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A methodology based on the use of asymmetrical flow field-flow fractionation (AF4) coupled to ICP-MS with size fraction-targeted isotope dilution analysis (IDA) has been developed, validated, and applied for the first time to determine the mass fraction of nanoscale silica (SiO2). For this purpose, 29Si-enriched SiO2 nanoparticles, to be used as an IDA spike/internal standard, were synthesized and characterized in-house. Double IDA was used to quantify an aqueous suspension of Stöber silica particles of similar characteristics to those of the 29SiO2 nanoparticle (NP) spike using a representative test material of natural Si isotopic composition as the calibrant. For fumed SiO2 NP in a highly complex food matrix, a methodology based on single IDA with AF4/ICP-MS using the same 29SiO2 NP spike was developed and validated. Relative expanded measurement uncertainties (k = 2) of 4% (double IDA) and 8% (single IDA) were achieved for nanoscale silica mass fractions of 5143 and 107 mg kg-1 in water suspension and food matrix, respectively. To assess the accuracy of AF4/ICP-IDMS for the characterization of SiO2 NP in a food matrix, standard addition measurements on samples spiked with Aerosil AF200, also in-house characterized for Si mass fraction, were undertaken, with an average recovery of 95.6 ± 4.1% (RSD, n = 3) obtained. The particle-specific IDA data obtained for both SiO2 NP-containing samples were also compared with that of post-AF4 channel external calibration using inorganic Si standards. The mass fractions obtained by IDA agreed well with those obtained by external calibration within their associated measurement uncertainties.
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Alcoholic-related liver disease (ALD) is one of the leading causes of chronic liver disease and morbidity. Unfortunately, the pathogenesis of ALD is still incompletely understood. StARD1 has emerged as a key player in other etiologies of chronic liver disease, and alcohol-induced liver injury exhibits zonal distribution. Here, we report that StARD1 is predominantly expressed in perivenous (PV) zone of liver sections from mice-fed chronic and acute-on-chronic ALD models compared to periportal (PP) area and is observed as early as 10 days of alcohol feeding. Ethanol and chemical hypoxia induced the expression of StARD1 in isolated primary mouse hepatocytes. The zonal-dependent expression of StARD1 resulted in the accumulation of cholesterol in mitochondria and increased lipid peroxidation in PV hepatocytes compared to PP hepatocytes, effects that were abrogated in PV hepatocytes upon hepatocyte-specific Stard1 KO mice. Transmission electron microscopy indicated differential glycogen and lipid droplets content between PP and PV areas, and alcohol feeding decreased glycogen content in both areas while increased lipid droplets content preferentially in PV zone. Moreover, transmission electron microscopy revealed that mitochondria from PV zone exhibited reduced length with respect to PP area, and alcohol feeding increased mitochondrial number, particularly, in PV zone. Extracellular flux analysis indicated lower maximal respiration and spared respiratory capacity in control PV hepatocytes that were reversed upon alcohol feeding. These findings reveal a differential morphology and functional activity of mitochondria between PP and PV hepatocytes following alcohol feeding and that StARD1 may play a key role in the zonal-dependent liver injury characteristic of ALD.
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Etanol , Fígado , Animais , Camundongos , Etanol/farmacologia , Hepatócitos , Fígado/metabolismo , Mitocôndrias Hepáticas , Estresse OxidativoRESUMO
Exchangeable copper (CuEXC), mainly comprised copper (Cu) bound to albumin, has been proposed as a specific marker of Cu overload in Wilson's disease (WD). To the author's knowledge, there are no methods capable of determining reliably CuEXC to meet the requirements and challenges faced by a clinical trial. The present work describes a novel speciation strategy for the determination of the main Cu-species in human serum by anion-exchange high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). A label-free protein quantification approach was conducted where the concentration of Cu associated to the protein fraction was based on its relative peak area distribution and the total Cu concentration in the sample. Such a methodology was characterized in terms of selectivity, sensitivity, precision, and robustness. Due to the lack of speciated Cu-reference materials, protein recovery was assessed by comparison with that of species-specific (SS) isotope dilution (ID). For this, a double SS HPLC-ICP-IDMS method for Cu-albumin was developed and presented here for the first time. Three human sera (two frozen LGC8211 and ERM®-DA250a, and the lyophilised Seronorm™ Human) were analyzed using both the relative and ID quantification methods. The validated relative approach, with relative expanded uncertainties (k = 2) between 5.7 and 10.1% for Cu-albumin concentrations ranging from 112 to 455 µg kg-1 Cu, was found to be able to discriminate between healthy and WD populations in terms of Cu-albumin content. Also, using such methodology, underestimation of CuEXC by the classical EDTA/ultrafiltration method was demonstrated. The methodology developed in this work will be invaluable for quality control assessment and WD drug monitoring. This work describes a Cu-protein quantification approach for the determination of exchangeable Cu relevant to Wilson's Disease.
