Abstract. The rise of the number and complexity of pervasive systems is a fact. Pervasive systems... more Abstract. The rise of the number and complexity of pervasive systems is a fact. Pervasive systems developers need advanced development meth-ods in order to build better systems in an easy way. Software Factories and the Model Driven Architecture (MDA) are two important ...
2010 IEEE International Conference on Industrial Technology, 2010
Page 1. A Novel Multi-Level Topology based on Current Source Power Cells for High Performance App... more Page 1. A Novel Multi-Level Topology based on Current Source Power Cells for High Performance Applications Carlos R. Baier ♣ Pedro E. Melín* José R. Espinoza* Javier Muñoz * Johan I. Guzmán* e-mail: cbaier@udec.cl ...
2010 IEEE International Symposium on Industrial Electronics, 2010
Page 1. A Novel Multi-Level CSI Based Topology with Inter-Cell Magnetic Couplings for Minimum DC ... more Page 1. A Novel Multi-Level CSI Based Topology with Inter-Cell Magnetic Couplings for Minimum DC Storage Components Carlos R. Baier ♣ José R. Espinoza ∗ Pedro E. Melin ∗ Eduardo Espinoza ∗ Javier Muñoz ∗ e-mail ...
This paper presents a three-phase unified powerquality conditioner based on current source conver... more This paper presents a three-phase unified powerquality conditioner based on current source converters (CSC-UPQC), including the design guidelines of the key components, an appropriate control scheme, and a selection procedure of the dc current level. Particularly, the ridethrough capability criterion is used to define a minimum dc current level so that the CSC-UPQC achieves the same characteristics as a UPQC based on voltage-source converters in terms of voltage disturbance compensation in the point of common coupling (PCC) and load power factor compensation. A 1.17 MVA load fed from a 3.3 kV system is used to show the proposed design procedure, and a laboratory prototype is implemented to show the system compensating sags and swells using low switching frequency in the CSC and maintaining a unitary displacement power factor in the PCC.
Current pervasive systems are developed ad-hoc or using implementation frameworks. These approach... more Current pervasive systems are developed ad-hoc or using implementation frameworks. These approaches could be not enough when dealing with large and complex pervasive systems. This paper introduces an implementation of a pervasive system for managing a meeting room. This system has been developed using a model driven method proposed by the authors. The system is specified using PervML, a UML-like modeling language. Then, a set of templates are applied to the specification in order to automatically produce Java code that uses an OSGi-based framework. The final application integrates several technologies like EIB and Web Services. Three different user interfaces are provided for interacting with the system. ⋆ This work has been developed with the support of MEC under the project DESTINO TIN2004-03534 and cofinanced by FEDER.
A discrete-time linear control scheme for a multilevel three-phase Unified Power Quality Conditio... more A discrete-time linear control scheme for a multilevel three-phase Unified Power Quality Conditioner (UPQC) based on Single-Phase Power Cells is presented. The multi-variable, non-linear, and coupled features of these topologies make the control strategy design a difficult task. Controlling this kind of systems with mono-variable linear controllers - as proposed in this work - presents significant advantages compared with other
Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from ... more Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3β and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely s...
Cellular reprogramming remodels the gene expression program by re-setting the epigenome of somati... more Cellular reprogramming remodels the gene expression program by re-setting the epigenome of somatic cells into an embryonic-like pluripotent state. Post-translational modifications of histones play an important role in this process. Previously, we found by ChIP-seq widespread changes of specific histone H3 marks in two divergent reprogramming routes leading to alternative pluripotent sates [1]. Here, using an unbiased middle-down proteomics approach we have identified 72 unique isoforms of histone H4 and quantified 56 of them in the same set of samples. We found substantial differences between somatic and late-phase reprogramming cells. Also, ESCs and iPSCs displayed higher levels of H4 acetylation and tri-methylation concomitantly with lower levels of mono- and di-methylation when compared to cells undergoing reprogramming. Our data shows that the epigenetic remodeling induced by the reprogramming process goes beyond histone H3 and reveals the importance of H4 modifications as well....
