Unveiling the spatial distribution of aflatoxin B1 and plant defense metabolites in maize using AP-SMALDI mass spectrometry imaging.

In order to cope with the presence of unfavorable compounds, plants can biotransform xenobiotics, translocate both parent compounds and metabolites, and perform compartmentation and segregation at the cellular or tissue level. Such a scenario also applies to mycotoxins, fungal secondary metabolites with a pre-eminent role in plant infection. In this work, we aimed to describe the effect of the interplay between Zea mays (maize) and aflatoxin B1 (AFB1) at the tissue and organ level. To address this challenge, we used atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) to investigate the biotransformation, localization and subsequent effects of AFB1 on primary and secondary metabolism of healthy maize plants, both in situ and from a metabolomics standpoint. High spatial resolution (5 µm) provided fine localization of AFB1, which was located within the root intercellular spaces, and co-localized with its phase-I metabolite aflatoxin M2. We provided a parallel visualization of maize metabolic changes, induced in different organs and tissues by an accumulation of AFB1. According to our untargeted metabolomics investigation, anthocyanin biosynthesis and chlorophyll metabolism in roots are most affected. The biosynthesis of these metabolites appears to be inhibited by AFB1 accumulation. On the other hand, metabolites found in above-ground organs suggest that the presence of AFB1 may also activate the biochemical response in the absence of an actual fungal infection; indeed, several plant secondary metabolites known for their antimicrobial or antioxidant activities were localized in the outer tissues, such as phenylpropanoids, benzoxazinoids, phytohormones and lipids.

Skeletal muscle fiber analysis by atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometric imaging at high mass and high spatial resolution.

Skeletal muscles are composed of heterogeneous muscle fibers with various fiber types. These fibers can be classified into different classes based on their different characteristics. MALDI mass spectrometric imaging (MSI) has been applied to study and visualize different metabolomics profiles of different fiber types. Here, skeletal muscles were analyzed by atmospheric pressure scanning microprobe MALDI-MSI at high spatial and high mass resolution.

Natural products in Glycyrrhiza glabra (licorice) rhizome imaged at the cellular level by atmospheric pressure matrix-assisted laser desorption/ionization tandem mass spectrometry imaging.

The rhizome of Glycyrrhiza glabra (licorice) was analyzed by high-resolution mass spectrometry imaging and tandem mass spectrometry imaging. An atmospheric pressure matrix-assisted laser desorption/ionization imaging ion source was combined with an orbital trapping mass spectrometer in order to obtain high-resolution imaging in mass and space. Sections of the rhizome were imaged with a spatial resolution of 10 µm in the positive ion mode, and a large number of secondary metabolites were localized and identified based on their accurate mass and MS/MS fragmentation patterns. Major tissue-specific metabolites, including free flavonoids, flavonoid glycosides and saponins, were successfully detected and visualized in images, showing their distributions at the cellular level. The analytical power of the technique was tested in the imaging of two isobaric licorice saponins with a mass difference of only 0.02 Da. With a mass resolving power of 140 000 and a bin width of 5 ppm in the image processing, the two compounds were well resolved in full-scan mode, and appeared with different distributions in the tissue sections. The identities of the compounds and their distributions were validated in a subsequent MS/MS imaging experiment, thereby confirming their identities and excluding possible analyte interference. The use of high spatial resolution, high mass resolution and tandem mass spectrometry in imaging experiments provides significant information about the biosynthetic pathway of flavonoids and saponins in legume species, combing the spatially resolved chemical information with morphological details at the microscopic level. Furthermore, the technique offers a scheme capable of high-throughput profiling of metabolites in plant tissues.

High resolution mass spectrometry imaging of plant tissues: towards a plant metabolite atlas.

Mass spectrometry (MS) imaging provides spatial and molecular information for a wide range of compounds. This tool can be used to investigate metabolic changes in plant physiology and environmental interactions. A major challenge in our study was to prepare tissue sections that were compatible with high spatial resolution analysis and therefore dedicated sample preparation protocols were established and optimized for the physicochemical properties of all major plant organs. We combined high spatial resolution (5 µm), in order to detect cellular features, and high mass accuracy (<2 ppm root mean square error), for molecular specificity. Mass spectrometry imaging experiments were performed in positive and negative ion mode. Changes in metabolite patterns during plant development were investigated for germination of oilseed rape. The detailed localization of more than 90 compounds allowed assignment to metabolic processes and indicated possible functions in plant tissues. The 'untargeted' nature of MS imaging allows the detection of marker compounds for the physiological status, as demonstrated for plant-pathogen interactions. Our images show excellent correlation with optical/histological examination. In contrast to previous MS imaging studies of plants, we present a complete workflow that covers multiple species, such as oilseed rape, wheat seed and rice. In addition, different major plant organs and a wide variety of compound classes were analyzed. Thus, our method could be used to develop a plant metabolite atlas as a reference to investigate systemic and local effects of pathogen infection or environmental stress.

