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1.
Int Wound J ; 2023 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-37846874

RESUMO

Toxic reactions can appear after pressurised flushing of soft tissue with octenidine (OCT) containing disinfectants. Their use for surgical disinfection could complicate the diagnosis of possible contamination. In patients with open lacerations of their hand's subcutaneous tissue samples were taken before and after surgical disinfection with Octenisept® and analysed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In 16 out of 20 tissue samples, OCT was detected after disinfection (lower limit of quantification (LLOQ)=10 pg/mL/mg). The concentration of OCT was below the LLOQ, estimation of mean of 0.6 pg/mL/mg (0.22-0.98 pg/mL/mg, 95%-CI) before disinfection and mean of 179.4 pg/mL/mg (13.35-432.0 pg/mL/mg, 95%-CI) after disinfection. This study shows that the disinfection of open wounds with Octenisept® leads to a quantifiable concentration of OCT in open wounds. In cases of suspected OCT-mediated toxic reaction, the use of antiseptics containing OCT should be avoided.

2.
Br J Clin Pharmacol ; 88(12): 5336-5347, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35831229

RESUMO

AIMS: Locally advanced rectal cancer (LARC) is an area of unmet medical need with one third of patients dying from their disease. With response to neoadjuvant chemo-radiotherapy being a major prognostic factor, trial SAKK 41/16 assessed potential benefits of adding regorafenib to capecitabine-amplified neoadjuvant radiotherapy in LARC patients. METHODS: Patients received regorafenib at three dose levels (40/80/120 mg once daily) combined with capecitabine 825 mg/m2 bidaily and local radiotherapy. We developed population pharmacokinetic models from plasma concentrations of capecitabine and its metabolites 5'-deoxy-5-fluorocytidine and 5'-deoxy-5-fluorouridine as well as regorafenib and its metabolites M-2 and M-5 as implemented into SAKK 41/16 to assess potential drug-drug interactions (DDI). After establishing parent-metabolite base models, drug exposure parameters were tested as covariates within the respective models to investigate for potential DDI. Simulation analyses were conducted to quantify their impact. RESULTS: Plasma concentrations of capecitabine, regorafenib and metabolites were characterized by one and two compartment models and absorption was described by parallel first- and zero-order processes and transit compartments, respectively. Apparent capecitabine clearance was 286 L/h (relative standard error [RSE] 14.9%, interindividual variability [IIV] 40.1%) and was reduced by regorafenib cumulative area under the plasma concentration curve (median reduction of 45.6%) as exponential covariate (estimate -4.10 × 10-4 , RSE 17.8%). Apparent regorafenib clearance was 1.94 L/h (RSE 12.1%, IIV 38.1%). Simulation analyses revealed significantly negative associations between capecitabine clearance and regorafenib exposure. CONCLUSIONS: This work informs the clinical development of regorafenib and capecitabine combination treatment and underlines the importance of studying potential DDI with new anticancer drug combinations.


Assuntos
Compostos de Fenilureia , Neoplasias Retais , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Capecitabina , Fluoruracila/uso terapêutico , Piridinas , Neoplasias Retais/tratamento farmacológico , Neoplasias Retais/induzido quimicamente
3.
Br J Haematol ; 193(6): 1203-1212, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33954979

RESUMO

A universal anti-Xa assay for the determination of rivaroxaban, apixaban and edoxaban drug concentrations would simplify laboratory procedures and facilitate widespread implementation. Following two pilot studies analysing spiked samples and material from 698 patients, we conducted a prospective multicentre cross-sectional study, including 867 patients treated with rivaroxaban, apixaban or edoxaban in clinical practice to comprehensively evaluate a simple, readily available anti-Xa assay that would accurately measure drug concentrations and correctly predict relevant levels in clinical practice. Anti-Xa activity was measured by an assay calibrated with low-molecular-weight heparin (LMWH) in addition to ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). As an external validation, LMWH-calibrated anti-Xa activity was also determined in nine external laboratories. The LMWH-calibrated anti-Xa activity correlated strongly with rivaroxaban, apixaban or edoxaban drug levels [rs  = 0·98, 95% confidence interval (CI) 0·98-0·98]. The sensitivity for the clinically relevant cut-off levels of 30, 50 and 100 µg/l was 96·2% (95% CI 94·4-97·4), 96·4% (95% CI 94·4-97·7) and 96·7% (95% CI 94·3-98·1) respectively. Concordant results were obtained in the external validation study. In conclusion, a universal, LMWH-calibrated anti-Xa assay accurately measured rivaroxaban, apixaban and edoxaban concentrations and correctly predicted relevant drug concentrations in clinical practice.


