Correlation between acoustic divergence and phylogenetic distance in soniferous European gobiids (Gobiidae; Gobius lineage)

In fish, species identity can be encoded by sounds, which have been thoroughly investigated in European gobiids (Gobiidae, Gobius lineage). Recent evolutionary studies suggest that deterministic and/or stochastic forces could generate acoustic differences among related animal species, though this has not been investigated in any teleost group to date. In the present comparative study, we analysed the sounds from nine soniferous gobiids and quantitatively assessed their acoustic variability. Our interspecific acoustic study, incorporating for the first time the representative acoustic signals from the majority of soniferous gobiids, suggested that their sounds are truly species-specific (92% of sounds correctly classified into exact species) and each taxon possesses a unique set of spectro-temporal variables. In addition, we reconstructed phylogenetic relationships from a concatenated molecular dataset consisting of multiple molecular markers to track the evolution of acoustic signals in soniferous gobiids. The results of this study indicated that the genus Padogobius is polyphyletic, since P. nigricans was nested within the Ponto-Caspian clade, while the congeneric P. bonelli turned out to be a sister taxon to the remaining investigated soniferous species. Lastly, by extracting the acoustic and genetic distance matrices, sound variability and genetic distance were correlated for the first time to assess whether sound evolution follows a similar phylogenetic pattern. The positive correlation between the sound variability and genetic distance obtained here emphasizes that certain acoustic features from representative sounds could carry the phylogenetic signal in soniferous gobiids. Our study was the first attempt to evaluate the mutual relationship between acoustic variation and genetic divergence in any teleost fish

Adenosine A2A-dopamine D2 receptor-receptor heteromerization: qualitative and quantitative assessment by fluorescence and bioluminescence energy transfer

There is evidence for strong functional antagonistic interactions between adenosine A2A receptors (A2ARs) and dopamine D2 receptors (D2Rs). Although a close physical interaction between both receptors has recently been shown using co-immunoprecipitation and co-localization assays, the existence of a A2AR-D2R protein-protein interaction still had to be demonstrated in intact living cells. In the present work, fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques were used to confirm the occurrence of A2AR-D2R interactions in co-transfected cells. The degree of A2AR-D2R heteromerization, measured by BRET, did not vary after receptor activation with selective agonists, alone or in combination. BRET competition experiments were performed using a chimeric D2R-D1R in which helices 5 and 6, the third intracellular loop (I3), and the third extracellular loop (E3) of the D2R were replaced by those of the dopamine D1 receptor (D1R). Although the wild type D2R was able to decrease the BRET signal, the chimera failed to achieve any effect. This suggests that the helix 5-I3-helix 6-E3 portion of D2R holds the site(s) for interaction with A2AR. Modeling of A2AR and D2R using a modified rhodopsin template followed by molecular dynamics and docking simulations gave essentially two different possible modes of interaction between D2R and A2AR. In the most probable one, helix 5 and/or helix 6 and the N-terminal portion of I3 from D2R approached helix 4 and the C-terminal portion of the C-tail from the A2AR, respectively

Altered splicing of the BIN1 muscle-specific exon in humans and dogs with highly progressive centronuclear myopathy

Amphiphysin 2, encoded by BIN1, is a key factor for membrane sensing and remodelling in different cell types. Homozygous BIN1 mutations in ubiquitously expressed exons are associated with autosomal recessive centronuclear myopathy (CNM), a mildly progressive muscle disorder typically showing abnormal nuclear centralization on biopsies. In addition, misregulation of BIN1 splicing partially accounts for the muscle defects in myotonic dystrophy (DM). However, the muscle-specific function of amphiphysin 2 and its pathogenicity in both muscle disorders are not well understood. In this study we identified and characterized the first mutation affecting the splicing of the muscle-specific BIN1 exon 11 in a consanguineous family with rapidly progressive and ultimately fatal centronuclear myopathy. In parallel, we discovered a mutation in the same BIN1 exon 11 acceptor splice site as the genetic cause of the canine Inherited Myopathy of Great Danes (IMGD). Analysis of RNA from patient muscle demonstrated complete skipping of exon 11 and BIN1 constructs without exon 11 were unable to promote membrane tubulation in differentiated myotubes. Comparative immunofluorescence and ultrastructural analyses of patient and canine biopsies revealed common structural defects, emphasizing the importance of amphiphysin 2 in membrane remodelling and maintenance of the skeletal muscle triad. Our data demonstrate that the alteration of the muscle-specific function of amphiphysin 2 is a common pathomechanism for centronuclear myopathy, myotonic dystrophy, and IMGD. The IMGD dog is the first faithful model for human BIN1-related CNM and represents a mammalian model available for preclinical trials of potential therapies

AAV-mediated intramuscular delivery of myotubularin corrects the myotubular myopathy phenotype in targeted murine muscle and suggests a function in plasma membrane homeostasis

