The cholesteryl ester transfer protein (CETP) raises cholesterol levels in the brain.

The cholesteryl ester transfer protein (CETP) is a lipid transfer protein responsible for the exchange of cholesteryl esters and triglycerides between lipoproteins. Decreased CETP activity is associated with longevity, cardiovascular health, and maintenance of good cognitive performance. Interestingly, mice lack the CETP-encoding gene and have very low levels of LDL particles compared with humans. Currently, the molecular mechanisms induced because of CETP activity are not clear. To understand how CETP activity affects the brain, we utilized CETP transgenic (CETPtg) mice that show elevated LDL levels upon induction of CETP expression through a high-cholesterol diet. CETPtg mice on a high-cholesterol diet showed up to 22% higher cholesterol levels in the brain. Using a microarray on mostly astrocyte-derived mRNA, we found that this cholesterol increase is likely not because of elevated de novo synthesis of cholesterol. However, cholesterol efflux is decreased in CETPtg mice along with an upregulation of the complement factor C1Q, which plays a role in neuronal cholesterol clearance. Our data suggest that CETP activity affects brain health through modulating cholesterol distribution and clearance. Therefore, we propose that CETPtg mice constitute a valuable research tool to investigate the impact of cholesterol metabolism on brain function.

Absolute quantification of cholesterol from thin tissue sections by silver-assisted laser desorption ionization mass spectrometry imaging.

Cholesterol is essential to all animal life, and its dysregulation is observed in many diseases. For some of these, the precise determination of cholesterol's histological location and absolute abundance at cellular length scales within tissue samples would open the door to a more fundamental understanding of the role of cholesterol in disease onset and progression. We have developed a fast and simple method for absolute quantification of cholesterol within brain samples based on the sensitive detection and mapping of cholesterol by silver-assisted laser desorption ionization mass spectrometry imaging (AgLDI MSI) from thin tissue sections. Reproducible calibration curves were generated by depositing a range of cholesterol-D7 concentrations on brain homogenate tissue sections combined with the homogeneous spray deposition of a non-animal steroid reference standard detectable by AgLDI MSI to minimize experimental variability. Results obtained from serial brain sections gave consistent cholesterol quantitative values in very good agreement with those obtained with other mass spectrometry-based methods.

Metal-Assisted Laser Desorption Ionization Imaging Mass Spectrometry.

Matrix-assisted laser desorption ionization (MALDI) remains the reference method to generate molecular images of proteins and lipids within thin tissue sections. However, traditional MALDI imaging mass spectrometry (IMS) suffers from low matrix homogeneity and high signal background in low mass range caused by matrix signals. To overcome these issues, alternative workflow and methods have been developed. Of these, metal-assisted laser desorption ionization (LDI) has become a reference technique to ionize low molecular weight compounds while allowing IMS at very high spatial resolutions with very low background signal in the low mass range. Silver and gold remain the two most used metals for the detection of neutral lipids including cholesterol, free fatty acids, and triglycerides. In this chapter, we demonstrate the potential of metal-assisted LDI IMS through the analysis of spinal cord and kidney thin tissue sections after silver and gold metal deposition. We also detail typical step-by-step workflows and discuss the strength of the methods.

Visualizing the distribution of strawberry plant metabolites at different maturity stages by MALDI-TOF imaging mass spectrometry.

This study aimed to visualize differences in the distribution of citric acid, soluble sugars, and anthocyanins in strawberries at four different maturity stages (green to red strawberries) by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF IMS). Results demonstrated citric acid and sugars are evenly distributed in the entire fruit at all maturity stages, while most of anthocyanins are mainly located in the periphery of fruit with increased abundance in red strawberries, indicating a correlation with the colour attributes. Sugar in red strawberries (11.92 brix) increased by two-fold compared to the green ones (6.23 brix). Finally, absolute quantitation of each compound from HPLC analyses support the quantitative results from MALDI-TOF IMS. The results provide a deeper understanding in the changes and distribution of phytochemicals during the growth of strawberries, and demonstrates the usefulness of IMS for plant breeding and postharvest technology.

