Data-centrism, precision health, data-science and open science are factors transforming health research, healthcare and health policy. The COVID-19 pandemic interacted with the former and revealed some deficiencies of the latter. This has in turn stimulated disciplinary clashes, interdisciplinary elaborations and political conflicts, concentrated around the techno-scientific, political and societal changes needed to cope with the complex health problems that were and will be challenging our societies (inequity, poverty, pandemics, climate change, reemerging diseases, etc). Herein, as a continuation of an interdisciplinary dialogue initiated in 2018 with the “Data and Information Sciences Applied to Human Health” project (CIDASH for short), we put forward the idea of planning a data-centric, open, health research infrastructure, interoperable with the integral national health system and with national health registries. We aimed to identify and analyze from interdisciplinary and trans-disciplinary perspectives and based on our experiences in Uruguay the opportunities, concerns and challenges regarding the implementation of an open health research infrastructure (OSHRI). Our specific objectives were: i) to provide a brief account of our experience in human health research in Uruguay; ii) to reflect on the perceived opportunities, challenges, issues and potential solutions in human health research from the scientific, methodological, technical, ethical and regulatory point of view; iii) to elaborate on and propose structuring pillars for future open data infrastructures for human health research in Uruguay. The methodology we followed to report our experience was qualitative, interpretative and deliberative. Results include a list of problems and design pillars identified in relation of the creation of an OSHRI. To discuss these results we took the Centre for the Integration of Health Data and Knowledge (CIDACS) in Salvador de Bahía (Brazil) as an example of an installed and running open science health research infrastructure that warrants access to population-size data with proper ethical and scientific standards. After deliberation on the above-mentioned experiences we found that this report could bring us a step closer in the way of engaging with participatory research and change from within the science community.

La datificación y las acciones de combate a la crisis pueden ser entendidas como un campo, que definimos en el sentido de Bourdieu como un sistema de relaciones sociales que funcionan respecto de un área donde se compite por lo mismo y cuya dinámicatiene una lógica interna propia(5). La posibilidad de dominancia en dicho campo, siguiendo al autor, estaría dada por la capacidad de apropiarse del espacio de forma simbólica o material, dependiendo del capital que posea cada agente. Esta conceptualización puede resultar útil para analizar la dinámica de la datificación de la pandemia en Uruguay.

Glucose-6-phosphate dehydrogenase (G6PDH) is the key enzyme supplying reducing power (NADPH) to the cells, by oxidation of glucose-6-phosphate (G6P), and in the process providing a precursor of ribose-5-phosphate. G6PDH is also a virulence factor of pathogenic trypanosomatid parasites. To uncover the biochemical and structural features that distinguish TcG6PDH from its human homolog, we have solved and analyzed the crystal structures of the G6PDH from Trypanosoma cruzi (TcG6PDH), alone and in complex with G6P. TcG6PDH crystallized as a tetramer and enzymatic assays further indicated that the tetramer is the active form in the parasite, in contrast to human G6PDH, which displays higher activity as a dimer. This quaternary structure was shown to be particularly stable. The molecular reasons behind this disparity were unveiled by structural analyses: a TcG6PDH-specific residue, R323, is located at the dimer–dimer interface, critically contributing with two salt bridges per subunit that are absent in the human enzyme. This explains why TcG6PDH dimerization impaired enzyme activity. The parasite protein is also distinct in displaying a 37-amino-acid extension at the N-terminus, which comprises the non-conserved C8 and C34 involved in the covalent linkage of two neighboring protomers. In addition, a cysteine triad (C53, C94 and C135) specific of Kinetoplastid G6PDHs proved critical for stabilization of TcG6PDH active site. Based on the structural and biochemical data, we posit that the N-terminal region and the catalytic site are highly dynamic. The unique structural features of TcG6PDH pave the way toward the design of efficacious and highly specific anti-trypanosomal drugs.

