RESUMEN
Targeted therapy to the tumor would greatly advance precision medicine. Many drug delivery vehicles have emerged, but liposomes are cited as the most successful to date. Recent efforts to develop liposomal drug delivery systems focus on drug distribution in tissues and ignore liposomal fate. In this study, we developed a novel method to elucidate both drug and liposomal bilayer distribution in a three-dimensional cell culture model using quantitative matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI qMSI) alongside fluorescence microscopy. Imaging liposomal distribution in a cell culture model is challenging, as lipids forming the bilayer are endogenous to the model system. To resolve this issue, we functionalized the bilayer by chemically cross-linking a fluorescent tag to the alkyne-containing lipid hexynoyl phosphoethanolamine (HPE). We synthesized liposomes incorporating the tagged HPE lipid and encapsulated within them doxorubicin, yielding a theranostic liposome capable of both drug delivery and monitoring liposomal uptake. We employed an "in-tissue" MALDI qMSI approach to generate a calibration curve with R2 = 0.9687, allowing for quantification of doxorubicin within spheroid sections at multiple time points. After 72 h of treatment with the theranostic liposomes, full doxorubicin penetration was observed. The metabolites doxorubicinone and 7-deoxydoxorubicinone were also detected after 48 h. Modification of the bilayer allowed for fluorescence microscopy tracking of liposomes, while MALDI MSI simultaneously permitted the imaging of drugs and metabolites. While we demonstrated the utility of our method with doxorubicin, this system could be applied to examine the uptake, release, and metabolism of many other liposome-encapsulated drugs.
Asunto(s)
Doxorrubicina , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Doxorrubicina/química , Doxorrubicina/administración & dosificación , Doxorrubicina/análogos & derivados , Humanos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Sistemas de Liberación de Medicamentos , Antibióticos Antineoplásicos/química , Antibióticos Antineoplásicos/administración & dosificación , Liposomas/química , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Polietilenglicoles/química , Microscopía Fluorescente , Línea Celular TumoralRESUMEN
Herein, we assess the complementarity and complexity of data that can be detected within mammalian lipidome mass spectrometry imaging (MSI) via matrix-assisted laser desorption ionization (MALDI) and nanospray desorption electrospray ionization (nano-DESI). We do so by employing 21 T Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) with absorption mode FT processing in both cases, allowing unmatched mass resolving power per unit time (≥613k at m/z 760, 1.536 s transients). While our results demonstrated that molecular coverage and dynamic range capabilities were greater in MALDI analysis, nano-DESI provided superior mass error, and all annotations for both modes had sub-ppm error. Taken together, these experiments highlight the coverage of 1676 lipids and serve as a functional guide for expected lipidome complexity within nano-DESI-MSI and MALDI-MSI. To further assess the lipidome complexity, mass splits (i.e., the difference in mass between neighboring peaks) within single pixels were collated across all pixels from each respective MSI experiment. The spatial localization of these mass splits was powerful in informing whether the observed mass splits were biological or artificial (e.g., matrix related). Mass splits down to 2.4 mDa were observed (i.e., sodium adduct ambiguity) in each experiment, and both modalities highlighted comparable degrees of lipidome complexity. Further, we highlight the persistence of certain mass splits (e.g., 8.9 mDa; double bond ambiguity) independent of ionization biases. We also evaluate the need for ultrahigh mass resolving power for mass splits ≤4.6 mDa (potassium adduct ambiguity) at m/z > 1000, which may only be resolved by advanced FTICR-MS instrumentation.