Assuntos
Ciclofosfamida/farmacocinética , Monitoramento de Medicamentos , Inibidores do Fator Xa/sangue , Pirazóis/farmacocinética , Piridonas/farmacocinética , Rivaroxabana/farmacocinética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Cromatografia Líquida de Alta Pressão , Estudos Transversais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Estudos Retrospectivos , Espectrometria de Massas em Tandem
4.
Metabolomics ; 15(9): 120, 2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31463683

RESUMO

INTRODUCTION: Non-targeted metabolic profiling using high-resolution mass spectrometry (HRMS) is a standard approach for pathway identification despite technical limitations. OBJECTIVES: To assess the performance of combining targeted quadrupole (QQQ) analysis with HRMS for in-depth pathway profiling. METHODS: Serum of exercising patients with type 1 diabetes (T1D) was profiled using targeted and non-targeted assays. RESULTS: Non-targeted analysis yielded a broad unbiased metabolic profile, targeted analysis increased coverage of purine metabolism (twofold) and TCA cycle (three metabolites). CONCLUSION: Our screening strategy combined the benefits of the unbiased full-scan HRMS acquisition with the deeper insight into specific pathways by large-scale QQQ analysis.


Assuntos
Diabetes Mellitus Tipo 1/sangue , Metaboloma , Metabolômica/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Ciclo do Ácido Cítrico , Diabetes Mellitus Tipo 1/metabolismo , Humanos , Limite de Detecção , Masculino , Metabolômica/normas , Condicionamento Físico Humano , Purinas/metabolismo , Espectrometria de Massas por Ionização por Electrospray/normas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/normas
5.
BMC Cancer ; 19(1): 1216, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842810

RESUMO

BACKGROUND: Little is known about the relationship between the metabolite profile of plasma from pre-operative prostate cancer (PCa) patients and the risk of PCa progression. In this study we investigated the association between pre-operative plasma metabolites and risk of biochemical-, local- and metastatic-recurrence, with the aim of improving patient stratification. METHODS: We conducted a case-control study within a cohort of PCa patients recruited between 1996 and 2015. The age-matched primary cases (n = 33) were stratified in low risk, high risk without progression and high risk with progression as defined by the National Comprehensive Cancer Network. These samples were compared to metastatic (n = 9) and healthy controls (n = 10). The pre-operative plasma from primary cases and the plasma from metastatic patients and controls were assessed with untargeted metabolomics by LC-MS. The association between risk of progression and metabolite abundance was calculated using multivariate Cox proportional-hazard regression and the relationship between metabolites and outcome was calculated using median cut-off normalized values of metabolite abundance by Log-Rank test using the Kaplan Meier method. RESULTS: Medium-chain acylcarnitines (C6-C12) were positively associated with the risk of PSA progression (p = 0.036, median cut-off) while long-chain acylcarnitines (C14-C16) were inversely associated with local (p = 0.034) and bone progression (p = 0.0033). In primary cases, medium-chain acylcarnitines were positively associated with suberic acid, which also correlated with the risk of PSA progression (p = 0.032, Log-Rank test). In the metastatic samples, this effect was consistent for hexanoylcarnitine, L.octanoylcarnitine and decanoylcarnitine. Medium-chain acylcarnitines and suberic acid displayed the same inverse association with tryptophan, while indoleacetic acid, a breakdown product of tryptophan metabolism was strongly associated with PSA (p = 0.0081, Log-Rank test) and lymph node progression (p = 0.025, Log-Rank test). These data were consistent with the increased expression of indoleamine 2,3 dioxygenase (IDO1) in metastatic versus primary samples (p = 0.014). Finally, functional experiments revealed a synergistic effect of long chain fatty acids in combination with dihydrotestosterone administration on the transcription of androgen responsive genes. CONCLUSIONS: This study strengthens the emerging link between fatty acid metabolism and PCa progression and suggests that measuring levels of medium- and long-chain acylcarnitines in pre-operative patient plasma may provide a basis for improving patient stratification.