High-resolution mass spectrometry and the use of stable isotopes have greatly improved our abilit... more High-resolution mass spectrometry and the use of stable isotopes have greatly improved our ability to quantify proteomes. Typically, the relative abundance of peptides is estimated by identifying the isotopic clusters and by comparing the peak intensities of peptide pairs. However, when the mass shift between the labeled peptides is small, there can be the possibility for overlap of the isotopic
Methods in molecular biology (Clifton, N.J.), 2011
Understanding the signaling pathways governing pluripotency and self-renewal is a prerequisite fo... more Understanding the signaling pathways governing pluripotency and self-renewal is a prerequisite for better controlling stem cell differentiation to specific fates. Reversible protein phosphorylation is one of the most important posttranslational modifications regulating signaling pathways in biological processes. Global analysis of dynamic changes in protein phosphorylation is, therefore, key to understanding signaling at the system level. Here, we describe a generic mass spectrometry (MS)-based phosphoproteomics strategy applied to monitor phosphorylation dynamics after bone morphogenetic protein 4 (BMP4)-induced differentiation of human embryonic stem cells (hESCs). Our method combines the use of strong cation exchange (SCX) and titanium dioxide (TiO(2)) for phosphopeptide enrichment, high-resolution MS for peptide and protein identification, and stable isotope labeling by amino acids in cell culture (SILAC) for quantification. This approach allows us to identify thousands of phosp...
Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry... more Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D subs...
To assess the interaction between intrinsic and externally applied positive end-expiratory pressu... more To assess the interaction between intrinsic and externally applied positive end-expiratory pressure (intrinsic PEEP and administered PEEP) in mechanically ventilated patients. Prospective study. Intensive care unit of a university hospital. Twelve consecutive critically ill patients. Application of an external PEEP of the same value as the intrinsic PEEP. We found that when the administered PEEP was increased from 0 to the baseline value of intrinsic PEEP, mean intrinsic PEEP decreased from 6.5 +/- 4.2 (SD) to 1.3 +/- 0.7 cm H2O (p = .001). The mean end-inspiratory pressure was increased from 20.3 +/- 4.6 to 23.1 +/- 6.1 cm H2O (p < .05). The difference between the modification of intrinsic PEEP and the change in the end-inspiratory pressure was not significantly > 0 cm H2O. Thus, the increase in end-inspiratory pressure may be directly attributable to the increase in total PEEP (administered PEEP plus intrinsic PEEP). None of the other measurements of pulmonary mechanics changed (peak pressure, inspiratory resistance, compliance, and trapped-gas volume). The administration of positive end-expiratory pressure equal to the intrinsic positive end-expiratory pressure causes the almost total disappearance of the intrinsic positive end-expiratory pressure. When the administered positive end-expiratory pressure does not exceed the intrinsic positive end-expiratory pressure, the former is applied almost in its entirety to the patient's external circuit. The administration of positive end-expiratory pressure without prior quantification of the intrinsic positive end-expiratory pressure results in an overestimation of the beneficial effects of the administered positive end-expiratory pressure on the quasi-static compliance.
Analysis of tyrosine (Tyr) phosphorylation by mass spectrometry (MS)-based proteomics remains cha... more Analysis of tyrosine (Tyr) phosphorylation by mass spectrometry (MS)-based proteomics remains challenging, due to the low occurrence of this post-translational modification compared to serine and threonine phosphorylation events in mammalian systems. Conventional metal-based affinity chromatography methods used to enrich phosphopeptides can nowadays isolate over 10,000 phosphopeptides. However, these approaches are not particularly suitable for the selective enrichment of low abundant Tyr phosphorylated peptides as the higher abundant co-enriched serine (Ser) and threonine (Thr) phosphorylated peptides typically obscure their detection. Therefore, a more targeted approach based on immuno-affinity precipitation at the peptide level has been introduced for the specific analysis of Tyr phosphorylated species. This method typically leads to the detection of a few hundreds of phosphopeptides, albeit typically over 70% of those are Tyr phosphorylated. Here, we evaluated and compared phosphotyrosine peptides enriched by a phospho-Tyr immuno-affinity enrichment (employing pY99 antibodies) and a multidimensional approach consisting of metal-affinity based enrichment (Ti(4+)-IMAC) followed by hydrophilic interaction liquid chromatography (HILIC) fractionation. Our aim was to assess differences and similarities in the set of Tyr phosphorylated peptides detected by each approach. Our data suggest that both strategies are not redundant but complementary and should ideally be combined for a more comprehensive view at phosphotyrosine signaling. Here we evaluated enabling tools for the global analysis of phosphotyrosine phosphorylation. Phosphotyrosine phosphorylation is a key protein modification driving cellular response also involved in disease/cancer molecular pathways.
