A <i>Caenorhabditis elegans</i> Locomotion Phenotype Caused by Transgenic Repeats of the <i>hlh-17</i> Promoter Sequence

<div><p>Transgene technology is one of the most heavily relied upon tools in modern biological research. Expression of an exogenous gene within cells, for research and therapeutic applications, nearly always includes promoters and other regulatory sequences. We found that repeats of a non-protein coding transgenic sequence produced profound changes to the behavior of the nematode <i>Caenorhabditis elegans</i>. These changes were produced by a glial promoter sequence but, unexpectedly, major deficits were observed specifically in backward locomotion, a neuron-driven behavior. We also present evidence that this behavioral phenotype is transpromoter copy number-dependent and manifests early in development and is maintained into adulthood of the worm.</p></div

In silico analysis of the <i>Phlh-17</i>.

<p>Cartoon showing possible binding sites of protein factors associated with backward ventral coiler phenotypes. The 2.5 kbp promoter (full ribbon length) was used in the VPR transgenic strains, while the shorter 2 kbp promoter (gray) was used to make line UL1713. Ribbon is pointing in the direction of transcription. A 100% match for an UNC-55 binding site (half-site for UNC-55 dimer; red arrowhead), along with possible (1 mismatch) other sites for UNC-55 binding (yellow arrowheads), a single CTGCTG site (green arrowhead), and exact site matches for the DNA version of the human MBNL1 binding consensus sequence YGCT(T/G)Y (blue arrowheads) are depicted. Arrowhead direction indicates a match to the top (right facing arrowhead) or bottom (left facing) DNA strand; arrowheads not drawn to scale of binding site relative to the hlh-17 promoter sequence.</p

VCP in adult worms is correlated to <i>Phlh-17</i> copy number (CN).

<p>(A) A <i>Phlh-17</i> primer pair was used to determine <i>Phlh-17</i> CN using genomic DNA extracts from individual worms (n=6-7 in each group). Bars represent means ± sem. * p<0.05, ** p<0.01; One way ANOVA, followed by Fisher’s least significant difference (LSD) post hoc test. (B) The <i>Phlh-17</i> CN (x) and the average VCP proportion (y) show statistically significant correlation (regression ANOVA, p = 0.0264). Equation and the coefficient of determination (R²) are shown in the lower right corner. </p

Description of utilized <i>C. elegans</i> strains listing the plasmid(s) used in the production of each line, if the transgene is carried as an extrachromosomal (<i>Ex</i>) or integrated (<i>Is</i>) array, and the presence of the ventral coiler phenotype (VCP).

<p>Description of utilized <i>C. elegans</i> strains listing the plasmid(s) used in the production of each line, if the transgene is carried as an extrachromosomal (<i>Ex</i>) or integrated (<i>Is</i>) array, and the presence of the ventral coiler phenotype (VCP).</p

Summary of plasmids by name, background vector, their promoter and gene content, and location of gene expression in transgenic <i>C. elegans</i> cells.

<p>Summary of plasmids by name, background vector, their promoter and gene content, and location of gene expression in transgenic <i>C. elegans</i> cells.</p

The t-<i>Phlh-17</i> produces the VCP without driving fluorescent protein expression.

<p>Average proportion of trials in which the VCP was displayed by strains N2, VPR163 (<i>Punc-54::mCherry</i> alone; n= 21) and VPR160 (<i>Phlh-17::none</i> + <i>Punc-54::mCherry</i>; n= 21). Bars represent means <u>+</u> sem. ** p<0.01, KWA followed by Dunn’s MCT.</p

A subset of <i>C. elegans</i> lines carrying transgenic arrays containing the trans-<i>hlh-17</i> promoter (t-<i>Phlh-17</i>) to drive expression of fluorescent proteins in the CEPsh glial cells display a ventral coiler phenotype (VCP) during backward movement.