Assuntos
Carnitina/análogos & derivados , Metabolômica , Neoplasias da Próstata/sangue , Idoso , Carnitina/sangue , Carnitina/metabolismo , Estudos de Casos e Controles , Cromatografia Líquida , Progressão da Doença , Ácidos Graxos/metabolismo , Humanos , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Prognóstico , Neoplasias da Próstata/diagnóstico , População Branca
6.
Respir Res ; 19(1): 7, 2018 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-29321022

RESUMO

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology. Patients present loss of lung function, dyspnea and dry cough. Diagnosis requires compatible radiologic imaging and, in undetermined cases, invasive procedures such as bronchoscopy and surgical lung biopsy. The pathophysiological mechanisms of IPF are not completely understood. Lung injury with abnormal alveolar epithelial repair is thought to be a major cause for activation of profibrotic pathways in IPF. Metabolic signatures might indicate pathological pathways involved in disease development and progression. Reliable serum biomarker would help to improve both diagnostic approach and monitoring of drug effects. METHOD: The global metabolic profiles measured by ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) of ten stable IPF patients were compared to the ones of ten healthy participants. The results were validated in an additional study of eleven IPF patients and ten healthy controls. RESULTS: We discovered 10 discriminative metabolic features using multivariate and univariate statistical analysis. Among them, we identified one metabolite at a retention time of 9.59 min that was two times more abundant in the serum of IPF patients compared to healthy participants. Based on its ion pattern, a lysophosphatidylcholine (LysoPC) was proposed. LysoPC is a precursor of lysophosphatidic acid (LPA) - a known mediator for lung fibrosis with its pathway currently being evaluated as new therapeutic drug target for IPF and other fibrotic diseases. CONCLUSIONS: We identified a LysoPC by UHPLC-HRMS as potential biomarker in serum of patients with IPF. Further validation studies in a larger cohort are necessary to determine its role in IPF. TRIAL REGISTRATION: Serum samples from IPF patients have been obtained within the clinical trial NCT02173145 at baseline and from the idiopathic interstitial pneumonia (IIP) cohort study. The study was approved by the Swiss Ethics Committee, Bern (KEK 002/14 and 246/15 or PB_2016-01524).


Assuntos
Fibrose Pulmonar Idiopática/sangue , Fibrose Pulmonar Idiopática/diagnóstico , Lisofosfatidilcolinas/sangue , Metabolômica/métodos , Metabolômica/normas , Adulto , Idoso , Biomarcadores/sangue , Estudos de Coortes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Reprodutibilidade dos Testes , Capacidade Vital/fisiologia
7.
J Biochem Mol Toxicol ; 29(1): 10-20, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25179160

RESUMO

Cellular induction of reductase enzymes can alter the susceptibility of cells toward drugs and chemicals. In this study, we compared the capacity of a single dose of sodium selenite and 3H-1,2-dithiole-3-thione (D3T) to influence the drug-relevant reducing capacity of HT29 cells over time, and defined the protein-specific contribution to this activity on the basis of selected reaction monitoring mass spectrometry. Thioredoxin reductase 1 (TrxR1) protein levels and activity were inducible up to 2.2-fold by selenium. In contrast, selenium had only a minor influence on prostaglandin reductase 1 (PTGR1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) activity and protein levels. D3T, a strong Nrf2 inducer, induced all the reductases and additionally increased the cytotoxicity of hydroxymethylacylfulvene, a bioreductive DNA-alkylating drug. The data and experimental approaches allow one to define induction potency for reductase enzymes PTGR1, TrxR1, and NQO1 in HT29 cells and link these to changes in drug cytotoxicity.


Assuntos
Antineoplásicos/farmacologia , Neoplasias do Colo/enzimologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , NAD(P)H Desidrogenase (Quinona)/biossíntese , Proteínas de Neoplasias/metabolismo , Selenito de Sódio/farmacologia , Tionas/farmacologia , Tiofenos/farmacologia , Tiorredoxina Redutase 1/biossíntese , Oligoelementos/farmacologia , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Indução Enzimática/efeitos dos fármacos , Humanos , Fator 2 Relacionado a NF-E2/metabolismo
8.
Chem Res Toxicol ; 27(3): 377-86, 2014 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-24383545