The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During dev... more The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα-GPR-LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα-GPR-LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division.
Cytokinesis has been extensively studied in different models, but the role of the extracellular c... more Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell...
ABSTRACT A herculean task: Determining the human proteome sets the ultimate challenge in cell bio... more ABSTRACT A herculean task: Determining the human proteome sets the ultimate challenge in cell biology as it is thought to consist of more than 1 000 000 proteoforms, in contrast to "only" 20 000 protein-coding genes. Two teams of researchers have now proved the translation of 18 000 proteins (and more than 27 000 isoforms) by mass spectrometry. They obtained important information on the extent of protein translation and alternative splicing.
The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust bi... more The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade.
Understanding the pathogenesis of small-for-size (SFS) syndrome is critical to expanding the appl... more Understanding the pathogenesis of small-for-size (SFS) syndrome is critical to expanding the applicability of partial liver transplantation. We aimed to characterize its acute presentation and association with alterations in hepatic hemodynamics, microstructure, and regeneration in a porcine model. Eighteen SFS liver transplants were performed. Donors underwent 70% hepatectomy. Partial grafts were implanted into larger recipients. Whole liver transplants were also performed (n ¼ 6). Recipients were followed until death or for 5 days. Hemodynamics were measured, and tissue was sampled intraoperatively and at the study end. Serum was sampled regularly during follow-up. Seventeen SFS transplants and 6 whole liver transplants were included. SFS grafts represented 23.2% (19.3%-25.3%) of the recipients' standard liver volume. The survival rate was 29% and 100% in the SFS and whole liver groups, respectively. The portal venous flow, pressure gradient, and resistance were significantly higher in recipients of SFS grafts versus whole livers after portal and arterial reperfusion. Arterial flow as a percentage of the total liver blood flow was significantly lower after reperfusion in SFS grafts and remained so when measured again after 5 days. Markers of endothelial cell injury increased soon after reperfusion, and those of hepatocellular injury increased later; both predicted the appearance of either graft failure or histological recovery. Proliferative activity peaked earlier and higher among nonsurvivors in the SFS group. Surviving grafts demonstrated a slower but maintained rise in regenerative activity, although metabolic activity failed to improve. In SFS transplantation in the acute setting, portal hyperperfusion is a stimulus for regeneration but may simultaneously cause irreparable endothelial injury. This porcine model not only helps to elucidate the inciting factors in SFS pathogenesis but also offers a clinically relevant means to study its prevention. Liver Transpl 16:364-374,
Abstract. The rise of the number and complexity of pervasive systems is a fact. Pervasive systems... more Abstract. The rise of the number and complexity of pervasive systems is a fact. Pervasive systems developers need advanced development meth-ods in order to build better systems in an easy way. Software Factories and the Model Driven Architecture (MDA) are two important ...
2010 IEEE International Conference on Industrial Technology, 2010
Page 1. A Novel Multi-Level Topology based on Current Source Power Cells for High Performance App... more Page 1. A Novel Multi-Level Topology based on Current Source Power Cells for High Performance Applications Carlos R. Baier ♣ Pedro E. Melín* José R. Espinoza* Javier Muñoz * Johan I. Guzmán* e-mail: cbaier@udec.cl ...
2010 IEEE International Symposium on Industrial Electronics, 2010
Page 1. A Novel Multi-Level CSI Based Topology with Inter-Cell Magnetic Couplings for Minimum DC ... more Page 1. A Novel Multi-Level CSI Based Topology with Inter-Cell Magnetic Couplings for Minimum DC Storage Components Carlos R. Baier ♣ José R. Espinoza ∗ Pedro E. Melin ∗ Eduardo Espinoza ∗ Javier Muñoz ∗ e-mail ...
This paper presents a three-phase unified powerquality conditioner based on current source conver... more This paper presents a three-phase unified powerquality conditioner based on current source converters (CSC-UPQC), including the design guidelines of the key components, an appropriate control scheme, and a selection procedure of the dc current level. Particularly, the ridethrough capability criterion is used to define a minimum dc current level so that the CSC-UPQC achieves the same characteristics as a UPQC based on voltage-source converters in terms of voltage disturbance compensation in the point of common coupling (PCC) and load power factor compensation. A 1.17 MVA load fed from a 3.3 kV system is used to show the proposed design procedure, and a laboratory prototype is implemented to show the system compensating sags and swells using low switching frequency in the CSC and maintaining a unitary displacement power factor in the PCC.