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Degeneração Hepatolenticular , Biomarcadores , Cobre , Degeneração Hepatolenticular/metabolismo , Humanos , Espectrometria de Massas/métodos , Análise EspectralRESUMO
BACKGROUND & AIMS: Besides their physiological role in bile formation and fat digestion, bile acids (BAs) synthesised from cholesterol in hepatocytes act as signalling molecules that modulate hepatocellular carcinoma (HCC). Trafficking of cholesterol to mitochondria through steroidogenic acute regulatory protein 1 (STARD1) is the rate-limiting step in the alternative pathway of BA generation, the physiological relevance of which is not well understood. Moreover, the specific contribution of the STARD1-dependent BA synthesis pathway to HCC has not been previously explored. METHODS: STARD1 expression was analyzed in a cohort of human non-alcoholic steatohepatitis (NASH)-derived HCC specimens. Experimental NASH-driven HCC models included MUP-uPA mice fed a high-fat high-cholesterol (HFHC) diet and diethylnitrosamine (DEN) treatment in wild-type (WT) mice fed a HFHC diet. Molecular species of BAs and oxysterols were analyzed by mass spectrometry. Effects of NASH-derived BA profiles were investigated in tumour-initiated stem-like cells (TICs) and primary mouse hepatocytes (PMHs). RESULTS: Patients with NASH-associated HCC exhibited increased hepatic expression of STARD1 and an enhanced BA pool. Using NASH-driven HCC models, STARD1 overexpression in WT mice increased liver tumour multiplicity, whereas hepatocyte-specific STARD1 deletion (Stard1ΔHep) in WT or MUP-uPA mice reduced tumour burden. These findings mirrored the levels of unconjugated primary BAs, ß-muricholic acid and cholic acid, and their tauroconjugates in STARD1-overexpressing and Stard1ΔHep mice. Incubation of TICs or PMHs with a mix of BAs mimicking this profile stimulated expression of genes involved in pluripotency, stemness and inflammation. CONCLUSIONS: The study reveals a previously unrecognised role of STARD1 in HCC pathogenesis, wherein it promotes the synthesis of primary BAs through the mitochondrial pathway, the products of which act in TICs to stimulate self-renewal, stemness and inflammation. LAY SUMMARY: Effective therapy for hepatocellular carcinoma (HCC) is limited because of our incomplete understanding of its pathogenesis. The contribution of the alternative pathway of bile acid (BA) synthesis to HCC development is unknown. We uncover a key role for steroidogenic acute regulatory protein 1 (STARD1) in non-alcoholic steatohepatitis-driven HCC, wherein it stimulates the generation of BAs in the mitochondrial acidic pathway, the products of which stimulate hepatocyte pluripotency and self-renewal, as well as inflammation.
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Ácidos e Sais Biliares/biossíntese , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/metabolismo , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais/genética , Adulto , Idoso , Animais , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Células Cultivadas , Estudos de Coortes , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Feminino , Deleção de Genes , Hepatócitos/metabolismo , Humanos , Fígado/patologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Hepatopatia Gordurosa não Alcoólica/genética , Fosfoproteínas/genética , Adulto JovemRESUMO
BACKGROUND & AIMS: Acetaminophen (APAP) overdose is a major cause of acute liver failure (ALF). Mitochondrial SH3BP5 (also called SAB) and phosphorylation of c-Jun N-terminal kinase (JNK) mediate the hepatotoxic effects of APAP. We investigated the involvement of steroidogenic acute regulatory protein (STARD1), a mitochondrial cholesterol transporter, in this process and sensitization by valproic acid (VPA), which depletes glutathione and stimulates steroidogenesis. METHODS: Nonfasted C57BL/6J mice (control) and mice with liver-specific deletion of STARD1 (Stard1ΔHep), SAB (SabΔHep), or JNK1 and JNK2 (Jnk1+2ΔHep) were given VPA with or without APAP. Liver tissues were collected and analyzed by histology and immunohistochemistry and for APAP metabolism, endoplasmic reticulum (ER) stress, and mitochondrial function. Adult human hepatocytes were transplanted into Fah-/-/Rag2-/-/Il2rg-/-/NOD (FRGN) mice to create mice with humanized livers. RESULTS: Administration of VPA before administration of APAP increased the severity of liver damage in control mice. The combination of VPA and APAP increased expression of CYP2E1, formation of NAPQI-protein adducts, and depletion of glutathione from liver tissues of control mice, resulting in ER stress and the upregulation of STARD1. Livers from control mice given VPA and APAP accumulated cholesterol in the mitochondria and had sustained mitochondrial depletion of glutathione and mitochondrial dysfunction. Inhibition of ER stress, by administration of tauroursodeoxycholic acid to control mice, prevented upregulation of STARD1 in liver and protected the mice from hepatoxicity following administration of VPA and APAP. Administration of N-acetylcysteine to control mice prevented VPA- and APAP-induced ER stress and liver injury. Stard1ΔHep mice were resistant to induction of ALF by VPA and APAP, despite increased mitochondrial levels of glutathione and phosphorylated JNK; we made similar observations in fasted Stard1ΔHep mice given APAP alone. SabΔHep mice or Jnk1+2ΔHep mice did not develop ALF following administration of VPA and APAP. The ability of VPA to increase the severity of APAP-induced liver damage was observed in FRGN mice with humanized liver. CONCLUSIONS: In studies of mice, we found that upregulation of STARD1 following ER stress mediates APAP hepatoxicity via SH3BP5 and phosphorylation of JNK1 and JNK2.
