Mutation-induced changes of transmembrane pore size revealed by combined ion-channel conductance and single vesicle permeabilization analyses

Permeabilization of the Endoplasmic Reticulum (ER) is instrumental in the progression of host-cell infection by many viral pathogens. We have described that permeabilization of ER model membranes by the pore-forming domain of the Classical Swine Fever Virus (CSFV) p7 protein depends on two sequence determinants: the C-terminal transmembrane helix, and the preceding polar loop that regulates its activity. Here, by combining ion-channel activity measurements in planar lipid bilayers with imaging of single Giant Unilamellar Vesicles (GUVs), we demonstrate that point substitutions directed to conserved residues within these regions affect ER-like membrane permeabilization following distinct mechanisms. Whereas the polar loop appeared to be involved in protein insertion and oligomerization, substitution of residues predicted to face the lumen of the pore inhibited large conducting channels (>1 nS) over smaller ones (120 pS). Quantitative analyses of the ER-GUV distribution as a function of the solute size revealed a selective inhibition for the permeation of solutes with sizes larger than 4 kDa, further demonstrating that the mutation targeting the transmembrane helix prevented formation of the large pores. Collectively, our data support the idea that the pore-forming domain of p7 may assemble into finite pores with approximate diameters of 1 and 5 nm. Moreover, the observation that the mutation interfering with formation of the larger pores can hamper virus production without affecting ER localization or homo-oligomerization, suggests prospective strategies to block/attenuate pestiviruses

Functional validation of CoQ deficiency fibroblast model with COQ7 mutations

Motivation: Coenzyme Q10 (CoQ10) deficiency syndrome comprises a heterogeneous group of mitochondrial disorders characterized by a decrease in CoQ10 content in cells and tissues. Primary CoQ deficiencies are rare genetic conditions caused by mutations in COQ genes, whose encoded proteins are directly linked to the final biochemical pathway of CoQ biosynthesis. Early diagnosis is both essential and one of the most challenging issues of this disease, mainly due to the variety of associated clinical manifestations. Here we present four clinical cases of primary CoQ10 deficiency, which is presumably caused by COQ7 mutations. The motivation for this work is to validate it in a cellular model based on primary cultures from patients' skin fibroblasts, in order to complete the previously started molecular diagnosis by whole-exome sequencing. Methods: Cultured patients fibroblasts was the biological starting material of our study. CoQ10 levels were measured by HPLC-ECD. In order to study mitochondrial function and respiration, oxygen consumption rate (OCR) was analysed using Seahorse technology. In addition, COQ7, several COQ proteins and other mitochondrial proteins expression were analysed by Western Blotting. Results: Patients' fibroblasts showed a basal level of CoQ10 lower than control fibroblasts (HDF). Moreover, the chromatogram revealed a peak corresponding to DMQ10, which was not seen in HDF. OCR showed mitochondrial respiration was affected in terms of maximal respiration and spare capacity with respect to control cells. Western blot analysis revealed the absence of COQ7 protein in patients' fibroblasts. Moreover, several COQ proteins, which are involved in the CoQ10 biosynthetic pathway, presented a moderate decreased expression. However, several mitochondrial related proteins maintained their physiological levels, such as VDAC, NDUFA9, UQCRCII or mtCOII. Conclusions: CoQ10 deficiency was confirmed in patients' fibroblasts. Since DMQ10 is the substrate of the reaction catalyzed by COQ7 protein, DMQ10 accumulation indicates that the COQ7 reaction is impaired. These results reveal that COQ7 mutation identified in patients' fibroblasts affects protein expression, CoQ10 levels and mitochondrial respiration. Finally, our data support the previous diagnosis obtained by exome analysis, proving that in these clinical cases, the CoQ10 deficiency is being produced by the absence of COQ7 protein

Specific gene correction of the AGXT gene and direct cell reprogramming for the treatment of Primary Hyperoxaluria Type 1