Current pervasive systems are developed ad-hoc or using implementation frameworks. These approach... more Current pervasive systems are developed ad-hoc or using implementation frameworks. These approaches could be not enough when dealing with large and complex pervasive systems. This paper introduces an implementation of a pervasive system for managing a meeting room. This system has been developed using a model driven method proposed by the authors. The system is specified using PervML, a UML-like modeling language. Then, a set of templates are applied to the specification in order to automatically produce Java code that uses an OSGi-based framework. The final application integrates several technologies like EIB and Web Services. Three different user interfaces are provided for interacting with the system. ⋆ This work has been developed with the support of MEC under the project DESTINO TIN2004-03534 and cofinanced by FEDER.
A discrete-time linear control scheme for a multilevel three-phase Unified Power Quality Conditio... more A discrete-time linear control scheme for a multilevel three-phase Unified Power Quality Conditioner (UPQC) based on Single-Phase Power Cells is presented. The multi-variable, non-linear, and coupled features of these topologies make the control strategy design a difficult task. Controlling this kind of systems with mono-variable linear controllers - as proposed in this work - presents significant advantages compared with other
Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from ... more Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naïve pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3β and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naïve pluripotent state, but the molecular underpinnings of this naïve transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naïve pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely s...
Cellular reprogramming remodels the gene expression program by re-setting the epigenome of somati... more Cellular reprogramming remodels the gene expression program by re-setting the epigenome of somatic cells into an embryonic-like pluripotent state. Post-translational modifications of histones play an important role in this process. Previously, we found by ChIP-seq widespread changes of specific histone H3 marks in two divergent reprogramming routes leading to alternative pluripotent sates [1]. Here, using an unbiased middle-down proteomics approach we have identified 72 unique isoforms of histone H4 and quantified 56 of them in the same set of samples. We found substantial differences between somatic and late-phase reprogramming cells. Also, ESCs and iPSCs displayed higher levels of H4 acetylation and tri-methylation concomitantly with lower levels of mono- and di-methylation when compared to cells undergoing reprogramming. Our data shows that the epigenetic remodeling induced by the reprogramming process goes beyond histone H3 and reveals the importance of H4 modifications as well....
High-resolution mass spectrometry and the use of stable isotopes have greatly improved our abilit... more High-resolution mass spectrometry and the use of stable isotopes have greatly improved our ability to quantify proteomes. Typically, the relative abundance of peptides is estimated by identifying the isotopic clusters and by comparing the peak intensities of peptide pairs. However, when the mass shift between the labeled peptides is small, there can be the possibility for overlap of the isotopic
Methods in molecular biology (Clifton, N.J.), 2011
Understanding the signaling pathways governing pluripotency and self-renewal is a prerequisite fo... more Understanding the signaling pathways governing pluripotency and self-renewal is a prerequisite for better controlling stem cell differentiation to specific fates. Reversible protein phosphorylation is one of the most important posttranslational modifications regulating signaling pathways in biological processes. Global analysis of dynamic changes in protein phosphorylation is, therefore, key to understanding signaling at the system level. Here, we describe a generic mass spectrometry (MS)-based phosphoproteomics strategy applied to monitor phosphorylation dynamics after bone morphogenetic protein 4 (BMP4)-induced differentiation of human embryonic stem cells (hESCs). Our method combines the use of strong cation exchange (SCX) and titanium dioxide (TiO(2)) for phosphopeptide enrichment, high-resolution MS for peptide and protein identification, and stable isotope labeling by amino acids in cell culture (SILAC) for quantification. This approach allows us to identify thousands of phosp...
Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry... more Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D subs...
