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Complement C1s association with the pathogenesis of several diseases cannot be simply explained only by considering its main role in activating the classical complement pathway. This suggests that non-canonical functions are to be deciphered for this protease. Here the focus is on C1s cleavage of HMGB1 as an auxiliary target. HMGB1 is a chromatin non-histone nuclear protein, which exerts in fact multiple functions depending on its location and its post-translational modifications. In the extracellular compartment, HMGB1 can amplify immune and inflammatory responses to danger associated molecular patterns, in health and disease. Among possible regulatory mechanisms, proteolytic processing could be highly relevant for HMGB1 functional modulation. The unique properties of HMGB1 cleavage by C1s are analyzed in details. For example, C1s cannot cleave the HMGB1 A-box fragment, which has been described in the literature as an inhibitor/antagonist of HMGB1. By mass spectrometry, C1s cleavage was experimentally identified to occur after lysine on position 65, 128 and 172 in HMGB1. Compared to previously identified C1s cleavage sites, the ones identified here are uncommon, and their analysis suggests that local conformational changes are required before cleavage at certain positions. This is in line with the observation that HMGB1 cleavage by C1s is far slower when compared to human neutrophil elastase. Recombinant expression of cleavage fragments and site-directed mutagenesis were used to confirm these results and to explore how the output of C1s cleavage on HMGB1 is finely modulated by the molecular environment. Furthermore, knowing the antagonist effect of the isolated recombinant A-box subdomain in several pathophysiological contexts, we wondered if C1s cleavage could generate natural antagonist fragments. As a functional readout, IL-6 secretion following moderate LPS activation of RAW264.7 macrophage was investigated, using LPS alone or in complex with HMGB1 or some recombinant fragments. This study revealed that a N-terminal fragment released by C1s cleavage bears stronger antagonist properties as compared to the A-box, which was not expected. We discuss how this fragment could provide a potent brake for the inflammatory process, opening the way to dampen inflammation.

Immunoglobulins type-M (IgMs) are one of the first antibody classes mobilized during immune responses against pathogens and tumor cells. Binding to specific target antigens enables the interaction with the C1 complex which strongly activates the classical complement pathway. This biological function is the basis for the huge therapeutic potential of IgMs. But, due to their high oligomeric complexity, in vitro production, biochemical characterization, and biophysical characterization are challenging. In this study, we present recombinant production of two IgM models (IgM617 and IgM012) in pentameric and hexameric states and the evaluation of their polymer distribution using different biophysical methods (analytical ultracentrifugation, size exclusion chromatography coupled to multi-angle laser light scattering, mass photometry, and transmission electron microscopy). Each IgM construct is defined by a specific expression and purification pattern with different sample quality. Nevertheless, both purified IgMs were able to activate complement in a C1q-dependent manner. More importantly, BioLayer Interferometry (BLI) was used for characterizing the kinetics of C1q binding to recombinant IgMs. We show that recombinant IgMs possess similar C1q-binding properties as IgMs purified from human plasma.

We examine some combinatorial properties of parallel cut elimination in multiplicative linear logic (MLL) proof nets. We show that, provided we impose a constraint on some paths, we can bound the size of all the nets satisfying this constraint and reducing to a fixed resultant net. This result gives a sufficient condition for an infinite weighted sum of nets to reduce into another sum of nets, while keeping coefficients finite. We moreover show that our constraints are stable under reduction. Our approach is motivated by the quantitative semantics of linear logic: many models have been proposed, whose structure reflect the Taylor expansion of multiplicative exponential linear logic (MELL) proof nets into infinite sums of differential nets. In order to simulate one cut elimination step in MELL, it is necessary to reduce an arbitrary number of cuts in the differential nets of its Taylor expansion. It turns out our results apply to differential nets, because their cut elimination is essentially multiplicative. We moreover show that the set of differential nets that occur in the Taylor expansion of an MELL net automatically satisfies our constraints. Interestingly, our nets are untyped: we only rely on the sequentiality of linear logic nets and the dynamics of cut elimination. The paths on which we impose bounds are the switching paths involved in the Danos--Regnier criterion for sequentiality. In order to accommodate multiplicative units and weakenings, our nets come equipped with jumps: each weakening node is connected to some other node. Our constraint can then be summed up as a bound on both the length of switching paths, and the number of weakenings that jump to a common node.

