Poster presentado en el XXXIV Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular... more Poster presentado en el XXXIV Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular SEBBM, celebrado del 5 al 8 de septiembre de 2011 en Barcelona (Espana)
Trabajo presentado al XXVth International Symposium on Cerebral Blood Flow, Metabolism and Functi... more Trabajo presentado al XXVth International Symposium on Cerebral Blood Flow, Metabolism and Function y a la Xth International Conference on Quantification of Brain Function with PET celebrados del 25 al 28 de mayo en Barcelona.
Comunicacion presentada en el IX Simposi de Neurobiologia Experimental, celebrado los dias 22 y 2... more Comunicacion presentada en el IX Simposi de Neurobiologia Experimental, celebrado los dias 22 y 23 de octubre de 2014 en Barcelona y organizado por la Societat Catalana de Biologia del Institut d'Estudis Catalans
Applied Immunohistochemistry & Molecular Morphology, Mar 3, 2020
Humanized antibodies targeting programmed death receptor 1 (PD-1) or its ligand (PD-L1) have been... more Humanized antibodies targeting programmed death receptor 1 (PD-1) or its ligand (PD-L1) have been approved for the treatment of different cancers. Some of these antibodies show a correlation between the tissue expression of PD-L1 and response. Evaluation of PD-L1 expression presents multiple challenges, but some preanalytical issues such as tissue fixation have been scarcely evaluated. With the hypothesis that immunohistochemical staining of PD-L1 may be impacted by the time of specimen fixation, we evaluated differences in its expression in tonsil samples exposed to predefined fixation times. Random nontumoral tonsillectomy specimens were blindly evaluated in tissue microarray slides after staining with SP142 and SP263 antibodies. With fixation times ranging from 12 to 72 hours, between 2.8% and 6.1% of the samples were considered to be suboptimally stained, with no differences between the 2 antibodies within these fixation times. A significantly higher proportion of samples exposed to a fixation time of 96 hours presented suboptimal immunostaining (15.6%, P<0.0001). In addition, suboptimally stained spots were 20.8% using SP142 and 10.4% using SP263 after 96 hours of fixation (P=0.046). In conclusion, the quality of staining for PD-L1 in tonsil samples decreased with overfixation of the specimen at times >72 hours. Samples exposed to formaldehyde for longer periods presented suboptimal results for both clones, but the SP142 antibody presented a significantly lower tolerance to formalin overexposure than SP263. These results indicate the relevance of a controlled preanalytical processing of samples and particularly the length of fixation of tumor specimens.
Applied Immunohistochemistry & Molecular Morphology
Antibodies targeting programmed death receptor 1 or programmed death ligand 1 (PD-L1) have become... more Antibodies targeting programmed death receptor 1 or programmed death ligand 1 (PD-L1) have become a standard of care to treat different cancers; for some of these tumors, there is a correlation between tissue expression of PD-L1 and response rates in patients. Although most of the analytical challenges in the evaluation of PD-L1 expression have been standardized, preanalytical issues have been less explored. The objective of this study was to evaluate the impact of time of ischemia on the performance of 2 commonly used antibodies against PD-L1. Sixteen tonsillectomy samples were kept in ischemia for <30 minutes from sample obtention (control) and 1, 3, 6, 12, and 24 hours at room temperature before formalin fixation and paraffin embedding. Selected areas were inserted into TMA paraffin recipient blocks stained with SP142 and SP263 antibodies and evaluated by 2 blind observers. The proportion of suboptimally stained samples was significantly higher for samples with cold ischemia t...
Background Valuable clone collections encoding the complete ORFeomes for some model organisms hav... more Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusi...
