Seeing is Believing: Developing Multimodal Metabolic Insights at the Molecular Level

This outlook explores how two different molecular imaging approaches might be combined to gain insight into dynamic, subcellular metabolic processes. Specifically, we discuss how matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and stimulated Raman scattering (SRS) microscopy, which have significantly pushed the boundaries of imaging metabolic and metabolomic analyses in their own right, could be combined to create comprehensive molecular images. We first briefly summarize the recent advances for each technique. We then explore how one might overcome the inherent limitations of each individual method, by envisioning orthogonal and interchangeable workflows. Additionally, we delve into the potential benefits of adopting a complementary approach that combines both MSI and SRS spectro-microscopy for informing on specific chemical structures through functional-group-specific targets. Ultimately, by integrating the strengths of both imaging modalities, researchers can achieve a more comprehensive understanding of biological and chemical systems, enabling precise metabolic investigations. This synergistic approach holds substantial promise to expand our toolkit for studying metabolites in complex environments

Imaging Mass Spectrometry Reveals Crosstalk between the Fallopian Tube and the Ovary that Drives Primary Metastasis of Ovarian Cancer

High grade serous ovarian cancer (HGSOC) is the fifth leading cause of cancer deaths among women. New evidence suggests that HGSOC arises in the fallopian tube and then colonizes the ovary before spreading into the peritoneal space. Therefore, due to the proximity of this metastasis, an experimental design was optimized using imaging mass spectrometry to capture the spatial composition of small molecules uniquely expressed when fallopian-tube-derived tumor cells were grown in the microenvironment of the ovary as a model of primary metastasis. The observed mass-to-charge ratios (m/z’s) that were induced specifically in coculture represent small molecules that may contribute to the metastasis of HGSOC selectively to the ovary. Human fallopian tube epithelial HGSOC and tumorigenic murine oviductal epithelial cells, but not normal cell types, repeatedly induced a signal from the ovary at m/z 170. This signal was identified as norepinephrine, which was confirmed to stimulate invasion of ovarian cancer cells lacking wild-type p53. These molecules may reveal pathways that contribute to metastasis and biological targets for therapeutic intervention to block ovarian metastasis of fallopian-tube-derived HGSOC. The developed mass spectrometry method can be adapted to other mammalian-based model systems for investigation of untargeted metabolomics that facilitate metastasis

Seeing is Believing: Developing Multimodal Metabolic Insights at the Molecular Level

This outlook explores how two different molecular imaging approaches might be combined to gain insight into dynamic, subcellular metabolic processes. Specifically, we discuss how matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and stimulated Raman scattering (SRS) microscopy, which have significantly pushed the boundaries of imaging metabolic and metabolomic analyses in their own right, could be combined to create comprehensive molecular images. We first briefly summarize the recent advances for each technique. We then explore how one might overcome the inherent limitations of each individual method, by envisioning orthogonal and interchangeable workflows. Additionally, we delve into the potential benefits of adopting a complementary approach that combines both MSI and SRS spectro-microscopy for informing on specific chemical structures through functional-group-specific targets. Ultimately, by integrating the strengths of both imaging modalities, researchers can achieve a more comprehensive understanding of biological and chemical systems, enabling precise metabolic investigations. This synergistic approach holds substantial promise to expand our toolkit for studying metabolites in complex environments

Fish Origins and Taxonomy, Microbial Isolates, and NCBI Closest Relatives.

<p>Fish Origins and Taxonomy, Microbial Isolates, and NCBI Closest Relatives.</p

Bioactivities of Fish Microbiome Isolates.

<p>A = Actinobacteria, F = Firmicutes, α = Alphaproteobacteria, γ = Gammaproteobacteria, (−) = Gram-negative, (+) = Gram-positive. Check mark indicates activity in growth inhibition assay.</p

Phylogenetic Relationships of Taxa Related to FI-1004.

<p>The evolutionary history was inferred using the Neighbor-Joining method. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Saitou1" target="_blank">[50]</a> The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Tamura2" target="_blank">[51]</a> and are in the units of the number of base substitutions per site. The analysis involved 24 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1353 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Tamura1" target="_blank">[30]</a> The sequence of <i>Brevibacterium linens</i> DSM 20425^T was used as an outgroup.</p

Chemical Structure for Sebastenoic Acid.

<p>a, b and c are subunits found using 2D NMR methods. HMBC correlations depicted by solid arrows, COSY correlations depicted by bold lines, NOESY correlations depicted by dashed arrows.</p

Molecular Phylogenetic Analysis by Maximum Likelihood for all Isolated, Culturable Strains of Bacteria Isolated from Fish Intestines.

<p>The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Kimura1" target="_blank">[48]</a> The bootstrap consensus tree inferred from 2000 replicates is taken to represent the evolutionary history of the taxa analyzed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) are shown next to the branches. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was <100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+<i>G</i>, parameter = 0.4869)). The rate variation model allowed for some sites to be evolutionarily invariable ([+<i>I</i>], 38.9340% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 53 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1244 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Tamura1" target="_blank">[30]</a> Strains identified as psychrophilic bacteria in NCBI denoted with blue diamonds. Strains whose closest published NCBI relatives are uncultured clones denoted with open triangles.</p

Sebastenoic Acid (1) MICs Against Bacterial Panel.

<p>Sebastenoic Acid (1) MICs Against Bacterial Panel.</p

Using Tumor Explants for Imaging Mass Spectrometry Visualization of Unlabeled Peptides and Small Molecules

Matrix assisted laser desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry has emerged as a powerful, label-free technique to visualize penetration of small molecules <i>in vivo</i> and <i>in vitro</i>, including in 3D cell culture spheroids; however, some spheroids do not grow sufficiently large to provide enough area for imaging mass spectrometry. Here, we describe an <i>ex vivo</i> method for visualizing unlabeled peptides and small molecules in tumor explants, which can be divided into pieces of desired size, thus circumventing the size limitations of many spheroids. As proof-of-concept, a small molecule drug (4-hydroxytamoxifen), as well as a peptide drug (cyclosporin A) and peptide chemical probe, can be visualized after <i>in vitro</i> incubation with tumor explants so that this technique may provide a solution to robing cell penetration by unlabeled peptides