LRP1 is a large endocytic modular receptor that plays a crucial role in the scavenging of apoptotic material through binding to pattern-recognition molecules. It is a membrane anchored receptor of the LDL receptor family with 4 extracellular clusters of ligand binding modules called cysteine rich complement-type repeats that are involved in the interaction of LRP1 with its numerous ligands. Complement C1q was shown to interact with LRP1 and to be implicated in the phagocytosis of apoptotic cells. The present work aimed at exploring how these two large molecules interact at the molecular level using a dissection strategy. For that purpose, recombinant LRP1 clusters II, III and IV were produced in mammalian HEK293F cells and their binding properties were investigated. Clusters II and IV were found to interact specifically and efficiently with C1q with KDs in the nanomolar range. The use of truncated C1q fragments and recombinant mutated C1q allowed to localize more precisely the binding site for LRP1 on the collagen-like regions of C1q (CLRs), nearby the site that is implicated in the interaction with the cognate protease tetramer C1r2s2. This site could be a common anchorage for other ligands of C1q CLRs such as sulfated proteoglycans and Complement receptor type 1. The use of a cellular model, consisting in CHO LRP1-null cells transfected with full-length LRP1 or a cluster IV minireceptor (mini IV) confirmed that mini IV interacts with C1q at the cell membrane as well as full-length LRP1. Further cellular interaction studies finally highlighted that mini IV can endorse the full-length LRP1 binding efficiency for apoptotic cells and that C1q has no impact on this interaction.

Resume Contexte Le cancer des testicules (CT) représente environ 1% de l’ensemble des cas de cancer chez les hommes, mais il demeure néanmoins le cancer le plus fréquent chez les adolescents et les jeunes adultes dans les pays industrialisés. Dans cette étude, nous avons évalué les variations temporelles des autoconservations de sperme réalisées par les hommes atteints d’un CT entre 1990 et 2013 en France. Méthodes Les données proviennent des autoconservations de sperme réalisées auprès de patients diagnostiqués avec CT, issues du réseau national français des banques de sperme. Les tendances de l’incidence des autoconservations de sperme ont été estimées à l’aide de deux modèles statistiques: la régression de Poisson, couramment utilisée, et le modèle de Verhulst. Résultats Entre 1990 et 2013, l’incidence globale des autoconservations de sperme est. passée de 1,73 à 5,57 pour 100,000 personnes-années. La régression de Poisson montre une estimation de l’incidence de près de 9 pour 100,000 [IC à 95% = 8,66-9,34] en 2020. Cependant, depuis 2005, le taux observé des autoconservations de sperme semble s’atténuer. Le modèle de Verhulst estime alors une incidence aux alentours de 6 pour 100,000 après 2020. Conclusions Les limites de cette étude comprennent l’impossibilité d’analyser l’incidence standardisée sur l’âge. Sur la base du modèle de Verhulst, les résultats suggèrent qu’il est. toujours et encore pertinent d’étudier l’évolution de l’incidence du cancer du testicule et des autoconservations de sperme afin de confirmer ou non la diminution/stagnation potentielle déjà observée dans cette maladie.

In France, control of chemotherapy preparations is highly recommended. Analytical control is a method of choice for identifying and quantifying drugs. Pediatric preparations, which often contain small quantities of drugs and are made in low final volumes were until then not analytically controlled. After the development and validation of a new sampling and assaying method for low volume chemotherapy preparations with an UV/Raman automaton (QCPrep +), the quality control results of the preparations intended for the patients were analyzed over a period of eighteen months

