143 research outputs found
Intrahepatic Cholestasis of Pregnancy: The Usefulness of Serum Bile Acid Profile for Diagnosis and Treatment
Coenzyme Q10 deficiency: an orphan disease in argentina?
En la actualidad, el estudio de la coenzima Q10 (CoQ10) representa un área de vacancia en Argentina. La CoQ10 es fundamental para la producción celular de energía y es considerada un potente antioxidante endógeno. Su deficiencia se asocia a diversas patologías que revierten parcial o totalmente con la suplementación terapéutica de CoQ10, especialmente si es administrada en niños. Sin embargo, en Argentina, la CoQ10 es considerada un suplemento dietario y para el tratamiento sólo se encuentra disponible comercialmente una formulación sólida que no sólo posee baja biodisponibilidad sino que además es inadecuada para su uso pediátrico, resultando en una pobre o inexistente respuesta terapéutica. Un mayor conocimiento de las patologías que involucran la deficiencia de CoQ10 facilitaría al equipo de salud el manejo de estas enfermedades que actualmente se conocen como “huérfanas” en Argentina.At present the study of coenzyme Q10 (CoQ10) is a large vacant area in Argentina. CoQ10 is essential for cellular energy production and is considered a potent endogenous antioxidant. Its deficiency is associated to different pathologies that partially or completely reversed with the therapeutic supplementation of CoQ10, particularly if it is administered in children. However, in Argentina, CoQ10 is considered a dietary supplement and for treatment is only available a solid formulation that not only has low bioavailability but also is unsuitable for pediatric use, resulting in poor or no therapeutic response. A greater understanding of diseases involving CoQ10 deficiency would facilitate the health team management of this disease that today are known as "orphan" in Argentina.Fil: Lucangioli, Silvia Edith. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Tripodi, Valeria Paula. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin
TGFβ impairs HNF1α functional activity in Epithelial-to-Mesenchymal Transition interfering with the recruitment of CBP/p300 acetyltransferases
The cytokine transforming growth factor β (TGFβ) plays a crucial role in the induction of both epithelial-to-mesenchymal transition (EMT) program and fibro-cirrhotic process in the liver, where it contributes also to organ inflammation following several chronic injuries. All these pathological situations greatly increase the risk of hepatocellular carcinoma (HCC) and contribute to tumor progression. In particular, late-stage HCCs are characterized by constitutive activation of TGFβ pathway and by an EMT molecular signature leading to the acquisition of invasive and metastatic properties. In these pathological conditions, the cytokine has been shown to induce the transcriptional downregulation of HNF1α, a master regulator of the epithelial/hepatocyte differentiation and of the EMT reverse process, the mesenchymal-to-epithelial transition (MET). Therefore, the restoration of HNF1α expression/activity has been proposed as targeted therapeutic strategy for liver fibro-cirrhosis and late-stage HCCs. In this study, TGFβ is found to trigger an early functional inactivation of HNF1α during EMT process that anticipates the effects of the transcriptional downregulation of its own gene. Mechanistically, the cytokine, while not affecting the HNF1α DNA-binding capacity, impaired its ability to recruit CBP/p300 acetyltransferases on target gene promoters and, consequently, its transactivating function. The loss of HNF1α capacity to bind to CBP/p300 and HNF1α functional inactivation have been found to correlate with a change of its posttranslational modification profile. Collectively, the results obtained in this work unveil a new level of HNF1α functional inactivation by TGFβ and contribute to shed light on the early events triggering EMT in hepatocytes. Moreover, these data suggest that the use of HNF1α as anti-EMT tool in a TGFβ-containing microenvironment may require the design of new therapeutic strategies overcoming the TGFβ-induced HNF1α inactivation
YAP integrates the regulatory Snail/HNF4α circuitry controlling epithelial/hepatocyte differentiation
Yes-associated protein (YAP) is a transcriptional co-factor involved in many cell processes, including development, proliferation, stemness, differentiation, and tumorigenesis. It has been described as a sensor of mechanical and biochemical stimuli that enables cells to integrate environmental signals. Although in the liver the correlation between extracellular matrix elasticity (greatly increased in the most of chronic hepatic diseases), differentiation/functional state of parenchymal cells and subcellular localization/activation of YAP has been previously reported, its role as regulator of the hepatocyte differentiation remains to be clarified. The aim of this study was to evaluate the role of YAP in the regulation of epithelial/hepatocyte differentiation and to clarify how a transducer of general stimuli can integrate tissue-specific molecular mechanisms determining specific cell outcomes. By means of YAP silencing and overexpression we demonstrated that YAP has a functional role in the repression of epithelial/hepatocyte differentiation by inversely modulating the expression of Snail (master