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While classical molecular biology assays can provide a measure of cellular response to chemical challenges, they rely on a single biological phenomenon to infer a broader measure of cellular metabolic response. These methods do not always afford the necessary sensitivity to answer questions of sub-cytotoxic effects, nor do they work for all cell types. Likewise, boutique assays such as cardiomyocyte beat rate may indirectly measure cellular metabolic response, but they too, are limited to measuring a specific biological phenomenon and are often limited to a single cell type. For these reasons, toxicological researchers need new approaches to determine metabolic changes across various doses in differing cell types, especially within the low-dose regime. The data collected herein demonstrate that LC-MS/MS-based untargeted metabolomics with a feature-agnostic view of the data, combined with a suite of statistical methods including an adapted environmental threshold analysis, provides a versatile, robust, and holistic approach to directly monitoring the overall cellular metabolomic response to pesticides. When employing this method in investigating two different cell types, human cardiomyocytes and neurons, this approach revealed separate sub-cytotoxic metabolomic responses at doses of 0.1 µM and 1 µM of chlorpyrifos and carbaryl. These findings suggest that this agnostic approach to untargeted metabolomics can provide a new tool for determining effective dose by metabolomics (EDm) of chemical challenges, such as pesticides, in a direct measurement of metabolomic response that is not cell type-specific or observable using traditional assays.
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In this work, we demonstrate rapid, high spatial, and high spectral resolution imaging of intact proteins by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on a hybrid quadrupole-reflectron time-of-flight (qTOF) mass spectrometer equipped with trapped ion mobility spectrometry (TIMS). Historically, untargeted MALDI IMS of proteins has been performed on TOF mass spectrometers. While advances in TOF instrumentation have enabled rapid, high spatial resolution IMS of intact proteins, TOF mass spectrometers generate relatively low-resolution mass spectra with limited mass accuracy. Conversely, the implementation of MALDI sources on high-resolving power Fourier transform (FT) mass spectrometers has allowed IMS experiments to be conducted with high spectral resolution with the caveat of increasingly long data acquisition times. As illustrated here, qTOF mass spectrometers enable protein imaging with the combined advantages of TOF and FT mass spectrometers. Protein isotope distributions were resolved for both a protein standard mixture and proteins detected from a whole-body mouse pup tissue section. Rapid (â¼10 pixels/s) 10 µm lateral spatial resolution IMS was performed on a rat brain tissue section while maintaining isotopic spectral resolution. Lastly, proof-of-concept MALDI-TIMS data was acquired from a protein mixture to demonstrate the ability to differentiate charge states by ion mobility. These experiments highlight the advantages of qTOF and timsTOF platforms for resolving and interpreting complex protein spectra generated from tissue by IMS.
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Diagnóstico por Imagem , Proteínas , Ratos , Camundongos , Animais , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Análise de FourierRESUMO
Introduction: Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers. Methods: S. aureus biofilms were grown statically, embedded in a mixture of carboxymethylcellulose/gelatin, and prepared for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass analysis. Results: Implementation of TIMS led to a â¼ threefold increase in the number of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) - CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p < 0.03). No horizontal layers were observed within biofilms grown anaerobically, and lipids were distributed homogenously. Conclusions: High spatial resolution analysis of S. aureus biofilm cross-sections by MALDI TIMS IMS revealed stark lipidomic heterogeneity between horizontal S. aureus biofilm layers demonstrating that each layer was molecularly distinct. Finally, this workflow uncovered an absence of layers in biofilms grown under anaerobic conditions, possibly indicating that oxygen contributes to the observed heterogeneity under aerobic conditions. Future applications of this workflow to study spatially localized molecular responses to antimicrobials could provide new therapeutic strategies.
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The human kidney is composed of many cell types that vary in their abundance and distribution from normal to diseased organ. As these cell types perform unique and essential functions, it is important to confidently label each within a single tissue to accurately assess tissue architecture and microenvironments. Towards this goal, we demonstrate the use of co-detection by indexing (CODEX) multiplexed immunofluorescence for visualizing 23 antigens within the human kidney. Using CODEX, many of the major cell types and substructures, such as collecting ducts, glomeruli, and thick ascending limb, were visualized within a single tissue section. Of these antibodies, 19 were conjugated in-house, demonstrating the flexibility and utility of this approach for studying the human kidney using custom and commercially available antibodies. We performed a pilot study that compared both fresh frozen and formalin-fixed paraffin-embedded healthy non-neoplastic and diabetic nephropathy kidney tissues. The largest cellular differences between the two groups was observed in cells labeled with aquaporin 1, cytokeratin 7, and α-smooth muscle actin. Thus, our data show the power of CODEX multiplexed immunofluorescence for surveying the cellular diversity of the human kidney and the potential for applications within pathology, histology, and building anatomical atlases.
