Mass Spectrometry Imaging on Porous Silicon: Investigating the Distribution of Bioactives in Marine Mollusc Tissues

Desorption/ionization on porous silicon-mass spectrometry (DIOS-MS) is an attractive alternative to conventional matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of low molecular weight compounds. Porous silicon (pSi) chips are also suitable as support for mass spectrometry imaging (MSI). Here, we report an implementation of DIOS-MSI using the biosynthetic organs of a marine mollusc for proof of principle. The tissue section is stamped onto a fluorocarbon-functionalized pSi chip, which extracts and traps small hydrophobic molecules from the tissue under retention of their relative spatial distribution. The section is subsequently removed and the chip is imaged without any remaining tissue. We apply this novel tissue contact printing approach to investigate the distribution of biologically active brominated precursors to Tyrian purple in the hypobranchial gland of the marine mollusc, <i>Dicathais orbita,</i> using DIOS–MSI. The tissue contact printing is also compatible with other types of desorption/ionization surfaces, such as nanoassisted laser desorption/ionization (NALDI) targets

Similarity of percentages (SIMPER) analysis showing the bacterial genus that contribute most to the differences between hypobranchial gland and foot of <i>Dicathais orbita</i> (Average dissimilarity = 68.51).

<p>Similarity of percentages (SIMPER) analysis showing the bacterial genus that contribute most to the differences between hypobranchial gland and foot of <i>Dicathais orbita</i> (Average dissimilarity = 68.51).</p

Venn diagram showing shared and non-shared bacterial species between the hypobranchial gland and foot of <i>Dicathais orbita</i>.

<p>The number of species that have biosynthetic capabilities relevant to Tyrian purple production are highlighted in different colours (Orange = indole producers; Blue = brominating enzymes; Purple = indole producers and brominating capabilities).</p

Phylogenetic tree of <i>Dicathais orbita</i> samples generated from 16S rRNA sequences by MEGAN.

<p>A = Female hypobranchial gland (F2H); B = Male hypobranchial gland (M1H); C = Female foot (F3F); D = Male foot (M3F). All these sample types have more than 15,000 reads.</p

Mean (+s.e.) number of OTUs in the hypobranchial gland and foot tissue of <i>Dicathais orbita</i>, showing the mean proportion unique to individuals samples of foot and hypobranchial gland tissue.

<p>(A) = OTUs richness, (B) = H index/diversity.</p

Alpha diversity showing the richness of bacterial community diversity within <i>Dicathais orbita</i> foot (F2F, F3F, M2F and M3F) and hypobranchial gland samples (F1H, F2H, M1H and M2H) (F = female; M = male).

<p>The phylogenetic diversity metric consists of genus richness based on 3585 observed OTUs at the 97% sequence similarity level and 443 possible observed genus. Sample with reads of more than 3000 are visible.</p

Principal Coordinates Ordination (PCO) of bacterial genus composition, based on a Bray Curtis similarity matrix of the relative abundance of OTUs at 97% sequence similarity level for the hypobranchial gland (purple) and foot (orange) of female (F) and male (M) <i>Dicathais orbita</i>.

<p>Principal Coordinates Ordination (PCO) of bacterial genus composition, based on a Bray Curtis similarity matrix of the relative abundance of OTUs at 97% sequence similarity level for the hypobranchial gland (purple) and foot (orange) of female (F) and male (M) <i>Dicathais orbita</i>.</p

Summary of <i>Dicathais orbita</i> hypobranchial gland and foot tissue 16S rRNA bacterial profiling.

<p>¹ The samples are labelled such that the first letter refers to the gender, the number to different replicate snails within each gender and the second letter to the tissue type.</p><p>² OTUs are shared among multiple samples and are based on 97% sequence similarity criteria in the Silva_119 database.</p><p>Summary of <i>Dicathais orbita</i> hypobranchial gland and foot tissue 16S rRNA bacterial profiling.</p

Proposed model of anti-inflammatory signalling pathway inhibition.

<p>6-Bromoisatin in <i>D</i>. <i>orbita</i> hypobranchial gland (HGB) extracts prevent acute lung damage caused by inflammatory neutrophils by reducing the synthesis of pro-inflammatory cytokines. This may occur due to blocking the LPS-induced NFκB translocation into the nucleus and activation of macrophages and direct inhibition of inflammatory mediators, such as TNFα and nitric oxide (NO), as has been previously demonstrated <i>in vitro</i> for 6-bromoisatin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186904#pone.0186904.ref016" target="_blank">16</a>]. Alternatively, it is possible that the HGB extracts and associated compounds also have an upstream effect by modulating the interaction of LPS with plasma membrane receptors. This figure was developed from known inflammatory pathways in lung macrophages [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186904#pone.0186904.ref003" target="_blank">3</a>].</p

Cellularity in the bronchoalveolar lavage fluids (BALF).

<p>Cell count in the BALF from mice stimulated with LPS and treated with 6-bromoisatin or a hypobranchial gland (HBG) extract from <i>Dicathais orbita</i>. A) Total cell counts; B) Neutrophil counts from the differential staining. *** = <i>p <</i> 0.001; **** <i>p <</i> 0.0001.</p