RNA and ribose are ligands for single Gr28 proteins.

<p><b>(</b>a) Terminal taste neurons expressing the Ca²⁺ sensor CaMPARI require <i>Gr28a</i> in order to respond to ribose and RNA (<i>n =</i> 4–9). Genotypes: <i>w</i>^<i>1118</i><i>; UAS-CaMPARI/+; Gr28a-GAL4/+</i> (<i>control</i>), <i>w</i>^<i>1118</i>; Δ<i>Gr28 UAS-CaMPARI/</i>Δ<i>Gr28; Gr28a-Gal4/+</i> (ΔGr28), and <i>w</i>^<i>1118</i>; Δ<i>Gr28 UAS-CaMPARI /</i>Δ<i>Gr28; Gr28a-GAL4/UAS-Gr28a</i> (<i>Gr28a rescue</i>). (b) Expression of single <i>Gr28</i> genes conveys ribose and RNA responses to fructose-sensing pharyngeal taste neurons (<i>n =</i> 4–13). Genotypes: <i>w</i>^<i>1118</i><i>; UAS-CaMPARI Gr43a</i>^<i>GAL4</i><i>/+</i> (<i>control</i>), <i>w</i>^<i>1118</i><i>; Gr43</i>^<i>GAL4</i> <i>UAS-CaMPARI/+; UAS-Gr28a/+</i> (<i>Gr28a</i>), <i>w</i>^<i>1118</i><i>; Gr43</i>^<i>GAL4</i> <i>UAS-CaMPARI/+; UAS-Gr28b</i>.<i>a/+</i> (<i>Gr28b</i>.<i>a</i>), <i>w</i>^<i>1118</i><i>; Gr43</i>^<i>GAL4</i> <i>UAS-CaMPARI/+; UAS-Gr28b</i>.<i>e/+</i> (<i>Gr28b</i>.<i>e</i>). Final concentration of all substrates was 100 mM in water except for RNA (0.5 mg/mL). Representative images of the indicated genotypes are shown above the graphs. Scale bar is 10 μm. Each bar represents the mean ± SEM of ratios of red and green fluorescence intensities. “*” represents significant differences between the preexposure (no PC light, no chemical) group and a substrate group (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s007" target="_blank">S3 Data</a>. PC, photoconversion.</p

Genes of the Gr28 locus mediate larval taste preference for ribose, RNA, and arabinose.

<p>Two-choice feeding assays of wild-type and <i>Gr28</i> mutant larvae. (a) Larvae require the <i>Gr28</i> genes for taste preference for arabinose (<i>n =</i> 12–51), ribose (<i>n =</i> 12–36), and RNA (<i>n =</i> 21–36); Genotypes: <i>w</i>^<i>1118</i> (<i>Control</i>), <i>w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28</i> (Δ<i>Gr28</i>) <i>and</i> Δ<i>Gr28/</i>Δ<i>Gr28; Gr28 genomic rescue/</i>+ (Δ<i>Gr28 g-rescue</i>). (b) Single <i>Gr28</i> genes rescue taste preference for ribose in Δ<i>Gr28</i> homozygous mutant larvae. Genotypes were <i>w</i>^<i>1118</i> (lane 1), <i>w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28</i> (2), <i>w</i>^<i>1118</i>;Δ<i>Gr28/</i>Δ<i>Gr28; Gr28a-GAL4/+</i> (3), <i>w</i>^<i>1118</i>;Δ<i>Gr28/</i>Δ<i>Gr28; Gr28a-GAL4/UAS</i> (4, 6, 8, 10, 12, 14), and Δ<i>Gr28/</i>Δ<i>GrGr28; +/UAS</i> (5, 7, 9, 11, 13, 15) such that <i>UAS</i> represents indicated <i>transgene</i> (<i>n =</i> 12–36). (c) <i>Gr28</i> mutant larvae expressing a single <i>Gr28</i> gene in fructose-sensing (<i>Gr43a</i>^<i>GAL</i> -expressing) neurons show preference for ribose. Genotypes: <i>w</i>^<i>1118</i> (lane 1), <i>w</i>^<i>1118</i>; Δ<i>Gr28 Gr43</i>^<i>GAL4</i><i>/</i>Δ<i>Gr28 Gr43</i>^<i>GAL4</i> (2), and <i>w</i>^<i>1118</i>; Δ<i>Gr28 Gr43</i>^<i>GAL4</i><i>/</i>Δ<i>Gr28 Gr43</i>^<i>GAL4</i><i>; UAS/+</i>, such that <i>UAS</i> represent indicated transgene (<i>n =</i> 12–30). Each bar represents the mean ± SEM of two-choice preference responses. Concentrations were 100 mM (arabinose and ribose) or 0.5 mg/mL (RNA) in 1% agarose. Red “*” represents significant difference between indicated genotype and <i>w</i>^<i>1118</i> control (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). Green “*” represents significant difference between indicated genotype and Δ<i>Gr28 Gr43a</i>^<i>GAL4</i> double mutant (<i>w</i>^<i>1118</i>; Δ<i>Gr28 Gr43</i>^<i>GAL4</i><i>/</i>Δ<i>Gr28 Gr43</i>^<i>GAL4</i>). Two-tailed Mann-Whitney U test, <i>p</i> < 0.05). The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s005" target="_blank">S1 Data</a>.</p