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Anticorpos , Rim , Imunofluorescência , Humanos , Rim/patologia , Projetos Piloto , Coloração e RotulagemRESUMO
Imaging mass spectrometry (IMS) technologies are capable of mapping a wide array of biomolecules in diverse cellular and tissue environments. IMS has emerged as an essential tool for providing spatially targeted molecular information due to its high sensitivity, wide molecular coverage, and chemical specificity. One of the major challenges for mapping the complex cellular milieu is the presence of many isomers and isobars in these samples. This challenge is traditionally addressed using orthogonal liquid chromatography (LC)-based analysis, though, common approaches such as chromatography and electrophoresis are not able to be performed at timescales that are compatible with most imaging applications. Ion mobility offers rapid, gas-phase separations that are readily integrated with IMS workflows in order to provide additional data dimensionality that can improve signal-to-noise, dynamic range, and specificity. Here, we highlight recent examples of ion mobility coupled to IMS and highlight their importance to the field.
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Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Massas/métodos , Imagem Molecular/métodos , Animais , Humanos , Rim/química , Rim/diagnóstico por imagem , Lipídeos/análise , Fígado/química , Fígado/diagnóstico por imagem , Camundongos , Proteínas/análise , Ratos , Imagem Corporal TotalRESUMO
Acinetobacter baumannii is a leading cause of ventilator-associated pneumonia and a critical threat due to multidrug resistance. The A. baumannii outer membrane is an asymmetric lipid bilayer composed of inner leaflet glycerophospholipids and outer leaflet lipooligosaccharides. Deleting mlaF of the maintenance of lipid asymmetry (Mla) system causes A. baumannii to become more susceptible to pulmonary surfactants and antibiotics and decreases bacterial survival in the lungs of mice. Spontaneous suppressor mutants isolated from infected mice contain an ISAba11 insertion upstream of the ispB initiation codon, an essential isoprenoid biosynthesis gene. The insertion restores antimicrobial resistance and virulence to ΔmlaF. The suppressor strain increases lipooligosaccharides, suggesting that the mechanism involves balancing the glycerophospholipids/lipooligosaccharides ratio on the bacterial surface. An identical insertion exists in an extensively drug-resistant A. baumannii isolate, demonstrating its clinical relevance. These data show that the stresses bacteria encounter during infection select for genomic rearrangements that increase resistance to antimicrobials.
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Acinetobacter baumannii/patogenicidade , Antibacterianos/metabolismo , Lipopolissacarídeos/metabolismo , Terpenos/metabolismo , HumanosRESUMO
Lipids are a structurally diverse class of molecules with important biological functions including cellular signaling and energy storage. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) allows for direct mapping of biomolecules in tissues. Fully characterizing the structural diversity of lipids remains a challenge due to the presence of isobaric and isomeric species, which greatly complicates data interpretation when only m/z information is available. Integrating ion mobility separations aids in deconvoluting these complex mixtures and addressing the challenges of lipid IMS. Here, we demonstrate that a MALDI quadrupole time-of-flight (Q-TOF) mass spectrometer with trapped ion mobility spectrometry (TIMS) enables a >250% increase in the peak capacity during IMS experiments. MALDI TIMS-MS separation of lipid isomer standards, including sn backbone isomers, acyl chain isomers, and double-bond position and stereoisomers, is demonstrated. As a proof of concept, in situ separation and imaging of lipid isomers with distinct spatial distributions were performed using tissue sections from a whole-body mouse pup.
