-1) were compared to a control set of 1000 random genes or 93 collagen genes that we expect to exert their expression independently of these three TFs. Each criterion that CisOrtho uses to rank potential TF target genes was calculated for each orthologous region between 5 nematode species in upstream, intronic, exonic, and downstream regions. Known TF target genes ranked significantly better for all criteria in upstream promoter regions while intronic, exonic, downstream, and total gene ranking criteria were not always significantly different. For in vivo validation of CisOrtho, six highly ranked potential UNC-3 target genes were chosen. Four of these genes are completely uncharacterized while two have published expression patterns in the ventral nerve cord where UNC-3 exerts its regulation as a terminal selector of cholinergic motor neurons. GFP promoter fusions were made for four of the genes that are previously uncharacterized in the literature. Each reporter was injected into wild type C. elegans then crossed into an UNC-3 mutant, unc-3(e151). The four uncharacterized genes showed ventral nerve cord neuron expression and all 6 genes had significant loss of ventral nerve cord neuron expression in the UNC-3 mutant suggesting UNC-3 dependence. CisOrtho predicted target genes are also enriched in neurogenesis pathway genes for all three terminal selector genes providing ample candidates for further in vivo experimentation. These lines of evidence suggest that CisOrtho is able to successfully predict novel targets of TFs.