Metabolite localization by atmospheric pressure high-resolution scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging in whole-body sections and individual organs of the rove beetle Paederus riparius.

Mass spectrometry imaging provides for non-targeted, label-free chemical imaging. In this study, atmospheric pressure high-resolution scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) was used for the first time to describe the chemical distribution of the defensive compounds pederin, pseudopederin, and pederon in tissue sections (16 µm thick) of the rove beetle Paederus riparius. The whole-insect tissue section was scanned with a 20-µm step size. Mass resolution of the orbital trapping mass spectrometer was set to 100,000 at m/z 200. Additionally, organ-specific compounds were identified for brain, nerve cord, eggs, gut, ovaries, and malpighian tubules. To confirm the distribution of the specific compounds, individual organs from the insect were dissected, and MSI experiments were performed on the dissected organs. Three ganglia of the nerve cord, with a dimension of 250-500 µm, were measured with 10-µm spatial resolution. High-quality m/z images, based on high spatial resolution and high mass accuracy were generated. These features helped to assign mass spectral peaks with high confidence. Mass accuracy of the imaging experiments was <3 ppm root mean square error, and mapping of different compound classes from a single experiment was possible. This approach improved the understanding of the biochemistry of P. riparius. Concentration differences and distributions of pederin and its analogues could be visualized in the whole-insect section. Without any labeling, we assigned key lipids for specific organs to describe their location in the body and to identify morphological structures with a specificity higher than with staining or immunohistology methods.

Analysis of cyathane-type diterpenoids from Cyathus striatus and Hericium erinaceus by high-resolution MALDI MS imaging.

Fungal secondary metabolites in both fruiting bodies and pellets from submerged cultures of basidiomycetes were analyzed by atmospheric pressure matrix-assisted laser desorption/ionization-mass spectrometry imaging at a lateral resolution of 15 µm, a mass resolution of 140,000 at m/z 200 and a mass accuracy of better than 2 ppm. The striatals A, B, C, and D, and a number of erinacine type metabolites were detected in the basidiomycetes Cyathus striatus and Hericium erinaceus, respectively. The two fungi were selected as model species, as they are well-known for efficient production of terpenoid secondary metabolites with interesting biological activities, e.g., antibacterial, fungicidal, cytotoxic properties, and stimulating effects on nerve growth factor synthesis. The localization of metabolites revealed a mostly homogeneous distribution of the striatals in the pellets of C. striatus, while a concentration gradient, increasing to the center, was observed in the pellets of H. erinaceus. A mostly homogeneous distribution of metabolites was also found in the fruiting body of H. erinaceus.

Evaluation of SARS-Cov-2 neutralizing antibody among Sinopharm Vero Cell (BBIBP-CorV) vaccinated medical students.

BACKGROUND: Information regarding seropositivity and vaccine efficacy among medical students is scarce. This study aims to detect the status of SARS-CoV-2 neutralizing antibodies among the Sinopharm's Vero Cell (BBIBP-CorV) vaccinated medical students. MATERIALS AND METHODS: A prospective, cross-sectional study was carried out among medical students of Gandaki Medical College Teaching Hospital, Pokhara, Nepal from March through August 2022. The level of SARS-CoV-2 serum- neutralizing IgG antibody was measured and its relation with participants' age and sex, duration of vaccination, and any comorbid condition was determined. RESULTS: A total of 110 medical students were included in the final analysis, the majority being females (65.5%) and the mean age is 23.1 ± 3.2 years. Most of the students (96.4%) had neutralizing antibodies against SARS-CoV-2. Among the 29 (26.36%) students who received a booster dose, the positivity rate was 100%. The mean IgG levels were 9.57 ± 9.58 µg/ml and 2.91 ± 2.47 µg/ml among students receiving an additional booster dose and among those not receiving it, respectively. In the cohort receiving a booster dose of the vaccine, the average value of neutralizing IgG antibodies was high. In contrast, the ones not receiving it, the titers were low and showed a declining trend. CONCLUSION: Though the dose strategy of the Sinopharm vaccine is effective, booster vaccination may be an important strategy to ensure protection among medical students, who are at high risk of COVID-19 due to constant patient exposure during their training. Further studies should assess vaccine efficacy among individuals who received other vaccines as well.