Asunto(s)
Lipidómica , Espectrometría de Masa por Ionización de Electrospray , Animales , Análisis de Fourier , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , MamíferosRESUMEN
Nanospray desorption electrospray ionization mass spectrometry, a powerful ambient sampling and imaging technique, is herein coupled as an isolated source with 21 Tesla (21T) Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). Absorption-mode data, enabled by an external data acquisition system, is applied for improved mass resolution, accuracy, and dynamic range without compromising spectral acquisition rates. Isotopic fine structure (IFS) information is obtained from the ambient sampling of living Bacillus and Fusarium species, allowing for high confidence in molecular annotations with a resolution >830 k (at m/z 825). Tandem mass spectrometry experiments for biological samples are shown to retain the IFS in addition to gained fragmentation information, providing a further degree of annotation confidence from ambient analyses. Rat brain was imaged by nanospray desorption electrospray ionization (nano-DESI) 21T FTICR MS in â¼5 h using 768 ms transients, producing over 800 molecular annotations using the METASPACE platform and low-parts-per-billion mass accuracy at a spatial resolution of â¼25 × 180 µm. Finally, nano-DESI 21T FTICR MS imaging is demonstrated to reveal images corresponding to the IFS, as well as hundreds of additional molecular features (including demonstrated differences as low as 8.96 mDa) that are otherwise undetected by a more conventional imaging methodology.
Asunto(s)
Ciclotrones , Espectrometría de Masa por Ionización de Electrospray , Animales , Diagnóstico por Imagen , Análisis de Fourier , Ratas , Espectrometría de Masa por Ionización de Electrospray/métodos , Espectrometría de Masas en Tándem/métodosRESUMEN
Three-dimensional cell cultures, or spheroids, are important model systems for cancer research because they recapitulate chemical and phenotypic aspects of in vivo tumors. Spheroids develop radially symmetric chemical gradients, resulting in distinct cellular populations. Stable isotopic labeling by amino acids in cell culture (SILAC) is a well-established approach to quantify protein expression and has previously been used in a pulse-chase format to evaluate temporal changes. In this article, we demonstrate that distinct isotopic signatures can be introduced into discrete spatial cellular populations, effectively tracking proteins to original locations in the spheroid, using a platform that we refer to as spatial SILAC. Spheroid populations were grown with light, medium, and heavy isotopic media, and the concentric shells of cells were harvested by serial trypsinization. Proteins were quantitatively analyzed by ultraperformance liquid chromatography-tandem mass spectrometry. The isotopic signatures correlated with the spatial location and the isotope position do not significantly impact the proteome of each individual layer. Spatial SILAC can be used to examine the proteomic changes in the different layers of the spheroid and to identify protein biomarkers throughout the structure. We show that SILAC labels can be discretely pulsed to discrete positions, without altering the spheroid's proteome, promising future combined pharmacodynamic and pharmacokinetic studies.
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Proteoma , Proteómica , Aminoácidos , Técnicas de Cultivo de Célula , Marcaje Isotópico , Esferoides CelularesRESUMEN
Accurate measurement and understanding of therapeutic uptake and metabolism is key in the drug development process. This work examines the amount of doxorubicin that can penetrate into spheroids after being encapsulated in a liposomal configuration in comparison with free drug. Through a process known as serial trypsinization, three distinct cellular populations of a spheroid were successfully separated and a small molecule extraction was used to isolate the chemotherapeutic. Doxorubicin showed a time-dependent permeability into spheroids with the most drug accumulating in the core at 24 h of treatment. Entrapment of the chemotherapeutic delayed the permeability of the drug and resulted in reduced amounts quantified at the earlier time points. These findings validate the claim that liposomal therapeutics have the ability to alter the pharmacokinetics and pharmacodynamics profiles of a drug while also demonstrating the combined power of mass spectrometry and three-dimensional cell cultures to evaluate drug penetration and metabolism. Graphical abstract.
