Preformulation evaluation of selumetinib for topical application: skin distribution and photodegradation analysis using MALDI imaging and LC-MS/MS.

Understanding drug behavior within the skin, especially for photosensitive compounds, is crucial for developing effective and safe topical therapies. This study employs Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) and Liquid Chromatography-Mass Spectrometry (LC-MS/MS) to investigate the skin permeation and photostability of selumetinib, a MEK inhibitor used in treating type 1 neurofibromatosis (NF1). The highest amounts of selumetinib in the skin sections were obtained when using the gel formulation, suggesting that it is to be preferred to cream formulations to achieve higher permeation of the drug. Our study also revealed that selumetinib is amenable to photodegradation in ex vivo skin explants, and yields one main degradation product, whose degradation is likely triggered by hydrogen abstraction. MALDI-MSI results showed selumetinib and its degradation product concentrate in skin appendages, indicating these structures might serve as drug reservoirs, potentially prolonging retention and efficacy. This study demonstrates that combining MALDI-MSI with LC/MS-MS can highly contribute to the characterization of the fate of photosensitive compounds in the skin, an essential prerequisite to the development of compound-specific photoprotective measures. It will also pave the way for innovative topical delivery strategies for NF1 treatment.

In situ imaging of LPMO action on plant tissues.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant polysaccharides such as cellulose. Several studies have reported LPMO action in synergy with other carbohydrate-active enzymes (CAZymes) for the degradation of lignocellulosic biomass but direct LPMO action at the plant tissue level remains challenging to investigate. Here, we have developed a MALDI-MS imaging workflow to detect oxidised oligosaccharides released by a cellulose-active LPMO at cellular level on maize tissues. Using this workflow, we imaged LPMO action and gained insight into the spatial variation and relative abundance of oxidised and non-oxidised oligosaccharides. We reveal a targeted action of the LPMO related to the composition and organisation of plant cell walls.

Is Lipid Metabolism of Value in Cancer Research and Treatment? Part I- Lipid Metabolism in Cancer.

For either healthy or diseased organisms, lipids are key components for cellular membranes; they play important roles in numerous cellular processes including cell growth, proliferation, differentiation, energy storage and signaling. Exercise and disease development are examples of cellular environment alterations which produce changes in these networks. There are indications that alterations in lipid metabolism contribute to the development and progression of a variety of cancers. Measuring such alterations and understanding the pathways involved is critical to fully understand cellular metabolism. The demands for this information have led to the emergence of lipidomics, which enables the large-scale study of lipids using mass spectrometry (MS) techniques. Mass spectrometry has been widely used in lipidomics and allows us to analyze detailed lipid profiles of cancers. In this article, we discuss emerging strategies for lipidomics by mass spectrometry; targeted, as opposed to global, lipid analysis provides an exciting new alternative method. Additionally, we provide an introduction to lipidomics, lipid categories and their major biological functions, along with lipidomics studies by mass spectrometry in cancer samples. Further, we summarize the importance of lipid metabolism in oncology and tumor microenvironment, some of the challenges for lipodomics, and the potential for targeted approaches for screening pharmaceutical candidates to improve the therapeutic efficacy of treatment in cancer patients.

Biofortified Rice Provides Rich Sakuranetin in Endosperm.