RESUMO

Diets enriched with bioactive food components trigger molecular changes in cells that may contribute to either health-promoting or adverse effects. Recent technological advances in high-throughput data generation allow for observing systems-wide molecular responses to cellular perturbations with nontoxic and dietary-relevant doses while considering the intrinsic differences between cancerous and noncancerous cells. In this chemical profile, we compared molecular responses of the colon cancer cell line HT29 and a noncancerous colon epithelial cell line (HCEC) to two widely encountered food components, sulforaphane and selenium. We conducted this comparison by generating new transcriptome data by microarray gene-expression profiling, analyzing them statistically on the single gene, network, and functional pathway levels, and integrating them with protein expression data. Sulforaphane and selenium, at doses that did not inhibit the growth of the tested cells, induced or repressed the transcription of a limited number of genes in a manner distinctly dependent on the chemical and the cell type. The genes that most strongly responded in cancer cells were observed after treatment with sulforaphane and were members of the aldo-keto reductase (AKR) superfamily. These genes were in high agreement in terms of fold change with their corresponding proteins (correlation coefficient r(2) = 0.98, p = 0.01). Conversely, selenium had little influence on the cancer cells. In contrast, in noncancerous cells, selenium induced numerous genes involved in apoptotic, angiogenic, or tumor proliferation pathways, whereas the influence of sulforaphane was very limited. These findings contribute to defining the significance of cell type in interpreting human cellular transcriptome-level responses to exposures to natural components of the diet.


Assuntos
Isotiocianatos/toxicidade , Selênio/toxicidade , Transcriptoma/efeitos dos fármacos , 20-Hidroxiesteroide Desidrogenases/genética , 20-Hidroxiesteroide Desidrogenases/metabolismo , Linhagem Celular , Colo/citologia , Regulação para Baixo/efeitos dos fármacos , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células HT29 , Humanos , Isotiocianatos/química , Marcação por Isótopo , Redes e Vias Metabólicas/efeitos dos fármacos , Análise de Componente Principal , Selênio/química , Sulfóxidos , Regulação para Cima/efeitos dos fármacos
9.
Cells ; 13(12)2024 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-38920664

RESUMO

Hepatitis C virus (HCV) is an oncogenic virus that causes chronic liver disease in more than 80% of patients. During the last decade, efficient direct-acting antivirals were introduced into clinical practice. However, clearance of the virus does not reduce the risk of end-stage liver diseases to the level observed in patients who have never been infected. So, investigation of HCV pathogenesis is still warranted. Virus-induced changes in cell metabolism contribute to the development of HCV-associated liver pathologies. Here, we studied the impact of the virus on the metabolism of polyamines and proline as well as on the urea cycle, which plays a crucial role in liver function. It was found that HCV strongly suppresses the expression of arginase, a key enzyme of the urea cycle, leading to the accumulation of arginine, and up-regulates proline oxidase with a concomitant decrease in proline concentrations. The addition of exogenous proline moderately suppressed viral replication. HCV up-regulated transcription but suppressed protein levels of polyamine-metabolizing enzymes. This resulted in a decrease in polyamine content in infected cells. Finally, compounds targeting polyamine metabolism demonstrated pronounced antiviral activity, pointing to spermine and spermidine as compounds affecting HCV replication. These data expand our understanding of HCV's imprint on cell metabolism.


Assuntos
Hepacivirus , Poliaminas , Prolina , Ureia , Replicação Viral , Prolina/metabolismo , Humanos , Hepacivirus/fisiologia , Hepacivirus/efeitos dos fármacos , Poliaminas/metabolismo , Ureia/metabolismo , Ureia/farmacologia , Replicação Viral/efeitos dos fármacos , Arginase/metabolismo , Antivirais/farmacologia , Antivirais/metabolismo , Hepatite C/metabolismo , Hepatite C/virologia , Linhagem Celular Tumoral , Prolina Oxidase/metabolismo
10.
Diagnostics (Basel) ; 13(12)2023 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-37371023

RESUMO

A universal calibrator for the determination of all anti-Xa inhibitors would support laboratory processes. We aimed to test the clinical performance of an anti-Xa assay utilizing a universal edoxaban calibrator to determine clinically relevant concentrations of all anti-Xa inhibitors. Following a pilot study, we enrolled 553 consecutive patients taking rivaroxaban, edoxaban, or apixaban from nine study centers in a prospective cross-sectional study. The Technochrom® anti-Xa assay was conducted using the Technoview® edoxaban calibrator. Using ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS), anti-Xa inhibitor drug concentrations were determined. Sensitivities and specificities to detect three clinically relevant drug concentrations (30 µgL-1, 50 µgL-1, 100 µgL-1) were determined. Overall, 300 patients treated with rivaroxaban, 221 with apixaban, and 32 with edoxaban were included. The overall correlation coefficient (rs) was 0.95 (95% CI 0.94, 0.96). An area under the receiver operating characteristic curve of 0.96 for 30 µgL-1, 0.98 for 50 µgL-1, and 0.99 for 100 µgL-1 was found. The sensitivities were 92.3% (95% CI 89.2, 94.6), 92.7% (89.4, 95.1), and 94.8% (91.1, 97.0), respectively (specificities 82.2%, 93.7%, and 94.4%). In conclusion, the clinical performance of a universal, edoxaban-calibrated anti-Xa assay was solid and most drug concentrations were predicted correctly.

