Selection of ovine housekeeping genes for normalisation by real-time RT-PCR; analysis of PrP gene expression and genetic susceptibility to scrapie

BACKGROUND: Cellular prion protein expression is essential for the development of transmissible spongiform encephalopathies (TSEs), and in sheep, genetic susceptibility to scrapie has been associated to PrP gene polymorphisms. To test the hypothetical linkage between PrP gene expression and genetic susceptibility, PrP mRNA levels were measured by real-time RT-PCR in six ovine tissues of animals with different genotypes. RESULTS: Previous to the PrP gene expression analysis the stability of several housekeeping (HK) genes was assessed in order to select the best ones for relative quantification. The normalisation of gene expression was carried out using a minimum of three HK genes in order to detect small expression differences more accurately than using a single control gene. The expression stability analysis of six HK genes showed a large tissue-associated variation reflecting the existence of tissue-specific factors. Thereby, a specific set of HK genes was required for an accurate normalisation of the PrP gene expression within each tissue. Statistical differences in the normalised PrP mRNA levels were found among the tissues, obtaining the highest expression level in obex, followed by ileum, lymph node, spleen, cerebellum and cerebrum. A tendency towards increased PrP mRNA levels and genetic susceptibility was observed in central nervous system. However, the results did not support the hypothesis that PrP mRNA levels vary between genotypes. CONCLUSION: The results on PrP gene expression presented here provide valuable baseline data for future studies on scrapie pathogenesis. On the other hand, the results on stability data of several HK genes reported in this study could prove very useful in other gene expression studies carried out in these relevant ovine tissues

Influencia de la geometría del indenter sobre el desarrollo de una cuña de pliegues y cabalgamientos arqueados: Resultados preliminares de modelos analógicos

Analogue models, with sand and silicone, have simulated the progressive development of an arcuate foldand- thrust belt around indenters of different geometry, which moved along a straight translation path. In the most external part of the model, the resulting geometry of the deformed wedge is roughly independent of the indenter form. Deformation affected only the frontal part of the indenter and the lateral deformation zone around the latter was very narro

Geolodía: a yearly successful organization of 54 geological fieldtrips in a weekend oriented to nongeologists Geolodía: Why and how to organize 54 geological fieldtrips in a weekend oriented to nongeologists?

Depto. de Mineralogía y PetrologíaFac. de Ciencias GeológicasTRUEpu

Poly(ionic liquid)-based engineered mixed matrix membranes for CO2/H2 separation

Unformatted preprintPoly(ionic liquid)s (PIL) have emerged as a class of versatile polyelectrolites, that can be used to prepare new materials able to achieve superior performances compared to conventional polymers. The combination of PILs with ionic liquids (ILs) may serve as a suitable matrix for the preparation of membranes for gas separation. In this work, mixed matrix membranes (MMMs) combining a pyrrolidinium-based PIL, an IL and three highly CO2-selective metal organic frameworks (MOFs) were prepared. The different MOFs (MIL-53, Cu3(BTC)2 and ZIF-8) were used as fillers, aiming to maximize the membranes performance towards the purification of syngas. The influence of different MOFs and loadings (0, 10, 20 and 30 wt.%) on the thermal and mechanical stabilities of the membranes and their performance in terms of CO2 permeability and CO2/H2 ideal selectivity was assessed. The compatibility between the materials was confirmed by SEM-EDS and FTIR spectroscopy. The prepared MMMs revealed to be thermally stable within the temperature range of the syngas stream, with a loss of mechanical stability upon the MOF incorporation. The increasing MOF content in the MMMs, resulted in an improvement of both CO2 permeability and CO2/H2 ideal selectivity. Among the three MOFs studied, membranes based on ZIF-8 showed the highest permeabilities (up to 97.2 barrer), while membranes based on MIL-53 showed the highest improvement in selectivity (up to 13.3). Remarkably, all permeation results surpass the upper bound limit for the CO2/H2 separation, showing the membranes potential for the desired gas separation.This work was partially supported by R&D Units UID/Multi/04551/2013 (Green-it), UID/QUI/00100/2013 (CQE), and the Associated Laboratory Research Unit for Green Chemistry, Technologies and Clean Processes, LAQV which is financed by national funds from FCT/MCTES(UID/QUI/50006/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007265). Ana R. Nabais, Luísa A. Neves and Liliana C. Tomé acknowledge FCT/MCTES for financial support through project PTDC/CTM-POL/2676/2014, FCT Investigator Contract IF/00505/2014 and Post-doctoral research grant SFRH/BDP/101793/2014, respectively. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 745734

