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Une enquête composée de 34 questions a été adressée à des chercheurs en chimie, physique, sciences de l'ingénieur et de l'environnement, en vue d'identifier leur rapport aux résultats de recherche infructueux ainsi que les freins et les leviers de leur diffusion. L'étude se fonde sur 310 réponses complètes émanant de participants affiliés à des établissements de recherche et d'enseignement français. Menée dans le cadre du projet Datacc, porté par les bibliothèques universitaires de Lyon et Grenoble Alpes, engagées dans l'accompagnement des chercheurs à l'ouverture des données de recherche, notre étude permet de combler le déficit de données sur le sujet au regard des disciplines concernées. Elle relève que 81% des chercheurs interrogés ont déjà produit des résultats négatifs pertinents et 75% se disent prêts à publier ce type de données. Pourtant, seuls 12,5% des répondants ont déjà eu l'occasion de le faire dans une revue scientifique. Ce contraste béant entre l'intention et la pratique soulève des interrogations sur les obstacles en présence et les solutions potentielles à apporter. Mots-clés : résultats négatifs, open data, biais de publication Points clés • Plus de 8 chercheurs sur 10 ont déjà produit des résultats négatifs qu'ils jugent utiles pour leur communauté, mais seule une petite fraction a été amenée à les partager. • Les résultats négatifs font l'objet de débats intenses au sein des équipes de recherche, mais leur communication demeure essentiellement interne et informelle, circulant peu au-delà des laboratoires. • Faute de diffusion adéquate, près de 30% des répondants rapportent avoir découvert qu'une autre équipe avait effectué les mêmes expériences aboutissant aux mêmes résultats infructueux. • Le manque de temps et la complexité de restitution et d'interprétation des résultats négatifs sont identifiés comme les freins principaux à leur diffusion. • L'appétence pour la création d'une plateforme en accès ouvert de diffusion des résultats négatifs peut varier en fonction des profils et des disciplines.

Neuroserpin (NS) is a serine protease inhibitor (SERPIN) involved in different neurological pathologies, including the Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), related to the aberrant polymerization of NS mutants. Here we present an in vitro and in silico characterization of native NS and its dysfunctional conformation isoforms: the proteolytically cleaved conformer, the inactive latent conformer, and the polymeric conformer. Using circular dichroism and fluorescence spectroscopy, we present an experimental validation of the latent model and highlight the main structural features of the different conformers. In par- ticular, emission spectra of aromatic residues yield a distinct conformational fingerprint, that provides a novel and simple spectroscopic tool for selecting serpin conformers in vitro. Based on the analogy between cleaved and latent serpins, we propose a structural model for latent neuroserpin, for which an experimental crystallographic structure is lacking. Molecular Dynamics simulations suggest that NS conformational stability and flexibility arise from the spatial pattern of intramolecular salt-bridges and hydrogen bonds.

JBIC Journal of Biological Inorganic Chemistry, 12 (6), ISSN:0949-8257, ISSN:1432-1327

Pulse EPR spectroscopy is used to investigate possible structural features of the copper(II) ion coordinated to poly(dG-dC).poly(dG-dC) in a frozen aqueous solution, and the structural changes of the polynucleotide induced by the presence of the metal ion. Two different copper species were identified and their geometry explained by a molecular model. According to this model, one species is exclusively coordinated to a single guanine with the N7 nitrogen atom forming a coordinative bond with the copper. In the other species, a guanine and a cytosine form a ternary complex together with the copper ion. A copper crosslink between the N7 of guanine and N3 of cytosine is proposed as the most probable coordination site. Moreover, no evidence was found for an interaction of either copper species with a phosphate group or equatorial water molecules. In addition, circular dichroism (CD) spectroscopy showed that the DNA of the CuII-poly(dG-dC)•poly(dG-dC) adducts resembles the left-handed Z-form. These results suggest that metal-mediated Hoogsteen base pairing, as previously proposed for a right-handed DNA duplex, can also occur in a double-stranded left-handed DNA (Santangelo et al. [1]).

