RESUMEN
Endoplasmic reticulum (ER)-endolysosome interactions regulate cholesterol exchange between the ER and the endolysosome. ER-endolysosome membrane contact sites mediate the ER-endolysosome interaction. VAP-ORP1L (vesicle-associated membrane protein-associated protein- OSBP-related protein 1L) interaction forms the major contact site between the ER and the lysosome, which is regulated by Rab7. RILP (Rab7-interacting lysosomal protein) is the downstream effector of Rab7, but its role in the organelle interaction between the ER and the lysosome is not clear. In this study, we found RILP interacts with ORP1L to competitively inhibit the formation of the VAP-ORP1L contact site. Immunofluorescence microscopy revealed that RILP induces late endosome/lysosome clustering, which reduces the contact of endolysosomes with the ER, interfering with the ER-endolysosome interaction. Further examination demonstrated that over-expression of RILP results in the accumulation of cholesterol in the clustered endolysosomes, which triggers cellular autophagy depending on RILP. Our results suggest that RILP interferes with the ER-endolysosome interaction to inhibit cholesterol flow from the endolysosome to the ER, which feedbacks to trigger autophagy.
Asunto(s)
Colesterol , Retículo Endoplásmico , Endosomas , Lisosomas , Lisosomas/metabolismo , Colesterol/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Endosomas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia , Células HeLa , Receptores de Esteroides/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Unión Proteica , Proteínas de Unión a GTP rab7 , Células HEK293RESUMEN
We describe a mass spectrometry (MS) analytical platform resulting from the novel integration of acoustic droplet ejection (ADE) technology, an open-port interface (OPI), and electrospray ionization (ESI)-MS that creates a transformative system enabling high-speed sampling and label-free analysis. The ADE technology delivers nanoliter droplets in a touchless manner with high speed, precision, and accuracy. Subsequent sample dilution within the OPI, in concert with the capabilities of modern ESI-MS, eliminates the laborious sample preparation and method development required in current approaches. This platform is applied to a variety of experiments, including high-throughput (HT) pharmacology screening, label-free in situ enzyme kinetics, in vitro absorption, distribution, metabolism, elimination, pharmacokinetic and biomarker analysis, and HT parallel medicinal chemistry.
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Ensayos Analíticos de Alto Rendimiento , Espectrometría de Masa por Ionización de Electrospray , AcústicaRESUMEN
High-throughput experimentation (HTE) has emerged as an important tool in drug discovery, providing a platform for preparing large compound libraries and enabling swift reaction screening over wide-ranging conditions. Recent advances in automated high-density, material-sparing HTE have necessitated the development of rapid analytics with sensitivity and resolution sufficient to identify products and/or assess reaction performance in a timely and data-rich manner. Combination of an ultrathroughput (UT) reader platform with Acoustic Droplet Ejection-Open Port Interface-Mass Spectrometry (ADE-OPI-MS) provides the requisite speed and sensitivity. Herein, we report the application of ADE-OPI-MS to HTE in the areas of parallel medicinal chemistry and reaction screening.
RESUMEN
Recently, object detection methods have developed rapidly and have been widely used in many areas. In many scenarios, helmet wearing detection is very useful, because people are required to wear helmets to protect their safety when they work in construction sites or cycle in the streets. However, for the problem of helmet wearing detection in complex scenes such as construction sites and workshops, the detection accuracy of current approaches still needs to be improved. In this work, we analyze the mechanism and performance of several detection algorithms and identify two feasible base algorithms that have complementary advantages. We use one base algorithm to detect relatively large heads and helmets. Also, we use the other base algorithm to detect relatively small heads, and we add another convolutional neural network to detect whether there is a helmet above each head. Then, we integrate these two base algorithms with an ensemble method. In this method, we first propose an approach to merge information of heads and helmets from the base algorithms, and then propose a linear function to estimate the confidence score of the identified heads and helmets. Experiments on a benchmark data set show that, our approach increases the precision and recall for base algorithms, and the mean Average Precision of our approach is 0.93, which is better than many other approaches. With GPU acceleration, our approach can achieve real-time processing on contemporary computers, which is useful in practice.
