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1.
Anal Chem ; 2024 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-39460701

RESUMO

We report the first demonstration of a microfluidics-based approach to measure lipids in single living cells using widely available liquid chromatography mass spectrometry (LC-MS) instrumentation. The method enables the rapid sorting of live cells into liquid chambers formed on standard Petri dishes and their subsequent dispensing into vials for analysis using LC-MS. This approach facilitates automated sampling, data acquisition, and analysis and carries the additional advantage of chromatographic separation, aimed at reducing matrix effects present in shotgun lipidomics approaches. We demonstrate that our method detects comparable numbers of features at around 200 lipids in populations of single cells versus established live single-cell capillary sampling methods and with greater throughput, albeit with the loss of spatial resolution. We also show the importance of optimization steps in addressing challenges from lipid contamination, especially in blanks, and demonstrate a 75% increase in the number of lipids identified. This work opens up a novel, accessible, and high-throughput way to obtain single-cell lipid profiles and also serves as an important validation of single-cell lipidomics through the use of different sampling methods.

2.
Anal Chem ; 96(18): 6922-6929, 2024 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-38653330

RESUMO

We report the development and validation of an untargeted single-cell lipidomics method based on microflow chromatography coupled to a data-dependent mass spectrometry method for fragmentation-based identification of lipids. Given the absence of single-cell lipid standards, we show how the methodology should be optimized and validated using a dilute cell extract. The methodology is applied to dilute pancreatic cancer and macrophage cell extracts and standards to demonstrate the sensitivity requirements for confident assignment of lipids and classification of the cell type at the single-cell level. The method is then coupled to a system that can provide automated sampling of live, single cells into capillaries under microscope observation. This workflow retains the spatial information and morphology of cells during sampling and highlights the heterogeneity in lipid profiles observed at the single-cell level. The workflow is applied to show changes in single-cell lipid profiles as a response to oxidative stress, coinciding with expanded lipid droplets. This demonstrates that the workflow is sufficiently sensitive to observing changes in lipid profiles in response to a biological stimulus. Understanding how lipids vary in single cells will inform future research into a multitude of biological processes as lipids play important roles in structural, biophysical, energy storage, and signaling functions.


Assuntos
Lipidômica , Lipídeos , Análise de Célula Única , Lipidômica/métodos , Humanos , Lipídeos/análise , Lipídeos/química , Animais , Cromatografia Líquida , Camundongos , Linhagem Celular Tumoral , Espectrometria de Massas , Macrófagos/metabolismo , Macrófagos/citologia
3.
Chem Res Toxicol ; 34(12): 2485-2499, 2021 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-34797640

RESUMO

Drug-induced liver injury is a leading cause of compound attrition during both preclinical and clinical drug development, and early strategies are in place to tackle this recurring problem. Human-relevant in vitro models that are more predictive of hepatotoxicity hazard identification, and that could be employed earlier in the drug discovery process, would improve the quality of drug candidate selection and help reduce attrition. We present an evaluation of four human hepatocyte in vitro models of increasing culture complexity (i.e., two-dimensional (2D) HepG2 monolayers, hepatocyte sandwich cultures, three-dimensional (3D) hepatocyte spheroids, and precision-cut liver slices), using the same tool compounds, viability end points, and culture time points. Having established the improved prediction potential of the 3D hepatocyte spheroid model, we describe implementing this model into an industrial screening setting, where the challenge was matching the complexity of the culture system with the scale and throughput required. Following further qualification and miniaturization into a 384-well, high-throughput screening format, data was generated on 199 compounds. This clearly demonstrated the ability to capture a greater number of severe hepatotoxins versus the current routine 2D HepG2 monolayer assay while continuing to flag no false-positive compounds. The industrialization and miniaturization of the 3D hepatocyte spheroid complex in vitro model demonstrates a significant step toward reducing drug attrition and improving the quality and safety of drugs, while retaining the flexibility for future improvements, and has replaced the routine use of the 2D HepG2 monolayer assay at GlaxoSmithKline.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/patologia , Hepatócitos/efeitos dos fármacos , Modelos Biológicos , Preparações Farmacêuticas/química , Esferoides Celulares/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células Hep G2 , Hepatócitos/patologia , Humanos , Masculino , Ratos , Ratos Wistar , Esferoides Celulares/patologia
4.
Child Youth Serv Rev ; 1262021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34483418