To assess the interaction between intrinsic and externally applied positive end-expiratory pressu... more To assess the interaction between intrinsic and externally applied positive end-expiratory pressure (intrinsic PEEP and administered PEEP) in mechanically ventilated patients. Prospective study. Intensive care unit of a university hospital. Twelve consecutive critically ill patients. Application of an external PEEP of the same value as the intrinsic PEEP. We found that when the administered PEEP was increased from 0 to the baseline value of intrinsic PEEP, mean intrinsic PEEP decreased from 6.5 +/- 4.2 (SD) to 1.3 +/- 0.7 cm H2O (p = .001). The mean end-inspiratory pressure was increased from 20.3 +/- 4.6 to 23.1 +/- 6.1 cm H2O (p < .05). The difference between the modification of intrinsic PEEP and the change in the end-inspiratory pressure was not significantly > 0 cm H2O. Thus, the increase in end-inspiratory pressure may be directly attributable to the increase in total PEEP (administered PEEP plus intrinsic PEEP). None of the other measurements of pulmonary mechanics changed (peak pressure, inspiratory resistance, compliance, and trapped-gas volume). The administration of positive end-expiratory pressure equal to the intrinsic positive end-expiratory pressure causes the almost total disappearance of the intrinsic positive end-expiratory pressure. When the administered positive end-expiratory pressure does not exceed the intrinsic positive end-expiratory pressure, the former is applied almost in its entirety to the patient's external circuit. The administration of positive end-expiratory pressure without prior quantification of the intrinsic positive end-expiratory pressure results in an overestimation of the beneficial effects of the administered positive end-expiratory pressure on the quasi-static compliance.
Analysis of tyrosine (Tyr) phosphorylation by mass spectrometry (MS)-based proteomics remains cha... more Analysis of tyrosine (Tyr) phosphorylation by mass spectrometry (MS)-based proteomics remains challenging, due to the low occurrence of this post-translational modification compared to serine and threonine phosphorylation events in mammalian systems. Conventional metal-based affinity chromatography methods used to enrich phosphopeptides can nowadays isolate over 10,000 phosphopeptides. However, these approaches are not particularly suitable for the selective enrichment of low abundant Tyr phosphorylated peptides as the higher abundant co-enriched serine (Ser) and threonine (Thr) phosphorylated peptides typically obscure their detection. Therefore, a more targeted approach based on immuno-affinity precipitation at the peptide level has been introduced for the specific analysis of Tyr phosphorylated species. This method typically leads to the detection of a few hundreds of phosphopeptides, albeit typically over 70% of those are Tyr phosphorylated. Here, we evaluated and compared phosphotyrosine peptides enriched by a phospho-Tyr immuno-affinity enrichment (employing pY99 antibodies) and a multidimensional approach consisting of metal-affinity based enrichment (Ti(4+)-IMAC) followed by hydrophilic interaction liquid chromatography (HILIC) fractionation. Our aim was to assess differences and similarities in the set of Tyr phosphorylated peptides detected by each approach. Our data suggest that both strategies are not redundant but complementary and should ideally be combined for a more comprehensive view at phosphotyrosine signaling. Here we evaluated enabling tools for the global analysis of phosphotyrosine phosphorylation. Phosphotyrosine phosphorylation is a key protein modification driving cellular response also involved in disease/cancer molecular pathways.
The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During dev... more The microtubule spindle apparatus dictates the plane of cell cleavage in animal cells. During development, dividing cells control the position of the spindle to determine the size, location, and fate of daughter cells. Spindle positioning depends on pulling forces that act between the cell periphery and astral microtubules. This involves dynein recruitment to the cell cortex by a heterotrimeric G-protein α subunit in complex with a TPR-GoLoco motif protein (GPR-1/2, Pins, LGN) and coiled-coil protein (LIN-5, Mud, NuMA). In this study, we searched for additional factors that contribute to spindle positioning in the one-cell Caenorhabditis elegans embryo. We show that cortical actin is not needed for Gα-GPR-LIN-5 localization and pulling force generation. Instead, actin accumulation in the anterior actually reduces pulling forces, possibly by increasing cortical rigidity. Examining membrane-associated proteins that copurified with GOA-1 Gα, we found that the transmembrane and coiled-coil domain protein 1 (TCC-1) contributes to proper spindle movements. TCC-1 localizes to the endoplasmic reticulum membrane and interacts with UNC-116 kinesin-1 heavy chain in yeast two-hybrid assays. RNA interference of tcc-1 and unc-116 causes similar defects in meiotic spindle positioning, supporting the concept of TCC-1 acting with kinesin-1 in vivo. These results emphasize the contribution of membrane-associated and cortical proteins other than Gα-GPR-LIN-5 in balancing the pulling forces that position the spindle during asymmetric cell division.