11.
Front Cardiovasc Med ; 9: 817826, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35369293

RESUMO

Background: Applying a single anti-Xa assay, calibrated to unfractionated heparin to measure rivaroxaban, apixaban, and edoxaban would simplify laboratory procedures and save healthcare costs. Aim: We hypothesized that a heparin-calibrated anti-Xa assay would accurately measure rivaroxaban, apixaban, and edoxaban drug concentrations and correctly predict clinically relevant drug levels. Methods: This analysis is part of the Simple-Xa study, a prospective multicenter cross-sectional study conducted in clinical practice. Patients treated with rivaroxaban, apixaban, or edoxaban were included. Anti-Xa activity was measured using the Siemens INNOVANCE® Heparin assay. Drug concentrations were determined using ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cut-off levels were determined in a derivation dataset (50% of patients) and sensitivities and specificities were calculated in a verification dataset (50% of patients). Results: Overall, 845 patients were available for analysis. Correlation coefficients (r s ) between the heparin-calibrated anti-Xa assay and drug concentrations were 0.97 (95% CI 0.97, 0.98) for rivaroxaban, 0.96 (0.96, 0.97) for apixaban, and 0.96 (0.94, 0.99) for edoxaban. The area under the receiver operating characteristics curve (ROC) was 0.99 for all clinically relevant drug concentrations. In the verification dataset, the sensitivity was 94.2% (95% CI 90.8-96.6) for 30 µg L-1, 95.8% (92.4-98.0) for 50 µg L-1, and 98.7% (95.5-99.9) for 100 µg L-1. Specificities were 86.3% (79.2-91.7), 89.8% (84.5-93.7), and 88.7% (84.2-92.2), respectively. Conclusion: In a large prospective study in clinical practice, a strong correlation of heparin-calibrated anti-Xa measurements with LC-MS/MS results was observed and clinically relevant drug concentrations were predicted correctly.

12.
Life (Basel) ; 12(7)2022 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-35888115

RESUMO

Prothrombinase-induced clotting time (PiCT) is proposed as a rapid and inexpensive laboratory test to measure direct oral anticoagulant (DOAC) drug levels. In a prospective, multicenter cross-sectional study, including 851 patients, we aimed to study the accuracy of PiCT in determining rivaroxaban, apixaban, and edoxaban drug concentrations and assessed whether clinically relevant drug levels could be predicted correctly. Citrated plasma samples were collected, and the Pefakit® PiCT was utilized. Ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to measure drug concentrations. Cut-off levels were established using receiver-operating characteristics curves. We calculated sensitivities and specificities with respect to clinically relevant drug concentrations. Spearman's correlation coefficient between PiCT and drug concentrations was 0.85 in the case of rivaroxaban (95% CI 0.82, 0.88), 0.66 for apixaban (95% CI 0.60, 0.71), and 0.78 for edoxaban (95% CI 0.65, 0.86). The sensitivity to detect clinically relevant drug concentrations was 85.1% in the case of 30 µg L-1 (95% CI 82.0, 87.7; specificity 77.9; 72.1, 82.7), 85.7% in the case of 50 µg L-1 (82.4, 88.4; specificity 77.3; 72.5, 81.5), and 85.1% in the case of 100 µg L-1 (80.9, 88.4; specificity 73.2%; 69.1, 76.9). In conclusion, the association of PiCT with DOAC concentrations was fair, and the majority of clinically relevant drug concentrations were correctly predicted.