Purpose: During sepsis and mechanical ventilation oxidative stress is generated by endothelial and inflammatory lung cells. Our main objective was to study pulmonary center dot NO (nitric oxide) production and nitroxidative stress in mechanically-ventilated septic patients. Methods: We study 69 mechanically ventilated patients, 36 with sepsis and 33 without sepsis within the first 48 h of ICU admission compared with 33 mechanically ventilated patients without sepsis (MV) plus eight operating room patients without lung disease served as control healthy group (ORCG). Nitrite plus nitrate (NOx-), 3-nitrotyrosine and malondialdehyde (MDA) in bronchoalveolar lavage fluid (BALF) were analyzed. Results: BALF NOx-, BALF 3-nitrotyrosine, BALF MDA and plasma NOx- were higher in the Sepsis than in MV patients (all p < .05). Both SG and MV patients had higher BALF NOx- than the healthy control group (p < .001). In the Sepsis patients, the ICU non-survivors had higher levels of BALF NOx- than ICU survivors 80(70-127) mu M versus 31(15-47) mu M, p < .001. Conclusions: We conclude that during early phases of sepsis there is an enhanced lung nitroxidative stress due to an increase of center dot NO production leading to secondary -NO-derived oxidants, which promote protein nitration and lipid peroxidation. (C) 2019 Published by Elsevier Inc.

Atherosclerosis is an epidemic worldwide disease and leading cause of death in developed countries. Two main pathogenic events are well recognized in the generation of the atheroma plaques: inflammation and lipid accumulation via LDL deposition/foam cells formation. Inflammation is a key event mediated by inflammasome activation by sterile signals (i.e. cholesterol crystals) and precedes massive lipid accumulation.

Human serum albumin (HSA) is the most abundant protein in plasma. Cys34, the only free Cys residue, is the predominant plasma thiol and a relevant sacrificial antioxidant. Both in vivo circulating HSA and pharmaceutical preparations are heterogeneous with respect to the oxidation state of Cys34. In this work, we developed an external pH gradient chromatofocusing procedure that allows the analysis of the oxidation status of HSA in human plasma and biopharmaceutical products based on the different apparent isoelectric points and chemical properties of the redox isoforms. Specifically, reduced-mercury blocked HSA (HSA-SHg(+)), HSA with Cys34 oxidized to sulfenic acid (HSA-SOH) and HSA oxidized to sulfinate anion (HSA-SO2(-)) can be separated with resolutions of 1.4 and 3.1 (first and last pair) and hence quantified and purified. In addition, an N-terminally degraded isoform (HSA3-585) in different redox states can be resolved as well. Confirmation of the identity of the chromatofocusing isolated isoforms was achieved by high resolution whole protein MS. It is proposed that the chromatofocusing procedure can be used to produce more exact and complete descriptions of the redox status of HSA in vivo and in vitro. Finally, the scalability capabilities of the chromatofocusing procedure allow for the preparation of highly pure standards of several redox isoforms of HSA.

ABSTRACT Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors. IMPORTANCE The ability to sense and respond to environmental variations is an essential property for cell survival. Two-component systems mediate key signaling pathways that allow bacteria to integrate extra- or intracellular signals. Here we focus on the DesK/DesR system, which acts as a molecular thermometer in B. subtilis, regulating the cell membrane’s fluidity. Using a combination of complementary approaches, including determination of the crystal structures of active and inactive forms of the response regulator DesR, we unveil novel molecular mechanisms of DesR’s activation switch. In particular, we show that the association of the cognate histidine kinase DesK triggers DesR activation beyond the transfer of the phosphoryl group. On the basis of sequence and structural analyses of other two-component systems, this activation mechanism appears to be used in a wide range of sensory systems, contributing a further level of specificity control among different signaling pathways.

Two-component systems (TCSs) are key players in bacterial signaling, to better understand signal-transmission with molecular detail. The TCS DesK/DesR controls fatty acid desaturation in Bacillus subtilis in response to cold shock and other membrane-altering effectors. We had previously put forward a model of signal-dependent allosteric control of the sensor kinase catalytic activity [1,2]. We have now turned our attention to the response regulator DesR. A canonical activation pathway has been widely accepted to explain phosphorylation-mediated control of response regulator function, allosterically coupling the phosphorylation site to the α4β5α5 surface. However, the structural evidence supporting the main hypotheses is still highly fragmentary. We are now reporting the crystal structure of full-length DesR, in complex with a phosphoryl-mimetic, showing the activated state [3]. Several crystal forms of the receiver domain were determined in the active and inactive configurations, revealing molecular details of the activation switch. Comparative small angle X ray scattering of full-length constructs, structure-guided point mutagenesis, as well as in vitro and in vivo functional analyses, allow us to propose an integral model of DesR activation. The phosphorylation of the receiver domain is allosterically coupled not to one, but two exposed surfaces, independently controlling its dimerization and tetramerization. Notably, a novel surface is shown to be essential for a non-canonical dimerization and activation mechanism. Direct coupling analysis highlights this interface as a shared feature of all NarL/LuxR regulators. This surface is further involved in cognate histidine kinase binding, disclosing a novel view of response regulator allosteric control. With the data we are now reporting, the DesK/DesR signaling pathway becomes, to the best of our knowledge, one of the most thoroughly studied examples of a thermosensor TCS at the molecular and biological levels.