Journal of Molecular and Cellular Cardiology, Apr 1, 2005
Cardiac hypertrophy and heart failure occur in association to alterations in glucose uptake and m... more Cardiac hypertrophy and heart failure occur in association to alterations in glucose uptake and metabolism. Phenylephrine, among other hypertrophic agonists, has been reported to increase expression of GLUT1 in neonatal rat cardiac myocytes by activating transcription. However, the specific cis- or trans-acting factors in the GLUT1 gene that are targeted by this agonist remain elusive. Here we describe that the activity of the -99/+134 basal promoter of rat GLUT1 is increased by phenylephrine. Nevertheless, this is not mediated by previously described binding sites (GC-box, MG1E) in the promoter. Rather, the TATA box is required by the agonist to activate transcription from the promoter. Interestingly, The Ras-ERK mitogen-activated protein (MAP) kinase pathway is involved in the actions of phenylephrine on GLUT1 transcription, and the effects of Ras on the activity of the promoter depend on the integrity of the TATA box. Our data indicate that phenylephrine induces the expression of the TBP-associated factor TAF(II)250 mRNA, which increases in parallel to the expression of GLUT1, suggesting that altering the expression of basal transcription factors could be one mechanism by which phenylephrine may regulate the activity of the GLUT1 promoter.
We report tripartite co-operation between MyoD, myocyte enhancer factor-2 (MEF2) and the thyroid ... more We report tripartite co-operation between MyoD, myocyte enhancer factor-2 (MEF2) and the thyroid hormone receptor (TRalpha1) that takes place in the context of an 82-bp muscle-specific enhancer in the rat insulin-responsive glucose transporter (GLUT4) gene that is active in both cardiac and skeletal muscle. In the L6E9 skeletal muscle cell line and in 10T1/2 fibroblasts, a powerful synergistic activation of the GLUT4 enhancer relied on the over-expression of MyoD, MEF2 and TRalpha1 and the integrity of their respective binding sites, and occurred when linked to either a heterologous promoter or in the context of the native GLUT4 promoter. In cardiac myocytes, enhancer activity was dependent on the binding sites for MEF2 and TRalpha1. Furthermore, we show that in 10T1/2 fibroblasts, the forced expression of MyoD, MEF2 and TRalpha1 induced the expression of the endogenous, otherwise silent, GLUT4 gene. In all, our results indicate a novel functional co-operation between these three factors which is required for full activation of GLUT4 transcription.
We report tripartite co-operation between MyoD, myocyte enhancer factor-2 (MEF2) and the thyroid ... more We report tripartite co-operation between MyoD, myocyte enhancer factor-2 (MEF2) and the thyroid hormone receptor (TRalpha1) that takes place in the context of an 82-bp muscle-specific enhancer in the rat insulin-responsive glucose transporter (GLUT4) gene that is active in both cardiac and skeletal muscle. In the L6E9 skeletal muscle cell line and in 10T1/2 fibroblasts, a powerful synergistic activation of the GLUT4 enhancer relied on the over-expression of MyoD, MEF2 and TRalpha1 and the integrity of their respective binding sites, and occurred when linked to either a heterologous promoter or in the context of the native GLUT4 promoter. In cardiac myocytes, enhancer activity was dependent on the binding sites for MEF2 and TRalpha1. Furthermore, we show that in 10T1/2 fibroblasts, the forced expression of MyoD, MEF2 and TRalpha1 induced the expression of the endogenous, otherwise silent, GLUT4 gene. In all, our results indicate a novel functional co-operation between these three factors which is required for full activation of GLUT4 transcription.
COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mit... more COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2 3' untranslated region, CR1 (conserved region 1), acts as a potent instability determinant, and mediates stabilization in response to p38 activation. A detailed structural and functional analysis of this element was performed in an attempt to identify RNA-binding proteins involved in the regulation of COX-2 mRNA stability. Destabilization of a beta-globin reporter mRNA was dependent upon two distinct AREs within CR1, each containing three copies of the sequence AUUUA. CR1 was shown to bind AUF-1 [ARE/poly(U)-binding/degradation factor-1] and/or AUF-2, HuR (Hu antigen R), TTP (tristetraprolin) and FBP1 (far-upstream-sequence-element-binding protein 1), yet these factors did not appear to account for the effects of CR1 upon mRNA stability. Mutant ...
Poster presentado en el XXXIV Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular... more Poster presentado en el XXXIV Congreso de la Sociedad Espanola de Bioquimica y Biologia Molecular SEBBM, celebrado del 5 al 8 de septiembre de 2011 en Barcelona (Espana)
Trabajo presentado al XXVth International Symposium on Cerebral Blood Flow, Metabolism and Functi... more Trabajo presentado al XXVth International Symposium on Cerebral Blood Flow, Metabolism and Function y a la Xth International Conference on Quantification of Brain Function with PET celebrados del 25 al 28 de mayo en Barcelona.
