Optical microscopy images of trypan blue and trypan blue combined with lutein/zeaxanthin in aqueous solution with no and after 120 hours of blue-light irradiation at 460 nm.

<p>In the trypan blue—lutein/zeaxanthin solution, fine homogenous yellow granules of lutein/zeaxanthin were observed, which changed into heterogeneous brown clots during the process of irradiation, as shown in the viewframe below. Scale bars represent 20 μm.</p

MALDI-TOF mass spectrometry of selected fragment ion peaks for the blue light irradiation series.

<p>Distinct mass signatures of trypan blue decomposition intermediates were formed subsequent to dissociation of methoxyamine (m/z = 825.6 [M– 47]⁺) <b>(a)</b> that accumulates over time (r = 0.917, p < 0.001); and following dissociation of sulfonyl arin (m/z = 671.1 [M– 47–155]⁺) <b>(b)</b> that is consumed in the course of irradiation (r = -0.488, p = 0.040).</p

Schematic illustration of proposed major/preferred trypan blue photochemical degradation pathways (primary and secondary steps, respectively).

<p>It is suggested that the self-sensitized photodegradation of trypan blue occurs under dissociation of dimethyl sulfate (m/z = 745.6 [M– 127.2]⁺), presumably followed by elimination of phenol (m/z = 651.4 [M– 127.2–94.2]⁺). In the presence of lutein/zeaxanthin, photochemical degradation of trypan blue is triggered and performs under presumed generation of methoxyamine (m/z 825.6 [M– 47]⁺), followed by dissociation of sulfonyl arin (m/z = 671.1 [M– 47–155]⁺).</p

Nuclear magnetic resonance spectra of trypan blue—Lutein/zeaxanthin mixture with no (lower graph) and after 120 hours (top graph) of blue light irradiation at 460 nm.

<p>Trypan blue and Lutein/zeaxanthin signals: ¹H NMR (700 MHz, DMSO-<i>d</i>₆, RT, ppm) <i>δ</i> = 8.08 (d, <i>J</i> = 8.4 Hz, 2H), 7.69 (d, <i>J</i> = 8.4 Hz, 1H), 7.68 (s, 2H), 7.32 (s, 2H), 7.06 (d, <i>J</i> = 1.5 Hz, 2H), 6.89 (d, <i>J</i> = 1.5 Hz, 2H), <i>δ</i> = 6.71 (d, <i>J</i> = 8.8 Hz, 2H), 6.69–6.60 (m, 2H), 6.39 (d, <i>J</i> = 14.9 Hz, 2H), 6.33 (d, <i>J</i> = 8.8 Hz, 2H), 6.22 (d, <i>J</i> = 11.5 Hz, 2H), 6.19–6.09 (m, 5H) ppm.</p

Nuclear magnetic resonance spectra of trypan blue with no (lower graph) and after 120 hours (top graph) of blue light irradiation at 460 nm.

<p>Trypan blue signals: ¹H NMR (700 MHz, DMSO-<i>d</i>₆, RT, ppm) <i>δ</i> = 8.08 (d, <i>J</i> = 8.4 Hz, 2H), 7.69 (d, <i>J</i> = 8.4 Hz, 1H), 7.68 (s, 2H), 7.32 (s, 2H), 7.06 (d, <i>J</i> = 1.5 Hz, 2H), 6.89 (d, <i>J</i> = 1.5 Hz, 2H) ppm. Dimethyl sulfate signal: ¹H NMR (700 MHz, DMSO-<i>d</i>₆) <i>δ</i> = 4.00 ppm (top graph).</p

Photometric spectra of trypan blue (dotted line) and trypan blue combined with lutein/zeaxanthin (black line) in aqueous solution.

<p>Characteristic absorption maxima are highlighted with marking lines: 379 nm (lutein/zeaxanthin), 580 nm (trypan blue) and 460 nm/505 nm (lutein/zeaxanthin diacetate).</p

MALDI-TOF spectra of examined dye solutions, comprising of 0.4 mg/mL trypan blue and 10.0 mg/mL lutein/zeaxanthin, with no (a) and after 120 hours of blue light irradiation at 460 nm (b).

<p>MALDI-TOF spectra of examined dye solutions, comprising of 0.4 mg/mL trypan blue and 10.0 mg/mL lutein/zeaxanthin, with no (a) and after 120 hours of blue light irradiation at 460 nm (b).</p

Results of MALDI-TOF-MS analysis.

<p>Average MALDI-TOF-MS N-glycan (A) relative intensities from 10 healthy donors under 16 preanalytical conditions (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200507#pone.0200507.g001" target="_blank">Fig 1</a>). The serum and plasma standard conditions are marked with thick lines. Small increase of N-glycans having low masses (B) can be observed for hemolysed samples (condition 16), while N-glycans having high masses show a small decrease (C).</p

Flowchart showing the preanalytical processing carried out in this study.

<p>Reference conditions (time frames between processing steps as short as possible) are marked in red. Samples were kept for 30 min at RT, were subsequently centrifuged for 15 minutes at 1500×g and 4°C, aliquoted immediately after centrifugation and stored at -80°C, unless stated otherwise. Conditions 1–15 were created for the first set of collection, conditions 1–16 were created for the second set of collection. It should be noted that tubes F and K were used for generating multiple conditions.</p

Data_Sheet_2_Consequences of GMPPB deficiency for neuromuscular development and maintenance.PDF

Guanosine diphosphate-mannose pyrophosphorylase B (GMPPB) catalyzes the conversion of mannose-1-phosphate and GTP to GDP-mannose, which is required as a mannose donor for the biosynthesis of glycan structures necessary for proper cellular functions. Mutations in GMPPB have been associated with various neuromuscular disorders such as muscular dystrophy and myasthenic syndromes. Here, we report that GMPPB protein abundance increases during brain and skeletal muscle development, which is accompanied by an increase in overall protein mannosylation. To model the human disorder in mice, we generated heterozygous GMPPB KO mice using CIRSPR/Cas9. While we were able to obtain homozygous KO mice from heterozygous matings at the blastocyst stage, homozygous KO embryos were absent beyond embryonic day E8.5, suggesting that the homozygous loss of GMPPB results in early embryonic lethality. Since patients with GMPPB loss-of-function manifest with neuromuscular disorders, we investigated the role of GMPPB in vitro. Thereby, we found that the siRNA-mediated knockdown of Gmppb in either primary myoblasts or the myoblast cell line C2C12 impaired myoblast differentiation and resulted in myotube degeneration. siRNA-mediated knockdown of Gmppb also impaired the neuron-like differentiation of N2A cells. Taken together, our data highlight the essential role of GMPPB during development and differentiation, especially in myogenic and neuronal cell types.</p