Larvae require <i>Gr28</i> genes for efficient growth and survival when presented with HM and HMΔ food.

<p>(a) About 40 eggs were deposited in 21-well microtiter plates containing 1 of 3 different foods: all wells containing HM (black; left), HMΔ (gray; middle), or a mixture of the two (12 HMΔ and 9 HM; right). Plates with either only HM or HMΔ medium were used to determine survival rate for complete (HM) or ribonucleoside-deficient (HMΔ) food. (b) Survival is displayed as percentage of flies hatched after eggs were deposited onto plate. For statistical analysis, survival in different foods was either compared across the same genotype (<i>Control</i> [black]: <i>w</i>^<i>1118</i>, Δ<i>Gr28</i> [red]: <i>w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28</i>, <i>and</i> Δ<i>Gr28 g-rescue</i> [green]: <i>w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28; genGr28/+</i>), or different genotypes were compared against the same mixed food (light–dark checkered pattern). Each bar represents the mean ± SEM (<i>n</i> = 5–6). Bars with different letters represent significant difference (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s008" target="_blank">S4 Data</a>. HM, holidic medium.</p

Larval preference for ribose and RNA is not mediated by sugar <i>Gr</i> genes.

<p><b>Two-choice preference assays for arabinose, ribose, deoxyribose, and RNA (panel a and c) and survival on these chemicals and nutritious sugars (panel b).</b> (a) Preference for arabinose is independent on various sugar <i>Gr</i> genes (<i>n</i> = 12–28). The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s005" target="_blank">S1 Data</a>. (b) Comparison of survival of <i>w</i>^<i>1118</i> larvae when kept on different substrates (<i>n</i> = 3–8). After 72 hours, approximately 50% of the larvae survive on agarose-only substrate (median survival, dashed line). For simplicity, significant differences are only indicated for median survival time. Data are represented as mean ± SEM. “*” represents significant difference between the larval survival on different substrates and agarose (two-tailed Mann-Whitney U test, <i>p</i> < 0.05). Reduced survival rate of larvae kept on arabinose and deoxyribose might be due to interference of these chemicals with sugar metabolism. The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s006" target="_blank">S2 Data</a>. (c) Larvae show strong preference for ribose (n = 12–36) and RNA (n = 6–36) when lacking <i>Gr43a</i> or the 8 <i>sGr</i>. Larvae are not attracted to deoxyribose (n = 6–24). As for fructose [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.ref004" target="_blank">4</a>], Δ<i>sGr</i> larvae showed stronger preference for ribose than wild-type larvae. Concentration of all substrates was 100 mM in 1% agarose, except RNA (0.5 mg/mL in 1% agarose). Genotypes: <i>w</i>^<i>1118</i> (control), <i>w</i>^<i>1118</i><i>; Gr43a</i>^<i>GAL4</i><i>/Gr43a</i>^{<i>GAL 4</i>}<i>(</i>Δ<i>Gr43a</i>), and <i>w</i>^<i>1118</i>; Δ<i>Gr61a</i> Δ<i>Gr64a-f/</i>Δ<i>Gr61a</i> Δ<i>Gr64a-f (</i>Δ<i>sGrs</i>). The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s005" target="_blank">S1 Data</a>. Gr, gustatory receptor; PREF, preference index; <i>sGr</i>, sugar <i>Gr</i> gene.</p

Expression of the 6 <i>Gr28</i> genes in third-instar larvae.