regulator of the epithelial-to-mesenchymal transition and liver stemness) and HNF4α (master regulator of hepatocyte differentiation) at transcriptional level, through the direct occupancy of their promoters. Furthermore, we found that Snail, in turn, is able to positively control YAP expression influencing protein level and subcellular localization and that HNF4α stably represses YAP transcription in differentiated hepatocytes both in cell culture and in adult liver. Overall, our data indicate YAP as a new member of the HNF4/Snail epistatic molecular circuitry previously demonstrated to control liver cell state. In this model, the dynamic balance between three main transcriptional regulators, that are able to control reciprocally their expression/activity, is responsible for the induction/maintenance of different liver cell differentiation states and its modulation could be the aim of therapeutic protocols for several chronic liver diseases
Development of Amine Capillary Column Applied to the Analysis of Basic Compounds by Electrochromatography
An amine capillary column was developed in a fused-silica capillary as stationary phase and appliedto separation of basic compounds by capillary electrochromatography (CEC). The functionalizedcapillary was prepared by inmobilization of 3-Aminopropyl)triethoxysilane (APTES). TheCEC conditions including APTES and buffer concentration, pH and applied voltage were investigatedto obtain the optimal CEC system for the separation of three anesthetics as basic test molecules.The capillary column provided an efficiency of up to 20,000 plates/m. Lidocaine, ketamineand xilacine were baseline separated under the running conditions with 10 mM Na2HPO4 pH 9.0 asBGE. The applied voltage was 5 kV temperature was set at 25˚C and UV detection was performed.The resolutions were 4.97 and 1.53 for ketamine, lidocaine and xilacine, respectively. The columnused in CEC mode showed better separation of the anesthetics compared with those used in thecapillary zone mode. The comparison with reversed stationary phases used in HPLC in terms ofresolution (Rs), sensitivity (LOD), efficiency (N), precision (RSD), asymmetry (T) was also performed.This study provided an alternative way for the CEC separation of basic compounds anddemonstrated the improvement in the chromatographic parametersFil: Flor, Sabrina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; ArgentinaFil: Huala, Juan Martin. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; ArgentinaFil: Tripodi, Valeria Paula. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; ArgentinaFil: Lucangioli, Silvia Edith. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentin
HDAC1 inhibition by MS-275 in mesothelial cells limits cellular invasion and promotes MMT reversal
Peritoneal fibrosis is a pathological alteration of the peritoneal membrane occurring in a variety of conditions including peritoneal dialysis (PD), post-surgery adhesions and peritoneal metastases. The acquisition of invasive and pro-fibrotic abilities by mesothelial cells (MCs) through induction of MMT, a cell-specific form of EMT, plays a main role in this process. Aim of this study was to evaluate possible effects of histone deacetylase (HDAC) inhibitors, key components of the epigenetic machinery, in counteracting MMT observed in MCs isolated from effluent of PD patients. HDAC inhibitors with different class/isoform selectivity have been used for pharmacological inhibition. While the effect of other inhibitors was limited to a partial E-cadherin re-expression, MS-275, a HDAC1-3 inhibitor, promoted: (i) downregulation of mesenchymal markers (MMP2, Col1A1, PAI-1, TGFβ1, TGFβRI) (ii) upregulation of epithelial markers (E-cadherin, Occludin), (iii) reacquisition of an epithelial-like morphology and (iv) marked reduction of cellular invasiveness. Results were confirmed by HDAC1 genetic silencing. Mechanistically, MS-275 causes: (i) increase of nuclear histone H3 acetylation (ii) rescue of the acetylation profile on E-cadherin promoter, (iii) Snail functional impairment. Overall, our study, pinpointing a role for HDAC1, revealed a new player in the regulation of peritoneal fibrosis, providing the rationale for future therapeutic opportunities
SILAC labeling coupled to shotgun proteomics analysis of membrane proteins of liver stem/hepatocyte allows to candidate the inhibition of TGF-beta pathway as causal to differentiation
International audienceDespite extensive research on hepatic cells precursors and their differentiated states, much remains to be learned about the mechanism underlying the self-renewal and differentiation. We apply the SILAC (stable isotope labeling by amino acids in cell culture) approach to quantitatively compare the membrane proteome of the resident liver stem cells (RLSCs) and their progeny spontaneously differentiated into epithelial/hepatocyte (RLSCdH). By means of nanoLC-MALDI-TOF/TOF approach, we identified and quantified 248 membrane proteins and 57 of them were found modulated during hepatocyte differentiation. Functional clustering of differentially expressed proteins by Ingenuity Pathway Analysis revealed that the most of membrane proteins found to be modulated are involved in cell-to-cell signaling/interaction pathways. Moreover, the upstream prediction analysis of proteins involved in cell-to-cell signaling and interaction unveiled that the activation of the mesenchymal to epithelial transition (MET), by the repression of TGFB1/Slug signaling, may be causal to hepatocyte differentiation. Taken together, this study increases the understanding of the underlying mechanisms modulating the complex biological processes of hepatic stem cell proliferation and differentiation