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Lipidômica , Lipídeos/análise , Animais , Espectrometria de Mobilidade Iônica , Camundongos , Camundongos Endogâmicos C57BL , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The human retina provides vision at light levels ranging from starlight to sunlight. Its supporting tissues regulate plasma-delivered lipophilic essentials for vision, including retinoids. The macula is an anatomic specialization for high-acuity and color vision that is also vulnerable to prevalent blinding diseases. The retina's exquisite architecture comprises numerous cell types that are aligned horizontally, yielding structurally distinct cell, synaptic, and vascular layers that are visible in histology and in diagnostic clinical imaging. MALDI imaging mass spectrometry (IMS) is now capable of uniting low micrometer spatial resolution with high levels of chemical specificity. In this study, a multimodal imaging approach fortified with accurate multi-image registration was used to localize lipids in human retina tissue at laminar, cellular, and subcellular levels. Multimodal imaging results indicate differences in distributions and abundances of lipid species across and within single cell types. Of note are distinct localizations of signals within specific layers of the macula. For example, phosphatidylethanolamine and phosphatidylinositol lipids were localized to central RPE cells, whereas specific plasmalogen lipids were localized to cells of the perifoveal RPE and Henle fiber layer. Subcellular compartments of photoreceptors were distinguished by PE(20:0_22:5) in the outer nuclear layer, PE(18:0_22:6) in outer and inner segments, and cardiolipin CL(70:5) in the mitochondria-rich inner segments. Several lipids, differing by a single double bond, have markedly different distributions between the central fovea and the ganglion cell and inner nuclear layers. A lipid atlas, initiated in this study, can serve as a reference database for future examination of diseased tissues.
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Lipídeos/análise , Retina/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Idoso de 80 Anos ou mais , Humanos , Microscopia/métodos , Imagem Óptica/métodosRESUMO
BACKGROUND AND AIMS: Studies have linked several metabolites to the risk of coronary heart disease (CHD) among Western populations, but prospective studies among Asian populations on the metabolite-CHD association remain limited. METHODS AND RESULTS: We evaluated the association of urinary metabolites with CHD risk among Chinese adults in a nested case-control study of 275 incident cases and 275 matched controls (127 pairs of men and 148 pairs of women). Fifty metabolites were measured by a predefined metabolomics panel and adjusted using urinary creatinine. Conditional logistic regression was used to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs). After adjusting for traditional CHD risk factors, urinary tryptophan showed a positive association with incident CHD: OR (95% CI) for the highest vs. lowest quartiles was 2.02 (1.15-3.56) among all study participants (p-trend = 0.02). The tryptophan-CHD association was more evident among individuals with dyslipidemia than among those without the condition (OR [95% CI] for the highest vs. lowest quartiles = 3.90 [1.86-8.19] and 0.74 [0.26-2.06], respectively; p-interaction<0.01). Other metabolites did not show significant associations with CHD risk among all study participants. However, a positive association of methionine with CHD risk was observed only among women (OR [95% CI] for the highest vs. lowest quartiles = 2.77 [1.17-6.58]; p-interaction = 0.03), and an inverse association of inosine with CHD risk was observed only among men (OR [95% CI] for the highest vs. lowest quartiles = 0.29 [0.11-0.81]; p-interaction = 0.04). CONCLUSION: Elevated urinary tryptophan may be related to CHD risk among Chinese adults, especially for those with dyslipidemia.
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Doença das Coronárias/urina , Triptofano/urina , Saúde da População Urbana , Adulto , Idoso , Biomarcadores/urina , Estudos de Casos e Controles , China/epidemiologia , Doença das Coronárias/diagnóstico , Doença das Coronárias/epidemiologia , Feminino , Humanos , Incidência , Masculino , Metabolômica , Pessoa de Meia-Idade , Projetos Piloto , Prevalência , Estudos Prospectivos , Medição de Risco , Fatores de Risco , Regulação para CimaRESUMO
Imaging mass spectrometry (IMS) enables the spatially targeted molecular assessment of biological tissues at cellular resolutions. New developments and technologies are essential for uncovering the molecular drivers of native physiological function and disease. Instrumentation must maximize spatial resolution, throughput, sensitivity, and specificity, because tissue imaging experiments consist of thousands to millions of pixels. Here, we report the development and application of a matrix-assisted laser desorption/ionization (MALDI) trapped ion-mobility spectrometry (TIMS) imaging platform. This prototype MALDI timsTOF instrument is capable of 10 µm spatial resolutions and 20 pixels/s throughput molecular imaging. The MALDI source utilizes a Bruker SmartBeam 3-D laser system that can generate a square burn pattern of <10 × 10 µm at the sample surface. General image performance was assessed using murine kidney and brain tissues and demonstrate that high-spatial-resolution imaging data can be generated rapidly with mass measurement errors <5 ppm and â¼40â¯000 resolving power. Initial TIMS-based imaging experiments were performed on whole-body mouse pup tissue demonstrating the separation of closely isobaric [PC(32:0) + Na]+ and [PC(34:3) + H]+ (3 mDa mass difference) in the gas phase. We have shown that the MALDI timsTOF platform can maintain reasonable data acquisition rates (>2 pixels/s) while providing the specificity necessary to differentiate components in complex mixtures of lipid adducts. The combination of high-spatial-resolution and throughput imaging capabilities with high-performance TIMS separations provides a uniquely tunable platform to address many challenges associated with advanced molecular imaging applications.