P428 Primary Hyperoxaluria Type 1 (PH1) is an inherited rare metabolic liver disease caused by the deficiency in the alanine: glyoxylate aminotransferase enzyme (AGXT), involved in the glyoxylate metabolism. The only potentially curative treatment is organ transplantation. Thus, the development of new therapeutic approaches for the treatment of these patients appears as a priority.We propose the combination of site-specific gene correction and direct cell reprogramming for the generation of autologous phenotypically healthy induced hepatocytes (iHeps) from skin-derived fibroblast of PH1 patients. For the correction of AGXT mutations, we have designed specific gene editing tools to address gene correction by two different strategies, assisted by CRISPR/Cas9 system. Accurate specific point mutation correction (c.853T-C) has been achieved by homologydirected repair (HDR) with ssODN harbouring wild-type sequence. In the second strategy, an enhanced version ofAGXTcDNAhas been inserted near the transcription start codon of the endogenous gene, constituting an almost universal correction strategy for PH1 mutations. Direct reprogramming of fibroblasts has been conducted by overexpression of hepatic transcription factors and in vitro culture in defined media. In vitro characterization of healthy induced hepatocytes (iHeps) has demonstrated hepatic function of the reprogrammed cells. PH1 patient fibroblasts and , ,

Relationship Between Glucocerebrosidase Activity and Clinical Response to Enzyme Replacement Therapy in Patients With Gaucher Disease Type I

The quantification of enzyme activity in the patient treated with enzyme replacement therapy (ERT) has been suggested as a tool for dosage individualization, so we conducted a study to evaluate the relationship between glucocerebrosidase activity and clinical response in patients with Gaucher disease type I (GD1) to ERT. The study included patients diagnosed with GD1, who were being treated with ERT, and healthy individuals. Markers based on glucocerebrosidase activity measurement in patients’ leucocytes were studied: enzyme activity at 15 min. post-infusion (Act75) reflects the amount of enzyme that is distributed in the body post-ERT infusion, and accumulated glucocerebrosidase activity during ERT infusion (Act75-0) indicates the total drug exposure during infusion. The clinical response was evaluated based on criteria established by Pastores et al. and Gaucher Severity Score Index. Statistical analysis included ROC analysis and area under the curve test. Act75 and Act75-0 were found to be moderate predictive markers of an optimal clinical response (area under the ROC of Act75 was 0.733 and Act75-0 was 0.817). Act75-0 showed statistical significance in its discriminative capacity (p < 0.05) for obtaining an optimal response to ERT. The cut-off point was 58% (RR = 1.800; 95% CI: 1.003–3.229; p < 0.05). Moreover, Act75 showed a significant and inverse correlation with the Gaucher Severity Score Index, and Act75 and Act75-0 presented a significant correlation with residual enzyme activity at diagnosis. Markers based on glucocerebrosidase activity have a good correlation with clinical response to ERT. Therefore, it could provide supporting clinical data for dose management in GD1 patients

Isothermal heat treatment influence on the interface of a powder metallurgy aluminium metal matrix composite reinforced with Ni₃Al intermetallics

Presentado al: 8º Congreso Nacional de Ciencia y Tecnología Metalúrgicas. Madrid 27 a 29 de mayo de 1998La mejora de las propiedades mecánicas de los composites de aluminio reforzados con partículas de Ni3Al se debe a la continuidad de la unión entre las partículas de refuerzo y la matriz, así como a la resistencia de las primeras. En este trabajo, se analiza la influencia que diferentes tratamientos térmicos tienen en la evolución de nuevas fases en la intercara matriz-partícula. Las muestras se prepararon por vía pulvimetalúrgica con una etapa de extrusión final. Se realizaron diferentes tratamientos térmicos abarcando un amplio espectro de temperaturas y tiempos, que dieron lugar al desarrollo de distintas fases alrededor de las partículas iniciales. Las muestras se analizaron mediante técnicas de microscopía óptica y electrónica de barrido con análisis de elementos por rayos X. Así mismo, se realizaron ensayos de microdureza en las distintas fases generadas.The improvement of the mechanical properties of aluminium MMCs reinforced with Ni3Al particles is based on the continuity of the matrix-particle interface as well as on the strength of these particles. This work deals with the influence of different heat treatments on the evolution of new phases in that interface. Samples were prepared following a powder metallurgy route with a final stage of extrusion. Several heat treatments encompassing a broad group of temperatures and times were applied, producing different phases around the primary particles. Samples were analysed via optical and scanning electron microscopy with energy dispersive X ray analysis. Microhardness tests were also conducted on the different phases generated.Este trabajo ha sido realizado con una subvención del proyecto del Plan Nacional de I+D de la CICYT con referencia MAT96-0722-C02-02