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Acetaminofen/toxicidade , Doença Hepática Induzida por Substâncias e Drogas/patologia , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Hepatócitos/patologia , Fosfoproteínas/metabolismo , Adulto , Animais , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Modelos Animais de Doenças , Overdose de Drogas/complicações , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hepatócitos/transplante , Humanos , Lipogênese/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoproteínas/genética , Esteroides/metabolismo , Quimeras de Transplante , Regulação para Cima , Ácido Valproico/administração & dosagemRESUMO
(1) Background: voltage-gated sodium channels (Navs) are integral membrane proteins that allow the sodium ion flux into the excitable cells and initiate the action potential. They comprise an α (Navα) subunit that forms the channel pore and are coupled to one or more auxiliary ß (Navß) subunits that modulate the gating to a variable extent. (2) Methods: after performing homology in silico modeling for all nine isoforms (Nav1.1α to Nav1.9α), the Navα and Navß protein-protein interaction (PPI) was analyzed chemometrically based on the primary and secondary structures as well as topological or spatial mapping. (3) Results: our findings reveal a unique isoform-specific correspondence between certain segments of the extracellular loops of the Navα subunits. Precisely, loop S5 in domain I forms part of the PPI and assists Navß1 or Navß3 on all nine mammalian isoforms. The implied molecular movements resemble macroscopic springs, all of which explains published voltage sensor effects on sodium channel fast inactivation in gating. (4) Conclusions: currently, the specific functions exerted by the Navß1 or Navß3 subunits on the modulation of Navα gating remain unknown. Our work determined functional interaction in the extracellular domains on theoretical grounds and we propose a schematic model of the gating mechanism of fast channel sodium current inactivation by educated guessing.
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Aminoácidos/química , Modelos Moleculares , Canais de Sódio Disparados por Voltagem/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Sódio/metabolismo , Canais de Sódio Disparados por Voltagem/químicaRESUMO
Fractionation data for cadmium in tobacco products, as obtained by sequential leaching of cadmium species with ICP-MS/MS analysis, and separately by X-ray absorption near edge structure (XANES) are presented here for the first time. The total amount of cadmium found in 3R4F cigarette cut tobacco was 1526 ± 42 µg kg-1, of which 5% was found in the smoke under ISO smoking conditions. XANES analysis showed that Cd in tobacco, cigarette smoke and ash was present in the + 2 oxidation state. Examination of the gas-particle partitioning of smoke cadmium suggests that Cd in mainstream smoke is best viewed as semi-volatile, existing in both particulate and gas phases. Sequential extraction of trapped tobacco smoke was carried out to get a deeper insight into the chemistry of cigarette smoke cadmium compounds. Consecutive extractions with ultrapure water, dilute (1%) nitric acid and 10% nitric acid led to extraction of a total amount of Cd which agreed with that obtained after microwave digestion of the whole sample, suggesting that cadmium was quantitatively leachable into aqueous/acidic solutions. Most Cd (~ 90% of the total Cd in the smoke condensate) was extracted into dilute nitric acid (likely as CdO, Cd(OH)2 and CdCO3) with a minor percentage (3%) extracted into water (likely as CdCl2) and in 10% nitric acid (likely as CdS). Extraction of trapped mainstream smoke with pentane, followed by ICP-MS/MS analysis, to examine the possible presence of organocadmium in 3R4F tobacco smoke, did not show the presence of organocadmium compounds above the method LOQ (2 µg kg-1), possibly due to their reactivity under the experimental conditions. The high selectivity with sufficient sensitivity achieved by ICP-MS/MS was invaluable to quantify Cd (at low µg kg-1levels) simultaneously with sulphur and chlorine in the tobacco smoke fractions of complex matrix. The cadmium chemistry in the smoke, identified in this study, is consistent with both relatively high lung absorption and DNA binding; both potentially important factors for disease progression in smokers. Graphical Abstract This paper provides quantitative fractionation data for cadmium in tobacco and smoke by using sequential leaching with ICPMS and XANES.