Mass Spectrometry Imaging Disclosed Spatial Distribution of Defense-Related Metabolites in Triticum spp.

Fusarium Head Blight is the most common fungal disease that strongly affects Triticum spp., reducing crop yield and leading to the accumulation of toxic metabolites. Several studies have investigated the plant metabolic response to counteract mycotoxins accumulation. However, information on the precise location where the defense mechanism is taking place is scarce. Therefore, this study aimed to investigate the specific tissue distribution of defense metabolites in two Triticum species and use this information to postulate on the metabolites' functional role, unlocking the "location-to-function" paradigm. To address this challenge, transversal cross-sections were obtained from the middle of the grains. They were analyzed using an atmospheric-pressure (AP) SMALDI MSI source (AP-SMALDI5 AF, TransMIT GmbH, Giessen, Germany) coupled to a Q Exactive HF (Thermo Fisher Scientific GmbH, Bremen, Germany) orbital trapping mass spectrometer. Our result revealed the capability of (AP)-SMALDI MSI instrumentation to finely investigate the spatial distribution of wheat defense metabolites, such as hydroxycinnamic acid amides, oxylipins, linoleic and α-linoleic acids, galactolipids, and glycerolipids.

5-n-Alkylresorcinol Profiles in Different Cultivars of Einkorn, Emmer, Spelt, Common Wheat, and Tritordeum.

5-n-Alkylresorcinols (AR) are bioactive compounds found in the edible parts of many cereals. Here, saturated and unsaturated homologues, including the oxidized forms 5-(2'-oxo) AR and their plant metabolites, were profiled by ultrahigh-performance liquid chromatography-ion mobility separation-high-resolution mass spectrometry in 18 cultivars of einkorn, emmer, spelt, common wheat, and tritordeum, cultivated in two consecutive years under uniform agronomic conditions. The average content of AR ranged between 672.5 ± 129.8 and 1408.9 ± 528.0 mg/kg, exceeding 2380 mg/kg in some samples and highlighting a superior content in tritordeum and in modern cultivars with respect to old wheat genotypes. By evaluating the effect of environmental and agronomic factors on the different variables, the harvest year resulted to be always significant, while location and variety influenced AR abundance only for some homologues. Furthermore, the spatial distribution of AR was investigated by mass spectrometry imaging using transversal cross sections of wheat kernels. Our results show that AR homologues are mainly localized in the testa and in the outer pericarp of wheat kernels.

Mycotoxin Uptake in Wheat - Eavesdropping Fusarium Presence for Priming Plant Defenses or a Trojan Horse to Weaken Them?

Fusarium mycotoxins represent a major threat for cereal crops and food safety. While previous investigations have described plant biotransforming properties on mycotoxins or metabolic relapses of fungal infections in plants, so far, the potential consequences of radical exposure in healthy crops are mostly unknown. Therefore, we aimed at evaluating whether the exposure to mycotoxins, deoxynivalenol (DON) and zearalenone (ZEN), at the plant-soil interface may be considered a form of biotic stress capable of inducing priming or a potential initiation of fungal attack. To address this, we used atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging to investigate the activation or the inhibition of specific biosynthetic pathways and in situ localization of primary and secondary metabolites in wheat. According to our untargeted metabolomics investigation, the translocation of plant defense metabolites (i.e., hydroxycinnamic acid amide and flavones) follows the mycotoxin accumulation organs, which is the root for ZEN-treated plantlet and culm for DON-treated sample, suggesting a local "defense-on-demand response." Therefore, it can be hypothesized that DON and ZEN are involved in the eavesdropping of Fusarium presence in soil and that wheat response based on secondary metabolites may operate on multiple organs with a potential interplay that involves masked mycotoxins.

Single Cell Analysis by High-Resolution Atmospheric-Pressure MALDI MS Imaging.

Complementary to other imaging techniques, MALDI MS imaging provides label-free analysis and chemical specificity, covering a wide range of molecules that can be visualized simultaneously. Here, we highlight a protocol to visualize lipids from single cardiac cells using high-resolution atmospheric-pressure matrix-assisted laser desorption/ionization coupled to an orbital trapping mass analyzer.

Spermidine and other functional phytochemicals in soybean seeds: Spatial distribution as visualized by mass spectrometry imaging.

