Miguel Sánchez

Updated clinical classification of pulmonary hypertension

In 1998, a clinical classification of pulmonary hypertension (PH) was established, categorizing PH into groups which share similar pathological and hemodynamic characteristics and therapeutic approaches. During the 5th World Symposium held in Nice, France, in 2013, the consensus was reached to maintain the general scheme of previous clinical classifications. However, modifications and updates especially for Group 1 patients (pulmonary arterial hypertension [PAH]) were proposed. The main change was to withdraw persistent pulmonary hypertension of the newborn (PPHN) from Group 1 because this entity carries more differences than similarities with other PAH subgroups. In the current classification, PPHN is now designated number 1. Pulmonary hypertension associated with chronic hemolytic anemia has been moved from Group 1 PAH to Group 5, unclear/multifactorial mechanism. In addition, it was decided to add specific items related to pediatric pulmonary hypertension in order to create a comprehensive, common classification for both adults and children. Therefore, congenital or acquired left-heart inflow/outflow obstructive lesions and congenital cardiomyopathies have been added to Group 2, and segmental pulmonary hypertension has been added to Group 5. Last, there were no changes for Groups 2, 3, and 4.

Diagnosis and Assessment of Pulmonary Arterial Hypertension

The diagnosis and assessment of pulmonary arterial hypertension is a rapidly evolving area, with changes occurring in the definition of the disease, screening and diagnostic techniques, and staging and follow-up assessment. The definition of pulmonary hypertension has been simplified, and is now based on currently available evidence. There has been substantial progress in advancing the imaging techniques and biomarkers used to screen patients for the disease and to follow up their response to therapy. The importance of accurate assessment of right ventricular function in following up the clinical course and response to therapy is more fully appreciated. As new therapies are developed for pulmonary arterial hypertension, screening, prompt diagnosis, and accurate assessment of disease severity become increasingly important. A clear definition of pulmonary hypertension and the development of a rational approach to diagnostic assessment and follow-up using both conventional and new tools will be essential to deriving maximal benefit from our expanding therapeutic armamentarium.

2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS)Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT)

Guidelines summarize and evaluate all available evidence on a particular issue at the time of the writing process, with the aim of assisting health professionals in selecting the best management strategies for an individual patient with a given condition, taking into account the impact on outcome, as well as the risk–benefit ratio of particular diagnostic or therapeutic means. Guidelines and recommendations should help health professionals to make decisions in their daily practice. However, the final decisions concerning an individual patient must be made by the responsible health professional(s) in consultation with the patient and caregiver as appropriate. 2015 ESC/ERS pulmonary hypertension guidelines incorporate changes and adaptations focusing on clinical management http://ow.ly/RiDLb

2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS)Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), Intern…

Guidelines summarize and evaluate all available evidence on a particular issue at the time of the writing process, with the aim of assisting health professionals in selecting the best management strategies for an individual patient with a given condition, taking into account the impact on outcome, as well as the risk–benefit ratio of particular diagnostic or therapeutic means. Guidelines and recommendations should help health professionals to make decisions in their daily practice. However, the final decisions concerning an individual patient must be made by the responsible health professional(s) in consultation with the patient and caregiver as appropriate. 2015 ESC/ERS pulmonary hypertension guidelines incorporate changes and adaptations focusing on clinical management http://ow.ly/RiDLb

Sequential Fractionation and Two-dimensional Gel Analysis Unravels the Complexity of the Dimorphic Fungus Candida albicans Cell Wall Proteome

The cell wall proteins of Candida albicans play a key role in morphogenesis and pathogenesis and might be potential target sites for new specific antifungal drugs. However, these proteins are difficult to analyze because of their high heterogeneity, interconnections with wall polysaccharides (mannan, glucan, and chitin), low abundance, low solubility, and hydrophobic nature. Here we report a subproteomic approach for the study of the cell wall proteins (CWPs) from C. albicans yeast and hyphal forms. Most of the mannoproteins present in this compartment were extracted by cell wall fractionation according to the type of interactions that they establish with other structural components. CWPs were solubilized from isolated cell walls by hot SDS and dithiothreitol treatment followed by extraction either by mild alkali conditions or by enzymatic treatment with glucanases and chitinases. These highly enriched cell wall fractions were analyzed by two-dimensional PAGE, showing that a large number of proteins are involved in cell wall construction and that the wall remodeling that occurs during germ tube formation is related to changes in the composition of CWPs. We suggest that the CWP-chitin linkage is an important retention mechanism of CWPs in C. albicans mycelial forms. This article also highlights the usefulness of the combination of sequential fractionation and two-dimensional PAGE followed by Western blotting using specific antibodies against known CWPs in the characterization of incorporation mechanisms of such CWPs into the cell wall and of their interactions with other wall components. Mass spectrometry analyses have allowed the identification of several cell surface proteins classically associated with both the cell wall and other compartments. The physiological significance of the dual location of these moonlighting proteins is also discussed. This approach is therefore a powerful tool for obtaining a comprehensive and integrated view of the cell wall proteome.