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Cádmio/análise , Nicotiana/química , Fumaça/análise , Espectrometria de Massas em Tandem/métodos , Produtos do Tabaco/análise , Espectroscopia por Absorção de Raios X/métodos , Adsorção , Cloro/análise , Etanol/química , Limite de Detecção , Oxirredução , Solventes/química , Enxofre/análiseRESUMO
A novel strategy for the absolute quantification of selenium (Se) included in selenoprotein P (SEPP1), an important biomarker for human nutrition and disease, including diabetes and cancer, is presented here for the first time. It is based on the use of species-specific double isotope dilution mass spectrometry (SSIDA) in combination with HPLC-ICP-MS/MS for the determination of protein bound Se down to the peptide level in a complex plasma matrix with a total content of Se of 105.5 µg kg(-1). The method enabled the selective Se speciation analysis of human plasma samples without the need of extensive cleanup or preconcentration steps as required for traditional protein mass spectrometric approaches. To assess the method accuracy, two plasma reference materials, namely, BCR-637 and SRM1950, for which literature data and a reference value for SEPP1 have been reported, were analyzed using complementary hyphenated methods and the species-specific approach developed in this work. The Se mass fractions obtained via the isotopic ratios (78)Se/(76)Se and (82)Se/(76)Se for each of the Se-peptides, namely, ENLPSLCSUQGLR (ENL) and AEENITESCQUR (AEE) (where U is SeCys), were found to agree within 2.4%. A relative expanded combined uncertainty (k = 2) of 5.4% was achieved for a Se (as SEPP1) mass fraction of approximately 60 µg kg(-1). This work represents a systematic approach to the accurate quantitation of plasma SEPP1 at clinical levels using SSIDA quantification. Such methodology will be invaluable for the certification of reference materials and the provision of reference values to clinical measurements and clinical trials.
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Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/química , Selênio/química , Selenoproteína P/sangue , Espectrometria de Massas em Tandem/métodos , Sequência de Aminoácidos , Humanos , Técnicas de Diluição do Indicador , Isótopos/química , Limite de Detecção , Selenoproteína P/análiseRESUMO
Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by impaired motor coordination due to neurological defects and cerebellar dysfunction caused by the accumulation of cholesterol in endolysosomes. Besides the increase in lysosomal cholesterol, mitochondria are also enriched in cholesterol, which leads to decreased membrane fluidity, impaired mitochondrial function and loss of GSH, and has been shown to contribute to the progression of NPC disease. S-Adenosyl-l-methionine (SAM) regulates membrane physical properties through the generation of phosphatidylcholine (PC) from phosphatidylethanolamine (PE) methylation and functions as a GSH precursor by providing cysteine in the transsulfuration pathway. However, the role of SAM in NPC disease has not been investigated. Here we report that Npc1-/- mice exhibit decreased brain SAM levels but unchanged S-adenosyl-l-homocysteine content and lower expression of Mat2a. Brain mitochondria from Npc1-/- mice display decreased mitochondrial GSH levels and liquid chromatography-high resolution mass spectrometry analysis reveal a lower PC/PE ratio in mitochondria, contributing to increased mitochondrial membrane order. In vivo treatment of Npc1-/- mice with SAM restores SAM levels in mitochondria, resulting in increased PC/PE ratio, mitochondrial membrane fluidity and subsequent replenishment of mitochondrial GSH levels. In vivo SAM treatment improves the decline of locomotor activity, increases Purkinje cell survival in the cerebellum and extends the average and maximal life spam of Npc1-/- mice. These findings identify SAM as a potential therapeutic approach for the treatment of NPC disease.
