Wong SS et al. (2012) Cell Calcium "Split-intein mediated re-assembly of genetically encoded Ca(2+) indicators."

Wong SS, Truong K, Mills E, Suzuki H, Kotera I

[Cell Calcium, 2012]

While genetically encoded Ca(2+) indicators (GECIs) allow Ca(2+) imaging in model organisms, the gene expression is often under the control of a single promoter that may drive expression beyond, the cell types of interest. To enable more cell-type specific targeting, GECIs can be brought under the, control of the intersecting expression from two promoters. Here, we present the splitting and, reassembly of two representative GECIs (TN-XL and GCaMP2) mediated by the split intein from Nostoc, punctiforme (NpuDnaE). While the split TN-XL biosensor offered ratiometric Ca(2+) imaging, it had a, diminished Ca(2+) response relative to the native TN-XL biosensor. In contrast, the split GCaMP2, biosensor retained similar Ca(2+) response to the native GCaMP2. The split GCaMP2 biosensor was, further targeted to the pharyngeal muscles of Caenorhabditis elegans where Ca(2+) signals from feeding C. elegans, were imaged. Thus, we envision that increased cell-type targetability of GECIs is feasible with two, complementary promoters.

Lim D et al. (2011) J Biomed Opt "Optically sectioned in vivo imaging with speckle illumination HiLo microscopy."

Mertz J, Lim D, Ford TN, Chu KK

[J Biomed Opt, 2011]

We present a simple wide-field imaging technique, called HiLo microscopy, that is capable of producing optically sectioned images in real time, comparable in quality to confocal laser scanning microscopy. The technique is based on the fusion of two raw images, one acquired with speckle illumination and another with standard uniform illumination. The fusion can be numerically adjusted, using a single parameter, to produce optically sectioned images of varying thicknesses with the same raw data. Direct comparison between our HiLo microscope and a commercial confocal laser scanning microscope is made on the basis of sectioning strength and imaging performance. Specifically, we show that HiLo and confocal 3-D imaging of a GFP-labeled mouse brain hippocampus are comparable in quality. Moreover, HiLo microscopy is capable of faster, near video rate imaging over larger fields of view than attainable with standard confocal microscopes. The goal of this paper is to advertise the simplicity, robustness, and versatility of HiLo microscopy, which we highlight with in vivo imaging of common model organisms including planaria, C. elegans, and zebrafish.

Frokjaer-Jensen C et al. (2016) Cell "An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C.elegans Germline."

Dupuy D, Fire AZ, Millet JR, Hansen L, Zhao D, Li Y, Liu X, Jain N, Rebora K, Jorgensen EM, Davis MW, Frokjaer-Jensen C, Kim SK

[Cell, 2016]

Cells benefit from silencing foreign genetic elements but must simultaneously avoid inactivating endogenous genes. Although chromatin modifications and RNAs contribute to maintenance of silenced states, the establishment of silenced regions will inevitably reflect underlying DNA sequence and/or structure. Here, we demonstrate that a pervasive non-coding DNA feature in Caenorhabditis elegans, characterized by 10-base pair periodic An/Tn-clusters (PATCs), can license transgenes for germline expression within repressive chromatin domains. Transgenes containing natural or synthetic PATCs are resistantto position effect variegation and stochastic silencing inthe germline. Among endogenous genes, intron length and PATC-character undergo dramatic changes as orthologs move from active to repressive chromatin over evolutionary time, indicating a dynamic character to the An/Tn periodicity. We propose that PATCs form the basis of a cellular immune system, identifying certain endogenous genes in heterochromatic contexts as privileged while foreign DNA can be suppressed with no requirement for a cellular memory of prior exposure.

McArdle K et al. (1998) J Cell Biol "Ca2+-dependent muscle dysfunction caused by mutation of the Caenorhabditis elegans troponin T-1 gene."