A matrix-assisted laser desorption/ionization imaging mass spectrometric approach to study weight-related changes within thyroid tissue.

Obesity is associated with numerous comorbidities along with abnormalities of the endocrine system, more commonly manifesting as dysfunctions of the thyroid gland such as goiter. Changes in weight, especially an increase, could lead to an increase in the incidence of thyroid dysfunction; however, its pathophysiology remains to be elucidated. In the present study, we aimed to interrogate the changes in the protein distribution and abundance between the lean patients and patients with obesity thyroid tissue sections through utilizing this technique. The FFPE-fixed thyroid tissue blocks from the selected cases and controls were identified and targeted for matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) analysis. Patients in the 30 to 75 years age group and undergoing total thyroidectomy for benign thyroid disease were recruited. Patients with thyroid cancers, autoimmune disorders, and other inflammatory conditions were excluded from the study. The selected patients were divided into two groups according to their BMIs: lean (BMI < 25) and obese (BMI > 35). An initial trial set was used as a pilot study for the optimization of the MALDI IMS protocol that was next applied to the selected thyroid tissues. MALDI IMS data from all the samples were aligned and statistical analysis carried out by k-means and linear discriminant analysis (LDA) classification model using principle component analysis (PCA) results were evaluated between the two groups: controls (lean) and cases (obese). Receiver operator characteristic (ROC) curves were alternatively used to calculate the variability of the identified peptides. The discriminating peptides were also independently identified and related to their corresponding proteins by using liquid chromatography and tandem mass spectrometry (LC-MS/MS) analyses. Eight peptides mainly from thyroglobulin were found to be upregulated whereas 10 others were found to be downregulated in the lean compared to the obese group. Through this technique, we will be able to better understand the relationship between the disease entity and obesity.

Multimodal Imaging Mass Spectrometry to Identify Markers of Pulmonary Arterial Hypertension in Human Lung Tissue Using MALDI-ToF, ToF-SIMS, and Hybrid SIMS.

Pulmonary arterial hypertension (PAH) is a rare and deadly disease affecting roughly 15-60 people per million in Europe with a poorly understood pathology. There are currently no diagnostic tools for early detection nor does a curative treatment exist. The lipid composition of arteries in lung tissue samples from human PAH and control patients were investigated using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) combined with time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging. Using random forests as an IMS data analysis technique, it was possible to identify the ion at m/z 885.6 as a marker of PAH in human lung tissue. The m/z 885.6 ion intensity was shown to be significantly higher around diseased arteries and was confirmed to be a diacylglycerophosphoinositol PI(C18:0/C20:4) via MS/MS using a novel hybrid SIMS instrument. The discovery of a potential biomarker opens up new research avenues which may finally lead to a better understanding of the PAH pathology and highlights the vital role IMS can play in modern biomedical research.

Minimizing Visceral Fat Delocalization on Tissue Sections with Porous Aluminum Oxide Slides for Imaging Mass Spectrometry.

A high correlation of bioanalytes with their corresponding histologies is the landmark feature of matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). Lipids are one of the most studied classes of biomolecules, and monitoring lipid distribution and abundance in tissue samples can lead to major inputs in the understanding of disease. Lipid delocalization and ion suppression are two major effects that can lead to misinterpretation of the IMS results to an unaware analyst. We and others have observed that tissue specimens containing high amounts of visceral fat are challenging to analyze because of fat delocalization on and off section leading to significant triacylglyceride and phospholipid delocalization and major ion suppression effects. In this work, we introduce a novel and easy to produce reusable porous aluminum oxide sample slide that minimizes visceral fat delocalization after thaw-mounting of tissue sections. Using fatty mouse kidneys and other tissues, we demonstrate its efficacy in minimizing delocalization of triacylglycerides, the primary constituents of fat, and the resulting beneficial effects on phospholipid MALDI IMS.

rMSIcleanup: an open-source tool for matrix-related peak annotation in mass spectrometry imaging and its application to silver-assisted laser desorption/ionization.