The polymerization of serpins is at the root of a large class of diseases; the molecular structure of serpin polymers has been recently debated. In this work, we study the polymerization kinetics of human neuroserpin by Fourier Transform Infra Red spectroscopy and by time-lapse Size Exclusion Chromatography. First, we show that two distinct neuroserpin polymers, formed at 45 and 85 °C, display the same isosbestic points in the Amide I band, and therefore share common secondary structure features. We also find a concentration independent polymerization rate at 45 °C suggesting that the polymerization rate limiting step is the formation of an activated monomeric species. The polymer structures are consistent with a model that predicts the bare insertion of portions of the reactive center loop into the A b-sheet of neighboring serpin molecule, although with different extents at 45 and 85 °C. Further studies are currently addressing the structure of monomeric neuroserpin conformers.

The Double Electron Electron Resonance (DEER) is an innovative technique that allows to measure the distance distribution between two spin labels within a range of 2-8 nm. This technique does not require crystalline samples, thus it is possible to determine the position of two different spin labelled domains of intrinsically flexible macromolecular systems. Our idea is to determinate the structural details of the intermediate conformers of human neuroserpin (hNS) by DEER. hNS is a protein that in the native state is folded in a metastable conformation. In particular conditions, hNS can either adopt a more stable conformation with the reactive centre loop (RCL) inserted into a β-sheet (latent conformer) or insert the RCL into the β-sheet of a neighbour hNS molecule thus forming dimers or polymers (polymeric conformers). This research project requires the production of spin labelled hNS with both a single and a double cysteine mutation via Site-Directed Spin Labelling (SDSL). We have already performed a first preliminary experiment on native hNS spin labelled at positions 157 and 296 (see Figure 1). The results are in agreement with the available crystallographic structure of native hNS and show the applicability of the DEER technique to the study of hNS conformers that have not been yet crystallized.

Neuroserpin is an inhibitory enzyme, belonging to the family of serpins and involved in several pathologies, such as ischemia, Alzheimer disease, and FENIB (Familial Encephalopathy with Neuroserpin Inclusion Body). Here, we study the mechanism of neuroserpin inactivation and polymerization by different experimental techniques (static and dynamic light scattering, liquid chromatography, Fourier transform infrared spectroscopy, emission spectroscopy). Our results show that at intermediate temperatures (45-55 °C) neuroserpin forms flexible polymers with a size from a few tens to a few hundreds of nanometers. At high temperatures, above 80 °C, our results reveal a different polymeric form, reached through an analogous loop-sheet mechanism, with considerably larger size and higher chemical stability. Our observations highlight the role of the different processes involved in serpin self-assembly, namely inactivating conformational changes, oligomer formation, polymer elongation and fragmentation.

Serpins are a wide class of proteins with high structural similarity, characterized by a unique substrate-like inhibitory mechanism that resembles a "molecular mousetrap". The active serpin is characterized by a main 5-stranded β-sheet and an exposed Reactive Centre Loop, which acts as a bait for the target protease. The cleavage of the loop by the protease triggers the insertion of the loop into the β-sheet as a strand and the disruptive translocation of the protease. This peculiar conformational mobility is achievable since serpins fold into a metastable native conformation. This feature gives a selective advantage to the serpin family to develop inhibitory activities, but leaves these proteins labile to misfolding and dysfunctional mutations, which cause a class of diseases known as serpinopathies. Indeed, the thermodynamically stable non-functional conformation can be reached without cleavage by intramolecular loop insertion (latent state) or by intermolecular insertion, leading to polymerization. Neuroserpin is a an inhibitor of tissue-type plasminogen activator that has a role in many pathologies, such as ischemia, Alzheimer disease, and FENIB (Familial Encephalopathy with Neuroserpin Inclusion Body). It is particularly suited to study the molecular basis of serpin inactivation and polymerization, since it may form latent conformers and polymers by thermal activation even in the wild type form. Here, we study the mechanism of neuroserpin unfolding and polymerization by different experimental techniques (static and dynamic light scattering, liquid chromatography, Fourier transform infrared spectroscopy) and by the support of Molecular Dynamics simulations. Our results show that at intermediate temperatures (45-55 °C) neuroserpin forms flexible polymers with a size from a few tens to a few hundreds of nanometers. At high temperatures, above 80 °C, our results reveal a different polymeric form, reached through an analogous loop-sheet mechanism, with considerably larger size and higher chemical stability. These observations highlight the fact that serpin polymerization is context-dependent, and that polymers accumulated in serpinopathies under physiological conditions may be different for different serpin and even for different pathological mutants of the same serpin.