2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension

Nazzareno Galiè (ESC Chairperson), Marc Humbert (ERS Chairperson), Jean-Luc Vachiery, Simon Gibbs, Irene Lang, Adam Torbicki, Gérald Simonneau, Andrew Peacock, Anton Vonk Noordegraaf, Maurice Beghetti, Ardeschir Ghofrani, Miguel Angel Gomez Sanchez, Georg Hansmann, Walter Klepetko, Patrizio Lancellotti, Marco Matucci, Theresa McDonagh, Luc A. Pierard, Pedro T. Trindade, Maurizio Zompatori and Marius Hoeper The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and of the European Respiratory Society (ERS)

Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity.

Mitogen-activated protein (MAP) kinases represent a group of serine/threonine protein kinases playing a central role in signal transduction processes in eukaryotic cells. Using a strategy based on the complementation of the thermosensitive autolytic phenotype of slt2 null mutants, we have isolated a Candida albicans homolog of Saccharomyces cerevisiae MAP kinase gene SLT2 (MPK1), which is involved in the recently outlined PKC1-controlled signalling pathway. The isolated gene, named MKC1 (MAP kinase from C. albicans), coded for a putative protein, Mkc1p, of 58,320 Da that displayed all the characteristic domains of MAP kinases and was 55% identical to S. cerevisiae Slt2p (Mpk1p). The MKC1 gene was deleted in a diploid Candida strain, and heterozygous and homozygous strains, in both Ura+ and Ura- backgrounds, were obtained to facilitate the analysis of the function of the gene. Deletion of the two alleles of the MKC1 gene gave rise to viable cells that grew at 28 and 37 degrees C but, nevertheless, displayed a variety of phenotypic traits under more stringent conditions. These included a low growth yield and a loss of viability in cultures grown at 42 degrees C, a high sensitivity to thermal shocks at 55 degrees C, an enhanced susceptibility to caffeine that was osmotically remediable, and the formation of a weak cell wall with a very low resistance to complex lytic enzyme preparations. The analysis of the functions downstream of the MKC1 gene should contribute to understanding of the connection of growth and morphogenesis in pathogenic fungi.

A protein kinase gene complements the lytic phenotype of Saccharomyces cerevisiae lyt2 mutants.

By genetic analysis of a thermosensitive autolytic mutant whose phenotype was complemented by osmotic stabilization with sorbitol, we identified gene LYT2 of Saccharomyces cerevisiae, which is probably involved in cell wall formation. A yeast gene complementing lyt2 strains was cloned and shown to carry an open reading frame coding for a 484‐amino‐acid protein exhibiting all the characteristic domains of serine/threonine protein kinases and highly homologous to other yeast protein kinases involved in control of the mitotic cycle. Mutants disrupted in the cloned gene also displayed an autolytic phenotype complemented by osmotic stabilization with sorbitol. However, genetic comparison of lyt2 mutants and disruptants of the protein kinase gene revealed that the cloned gene is not the structural gene LVT2 but a suppressor of the lytic phenotype, named gene SLT2, that was mapped to chromosome V. The product of gene SLT2 is the first protein kinase to be described in relation to the yeast cell‐wall functions.

Activity of the yeast MAP kinase homologue Slt2 is critically required for cell integrity at 37° C

Deletion of the SLT2 gene of Saccharomyces cerevisiae, which codes for a homologue of MAP (mitogen-activated) protein kinases, causes an autolytic lethal phenotype in cells grown at 37° C. The gene encodes domains characteristic of protein kinases, which include a lysine (at position 54) that lies 19 residues from a glycine-rich cluster, considered to be the putative ATP binding site. The ability of three mutant alleles of SLT2 generated by site-directed mutagenesis, namely E54 (glutamic acid), R54 (arginine) and F54 (phenylalanine), to complement slt2 mutants was tested. All three failed to complement the autolytic phenotype and were unable to restore growth and viability of cells. A strain obtained by transplacement of slt2-F54 also behaved as a thermosensitive autolytic mutant. By immunoprecipitation with polyclonal antibodies raised against Slt2 protein expressed in Escherichia coli, it was possible to confirm that alteration of the lysine-54 residue did not affect the stability of the protein, thus allowing us to conclude that activity of the Slt2 protein kinase is critically required for growth and morphogenesis of S. cerevisiae at 37° C. A significant fraction of the mutant cell population lysed at 24° C and the cells displayed a characteristic alteration of the surface consisting of a typical depression in an area of the cell wall. At 37° C, the cell surface was clearly disorganized.

Two-Dimensional analysis of proteins secreted by Saccharomyces cerevisiae regenerating protoplasts: a novel approach to study the cell wall

Protoplasts of Saccharomyces cerevisiae incubated in regenerating conditions secrete cell wall components in order to allow the biosynthesis of this structure. During the first hours of incubation, many of these are not retained in the forming cell wall but remain in the medium. We have developed a method for collecting the secreted proteins and have analysed these by two‐dimensional electrophoresis to obtain a reference map of putative cell wall proteins. Several proteins were identified by microsequencing or immunoblotting; namely, cell wall hydrolytic enzymes, heat shock proteins, glycolytic enzymes and others. Some β‐1,3‐ and β‐1,6‐glucosylation was detected in the proteins secreted by regenerating protoplasts. Copyright