Comparative studies of ultrasound and membrane emulsification for the production of stable Perfluorocarbon-in-water nanoemulsions

Low-molecular weight perfluorocarbons (PFCs) are usually chemically and biologically inert, clear, colorless liquids, presenting a high affinity for many gases, namely for O2, NO and CO2, which turn them particularly suitable in various biomedical applications involving gas capture, transport and release. In fact, PFC-in-Water emulsions were one of the two major classes of systems proposed as blood substitutes and for O2/NO therapeutics. However there are still some important issues concerning this type of systems which have limited their efficiency, approval and commercial success, namely those related to emulsion stability, hydrodynamic size distribution and reduced shelf-lives. Stability issues can easily lead to PFC diffusion in water, to aggregation and to the consequent hydrodynamic size increase and emulsion degradation [1]. Nanoemulsions (typically within a range of hydrodynamic sizes of 10-100 nm) exhibit various advantages over typical microemulsions [2]. Hence, our aim is to produce monodisperse PFC nanoemulsions presenting larger surface-to-volume ratios, enhanced stabilities and more efficient gas capture/delivery properties. A first approach to achieve these goals is to prepare and to study a mixed surfactant system based on Tween 80 and on a perfluorinated surfactant (perfluorooctyl phosphocholine) at different relative compositions. PFC-in-Water nanoemulsions were produced by using the traditional ultrasound emulsification method (500W). The effects of co-surfactants relative compositions on CMC values, on the kinetics of emulsion formation and on the corresponding stabilities of prepared nanoemulsions were evaluated. Hydrodynamic sizes and Zeta-potentials were also assessed, being able to obtain stable nanoemulsions with hydrodynamic sizes between 150 and 200 nm. In a comparative study, PFC-in-Water nanoemulsions were also produced by membrane emulsification. This low energy-intensive technique has received increasing interest as it allows more flexible operating conditions. Regenerated cellulose membranes such as Nadir UC500 and Millipore Ultracel RC100, polyethersulphone membranes such as Nadir UP150 and Millipore PBHK04310, and a promising polycarbonate Whatman Track-ethched 30 nm Nuclepore membrane, were employed to produce nanoemulsions, and using the same mixed surfactant system and relative compositions. The energy inputs of these two methods were compared and discussed along with their efficiencies in terms of producing nanoemulsions presenting improved stabilities, smaller hydrodynamic sizes and narrower hydrodynamic size distributions. References [1] M.P. Krafft, A. Chittofrati, J.G. Riess, Curr Opin in Colloid Interface Sci., 8 (2003) 251–258 [2] E. Piacentini, E. Drioli, L. Giorno, J. of Membr. Sci., 468 (2014) 410–42

Efficacy of aldose reductase inhibitors is affected by oxidative stress induced under X-ray irradiation

Human aldose reductase (hAR, AKR1B1) has been explored as drug target since the 1980s for its implication in diabetic complications. An activated form of hAR was found in cells from diabetic patients, showing a reduced sensitivity to inhibitors in clinical trials, which may prevent its pharmacological use. Here we report the conversion of native hAR to its activated form by X-ray irradiation simulating oxidative stress conditions. Upon irradiation, the enzyme activity increases moderately and the potency of several hAR inhibitors decay before global protein radiation damage appears. The catalytic behavior of activated hAR is also reproduced as the KM increases dramatically while the kcat is not much affected. Consistently, the catalytic tetrad is not showing any modification. The only catalytically-relevant structural difference observed is the conversion of residue Cys298 to serine and alanine. A mechanism involving electron capture is suggested for the hAR activation. We propose that hAR inhibitors should not be designed against the native protein but against the activated form as obtained from X-ray irradiation. Furthermore, since the reactive species produced under irradiation conditions are the same as those produced under oxidative stress, the described irradiation method can be applied to other relevant proteins under oxidative stress environments.This work was started, and partly supported by a grant from the Spanish Nuclear Council (CSN)

Stimulation of an α1-adrenergic receptor downregulates ecto-5′ nucleotidase activity on the apical membrane of RPE cells