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Antibióticos Antineoplásicos/metabolismo , Doxorrubicina/análogos & derivados , Esferoides Celulares/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Doxorrubicina/metabolismo , Doxorrubicina/farmacocinética , Células HCT116 , Humanos , Espectrometría de Masas , Polietilenglicoles/metabolismo , Polietilenglicoles/farmacocinética , Tripsina/metabolismoRESUMEN
Immunotherapies are treatments that use a patient's immune system to combat disease. One important type of immunotherapy employed in cancer treatments is the delivery of monoclonal antibodies to block growth receptors. In this manuscript, we develop a methodology that enables accurate and simple evaluation of antibody-type drug delivery using MALDI-MSI. To overcome the mass-range limitation that prevents the detection of large therapeutic antibodies, we used in situ reduction and alkylation to break disulfide bonds to generate smaller fragments. These smaller fragments are more readily ionized and detected by MALDI-MSI without loss of spatial information on the parent drug. As a proof of concept study, we evaluated the distribution of cetuximab in 3D colon cell cultures. Cetuximab is a monoclonal antibody that binds to the extracellular domain of epidermal-growth-factor receptor (EGFR), which is often overexpressed in colorectal cancer (CRC) and mediates cell differentiation, proliferation, migration, and angiogenesis. Cetuximab directly inhibits tumor growth and metastasis and induces apoptosis. By performing on-tissue reduction followed by MALDI-MSI analysis, we successfully mapped the time-dependent penetration and distribution of cetuximab in spheroids derived from two different colon-cancer cell lines (HT-29 and DLD-1). The localization patterns were further confirmed with IF staining of the drug. Changes in other biomolecules following drug treatment were also observed, including the elevation of ATP in spheroids. The developed method has also been applied to map cetuximab distribution in patient-derived colorectal-tumor organoids (CTOs). Overall, we believe this powerful label-free approach will be useful for visualizing the heterogeneous distribution of antibody drugs in tissues and tumors and will help to monitor and optimize their use in the clinic.
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Cetuximab/inmunología , Receptores ErbB/inmunología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Adenosina Trifosfato/metabolismo , Área Bajo la Curva , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cetuximab/análisis , Cetuximab/farmacología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Neoplasias del Colon/terapia , Humanos , Inmunoterapia , Microscopía Fluorescente , Curva ROC , Esferoides Celulares/efectos de los fármacosRESUMEN
Cancer chemotherapeutics often fail to reach all diseased cells. To help solve this problem, researchers are investigating novel drug delivery systems. Liposomes are an attractive option due to their low toxicity, high biocompatibility, and potential to carry a large amount of a drug to the tumor site, all while avoiding being eliminated from the body. This study evaluates the penetration of doxorubicin-encased liposomes into three-dimensional cell cultures, or spheroids. Liposomes composed of lipids containing head groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cholesterol were created by extrusion. Doxorubicin is encapsulated within the hydrophilic core of the liposome. The drug is actively released in the spheroid as the lipids bind to cellular lipid bilayers. Spheroids were dosed with liposomal doxorubicin, free doxorubicin, or media control to assess drug distribution over the course of 72 h. Drug penetration was visualized by Matrix-Assisted Laser Desorption/Ionization-Imaging Mass Spectrometry (MALDI-IMS) with confirmation by steady state fluorescence microscopy, creating a comprehensive picture of drug distribution. This technique is able to identify both free and liposomal doxorubicin throughout the spheroid after just 12 hours of treatment. Additionally, MALDI-IMS is able to detect three metabolites of doxorubicin, indicating that cells actively metabolize the drug during treatment. Steady state fluorescence microscopy cannot distinguish the drug from its metabolites as they have the same emission spectra. This report summarizes the first study to use MALDI-IMS to analyze drug penetration of a liposomal drug carrier as well as its metabolites.