RESUMO

Although there is a significant link between maternal substance use and child maltreatment risk, extant literature has not investigated this link specifically among the growing number of parents abusing opioids. Underreporting of opioid use within child welfare presents further challenges in elucidating relations between maternal opioid use and child maltreatment. The purpose of the current study is to examine the link between maternal opioid use in women in substance use treatment and self-reported rates of child maltreatment and child welfare involvement of their children. We examined maternal substance use, severity of substance use, severity and type of child maltreatment of their children, and child welfare involvement across mothers who misuse opioids and misuse other substances using self-report surveys with 89 mothers. Results suggest similarities and differences among mothers who use opioids and other substances. Mothers who use opioids endorsed more significant and prolonged involvement with child welfare than mothers who use other substances. Participants did not endorse significant differences between rates of child maltreatment, and treatment engagement across groups. Given increased awareness of significant risks associated with opioid abuse, including greater risk for child maltreatment, a better understanding of its intersection with child welfare is necessary.

5.
Nat Methods ; 14(12): 1175-1183, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29131162

RESUMO

We report the development of a 3D OrbiSIMS instrument for label-free biomedical imaging. It combines the high spatial resolution of secondary ion mass spectrometry (SIMS; under 200 nm for inorganic species and under 2 µm for biomolecules) with the high mass-resolving power of an Orbitrap (>240,000 at m/z 200). This allows exogenous and endogenous metabolites to be visualized in 3D with subcellular resolution. We imaged the distribution of neurotransmitters-gamma-aminobutyric acid, dopamine and serotonin-with high spectroscopic confidence in the mouse hippocampus. We also putatively annotated and mapped the subcellular localization of 29 sulfoglycosphingolipids and 45 glycerophospholipids, and we confirmed lipid identities with tandem mass spectrometry. We demonstrated single-cell metabolomic profiling using rat alveolar macrophage cells incubated with different concentrations of the drug amiodarone, and we observed that the upregulation of phospholipid species and cholesterol is correlated with the accumulation of amiodarone.


Assuntos
Dopamina/análise , Hipocampo/metabolismo , Imagem Molecular/métodos , Serotonina/análise , Frações Subcelulares/metabolismo , Ácido gama-Aminobutírico/análise , Amiodarona/metabolismo , Animais , Células Cultivadas , Desenho de Equipamento , Feminino , Glicerofosfolipídeos/análise , Imageamento Tridimensional , Macrófagos Alveolares/metabolismo , Metabolômica/instrumentação , Metabolômica/métodos , Camundongos , Imagem Molecular/instrumentação , Sensibilidade e Especificidade , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Sulfoglicoesfingolipídeos/análise , Espectrometria de Massas em Tandem
6.
J Pharmacol Exp Ther ; 369(3): 443-453, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30940692

RESUMO

This study describes the pharmacokinetic (PK) and pharmaco-dynamic (PD) profile of N-(5-(4-(5-(((2R,6S)-2,6-dimethylmorpholino)methyl)oxazol-2-yl)-1H-indazol-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide (GSK2292767A), a novel low-solubility inhaled phosphoinositide 3-kinase delta (PI3Kδ) inhibitor developed as an alternative to 2-(6-(1H-indol-4-yl)-1H-indazol-4-yl)-5-((4-isopropylpiperazin-1-yl)methyl)oxazole (nemiralisib), which is a highly soluble inhaled inhibitor of PI3Kδ with a lung profile consistent with once-daily dosing. GSK2292767A has a similar in vitro cellular profile to nemiralisib and reduces eosinophilia in a murine PD model by 63% (n = 5, P < 0.05). To explore whether a low-soluble compound results in effective PI3Kδ inhibition in humans, a first time in human study was conducted with GSK2292767A in healthy volunteers who smoke. GSK2292767A was generally well tolerated, with headache being the most common reported adverse event. PD changes in induced sputum were measured in combination with drug concentrations in plasma from single (0.05-2 mg, n = 37), and 14-day repeat (2 mg, n = 12) doses of GSK2292767A. Trough bronchoalveolar lavage (BAL) for PK was taken after 14 days of repeat dosing. GSK2292767A displayed a linear increase in plasma exposure with dose, with marginal accumulation after 14 days. Induced sputum showed a 27% (90% confidence interval 15%, 37%) reduction in phosphatidylinositol-trisphosphate (the product of phosphoinositide 3-kinase activation) 3 hours after a single dose. Reduction was not maintained 24 hours after single or repeat dosing. BAL analysis confirmed the presence of GSK2292767A in lung at 24 hours, consistent with the preclinical lung retention profile. Despite good lung retention, target engagement was only present at 3 hours. This exposure-response disconnect is an important observation for future inhaled drug design strategies considering low solubility to drive lung retention.