Soybean seeds contain phytochemicals such as polyamines and isoflavones, which have been identified as functional components mediating health benefits in association with the consumption of soy foods. While a clear picture of the spatial distribution of these components within the seed is lacking, such information would be important to enhance or reduce their concentration in respective foods through processing. Thus, the objective of the present study was to visualize the most relevant components with respect to their distribution in soybean seeds. Mature soybean seeds were subject to atmospheric-pressure scanning-microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) combined with a Fourier-transform orbital trapping mass spectrometer to generate high-resolution chemical images of phytochemical distribution. Based on seed cross sections, differential distributions of functional components were found between soybean cotyledon and germ (shoot, hypocotyl, root) regions. Spermidine and spermine were present in higher concentrations in the germ rather than in cotyledons with highest concentrations in root and shoot meristem tissues. Differential concentrations of spermidine and other components between the germ and cotyledon regions were confirmed by seed fractioning. In contrast to polyamines spermidine and spermine, the different types of daidzein, glycitein, and genistein isoflavones were all visualized in root parenchyma tissue exclusively. Overall, mass spectrometry imaging of soybean seeds revealed clear insights into the differential distribution of functional phytochemicals. Based on their distribution and depending on specific needs, spermidine and isoflavones can either be enriched or reduced during food processing by separating cotyledon and germ fractions.

High-resolution atmospheric-pressure MALDI mass spectrometry imaging workflow for lipidomic analysis of late fetal mouse lungs.

Mass spectrometry imaging (MSI) provides label-free, non-targeted molecular and spatial information of the biomolecules within tissue. Lipids play important roles in lung biology, e.g. as surfactant, preventing alveolar collapse during normal and forced respiration. Lipidomic characterization of late fetal mouse lungs at day 19 of gestation (E19) has not been performed yet. In this study we employed high-resolution atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization MSI for the lipidomic analysis of E19 mouse lungs. Molecular species of different lipid classes were imaged in E19 lung sections at high spatial and mass resolution in positive- and negative-ion mode. Lipid species were characterized based on accurate mass and on-tissue tandem mass spectrometry. In addition, a dedicated sample preparation protocol, homogenous deposition of matrices on tissue surfaces and data processing parameters were optimized for the comparison of signal intensities of lipids between different tissue sections of E19 lungs of wild type and Pex11ß-knockout mice. Our study provides lipid information of E19 mouse lungs, optimized experimental and data processing strategies for the direct comparison of signal intensities of metabolites (lipids) among the tissue sections from MSI experiments. To best of our knowledge, this is the first MSI and lipidomic study of E19 mouse lungs.

Visualizing and Profiling Lipids in the OVLT of Fat-1 and Wild Type Mouse Brains during LPS-Induced Systemic Inflammation Using AP-SMALDI MSI.

Lipids, including omega-3 polyunsaturated fatty acids (n-3-PUFAs), modulate brain-intrinsic inflammation during systemic inflammation. The vascular organ of the lamina terminalis (OVLT) is a brain structure important for immune-to-brain communication. We, therefore, aimed to profile the distribution of several lipids (e.g., phosphatidyl-choline/ethanolamine, PC/PE), including n-3-PUFA-carrying lipids (esterified in phospholipids), in the OVLT during systemic lipopolysaccharide(LPS)-induced inflammation. We injected wild type and endogenously n-3-PUFA producing fat-1 transgenic mice with LPS (i.p., 2.5 mg/kg) or PBS. Brain samples were analyzed using immunohistochemistry and high-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization orbital trapping mass spectrometry imaging (AP-SMALDI-MSI) for spatial resolution of lipids. Depending on genotype and treatment, several distinct distribution patterns were observed for lipids [e.g., lyso(L)PC (16:0)/(18:0)] proposed to be involved in inflammation. The distribution patterns ranged from being homogeneously disseminated [LPC (18:1)], absent/reduced signaling within the OVLT relative to adjacent preoptic tissue [PE (38:6)], either treatment- and genotype-dependent or independent low signal intensities [LPC (18:0)], treatment- and genotype-dependent [PC 38:6)] or independent accumulation in the OVLT [PC (38:7)], and accumulation in commissures, e.g., nerve fibers like the optic nerve [LPE (18:1)]. Overall, screening of lipid distribution patterns revealed distinct inflammation-induced changes in the OVLT, highlighting the prominent role of lipid metabolism in brain inflammation. Moreover, known and novel candidates for brain inflammation and immune-to-brain communication were detected specifically within this pivotal brain structure, a window between the periphery and the brain. The biological significance of these newly identified lipids abundant in the OVLT and the adjacent preoptic area remains to be further analyzed.