Mass spectrometry imaging (MSI) has become a mature, widespread analytical technique to perform non-targeted spatial metabolomics. However, the compounds used to promote desorption and ionization of the analyte during acquisition cause spectral interferences in the low mass range that hinder downstream data processing in metabolomics applications. Thus, it is advisable to annotate and remove matrix-related peaks to reduce the number of redundant and non-biologically-relevant variables in the dataset. We have developed rMSIcleanup, an open-source R package to annotate and remove signals from the matrix, according to the matrix chemical composition and the spatial distribution of its ions. To validate the annotation method, rMSIcleanup was challenged with several images acquired using silver-assisted laser desorption ionization MSI (AgLDI MSI). The algorithm was able to correctly classify m/z signals related to silver clusters. Visual exploration of the data using Principal Component Analysis (PCA) demonstrated that annotation and removal of matrix-related signals improved spectral data post-processing. The results highlight the need for including matrix-related peak annotation tools such as rMSIcleanup in MSI workflows.

Probiotics Modulate a Novel Amphibian Skin Defense Peptide That Is Antifungal and Facilitates Growth of Antifungal Bacteria.

Probiotics can ameliorate diseases of humans and wildlife, but the mechanisms remain unclear. Host responses to interventions that change their microbiota are largely uncharacterized. We applied a consortium of four natural antifungal bacteria to the skin of endangered Sierra Nevada yellow-legged frogs, Rana sierrae, before experimental exposure to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). The probiotic microbes did not persist, nor did they protect hosts, and skin peptide sampling indicated immune modulation. We characterized a novel skin defense peptide brevinin-1Ma (FLPILAGLAANLVPKLICSITKKC) that was downregulated by the probiotic treatment. Brevinin-1Ma was tested against a range of amphibian skin cultures and found to inhibit growth of fungal pathogens Bd and B. salamandrivorans, but enhanced the growth of probiotic bacteria including Janthinobacterium lividum, Chryseobacterium ureilyticum, Serratia grimesii, and Pseudomonas sp. While commonly thought of as antimicrobial peptides, here brevinin-1Ma showed promicrobial function, facilitating microbial growth. Thus, skin exposure to probiotic bacterial cultures induced a shift in skin defense peptide profiles that appeared to act as an immune response functioning to regulate the microbiome. In addition to direct microbial antagonism, probiotic-host interactions may be a critical mechanism affecting disease resistance.

Silver spray deposition for AgLDI imaging MS of cholesterol and other olefins on thin tissue sections.

Olefins such as cholesterol and unsaturated fatty acids play important biological roles. Silver-assisted laser desorption ionization (AgLDI) takes advantage of the strong affinity of silver to conjugate with double bonds to selectively ionize these molecules for imaging mass spectrometry (IMS) experiments. For IMS studies, two main approaches for silver deposition have been described in the literature: fine coating by silver sputtering and spray deposition of silver nanoparticles. While these approaches allow for extremely high resolution IMS experiments to be conducted, they are not readily available to all laboratories. Herein, we present a silver nitrate spray deposition approach as an alternative to silver sputtering and nanoparticle deposition for routine IMS analysis. The silver nitrate spray has the same level of specificity and sensitivity for olefins, particularly cholesterol, and has shown to be capable of IMS experiments down to 10-µm spatial resolution. Minimal sample preparation and the affordability of silver nitrate make this a convenient and accessible technique worth considering.

Mapping the fly Malpighian tubule lipidome by imaging mass spectrometry.