13.
Front Cardiovasc Med ; 8: 717939, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34568459

RESUMO

Background: The thrombin generation assay (TG) is a promising approach to measure the degree of anticoagulation in patients treated with direct oral anticoagulants (DOAC). A strong association with plasma drug concentrations would be a meaningful argument for the potential use to monitor DOAC. Objectives: We aimed to study the correlation of TG with rivaroxaban, apixaban, and edoxaban drug concentrations in a large, prospective multicenter cross-sectional study. Methods: Five-hundred and fifty-nine patients were included in nine tertiary hospitals. The Technothrombin® TG was conducted in addition to an anti-Xa assay; LC-MS/MS was performed as the reference standard. Results: Correlation (rs) between thrombin generation measurements and drug concentrations was -0.72 for peak thrombin generation (95% confidence interval, CI, -0.77, -0.66), -0.55 for area under the curve (AUC; 95% CI -0.61, -0.48), and 0.80 for lag time (95% CI 0.75, 0.84). In contrast, rs was 0.96 with results of the anti-Xa activity (95% CI 0.95-0.97). Sensitivity with regard to the clinically relevant cut-off value of 50 µgL-1 was 49% in case of peak thrombin generation (95% CI, 44, 55), 29% in case of AUC (95% CI, 24, 34), and 64% in case of lag time (95% CI, 58, 69). Sensitivity of the anti-Xa assay was 95% (95% CI, 92, 97). Conclusions: The correlation of thrombin generation measurements with DOAC drug concentrations was weak, and clinically relevant drug levels were not predicted correctly. Our results do not support an application of TG in the monitoring of DOAC.

14.
Cell Death Differ ; 27(6): 1965-1980, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31844253

RESUMO

In contrast to the "Warburg effect" or aerobic glycolysis earlier generalized as a phenomenon in cancer cells, more and more recent evidence indicates that functional mitochondria are pivotal for ensuring the energy supply of cancer cells. Here, we report that cancer cells with reduced autophagy-related protein 12 (ATG12) expression undergo an oncotic cell death, a phenotype distinct from that seen in ATG5-deficient cells described before. In addition, using untargeted metabolomics with ATG12-deficient cancer cells, we observed a global reduction in cellular bioenergetic pathways, such as ß-oxidation (FAO), glycolysis, and tricarboxylic acid cycle activity, as well as a decrease in mitochondrial respiration as monitored with Seahorse experiments. Analyzing the biogenesis of mitochondria by quantifying mitochondrial DNA content together with several mitochondrion-localizing proteins indicated a reduction in mitochondrial biogenesis in ATG12-deficient cancer cells, which also showed reduced hexokinase II expression and the upregulation of uncoupling protein 2. ATG12, which we observed in normal cells to be partially localized in mitochondria, is upregulated in multiple types of solid tumors in comparison with normal tissues. Strikingly, mouse xenografts of ATG12-deficient cells grew significantly slower as compared with vector control cells. Collectively, our work has revealed a previously unreported role for ATG12 in regulating mitochondrial biogenesis and cellular energy metabolism and points up an essential role for mitochondria as a failsafe mechanism in the growth and survival of glycolysis-dependent cancer cells. Inducing oncosis by imposing an ATG12 deficiency in solid tumors might represent an anticancer therapy preferable to conventional caspase-dependent apoptosis that often leads to undesirable consequences, such as incomplete cancer cell killing and a silencing of the host immune system.


Assuntos
Proteína 12 Relacionada à Autofagia/fisiologia , Mitocôndrias/metabolismo , Neoplasias/metabolismo , Animais , Linhagem Celular Tumoral , Metabolismo Energético , Glicólise , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID
15.
Toxicol In Vitro ; 23(4): 704-9, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19233257

RESUMO

High-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking has the ability to monitor the ligand-dependent dimerization of the human estrogen receptor alpha ligand binding domain (hERalpha LBD) in solution. Because only ER ligands enhance the homodimer abundance, we evaluated the ability of this label-free approach for identifying endocrine disrupting compounds (EDCs) in a high-throughput manner. This was achieved by combining an automated liquid handler with an automated MS acquisition procedure, which allowed a five-fold gain in operator time compared to a fully manual approach. To detect ligand binding with enough confidence, the receptor has to be incubated with at least a 10 microM concentration of the test compound. Based on the increase of the measured homodimer intensity, eight compounds with a relative binding affinity (RBA, relative to the natural hormone estradiol) >7% were identified as ER ligands among the 28 chemicals tested. Two other compounds, quercetin and 4-tert-amylphenol, were also identified as ER ligands, although their RBAs have been reported to be only 0.01% and 0.000055%, respectively. This suggests that these two ligands have a higher affinity for hERalpha LBD than reported in the literature. The high-mass MALDI approach thus allows identifying high affinity EDCs in an efficient way.