Histology-guided high-resolution AP-SMALDI mass spectrometry imaging of wheat-Fusarium graminearum interaction at the root-shoot junction.

BACKGROUND: Fungal pathogens like Fusarium graminearum can cause severe yield losses and mycotoxin contamination of food and feed worldwide. We recently showed its ability to systemically colonize wheat via root infection. However, the molecular response of wheat to Fusarium root rot (FRR) infection and systemic spread is still unknown. As a molecular camera, mass spectrometry (MS) imaging combines label-free and multiplex metabolite profiling with histopathology. RESULTS: Atmospheric-pressure (AP)-SMALDI-MS imaging was combined with optical microscopy to study wheat-F. graminearum interaction at the root-shoot junction, which is a crucial line of defense against a pathogen that can invade all distal plant parts. To scope the functional, temporal and local aspects of FRR disease spread, metabolic changes were simultaneous visualized in diseased and healthy stem bases of the resistant cultivar Florence-Aurore at 10, 14 and 21 days after root inoculation. Histological information was used to identify disease relevant tissues and to assist the interpretation of molecular images. Detected mycotoxin compounds secreted by F. graminearum showed a route of stem infection that was consistent with observations made by microscopy. The outer epidermis and vasculature of leaf sheath were, at different disease stages, identified as prominent sites of pathogen migration and wheat protection. Wheat metabolites mapped to these relatively small tissues indicated cell wall strengthening and antifungal activity as direct defenses as well as conservation in the wheat reactions to F. graminearum diseases that affect different plant organs. CONCLUSIONS: AP-SMALDI-MS imaging at high spatial resolution is a versatile technique that can be applied to basic and applied aspects of agricultural research. Combining the technology with optical microscopy was found to be a powerful tool to gain in-depth information on almost unknown crop disease. Moreover, the approach allowed studying metabolism at the host-pathogen interface. The results provide important hints to an understanding of the complex spatio-temporal organization of plant resistance. Defense-on-demand responses to pathogen ingress were found, which provide opportunities for future research towards an improved resistance that does not negatively impact yield development in the field by saving plant resources and, moreover, may control different Fusarium diseases.

High-resolution MALDI mass spectrometry imaging of gallotannins and monoterpene glucosides in the root of Paeonia lactiflora.

High-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-SMALDI MSI) at 10 µm pixel size was performed to unravel the spatio-chemical distribution of major secondary metabolites in the root of Paeonia lactiflora. The spatial distributions of two major classes of bioactive components, gallotannins and monoterpene glucosides, were investigated and visualized at the cellular level in tissue sections of P. lactiflora roots. Accordingly, other primary and secondary metabolites were imaged, including amino acids, carbohydrates, lipids and monoterpenes, indicating the capability of untargeted localization of metabolites by using high-resolution MSI platform. The employed AP-SMALDI MSI system provides significant technological advancement in the visualization of individual molecular species at the cellular level. In contrast to previous histochemical studies of tannins using unspecific staining reagents, individual gallotannin species were accurately localized and unequivocally discriminated from other phenolic components in the root tissues. High-quality ion images were obtained, providing significant clues for understanding the biosynthetic pathway of gallotannins and monoterpene glucosides and possibly helping to decipher the role of tannins in xylem cells differentiation and in the defence mechanisms of plants, as well as to investigate the interrelationship between tannins and lignins.

Nepalese pharmacy students' perceptions regarding mental disorders and pharmacy education.

OBJECTIVE: To determine Nepalese pharmacy students' perceptions of whether mental disorders impact performance in pharmacy school. METHOD: All first- and third-year undergraduate pharmacy students (n=226) in Nepal were invited to complete a modified version of the Mental Illness Performance Scale. RESULTS: Among the 200 respondents (response rate 88.5%), 14% reported that they had a mental disorder. The majority (92%) of third-year students agreed or strongly agreed that depression would interfere with a student's academic performance. Almost half of first-year students agreed or strongly agreed that alcohol or drug abuse would be grounds for both rejecting an applicant from pharmacy school (49%) and dismissal of a student from pharmacy school (46%). CONCLUSIONS: Students perceived a high level of academic impairment associated with mental disorders, but the majority did not perceive that mental disorders were grounds for dismissal from or rejection of entry to pharmacy school. Students' attitudes may discourage them from seeking help or providing mental health support to others.