Ehlers-Danlos syndromes (EDS) are clinically and genetically heterogeneous disorders characterized by soft connective tissue alteration like joint hypermobility and skin hyper-extensibility. We previously identified heterozygous missense mutations in the C1R and C1S genes, coding for the complement C1 proteases, in patients affected by periodontal EDS, a specific EDS subtype hallmarked by early severe periodontitis leading to premature loss of teeth and connective tissue alterations. Up to now, there is no clear molecular link relating the nominal role of the C1r and C1s proteases, which is to activate the classical complement pathway, to these heterogeneous symptoms of periodontal EDS syndrome. We aim therefore to elucidate the functional effect of these mutations, at the molecular and enzymatic levels. To explore the molecular consequences, a set of cell transfection experiments, recombinant protein purification, mass spectroscopy and N-terminal analyses have been performed. Focusing on the results obtained on two different C1S variants, namely p.Val316del and p.Cys294Arg, we show that HEK293-F cells stably transfected with the corresponding C1s variant plasmids, unexpectedly, do not secrete the full-length mutated C1s, but only a truncated Fg40 fragment of 40 kDa, produced at very low levels. Detailed analyses of the Fg40 fragments purified for the two C1s variants show that they are identical, which was also unexpected. This suggests that local misfolding of the CCP1 module containing the patient mutation exposes a novel cleavage site, between Lys353 and Cys354, which is not normally accessible. The mutation-induced Fg40 fragment contains the intact C-terminal serine protease domain but not the N-terminal domain mediating C1s interaction with the other C1 subunits, C1r, and C1q. Thus, Fg40 enzymatic activity escapes the normal physiological control of C1s activity within C1, potentially providing a loss-of-control. Comparative enzymatic analyses show that Fg40 retains the native esterolytic activity of C1s, as well as its cleavage efficiency toward the ancillary alarmin HMGB1 substrate, for example, whereas the nominal complement C4 activation cleavage is impaired. These new results open the way to further molecular explorations possibly involving subsidiary C1s targets.

Calreticulin (CRT) is a multifaceted protein, initially discovered as an endoplasmic reticulum (ER) chaperone protein, that is essential in calcium metabolism. Various implications in cancer, early development and immunology have been discovered more recently for CRT, as well as its role as a dominant `eat-me' prophagocytic signal. Intriguingly, cell-surface exposure/secretion of CRT is among the infective strategies used by parasites such as Trypanosoma cruzi, Entamoeba histolytica, Taenia solium, Leishmania donovani and Schistosoma mansoni. Because of the inherent flexibility of CRTs, their analysis by X-ray crystallography requires the design of recombinant constructs suitable for crystallization, and thus only the structures of two very similar mammalian CRT lectin domains are known. With the X-ray structures of two distant parasite CRTs, insights into species structural determinants that might be harnessed to fight against the parasites without affecting the functions of the host CRT are now provided. Moreover, although the hypothesis that CRT can exhibit both open and closed conformations has been proposed in relation to its chaperone function, only the open conformation has so far been observed in crystal structures. The first evidence is now provided of a complex conformational transition with the junction reoriented towards P-domain closure. SAXS experiments also provided additional information about the flexibility of T. cruzi CRT in solution, thus complementing crystallographic data on the open conformation. Finally, regarding the conserved lectin-domain structure and chaperone function, evidence is provided of its dual carbohydrate/protein specificity and a new scheme is proposed to interpret such unusual substrate-binding properties. These fascinating features are fully consistent with previous experimental observations, as discussed considering the broad spectrum of CRT sequence conservations and differences.

Background: Pediatric chemotherapy preparations are usually not analytically controlled, for several reasons. First, they are generally made in syringe, which does not allow to take a sample without changing the final volume. Secondly, the percentage of the dose consecrated to control is important and finally low concentrations can cause sensitivity problems. This lack of quality control, greatly reduces the security of the chemotherapy circuit.

Se abordó el presente estudio con el objetivo de desarrollar estrategias que incrementaran la calidad de los aprendizajes y la evaluación, además de la motivación de los alumnos en la materia de Música en Educación Secundaria Obligatoria. Para ello se diseñó una experiencia en la que se eliminó el libro de texto y se utilizó el portafolio a lo largo del curso académico 2013-2014, complementando dicho uso con trabajos cooperativos, autoevaluaciones y co-evaluaciones. Se utilizó un diseño de dos grupos para comparar los resultados en cuanto a satisfacción con la asignatura y medias académicas. Se completó la investigación con los datos relativos a la evaluación del clima de aula por parte del alumnado. Los resultados muestran un aumento significativo del interés del alumnado por la materia y opiniones favorables acerca de la mejora personal en procesos de trabajo en equipo entre otros, la capacidad de apreciar estilos nuevos o la capacidad de síntesis.