Comunicacion presentada en el IX Simposi de Neurobiologia Experimental, celebrado los dias 22 y 2... more Comunicacion presentada en el IX Simposi de Neurobiologia Experimental, celebrado los dias 22 y 23 de octubre de 2014 en Barcelona y organizado por la Societat Catalana de Biologia del Institut d'Estudis Catalans
Applied Immunohistochemistry & Molecular Morphology, Mar 3, 2020
Humanized antibodies targeting programmed death receptor 1 (PD-1) or its ligand (PD-L1) have been... more Humanized antibodies targeting programmed death receptor 1 (PD-1) or its ligand (PD-L1) have been approved for the treatment of different cancers. Some of these antibodies show a correlation between the tissue expression of PD-L1 and response. Evaluation of PD-L1 expression presents multiple challenges, but some preanalytical issues such as tissue fixation have been scarcely evaluated. With the hypothesis that immunohistochemical staining of PD-L1 may be impacted by the time of specimen fixation, we evaluated differences in its expression in tonsil samples exposed to predefined fixation times. Random nontumoral tonsillectomy specimens were blindly evaluated in tissue microarray slides after staining with SP142 and SP263 antibodies. With fixation times ranging from 12 to 72 hours, between 2.8% and 6.1% of the samples were considered to be suboptimally stained, with no differences between the 2 antibodies within these fixation times. A significantly higher proportion of samples exposed to a fixation time of 96 hours presented suboptimal immunostaining (15.6%, P&lt;0.0001). In addition, suboptimally stained spots were 20.8% using SP142 and 10.4% using SP263 after 96 hours of fixation (P=0.046). In conclusion, the quality of staining for PD-L1 in tonsil samples decreased with overfixation of the specimen at times &gt;72 hours. Samples exposed to formaldehyde for longer periods presented suboptimal results for both clones, but the SP142 antibody presented a significantly lower tolerance to formalin overexposure than SP263. These results indicate the relevance of a controlled preanalytical processing of samples and particularly the length of fixation of tumor specimens.
Applied Immunohistochemistry & Molecular Morphology
Antibodies targeting programmed death receptor 1 or programmed death ligand 1 (PD-L1) have become... more Antibodies targeting programmed death receptor 1 or programmed death ligand 1 (PD-L1) have become a standard of care to treat different cancers; for some of these tumors, there is a correlation between tissue expression of PD-L1 and response rates in patients. Although most of the analytical challenges in the evaluation of PD-L1 expression have been standardized, preanalytical issues have been less explored. The objective of this study was to evaluate the impact of time of ischemia on the performance of 2 commonly used antibodies against PD-L1. Sixteen tonsillectomy samples were kept in ischemia for <30 minutes from sample obtention (control) and 1, 3, 6, 12, and 24 hours at room temperature before formalin fixation and paraffin embedding. Selected areas were inserted into TMA paraffin recipient blocks stained with SP142 and SP263 antibodies and evaluated by 2 blind observers. The proportion of suboptimally stained samples was significantly higher for samples with cold ischemia t...
Background Valuable clone collections encoding the complete ORFeomes for some model organisms hav... more Background Valuable clone collections encoding the complete ORFeomes for some model organisms have been constructed following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORF) into any suitable destination vector. The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway destination expression vectors allow the construction of fusion proteins from ORFeome-derived sequences, but they are restricted to the possibilities offered by their inbuilt functional modules and their pre-defined model organism-specificity. Thus, the availability of cloning systems that overcome these limitations would be highly advantageous. Results We present a versatile cloning toolkit for constructing fully-customizable three-part fusi...