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Antibióticos Antineoplásicos/farmacología , Técnicas de Cultivo de Célula , Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos , Antibióticos Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Doxorrubicina/química , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Humanos , Liposomas/química , Microscopía Fluorescente , Estructura Molecular , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Células Tumorales CultivadasRESUMEN
Cell cultures are widely used model systems. Some immortalized cell lines can be grown in either two-dimensional (2D) adherent monolayers or in three-dimensional (3D) multicellular aggregates, or spheroids. Here, the quantitative proteome and phosphoproteome of colon carcinoma HT29 cells cultures in 2D monolayers and 3D spheroids were compared with a stable isotope labeling of amino acids (SILAC) labeling strategy. Two biological replicates from each sample were examined, and notable differences in both the proteome and the phosphoproteome were determined by nanoliquid chromatography tandem mass spectrometry (LC-MS/MS) to assess how growth configuration affects molecular expression. A total of 5867 protein groups, including 2523 phosphoprotein groups and 8733 phosphopeptides were identified in the samples. The Gene Ontology analysis revealed enriched GO terms in the 3D samples for RNA binding, nucleic acid binding, enzyme binding, cytoskeletal protein binding, and histone binding for their molecular functions (MF) and in the process of cell cycle, cytoskeleton organization, and DNA metabolic process for the biological process (BP). The KEGG pathway analysis indicated that 3D cultures are enriched for oxidative phosphorylation pathways, metabolic pathways, peroxisome pathways, and biosynthesis of amino acids. In contrast, analysis of the phosphoproteomes indicated that 3D cultures have decreased phosphorylation correlating with slower growth rates and lower cell-to-extracellular matrix interactions. In sum, these results provide quantitative assessments of the effects on the proteome and phosphoproteome of culturing cells in 2D versus 3D cell culture configurations.
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Neoplasias del Colon/patología , Modelos Biológicos , Fosfoproteínas/análisis , Proteómica/métodos , Esferoides Celulares/química , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Humanos , Marcaje Isotópico , Fosfoproteínas/fisiología , Fosforilación , Proteómica/instrumentación , Esferoides Celulares/patología , Células Tumorales CultivadasRESUMEN
The transition between dormant and active Mycobacterium tuberculosis infection requires reorganization of its lipid metabolism and activation of a battery of serine hydrolase enzymes. Among these serine hydrolases, Rv0045c is a mycobacterial-specific serine hydrolase with limited sequence homology outside mycobacteria but structural homology to divergent bacterial hydrolase families. Herein, we determined the global substrate specificity of Rv0045c against a library of fluorogenic hydrolase substrates, constructed a combined experimental and computational model for its binding pocket, and performed comprehensive substitutional analysis to develop a structural map of its binding pocket. Rv0045c showed strong substrate selectivity toward short, straight chain alkyl esters with the highest activity toward four atom chains. This strong substrate preference was maintained through the combined action of residues in a flexible loop connecting the cap and α/ß hydrolase domains and in residues close to the catalytic triad. Two residues bracketing the substrate-binding pocket (Gly90 and His187) were essential to maintaining the narrow substrate selectivity of Rv0045c toward various acyl ester substituents, as independent conversion of these residues significantly increased its catalytic activity and broadened its substrate specificity. Focused saturation mutagenesis of position 187 implicated this residue, as the differentiation point between the substrate specificity of Rv0045c and the structurally homologous ybfF hydrolase family. Insertion of the analogous tyrosine residue from ybfF hydrolases into Rv0045c increased the catalytic activity of Rv0045 by over 20-fold toward diverse ester substrates. The unique binding pocket structure and selectivity of Rv0045c provide molecular indications of its biological role and evidence for expanded substrate diversity in serine hydrolases from M. tuberculosis.
