RNA-Seq identifies SPGs as a ventral skeletal patterning cue in sea urchins

The sea urchin larval skeleton offers a simple model for formation of developmental patterns. The calcium carbonate skeleton is secreted by primary mesenchyme cells (PMCs) in response to largely unknown patterning cues expressed by the ectoderm. To discover novel ectodermal cues, we performed an unbiased RNA-Seq-based screen and functionally tested candidates; we thereby identified several novel skeletal patterning cues. Among these, we show that SLC26a2/7 is a ventrally expressed sulfate transporter that promotes a ventral accumulation of sulfated proteoglycans, which is required for ventral PMC positioning and skeletal patterning. We show that the effects of SLC perturbation are mimicked by manipulation of either external sulfate levels or proteoglycan sulfation. These results identify novel skeletal patterning genes and demonstrate that ventral proteoglycan sulfation serves as a positional cue for sea urchin skeletal patterning

Le Forum, Vol. 40 No. 3

https://digitalcommons.library.umaine.edu/francoamericain_forum/1088/thumbnail.jp

Bottom-Up Organizing with Tools from On High: Understanding the Data Practices of Labor Organizers

This paper provides insight into the use of data tools in the American labor movement by analyzing the practices of staff employed by unions to organize alongside union members. We interviewed 23 field-level staff organizers about how they use data tools to evaluate membership. We find that organizers work around and outside of these tools to develop access to data for union members and calibrate data representations to meet local needs. Organizers mediate between local and central versions of the data, and draw on their contextual knowledge to challenge campaign strategy. We argue that networked data tools can compound field organizers' lack of discretion, making it more difficult for unions to assess and act on the will of union membership. We show how the use of networked data tools can lead to less accurate data, and discuss how bottom-up approaches to data gathering can support more accurate membership assessments

Alpha-Class Glutathione S-Transferases in Wild Turkeys (<i>Meleagris gallopavo</i>): Characterization and Role in Resistance to the Carcinogenic Mycotoxin Aflatoxin B₁

<div><p>Domestic turkeys (<i>Meleagris gallopavo</i>) are one of the most susceptible animals known to the toxic effects of the mycotoxin aflatoxin B₁ (AFB₁), a potent human hepatocarcinogen, and universal maize contaminant. We have demonstrated that such susceptibility is associated with the inability of hepatic glutathione S-transferases (GSTs) to detoxify the reactive electrophilic metabolite <i>exo</i>-AFB₁-8,9-epoxide (AFBO). Unlike their domestic counterparts, wild turkeys, which are relatively AFB₁-resistant, possess hepatic GST-mediated AFBO conjugating activity_. Here, we characterized the molecular and functional properties of hepatic alpha-class GSTs (GSTAs) from wild and domestic turkeys to shed light on the differences in resistance between these closely related strains. Six alpha-class GST genes (<i>GSTA</i>) amplified from wild turkeys (Eastern and Rio Grande subspecies), heritage breed turkeys (Royal Palm) and modern domestic (Nicholas strain) turkeys were sequenced, and catalytic activities of heterologously-expressed recombinant enzymes determined. Alpha-class identity was affirmed by conserved GST domains and four signature motifs. All <i>GSTAs</i> contained single nucleotide polymorphisms (SNPs) in their coding regions: <i>GSTA1.1</i> (5 SNPs), <i>GSTA1.2</i> (7), <i>GSTA1.3</i> (3), <i>GSTA2</i> (3), <i>GSTA3</i> (1) and <i>GSTA4</i> (2). <i>E. coli</i>-expressed GSTAs possessed varying activities toward GST substrates 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene (DCNB), ethacrynic acid (ECA), cumene hydroperoxide (CHP). As predicted by their relative resistance, livers from domestic turkeys lacked detectable GST-mediated AFBO detoxification activity, whereas those from wild and heritage birds possessed this critical activity, suggesting that intensive breeding and selection resulted in loss of AFB₁-protective alleles during domestication. Our observation that recombinant tGSTAs detoxify AFBO, whereas their hepatic forms do not, implies that the hepatic forms of these enzymes are down-regulated, silenced, or otherwise modified by one or more mechanisms. These data may inform of possible molecular mechanisms of resistance to AFB₁, and may also have the benefit of identifying genetic markers which could be used to enhance AFB₁ resistance in modern domestic strains.</p></div

Primers with restriction sites for cloning and the 6X His-recombinant constructs of alpha-class <i>GSTAs</i> from wild and heritage turkey cDNA.