Complement C1s association with the pathogenesis of several diseases cannot be simply explained only by considering its main role in activating the classical complement pathway. This suggests that non-canonical functions are to be deciphered for this protease. Here the focus is on C1s cleavage of HMGB1 as an auxiliary target. HMGB1 is a chromatin non-histone nuclear protein, which exerts in fact multiple functions depending on its location and its post-translational modifications. In the extracellular compartment, HMGB1 can amplify immune and inflammatory responses to danger associated molecular patterns, in health and disease. Among possible regulatory mechanisms, proteolytic processing could be highly relevant for HMGB1 functional modulation. The unique properties of HMGB1 cleavage byC1s are analyzed in details. For example, C1s cannot cleave the HMGB1 A-box fragment, which has been described in the literature as an inhibitor/ antagonist of HMGB1. By mass spectrometry, C1s cleavage was experimentally identified to occur after lysine on position 65, 128 and 172 in HMGB1. Compared to previously identified C1s cleavage sites, the ones identified here are uncommon, and their analysis suggests that local conformational changes are required before cleavage at certain positions. This is in line with the observation that HMGB1 cleavage by C1s is far slower when compared to human neutrophil elastase. Recombinant expression of cleavage fragments and site-directed mutagenesis were used to confirm these results and to explore how the output of C1s cleavage on HMGB1 is finely modulated by the molecular environment. Furthermore, knowing the antagonist effect of the isolated recombinant A-box subdomain in several pathophysiological contexts, we wondered if C1s cleavage could generate natural antagonist fragments. As a functional readout, IL-6 secretion following moderate LPS activation of RAW264.7 macrophage was investigated, using LPS alone or in complex with HMGB1 or some recombinant fragments. This study revealed that a N-terminal fragment released by C1s cleavage bears stronger antagonist properties as compared to the A-box, which was not expected. We discuss how this fragment could provide a potent brake for the inflammatory process, opening the way to dampen inflammation. [ABSTRACT FROM AUTHOR]

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca(2+)-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 Å resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism.

Aims Hypertrophic cardiomyopathy (HCM) may be associated with very narrow QRS, while left ventricular hypertrophy (LVH) may increase QRS duration. We investigated the relationships between QRS duration and LV mass (LVM) in subtypes of abnormal LV wall thickness. Methods and results Automated measurement of LVM on MRI was correlated to automated measurement of QRS duration on ECG in HCM, left ventricular non compaction (LVNC), left ventricular hypertrophy (LVH), and controls with healthy hearts. Uni and multivariate analyses were performed between groups including explanatory variables expected to influence LVM and QRS duration. The relationships between QRS duration and LVM were further studied within each group. Two hundred and twenty-one HCM, 28 LVNC, 16 LVH, and 40 controls were retrospectively included. Mean QRS duration was 92 ms for HCM, 104 for LVNC, 110 for LVH, and 92 for controls (P  < 0.01). Mean LVM was 100, 90, 108, and 68 g/m2 (P  < 0.01). QRS duration, LVM, hypertension, maximal wall thickness, and late gadolinium enhancement were significantly linked to HCM in multivariate analysis (w/wo bundle branch block). An independent negative correlation was found between LVM and QRS duration in the HCM group, while the relationship was reverse in LVNC, LVH, and controls. Conclusion QRS duration increases with LVM in LVNC, LVH, or in healthy hearts, while reverse relationship is present in HCM. These relationships were independent from other parameters. These results warrant additional investigations for refining diagnosis criteria for HCM in the future. [ABSTRACT FROM AUTHOR]

We examine some combinatorial properties of parallel cut elimination in multiplicative linear logic (MLL) proof nets. We show that, provided we impose a constraint on some paths, we can bound the size of all the nets satisfying this constraint and reducing to a fixed resultant net. This result gives a sufficient condition for an infinite weighted sum of nets to reduce into another sum of nets, while keeping coefficients finite. We moreover show that our constraints are stable under reduction. Our approach is motivated by the quantitative semantics of linear logic: many models have been proposed, whose structure reflect the Taylor expansion of multiplicative exponential linear logic (MELL) proof nets into infinite sums of differential nets. In order to simulate one cut elimination step in MELL, it is necessary to reduce an arbitrary number of cuts in the differential nets of its Taylor expansion. It turns out our results apply to differential nets, because their cut elimination is essentially multiplicative. We moreover show that the set of differential nets that occur in the Taylor expansion of an MELL net automatically satisfies our constraints. Interestingly, our nets are untyped: we only rely on the sequentiality of linear logic nets and the dynamics of cut elimination. The paths on which we impose bounds are the switching paths involved in the Danos-Regnier criterion for sequentiality. In order to accommodate multiplicative units and weakenings, our nets come equipped with jumps: each weakening node is connected to some other node. Our constraint can then be summed up as a bound on both the length of switching paths, and the number of weakenings that jump to a common node. [ABSTRACT FROM AUTHOR]

