Phylogenetic tree of protostome RNase T2 proteins.

<p>Tree was obtained by the Neighbor-Joining method using only conserved regions. Bootstrap percentages (for 1,000 replications) greater than 50 are shown on interior branches. The tree was rooted using bacteria sequences. Groups discussed in the text are labeled on the right.</p

Identification of mutations in conserved active site residues in protostome RNase T2 proteins.

<p>The alignment shows the conserved CAS I and CAS II regions characteristic of RNase T2 enzymes. The catalytic histidines are marked with asterisks. Mutations in the catalytic histidine in CAS I should result in complete loss of activity (shown in red). Mutations in the additional histidine in CAS II, implicated in binding to the substrate or stabilization of the pentacovalent intermediate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105444#pone.0105444-MacIntosh1" target="_blank">[2]</a>, should result in enzymes with reduced activity (shown in green or yellow). The active sites of human RNASET2 and RNase X25, two active RNases, are shown for comparison.</p

Starvation induces expression of the autophagy marker, <i>Atg 5</i> and <i>Amyrel, Lip3</i> and <i>RNase X25</i> in larvae.

<p>RNA was isolated from whole 3^rd instar larvae, 14 h after transfer to control (C) or starvation (S) conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105444#s2" target="_blank">Materials and methods</a>). qPCR quantification of the relative level of (A) autophagy marker <i>Atg5,</i> (B) starvation markers <i>Amyrel</i>, and <i>Lip3</i>, and (C) <i>RNase X25</i> mRNAs in these samples was carried out using the ribosomal protein L3 (<i>RpL3</i>) transcript as internal standard control for normalization. Increased levels of <i>Atg5, Amyrel, Lip3</i>, and <i>RNase X25</i> transcripts were apparent in samples after starvation as compared with fed-control animals. Data are representative of 3 independent experiments and are means and S.E. of triplicates. *, <i>P</i><0.05; **, <i>P</i><0.01 (<i>t</i>-test). (D) Protein extracts from 14 h starved (S) and fed-control (C) whole 3^rd instar larvae were analyzed using RNase <i>in gel</i> activity assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105444#pone-0105444-g001" target="_blank">Figure 1</a>. RNase activity in the size range corresponding to RNase T2 enzymes was evident in starved as compared with fed-control animals. Each lane in both gels contains 80 µg of protein. “F 4–24” denotes extracts from animals nourished with Formula 4–24 instant <i>D. melanogaster</i> diet without yeast; “Rich” denotes extracts from animals fed a yeast rich diet (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105444#s2" target="_blank">Materials and methods</a>).</p

Effect of pH on <i>Drosophila</i> RNase activities.

<p>Protein extracts from ovaries and embryos were analyzed using RNase <i>in gel</i> activity assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105444#pone-0105444-g001" target="_blank">Figure 1</a>, with incubations at neutral (pH 7.0; upper panel) and acidic (pH 6.0; lower panel) conditions. RNase activity in the size range corresponding to RNase T2 enzymes was abundant after incubation at pH 6, while almost no activity was observed at neutral pH. Each lane in both gels contains 20 µg of protein. A plant protein that is active at the two pH conditions, <i>Arabidopsis thaliana</i> RNS2 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105444#pone.0105444-Hillwig1" target="_blank">[7]</a>, was used as control.</p

Effect of starvation on the accumulation of Lysotracker-positive vesicles in larval fat body.

<p>(A and B) A high level of bright red Lysotracker-positive vesicles accumulate in fat body cells isolated from (B) 14 h starved 3^rd instar larvae, with few observed for (A) fed-control larvae. (C and D) Hoescht 33342 staining of DNA, and (E and F) merged images. Scale bar = 20 µm.</p

Reduced RNase activity and expression correlates with reduced <i>RNase X25</i> gene dose.

<p>Ovarian extracts were prepared from wild type control (<i>+/+</i>), or deletion mutant <i>Df(3L)Excel6279/+</i> females (<i>+/−</i>), carrying two or one copy of the <i>RNase X25</i> gene, respectively. Protein samples were analyzed using (A) <i>in gel</i> RNase activity assay, or (B) standard SDS/PAGE analysis. Compared to the control (<i>+/+</i>), RNase activity was reduced in ovaries dissected from females with one copy of the <i>RNase X25</i> gene (<i>+/−</i>). Each lane in both gels contains 20 µg of protein. (C) RNA was isolated from ovaries and qPCR quantification of the relative level of <i>RNase X25</i> mRNAs in these samples was carried out using the ribosomal protein L3 (<i>RpL3</i>) transcript as internal standard control for normalization. <i>RNase X25</i> expression levels were reduced in tissue samples from mutant <i>Df(3L)Excel6279/+</i> females (<i>+/−</i>), compared to control females (<i>+/+</i>). Data are representative of 3 independent experiments and are means and S.E. of triplicates. **, <i>P</i><0.01 (<i>t</i>-test).</p

Developmental profile of <i>Drosophila</i> RNase activities.

<p>Protein extracts were produced from embryos at 0–2 hours (h), 2–6 h, and 0–16 h after egg deposition and from animals at 3rd instar larval (L3), white prepupal (WPP), pupal (P), and adult male (M) or female (F) stages of development. Ovarian tissue (ovary) was prepared from 3–5 day old females. (Upper panel) Protein was fractionated by electrophoresis through a 12% polyacrylamide gel containing 3 mg/ml Torula yeast RNA, washed to remove SDS, incubated in 100 mM Tris-HCl at pH 6.0 and stained with toluidine blue to visualize regions of nuclease activity. Low molecular weight (∼25–30 kD) activities in the size range of the RNase T2 family were detected at all developmental stages assayed. High molecular weight (∼200 kD) activities were also apparent (arrow), but absent from embryos. (Lower panel) Protein extracts were analyzed by SDS/PAGE and stained with Coomassie Blue R-250 to control for equal loading and protein integrity. Each lane in both gels contains 20 µg of protein.</p

<i>RNase X25</i> gene expression is regulated by nutritional and oxidative stress.

<p>RNA was isolated from whole 3^rd instar larvae, 14 h after transfer to control or experimental media (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105444#s2" target="_blank">Materials and methods</a>). qPCR quantification of the relative level of <i>RNase X25</i> mRNAs in these samples was carried out using the ribosomal protein L3 (<i>RpL3</i>) transcript as internal standard control for normalization. Increased levels of <i>RNase X25</i> transcripts were apparent in samples after (A) starvation and treatments with 1% [w/w] wheat germ agglutinin (WGA), and (B) 0.1% [w/w] or 0.5% [w/w] hydrogen peroxide. Data are representative of 3 independent experiments and are means and S.E. of triplicates. *, <i>P</i><0.05; **, <i>P</i><0.01 (<i>t</i>-test).</p

The Insulin-Only Bionic Pancreas Improves Glycemic Control in Non-Hispanic White and Minority Adults and Children with Type 1 Diabetes

   Objective: We evaluated the performance of the iLet® bionic pancreas (BP) in non-Hispanic Whites (‘Whites’) and in Blacks, Hispanics, and others (‘Minorities’). Research Design and Methods: A multicenter, randomized controlled trial evaluated glycemic management with the BP versus standard-of-care (SC) in 161 adult and 165 pediatric participants with type 1 diabetes over 13 weeks.  Results: In Whites (N=240), the mean baseline-adjusted difference in 13-week HbA1c between the BP and SC groups was -0.45% (95% CI -0.61% to -0.29% [-4.9, -6.6 to -3.1 mmol/mol]; P Conclusions: The BP improves glycemic control in both Whites and Minorities and offers promise in decreasing health care disparities. </p