Matrix-assisted laser/desorption ionization imaging mass spectrometry (MALDI IMS) is an analytical technique for understanding the spatial distribution of biomolecules across a sample surface. Originally employed for mammalian tissues, this technology has been adapted to study specimens as diverse as microbes and cell cultures, food such as strawberries, and invertebrates including the vinegar fly Drosophila melanogaster. As an ideal model organism, Drosophila has brought greater understanding about conserved biological processes, organism development, and diseased states and even informed management practices of agriculturally and environmentally important species. Drosophila displays anatomically separated renal (Malpighian) tubules that are the physiological equivalent to the vertebrate nephron. Insect Malpighian tubules are also responsible for pesticide detoxification. In this article, we first describe an effective workflow and sample preparation method to study the phospholipid distribution of the Malpighian tubules that initially involves the manual microdissection of the tubules in saline buffer followed by a series of washes to remove excess salt and enhances the phospholipid signals prior to matrix deposition and IMS at 25-µm spatial resolution. We also established a complementary methodology for lipid IMS analysis of whole-body fly sections using a dual-polarity data acquisition approach at the same spatial resolution after matrix deposition by sublimation. Both procedures yield rich signal profiles from the major phospholipid classes. The reproducibility and high-quality results offered by these methodologies enable cohort studies of Drosophila through MALDI IMS.

Co-registration and analysis of multiple imaging mass spectrometry datasets targeting different analytes.

MOTIVATION: MALDI imaging mass spectrometry (IMS) has been successfully used to image a variety of biomolecules. Imaging of the many classes of biomolecules is often achieved through several incompatible sample preparations. Thus, multiple datasets must be acquired from multiple tissue sections to obtain a total molecular overview of a single sample. Addressing the need for single datasets from multiple IMS analyses, we developed the R package RegCombIMS as an extension of R package Cardinal to co-register, combine and create single IMS datasets acquired from serial sections of tissue. RESULTS: Dataset recombination and analysis is achieved by registration of the IMS datasets to a single coordinate space. The workflow allows for correlation of ions from IMS acquisitions that require incompatible sample preparations as well as multivariate analysis to mine the combined dataset for rapid and more thorough molecular query. AVAILABILITY AND IMPLEMENTATION: The source code and example data are freely available at https://github.com/NHPatterson/RegCombIMS. All code was implemented in R. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Mild Methylenetetrahydrofolate Reductase Deficiency Alters Inflammatory and Lipid Pathways in Liver.

SCOPE: Dietary and genetic folate disturbances can lead to nonalcoholic fatty liver disease (NAFLD). A common variant in methylenetetrahydrofolate reductase (MTHFR 677C→T) causes mild MTHFR deficiency with lower 5-methyltetrahydrofolate for methylation reactions. The goal is to determine whether mild murine MTHFR deficiency contributes to NAFLD-related effects. METHODS AND RESULTS: Wild-type and Mthfr+/- mice, a model for the human variant, are fed control (CD) or high-fat (HFAT) diets for 8 weeks. On both diets, MTHFR deficiency results in decreased S-adenosylmethionine, increased S-adenosylhomocysteine, and decreased betaine with reduced methylation capacity, and changes in expression of several inflammatory or anti-inflammatory mediators (Saa1, Apoa1, and Pon1). On CD, MTHFR deficiency leads to microvesicular steatosis with expression changes in lipid regulators Xbp1s and Cyp7a1. The combination of MTHFR deficiency and HFAT exacerbates changes in inflammatory mediators and introduces additional effects on inflammation (Saa2) and lipid metabolism (Nr1h4, Srebf1c, Ppara, and Crot). These effects are consistent with increased expression of pro-inflammatory HDL precursors and greater lipid accumulation. MTHFR deficiency may enhance liver injury through alterations in methylation capacity, inflammatory response, and lipid metabolism. CONCLUSION: Individuals with the MTHFR variant may be at increased risk for liver disease and related complications, particularly when consuming high-fat diets.

Mapping the triglyceride distribution in NAFLD human liver by MALDI imaging mass spectrometry reveals molecular differences in micro and macro steatosis.

