Cellular glutathione content in the organ of Corti and its role during ototoxicity.

Glutathione (GSH) is the major scavenger of reactive oxygen species (ROS) inside cells. We used live confocal imaging in order to clarify the role of GSH in the biology of the organ of Corti, the sensory epithelium of the cochlea, before, during and after the onset of hearing and in ~1 year old mice. GSH content was measured using monochlorobimane (MCB), a non-fluorescent cell permeant bimane that reacts with GSH, forming a fluorescent adduct through a reaction catalyzed by glutathione-S-transferase. GSH content increased significantly in inner hair cells during maturation in young adult animals, whereas there was no significant change in the outer hair cells. However, the GSH content in inner hair cells was significantly reduced in ~1 year old mice. The GSH content of supporting cells was comparatively stable over these ages. To test whether the GSH content played a significant protective role during ototoxicity, GSH synthesis was inhibited by buthionine sulfoximine (BSO) in organotypic cochlear explant cultures from immature mice. BSO treatment alone, which reduced GSH by 65 and 85% in inner hair cells and outer hair cells respectively, did not cause any significant cell death. Surprisingly, GSH depletion had no significant effect on hair cell survival even during exposure to the ototoxic aminoglycoside neomycin. These data suggest that the involvement of ROS during aminoglycoside-induced hair cell death is less clear than previously thought and requires further investigation

Linking micro and macroevolution in the presence of migration

Understanding macroevolutionary patterns is central to evolutionary biology. This involves the process of divergence within a species, which starts at the microevolutionary level, for instance, when two sub populations evolve towards different phenotypic optima. The speed at which these optima are reached is controlled by the degree of stabilising selection, which pushes the mean trait towards different optima in the different subpopulations, and ongoing migration that pulls the mean phenotype away from that optimum. Traditionally, macro phenotypic evolution is modelled by directional selection processes, but these models usually ignore the role of migration within species. Here, our goal is to reconcile the processes of micro and macroevolution by modelling migration as part of the speciation process. More precisely, we introduce an Ornstein-Uhlenbeck (OU) model where migration happens between two subpopulations within a branch of a phylogeny and this migration decreases over time as it happens during speciation. We then use this model to study the evolution of trait means along a phylogeny, as well as the way phenotypic disparity between species changes with successive epochs. We show that ignoring the effect of migration in sampled time-series data biases significantly the estimation of the selective forces acting upon it. We also show that migration decreases the expected phenotypic disparity between species and we analyse the effect of migration in the particular case of niche filling. We further introduce a method to jointly estimate selection and migration from time-series data. Our model extends traditional quantitative genetics results of selection and migration from a microevolutionary time frame to multiple speciation events at a macroevolutionary scale. Our results further support that not accounting for gene flow has important consequences in inferences at both the micro and macroevolutionary scale. (C) 2019 The Authors. Published by Elsevier Ltd

Imaging the Neutrophil Phagosome and Cytoplasm Using a Ratiometric pH Indicator.

Neutrophils are crucial to host innate defense and, consequently, constitute an important area of medical research. The phagosome, the intracellular compartment where the killing and digestion of engulfed particles take place, is the main arena for neutrophil pathogen killing that requires tight regulation. Phagosomal pH is one aspect that is carefully controlled, in turn regulating antimicrobial protease activity. Many fluorescent pH-sensitive dyes have been used to visualize the phagosomal environment. S-1 has several advantages over other pH-sensitive dyes, including its dual emission spectra, its resistance to photo-bleaching, and its high pKa. Using this method, we have demonstrated that the neutrophil phagosome is unusually alkaline in comparison to other phagocytes. By using different biochemical conjugations of the dye, the phagosome can be delineated from the cytoplasm so that changes in the size and shape of the phagosome can be assessed. This allows for further monitoring of ionic movement

On the Effect of Asymmetrical Trait Inheritance on Models of Trait Evolution.