Assuntos
Disruptores Endócrinos/farmacologia , Receptor alfa de Estrogênio/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sítios de Ligação , Dimerização , Receptor alfa de Estrogênio/metabolismo , Humanos , Ligantes
16.
Sci Rep ; 9(1): 9269, 2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239498

RESUMO

Vascularized composite allotransplantation (VCA), such as hand and face transplantation, is emerging as a potential solution in patients that suffered severe injuries. However, adverse effects of chronic high-dose immunosuppression regimens strongly limit the access to these procedures. In this study, we developed an in situ forming implant (ISFI) loaded with rapamycin to promote VCA acceptance. We hypothesized that the sustained delivery of low-dose rapamycin in proximity to the graft may promote graft survival and induce an immunoregulatory microenvironment, boosting the expansion of T regulatory cells (Treg). In vitro and in vivo analysis of rapamycin-loaded ISFI (Rapa-ISFI) showed sustained drug release with subtherapeutic systemic levels and persistent tissue levels. A single injection of Rapa-ISFI in the groin on the same side as a transplanted limb significantly prolonged VCA survival. Moreover, treatment with Rapa-ISFI increased the levels of multilineage mixed chimerism and the frequency of Treg both in the circulation and VCA-skin. Our study shows that Rapa-ISFI therapy represents a promising approach for minimizing immunosuppression, decreasing toxicity and increasing patient compliance. Importantly, the use of such a delivery system may favor the reprogramming of allogeneic responses towards a regulatory function in VCA and, potentially, in other transplants and inflammatory conditions.


Assuntos
Aloenxertos Compostos/efeitos dos fármacos , Rejeição de Enxerto/prevenção & controle , Sobrevivência de Enxerto/efeitos dos fármacos , Membro Posterior/transplante , Sirolimo/farmacologia , Linfócitos T Reguladores/imunologia , Alotransplante de Tecidos Compostos Vascularizados/efeitos adversos , Animais , Aloenxertos Compostos/imunologia , Aloenxertos Compostos/patologia , Sistemas de Liberação de Medicamentos , Imunossupressores/farmacologia , Masculino , Ratos , Ratos Endogâmicos Lew , Quimeras de Transplante , Tolerância ao Transplante/imunologia
17.
Anal Chem ; 80(20): 7833-9, 2008 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-18778086

RESUMO

Many drugs and chemicals exert their biological effect by modulating protein-protein interactions. In vitro approaches to characterize these mechanisms are often based on indirect measurements (e.g., fluorescence). Here, we used mass spectrometry (MS) to directly monitor the effect of small-molecule ligands on the binding of a coactivator peptide (SRC1) by the human estrogen receptor alpha ligand binding domain (hERalpha LBD). Nanoelectrospray mass spectrometry (nanoESI-MS) and high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking were employed to follow these processes. The chemical cross-linking protocol used prior to high-mass MALDI analysis allows detection of intact noncovalent complexes. The binding of intact hERalpha LBD homodimer with two coactivator peptides was detected with nanoESI-MS and high-mass MALDI-MS only in the presence of an agonist ligand. Furthermore, high-mass MALDI-MS revealed an increase of the homodimer abundance after incubating the receptor with a ligand, independent of the ligand character (i.e., agonist, antagonist). The binding characteristics of the compounds tested by MS correlate very well with their biological activity reported by cell-based assays. High-mass MALDI appears to be an efficient and simple tool for directly monitoring ligand regulation mechanisms involved in protein-protein interactions. Furthermore, the combination of both MS methods allows identifying and characterizing endocrine-disrupting compounds or new drug compounds in an efficient way.


Assuntos
Disruptores Endócrinos/metabolismo , Disruptores Endócrinos/farmacologia , Receptor alfa de Estrogênio/metabolismo , Sequência de Aminoácidos , Dimerização , Disruptores Endócrinos/química , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/química , Humanos , Ligantes , Espectrometria de Massas , Peptídeos/química , Peptídeos/metabolismo , Farmacologia , Ligação Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
18.
J Am Soc Mass Spectrom ; 19(3): 332-43, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18083584