RESUMEN
11ß-Hydroxysteroid dehydrogenase 1 (11ß-HSD1) is distributed mainly in the human liver, with no detectable levels in the intestine or kidney, based on a newly developed proteomic approach. 11ß-HSD1 is mostly membrane-bound and retained in the liver microsomal fraction. Interindividual variability of 11ß-HSD1 is relatively low, with about a 3-fold difference. A significant correlation was not observed between various demographic variables (ethnicity, gender, age, weight, smoking, and alcohol use) and 11ß-HSD1 protein expression or activity based on data from 31 donors. PF-915275 has been identified as a selective 11ß-HSD1 inhibitor with minimal effects on carbonyl reductase 1 and major cytochrome P450 enzymes. 11ß-HSD1 has been shown, for the first time, to be involved in doxorubicin metabolism, accounting for approximately 30% of doxorubicinol formation in human hepatocytes.
Asunto(s)
11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Doxorrubicina/metabolismo , Distribución Tisular/fisiología , Aminopiridinas/metabolismo , Carbonil Reductasa (NADPH)/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Femenino , Hepatocitos/metabolismo , Humanos , Inactivación Metabólica/fisiología , Mucosa Intestinal/metabolismo , Riñón/metabolismo , Hígado/metabolismo , Masculino , Microsomas Hepáticos/metabolismo , Proteómica/métodos , Sulfonamidas/metabolismoRESUMEN
The ability to predict human liver-to-plasma unbound partition coefficient (Kpuu) is of great importance to estimate unbound liver concentration, develop PK/PD relationships, predict efficacy and toxicity in the liver, and model the drug-drug interaction potential for drugs that are asymmetrically distributed into the liver. A novel in vitro method has been developed to predict in vivo Kpuu with good accuracy using cryopreserved suspension hepatocytes in InVitroGRO HI media with 4% BSA. Validation was performed using six OATP substrates with rat in vivo Kpuu data from i.v. infusion studies where a steady state was achieved. Good in vitro-in vivo correlation (IVIVE) was observed as the in vitro Kpuu values were mostly within 2-fold of in vivo Kpuu Good Kpuu IVIVE in human was also observed with in vivo Kpuu data of dehydropravastatin from positron emission tomography and in vivo Kpuu data from PK/PD modeling for pravastatin and rosuvastatin. Under the specific Kpuu assay conditions, the drug-metabolizing enzymes and influx/efflux transporters appear to function at physiologic levels. No scaling factors are necessary to predict in vivo Kpuu from in vitro data. The novel in vitro Kpuu method provides a useful tool in drug discovery to project in vivo Kpuu.
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Descubrimiento de Drogas/métodos , Hepatocitos/metabolismo , Hígado/metabolismo , Modelos Biológicos , Transportadores de Anión Orgánico/metabolismo , Preparaciones Farmacéuticas/sangre , Animales , Células Cultivadas , Hepatocitos/efectos de los fármacos , Humanos , Masculino , Preparaciones Farmacéuticas/metabolismo , Valor Predictivo de las Pruebas , Ratas , Ratas Wistar , Especificidad por SustratoRESUMEN
Human tissue distribution of carbonyl reductase 1 (CBR1) is quite controversial in the literature. To understand the differences, CBR1 protein abundance in human intestine, liver, and kidney has been determined using a proteomic approach with liquid chromatography-tandem mass spectrometry. The results show that CBR1 distribution in the 3 tissues is relatively similar, within 2- to 3-folds of each other. Intestine has the highest CBR1 enzyme level (106 pmol/mg protein) followed by liver (76 pmol/mg protein) and kidney (39 pmol/mg protein). The high abundance of CBR1 in the intestine and kidney suggests the critical role of this enzyme in gut first-pass metabolism and extra-hepatic clearance. CBR1 is also detected, for the first time, in the microsomal fractions with 5- to 17-fold lower amounts than cytosols of the corresponding tissues. The average CBR1 protein amount is 14 pmol/mg protein in human hepatocytes. Individual variability of CBR1 is about 4-fold based on 13 lots of cryopreserved human hepatocytes. The relative expression factor between human liver cytosol and human recombinant CBR1 enzyme (hr-CBR1) is 0.0042. The relative activity factor between human liver cytosol and hr-CBR1 is 0.040, suggesting that significant amount of CBR1 protein in the hr-CBR1 has no catalytic activity.