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Encéfalo , Glutationa , Fluidez de Membrana , Membranas Mitocondriais , Doença de Niemann-Pick Tipo C , S-Adenosilmetionina , Animais , Camundongos , S-Adenosilmetionina/metabolismo , Membranas Mitocondriais/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , Glutationa/metabolismo , Encéfalo/metabolismo , Mitocôndrias/metabolismo , Proteína C1 de Niemann-Pick , Modelos Animais de Doenças , Camundongos Knockout , Fosfatidilcolinas/metabolismoRESUMO
BACKGROUND AND OBJECTIVES: Fibrosis contributes to 45% of deaths in industrialized nations and is characterized by an abnormal accumulation of extracellular matrix (ECM). There are no specific anti-fibrotic treatments for liver fibrosis, and previous unsuccessful attempts at drug development have focused on preventing ECM deposition. Because liver fibrosis is largely acknowledged to be reversible, regulating fibrosis resolution could offer novel therapeutical options. However, little is known about the mechanisms controlling ECM remodeling during resolution. Changes in proteolytic activity are essential for ECM homeostasis and macrophages are an important source of proteases. Herein, in this study we evaluate the role of macrophage-derived cathepsin D (CtsD) during liver fibrosis. METHODS: CtsD expression and associated pathways were characterized in single-cell RNA sequencing and transcriptomic datasets in human cirrhosis. Liver fibrosis progression, reversion and functional characterization were assessed in novel myeloid-CtsD and hepatocyte-CtsD knock-out mice. RESULTS: Analysis of single-cell RNA sequencing datasets demonstrated CtsD was expressed in macrophages and hepatocytes in human cirrhosis. Liver fibrosis progression, reversion and functional characterization were assessed in novel myeloid-CtsD (CtsDΔMyel) and hepatocyte-CtsD knock-out mice. CtsD deletion in macrophages, but not in hepatocytes, resulted in enhanced liver fibrosis. Both inflammatory and matrisome proteomic signatures were enriched in fibrotic CtsDΔMyel livers. Besides, CtsDΔMyel liver macrophages displayed functional, phenotypical and secretomic changes, which resulted in a degradomic phenotypical shift, responsible for the defective proteolytic processing of collagen I in vitro and impaired collagen remodeling during fibrosis resolution in vivo. Finally, CtsD-expressing mononuclear phagocytes of cirrhotic human livers were enriched in lysosomal and ECM degradative signaling pathways. CONCLUSIONS: Our work describes for the first-time CtsD-driven lysosomal activity as a central hub for restorative macrophage function during fibrosis resolution and opens new avenues to explore their degradome landscape to inform drug development.
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Catepsina D , Cirrose Hepática , Macrófagos , Camundongos Knockout , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Cirrose Hepática/genética , Animais , Catepsina D/metabolismo , Catepsina D/genética , Macrófagos/metabolismo , Camundongos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Matriz Extracelular/metabolismo , Hepatócitos/metabolismoRESUMO
Alcoholic (ASH) and nonalcoholic. (NASH).steatohepatitis are advanced.stages.of.fatty.liver.disease.Methionine adenosyltransferase 1A (MAT1A) plays a key role in hepatic methionine metabolism and germline Mat1a deletion in mice promotes NASH. Acid sphingomyelinase (ASMase) triggers hepatocellular apoptosis and liver fibrosis and has been shown to downregulate MAT1A expression in the context of fulminant liver failure. Given the role of ASMase in steatohepatitis development, we investigated the status of ASMase in Mat1a-/- mice and the regulation of ASMase by SAM/SAH. Consistent with its role in NASH, Mat1a-/- mice fed a choline-deficient (CD) diet exhibited macrosteatosis, inflammation, fibrosis and liver injury as well as reduced total and mitochondrial GSH levels. Our data uncovered an increased basal expression and activity of ASMase but not neutral SMase in Mat1a-/- mice, which further increased upon CD feeding. Interestingly, adenovirus-mediated shRNA expression targeting ASMase reduced ASMase activity and protected Mat1a-/- mice against CD diet-induced NASH. Similar results were observed in CD fed Mat1a-/- mice by pharmacological inhibition of ASMase with amitriptyline. Moreover, Mat1a/ASMase double knockout mice were resistant to CD-induced NASH. ASMase knockdown protected wild type mice against NASH induced by feeding a diet deficient in methionine and choline. Furthermore, Mat1a-/- mice developed acute-on-chronic ASH and this outcome was ameliorated by amitriptyline treatment. In vitro data in primary mouse hepatocytes revealed that decreased SAM/SAH ratio increased ASMase mRNA level and activity. MAT1A and ASMase mRNA levels exhibited an inverse correlation in liver samples from patients with ASH and NASH. Thus, disruption of methionine metabolism sensitizes to steatohepatitis by ASMase activation via decreased SAM/SAH. These findings imply that MAT1A deletion and ASMase activation engage in a self-sustained loop of relevance for steatohepatitis.