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Encéfalo/diagnóstico por imagem , Rim/diagnóstico por imagem , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Lipídeos/análise , Camundongos Endogâmicos C57BL , Estudo de Prova de Conceito , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/instrumentaçãoRESUMO
The reverse transsulfuration pathway is the major route for the metabolism of sulfur-containing amino acids. The role of this metabolic pathway in macrophage response and function is unknown. We show that the enzyme cystathionine γ-lyase (CTH) is induced in macrophages infected with pathogenic bacteria through signaling involving phosphatidylinositol 3-kinase (PI3K)/MTOR and the transcription factor SP1. This results in the synthesis of cystathionine, which facilitates the survival of pathogens within myeloid cells. Our data demonstrate that the expression of CTH leads to defective macrophage activation by (i) dysregulation of polyamine metabolism by depletion of S-adenosylmethionine, resulting in immunosuppressive putrescine accumulation and inhibition of spermidine and spermine synthesis, and (ii) increased histone H3K9, H3K27, and H3K36 di/trimethylation, which is associated with gene expression silencing. Thus, CTH is a pivotal enzyme of the innate immune response that disrupts host defense. The induction of the reverse transsulfuration pathway by bacterial pathogens can be considered an unrecognized mechanism for immune escape.IMPORTANCE Macrophages are professional immune cells that ingest and kill microbes. In this study, we show that different pathogenic bacteria induce the expression of cystathionine γ-lyase (CTH) in macrophages. This enzyme is involved in a metabolic pathway called the reverse transsulfuration pathway, which leads to the production of numerous metabolites, including cystathionine. Phagocytized bacteria use cystathionine to better survive in macrophages. In addition, the induction of CTH results in dysregulation of the metabolism of polyamines, which in turn dampens the proinflammatory response of macrophages. In conclusion, pathogenic bacteria can evade the host immune response by inducing CTH in macrophages.
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Bactérias/imunologia , Bactérias/metabolismo , Imunidade Inata , Macrófagos/metabolismo , Redes e Vias Metabólicas/fisiologia , Enxofre/metabolismo , Animais , Bactérias/patogenicidade , Inativação Gênica , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , Histonas/metabolismo , Humanos , Evasão da Resposta Imune , Imunoglobulinas , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico Sintase Tipo II/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Poliaminas/metabolismo , Células RAW 264.7 , Espermidina/metabolismo , Espermina/metabolismo , Fatores de TranscriçãoRESUMO
Background Trimethylamine-N-oxide ( TMAO ), a diet-derived, gut microbial-host cometabolite, has been associated with adverse cardiovascular outcomes in patient populations; however, evidence is lacking from prospective studies conducted in general populations and non-Western populations. Methods and Results We evaluated urinary levels of TMAO and its precursor metabolites (ie, choline, betaine, and carnitine) in relation to risk of coronary heart disease ( CHD ) among Chinese adults in a nested case-control study, including 275 participants with incident CHD and 275 individually matched controls. We found that urinary TMAO , but not its precursors, was associated with risk of CHD . The odds ratio for the highest versus lowest quartiles of TMAO was 1.91 (95% CI, 1.08-3.35; Ptrend=0.008) after adjusting for CHD risk factors including obesity, diet, lifestyle, and metabolic diseases and 1.75 (95% CI, 0.96-3.18; Ptrend=0.03) after further adjusting for potential confounders or mediators including central obesity, dyslipidemia, inflammation, and intake of seafood and deep-fried meat or fish, which were associated with TMAO level in this study. The odds ratio per standard deviation increase in log- TMAO was 1.30 (95% CI, 1.03-1.63) in the fully adjusted model. A history of diabetes mellitus modified the TMAO - CHD association. A high TMAO level (greater than or equal to versus lower than the median) was associated with odds ratios of 6.21 (95% CI, 1.64-23.6) and 1.56 (95% CI, 1.00-2.43), respectively, among diabetic and nondiabetic participants ( Pinteraction=0.02). Diabetes mellitus status also modified the associations of choline, betaine, and carnitine with risk of CHD ; significant positive associations were found among diabetic participants, but null associations were noted among total and nondiabetic participants. Conclusions Our study suggests that TMAO may accelerate the development of CHD , highlighting the importance of diet-gut microbiota-host interplay in cardiometabolic health.