<p>(a) Graphic summary of <i>Gr28</i> gene expression. Cells and neurons with their axons expressing the respective GAL4 driver are all shown in green. Brain is shown in grey, and the digestive system—including the pharynx, PV, and gut—are outlined. (b) Live GFP imaging of the larval head, showing expression of 3 genes (<i>Gr28a</i>, <i>Gr28b.a</i>, and <i>Gr28b.e</i>) in neurons of the TO. <i>Gr28b.d</i> is expressed in neurons of the DPS and VPS organs, while <i>Gr28a</i> is also expressed in the PPS organ. Neither <i>Gr28a</i> nor <i>Gr28b.d</i> are co-expressed with <i>Gr43^GAL4</i> (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s002" target="_blank">S1 Fig</a>). Number of larvae with GFP positive taste neurons/total number of larvae analyzed were 7/7 for <i>Gr28a</i>, 4/4 for <i>Gr28b.a</i>, 0/5 for <i>Gr28b.b</i>, 0/7 for <i>Gr28b.c</i>, 5/5 for <i>Gr28b.d</i>, and 5/5 for <i>Gr28b.e</i>. (c) View of the brain and parts of the ventral nerve cord, showing different degrees of expression for each of the 6 <i>Gr28</i> genes. The brains were stained with anti-GFP antibody (green) and counterstained with nc82 antibody (red). Number of larvae with GFP antibody–positive staining in the brain-VNC/number of brains analyzed were 3/3 for <i>Gr28a</i>, 5/5 for <i>Gr28b.a</i>, 3/3 for <i>Gr28b.b</i>, 5/5 for <i>Gr28b.c</i>, 3/3 for <i>Gr28b.d</i>, and 6/6 for <i>Gr28b.e</i>. (d) Live GFP imaging of the PV and midgut, showing expression of all <i>Gr28</i> genes with the exception of <i>Gr28b.d</i>. Expression of <i>Gr28b.a</i> and <i>Gr28b.e</i> is broad and includes the PV and midgut, while expression of <i>Gr28a</i> and <i>Gr28b.b</i> is defined to a smaller area of the gut only. Number of larvae with GFP-positive cells/total number of larvae analyzed were 5/5 for <i>Gr28a</i>, 4/4 for <i>Gr28b.a</i>, 3/3 for <i>Gr28b.b</i>, 5/5 for <i>Gr28b.c</i>, 0/5 for <i>Gr28b.d</i>, and 4/4 for <i>Gr28b.e</i>. (e) Summary of tissues expressing each of the 6 <i>Gr28</i> genes. Genotypes were <i>w</i>; <i>UAS-mCD8GFP/Gr28x-GAL4</i>, such that x refers to indicated <i>Gr-Gal4</i> driver. Scale bar is 100 μm. For live imaging (panel b and d), at least 5 larvae for each genotype were analyzed, and GFP cells in taste sensilla and the gut were observed in each case for <i>Gr28a</i>, <i>Gr28ba</i>, <i>Gr28b.e</i>, <i>Gr28b.d</i>, and <i>Gr28b.e</i>; for staining (panel c), at least 3 brains for each genotype were analyzed, with GFP-positive neurons observed in each case. The images are good representatives of these experiments. DPS, dorsal pharyngeal sensory; GFP, green fluorescent protein; <i>Gr28</i>, gustatory receptor subfamily 28; PPS, posterior pharyngeal sensory; PV, proventriculus; TO, terminal organ; VPS, ventral pharyngeal sensory.</p

Ribonucleosides are essential nutrients for rapid larval growth and survival.