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Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/metabolismo , Proteómica/métodos , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Animales , Cromatografía Liquida/métodos , Femenino , Hepatocitos/enzimología , Humanos , Intestinos/enzimología , Riñón/enzimología , Hígado/enzimología , Masculino , Ratones , Distribución Tisular/fisiologíaRESUMEN
BACKGROUND: Quantitation of CAM H4 in biological matrix has been a challenge due to low concentrations, instability and the existence of four CAM diastereomers. Historically, either inactive clopidogrel acid or CAM diastereomers without separation were measured for exposure and PK parameters. RESULTS: This study presents a sensitive and fast UHPLC-MS/MS method for simultaneous quantitation of clopidogrel, clopidogrel acid and active metabolite H4 in human plasma. The method demonstrated the separation of H4 from other isomers and yet retained clopidogrel acid. Matrix stabilities, accuracy and precision, recovery and matrix effect were evaluated during development. CONCLUSION: With LLOQ of 0.05 ng/ml for clopidogrel and H4, and 5.5 min LC gradient, it is the most sensitive and fastest method to our knowledge.
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Análisis Químico de la Sangre/métodos , Antagonistas del Receptor Purinérgico P2Y/sangre , Espectrometría de Masas en Tándem , Ticlopidina/análogos & derivados , Análisis Químico de la Sangre/normas , Calibración , Cromatografía Líquida de Alta Presión/normas , Clopidogrel , Humanos , Microsomas Hepáticos/metabolismo , Antagonistas del Receptor Purinérgico P2Y/metabolismo , Antagonistas del Receptor Purinérgico P2Y/normas , Sensibilidad y Especificidad , Estereoisomerismo , Espectrometría de Masas en Tándem/normas , Temperatura , Ticlopidina/sangre , Ticlopidina/metabolismo , Ticlopidina/normasRESUMEN
A highly sensitive, selective, and rugged quantification method was developed and validated for decitabine (5-aza-2'-deoxycytidine) in human plasma treated with 100µg/mL of tetrahydrouridine (THU). Chromatographic separation was accomplished using hydrophilic interaction liquid chromatography (HILIC) and detection used electrospray ionization (ESI) tandem mass spectrometry (MS/MS) by monitoring lithiated adducts of the analytes as precursor ions. The method involves simple acetonitrile precipitation steps (in an ice bath) followed by injection of the supernatant onto a Thermo Betasil Silica-100, 100×3.0mm, 5µm LC column. Protonated ([M+H](+)), sodiated ([M+Na](+)), and lithiated ([M+Li](+)) adducts as precursor ions for MS/MS detection were evaluated for best sensitivity and assay performance. During initial method development abundant sodium [M+Na](+) and potassium [M+K](+) adducts were observed while the protonated species [M+H](+) was present at a relative abundance of less than 5% in Q1. The alkali adducts were not be able to be minimized by the usual approach of increasing acid content in mobile phases. Significant analyte/internal standard (IS) co-suppression and inter-lot response differences were observed when using the sodium adduct as the precursor ion for quantification. By adding 2mM lithium acetate in aqueous mobile phase component, the lithium adduct effectively replaced other cationic species and was successfully used as the precursor ion for selected reaction monitoring (SRM) detection. The method demonstrated the separation of anomers and from other endogenous interferences using a 3-min gradient elution. Decitabine stock, working solution stabilities were investigated during method development. Three different peaks, including one from anomerization, were observed in the SRM transition of the analyte when it was in neutral aqueous solution. The assay was validated over a concentration range of 0.5-500ng/mL (or 0.44-440pg injected on column) in 50µL of human plasma. The accuracy and precision were within 8.6% relative error and 6.3% coefficient of variation, respectively. Decitabine was stable in THU treated human plasma for at least 68 days and after 5 freeze-thaw cycles when stored at -70°C. Stability of decitabine in THU treated human whole blood, matrix factor and recovery were also evaluated during method validation. The method was successfully used for clinical sample analysis.
