Development by "simple consensus" of a tool to assist the activation of the helicopter at SAMU 14 / Élaboration par méthode du consensus simple d'un outil d'aide au déclenchement d'un hélicoptère au SAMU 14

The main objective of this work was to develop a tool to assist the activation of a helicopter emergency medical service (HEMS) for the SAMU 14. We opted for a methodology based on "guidelines of good professional practice." Simple consensus was used. A multidisciplinary working group (pilots, medical regulation assistants, doctors) was created. Subgroup meetings (pilots, medical regulation assistants, doctors) developed subparts of the tool. The assembly of the tool's subparts was reviewed by the working group and then by an independent reading group. This work enabled the consensual creation of a tool to support the use of the helicopter emergency medical service (HEMS) for the SAMU 14. It is composed of maps, a protocol, and a written procedure of activation. This methodology by "simple consensus" allowed the development of a tool rationalizing the activation of the helicopter emergency medical service (HEMS) for the SAMU 14. It was the first work of this type within the SAMU 14. This simple and transposable methodology could be used in other emergency centers or for other multidisciplinary protocols.

L'objectif principal de ce travail était la création d'un outil d'aide au déclenchement d'un SMUR héliporté au SAMU 14. Nous avons opté pour une méthodologie type « recommandations de bonnes pratiques professionnelles ¼ (RBPP). Le consensus simple a été utilisé. Un groupe de travail multidisciplinaire (pilotes, assistant de régulation médicale [ARM], médecins) a été créé. Des réunions en sous-groupe (pilote, ARM et médecins) ont permis d'élaborer des sous-parties de l'outil. L'assemblage des sous-parties de l'outil a été relu par le groupe de travail puis par un groupe de lecture autonome et validé en réunion de service. Ce travail a permis la création consensuelle d'un outil d'aide à l'emploi du vecteur héliporté en SMUR primaire au sein du SAMU 14. Il est composé de cartes, d'une fiche réflexe et d'une procédure écrite de déclenchement. Cette méthodologie par consensus simple a permis la création d'un outil rationalisant le déclenchement du vecteur héliporté pour le SAMU 14. Il s'agissait du premier travail de ce type au SAMU 14. Cette méthodologie simple et transposable pourrait être utilisée dans d'autres centres 15 ou pour d'autres protocoles multidisciplinaires.

Retracing the evolution of a modern periplasmic binding protein.

Investigating the evolution of structural features in modern multidomain proteins helps to understand their immense diversity and functional versatility. The class of periplasmic binding proteins (PBPs) offers an opportunity to interrogate one of the main processes driving diversification: the duplication and fusion of protein sequences to generate new architectures. The symmetry of their two-lobed topology, their mechanism of binding, and the organization of their operon structure led to the hypothesis that PBPs arose through a duplication and fusion event of a single common ancestor. To investigate this claim, we set out to reverse the evolutionary process and recreate the structural equivalent of a single-lobed progenitor using ribose-binding protein (RBP) as our model. We found that this modern PBP can be deconstructed into its lobes, producing two proteins that represent possible progenitor halves. The isolated halves of RBP are well folded and monomeric proteins, albeit with a lower thermostability, and do not retain the original binding function. However, the two entities readily form a heterodimer in vitro and in-cell. The x-ray structure of the heterodimer closely resembles the parental protein. Moreover, the binding function is fully regained upon formation of the heterodimer with a ligand affinity similar to that observed in the modern RBP. This highlights how a duplication event could have given rise to a stable and functional PBP-like fold and provides insights into how more complex functional structures can evolve from simpler molecular components.

Structures of permuted halves of a modern ribose-binding protein.

Periplasmic binding proteins (PBPs) are a class of proteins that participate in the cellular transport of various ligands. They have been used as model systems to study mechanisms in protein evolution, such as duplication, recombination and domain swapping. It has been suggested that PBPs evolved from precursors half their size. Here, the crystal structures of two permuted halves of a modern ribose-binding protein (RBP) from Thermotoga maritima are reported. The overexpressed proteins are well folded and show a monomer-dimer equilibrium in solution. Their crystal structures show partially noncanonical PBP-like fold type I conformations with structural deviations from modern RBPs. One of the half variants forms a dimer via segment swapping, suggesting a high degree of malleability. The structural findings on these permuted halves support the evolutionary hypothesis that PBPs arose via a duplication event of a flavodoxin-like protein and further support a domain-swapping step that might have occurred during the evolution of the PBP-like fold, a process that is necessary to generate the characteristic motion of PBPs essential to perform their functions.