What have we learnt from EUPORIAS climate service prototypes?

The international effort toward climate services, epitomised by the development of the Global Framework for Climate Services and, more recently the launch of Copernicus Climate Change Service has renewed interest in the users and the role they can play in shaping the services they will eventually use. Here we critically analyse the results of the five climate service prototypes that were developed as part of the EU funded project EUPORIAS. Starting from the experience acquired in each of the projects we attempt to distil a few key lessons which, we believe, will be relevant to the wider community of climate service developers

Bias adjustment and ensemble recalibration methods for seasonal forecasting: a comprehensive intercomparison using the C3S dataset

This work presents a comprehensive intercomparison of diferent alternatives for the calibration of seasonal forecasts, ranging from simple bias adjustment (BA)-e.g. quantile mapping-to more sophisticated ensemble recalibration (RC) methods- e.g. non-homogeneous Gaussian regression, which build on the temporal correspondence between the climate model and the corresponding observations to generate reliable predictions. To be as critical as possible, we validate the raw model and the calibrated forecasts in terms of a number of metrics which take into account diferent aspects of forecast quality (association, accuracy, discrimination and reliability). We focus on one-month lead forecasts of precipitation and temperature from four state-of-the-art seasonal forecasting systems, three of them included in the Copernicus Climate Change Service dataset (ECMWF-SEAS5, UK Met Ofce-GloSea5 and Météo France-System5) for boreal winter and summer over two illustrative regions with diferent skill characteristics (Europe and Southeast Asia). Our results indicate that both BA and RC methods efectively correct the large raw model biases, which is of paramount importance for users, particularly when directly using the climate model outputs to run impact models, or when computing climate indices depending on absolute values/thresholds. However, except for particular regions and/or seasons (typically with high skill), there is only marginal added value-with respect to the raw model outputs-beyond this bias removal. For those cases, RC methods can outperform BA ones, mostly due to an improvement in reliability. Finally, we also show that whereas an increase in the number of members only modestly afects the results obtained from calibration, longer hindcast periods lead to improved forecast quality, particularly for RC methods.This work has been funded by the C3S activity on Evaluation and Quality Control for seasonal forecasts. JMG was partially supported by the project MULTI-SDM (CGL2015-66583-R, MINECO/FEDER). FJDR was partially funded by the H2020 EUCP project (GA 776613)

Natural images from the birthplace of the human eye

Here we introduce a database of calibrated natural images publicly available through an easy-to-use web interface. Using a Nikon D70 digital SLR camera, we acquired about 5000 six-megapixel images of Okavango Delta of Botswana, a tropical savanna habitat similar to where the human eye is thought to have evolved. Some sequences of images were captured unsystematically while following a baboon troop, while others were designed to vary a single parameter such as aperture, object distance, time of day or position on the horizon. Images are available in the raw RGB format and in grayscale. Images are also available in units relevant to the physiology of human cone photoreceptors, where pixel values represent the expected number of photoisomerizations per second for cones sensitive to long (L), medium (M) and short (S) wavelengths. This database is distributed under a Creative Commons Attribution-Noncommercial Unported license to facilitate research in computer vision, psychophysics of perception, and visual neuroscience.Comment: Submitted to PLoS ON