General anesthesia is frequently associated to transient hypoxemia and lung atelectasis. Although volatile anesthetics are safe and widely used, their potential role on anesthesia-induced pulmonary impairment has not been fully explored. In this study, we investigated the effect of volatile anesthetic sevoflurane on pulmonary surfactant composition and structure that could contribute to atelectasis. After 30 min of sevoflurane anesthesia, Sprague-Dawley rats showed increased levels of lyso-phosphatidylcholine and decreased levels of phosphatidylcholine associated with significant impairment in lung mechanics and alveolar collapse, but showed no deterioration of alveolar fluid reabsorption when compared to control group of rats anesthetized with pentobarbital. Exposure to sevoflurane altered the thermotropic profile of surfactant model membranes, as detected by fluorescence anisotropy. In this sense, sevoflurane-promoted fluidification of condensed phases could potentially impair the ability of surfactant films to sustain the lowest surface tensions.In conclusion, the observed changes in surfactant composition and viscosity properties suggest a direct effect of sevoflurane on surfactant function, a factor potentially involved in anesthetic-induced alterations in lung mechanics. (C) 2013 Elsevier Ltd. All rights reserved.

Several Leptospira species cause leptospirosis, the most extended zoonosis worldwide. In bacteria, two-component systems constitute key signalling pathways, some of which are involved in pathogenesis. The physiological roles of two-component systems in Leptospira are largely unknown, despite identifying several dozens within their genomes. Biochemical confirmation of an operative phosphorelaying two-component system has been obtained so far only for the Hklep/Rrlep pair. It is known that hklep/rrlep knockout strains of Leptospira biflexa result in haem auxotrophy, although their de novo biosynthesis machinery remains fully functional. Haem is essential for Leptospira, but information about Hklep/Rrlep effector function(s) and target(s) is still lacking. We are now reporting a thorough molecular characterization of this system, which we rename HemK/HemR. The DNA HemR-binding motif was determined, and found within the genomes of saprophyte and pathogenic Leptospira. In this way, putative HemR-regulated genes were pinpointed, including haem catabolism-related (hmuO - haem oxygenase) and biosynthesis-related (the hemA/C/D/B/L/E/N/G operon). Specific HemR binding to these two promoters was quantified, and a dual function was observed in vivo, inversely repressing the hmuO, while activating the hemA operon transcription. The crystal structure of HemR receiver domain was determined, leading to a mechanistic model for its dual regulatory role.

Sulfenic acid is formed upon oxidation of thiols and is a central intermediate in the redox modulation of an increasing number of proteins. Methods for quantifying or even detecting sulfenic acid are scarce. Herein, the reagent 7-chloro-4-nitrobenz-2-oxa-1,3-diazole was determined not to be suitable as a chromophoric probe for sulfenic acid in human serum albumin (HSA-SOH) because of lack of specificity. Thionitrobenzoate (TNB) reacted with HSA exposed to hydrogen peroxide, but not control or thiol-blocked HSA. The reaction was biphasic. The first phase was approximately 20-fold faster than the second phase and first order in HSA-SOH and TNB (105 +/- 11 M-1 s-1, 25 degrees C, pH 7.4), allowing quantitative data on HSA-SOH formation and reactivity to be obtained. Exposure of reduced HSA (0.5 mM) to hydrogen peroxide (4 mM, 37 degrees C, 4 min) yielded 0.18 +/- 0.02 mol of HSA-SOH per mol of HSA. HSA-SH reacted with hydrogen peroxide at 2.7 +/- 0.7 M-1 s-1 (37 degrees C, pH 7.4), while HSA-SOH reacted at 0.4 +/- 0.2 M-1 s-1, yielding sulfinic acid (HSA-SO2H), as detected by mass spectrometry. The rate constants of HSA-SOH with targets of analytical interest such as dimedone and sodium arsenite were determined. HSA-SOH did not react appreciably with the plasma reductants ascorbate or urate, nor with free basic amino acids. In contrast, HSA-SOH reacted rapidly with the plasma thiols cysteine, glutathione, homocysteine, and cysteinylglycine at 21.6 +/- 0.2, 2.9 +/- 0.5, 9.3 +/- 0.9, and 55 +/- 3 M-1 s-1 (25 degrees C, pH 7.4), respectively, supporting a role for HSA-SOH in the formation of mixed disulfides.