Sakuranetin plays a key role as a phytoalexin in plant resistance to biotic and abiotic stresses, and possesses diverse health-promoting benefits. However, mature rice seeds do not contain detectable levels of sakuranetin. In the present study, a transgenic rice plant was developed in which the promoter of an endosperm-specific glutelin gene OsGluD-1 drives the expression of a specific enzyme naringenin 7-O-methyltransferase (NOMT) for sakuranetin biosynthesis. The presence of naringenin, which serves as the biosynthetic precursor of sakuranetin made this modification feasible in theory. Liquid chromatography tandem mass spectrometry (LC-MS/MS) validated that the seeds of transgenic rice accumulated remarkable sakuranetin at the mature stage, and higher at the filling stage. In addition, the panicle blast resistance of transgenic rice was significantly higher than that of the wild type. Specially, the matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging was performed to detect the content and spatial distribution of sakuranetin and other nutritional metabolites in transgenic rice seeds. Notably, this genetic modification also did not change the nutritional and quality indicators such as soluble sugars, total amino acids, total flavonoids, amylose, total protein, and free amino acid content in rice. Meanwhile, the phenotypes of the transgenic plant during the whole growth and developmental periods and agricultural traits such as grain width, grain length, and 1000-grain weight exhibited no significant differences from the wild type. Collectively, the study provides a conceptual advance on cultivating sakuranetin-rich biofortified rice by metabolic engineering. This new breeding idea may not only enhance the disease resistance of cereal crop seeds but also improve the nutritional value of grains for human health benefits.

Saturated Fatty Acid Emulsions Open the Blood-Brain Barrier and Promote Drug Delivery in Rat Brains.

We performed this study to evaluate whether saturated fatty acid (SFA) emulsions affect the BBB and determine the duration of BBB opening, thereby promoting drug delivery to the brain. Butyric, valeric, caproic, enanthic, and caprylic acid emulsions were infused into the carotid artery of the rat model. We evaluated the BBB opening and drug delivery over time. The trypan blue and doxorubicin delivery studies were repeated from 30 min to 6 h. In the 1 h rats in each group, transmission electron microscopy (TEM) was performed to morphologically evaluate tight junctions, and the delivery of temozolomide was assessed by desorption electrospray ionization mass spectrometry. The ipsilateral hemisphere was positive for trypan blue staining in all the five SFA emulsion groups. In the valeric, enanthic, and caprylic acid emulsion groups, RGB ratios were significantly higher at 30 min and decreased thereafter. Doxorubicin delivery increased in all emulsion groups at all time points. Tight junctions were observed to be open in all groups. TMZ delivery was significantly higher in the ipsilateral hemisphere. In conclusion, intra-arterially infused SFA emulsions opened the BBB and promoted drug delivery within 30 min, which decreased thereafter. Therefore, SFA emulsions may aid BBB research and promote drug delivery to the brain.

Assessing Molecular Localization of Symbiont Microalgae in Coral Branches Through Mass Spectrometry Imaging.

Reef-building corals are a fundamental pillar of coral reef ecosystems in tropical and subtropical shallow environments. Corals harbor symbiotic dinoflagellates belonging to the family Symbiodiniaceae, commonly known as zooxanthellae. Extensive research has been conducted on this symbiotic relationship, yet the fundamental information about the distribution and localization of Symbiodiniaceae cells in corals is still limited. This information is crucial to understanding the mechanism underlying the metabolite exchange between corals and their algal symbionts, as well as the metabolic flow within holobionts. To examine the distribution of Symbiodiniaceae cells within corals, in this study, we used fluorescence imaging and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MS-Imaging) on branches of the Acropora tenuis coral. We successfully prepared frozen sections of the coral for molecular imaging without fixing or decalcifying the coral branches. By combining the results of MS-Imaging with that of the fluorescence imaging, we determined that the algal Symbiodiniaceae symbionts were not only localized in the tentacle and surface region of the coral branches but also inhabited the in inner parts. Therefore, the molecular imaging technique used in this study could be valuable to further investigate the molecular dynamics between corals and their symbionts.

Development of Mass Spectrometry Imaging on skeletal muscle to characterize the local pro-inflammatory and pro-resolution lipid responses in a vaccination context.