Cholestasis: The Close Relationship between Bile Acids and Coenzyme Q10
Cholestasis is defined as the impairment in formation or excretion of bile from the liver to the intestine. It may result from defects in intrahepatic production of bile, impairment of hepatic transmembrane transporters, or mechanical obstruction to bile flow. In cholestasis, hepatocytes are exposed to high levels of bile acids, particularly those bearing hydrophobic properties. The increase in bile acids induces oxidative stress, leading to an imbalance in the prooxidant:antioxidant ratio which determines the final cellular redox status. This chapter will focus on the close relationship between bile acids and the most powerful endogenous antioxidant, coenzyme Q10 in cholestasis, and the eventual alternative therapeutic option of CoQ10 supplementation to current traditional therapies
Epigenetic control of EMT/MET dynamics: HNF4α impacts DNMT3s through miRs-29
Background and aims: Epithelial-to-mesenchymal transition (EMT) and the reverse mesenchymal-to-epithelial
transition (MET) are manifestations of cellular plasticity that imply a dynamic and profound gene expression
reprogramming. While a major epigenetic code controlling the coordinated regulation of a whole transcriptional
profile is guaranteed by DNA methylation, DNA methyltransferase (DNMT) activities in EMT/MET dynamics are
still largely unexplored.
Here, we investigated the molecular mechanisms directly linking HNF4α, the master effector of MET, to the
regulation of both de novo of DNMT 3A and 3B.
Methods: Correlation among EMT/MET markers, microRNA29 and DNMT3s expression was evaluated by
RT-qPCR, Western blotting and immunocytochemical analysis. Functional roles of microRNAs and DNMT3s
were tested by anti-miRs, microRNA precursors and chemical inhibitors. ChIP was utilized for investigating
HNF4α DNA binding activity.
Results: HNF4α silencing was sufficient to induce positive modulation of DNMT3B, in in vitro differentiated
hepatocytes as well as in vivo hepatocyte-specific Hnf4α knockout mice, and DNMT3A, in vitro, but not
DNMT1. In exploring the molecular mechanisms underlying these observations, evidence have been gathered
for (i) the inverse correlation between DNMT3 levels and the expression of their regulators miR-29a and miR-
29b and (ii) the role of HNF4α as a direct regulator of miR-29a-b transcription. Notably, during TGFβ-induced
EMT, DNMT3s' pivotal function has been proved, thus suggesting the need for the repression of these DNMTs
in the maintenance of a differentiated phenotype.
Conclusions: HNF4α maintains hepatocyte identity by regulating miR-29a and -29b expression, which in turn
control epigenetic modifications by limiting DNMT3A and DNMT3B levels
Miniaturized imprinted solid phase extraction to the selective analysis of Coenzyme Q10 in urine
Coenzyme Q10 (CoQ10) is an important cofactor in the mitochondrial respiratory chain and a potent endogenous antioxidant. CoQ10 deficiency is currently associated with numerous diseases like mitochondrial and neurodegenerative pathologies, in which the earliest diagnosis and treatment with CoQ10 supplementation becomes paramount for patient's treatment. Consequently, the determination of CoQ10 levels in different biological matrices positions as a fundamental tool. Urine is an attractive and non-invasive alternative source to tissue, blood or other biofluids for CoQ10 analysis. However, it poses an analytical challenge, as it generally requires a complex sample preparation, with multiple steps. In this work we developed and validated a molecularly imprinted polymer solid phase extraction (MIP-SPE) followed by a HPLC-MS/MS method for the analysis of CoQ10 in urine. The MIP-SPE method developed is simple and fast compared to previously traditional reported methods, with reduced processing time, improved sample cleaning and excellent recovery values, along with its inherent high selectivity. The developed chromatographic method was validated according to FDA guidelines, and demonstrated to be suitable for the analysis of CoQ10 in urine samples with LOQ and LOD values of 0.6 ng/mL and 0.2 ng/mL of CoQ10 in urine respectively. Recovery values at three concentration levels were higher than 90.0%.The proposed method is amenable to be applied in pediatric patients due to the low sample requirement and useful for diagnosis and post-treatment control.Fil: García Becerra, Cristian Ezequiel. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; ArgentinaFil: Baez, Francisco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Lucangioli, Silvia Edith. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; ArgentinaFil: Flor, Sabrina Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Química Analítica y Fisicoquímica; ArgentinaFil: Tripodi, Valeria Paula. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Tecnología Farmacéutica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin
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