Asunto(s)
Hidrolasas/metabolismo , Mycobacterium tuberculosis/enzimología , Dominio Catalítico , Hidrolasas/química , Cinética , Modelos Moleculares , Serina , Especificidad por SustratoRESUMEN
Epithelial ovarian cancer (EOC) is the most common form of ovarian cancer. The poor prognosis generally associated with this disease has led to the search for improved therapies such as ferroptosis-inducing agents. Ferroptosis is a form of regulated cell death that is dependent on iron and is characterized by lipid peroxidation. Precise mapping of lipids and iron within tumors exposed to ferroptosis-inducing agents may provide insight into processes of ferroptosis in vivo and ultimately assist in the optimal deployment of ferroptosis inducers in cancer therapy. In this work, we present a method for combining matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) with secondary ion mass spectrometry (SIMS) to analyze changes in spatial lipidomics and metal composition, respectively, in ovarian tumors following exposure to a ferroptosis inducer. Tumors were obtained by injecting human ovarian cancer tumor-initiating cells into mice, followed by treatment with the ferroptosis inducer erastin. SIMS imaging detected iron accumulation in the tumor tissue, and sequential MALDI-MS imaging of the same tissue section displayed two chemically distinct regions of lipids. One region was associated with the iron-rich area detected with SIMS, and the other region encompassed the remainder of the tissue section. Bulk lipidomics confirmed the lipid assignments putatively assigned from the MALDI-MS data. Overall, we demonstrate the ability of multimodal MSI to identify the spatial locations of iron and lipids in the same tissue section and associate these regions with clinical pathology.
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Ferroptosis , Neoplasias Ováricas , Humanos , Animales , Ratones , Femenino , Lípidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Neoplasias Ováricas/tratamiento farmacológico , HierroRESUMEN
Human disease states are biomolecularly multifaceted and can span across phenotypic states, therefore it is important to understand diseases on all levels, across cell types, and within and across microanatomical tissue compartments. To obtain an accurate and representative view of the molecular landscape within human lungs, this fragile tissue must be inflated and embedded to maintain spatial fidelity of the location of molecules and minimize molecular degradation for molecular imaging experiments. Here, we evaluated agarose inflation and carboxymethyl cellulose embedding media and determined effective tissue preparation protocols for performing bulk and spatial mass spectrometry-based omics measurements. Mass spectrometry imaging methods were optimized to boost the number of annotatable molecules in agarose inflated lung samples. This optimized protocol permitted the observation of unique lipid distributions within several airway regions in the lung tissue block. Laser capture microdissection of these airway regions followed by high-resolution proteomic analysis allowed us to begin linking the lipidome with the proteome in a spatially resolved manner, where we observed proteins with high abundance specifically localized to the airway regions. We also compared our mass spectrometry results to lung tissue samples preserved using two other inflation/embedding media, but we identified several pitfalls with the sample preparation steps using this preservation method. Overall, we demonstrated the versatility of the inflation method, and we can start to reveal how the metabolome, lipidome, and proteome are connected spatially in human lungs and across disease states through a variety of different experiments.
RESUMEN
Biological systems are composed of heterogeneous populations of cells that intercommunicate to form a functional living tissue. Biological function varies greatly across populations of cells, as each single cell has a unique transcriptome, proteome, and metabolome that translates to functional differences within single species and across kingdoms. Over the past decade, substantial advancements in our ability to characterize omic profiles on a single cell level have occurred, including in multiple spectroscopic and mass spectrometry (MS)-based techniques. Of these technologies, spatially resolved mass spectrometry approaches, including mass spectrometry imaging (MSI), have shown the most progress for single cell proteomics and metabolomics. For example, reporter-based methods using heavy metal tags have allowed for targeted MS investigation of the proteome at the subcellular level, and development of technologies such as laser ablation electrospray ionization mass spectrometry (LAESI-MS) now mean that dynamic metabolomics can be performed in situ. In this Perspective, we showcase advancements in single cell spatial metabolomics and proteomics over the past decade and highlight important aspects related to high-throughput screening, data analysis, and more which are vital to the success of achieving proteomic and metabolomic profiling at the single cell scale. Finally, using this broad literature summary, we provide a perspective on how the next decade may unfold in the area of single cell MS-based proteomics and metabolomics.
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Espectrometría de Masas/métodos , Metabolómica/métodos , Proteómica/métodos , Análisis de la Célula Individual/métodos , Animales , Predicción , Ensayos Analíticos de Alto Rendimiento/métodos , Rayos Láser , Mamíferos , Metabolómica/tendencias , Proteómica/tendencias , Espectrometría de Masa por Ionización de Electrospray/métodosRESUMEN
Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial (â¼67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels.
