Automatic Morphological Subtyping Reveals New Roles of Caspases in Mitochondrial Dynamics

Morphological dynamics of mitochondria is associated with key cellular processes related to aging and neuronal degenerative diseases, but the lack of standard quantification of mitochondrial morphology impedes systematic investigation. This paper presents an automated system for the quantification and classification of mitochondrial morphology. We discovered six morphological subtypes of mitochondria for objective quantification of mitochondrial morphology. These six subtypes are small globules, swollen globules, straight tubules, twisted tubules, branched tubules and loops. The subtyping was derived by applying consensus clustering to a huge collection of more than 200 thousand mitochondrial images extracted from 1422 micrographs of Chinese hamster ovary (CHO) cells treated with different drugs, and was validated by evidence of functional similarity reported in the literature. Quantitative statistics of subtype compositions in cells is useful for correlating drug response and mitochondrial dynamics. Combining the quantitative results with our biochemical studies about the effects of squamocin on CHO cells reveals new roles of Caspases in the regulatory mechanisms of mitochondrial dynamics. This system is not only of value to the mitochondrial field, but also applicable to the investigation of other subcellular organelle morphology

Index-Based Cost-Effectiveness Analysis vs. Least-Cost River Basin Optimization Model: Comparison in the Selection of a Programme of Measures at the River Basin Scale

Increasing water scarcity challenges conventional approaches to managing water resources. More holistic tools and methods are required to support the integrated planning and management of fresh water resources at the river basin level. This paper compares an index-based cost-effectiveness analysis (IBCEA) with a least-cost river basin optimization model (LCRBOM). Both methods are applied to a real case study to design a cost-effective portfolio of water demand and supply management measures that ensures compliance with water supply and environmental targets. The IBCEA is a common approach to select programmes of measures in the implementation of the EU Water Framework Directive. We describe its limitations in finding a least-cost solution at the river basin level and highlight the benefits from implementing a LCRBOM. Both methods are compared in a real case study, the Orb river basin, in the south of France. The performances of the programmes of measures selected by the two methods are compared for the same annual equivalent cost. By ignoring the spatial and temporal variability of water availability and water demands in the river basin and the interconnection among its elements, the aggregated approach used in the standard IBCEA can miss more cost-effective solutions at the river basin scale.This paper is based on work conducted as part of several projects over more than 6 years. It benefited from the financial and technical support of the Agence de l'Eau Rhone Mediteranee et Corse; Conseil General de l'Herault; Conseil Regional du Languedoc Roussillon et ONEMA. Funding was partly provided by the IMPADAPT project /CGL2013-48424-C2-1-R) from the Spanish ministry MINECO (Ministerio de Economia y Competitividad) and European FEDER funds. Corentin Girard is supported by a grant from the University Lecturer Training Programme (FPU12/03803) of the Ministry of Education, Culture and Sports of Spain. We are very grateful to Y. Caballero (BRGM), S. Chazot (BRLi), E. Vier and F. Aigoui (GINGERGROUP) and L. Rippert and his team from the SMVOL for their help during the project and for the data provided. We thank as well the two anonymous reviewers, the Associated Editor and Editor-in-Chief of Water Resources Management, for their useful and encouraging comments during the review process.Girard-Martin, CDP.; Rinaudo, J.; Pulido-Velazquez, M. (2015). Index-Based Cost-Effectiveness Analysis vs. Least-Cost River Basin Optimization Model: Comparison in the Selection of a Programme of Measures at the River Basin Scale. 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Rac1 selective activation improves retina ganglion cell survival and regeneration

Contains fulltext : 117529.pdf (publisher's version ) (Open Access)In adult mammals, after optic nerve injury, retinal ganglion cells (RGCs) do not regenerate their axons and most of them die by apoptosis within a few days. Recently, several strategies that activate neuronal intracellular pathways were proposed to prevent such degenerative processes. The rho-related small GTPase Rac1 is part of a complex, still not fully understood, intracellular signaling network, mediating in neurons many effects, including axon growth and cell survival. However, its role in neuronal survival and regeneration in vivo has not yet been properly investigated. To address this point we intravitreally injected selective cell-penetrating Rac1 mutants after optic nerve crush and studied the effect on RGC survival and axonal regeneration. We injected two well-characterized L61 constitutively active Tat-Rac1 fusion protein mutants, in which a second F37A or Y40C mutation confers selectivity in downstream signaling pathways. Results showed that, 15 days after crush, both mutants were able to improve survival and to prevent dendrite degeneration, while the one harboring the F37A mutation also improved axonal regeneration. The treatment with F37A mutant for one month did not improve the axonal elongation respect to 15 days. Furthermore, we found an increase of Pak1 T212 phosphorylation and ERK1/2 expression in RGCs after F37A treatment, whereas ERK1/2 was more activated in glial cells after Y40C administration. Our data suggest that the selective activation of distinct Rac1-dependent pathways could represent a therapeutic strategy to counteract neuronal degenerative processes in the retina

Comparison of Blood RNA Extraction Methods Used for Gene Expression Profiling in Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that causes death within a mean of 2-3 years from symptom onset. There is no diagnostic test and the delay from symptom onset to diagnosis averages 12 months. The identification of prognostic and diagnostic biomarkers in ALS would facilitate earlier diagnosis and faster monitoring of treatments. Gene expression profiling (GEP) can help to identify these markers as well as therapeutic targets in neurological diseases. One source of genetic material for GEP in ALS is peripheral blood, which is routinely accessed from patients. However, a high proportion of globin mRNA in blood can mask important genetic information. A number of methods allow safe collection, storage and transport of blood as well as RNA stabilisation, including the PAXGENE and TEMPUS systems for the collection of whole blood and LEUKOLOCK which enriches for the leukocyte population. Here we compared these three systems and assess their suitability for GEP in ALS. We collected blood from 8 sporadic ALS patients and 7 controls. PAXGENE and TEMPUS RNA extracted samples additionally underwent globin depletion using GlobinClear. RNA was amplified and hybridised onto Affymetrix U133 Plus 2.0 arrays. Lists of genes differentially regulated in ALS patients and controls were created for each method using the R package PUMA, and RT-PCR validation was carried out on selected genes. TEMPUS/GlobinClear, and LEUKOLOCK produced high quality RNA with sufficient yield, and consistent array expression profiles. PAXGENE/GlobinClear yield and quality were lower. Globin depletion for PAXGENE and TEMPUS uncovered the presence of over 60% more transcripts than when samples were not depleted. TEMPUS/GlobinClear and LEUKOLOCK gene lists respectively contained 3619 and 3047 genes differentially expressed between patients and controls. Real-time PCR validation revealed similar reliability between these two methods and gene ontology analyses revealed similar pathways differentially regulated in disease compared to controls