<p>A-B) Transgenic worm lines expressing fluorescent proteins in the CEPsh glial cells driven by the t-<i>Phlh-17</i>. Confocal images show anterior, head, portion of worms. (A) VPR156 shows cytosolic monomericDsRed expression. Dashed circle indicates the CEPsh glial cell bodies and membrane extensions. The arrowhead indicates the thin processes emanating to the anterior sensory structures. (B) VPR839 shows similar GFP expression in the CEPsh glial cell. Scale bar, 20 μm. (C) Image series depicting normal spontaneous reverse movement (WT, left column) and spontaneous VCP in line VPR156 (VCP, right column). Worms are shown crawling with right lateral side of the body on the agar surface. Numbers indicate time in seconds. Scale bar, 100 μm. (D) Proportion of trials in which the touch-induced VCP was displayed by N2 (non-transgenic, Bristol strain) and t-<i>Phlh-17</i> transgenic (VPR839, VPR128, VPR156, VPR127, and VPR157) strains. The matrix below the graph indicates the composition of transgenes in each line (E, DsRedExpress2; m, monomericDsRed). Bars represent means <u>+</u> sem; n=53 for all groups; ** indicates a significant difference at p<0.01, Kruskal-Wallis one-way ANOVA (KWA) followed by Newman-Keuls multiple comparisons test (MCT). </p

The severity of the VCP depends on copy number of the trans-promoter and is maintained from early larval stages through adulthood.

<p>(A) Averaged VCP severity rating assigned to worms (parental N2 and VPR 156 lines, as well as their cross, X, heterozygous for the integrated transgenic array) based on a 1-10 counting number scale (1, normal backward crawling; 10, full VCP). While VPR156 line shows VCP (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081771#pone.0081771.s002" target="_blank">Movie S2</a>), its cross to N2 does not (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081771#pone.0081771.s003" target="_blank">Movie S3</a>). Dashed line indicates baseline score for N2. Number of worms tested for each category is shown within each column. Groups were compared by KWA followed by Dunn’s MCT; **p<0.01, * p<0.05. (B) The VCP is maintained from early larval stages through adulthood. Average proportion of trials in which the VCP was displayed by N2 and VPR156 lines at various life stages. L, larval stages 2-4; A, adult. Note that L1 worms were not tested due to their size. Data for adults is sourced from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081771#pone-0081771-g001" target="_blank">Figure 1D</a> (n=53); n=18 for both genotypes for all other life stages shown. Groups at each life stage were compared by Mann-Whitney U-test; ** p<0.01. Bars in A and B represent means <u>+</u> sem.</p

Nanopore Sensing of Botulinum Toxin Type B by Discriminating an Enzymatically Cleaved Peptide from a Synaptic Protein Synaptobrevin 2 Derivative

Botulinum neurotoxins (BoNTs) are the most lethal toxin known to human. Biodefense requires early and rapid detection of BoNTs. Traditionally, BoNTs can be detected by looking for signs of botulism in mice that receive an injection of human material, serum or stool. While the living animal assay remains the most sensitive approach, it is costly, slow and associated with legal and ethical constrains. Various biochemical, optical and mechanical methods have been developed for BoNTs detection with improved speed, but with lesser sensitivity. Here, we report a novel nanopore-based BoNT type B (BoNT-B) sensor that monitors the toxin’s enzymatic activity on its substrate, a recombinant synaptic protein synaptobrevin 2 derivative. By analyzing the modulation of the pore current caused by the specific BoNT-B-digested peptide as a marker, the presence of BoNT-B at a subnanomolar concentration was identified within minutes. The nanopore detector would fill the niche for a much needed rapid and highly sensitive detection of neurotoxins, and provide an excellent system to explore biophysical mechanisms for biopolymer transportation

Chemically Functionalized Water-Soluble Single-Walled Carbon Nanotubes Modulate Morpho-Functional Characteristics of Astrocytes

We report the use of chemically functionalized water-soluble single-walled carbon nanotubes (ws-SWCNTs) for the modulation of morpho-functional characteristics of astrocytes. When added to the culturing medium, ws-SWCNTs were able to make astrocytes larger and stellate/mature, changes associated with the increase in glial fibrillary acidic protein immunoreactivity. Thus, ws-SWCNTs could have more beneficial effects at the injury site than previously thought; by affecting astrocytes, they could provide for a more comprehensive re-establishment of the brain computational power