Vaccine reactogenicity is well documented at the clinical level but the mechanism involved at the local or systemic level are still poorly understood. Muscular tissue where most vaccines are administered is the first place of interaction between the vaccine formulation and the host's immune cells. So far, this site of vaccine administration is not well documented from a mechanistic standpoint. The study of early molecular events at the injection site is crucial to understand the local response to vaccines. In this paper, we report a standardized workflow, from the injection of vaccine formulations in rabbit muscle, to the analysis by desorption electrospray ionization and histology staining to understand the role of lipids involved in the inflammation and its resolution on striated muscular tissue. The analysis of lipid mediators was optimized at the site of needle insertion to allow the spatial comparison of cellular infiltrates at the injection site. We showed that lipids were distributed across the spatial tissue morphology in a time-dependent manner. The MS imaging applied to vaccinology could pave the way to a better understanding of vaccine reactogenicity and mechanism of action.

Immunolabelling perturbs the endogenous and antibody-conjugated elemental concentrations during immuno-mass spectrometry imaging.

Immuno-mass spectrometry imaging uses lanthanide-conjugated antibodies to spatially quantify biomolecules via laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The multi-element capabilities allow for highly multiplexed analyses that may include both conjugated antibodies and endogenous metals to reveal relationships between disease and chemical composition. Sample handling is known to perturb the composition of the endogenous elements, but there has been little investigation into the effects of immunolabelling and coverslipping. Here, we used cryofixed muscle sections to examine the impact of immunolabelling steps on the concentrations of a Gd-conjugated anti-dystrophin primary antibody, and the endogenous metals Cu and Zn. Primary antibody incubation resulted in a decrease in Zn, and an increase in Cu. Zn was removed from the cytoplasm where it was hypothesised to be more labile, whereas concentrated locations of Zn remained in the cell membrane in all samples that underwent the immunostaining process. Cu increased in concentration and was found mostly in the cell membrane. The concentration of the Gd-conjugated antibody when compared to the standard air-dried sample was not significantly different when coverslipped using an organic mounting medium, whereas use of an aqueous mounting medium significantly reduced the concentration of Gd. These results build on the knowledge of how certain sample handling techniques change elemental concentrations and distributions in tissue sections. Immunolabelling steps impact the concentration of endogenous elements, and separate histological sections are required for the quantitative analysis of endogenous elements and biomolecules. Additionally, coverslipping tissue sections for complementary immunohistochemical/immunofluorescent imaging may compromise the integrity of the elemental label, and organic mounting media are recommended over aqueous mounting media.

Investigation of the active ingredients of Shuangshen Ningxin Fomula and the mechanism underlying their protective effects against myocardial ischemia-reperfusion injury by mass spectrometric imaging.

BACKGROUND: Traditional Chinese medicine, particularly Shuangshen Ningxin Capsule (SSNX), has been studied intensely. SSNX includes total ginseng saponins (from Panax ginseng Meyer), total phenolic acids from Salvia miltiorrhiza Bunge, and total alkaloids from Corydalis yanhusuo W. T. Wang. It has been suggested to protect against myocardial ischemia by a mechanism that has not been fully elucidated. METHODS: The composition and content of SSNX were determined by UHPLC-Q-TOFQ-TOF / MS. Then, a rat model of myocardial ischemia-reperfusion injury was established, and the protective effect of SSNX was measured. The protective mechanism was investigated using spatial metabolomics. RESULTS: We found that SSNX significantly improved left ventricular function and ameliorated pathological damages in rats with myocardial ischemia-reperfusion injury. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), the protective mechanism of SSNX was examined by comparing the monomer components of drugs targeted in myocardial tissue with the distribution of myocardial energy metabolism-related molecules and phospholipids. Interestingly, some lipids display inconsistent content distribution in the myocardial ischemia risk and non-risk zones. These discrepancies reflect the degree of myocardial injury in different regions. CONCLUSION: These findings suggest that SSNX protects against myocardial ischemia-reperfusion injury by correcting abnormal myocardial energy metabolism, changing the levels and distribution patterns of phospholipids, and stabilizing the structure of the myocardial cell membrane. MALDI-TOF MS can detect the spatial distribution of small molecule metabolites in the myocardium and can be used in pharmacological research.

Unveiling targeted spatial metabolome of rice seed at the dough stage using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry imaging.