Élaboration par méthode du consensus simple d'un outil d'aide au déclenchement d'un hélicoptère au SAMU 14.

The main objective of this work was to develop a tool to assist the activation of a helicopter emergency medical service (HEMS) for the SAMU 14. We opted for a methodology based on "guidelines of good professional practice." Simple consensus was used. A multidisciplinary working group (pilots, medical regulation assistants, doctors) was created. Subgroup meetings (pilots, medical regulation assistants, doctors) developed subparts of the tool. The assembly of the tool's subparts was reviewed by the working group and then by an independent reading group. This work enabled the consensual creation of a tool to support the use of the helicopter emergency medical service (HEMS) for the SAMU 14. It is composed of maps, a protocol, and a written procedure of activation. This methodology by "simple consensus" allowed the development of a tool rationalizing the activation of the helicopter emergency medical service (HEMS) for the SAMU 14. It was the first work of this type within the SAMU 14. This simple and transposable methodology could be used in other emergency centers or for other multidisciplinary protocols.

Mitogen-activated protein kinase signaling in childhood asthma development and environment-mediated protection.

BACKGROUND: While childhood asthma prevalence is rising in Westernized countries, farm children are protected. The mitogen-activated protein kinase (MAPK) pathway with its negative regulator dual-specificity phosphatase-1 (DUSP1) is presumably associated with asthma development. OBJECTIVES: We aimed to investigate the role of MAPK signaling in childhood asthma and its environment-mediated protection, including a representative selection of 232 out of 1062 children from two cross-sectional cohorts and one birth cohort study. METHODS: Peripheral blood mononuclear cells (PBMC) from asthmatic and healthy children were cultured upon stimulation with farm-dust extracts or lipopolysaccharide. In subgroups, gene expression was analyzed by qPCR (PBMCs, cord blood) and NanoString technology (dendritic cells). Protein expression of phosphorylated MAPKs was measured by mass cytometry. Histone acetylation was investigated by chromatin immunoprecipitation. RESULTS: Asthmatic children expressed significantly less DUSP1 (p = .006) with reduced acetylation at histone H4 (p = .012) compared with healthy controls. Farm-dust stimulation upregulated DUSP1 expression reaching healthy levels and downregulated inflammatory MAPKs on gene and protein levels (PBMCs; p ≤ .01). Single-cell protein analysis revealed downregulated pMAPKs upon farm-dust stimulation in B cells, NK cells, monocytes, and T-cell subpopulations. CONCLUSION: Lower DUSP1 baseline levels in asthmatic children and anti-inflammatory regulation of MAPK in several immune cell types by farm-dust stimulation indicate a regulatory function for DUSP1 for future therapy contributing to anti-inflammatory characteristics of farming environments.

Fuzzle 2.0: Ligand Binding in Natural Protein Building Blocks.

Modern proteins have been shown to share evolutionary relationships via subdomain-sized fragments. The assembly of such fragments through duplication and recombination events led to the complex structures and functions we observe today. We previously implemented a pipeline that identified more than 1,000 of these fragments that are shared by different protein folds and developed a web interface to analyze and search for them. This resource named Fuzzle helps structural and evolutionary biologists to identify and analyze conserved parts of a protein but it also provides protein engineers with building blocks for example to design proteins by fragment combination. Here, we describe a new version of this web resource that was extended to include ligand information. This addition is a significant asset to the database since now protein fragments that bind specific ligands can be identified and analyzed. Often the mode of ligand binding is conserved in proteins thereby supporting a common evolutionary origin. The same can now be explored for subdomain-sized fragments within this database. This ligand binding information can also be used in protein engineering to graft binding pockets into other protein scaffolds or to transfer functional sites via recombination of a specific fragment. Fuzzle 2.0 is freely available at https://fuzzle.uni-bayreuth.de/2.0.

Evolution, folding, and design of TIM barrels and related proteins.

Proteins are chief actors in life that perform a myriad of exquisite functions. This diversity has been enabled through the evolution and diversification of protein folds. Analysis of sequences and structures strongly suggest that numerous protein pieces have been reused as building blocks and propagated to many modern folds. This information can be traced to understand how the protein world has diversified. In this review, we discuss the latest advances in the analysis of protein evolutionary units, and we use as a model system one of the most abundant and versatile topologies, the TIM-barrel fold, to highlight the existing common principles that interconnect protein evolution, structure, folding, function, and design.