<p>(a) Growth time in days from hatching of the first-instar larvae to eclosion (left) and survival rate (right) of larvae raised in different media shows that inosine and uridine are essential components. Larvae raised on HM grow slightly slower than, but have the same survival rate as, larvae raised on SCF. Replacing ribonucleosides with RNA (0.5 mg/mL) in HMΔ restores both growth time and survival rate, while replacing it with equimolar concentration of ribose fails to do so. Each bar represents the mean ± SEM (<i>n</i> = 4). Bars with different letters represent significant differences (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). Genotype: <i>w</i>^<i>1118</i>. The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s008" target="_blank">S4 Data</a>. (b) CaMPARI imaging of TO taste neurons shows that inosine and uridine, but none of the 3 other ribonucleosides, are potent ligands for Gr28 neurons. Uridine, cytidine (100 mM), and inosine (50 mM) were dissolved in water, while guanosine and adenosine were dissolved in DMSO and presented at concentration of 25 mM and 50 mM in water containing 25% and 10% f.c. DMSO, respectively. Each bar represents the mean ± SEM of ratios of red and green fluorescence intensities (<i>n</i> = 5–19). “*” represents significant differences between the preexposure (untreated) group to the groups with the indicated ligands applied (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). Genotype: <i>w</i>^<i>1118</i><i>; UAS-CaMPARI/Gr28a-GAL4</i>. The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s007" target="_blank">S3 Data</a>. (c) Two-choice preference assay shows that larvae require the <i>Gr28</i> genes to exhibit preference for uridine (50 mM, <i>n =</i> 12–24) and inosine (100 mM, <i>n</i> = 12–18). “*” represents significant difference between the genotypes (two-tailed Mann-Whitney U test, <i>p <</i> 0.05). All the genotypes are compared to control. Genotypes: <i>w</i>^<i>1118</i>, <i>w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28 and w</i>^<i>1118</i>; Δ<i>Gr28/</i>Δ<i>Gr28; genGr28/+</i>. The underlying data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005570#pbio.2005570.s005" target="_blank">S1 Data</a>. f.c., final concentration; HM, holidic medium; SCF, standard cornmeal food; TO, terminal organ.</p

Additional file 1: Supplemental figures. of Neural stem cells for disease modeling of Wolman disease and evaluation of therapeutics

Figure S1. Immunocytochemical characterization of WD iPSCs. Figure S2. STR DNA analysis of WD fibroblasts, iPSCs, and NSCs. Figure S3. LysoTracker staining and Nile red staining of LDL loaded NSCs. Figure S4. HT144B NSCs show increased LysoTracker staining. Figure S5. Chemical structures. Figure S6. DT and HPBCD treatment reduces lysosomal staining in HT144B NSCs. Figure S7. DT and HPBCD do not significantly reduce neutral lipid accumulation in WD NSCs. Figure S8. High concentrations of DT and HPBCD affect cell viability. Figure S9. DT and HPBCD combination treatment have an additive effect on reducing lysosomal staining in HT144B NSCs. (PDF 652 kb

The Zn²⁺ binding site of PyAeADHII.

<p><b>a) Zn²⁺ binding in the active site of ADH-WT subunit A.</b> The phased anomalous map contoured at 5σ calculated using the Zn-peak data, is shown as blue mesh. b) Metal binding site for Co-substituted PyAeADHII with the cobalt ion drawn as a red sphere. Phased anomalous difference maps contoured at 5σ were calculated using Co-peak data (orange) and Zn²⁺-peak data (blue) and revealed that both ions were present.</p

Active site view of ADH-NADPH (green) with YADH (coral, PDB 2HCY) and HADH (blue, PDB 5ADH) (a).

<p>YADH and HADH have adenosine-5-diphosphoribose and nicotinamide-8-iodo-adenine-dinucleotide bound in the active site respectively. Active site ligands are drawn as cylinders and active site Zn²⁺ ions for YADH and HADH are represented as spheres. b) Superposition of ADH-WT (magenta) and ADH-NADPH (green). The NADPH molecule and Cys residues in the metal binding site are drawn as cylinders. The Zn²⁺ ions associated with ADH-WT and ADH-NADPH are drawn as grey and blue spheres respectively. Regions with the largest conformational differences are highlighted.</p

Binding interaction of α-tetralone in the substrate binding site of PyAeADHII/NADPH predicted by AutoDock.

<p>a) Binding mode 1, α-tetralone was positioned on top of the nicotinamide ring as a stacking interaction, and the oxygen atom formed a H-bonding interaction with the side chain of residue Asn-39 (3.5 Å). b) α-tetralone again forms stacking interaction with nicotinamide ring, but the oxygen atom was orientated towards residue Arg-88 forming a H-bonding interaction (3.0 Å). PyAeADHII is shown as a cartoon (green) and residues in the substrate binding site are shown as sticks (carbon colored in yellow, nitrogen in blue, oxygen in red). NADPH (carbons are in grey) and the substrate α-tetralone (carbon are in orange) are shown as sticks.</p