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Bacillus subtilis/química , Espectrometría de Masas/métodos , Imagen Molecular/métodos , Bacillus subtilis/metabolismo , Biopelículas , Rayos Láser , Metabolismo de los Lípidos , Lípidos/química , Espectrometría de Masas/instrumentación , Imagen Molecular/instrumentaciónRESUMEN
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, encoding for a chloride ion channel. Membrane expression of CFTR is negatively regulated by CFTR-associated ligand (CAL). We previously showed that inhibition of the CFTR/CAL interaction with a cell-permeable peptide improves the function of rescued F508del-CFTR. In this study, optimization of the peptidyl inhibitor yielded PGD97, which exhibits a KD value of 6 nM for the CAL PDZ domain, ≥ 130-fold selectivity over closely related PDZ domains, and a serum t1/2 of >24 h. In patient-derived F508del homozygous cells, PGD97 (100 nM) increased short-circuit currents by â¼3-fold and further potentiated the therapeutic effects of small-molecule correctors (e.g., VX-661) by â¼2-fold (with an EC50 of â¼10 nM). Our results suggest that PGD97 may be used as a novel treatment for CF, either as a single agent or in combination with small-molecule correctors/potentiators.
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Regulador de Conductancia de Transmembrana de Fibrosis Quística/antagonistas & inhibidores , Péptidos Cíclicos/química , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fibrosis Quística/tratamiento farmacológico , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Estabilidad de Medicamentos , Humanos , Cinética , Ligandos , Simulación del Acoplamiento Molecular , Mutación , Dominios PDZ , Péptidos Cíclicos/metabolismo , Péptidos Cíclicos/farmacología , Permeabilidad/efectos de los fármacos , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno/química , Intercambiadores de Sodio-Hidrógeno/metabolismoRESUMEN
Lipids often are labile, unstable, and tend to degrade overtime, so it is of the upmost importance to study these molecules in their most native state. We sought to understand the optimal storage conditions for spatial lipidomic analysis of human kidney tissue sections. Specifically, we evaluated human kidney tissue sections on several different days throughout the span of a week using our established protocol for elucidating lipids using high mass resolution matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). We studied kidney tissue sections stored under five different conditions: open stored at -80 °C, vacuumed sealed and stored at -80 °C, with matrix preapplied before storage at -80 °C, under a nitrogen atmosphere and stored at -80 °C, and at room temperature in a desiccator. Results were compared to data obtained from kidney tissue sections that were prepared and analyzed immediately after cryosectioning. Data was processed using METASPACE. After a week of storage, the sections stored at room temperature showed the largest amount of lipid degradation, while sections stored under nitrogen and at -80 °C retained the greatest number of overlapping annotations in relation to freshly cut tissue. Overall, we found that molecular degradation of the tissue sections was unavoidable over time, regardless of storage conditions, but storing tissue sections in an inert gas at low temperatures can curtail molecular degradation within tissue sections.
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Riñón/química , Lipidómica/métodos , Lípidos/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Preservación de Órganos/métodosRESUMEN
Acute respiratory distress syndrome (ARDS) is an inflammatory lung disease with a high morbidity and mortality rate, for which no pharmacologic treatment is currently available. Our previous studies discovered that a pivotal step in the disease process is the activation of the nuclear factor of activated T cells (NFAT) c3 in lung macrophages, suggesting that inhibitors against the upstream protein phosphatase calcineurin should be effective for prevention/treatment of ARDS. Herein, we report the development of a highly potent, cell-permeable, and metabolically stable peptidyl inhibitor, CNI103, which selectively blocks the interaction between calcineurin and NFATc3, through computational and medicinal chemistry. CNI103 specifically inhibited calcineurin signaling in vitro and in vivo and exhibited a favorable pharmacokinetic profile, broad tissue distribution following different routes of administration, and minimal toxicity. Our data indicate that CNI103 is a promising novel treatment for ARDS and other inflammatory diseases.