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Azacitidina/análogos & derivados , Cromatografía Líquida de Alta Presión/métodos , Litio/química , Espectrometría de Masas en Tándem/métodos , Azacitidina/sangre , Azacitidina/química , Decitabina , Estabilidad de Medicamentos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Modelos Lineales , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
The depletion and degradation of pharmacologically active compounds (PhACs) and pesticides as a function of ozonation in drinking water treatment processes is not well studied. The A.H. Weeks drinking water treatment plant (DWTP) serves the City of Windsor, Ontario Canada, and incorporates ozone treatment into the production of drinking water. This DWTP also operates a real-time, scaled down pilot plant, which has two parallel streams, conventional and ozone plus conventional treatments. In this study water samples were collected from key points in the two streams of the pilot plant system to determine the depletion and influence of seasonal changes in water processing parameters on eighteen major PhACs (and metabolites) and seven s-triazines herbicides. However, only carbamazepine (antiepileptic), caffeine (stimulant), cotinine (metabolite of nicotine) and atrazine were consistently detectable in the raw water intake (low to sub-ng/L level). Regardless of the seasonality, the flocculation-coagulation and dual media filtration steps without ozone treatment resulted in no decrease in analyte concentrations, while decreases of 66-100% (undetectable, method detection limits 0.05-1 ng/L) of the analyte concentrations were observed when ozone treatment was part of the water processing. These findings demonstrate that ozone treatment is highly effective in depleting carbamazepine, caffeine, cotinine, and atrazine, and thus is highly influential in the fate of these compounds in drinking water treatment regardless of the seasonal time frame. Currently very few Canadian DWTPs incorporate ozonation into conventional treatment, which suggests that human exposure to these compounds via drinking water consumption may be an issue in affected communities.
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Atrazina/metabolismo , Herbicidas/metabolismo , Oxidantes Fotoquímicos/farmacología , Ozono/farmacología , Preparaciones Farmacéuticas/metabolismo , Contaminantes Químicos del Agua/metabolismo , Purificación del Agua/métodos , Canadá , Humanos , Cinética , Ontario , Oxidación-Reducción , Ríos , Abastecimiento de AguaRESUMEN
Triclosan (5-chloro-2-(2,4-dichlorophenoxy)phenol) is an antimicroibial disinfectant agent used in a wide array of consumer products. An analytical method based on solid-phase extraction (SPE) followed by reverse phase, liquid chromatography coupled with electrospray ionization (negative)-tandem quadrupole mass spectrometry (LC-ESI(-)-MS/MS; in the multiple reaction monitoring (MRM) mode) was developed, optimized and validated for the determination of triclosan in wastewater/sewage treatment plant (WSTP) effluent and surface waters from the upper Detroit River (Canada). The mean recoveries (+/-%RSD) of triclosan and the internal standard 2'-HO-tribromodiphenyl ether (2'-HO-BDE-28) spiked to surface water and WSTP effluent samples ranged similarly from 104+/-8% and 91+/-10%, respectively, and method limits of quantification were in the low ppb/high ppt range. However, ESI(-) enhancement was found for both analytes due to sample matrix effects, as indicated by % process efficiencies (a measure of ESI(-) enhancement/suppression), which were in the 111-128% range. We report the first known assessment of triclosan in the Detroit River watershed of the Great Lakes, which preliminarily examined the presence and UV-treatment effects on triclosan in effluent from the major WSTP serving the City of Windsor, Ontario, Canada, and fate in surface waters of the upper Detroit River. Triclosan in WSTP effluent decreased 22% after final UV treatment to a mean concentration of 63 ng/L. Concentrations were further reduced to 4 and 8 ng/L (a >90% reduction) in surface water samples from sites downstream on the Canadian shoreline of the Detroit River, which was due to dilution. Two hydroxylated tribrominated diphenyl ethers, which are structurally similar to triclosan, were also detected in WSTP effluent and surface water samples.