MicroRNA-204 Is Necessary for Aldosterone-Stimulated T-Type Calcium Channel Expression in Cardiomyocytes.

Activation of the mineralocorticoid receptor (MR) in the heart is considered to be a cardiovascular risk factor. MR activation leads to heart hypertrophy and arrhythmia. In ventricular cardiomyocytes, aldosterone induces a profound remodeling of ion channel expression, in particular, an increase in the expression and activity of T-type voltage-gated calcium channels (T-channels). The molecular mechanisms immediately downstream from MR activation, which lead to the increased expression of T-channels and, consecutively, to an acceleration of spontaneous cell contractions in vitro, remain poorly investigated. Here, we investigated the putative role of a specific microRNA in linking MR activation to the regulation of T-channel expression and cardiomyocyte beating frequency. A screening assay identified microRNA 204 (miR-204) as one of the major upregulated microRNAs after aldosterone stimulation of isolated neonatal rat cardiomyocytes. Aldosterone significantly increased the level of miR-204, an effect blocked by the MR antagonist spironolactone. When miR-204 was overexpressed in isolated cardiomyocytes, their spontaneous beating frequency was significantly increased after 24 h, like upon aldosterone stimulation, and messenger RNAs coding T-channels (CaV3.1 and CaV3.2) were increased. Concomitantly, T-type calcium currents were significantly increased upon miR-204 overexpression. Specifically repressing the expression of miR-204 abolished the aldosterone-induced increase of CaV3.1 and CaV3.2 mRNAs, as well as T-type calcium currents. Finally, aldosterone and miR-204 overexpression were found to reduce REST-NRSF, a known transcriptional repressor of CaV3.2 T-type calcium channels. Our study thus strongly suggests that miR-204 expression stimulated by aldosterone promotes the expression of T-channels in isolated rat ventricular cardiomyocytes, and therefore, increases the frequency of the cell spontaneous contractions, presumably through the inhibition of REST-NRSF protein.

Metal ions and phosphatidylinositol 4,5-bisphosphate as interacting effectors of α-type plant phospholipase D.

Plant phospholipases D (PLD) are typically characterized by a C2 domain with at least two Ca2+ binding sites. In vitro, the predominantly expressed α-type PLDs need 20-100 mM CaCl2 for optimum activity, whereas the essential activator of ß- or γ-type PLDs, phosphatidylinositol 4,5-bisphosphate (PIP2), plays a secondary role. In the present paper, we have studied the interplay between PIP2 and metal ion activation of the well-known α-type PLD from cabbage (PLDα). With mixed micelles containing phosphatidyl-p-nitrophenol as substrate, PIP2-concentrations in the nanomolar range are able to activate the enzyme in addition to the essential Ca2+ activation. Mg2+ ions are able to replace Ca2+ ions but they do not activate PLDα. Rather, they abolish the activation of the enzyme by Ca2+ ions in the absence, but not in the presence, of PIP2. The presence of PIP2 causes a shift in the pH optimum of PLDα activity to the acidic range. Employing fluorescence measurements and replacing Ca2+ by Tb3+ ions, confirmed the presence of two metal ion-binding sites, in which the one of lower affinity proved crucial for PLD activation. Moreover, we have generated a homology model of the C2 domain of this enzyme, which was used for Molecular Dynamics (MD) simulations and docking studies. As is common for C2 domains, it shows two antiparallel ß-sheets consisting of four ß-strands each and loop regions that harbor two Ca2+ binding sites. Based on the findings of the MD simulation, one of the bound Ca2+ ions is coordinated by five amino acid residues. The second Ca2+ ion induces a loop movement upon its binding to three amino acid residues. Docking studies with PIP2 reveal, in addition to the previously postulated PIP2-binding site in the middle of the ß-sheet structure, another PIP2-binding site near the two Ca2+ ions, which is in accordance with the experimental interplay of PIP2, Ca2+ and Mg2+ ions.

Investigations of the polarization behavior of quantum cascade lasers by Stokes parameters.