Rice (Oryza sativa) seeds contain a variety of metabolites, which not only provide energy for their own growth and development, but also are an important source of nutrition for humans. It is crucial to study the distribution of metabolites in rice seeds, but the spatial metabolome of rice seeds is rarely investigated. In this study, Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) imaging was used to reveal the spatial distribution of free soluble sugars (glucose, fructose, sucrose, and maltose), amino acids (9 essential amino acids and 2 amino acids affecting rice eating quality: L-aspartic acid and L-glutamic acid), and 4 metabolites in the flavonoids synthesis pathway (cinnamic acid, naringenin chalcone, naringenin, and dihydrokaempferol) in rice seed at the dough stage. It was found that the 4 free soluble sugars present similar spatial distribution, mainly distributed in the seed cortex and embryo with high abundance. The majority of amino acids are also concentrated in the rice cortex and embryo, while the others are abundant in the whole seed. Besides cinnamic acid distributed in the seed cortex and embryo, the naringenin chalcone, naringenin, and dihydrokaempferol were also found in the endosperm and had lower content. Furthermore, a colocalization phylogenetic tree according to the spatial distribution imaging of each metabolite was constructed. This study revealed the distribution diversity of metabolites in different segmentations of rice seed at the dough stage, providing clues for the nutritional differences between brown rice and white rice, and serving as a reference for people to target a healthy diet.

Imaging Analysis of Phosphatidylcholines and Diacylglycerols Using Surface-Assisted Laser Desorption/Ionization Mass Spectrometry with Metal Film Formed by Mist Chemical Vapor Deposition.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) has been widely used for analyses of biomolecules and industrial materials. Surface-assisted laser desorption/ionization (SALDI) is studied to complement the ionization ability for the MALDI/MS. In this study, lab-made mist chemical vapor deposition (mist CVD) system was used to produce metal films as ionization assistance materials for SALDI/MS. The system could give Ag film from inexpensive silver trifluoroacetate solution rapidly and simply under atmospheric pressure. Phosphatidylcholines could be detected high sensitively and diacylglycerols (DAGs) could not be detected in MALDI/MS. In the SALDI/MS and the MS imaging with Ag film by mist CVD, both the phosphatidylcholines and the DAGs could be detected and the localized images. In the Ag film-SALDI/MS of lipids, not only Ag-adducted ions but also Na- and K-adducted ions were detected. The Ag film formed by the mist CVD to act as an ionization-assistance material and a cationization agent in SALDI would be useful in MS imaging of biological tissue sections.

Effects of In Utero PFOS Exposure on Epigenetics and Metabolism in Mouse Fetal Livers.

Prenatal exposure to perfluorooctanesulfonate (PFOS) increases fetus' metabolic risk; however, the investigation of the underlying mechanism is limited. In this study, pregnant mice in the gestational days (GD, 4.5-17.5) were exposed to PFOS (0.3 and 3 µg/g of body weight). At GD 17.5, PFOS perturbed maternal lipid metabolism and upregulated metabolism-regulating hepatokines (Angptl4, Angptl8, and Selenop). Mass-spectrometry imaging and whole-genome bisulfite sequencing revealed, respectively, selective PFOS localization and deregulation of gene methylation in fetal livers, involved in inflammation, glucose, and fatty acid metabolism. PCR and Western blot analysis of lipid-laden fetal livers showed activation of AMPK signaling, accompanied by significant increases in the expression of glucose transporters (Glut2/4), hexose-phosphate sensors (Retsat and ChREBP), and the key glycolytic enzyme, pyruvate kinase (Pk) for glucose catabolism. Additionally, PFOS modulated the expression levels of PPARα and PPARγ downstream target genes, which simultaneously stimulated fatty acid oxidation (Cyp4a14, Acot, and Acox) and lipogenesis (Srebp1c, Acaca, and Fasn). Using human normal hepatocyte (MIHA) cells, the underlying mechanism of PFOS-elicited nuclear translocation of ChREBP, associated with a fatty acid synthesizing pathway, was revealed. Our finding implies that in utero PFOS exposure altered the epigenetic landscape associated with dysregulation of fetal liver metabolism, predisposing postnatal susceptibility to metabolic challenges.