RESUMO

We present a comparison of three different electrospray-based ionization techniques for the investigation of noncovalent complexes with mass spectrometry. The features and characteristics of standard electrospray ionization (ESI), chip-based nanoESI, and electrosonic spray ionization (ESSI) mounted onto a hybrid quadrupole time-of-flight mass spectrometer were compared in their performance to determine the dissociation constant (KD) of the model system hen egg white lysozyme (HEWL) binding to N,N',N''-triacetylchitotriose (NAG3). The best KD value compared with solution data were found for ESSI, 19.4 +/- 3.6 microM. Then, we determined the KDs of the two nucleotide binding sites of adenylate kinase (AK), where we obtained KDs of 2.2 +/- 0.8 microM for the first and 19.5 +/- 8.0 microM for the second binding site using ESSI. We found a weak charge state dependence of the KD for both protein-ligand systems, where for all ionization techniques the KD value decreases with increasing charge state. We demonstrate that ESSI is very gentle and insensitive to instrumental parameters, and the KD obtained is in good agreement with solution phase results from the literature. In addition, we tried to determine the KD for the lymphocyte-specific kinase LCK binding to a kinase inhibitor using nanoESI due to the very low amount of sample available. In this case, we found KD values with a strong charge state dependence, which were in no case close to literature values for solution phase.


Assuntos
Ligantes , Proteínas/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Adenilato Quinase/química , Adenilato Quinase/metabolismo , Animais , Galinhas , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Muramidase/química , Muramidase/metabolismo , Ligação Proteica , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas/metabolismo , Titulometria , Trissacarídeos/química , Trissacarídeos/metabolismo
19.
Protein Sci ; 16(5): 938-46, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17400923

RESUMO

In the present report, a method based on chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor alpha ligand-binding domain (hERalpha LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine disruptors, can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. Using proper experimental conditions, the nanoESI-MS approach allowed for the detection of specific ligand interactions with hERalpha LBD. The relative gas-phase stability of selected hERalpha LBD-ligand complexes did not mirror the binding affinity in solution, a result that demonstrates the prominent role of hydrophobic contacts for stabilizing ER-ligand complexes in solution. The best approach to evaluate relative solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17beta-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERalpha LBD measured by nanoESI-MS was 4-hydroxtamoxifen approximately diethylstilbestrol > E2 >> genistein >> bisphenol A, consistent with the order of the binding affinities in solution. The limited reproducibility of the bound to free protein ratio measured by nanoESI-MS for this system only allowed the binding constants (K(d)) to be estimated (low nanomolar range for E2). The specificity of nanoESI-MS combined with its speed (1 min/ligand), low sample consumption (90 pmol protein/ligand), and its sensitivity for ligand (30 ng/mL) demonstrates that this technique is a promising method for screening suspected endocrine disrupting compounds and to qualitatively evaluate their binding affinity.


Assuntos
Disruptores Endócrinos/química , Receptor alfa de Estrogênio/química , Ligantes , Espectrometria de Massas por Ionização por Electrospray/métodos , Disruptores Endócrinos/análise , Disruptores Endócrinos/metabolismo , Receptor alfa de Estrogênio/metabolismo , Humanos , Nanotecnologia/métodos , Ligação Proteica
20.
J Breath Res ; 12(1): 016003, 2017 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-28775244

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a devastating lung disease with poor survival. There is an urgent need to better diagnose and monitor IPF patients as new treatments which slow down disease progression are now available. Exhaled breath condensate (EBC) is easily and non-invasively collected, but analysis of potential biomarkers is difficult due to low concentrations and methodological limitations. We used a non-targeted metabolomics approach to identify discriminatory metabolic profiles that distinguish IPF patients from healthy controls. For the pilot study set, we collected EBC from 10 stable IPF patients and 10 lung healthy controls. Samples were analyzed by ultra high-performance liquid chromatography coupled to high-resolution mass spectrometry in positive and negative ion mode. After data processing and statistical analysis, 58 metabolites were found to be discriminative between IPF patients and controls in the positive ion mode. One metabolite candidate m/z = 341.3514 at a retention time of 9.94 min was 2.5-fold up-regulated in IPF patients compared to healthy controls and validated in a second set of eight IPF patients and healthy controls. The identity of this metabolic feature still remains elusive. Our preliminary results identified a distinguished EBC profile of IPF patients compared to controls. Although these results need to be confirmed in additional individuals, EBC sampling for diagnosis and/or monitoring of IPF patients is a promising new method, which should be further explored. The EBC samples have been obtained within the clinical trial NCT02173145 at baseline.


Assuntos
Biomarcadores/análise , Testes Respiratórios/métodos , Expiração , Fibrose Pulmonar Idiopática/diagnóstico , Idoso , Análise Discriminante , Feminino , Humanos , Análise dos Mínimos Quadrados , Masculino , Metaboloma , Metabolômica , Projetos Piloto , Reprodutibilidade dos Testes
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