Assuntos
Indazóis/farmacologia , Indazóis/farmacocinética , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Inibidores de Fosfoinositídeo-3 Quinase/farmacocinética , Sulfonamidas/farmacologia , Sulfonamidas/farmacocinética , Pesquisa Translacional Biomédica , Administração por Inalação , Adulto , Animais , Lavagem Broncoalveolar , Eosinofilia/tratamento farmacológico , Feminino , Humanos , Indazóis/administração & dosagem , Indazóis/efeitos adversos , Pulmão/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Permeabilidade , Inibidores de Fosfoinositídeo-3 Quinase/administração & dosagem , Inibidores de Fosfoinositídeo-3 Quinase/efeitos adversos , Segurança , Solubilidade , Escarro/efeitos dos fármacos , Escarro/metabolismo , Sulfonamidas/administração & dosagem , Sulfonamidas/efeitos adversos
7.
J Allergy Clin Immunol ; 140(5): 1299-1309, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28479159

RESUMO

BACKGROUND: Atopic eczema (AE) is characterized by skin barrier and immune dysfunction. Null mutations in filaggrin (FLG), a key epidermal barrier protein, strongly predispose to AE; however, the precise role of FLG deficiency in AE pathogenesis remains incompletely understood. OBJECTIVES: We sought to identify global proteomic changes downstream of FLG deficiency in human epidermal living skin-equivalent (LSE) models and validate findings in skin of patients with AE. METHODS: Differentially expressed proteins from paired control (nontargeting control short hairpin RNA [shNT]) and FLG knockdown (FLG knockdown short hairpin RNA [shFLG]) LSEs were identified by means of proteomic analysis (liquid chromatography-mass spectrometry) and Ingenuity Pathway Analysis. Expression of key targets was validated in independent LSE samples (quantitative RT-PCR and Western blotting) and in normal and AE skin biopsy specimens (immunofluorescence). RESULTS: Proteomic analysis identified 17 (P ≤ .05) differentially expressed proteins after FLG knockdown, including kallikrein-7 (KLK7; 2.2-fold), cyclophilin A (PPIA; 0.9-fold), and cofilin-1 (CFL1, 1.3-fold). Differential protein expression was confirmed in shNT/shFLG LSEs; however, only KLK7 was transcriptionally dysregulated. Molecular pathways overrepresented after FLG knockdown included inflammation, protease activity, cell structure, and stress. Furthermore, KLK7 (1.8-fold) and PPIA (0.65-fold) proteins were differentially expressed in lesional biopsy specimens from patients with AE relative to normal skin. CONCLUSIONS: For the first time, we show that loss of FLG in the absence of inflammation is sufficient to alter the expression level of proteins relevant to the pathogenesis of AE. These include proteins regulating inflammatory, proteolytic, and cytoskeletal functions. We identify PPIA as a novel protein with levels that are decreased in clinically active AE skin and show that the characteristic upregulation of KLK7 expression in patients with AE occurs downstream of FLG loss. Importantly, we highlight disconnect between the epidermal proteome and transcriptome, emphasizing the utility of global proteomic studies.