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Hepatite , Metionina , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Amitriptilina/farmacologia , Amitriptilina/metabolismo , Colina , Dieta , Modelos Animais de Doenças , Fígado/metabolismo , Metionina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Racemetionina/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , Hepatite/metabolismoRESUMO
Abnormal cholesterol metabolism is linked to many neurodegenerative disorders. Here, we present a protocol for the production of a recombinant protein consisting of a Glutathione-S-Transferase tag fused with the Perfringolysin O (PFO). The GST-PFO tag enables analysis of the localization of cholesterol in subcellular membranes of human and mice brain and liver tissues. We have used this approach for samples from Niemann-Pick type C disease and non-alcoholic steatohepatitis models. The construct may also have applications for the diagnosis of cholesterol-accumulating disorders. For complete details on the use and execution of this protocol, please refer to Kwiatkowska et al. (2014).
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Colesterol , Proteínas Hemolisinas , Animais , Toxinas Bacterianas , Encéfalo/metabolismo , Colesterol/metabolismo , Proteínas Hemolisinas/genética , Humanos , Fígado/metabolismo , CamundongosRESUMO
We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles.
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PURPOSE: A case of cauda equina lesion as a result of recurrent adjacent segment degeneration (ASD) after multiple lumbar fusions is reported. ASD might be a consequence of biomechanical overload or simply a normal degenerative process. The reported clinical relevance of ASD is rather low. We describe an unusual case of cauda equina compression at L1-L2 in a patient who had undergone L2-L4 fusion 8 years previously and 2 decompression-fusion surgeries 16 years before. MATERIALS AND METHODS: A 72-year-old man, who had two previous lumbar fusion-decompression procedures, underwent a third lumbar surgery in December 2000 to treat symptomatic spinal canal stenosis associated with L3-L4 pseudoarthrosis. After a symptom-free period of 8 years, the patient experienced low back pain radiating to both legs while standing, associated with saddle sensory disturbances and incontinence. Physical examination ruled out significant motor deficits. Plain radiographs showed solid fusion from L2 to L4, good spinal alignment, and low-grade L1-L2 retrolisthesis. Stainless steel pedicular instrumentation distorted magnetic resonance imaging, preventing adequate spinal canal evaluation. Electromyography demonstrated signs of cauda equina compression (bilateral L3-S2). CT myelography showed a stop at L1-L2, due to a severe spinal canal stenosis. L1-L2 decompression and fusion were performed. RESULTS: After an uneventful surgery with no complications, the symptoms abated and incontinence recovered. CONCLUSIONS: Even if the reported clinical relevance of ASD is very low, fused patients with a constitutional narrow spinal canal are at risk of developing severe neural compression at the level adjacent to the fusion.
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Dor Lombar/cirurgia , Vértebras Lombares/cirurgia , Polirradiculopatia/cirurgia , Estenose Espinal/cirurgia , Idoso , Cauda Equina/diagnóstico por imagem , Cauda Equina/cirurgia , Descompressão Cirúrgica , Humanos , Dor Lombar/diagnóstico por imagem , Vértebras Lombares/diagnóstico por imagem , Masculino , Polirradiculopatia/diagnóstico por imagem , Radiografia , Recidiva , Fusão Vertebral , Estenose Espinal/diagnóstico por imagem , Resultado do TratamentoRESUMO
INTRODUCTION: The aim of this study is to explore the effectiveness of a training programme aimed at managing patients' chronic pain in physiotherapy students in Spain. The programme addressed providing them with efficient skills to manage patients' chronic pain from psychological flexibility (PF) perspective. METHODS: The programme integrates communication skills training into PF-based training. It sought to contribute to better recognise the role of psychosocial factors in chronic pain and to better promote adherence to treatment. This is an observational study with a pre- and post-training programme design and a 2-month follow-up. A total of 35 physiotherapy students, divided into three groups, participated in a 10-hour training course. Training focused on three areas: (1) communication skills, (2) therapeutic adherence and (3) managing distress and pain. The three areas were addressed from the PF point of view. Impact of training was measured through standardised questionnaires that assessed attitudes towards chronic pain, an ad hoc questionnaire that assessed responses to difficult communicative situations and a training satisfaction scale. RESULTS: Final analyses showed that attitudes changed significantly after training, biomedical attitude scores decreased and biopsychosocial attitude increased, while pain was considered less disabling, and informed empathic responses in communication situations increased. These changes were maintained at 2-month follow-up. Satisfaction with the training was high. CONCLUSION: We conclude that a brief training programme based on the PF model may help students develop a more comprehensive approach and improve their skills for managing chronic pain.