Current phylogenetic comparative methods modeling quantitative trait evolution generally assume that, during speciation, phenotypes are inherited identically between the two daughter species. This, however, neglects the fact that species consist of a set of individuals, each bearing its own trait value. Indeed, because descendent populations after speciation are samples of a parent population, we can expect their mean phenotypes to randomly differ from one another potentially generating a "jump" of mean phenotypes due to asymmetrical trait inheritance at cladogenesis. Here, we aim to clarify the effect of asymmetrical trait inheritance at speciation on macroevolutionary analyses, focusing on model testing and parameter estimation using some of the most common models of quantitative trait evolution. We developed an individual-based simulation framework in which the evolution of phenotypes is determined by trait changes at the individual level accumulating across generations, and cladogenesis occurs then by separation of subsets of the individuals into new lineages. Through simulations, we assess the magnitude of phenotypic jumps at cladogenesis under different modes of trait inheritance at speciation. We show that even small jumps can strongly alter both the results of model selection and parameter estimations, potentially affecting the biological interpretation of the estimated mode of evolution of a trait. Our results call for caution when interpreting analyses of trait evolution, while highlighting the importance of testing a wide range of alternative models. In the light of our findings, we propose that future methodological advances in comparative methods should more explicitly model the intraspecific variability around species mean phenotypes and how it is inherited at speciation

Mitochondrial dynamics and quality control in Huntington's disease

Huntington's disease (HD) is an inherited neurodegenerative disorder caused by polyglutamine expansion mutations in the huntingtin protein. Despite its ubiquitous distribution, expression of mutant huntingtin (mHtt) is particularly detrimental to medium spiny neurons within the striatum. Mitochondrial dysfunction has been associated with HD pathogenesis. Here we review the current evidence for mHtt-induced abnormalities in mitochondrial dynamics and quality control, with a particular focus on brain and neuronal data pertaining to striatal vulnerability. We address mHtt effects on mitochondrial biogenesis, protein import, complex assembly, fission and fusion, mitochondrial transport, and on the degradation of damaged mitochondria via autophagy (mitophagy). For an integrated perspective on potentially converging pathogenic mechanisms, we also address impaired autophagosomal transport and abnormal mHtt proteostasis in HD

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering

Summary: Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development. : Maffioletti et al. generate human 3D artificial skeletal muscles from healthy donors and patient-specific pluripotent stem cells. These human artificial muscles accurately model severe genetic muscle diseases. They can be engineered to include other cell types present in skeletal muscle, such as vascular cells and motor neurons. Keywords: skeletal muscle, pluripotent stem cells, iPS cells, myogenic differentiation, tissue engineering, disease modeling, muscular dystrophy, organoid

Decellularised cartilage directs chondrogenic differentiation: creation of a fracture callus mimetic

Complications that arise from impaired fracture healing have considerable socioeconomic implications. Current research in the field of bone tissue engineering predominantly aims to mimic the mature bone tissue microenvironment. This approach, however, may produce implants that are intrinsically unresponsive to the cues present during the initiation of fracture repair. As such, this study describes the development of decellularised xenogeneic hyaline cartilage matrix in an attempt to mimic the initial reparative phase of fracture repair. Three approaches based on vacuum-assisted osmotic shock (Vac-OS), Triton X (Vac-Stx) and SDS (Vac-SDS) were investigated. The Vac-OS methodology reduced DNA content below 50ng/mg of tissue, whilst retaining 85% of the sGAG content and as such was selected as the optimal methodology for decellularisation. The resultant Vac-OS scaffolds (dcECM) were also devoid of the immunogenic alpha-gal epitope. Furthermore, minimal disruption to the structural integrity of the dcECM was demonstrated using differential scanning calorimetry (DSC) and fluorescence lifetime imaging microscopy (FLIM). The biological integrity of the dcECM was confirmed by its ability to drive the chondrogenic commitment and differentiation of human chondrocytes and periosteum-derived cells respectively. Furthermore, histological examination of dcECM constructs implanted in immunocompetent mice revealed a predominantly M2-macrophage driven regenerative response both at 2 and 8 weeks post-implantation. These findings contrasted with the implanted native costal cartilage that elicited a predominantly M1-macrophage mediated inflammatory response. This study highlights the capacity of dcECM from the Vac-OS methodology to direct the key biological processes of endochondral ossification, thus potentially recapitulating the callus phase of fracture repair

Diabetes causes marked inhibition of mitochondrial metabolism in pancreatic β-cells