Journal of Molecular and Cellular Cardiology, Apr 1, 2005
Cardiac hypertrophy and heart failure occur in association to alterations in glucose uptake and m... more Cardiac hypertrophy and heart failure occur in association to alterations in glucose uptake and metabolism. Phenylephrine, among other hypertrophic agonists, has been reported to increase expression of GLUT1 in neonatal rat cardiac myocytes by activating transcription. However, the specific cis- or trans-acting factors in the GLUT1 gene that are targeted by this agonist remain elusive. Here we describe that the activity of the -99/+134 basal promoter of rat GLUT1 is increased by phenylephrine. Nevertheless, this is not mediated by previously described binding sites (GC-box, MG1E) in the promoter. Rather, the TATA box is required by the agonist to activate transcription from the promoter. Interestingly, The Ras-ERK mitogen-activated protein (MAP) kinase pathway is involved in the actions of phenylephrine on GLUT1 transcription, and the effects of Ras on the activity of the promoter depend on the integrity of the TATA box. Our data indicate that phenylephrine induces the expression of the TBP-associated factor TAF(II)250 mRNA, which increases in parallel to the expression of GLUT1, suggesting that altering the expression of basal transcription factors could be one mechanism by which phenylephrine may regulate the activity of the GLUT1 promoter.
We report tripartite co-operation between MyoD, myocyte enhancer factor-2 (MEF2) and the thyroid ... more We report tripartite co-operation between MyoD, myocyte enhancer factor-2 (MEF2) and the thyroid hormone receptor (TRalpha1) that takes place in the context of an 82-bp muscle-specific enhancer in the rat insulin-responsive glucose transporter (GLUT4) gene that is active in both cardiac and skeletal muscle. In the L6E9 skeletal muscle cell line and in 10T1/2 fibroblasts, a powerful synergistic activation of the GLUT4 enhancer relied on the over-expression of MyoD, MEF2 and TRalpha1 and the integrity of their respective binding sites, and occurred when linked to either a heterologous promoter or in the context of the native GLUT4 promoter. In cardiac myocytes, enhancer activity was dependent on the binding sites for MEF2 and TRalpha1. Furthermore, we show that in 10T1/2 fibroblasts, the forced expression of MyoD, MEF2 and TRalpha1 induced the expression of the endogenous, otherwise silent, GLUT4 gene. In all, our results indicate a novel functional co-operation between these three factors which is required for full activation of GLUT4 transcription.
We report tripartite co-operation between MyoD, myocyte enhancer factor-2 (MEF2) and the thyroid ... more We report tripartite co-operation between MyoD, myocyte enhancer factor-2 (MEF2) and the thyroid hormone receptor (TRalpha1) that takes place in the context of an 82-bp muscle-specific enhancer in the rat insulin-responsive glucose transporter (GLUT4) gene that is active in both cardiac and skeletal muscle. In the L6E9 skeletal muscle cell line and in 10T1/2 fibroblasts, a powerful synergistic activation of the GLUT4 enhancer relied on the over-expression of MyoD, MEF2 and TRalpha1 and the integrity of their respective binding sites, and occurred when linked to either a heterologous promoter or in the context of the native GLUT4 promoter. In cardiac myocytes, enhancer activity was dependent on the binding sites for MEF2 and TRalpha1. Furthermore, we show that in 10T1/2 fibroblasts, the forced expression of MyoD, MEF2 and TRalpha1 induced the expression of the endogenous, otherwise silent, GLUT4 gene. In all, our results indicate a novel functional co-operation between these three factors which is required for full activation of GLUT4 transcription.
COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mit... more COX-2 (cyclo-oxygenase-2) mRNA is degraded rapidly in resting cells, but is stabilized by the mitogen-activated protein kinase p38 signalling pathway in response to pro-inflammatory stimuli. A conserved ARE (AU-rich element) of the COX-2 3' untranslated region, CR1 (conserved region 1), acts as a potent instability determinant, and mediates stabilization in response to p38 activation. A detailed structural and functional analysis of this element was performed in an attempt to identify RNA-binding proteins involved in the regulation of COX-2 mRNA stability. Destabilization of a beta-globin reporter mRNA was dependent upon two distinct AREs within CR1, each containing three copies of the sequence AUUUA. CR1 was shown to bind AUF-1 [ARE/poly(U)-binding/degradation factor-1] and/or AUF-2, HuR (Hu antigen R), TTP (tristetraprolin) and FBP1 (far-upstream-sequence-element-binding protein 1), yet these factors did not appear to account for the effects of CR1 upon mRNA stability. Mutant ...
Uploads