We experimentally investigate the full polarization behavior of mid-infrared emitting quantum cascade lasers (QCLs) in terms of measuring the complete Stokes parameters, instead of only projecting them on a linear polarization basis. We demonstrate that besides the pre-dominant linear TM polarization of the emitted light as governed by the selection rules of the intersubband transition, small non-TM contributions, e.g., circularly polarized light, are present reflecting the birefringent behavior of the semiconductor quantum well waveguide. Surprisingly unique is the persistence of these polarization properties well below laser threshold. These investigations give further insight into understanding, manipulating, and exploiting the polarization properties of QCLs, both from a laser point of view and with respect toward applications.

In vivo and in vitro evidences of dehydroepiandrosterone protective role on the cardiovascular system.

CONTEXT: Dehydroepiandrosterone (DHEA) and its sulfate ester, Dehydroepiandrosterone Sulfate (DHEA-S) have been considered as putative anti-aging hormones for many years. Indeed, while DHEAS is the most abundant circulating hormone, its concentration is markedly decreased upon aging and early epidemiologic trials have revealed a strong inverse correlation between the hormone concentrations and the occurrence of several dysfunctions frequently encountered in the elderly. Naturally, hormonal supplementation has been rapidly suggested to prevent DHEA (S) deficiency and therefore, age-related development of these pathologies, using the same strategy as estrogen replacement therapy proposed in postmenopausal women. EVIDENCE ACQUISITION: All references were searched using PubMed and the following strategy: our initial selection included all articles in English and we sorted them with the following keywords: "DHEA or DHEA-S" and "heart or vascular or endothelium or cardiovascular disease". The search was limited to neither the publication date nor specific journals. The final selection was made according to the relevance of the article content with the aims of the review. According to these criteria, fewer than 10% of the articles retrieved at the first step were discarded. RESULTS: In this short review, we have focused on the cardiovascular action of DHEA. We started by analyzing evidences in favor of a strong inverse association between DHEA (S) levels and the cardiovascular risk as demonstrated in multiple observational epidemiologic studies for several decades. Then we discussed the different trials aimed at supplementing DHEA (S), both in animals and human, for preventing cardiovascular diseases and we analyzed the possible reasons for the discrepancy observed among the results of some studies. Finally, we presented putative molecular mechanisms of action for DHEA (S), demonstrated in vitro in different models of vascular and cardiac cells, highlighting the complexity of the involved signaling pathways. CONCLUSIONS: The identification of the beneficial cardiovascular effects of DHEA (S) and a better understanding of the involved mechanisms should be helpful to develop new strategies or pharmacologic approaches for many lethal diseases in Western countries.

30% improvement in absorption spectroscopy detectivity achieved by the detuned loading of a quantum cascade laser.

We perform a direct absorption spectroscopy experiment of carbon monoxide at 2193 cm(-1) by exploring the detectivity improvement potential of an intensity noise (IN)-reduced distributed feedback (DFB) quantum cascade laser. This was achieved by a detuned loading approach via a short, phase-sensitive optical feedback cavity. Under optimum IN reduction conditions, we obtain an improvement in signal-to-noise ratio from 733 to 1048, which transfers into a detection limit improvement from 1.2 ppm to 840 ppb. Therefore, we achieve a 30% lower detection limit, with the IN reduced when compared to the free-running case.

The DEX/CRH neuroendocrine test and the prediction of depressive relapse in remitted depressed outpatients.

Hypothalamo-pituitary-adrenal (HPA) system dysfunction is the most characteristic biological alteration found in a majority of depressed patients. Accumulating evidence suggests that the combined dexamethasone (DEX)/corticotropin-releasing hormone (CRH) test is highly sensitive to detect HPA system abnormalities. The aim of this study was to evaluate whether the DEX/CRH test has a predictive value for the risk of depressive relapse in outpatients who are in clinical remission from a major depressive episode. Thirty-eight depressed outpatients (23 women, 15 men) in remission (MADRS score < or =8) underwent the DEX/CRH test and were followed up for 12 months regarding the occurrence of a new depressive episode. In parallel we recruited 24 controls (13 men and 11 women). The main result is a statistically significant difference concerning the delta and AUC numbers for cortisol plasmatic values between the group of patients who relapsed during the 1-year follow-up and control subjects, but not between the group of patients with prolonged remission and controls. These results suggest that in outpatients who are in clinical remission from a major depressive episode, high delta and AUC values in the DEX/CRH test compared to controls subjects can be associated with a higher risk of relapse.