Diabetes is a global health problem caused primarily by the inability of pancreatic β-cells to secrete adequate levels of insulin. The molecular mechanisms underlying the progressive failure of β-cells to respond to glucose in type-2 diabetes remain unresolved. Using a combination of transcriptomics and proteomics, we find significant dysregulation of major metabolic pathways in islets of diabetic βV59M mice, a non-obese, eulipidaemic diabetes model. Multiple genes/proteins involved in glycolysis/gluconeogenesis are upregulated, whereas those involved in oxidative phosphorylation are downregulated. In isolated islets, glucose-induced increases in NADH and ATP are impaired and both oxidative and glycolytic glucose metabolism are reduced. INS-1 β-cells cultured chronically at high glucose show similar changes in protein expression and reduced glucose-stimulated oxygen consumption: targeted metabolomics reveals impaired metabolism. These data indicate hyperglycaemia induces metabolic changes in β-cells that markedly reduce mitochondrial metabolism and ATP synthesis. We propose this underlies the progressive failure of β-cells in diabetes.Peer reviewe

Characterization of Membrane Potential Dependency of Mitochondrial Ca2+ Uptake by an Improved Biophysical Model of Mitochondrial Ca2+ Uniporter

Mitochondrial Ca2+ uniporter is the primary influx pathway for Ca2+ into respiring mitochondria, and hence plays a key role in mitochondrial Ca2+ homeostasis. Though the mechanism of extra-matrix Ca2+ dependency of mitochondrial Ca2+ uptake has been well characterized both experimentally and mathematically, the mechanism of membrane potential (ΔΨ) dependency of mitochondrial Ca2+ uptake has not been completely characterized. In this paper, we perform a quantitative reevaluation of a previous biophysical model of mitochondrial Ca2+ uniporter that characterized the possible mechanism of ΔΨ dependency of mitochondrial Ca2+ uptake. Based on a model simulation analysis, we show that model predictions with a variant assumption (Case 2: external and internal Ca2+ binding constants for the uniporter are distinct), that provides the best possible description of the ΔΨ dependency, are highly sensitive to variation in matrix [Ca2+], indicating limitations in the variant assumption (Case 2) in providing physiologically plausible description of the observed ΔΨ dependency. This sensitivity is attributed to negative estimate of a biophysical parameter that characterizes binding of internal Ca2+ to the uniporter. Reparameterization of the model with additional nonnengativity constraints on the biophysical parameters showed that the two variant assumptions (Case 1 and Case 2) are indistinguishable, indicating that the external and internal Ca2+ binding constants for the uniporter may be equal (Case 1). The model predictions in this case are insensitive to variation in matrix [Ca2+] but do not match the ΔΨ dependent data in the domain ΔΨ≤120 mV. To effectively characterize this ΔΨ dependency, we reformulate the ΔΨ dependencies of the rate constants of Ca2+ translocation via the uniporter by exclusively redefining the biophysical parameters associated with the free-energy barrier of Ca2+ translocation based on a generalized, non-linear Goldman-Hodgkin-Katz formulation. This alternate uniporter model has all the characteristics of the previous uniporter model and is also able to characterize the possible mechanisms of both the extra-matrix Ca2+ and ΔΨ dependencies of mitochondrial Ca2+ uptake. In addition, the model is insensitive to variation in matrix [Ca2+], predicting relatively stable physiological operation. The model is critical in developing mechanistic, integrated models of mitochondrial bioenergetics and Ca2+ handling

The impact of taxonomic change on conservation: Does it kill, can it save, or is it just irrelevant?

a b s t r a c t The important question of taxonomy and its impact on conservation efforts was brought to general attention by Robert May in 1990 with a News and Views article in Nature entitled ''Taxonomy as destiny." Taxonomy, however, has built-in instabilities that result in name changes, raising the question of whether name changes have a consistent impact on conservation efforts. Our review investigates three possible outcomes of taxonomic change, namely a positive impact on protection efforts, a hampering impact, or no measurable impact. We address these cases with a review of the relevant literature: specifically, government and conservation agency reports, scientific papers, and the general press, as well as correspondence with biologists active in plant and animal conservation. We found no evidence of a consistent effect of taxonomic change on conservation, although splitting taxa may tend to increase protection, and name changes may have the least effect where they concern charismatic organisms