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The association of nonalcoholic steatohepatitis (NASH) with obesity and type 2 diabetes is a major determinant factor for the continued rise of NASH-driven HCC. Unfortunately, the mechanisms underlying the progression from NASH to HCC are not well-understood. Steatosis is characterized by the accumulation of different lipid species, and cholesterol has emerged as an important player in NASH development, which has been shown to promote NASH-driven HCC. However, recent findings indicated a tumor suppressor role of cholesterol in liver carcinogenesis and HCC development. Thus, we examined the contribution of hepatic steatosis with or without cholesterol accumulation induced by dietary or genetic approaches in liver tumorigenesis and whether the role of cholesterol in NASH-driven HCC is species-dependent. While diethylnitrosamine (DEN) treatment to rats or mice fed a choline-deficient diet decreased the hepatic steatosis, feeding an atherogenic diet enriched in cholesterol potentiated the liver tumor markers. Similar effects were observed in DEN-treated transgenic SREBP-2 mice but not wild-type (WT) mice fed a regular chow diet. Remarkably, long-term feeding of a high-fat high-cholesterol diet (HFHC) but not a high-fat diet (HFD) to WT mice caused severe NASH with spontaneous progression to HCC. A similar outcome was observed in MUP-uPA transgenic mice fed a HFHC diet, which resulted in increased liver tumors and expression of the genes involved in the immune checkpoints. Ezetimibe treatment ameliorated chronic liver disease and, more importantly, tumor multiplicity in HFHC-fed MUP-uPA mice or DEN-treated WT mice. Thus, these results revealed a differential role of steatosis and cholesterol in NASH-driven HCC and indicated that the tumor-promoter role of cholesterol is species-independent and associated with impaired immunosurveillance.
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Niemann-Pick type C (NPC) disease, a lysosomal storage disorder caused by defective NPC1/NPC2 function, results in the accumulation of cholesterol and glycosphingolipids in lysosomes of affected organs, such as liver and brain. Moreover, increase of mitochondrial cholesterol (mchol) content and impaired mitochondrial function and GSH depletion contribute to NPC disease. However, the underlying mechanism of mchol accumulation in NPC disease remains unknown. As STARD1 is crucial in intramitochondrial cholesterol trafficking and acid ceramidase (ACDase) has been shown to regulate STARD1, we explored the functional relationship between ACDase and STARD1 in NPC disease. Liver and brain of Npc1-/- mice presented a significant increase in mchol levels and STARD1 expression. U18666A, an amphiphilic sterol that inhibits lysosomal cholesterol efflux, increased mchol levels in hepatocytes from Stard1f/f mice but not Stard1ΔHep mice. We dissociate the induction of STARD1 expression from endoplasmic reticulum stress, and establish an inverse relationship between ACDase and STARD1 expression and LRH-1 levels. Hepatocytes from Npc1+/+ mice treated with U18666A exhibited increased mchol accumulation, STARD1 upregulation and decreased ACDase expression, effects that were reversed by cholesterol extraction with 2-hydroxypropyl-ß-cyclodextrin. Moreover, transfection of fibroblasts from NPC patients with ACDase, decreased STARD1 expression and mchol accumulation, resulting in increased mitochondrial GSH levels, improved mitochondrial functional performance, decreased oxidative stress and protected NPC fibroblasts against oxidative stress-mediated cell death. Our results demonstrate a cholesterol-dependent inverse relationship between ACDase and STARD1 and provide a novel approach to target the accumulation of cholesterol in mitochondria in NPC disease.