Detection of proteomic alterations at different stages in a Huntington's disease mouse model via matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging.

Huntington's disease (HD) is a progressive and irreversible neurodegenerative disease leading to the inability to carry out daily activities and for which no cure exists. The underlying mechanisms of the disease have not been fully elucidated yet. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) allows the spatial information of proteins to be obtained upon the tissue sections without homogenisation. In this study, we aimed to examine proteomic alterations in the brain tissue of an HD mouse model with MALDI-MSI coupled to LC-MS/MS system. We used 3-, 6- and 12-month-old YAC128 mice representing pre-stage, mild stage and pathological stage of the HD and their non-transgenic littermates, respectively. The intensity levels of 89 proteins were found to be significantly different in YAC128 in comparison to their control mice in the pre-stage, 83 proteins in the mild stage, and 82 proteins in the pathological stage. Among them, Tau, EF2, HSP70, and NogoA proteins were validated with western blot analysis. In conclusion, the results of this study have provided remarkable new information about the spatial proteomic alterations in the HD mouse model, and we suggest that MALDI-MSI is an excellent technique for identifying such regional proteomic changes and could offer new perspectives in examining complex diseases.

Cytological Cytospin Preparation for the Spatial Proteomics Analysis of Thyroid Nodules Using MALDI-MSI.

The application of innovative spatial omics approaches in the context of cytological specimens may open new frontiers for their diagnostic assessment. In particular, spatial proteomics using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) represents one of the most promising avenues, owing to its capability to map the distribution of hundreds of proteins within a complex cytological background in a multiplexed and relatively high-throughput manner. This approach may be particularly beneficial in the heterogeneous context of thyroid tumors where certain cells may not present clear-cut malignant morphology upon fine-needle aspiration biopsy, highlighting the necessity for additional molecular tools which are able to improve their diagnostic performance.This chapter aims to provide a detailed overview of a cytospin-based preparation workflow that has been optimized to facilitate the reliable spatial proteomics analysis of cytological thyroid specimens using MALDI-MSI, indicating the key aspects which should be considered when handling such samples.

Detection of early proteomic alterations in 5xFAD Alzheimer's disease neonatal mouse model via MALDI-MSI.

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder, characterized by memory deficit and dementia. AD is considered a multifactorial disorder where multiple processes like amyloid-beta and tau accumulation, axonal degeneration, synaptic plasticity, and autophagic processes plays an important role. In this study, the spatial proteomic differences in the neonatal 5xFAD brain tissue were investigated using MALDI-MSI coupled to LC-MS/MS, and the statistically significantly altered proteins were associated with AD. Thirty-five differentially expressed proteins (DEPs) between the brain tissues of neonatal 5xFAD and their littermate mice were detected via MALDI-MSI technique. Among the 35 proteins identified, 26 of them were directly associated with AD. Our results indicated a remarkable resemblance in the protein expression profiles of neonatal 5xFAD brain when compared to AD patient specimens or AD mouse models. These findings showed that the molecular alterations in the AD brain existed even at birth and that some proteins are neurodegenerative presages in neonatal AD brain. HIGHLIGHTS: Spatial proteomic alterations in the 5xFAD mouse brain compared to the littermate. 26 out of 35 differentially expressed proteins associated with Alzheimer's disease (AD). Molecular alterations and neurodegenerative presages in neonatal AD brain. Alterations in the synaptic function an early and common neurobiological thread.

Bioorthogonal nanozymes for breast cancer imaging and therapy.