<p>Primers with restriction sites for cloning and the 6X His-recombinant constructs of alpha-class <i>GSTAs</i> from wild and heritage turkey cDNA.</p

6X His-tagged purified alpha-class turkey GSTSA1.2 separated by SDS-PAGE gel (15%), visualized by Coomassie R-250 blue.

<p>Marker: molecular weight marker (MW); Lane 1, Empty vector (negative control); Lane 2, tGSTA1.2 (domestic); Lane 3, EWtGSTA1.2 (Eastern Wild); Lane 4, RGWtGSTA1.2 (Rio Grande Wild); Lane 5, RPtGSTA1.2 (Royal Palm heritage); Lane 6.</p

Multiple amino acid sequence alignments of six alpha-class GSTs from the wild, heritage and domestic turkeys with alpha-class GSTs. Underlines indicate alpha helices (black background) and arrows indicate beta strands (grey background). * indicates alpha-class signature motifs and specific conserved residues.

<p>The sequences of amino acid are (species name, GenBank accession no.): tGSTA1.1 (<i>M. gallopavo</i>, ACU44693), tGSTA1.2 (<i>M. gallopavo</i>, ACU44694), tGSTA1.3 (<i>M. gallopavo</i>, ACU44695), tGSTA2 (<i>M. gallopavo</i>, ACU44696), tGSTA3 (<i>M. gallopavo</i>, ACU44697), tGSTA4 (<i>M. gallopavo</i>, ACU44698), EWtGSTA1.1 (<i>M. g. silvestris</i>, AET31416), EWtGSTA1.2 (<i>M. g. silvestris</i>, AET31413JN575077), EWtGSTA1.3 (<i>M. g. silvestris</i>, AET31410), EWtGSTA2 (<i>M. g. silvestris</i>, AET31407), EWtGSTA3 (<i>M. g. silvestris</i>, AET31404), EWtGSTA4 (<i>M. g. silvestris</i>, AET31401), RGWtGSTA1.1 (<i>M. g. intermedia</i>, AET31417), RGWtGSTA1.2 (<i>M. g. intermedia,</i> AET31414), RGWtGSTA1.3 (<i>M. g. intermedia</i>, AET31411), RGWtGSTA2 (<i>M. g. intermedia</i>, AET31408), RGWtGSTA3 (<i>M. g. intermedia</i>, AET31405), RGWtGSA4 (<i>M. g. intermedia</i>, AET31402). RPtGSTA1.1 (<i>M. gallopavo</i>, AET31418), RPtGSTA1.2 (<i>M. gallopavo</i>, AET31415), RPtGSTA1.3 (<i>M. gallopavo</i>, AET31412), RPtGSTA2 (<i>M. gallopavo</i>, AET31409), RPtGSTA3 (<i>M. gallopavo</i>, AET31406), RPtGSTA4 (<i>M. gallopavo</i>, AET31403).</p

Genetic diversity of six Alpha class <i>GST</i> genes in four subspecies of turkeys.

<p>Domestic turkey was used for the reference to compare three subspecies. The <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060662#pone-0060662-g001" target="_blank">Figure 1</a> shows the number of SNPs between domestic turkey and wild/heritage turkey. <b>DT</b>: domestic turkey, <b>EW</b>: Eastern Wild, <b>RGW</b>: Rio Grande Wild, <b>RP</b>: Royal Palm. Parenthesis (): the size of alpha class GST genes. The alpha class GST gene in subspecies showed the same nucleic acid sequences: <b><i>tGSTA1.1</i></b>: DT = RGW, EW = RP; <b><i>tGSTA1.3</i></b>: EW = RGW = RP; <b><i>tGSTA2</i></b>: EW = RP; <b><i>tGSTA3</i></b>: DT = RGW, EW = RP; <b><i>tGSTA4</i></b>: DT = RP. <i>tGSTA1.2</i> showed the most diversity.</p

Western immunoblot showing expression of His-tag recombinant purified soluble alpha-class turkey GSTA1.2.

<p>Anti-his primary antibody and HRP-conjugated secondary antibody were used and bands were detected by chemiluminescence analysis. MW: molecular weight marker; Lane 1, Empty vector (negative control); Lane 2, tGSTA1.2 (domestic turkey); Lane 3, EWtGSTA1.2 (Eastern Wild); Lane 4, RGWtGSTA1.2 (Rio Grande Wild); Lane 5, RPtGSTA1.2 (Royal Palm heritage); Lane 6.</p

Summary of primers used to amplify the open reading frame of Alpha-class <i>GST</i> fragments from wild and heritage turkey hepatic cDNA.

<p>Summary of primers used to amplify the open reading frame of Alpha-class <i>GST</i> fragments from wild and heritage turkey hepatic cDNA.</p