Assuntos
Doença de Niemann-Pick Tipo C , Fosfoproteínas/metabolismo , Ceramidase Ácida/metabolismo , Animais , Colesterol/metabolismo , Humanos , Lisossomos/metabolismo , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , Estresse OxidativoRESUMO
A method based on asymmetric flow field-flow fractionation (AF4) coupled to ultraviolet-visible (UV-vis) spectroscopy and inductively coupled plasma mass spectrometry (ICP-MS) has been developed for silver nanoparticles (Ag NPs) detection and quantification in bivalve molluscs. Samples were pre-treated using a conventional enzymatic (pancreatin and lipase) hydrolysis procedure (37 °C, 12 h). AF4 was performed using a regenerated cellulose (RC) membrane (10 kDa, 350 µm spacer) and aqueous 5 mM Tris-HCl pH = 7.4 as carrier. AF4 separation was achieved with a program that included a focusing step with tip and focus flows of 0.20 and 3.0 mL min-1, respectively, and an injection time of 4.0 min. Elution of different size fractions was performed using a cross flow of 3.0 mL min-1 for 15 min, followed by linear cross flow decrease for 7.5 min, and a washing step for 9.4 min with no cross flow. Several bivalve molluscs (clams, oysters and variegated scallops) were analysed for total Ag content (ICP-MS after microwave assisted acid digestion), and for Ag NPs by the method presented here. Results show that Ag NPs are detected at the same elution time than proteins (UV monitoring at 280 and 405 nm), which suggests a certain interaction occurred between Ag NPs with proteins in the enzymatic extracts. AF4-UV-ICP-MS fractograms also suggest different Ag NPs size distributions for selected samples. Membrane recoveries, determined by peak area comparison of fractograms with and without application of cross flow, were within the 49-121% range. Confirmation of the presence Ag NPs in the investigated enzymatic extracts was demonstrated by SEM after an oxidative pre-treatment based on hydrogen peroxide and microwave irradiation.
Assuntos
Fracionamento por Campo e Fluxo , Nanopartículas Metálicas , Hidrólise , Espectrometria de Massas , Nanopartículas Metálicas/análise , Tamanho da Partícula , Alimentos Marinhos , Prata , Análise EspectralRESUMO
BACKGROUND: Safe and rational development of nanomaterials for clinical translation requires the assessment of potential biocompatibility. Autophagy, a critical homeostatic pathway intrinsically linked to cellular health and inflammation, has been shown to be affected by nanomaterials. It is, therefore, important to be able to assess possible interactions of nanomaterials with autophagic processes. RESULTS: CEM (T cell), Raji (B lymphocyte), and THP-1 (human monocyte) cell lines were subject to treatment with rapamycin and chloroquine, known to affect the autophagic process, in order to evaluate cell line-specific responses. Flow cytometric quantification of a fluorescent autophagic vacuole stain showed that maximum observable effects (105%, 446%, and 149% of negative controls) were achieved at different exposure durations (8, 6, and 24 h for CEM, Raji, and THP-1, respectively). THP-1 was subsequently utilised as a model to assess the autophagic impact of a small library of nanomaterials. Association was observed between hydrodynamic size and autophagic impact (r2 = 0.11, p = 0.004). An ELISA for p62 confirmed the greatest impact by 10 nm silver nanoparticles, abolishing p62, with 50 nm silica and 180 nm polystyrene also lowering p62 to a significant degree (50%, 74%, and 55%, respectively, p < 0.05). CONCLUSIONS: This data further supports the potential for a variety of nanomaterials to interfere with autophagic processes which, in turn, may result in altered cellular function and viability. The association of particle size with impact on autophagy now warrants further investigation.
RESUMO
Alzheimer´s disease (AD) is a progressive neurodegenerative disorder of complex etiology, while Down syndrome (DS) is considered a genetically determined form of AD. Alterations in cholesterol homeostasis have been linked to AD although the role in this association is not well understood. Increased expression of STARD1 and NPC1, which are involved in intracellular cholesterol trafficking, has been reported in experimental AD models but not in patients with AD. Here we analyzed endolysosomal/mitochondrial cholesterol homeostasis, expression of NPC1 and STARD1 and correlation with pathological markers of AD in cortex and hippocampus from post-mortem brains from patients with AD and DS. NPC1 expression was observed in hippocampus from patients with AD and DS. Moreover, STARD1 expression increased in hippocampus and cortex from patients with AD and DS, respectively, and its immunoreactivity discriminated controls from AD or DS with a better accuracy than Aß42. Hippocampal areas stained with the recombinant GST-PFO probe showed increased mitochondrial cholesterol within astrocytes of brains from patients with AD and DS-brains compared to controls. Lysosomal cholesterol accumulation within hippocampal astrocytes was higher in DS than in AD. These data revealed increased intracellular cholesterol loading in hippocampus from patient with AD and DS and suggest that STARD1 could be a potential pre-clinical marker associated with early stages of AD pathology.