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

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

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

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

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

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 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

Increases in brain white matter abnormalities and subcortical gray matter are linked to CD4 recovery in HIV infection

MRI alterations in the cerebral white (WM) and gray matter (GM) are common in HIV infection, even during successful combination antiretroviral therapy (CART), and their pathophysiology and clinical significance are unclear. We evaluated the association of these alterations with recovery of CD4+ T-cells. Seventy-five HIV-infected (HIV+) volunteers in the CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) study underwent brain MRI at two visits. Multi-channel morphometry yielded volumes of total cerebral WM, abnormal WM, cortical and subcortical GM, and ventricular and sulcal CSF. Multivariable linear regressions were used to predict volumetric changes with change in current CD4 and detectable HIV RNA. On average, the cohort (79% initially on CART) demonstrated loss of total cerebral WM alongside increases in abnormal WM and ventricular volumes. A greater extent of CD4 recovery was associated with increases in abnormal WM and subcortical GM volumes. Virologic suppression was associated with increased subcortical GM volume, independent of CD4 recovery. These findings suggest a possible link between brain alterations and immune recovery, distinct from the influence of virologic suppression. The association of increasing abnormal WM and subcortical GM volumes with CD4+ T-cell recovery suggests that neuroinflammation may be one mechanism in CNS pathogenesis