Résumé: Le département de pharmacie de Gustave Roussy gère près de 600 essais cliniques de phase 1 à 3 et a dispensé, en 2019, 15 500 médicaments expérimentaux. Suite à de nombreuses non-conformités, une réorganisation du secteur basée sur une approche par processus tiré du Lean santé a été débutée. Les objectifs de ce travail étaient de rapporter une expérience et de mettre en avant les leçons apprises dans la mise en place de ce type d'approche. Le projet s'est déroulé en deux étapes distinctes. La première consistait en une cartographie des processus utilisant les bases du Lean Healthcare et aboutissant à l'élaboration d'un plan d'actions prioritaires. La seconde correspondait à la poursuite de la restructuration du secteur par une méthode collaborative utilisant une approche « bottom-up », basée sur la méthode EPEHo et utilisant des ateliers de résolution de problème. La cartographie des macro-processus a abouti à un plan de 7 actions prioritaires. Suite à la formation de deux pharmaciens aux méthodes d'animation et à l'acquisition de compétences d'écoute, reformulation et de synthèse, 14 séances ont été réalisées permettant de mettre en place 18 solutions. Ces ateliers ont permis d'apporter une vision nouvelle de l'amélioration des processus et de la résolution des problèmes. Ce travail illustre en vie réelle une expérience réussie d'amélioration continue par une approche collaborative. Les solutions mises en place au travers de ces ateliers sont un succès tant vis-à-vis de l'amélioration de la performance de la prestation pharmaceutique que de la transformation d'attitude de certains collaborateurs. The Clinical Pharmacy Department manages nearly 600 phase 1 to 3 CT clinical trials and, in 2019, dispensed up to 15,500 experimental drugs. Following numerous non-conformities, a reorganization of the CT unit based on a process oriented management approach according to Lean healthcare was started. The objectives of this work were to report an experience and to highlight the lessons learned in the implementation of this type of approach. Two phases were considered to conduct this transformation. The first consisted of a process mapping using the basics of Lean Healthcare and resulting in the development of a priority action plan. The second corresponded to the continued restructuring of the CT Unit by a collaborative method using a "bottom-up" approach, based on the EPEHo method and using problem-solving workshops. The mapping of macro-processes resulted in a plan of 7 priority actions. Some pharmacists were trained to facilitate in an appropriate manner the workshops. In total, 14 sessions were carried out allowing 18 solutions to be implemented. These workshops provided a new perspective on process improvement and problem solving. This work illustrates in real life a successful experience of continuous improvement through a collaborative approach. The solutions put in place through these workshops are a success both in terms of improving the performance of the pharmaceutical service and in transforming the adherence to work of collaborators. [ABSTRACT FROM AUTHOR]

LRP1 is a large endocytic modular receptor that plays a crucial role in the scavenging of apoptotic material through binding to pattern-recognition molecules. It is a membrane anchored receptor of the LDL receptor family with 4 extracellular clusters of ligand binding modules called cysteine rich complement-type repeats that are involved in the interaction of LRP1 with its numerous ligands. Complement C1q was shown to interact with LRP1 and to be implicated in the phagocytosis of apoptotic cells. The present work aimed at exploring how these two large molecules interact at the molecular level using a dissection strategy. For that purpose, recombinant LRP1 clusters II, III and IV were produced in mammalian HEK293F cells and their binding properties were investigated. Clusters II and IV were found to interact specifically and efficiently with C1q with K Ds in the nanomolar range. The use of truncated C1q fragments and recombinant mutated C1q allowed to localize more precisely the binding site for LRP1 on the collagen-like regions of C1q (CLRs), nearby the site that is implicated in the interaction with the cognate protease tetramer C1r2s2. This site could be a common anchorage for other ligands of C1q CLRs such as sulfated proteoglycans and Complement receptor type 1. The use of a cellular model, consisting in CHO LRP1-null cells transfected with full-length LRP1 or a cluster IV minireceptor (mini IV) confirmed that mini IV interacts with C1q at the cell membrane as well as full-length LRP1. Further cellular interaction studies finally highlighted that mini IV can endorse the full-length LRP1 binding efficiency for apoptotic cells and that C1q has no impact on this interaction. [ABSTRACT FROM AUTHOR]