Allen TS, Bucher EA, McArdle K

[J Cell Biol, 1998]

We have investigated the functions of troponin T (CeTnT-1) in Caenorhabditis elegans embryonic body wall muscle. TnT tethers troponin I (TnI) and troponin C (TnC) to the thin filament via tropomyosin (Tm), and TnT/Tm regulates the activation and inhibition of myosin-actin interaction in response to changes in intracellular [Ca2+]. Loss of CeTnT-1 function causes aberrant muscle trembling and tearing of muscle cells from their exoskeletal attachment sites (Myers, C.D., P.-Y. Goh, T. StC. Allen, E.A. Bucher, and T. Bogaert. 1996. J. Cell Biol. 132:1061-1077). We hypothesized that muscle tearing is a consequence of excessive force generation resulting from defective tethering of Tn complex proteins. Biochemical studies suggest that such defective tethering would result in either (a) Ca2+-independent activation, due to lack of Tn complex binding and consequent lack of inhibition, or (b) delayed reestablishment of TnI/TnC binding to the thin filament after Ca2+ activation and consequent abnormal duration of force. Analyses of animals doubly mutant for CeTnT-1 and for genes required for Ca2+ signaling support that CeTnT-1 phenotypes are dependent on Ca2+ signaling, thus supporting the second model and providing new in vivo evidence that full inhibition of thin filaments in low [Ca2+] does not require TnT.

Lladser ME et al. (2014) J Math Biol "Approximation of sojourn-times via maximal couplings: motif frequency distributions."

Chestnut SR, Lladser ME

[J Math Biol, 2014]

Sojourn-times provide a versatile framework to assess the statistical significance of motifs in genome-wide searches even under non-Markovian background models. However, the large state spaces encountered in genomic sequence analyses make the exact calculation of sojourn-time distributions computationally intractable in long sequences. Here, we use coupling and analytic combinatoric techniques to approximate these distributions in the general setting of Polish state spaces, which encompass discrete state spaces. Our approximations are accompanied with explicit, easy to compute, error bounds for total variation distance. Broadly speaking, if Tn is the random number of times a Markov chain visits a certain subset T of states in its first n transitions, then we can usually approximate the distribution of Tn for n of order (1 )(m), where m is the largest integer for which the exact distribution of Tm is accessible and 0 1 is an ergodicity coefficient associated with the probability transition kernel of the chain. This gives access to approximations of sojourn-times in the intermediate regime where n is perhaps too large for exact calculations, but too small to rely on Normal approximations or stationarity assumptions underlying Poisson and compound Poisson approximations. As proof of concept, we approximate the distribution of the number of matches with a motif in promoter regions of C.

Natsuka S et al. (2005) J Biochem "Characterization of wheat germ agglutinin ligand on soluble glycoproteins in Caenorhabditis elegans."

Natsuka S, Kawaguchi M, Wada Y, Ichikawa A, Hase S, Ikura K

[J Biochem, 2005]

Some mutants of Caenorhabditis elegans show altered patterns of ectopic binding with wheat germ agglutinin (WGA). Some of these mutants also have defects of morphogenesis and movement during development. To clarify the structures of WGA-ligands in C. elegans that may be involved in developmental events, we have analyzed glycan structures capable of binding WGA. We isolated glycoproteins from wild-type C. elegans by WGA-affinity chromatography, and analyzed their glycan structures by a combination of hydrazine degradation and fluorescent labeling. The glycoproteins had oligomannose-type and complex-type N-glycans that included agalacto-biantenna and agalacto-tetraantenna glycans. Although the complex-type glycans carried beta-GlcNAc residues at their non-reducing ends, they did not bind to the WGA-agarose-resin. Thus, it was suggested that these N-glycans were not responsible for WGA-binding of the isolated glycoproteins. Hydrazinolysis of the glycoproteins also released a considerable amount of GalNAc monosaccharide. It was surmised that N-acetylgalactosamine was derived from mucin-type O-glycans with the Tn-antigen structure (GalNAcalpha1-O-Ser/Thr). WGA-blotting assay of neoglycoproteins revealed that a cluster of Tn-antigens was a good ligand for WGA. These results suggested that the WGA-ligand in C. elegans is a cluster of alpha-GalNAc monosaccharides linked to mucin-like glycoprotein(s). The observations reported in this paper emphasize the possible significance of mucin-type O-glycans in the development of a multicellular organism.

Zhang D et al. (2018) Science "The piRNA targeting rules and the resistance to piRNA silencing in endogenous genes."