Pulmonary surfactant (PS) is a lipid-protein mixture lining the alveolar air-water interface. By lowering surface tension, PS stabilizes the respiratory surface, thereby impeding alveolar collapse. Lack or alterations of PS properties are associated with acute lung injury (ALI). Sevoflurane is widely used in medical practice; however, some adverse properties were described in the lung function and PS [1,2]. The hypothesis of this study is that Sevoflurane affects native PS (NPS) system, which may contribute to ALI during anesthesia. In the present work, we have analyzed the functional, structural, and thermodynamic properties of NPS and its membranes reconstituted from the organic lipid extract (EPS) upon exposure to Sevoflurane. The compression-expansion properties of NPS films exposed to Sevoflurane were analyzed in a captive bubble surfactometer (CBS) as well as the effects of the anesthetic on the thermotropic properties of NPS and EPS membranes. Q-Static and dynamic cycles of NPS films were strongly affected upon exposure to Sevoflurane, which diminished the ability of the films to reach very low surface tension during compression. However, the equilibrium surface tension after adsorption and the maximum surface tension at the end of the expansion moieties of cycling isotherms were not affected by the presence of Sevoflurane. Spectroscopic analysis of surfactant complexes doped with LAURDAN or diphenylhexatriene indicate fluidification of EPS membranes upon exposure to Sevoflurane at room temperature. DSC experiments indicated that Sevoflurane induces a decrease of Tm and ΔH associated with the main thermotropic transition in PS membranes. In conclusion, the present results indicate that Sevoflurane impairs the biophysical properties of PS through perturbation of the lateral order of segregated phases, compromising correct compression along the respiratory cycles. [1] L. Malacrida et al. Am. J. Respir. Crit. Care Med.181;2010:A3631. [2] L. Malacrida et al. Biophysical J. 100(3);2011:505a-506a.

symmetrically (a) and each recognized by the specific interactions (b).

Sessions C594comprehensive implementation of the fundamentals of crystallography (symmetries, Fourier, scattering, etc).As the foundation of the macromolecular suite PHENIX, it has a certain connotation which is undeserved since the algorithms and data structures it features are correct for any crystal structure.As part of the EPSRC grant "Age Concern" we developed a companion library, the Small Molecule Toolbox (smtbx).It shares the same philosophy as the cctbx: it is designed to make the writing of short scripts easy as well as to make it possible to build or to integrate it into large programs.It provides tools covering the whole workflow of small molecule work but we will focus on refinement in this talk.The smtbx provides full matrix least-squares, restraints on bond lengths, angles, or dihedral angles, and special position constraints as well as the wealth of geometrical constraints available in ShelX; the both of merohedral and non-merohedral twin refinement; a solvent disorder modelling similar to the SQUEEZE procedure in PLATON.More importantly, its modular and open design ease the addition of new features.We will present one such new tool to model water molecules.

The structural comparison of two crystallographic models representing different functional and/or physical states of the same protein might seem easy if big conformational differences are detected by simple visual inspection. The precision of the models must be immediately considered if subtle structural changes are at play or if the aim is to evaluate small internal changes that occur in combination with large scale rigid-body changes. While the estimation of the positional uncertainty of crystallographic models has been extensively studied and a few methods have been developed to perform error-inclusive coordinate comparisons, the comparison of isotropic atomic B factors (Bi) has received less attention. It is known that direct quantitative comparisons of Bi are flawed by a scaling problem. Precise and accurate comparison of Bi might be especially important to tackle the problem of protein “allostery without conformational change”. In this work we introduce a method for the pair-wise structural comparison of crystallographic B factors which is based on the linear correlation between positional uncertainty (σ(r,Bavg)) and the average B factor (Bavg) in restrained refined models observed by Cruickshank1. If this is combined with the calculated standard average uncertainty of Bi for each model, available at the end of the model refinement process, and after consideration of non-atomic model parameters and refinement details, the method can be used to perform statistically meaningful comparisons. The method is validated by finding previously overlooked relationships between Bavg and σ(r,Bavg) and Rfree for models of different protein states. The method is used to analyze the redox-dependent allosteric communication between Cys34 thiol site and Sudlow's drug binding site I in Human Serum Albumin. 1. Cruickshank, D. W. Acta Crystallogr D Biol Crystallogr 1999, 55, 583.