Hepatic lipid accumulation, mainly in the form of triglycerides (TGs), is the hallmark of non-alcoholic fatty liver disease (NAFLD). To date, the spatial distribution of individual lipids in NAFLD-affected livers is not well characterized. This study aims to map the triglyceride distribution in normal human liver samples and livers with NAFLD and cirrhosis with imaging mass spectrometry (MALDI IMS). Specifically, whether individual triglyceride species differing by fatty acid chain length and degree of saturation correlate with the histopathological features of NAFLD as identified with classical H&E. Using a recently reported sodium-doped gold-assisted laser desorption/ionization IMS sample preparation, 20 human liver samples (five normal livers, five samples with simple steatosis, five samples with steatohepatitis, and five samples with cirrhosis) were analyzed at 10-µm lateral resolution. A total of 24 individual lipid species, primarily neutral lipids, were identified (22 TGs and two phospholipids). In samples with a low level of steatosis, TGs accumulated around the pericentral zone. In all samples, TGs with different degrees of side-chain saturation and side-chain length demonstrated differential distribution. Furthermore, hepatocytes containing macro lipid droplets were highly enriched in fully saturated triglycerides. This enrichment was also observed in areas of hepatocyte ballooning in samples with steatohepatitis and cirrhosis. In conclusion, macro lipid droplets in NAFLD are enriched in fully saturated triglycerides, indicating a possible increase in de novo lipogenesis that leads to steatohepatitis and cirrhosis.

Detection of exogenous substances in latent fingermarks by silver-assisted LDI imaging MS: perspectives in forensic sciences.

For over one hundred years, the fingerprint has reigned as one of the most trusted pieces of forensic evidence for suspect identification. In the last few decades, the modernization of chemical analysis technologies led scientists to explore new possibilities to further analyse fingermarks sampled from a crime scene. Indeed, the detection of chemicals a suspect has been in contact with before or during the crime can provide valuable insights into criminal investigations. In this regard, imaging mass spectrometry (IMS) has shown to be a powerful tool for the analysis of fingermarks by combining suspect identification and the detection of numerous endogenous and exogenous compounds. A novel approach developed in our laboratory, silver-assisted laser desorption ionization (AgLDI), was adopted to allow for the chemical analysis of latent fingermarks left on nonconductive surfaces (such as paper, cardboard, plastic and forensic lifting tape) with a time-of-flight mass spectrometer. In this study, we continue to evaluate the potential of AgLDI IMS to provide circumstantial evidence by detecting exogenous substances. We first demonstrate that owner-specific chemical signatures can be recovered from fingermarks based on the presence of several cosmetics and personal care products. We then show the possibility of detecting and imaging fingermarks containing three common illicit drugs, namely tetrahydrocannabinol, cocaine and heroin. Finally, we demonstrate that the methodology also allows us to successfully image bloody fingermarks after appropriate forensic enhancement treatments. Overall, we believe that AgLDI IMS has significant potential that could positively contribute to forensic investigations.

Insights into the MALDI Process after Matrix Deposition by Sublimation Using 3D ToF-SIMS Imaging.

Imaging mass spectrometry (IMS) has become a powerful tool to characterize the spatial distribution of biomolecules in thin tissue sections. In the case of matrix-assisted laser desorption ionization (MALDI) IMS, homogeneous matrix deposition is critical to produce high-quality ion images, and sublimation in particular has shown to be an excellent matrix deposition method for the imaging of lipids. Matrix deposition by sublimation is, however, a completely solvent-free system, which ought to prevent the mixing of matrix and analytes thought to be necessary for successful MALDI. Using 3D time-of-flight secondary ion imaging mass spectrometry, we have studied the matrix-tissue interface in 3D with high resolution to understand the MALDI process of lipids after matrix deposition by sublimation. There is a strong indication that diffusion is the process by which lipids migrate from the tissue to the matrix layer. We show that triacylglycerols and phospholipids have a delayed migratory trend as compared to diacylglycerols and monoacylglycerols, which is dependent on time and matrix thickness. Additional experiments show that a pure lipid's capacity to migrate into the matrix is dependent on its fluidity at room temperature. Furthermore, it is shown that cholesterol can only migrate in the presence of a (fluid) lipid and appears to fluidize lipids, which could explain its colocalization with the diacylglycerols and monoacylglycerols in the matrix.

A Proteomics Based Approach Reveals Differential Regulation of Visceral Adipose Tissue Proteins between Metabolically Healthy and Unhealthy Obese Patients.