Cytokinesis has been extensively studied in different models, but the role of the extracellular c... more Cytokinesis has been extensively studied in different models, but the role of the extracellular cell wall is less understood. Here we studied this process in fission yeast. The essential protein Bgs4 synthesizes the main cell wall β(1,3)glucan. We show that Bgs4-derived β(1,3)glucan is required for correct and stable actomyosin ring positioning in the cell middle, before the start of septum formation and anchorage to the cell wall. Consequently, β(1,3)glucan loss generated ring sliding, oblique positioned rings and septa, misdirected septum synthesis indicative of relaxed rings, and uncoupling between a fast ring and membrane ingression and slow septum synthesis, suggesting that cytokinesis can progress with defective septum pushing and/or ring pulling forces. Moreover, Bgs4-derived β(1,3)glucan is essential for secondary septum formation and correct primary septum completion. Therefore, our results show that extracellular β(1,3)glucan is required for cytokinesis to connect the cell...
ABSTRACT A herculean task: Determining the human proteome sets the ultimate challenge in cell bio... more ABSTRACT A herculean task: Determining the human proteome sets the ultimate challenge in cell biology as it is thought to consist of more than 1 000 000 proteoforms, in contrast to "only" 20 000 protein-coding genes. Two teams of researchers have now proved the translation of 18 000 proteins (and more than 27 000 isoforms) by mass spectrometry. They obtained important information on the extent of protein translation and alternative splicing.
The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust bi... more The scarcity of primordial germ cells (PGCs) in the developing mammalian embryo hampers robust biochemical analysis of the processes that underlie early germ cell formation. Here, we demonstrate that DAZL, a germ cell-specific RNA binding protein, is a robust PGC marker during in vitro germ cell development. Using Dazl-GFP reporter ESCs, we demonstrate that DAZL plays a central role in a large mRNA/protein interactive network that blocks the translation of core pluripotency factors, including Sox2 and Sall4, as well as of Suz12, a polycomb family member required for differentiation of pluripotent cells. Thus, DAZL limits both pluripotency and somatic differentiation in nascent PGCs. In addition, we observed that DAZL associates with mRNAs of key Caspases and similarly inhibits their translation. This elegant fail-safe mechanism ensures that, whereas loss of DAZL results in prolonged expression of pluripotency factors, teratoma formation is avoided due to the concomitant activation of the apoptotic cascade.
Understanding the pathogenesis of small-for-size (SFS) syndrome is critical to expanding the appl... more Understanding the pathogenesis of small-for-size (SFS) syndrome is critical to expanding the applicability of partial liver transplantation. We aimed to characterize its acute presentation and association with alterations in hepatic hemodynamics, microstructure, and regeneration in a porcine model. Eighteen SFS liver transplants were performed. Donors underwent 70% hepatectomy. Partial grafts were implanted into larger recipients. Whole liver transplants were also performed (n ¼ 6). Recipients were followed until death or for 5 days. Hemodynamics were measured, and tissue was sampled intraoperatively and at the study end. Serum was sampled regularly during follow-up. Seventeen SFS transplants and 6 whole liver transplants were included. SFS grafts represented 23.2% (19.3%-25.3%) of the recipients' standard liver volume. The survival rate was 29% and 100% in the SFS and whole liver groups, respectively. The portal venous flow, pressure gradient, and resistance were significantly higher in recipients of SFS grafts versus whole livers after portal and arterial reperfusion. Arterial flow as a percentage of the total liver blood flow was significantly lower after reperfusion in SFS grafts and remained so when measured again after 5 days. Markers of endothelial cell injury increased soon after reperfusion, and those of hepatocellular injury increased later; both predicted the appearance of either graft failure or histological recovery. Proliferative activity peaked earlier and higher among nonsurvivors in the SFS group. Surviving grafts demonstrated a slower but maintained rise in regenerative activity, although metabolic activity failed to improve. In SFS transplantation in the acute setting, portal hyperperfusion is a stimulus for regeneration but may simultaneously cause irreparable endothelial injury. This porcine model not only helps to elucidate the inciting factors in SFS pathogenesis but also offers a clinically relevant means to study its prevention. Liver Transpl 16:364-374,
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