Green fluorescent protein expression in the symbiotic basidiomycete fungus Hebeloma cylindrosporum

The symbiotic basidiomycete Hebeloma cylindrosporum is a model fungal species used to study ectomycorrhizal symbiosis at the molecular level. In order to have a vital marker, we developed a green fluorescent protein (GFP) reporter system efficiently expressed in H. cylindrosporum using the sgfp coding region bordered by two introns fused to the saprophytic basidiomycete Coprinopsis cinerea cgl1 promoter. Expression of this reporter system was tested under different environmental conditions in two transformants, and glucose was shown to repress gfp expression. Such a reporter system will be used in plant-fungus interaction to evaluate sugar supply by the plant to the compatible mycorrhizal symbiont and to compare the expression of various genes of interest in the free-living mycelia, in the symbiotic (mycorrhizas) and the reproductive (fruit bodies) structures formed by H. cylindrosporu

µ-Calpain Conversion of Antiapoptotic Bfl-1 (BCL2A1) into a Prodeath Factor Reveals Two Distinct alpha-Helices Inducing Mitochondria-Mediated Apoptosis

Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance

Characterization of the signaling pathways leading to ectopic osteogenesis after spinal cord injury in a mouse model of neurogenic heterotopic ossification

La morbi-mortalité associée aux lésions vertébro-médullaires traumatiques (SCI) reste significative malgré les progrès réalisés en matière de prise en charge médicale et rééducative. Des complications secondaires musculosquelettiques, spasticité, amyotrophie ou ossifications hétérotopiques (HO), continuent de grever le pronostic fonctionnel de ces patients faute de thérapies préventives efficaces. Si les perturbations neuro-immunes et neuroendocrines engendrées par les lésions du système nerveux central (CNS) font l’objet d’un intérêt croissant, leur rôle dans la perte de l’homéostasie tissulaire, en particulier au niveau du muscle strié squelettique demeure mal compris.L’objectif de ce travail était de caractériser la nature et la cinétique de l’effet pro-ostéogénique observé après SCI dans un modèle murin d’HO neurogènes (NHO). Différentes procédures expérimentales in-vivo ont été utilisées pour évaluer l’influence de la systématisation et de la sévérité de l’atteinte neurologique sur la formation des NHO, ainsi que pour préciser la chronologie des processus physiopathologiques impliqués. Des techniques d’ablation pharmacologiques et de quantification moléculaire par qPCR et ELISA ont permis de mieux comprendre le rôle des hormones de stress et des neuropeptides nociceptifs dans ce modèle.Les principaux résultats obtenus ont mis en évidence l’aspect multiphasique des perturbations induites par la lésion médullaire sur le muscle en cours de régénération. Le relargage rapide de neuropeptides crée des conditions micro-environnementales altérant la clairance des débris nécrotiques et favorisant l’expression ectopique du programme ostéogénique. Le processus d’ossification intra-membraneuse s’organisant autour des débris minéralisés est secondairement régulé par une signalisation adrénergique spécifique impliquant les récepteurs beta de type1.Au-delà de l’amélioration de la compréhension mécanistique d’un modèle préclinique d’HO neurogène, ce travail supporte le développement de stratégies thérapeutiques séquentielles et ciblées dans la prévention des complications secondaires chez le patient blessé médullaire.Despite promising advances in experimental therapies and robotic rehabilitation, traumatic spinal cord injury (SCI) is still a common cause of potentially life-threatening and/or severely disabling complications. Besides the loss of motor function, SCI patients can secondarily experience muscle spasticity, atrophy and heterotopic ossifications (HO) that negatively impact on both their recovery and quality of life. Increasing evidence suggests that neuro-immune and/or neuro-metabolic dysfunction after SCI may interfere with peripheral organ homeostasis and tissue repair. Whether this can also drive the development of ectopic osteogenesis remains unknown.In this work, we investigated the nature and the kinetics of SCI-induced effects that derail the fate of muscle progenitors in a mouse model of acquired neurogenic HO. We conducted different types of spinal cord lesions to explore the influence of neurological injury level and severity. We then clarified the role of neuropeptides and stress hormones signals in the development of NHO through tissular quantification (ELISA, qPCR), surgical approaches (e.g. adrenalectomy), pharmacologic studies and chemical ablation.Our main results are that spinal cord lesions can interfere with muscular regeneration at various levels of a complex multi-steps process, beginning within the first hours of combined insults. This cascade of interconnected events starts with immediate and critical metabolic changes affecting the endogenous stem/progenitor cell niche. Secondarily, plasmatic release of epinephrine further impairs muscle regeneration, promoting the development of an ectopic intramembranous ossification process.Better understanding of the kinetics governing SCI-induced deregulations in skeletal muscle maintenance is crucial to develop effective preventive strategies against wasting disuse and aberrant repair in paralysed patients. Given the unravelled importance of timing, sequential targeted therapies could prove a promising path forward to maximally reduce and/or prevent NHO in at-risk patients