Zhang D, Wu WS, Tu S, Huang WC, Weng Z, Lee HC, Stubna M

[Science, 2018]

Piwi-interacting RNAs (piRNAs) silence transposons to safeguard genome integrity in animals. However, the functions of the many piRNAs that do not map to transposons remain unknown. Here, we show that piRNA targeting in Caenorhabditis elegans can tolerate a few mismatches but prefer perfect pairing at the seed region. The broad targeting capacity of piRNAs underlies the germline silencing of transgenes in C. elegans Transgenes engineered to avoid piRNA recognition are stably expressed. Many endogenous germline-expressed genes also contain predicted piRNA targeting sites, and periodic An/Tn clusters (PATCs) are an intrinsic signal that provides resistance to piRNA silencing. Together, our study revealed the piRNA targeting rules and highlights a distinct strategy that C. elegans uses to distinguish endogenous from foreign nucleic acids.

Zhang H et al. (2012) Water Res "Degradation of microcystin-LR in water by glow discharge plasma oxidation at the gas-solution interface and its safety evaluation."

Zhang H, Yang L, Huang Q, Wang X, Ke Z, Yu Z

[Water Res, 2012]

Microcystin-LR (MC-LR) is one of the most commonly found microcystins (MCs) in fresh water and it poses danger to human health due to its potential hepatotoxicity. In the present study, we employed a novel method by using discharge plasma taking place at the gas-solution interface in gas atmosphere to degrade MC-LR in aqueous solution. The initial degradation rate of MC-LR was fastest under acidic conditions (5.41 +/- 0.17 x 10(-3) mM min(-1) at pH 3.04) and decreased to 2.22 +/- 0.11 x 10(-3) mM min(-1) and 0.912 +/- 0.02 x 10(-3) mM min(-1) at pH 4.99 and 7.02, respectively. The effects of total soluble nitrogen (TN), total soluble phosphorus (TP) and natural organic matter (NOM) on the degradation efficiency were studied. The degradation rate was remarkably affected by TP and TN. Mass spectrometry was applied to identify the products of the reactions. Major degradation pathways are proposed according to the results of liquid chromatography/mass spectrometry (LC/MS) results. It suggests that the degradation of MC-LR is initiated via the attack of hydroxyl radicals on the conjugated carbon double bonds of Adda and on the benzene ring of Adda. Finally, the toxicity of intermediates or end-products from MC-LR degraded by this method was assessed using Caenorhabditis elegans. Our findings demonstrates that discharge plasma oxidation is a promising technology for degradation and removal of MC-LR and it may lead us to a new route to efficient treatment of other cyanotoxins from aqueous solutions.

Abad P et al. (1993) Biochem Biophys Res Commun "Expression and biochemical characterization of the DNA binding activity of TcA, the putative transposase of Caenorhabditis elegans transposable element Tc1."

Abad P, Cerutti M, Palin B, Dalmasso A, Pauron D, Devauchelle G, Quiles C

[Biochem Biophys Res Commun, 1993]

The TcA protein is one of the proteins essential for Tc1 transposition. In order to study the biochemical parameters of Tc1 transposition mechanism, we used TcA protein overproduced in baculovirus system for DNA binding experiments. We show that, despite its relatively strong non specific affinity for DNA, TcA exhibits a better affinity for its Tc1 specific binding sites. The K0.5 is 3.8 nM for the Tc1 whereas in the same type of experiment the K0.5 is 24 nM for calf thymus DNA. The ratio value between specific and non specific DNA binding activity of the TcA protein was also exhibited by other transposases such as those of the bacteriophage Mu, Tn 10 and the Drosophila P element. This nonspecific DNA binding activity may be involved in determining sites of transposable element insertion.

Fire A et al. (2006) Genetics "Unusual DNA structures associated with germline genetic activity in Caenorhabditis elegans."

Fire A, Tan F, Alcazar R

[Genetics, 2006]

We describe a surprising long-range periodicity which underlies a substantial fraction of C. elegans genomic sequence. Extended segments (up to several hundred nucleotides) of the C. elegans genome show a strong bias toward occurrence of AA/TT dinucleotides along one face of the helix while little or no such constraint is evident on the opposite helical face. Segments with this characteristic periodicity are highly overrepresented in intron sequences and are associated with a large fraction of genes with known germline expression in C. elegans. In addition to altering the path and flexibility of DNA in vitro, sequences of this character have been shown by others to constrain DNA::nucleosome interactions, potentially producing a structure which could resist the assembly of highly ordered (phased) nucleosome arrays that have been proposed as a precursor to heterochromatin. We propose a number of ways that the periodic occurrence of An/Tn clusters could reflect evolution and function of genes which express in the germ cell lineage of C. elegans.