Myotubular myopathy (XLMTM, OMIM 310400) is a severe congenital muscular disease due to mutations in the myotubularin gene (MTM1) and characterized by the presence of small myofibers with frequent occurrence of central nuclei. Myotubularin is a ubiquitously expressed phosphoinositide phosphatase with a muscle-specific role in man and mouse that is poorly understood. No specific treatment exists to date for patients with myotubular myopathy. We have constructed an adeno-associated virus (AAV) vector expressing myotubularin in order to test its therapeutic potential in a XLMTM mouse model. We show that a single intramuscular injection of this vector in symptomatic Mtm1-deficient mice ameliorates the pathological phenotype in the targeted muscle. Myotubularin replacement in mice largely corrects nuclei and mitochondria positioning in myofibers and leads to a strong increase in muscle volume and recovery of the contractile force. In addition, we used this AAV vector to overexpress myotubularin in wild-type skeletal muscle and get insight into its localization and function. We show that a substantial proportion of myotubularin associates with the sarcolemma and I band, including triads. Myotubularin overexpression in muscle induces the accumulation of packed membrane saccules and presence of vacuoles that contain markers of sarcolemma and T-tubules, suggesting that myotubularin is involved in plasma membrane homeostasis of myofibers. This study provides a proof-of-principle that local delivery of an AAV vector expressing myotubularin can improve the motor capacities of XLMTM muscle and represents a novel approach to study myotubularin function in skeletal muscle

Health system readiness and the implementation of rectal artesunate for severe malaria in sub-Saharan Africa: an analysis of real-world costs and constraints

BACKGROUND: Rectal artesunate, an efficacious pre-referral treatment for severe malaria in children, was deployed at scale in Uganda, Nigeria, and DR Congo. In addition to distributing rectal artesunate, implementation required additional investments in crucial but neglected components in the care for severe malaria. We examined the real-world costs and constraints to rectal artesunate implementation. METHODS: We collected primary data on baseline health system constraints and subsequent rectal artesunate implementation expenditures. We calculated the equivalent annual cost of rectal artesunate implementation per child younger than 5 years at risk of severe malaria, from a health system perspective, separating neglected routine health system components from incremental costs of rectal artesunate introduction. FINDINGS: The largest baseline constraints were irregular health worker supervisions, inadequate referral facility worker training, and inadequate malaria commodity supplies. Health worker training and behaviour change campaigns were the largest startup costs, while supervision and supply chain management accounted for most annual routine costs. The equivalent annual costs of preparing the health system for managing severe malaria with rectal artesunate were US$2.63,$2.20, and $4.19 per child at risk and$322, $219, and$464 per child treated in Uganda, Nigeria, and DR Congo, respectively. Strengthening the neglected, routine health system components accounted for the majority of these costs at 71.5%, 65.4%, and 76.4% of per-child costs, respectively. Incremental rectal artesunate costs accounted for the minority remainder. INTERPRETATION: Although rectal artesunate has been touted as a cost-effective pre-referral treatment for severe malaria in children, its real-world potential is limited by weak and under-financed health system components. Scaling up rectal artesunate or other interventions relying on community health-care providers only makes sense alongside additional, essential health system investments sustained over the long term. FUNDING: Unitaid. TRANSLATION: For the French translation of the abstract see Supplementary Materials section

GDNF Selectively Induces Microglial Activation and Neuronal Survival in CA1/CA3 Hippocampal Regions Exposed to NMDA Insult through Ret/ERK Signalling

The glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for several neuronal populations in different brain regions, including the hippocampus. However, no information is available on the: (1) hippocampal subregions involved in the GDNF-neuroprotective actions upon excitotoxicity, (2) identity of GDNF-responsive hippocampal cells, (3) transduction pathways involved in the GDNF-mediated neuroprotection in the hippocampus. We addressed these questions in organotypic hippocampal slices exposed to GDNF in presence of N-methyl-D-aspartate (NMDA) by immunoblotting, immunohistochemistry, and confocal analysis. In hippocampal slices GDNF acts through the activation of the tyrosine kinase receptor, Ret, without involving the NCAM-mediated pathway. Both Ret and ERK phosphorylation mainly occurred in the CA3 region where the two activated proteins co-localized. GDNF protected in a greater extent CA3 rather than CA1 following NMDA exposure. This neuroprotective effect targeted preferentially neurons, as assessed by NeuN staining. GDNF neuroprotection was associated with a significant increase of Ret phosphorylation in both CA3 and CA1. Interestingly, confocal images revealed that upon NMDA exposure, Ret activation occurred in microglial cells in the CA3 and CA1 following GDNF exposure. Collectively, this study shows that CA3 and CA1 hippocampal regions are highly responsive to GDNF-induced Ret activation and neuroprotection, and suggest that, upon excitotoxicity, such neuroprotection involves a GDNF modulation of microglial cell activity

Defective Membrane Remodeling in Neuromuscular Diseases: Insights from Animal Models

Proteins involved in membrane remodeling play an essential role in a plethora of cell functions including endocytosis and intracellular transport. Defects in several of them lead to human diseases. Myotubularins, amphiphysins, and dynamins are all proteins implicated in membrane trafficking and/or remodeling. Mutations in myotubularin, amphiphysin 2 (BIN1), and dynamin 2 lead to different forms of centronuclear myopathy, while mutations in myotubularin-related proteins cause Charcot-Marie-Tooth neuropathies. In addition to centronuclear myopathy, dynamin 2 is also mutated in a dominant form of Charcot-Marie-Tooth neuropathy. While several proteins from these different families are implicated in similar diseases, mutations in close homologues or in the same protein in the case of dynamin 2 lead to diseases affecting different tissues. This suggests (1) a common molecular pathway underlying these different neuromuscular diseases, and (2) tissue-specific regulation of these proteins. This review discusses the pathophysiology of the related neuromuscular diseases on the basis of animal models developed for proteins of the myotubularin, amphiphysin, and dynamin families. A better understanding of the common mechanisms between these neuromuscular disorders will lead to more specific health care and therapeutic approaches