Assuntos
Cofilina 1/metabolismo , Ciclofilina A/metabolismo , Citoesqueleto/metabolismo , Dermatite Atópica/genética , Inflamação/genética , Calicreínas/metabolismo , Queratinócitos/metabolismo , Células Cultivadas , Cromatografia Líquida , Cofilina 1/genética , Ciclofilina A/genética , Dermatite Atópica/imunologia , Proteínas Filagrinas , Regulação da Expressão Gênica , Humanos , Inflamação/imunologia , Proteínas de Filamentos Intermediários/genética , Calicreínas/genética , Queratinócitos/patologia , Mutação com Perda de Função/genética , Espectrometria de Massas , Proteólise , Proteoma , RNA Interferente Pequeno/genética , Transcriptoma
8.
Anal Chem ; 89(22): 11944-11953, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29039651

RESUMO

ToF-SIMS is a label-free imaging method that has been shown to enable imaging of amiodarone in single rat macrophage (NR8383) cells. In this study, we show that the method extends to three other cell lines relevant to drug discovery: human embryonic kidney (HEK293), cervical cancer (HeLa), and liver cancer (HepG2). There is significant interest in the variation of drug uptake at the single cell level, and we use ToF-SIMS to show that there is great diversity between individual cells and when comparing each of the cell types. These single cell measurements are compared to quantitative measurements of cell-associated amiodarone for the population using LC/MS/MS and cell counting with flow cytometry. NR8383 and HepG2 cells uptake the greatest amount of amiodarone with an average of 2.38 and 2.60 pg per cell, respectively, and HeLa and Hek 293 have a significantly lower amount of amiodarone at 0.43 and 0.36 pg per cell, respectively. The amount of cell-associated drug for the ensemble population measurement (LC/MS/MS) is compared with the ToF-SIMS single cell data: a similar amount of drug was detected per cell for the NR8383, and HepG2 cells at a greater level than that for the HEK293 cells. However, the two techniques did not agree for the HeLa cells, and we postulate potential reasons for this.


Assuntos
Amiodarona/farmacocinética , Espectrometria de Massa de Íon Secundário , Amiodarona/análise , Animais , Linhagem Celular , Cromatografia Líquida , Citometria de Fluxo , Células HEK293 , Células HeLa , Células Hep G2 , Humanos , Ratos , Espectrometria de Massas em Tandem , Fatores de Tempo
9.
Anal Chem ; 87(13): 6696-702, 2015 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-26023862

RESUMO

Detecting metabolites and parent compound within a cell type is now a priority for pharmaceutical development. In this context, three-dimensional secondary ion mass spectrometry (SIMS) imaging was used to investigate the cellular uptake of the antiarrhythmic agent amiodarone, a phospholipidosis-inducing pharmaceutical compound. The high lateral resolution and 3D imaging capabilities of SIMS combined with the multiplex capabilities of ToF mass spectrometric detection allows for the visualization of pharmaceutical compound and metabolites in single cells. The intact, unlabeled drug compound was successfully detected at therapeutic dosages in macrophages (cell line: NR8383). Chemical information from endogenous biomolecules was used to correlate drug distributions with morphological features. From this spatial analysis, amiodarone was detected throughout the cell, with the majority of the compound found in the membrane and subsurface regions and absent in the nuclear regions. Similar results were obtained when the macrophages were doped with amiodarone metabolite, desethylamiodarone. The fwhm lateral resolution measured across an intracellular interface in high lateral resolution ion images was approximately 550 nm. Overall, this approach provides the basis for studying cellular uptake of pharmaceutical compounds and their metabolites on the single cell level.


Assuntos
Espectrometria de Massas/métodos , Farmacocinética , Análise de Célula Única , Animais , Linhagem Celular Transformada , Ratos , Ratos Sprague-Dawley
10.
Lab Chip ; 24(19): 4594-4608, 2024 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-39258913