Bioorthogonal catalysis via transition metal catalysts (TMCs) enables the generation of therapeutics locally through chemical reactions not accessible by biological systems. This localization can enhance the efficacy of anticancer treatment while minimizing off-target effects. The encapsulation of TMCs into nanomaterials generates "nanozymes" to activate imaging and therapeutic agents. Here, we report the use of cationic bioorthogonal nanozymes to create localized "drug factories" for cancer therapy in vivo. These nanozymes remained present at the tumor site at least seven days after a single injection due to the interactions between cationic surface ligands and negatively charged cell membranes and tissue components. The prodrug was then administered systemically, and the nanozymes continuously converted the non-toxic molecules into active drugs locally. This strategy substantially reduced the tumor growth in an aggressive breast cancer model, with significantly reduced liver damage compared to traditional chemotherapy.

A Caged In-Source Laser-Cleavable MALDI Matrix with High Vacuum Stability for Extended MALDI-MS Imaging.

Insufficient vacuum stability of matrix chemicals is a major limitation in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) of large tissue sample cohorts. Here, we designed and synthesized the photo-cleavable caged molecule 4,5-dimethoxy-2-nitrobenzyl-2,5-dihydroxyacetophenone (DMNB-2,5-DHAP) and employed it for lipid MALDI-MSI of mouse brain tissue sections. DMNB-2,5-DHAP is vacuum-stable in a high vacuum MALDI ion source for at least 72 h. Investigation of the uncaging process suggested that the built-in laser (355 nm) in the MALDI ion source promoted the in situ generation of 2,5-DHAP. A caging group is used for the first time in designing a MALDI matrix that is vacuum-stable, uncaged upon laser irradiation during the measurement process, and that boosts lipid ion intensity with MALDI-2 laser-induced postionization.

Application of Teeth in Toxicological Analysis of Decomposed Cadavers Using a Carbamazepine-Administered Rat Model.

In a regular autopsy, blood and organs are used to quantify drug and toxicant concentrations; however, specimens such as blood cannot be collected from highly decomposed corpses, making the quantification of drug and toxicants impossible. This study aimed to estimate the blood carbamazepine (CBZ) concentration from teeth, a part of the human body that is best preserved after death. We sampled teeth and blood of rats administered CBZ. The correlation between the tooth and serum CBZ concentrations was analyzed. Rats were euthanized after CBZ administration and kept at 22 °C for 0 to 15 days before sampling the teeth and measuring the CBZ concentration. Undecalcified, fresh, frozen sections of rat teeth were prepared, and CBZ localization was evaluated. CBZ concentrations in both teeth and cardiac blood peaked at 60 min after administration and increased in a dose-dependent manner. CBZ concentration in teeth did not substantially change after death, with high CBZ distribution being observed in the pulp cavity. The tooth and serum CBZ concentrations were highly correlated, suggesting that the measurement of toxicant concentration in sampled teeth would allow for the estimation of blood toxicant concentration in highly decomposed corpses.

FT-ICR-MS-based metabolomics: A deep dive into plant metabolism.

Metabolomics involves the identification and quantification of metabolites to unravel the chemical footprints behind cellular regulatory processes and to decipher metabolic networks, opening new insights to understand the correlation between genes and metabolites. In plants, it is estimated the existence of hundreds of thousands of metabolites and the majority is still unknown. Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) is a powerful analytical technique to tackle such challenges. The resolving power and sensitivity of this ultrahigh mass accuracy mass analyzer is such that a complex mixture, such as plant extracts, can be analyzed and thousands of metabolite signals can be detected simultaneously and distinguished based on the naturally abundant elemental isotopes. In this review, FT-ICR-MS-based plant metabolomics studies are described, emphasizing FT-ICR-MS increasing applications in plant science through targeted and untargeted approaches, allowing for a better understanding of plant development, responses to biotic and abiotic stresses, and the discovery of new natural nutraceutical compounds. Improved metabolite extraction protocols compatible with FT-ICR-MS, metabolite analysis methods and metabolite identification platforms are also explored as well as new in silico approaches. Most recent advances in MS imaging are also discussed.