Viewing a DSSA in Context: Problems versus Solutions

Software architectures are solution structures for problem families. Corollary to this definition is the observation that software architectures do not directly address the nature or simply the definition of the problems for which they are supposed to provide solutions. Yet, we construct software system because we have problems to model and solve. Hence, it would be beneficial to provide both problem characterizations and mappings to solution structures, if only to help match problems to solutions. In this position paper, we outline a domain oriented method for the defintion of problems and their mapping to architectures

Ostéogenèse ectopique associée aux traumatismes vertébro-médullaires : étude des voies d’induction dans un modèle murin d’ossification hétérotopiques neurogènes

Despite promising advances in experimental therapies and robotic rehabilitation, traumatic spinal cord injury (SCI) is still a common cause of potentially life-threatening and/or severely disabling complications. Besides the loss of motor function, SCI patients can secondarily experience muscle spasticity, atrophy and heterotopic ossifications (HO) that negatively impact on both their recovery and quality of life. Increasing evidence suggests that neuro-immune and/or neuro-metabolic dysfunction after SCI may interfere with peripheral organ homeostasis and tissue repair. Whether this can also drive the development of ectopic osteogenesis remains unknown.In this work, we investigated the nature and the kinetics of SCI-induced effects that derail the fate of muscle progenitors in a mouse model of acquired neurogenic HO. We conducted different types of spinal cord lesions to explore the influence of neurological injury level and severity. We then clarified the role of neuropeptides and stress hormones signals in the development of NHO through tissular quantification (ELISA, qPCR), surgical approaches (e.g. adrenalectomy), pharmacologic studies and chemical ablation.Our main results are that spinal cord lesions can interfere with muscular regeneration at various levels of a complex multi-steps process, beginning within the first hours of combined insults. This cascade of interconnected events starts with immediate and critical metabolic changes affecting the endogenous stem/progenitor cell niche. Secondarily, plasmatic release of epinephrine further impairs muscle regeneration, promoting the development of an ectopic intramembranous ossification process.Better understanding of the kinetics governing SCI-induced deregulations in skeletal muscle maintenance is crucial to develop effective preventive strategies against wasting disuse and aberrant repair in paralysed patients. Given the unravelled importance of timing, sequential targeted therapies could prove a promising path forward to maximally reduce and/or prevent NHO in at-risk patients.La morbi-mortalité associée aux lésions vertébro-médullaires traumatiques (SCI) reste significative malgré les progrès réalisés en matière de prise en charge médicale et rééducative. Des complications secondaires musculosquelettiques, spasticité, amyotrophie ou ossifications hétérotopiques (HO), continuent de grever le pronostic fonctionnel de ces patients faute de thérapies préventives efficaces. Si les perturbations neuro-immunes et neuroendocrines engendrées par les lésions du système nerveux central (CNS) font l’objet d’un intérêt croissant, leur rôle dans la perte de l’homéostasie tissulaire, en particulier au niveau du muscle strié squelettique demeure mal compris.L’objectif de ce travail était de caractériser la nature et la cinétique de l’effet pro-ostéogénique observé après SCI dans un modèle murin d’HO neurogènes (NHO). Différentes procédures expérimentales in-vivo ont été utilisées pour évaluer l’influence de la systématisation et de la sévérité de l’atteinte neurologique sur la formation des NHO, ainsi que pour préciser la chronologie des processus physiopathologiques impliqués. Des techniques d’ablation pharmacologiques et de quantification moléculaire par qPCR et ELISA ont permis de mieux comprendre le rôle des hormones de stress et des neuropeptides nociceptifs dans ce modèle.Les principaux résultats obtenus ont mis en évidence l’aspect multiphasique des perturbations induites par la lésion médullaire sur le muscle en cours de régénération. Le relargage rapide de neuropeptides crée des conditions micro-environnementales altérant la clairance des débris nécrotiques et favorisant l’expression ectopique du programme ostéogénique. Le processus d’ossification intra-membraneuse s’organisant autour des débris minéralisés est secondairement régulé par une signalisation adrénergique spécifique impliquant les récepteurs beta de type1.Au-delà de l’amélioration de la compréhension mécanistique d’un modèle préclinique d’HO neurogène, ce travail supporte le développement de stratégies thérapeutiques séquentielles et ciblées dans la prévention des complications secondaires chez le patient blessé médullaire

Using Executable Domain Models to Implement Legacy Software Re-Engineering Position Paper

In this position paper, we advocate a domain-centric approach to the evolution of legacy systems. The migration of legacy systems is a difficult endeavor because traditional methods have two principal deficiencies. First, they fail to capture the context of a system, i.e., its domain. Second, the legacy system’s comprehension results are not directly usable for the system evolution. We propose the construction of executable domain models to alleviate both problems. The construction of an executable domain model entails a process of domain analysis that leads to a domain model, as well as the transition of the former to an executable state. The domain model provides domain expectations that drive legacy system understanding. The executable domain model provides a medium in which the result of the legacy system comprehension can be recorded. In fact, the executable domain model is instantiated using the system requirements derived during program comprehension. The artifact thus created takes the role of the re-engineered program. Our work uses the technique of object-oriented frameworks (OOF) as the executable domain model representation. 1