RESUMO

A liver-on-a-chip model is an advanced complex in vitro model (CIVM) that incorporates different cell types and extracellular matrix to mimic the microenvironment of the human liver in a laboratory setting. Given the heterogenous and complex nature of liver-on-a-chip models, brightfield and fluorescence-based imaging techniques are widely utilized for assessing the changes occurring in these models with different treatment and environmental conditions. However, the utilization of optical microscopy techniques for structural and functional evaluation of the liver CIVMs have been limited by the reduced light penetration depth and lack of 3D information obtained using these imaging techniques. In this study, the potential of both labelled as well as label-free multimodal optical imaging techniques for visualization and characterization of the cellular and sub-cellular features of a liver-on-a-chip model was investigated. (1) Cellular uptake and distribution of Alexa 488 (A488)-labelled non-targeted and targeted antisense oligonucleotides (ASO and ASO-GalNAc) in the liver-on-a-chip model was determined using multiphoton microscopy. (2) Hyperspectral stimulated Raman scattering (SRS) microscopy of the C-H region was used to determine the heterogeneity of chemical composition of circular and cuboidal hepatocytes in the liver-on-a-chip model in a label-free manner. Additionally, the spatial overlap between the intracellular localization of ASO and lipid droplets was explored using simultaneous hyperspectral SRS and fluorescence microscopy. (3) The capability of light sheet fluorescence microscopy (LSFM) for full-depth 3D visualization of sub-cellular distribution of A488-ASO and cellular phenotypes in the liver-on-a-chip model was demonstrated. In summary, multimodal optical microscopy is a promising platform that can be utilized for visualization and quantification of 3D cellular organization, drug distribution and functional changes occurring in liver-on-a-chip models, and can provide valuable insights into liver biology and drug uptake mechanisms by enabling better characterization of these liver models.


Assuntos
Dispositivos Lab-On-A-Chip , Fígado , Humanos , Fígado/diagnóstico por imagem , Fígado/metabolismo , Fígado/citologia , Imagem Multimodal , Imagem Óptica , Hepatócitos/metabolismo , Hepatócitos/citologia , Microscopia/métodos , Modelos Biológicos
11.
Clin Pediatr (Phila) ; 58(11-12): 1239-1249, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31248263

RESUMO

This study examined the feasibility and outcomes of a training designed to enhance pediatric residents' trauma-informed practices in primary care. Paired samples t tests examined changes in 33 residents' attitudes, perceived competence, and perceived barriers toward trauma-informed care after a 2-hour training. Fisher's exact tests measured changes in residents' screening and referral behaviors. A subsample (n = 9) of residents were interviewed about the training. Residents reported increases in favorable attitudes (P = .065) and perceived competence (P < .001) and decreases in perceived barriers (P = .001 to .521) to implementing trauma-informed care practices. Chart reviews revealed a significant increase in completed trauma screens (0% to 8.0%, P < .001) but no difference in referrals for psychology/psychiatry services (1.9% to 4.2%, P = .200). Residents reported finding the training helpful. Although residents were willing and understood the utility of assessing for trauma, they faced substantial barriers.


Assuntos
Atitude do Pessoal de Saúde , Competência Clínica/estatística & dados numéricos , Internato e Residência/métodos , Pediatria/educação , Atenção Primária à Saúde/métodos , Ferimentos e Lesões/diagnóstico , Adulto , Estudos de Viabilidade , Feminino , Humanos , Masculino , Encaminhamento e Consulta/estatística & dados numéricos , Sudeste dos Estados Unidos , Inquéritos e Questionários
12.
Nat Commun ; 10(1): 338, 2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30659183

RESUMO

Macrophages undergo metabolic changes during activation that are coupled to functional responses. The gram negative bacterial product lipopolysaccharide (LPS) is especially potent at driving metabolic reprogramming, enhancing glycolysis and altering the Krebs cycle. Here we describe a role for the citrate-derived metabolite malonyl-CoA in the effect of LPS in macrophages. Malonylation of a wide variety of proteins occurs in response to LPS. We focused on one of these, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In resting macrophages, GAPDH binds to and suppresses translation of several inflammatory mRNAs, including that encoding TNFα. Upon LPS stimulation, GAPDH undergoes malonylation on lysine 213, leading to its dissociation from TNFα mRNA, promoting translation. We therefore identify for the first time malonylation as a signal, regulating GAPDH mRNA binding to promote inflammation.


Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Mediadores da Inflamação/farmacologia , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Animais , Citocinas/metabolismo , Células HEK293 , Humanos , Lipopolissacarídeos/farmacologia , Lisina/metabolismo , Malonil Coenzima A/metabolismo , Camundongos Endogâmicos C57BL , Mutagênese , Polirribossomos , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
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