Obesity and the metabolic disorders that constitute metabolic syndrome are a primary cause of morbidity and mortality in the world. Nonetheless, the changes in the proteins and the underlying molecular pathways involved in the relevant pathogenesis are poorly understood. In this study a proteomic analysis of the visceral adipose tissue isolated from metabolically healthy and unhealthy obese patients was used to identify presence of altered pathway(s) leading to metabolic dysfunction. Samples were obtained from 18 obese patients undergoing bariatric surgery and were subdivided into two groups based on the presence or absence of comorbidities as defined by the International Diabetes Federation. Two dimensional difference in-gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was carried out. A total of 28 proteins were identified with a statistically significant difference in abundance and a 1.5-fold change (ANOVA, p ≤ 0.05) between the groups. 11 proteins showed increased abundance while 17 proteins were decreased in the metabolically unhealthy obese compared to the healthy obese. The differentially expressed proteins belonged broadly to three functional categories: (i) protein and lipid metabolism (ii) cytoskeleton and (iii) regulation of other metabolic processes. Network analysis by Ingenuity pathway analysis identified the NFκB, IRK/MAPK and PKC as the nodes with the highest connections within the connectivity map. The top network pathway identified in our protein data set related to cellular movement, hematological system development and function, and immune cell trafficking. The VAT proteome between the two groups differed substantially between the groups which could potentially be the reason for metabolic dysfunction.

Multimodal detection of GM2 and GM3 lipid species in the brain of mucopolysaccharidosis type II mouse by serial imaging mass spectrometry and immunohistochemistry.

Mucopolysaccharidosis type II (Hunter's disease) mouse model (IdS-KO) was investigated by both imaging mass spectrometry (IMS) and immunohistochemistry (IHC) performed on the same tissue sections. For this purpose, IdS-KO mice brain sections were coated with sublimated 1,5-diaminonaphtalene and analyzed by high spatial resolution IMS (5 µm) and anti-GM3 IHC on the same tissue sections to characterize the ganglioside monosialated ganglioside (GM) deposits found in Hunter's disease. IMS analysis have found that two species of GM3 and GM2 that are only different due to the length of their fatty acid residue (stearic or arachidic residue) were overexpressed in the IdS-KO mice compared to a control mouse. GM3 and GM2 were characterized by on-tissue exact mass and MS/MS compared to a GM3 standard. Realignment of both IMS and IHC data sets further confirmed the observed regioselective signal previously detected by providing direct correlation of the IMS image for the two GM3 overly expressed MS signals with the anti-GM3 IHC image. Furthermore, these regioselective GM MS signals were also found to have highly heterogeneous distributions within the GM3-IHC staining. Some deposits showed high content in GM3 and GM2 stearic species (r = 0.74) and others had more abundant GM3 and GM2 arachidic species (r = 0.76). Same-section analysis of Hunter's disease mouse model by both high spatial resolution IMS and IHC provides a more in-depth analysis of the composition of the GM aggregates while providing spatial distribution of the observed molecular species. Graphical Abstract Ganglioside imaging mass spectrometry followed by immunohistochemistry performed on the same tissue section.

Life history linked to immune investment in developing amphibians.

The broad diversity of amphibian developmental strategies has been shaped, in part, by pathogen pressure, yet trade-offs between the rate of larval development and immune investment remain poorly understood. The expression of antimicrobial peptides (AMPs) in skin secretions is a crucial defense against emerging amphibian pathogens and can also indirectly affect host defense by influencing the composition of skin microbiota. We examined the constitutive or induced expression of AMPs in 17 species at multiple life-history stages. We found that AMP defenses in tadpoles of species with short larval periods (fast pace of life) were reduced in comparison with species that overwinter as tadpoles and grow to a large size. A complete set of defensive peptides emerged soon after metamorphosis. These findings support the hypothesis that species with a slow pace of life invest energy in AMP production to resist potential pathogens encountered during the long larval period, whereas species with a fast pace of life trade this investment in defense for more rapid growth and development.