The purines ATP and adenosine play an important role in the communication between the photoreceptors and the retinal pigment epithelium (RPE). While the RPE is known to release ATP into subretinal space, the source of extracellular adenosine is unclear. In other tissues, ecto-nucleotidases mediate the consecutive dephosphorylation of ATP to AMP, and AMP is converted to adenosine by ecto-5′ nucleotidase (CD73). This study identifies ecto-5′ nucleotidase on RPE cells and investigates modulation of enzyme activity. The RPE was the most active site of 5′AMP dephosphorylation in the posterior rat eye. The ecto-5′ nucleotidase inhibitor αβmADP prevented the production adenosine by the apical membrane of the bovine RPE. Cultured human ARPE-19 cells expressed mRNA and protein for ecto-5′ nucleotidase. The production of phosphate from 5′AMP by ARPE-19 cells was inhibited by αβmADP, but the ecto-alkaline phosphatase inhibitor levamisole had no effect. Degradation of 5′AMP was blocked by norepinephrine, epinephrine and phenylephrine, with inhibition by antagonists prazosin and corynanthine implicating the α1 adrenergic receptor. The block of enzyme activity by norepinephrine was rapid, occurring within 1 min, and was similar at both 4 and 37°C, consistent with cleavage of the enzyme from its GPI anchor. HPLC measurements indicated norepinephrine reduced levels of adenosine in the bath. In the apical face of the bovine-RPE eyecup, norepinephrine reduced the production of phosphate from 5′AMP, suggesting that both receptor and enzyme face sub-retinal space. In conclusion, RPE cells express ecto-5′ nucleotidase, with activity on the apical membrane, and stimulation of α-1 adrenergic receptors downregulates activity. As epinephrine is released at light onset, and adenosine can inhibit phagocytosis, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced the phagocytosis of rod outer segments that occurs at this time

Rapid decrease in titer and breadth of neutralizing anti-HCV antibodies in HIV/HCV-coinfected patients who achieved SVR

The main targets for neutralizing anti-hepatitis C virus (HCV) antibodies (HCV-nAbs) are the E1 and E2 envelope glycoproteins. We have studied the characteristics of HCV-nAbs through a retrospective study involving 29 HIV/HCV-coinfected patients who achieved sustained virological response (SVR) with pegIFNα+ribavirin anti-HCV therapy. Plasma samples at baseline and week 24 after SVR were used to perform neutralization assays against fve JFH1-based HCV recombinant viruses coding for E1 and E2 from genotypes 1a (H77), 1b (J4), 2a (JFH1), 3a (S52) and 4a (ED43). At baseline, the majority of plasma samples neutralized 1a, 1b, 2a, and 4a, but not 3a, genotypes. Twenty-four weeks following SVR, most neutralizing titers declined substantially. Furthermore, titers against 3a and 2a were not detected in many patients. Plasma samples with high HCV-nAb titers neutralized all genotypes, and the highest titers at the starting point correlated with the highest titers at week 24 after SVR. In conclusion, high titers of broad-spectrum HCV-nAbs were detected in HIV/HCV-coinfected individuals, however, those titers declined soon after SVRThis study was supported by grants from Instituto de Salud Carlos III (ISCIII; grant numbers PI14/01094 and PI17/00657 to JB, PI17/00903 to JGG, PI14CIII/00011 and PI17CIII/00003 to SR) and Ministerio de Sanidad, Servicios Sociales e Igualdad (grant number EC11-241). Te study was also funded by the RD16CIII/0002/0002, RD16/0025/0018, and RD16/0025/0017 projects as part of the Plan Nacional R+D+I and co-funded by ISCIII- Subdirección General de Evaluación and the Fondo Europeo de Desarrollo Regional (FEDER

Asymmetric organocatalytic synthesis of tertiary azomethyl alcohols: key intermediates towards azoxy compounds and α-hydroxy-β-amino esters

A series of peracylated glycosamine-derived thioureas have been synthesized and their behavior as bifunctional organocatalysts has been tested in the enantioselective nucleophilic addition of formaldehyde tert-butyl hydrazone to aliphatic α-keto esters for the synthesis of tertiary azomethyl alcohols. Using the 1,3,4,6-tetra-O-acetyl-2-amino-2-deoxy-β-d-glucosamine derived 3,5-bis-(trifluoromethyl)phenyl thiourea the reaction could be accomplished with high yields (75-98%) and moderate enantioselectivities (50-64% ee). Subsequent high-yielding and racemization-free tranformations of both aromatic- and aliphatic-substituted diazene products in a one pot fashion provide a direct entry to valuable azoxy compounds and α-hydroxy-β-amino esters.Ministerio de Economía y Competitividad CTQ2016-76908-C2-1-P, CTQ2016- 76908-C2-2-P, contract RYC-2012-10014 for G. de G.European Funds FEDERJunta de Andalucía 2012/FQM 107