In vivo electrophysiological characterization of TASK-1 deficient mice

Background/Aims: TASK-1 is a potassium channel predominantly expressed in heart and brain. We have previously shown that anesthetized TASK-1(-/-) mice have prolonged QT intervals in surface electrocardiograms (ECGs). In addition, heart rate variability quantified by time and frequency domain parameters was significantly altered in TASK-1(-/-) mice with a sympathetic preponderance. Aims of the present study were the analysis of QT intervals by telemetric ECGs, to determine potential influences of anesthesia and beta-adrenergic stimulation on repolarization in surface ECGs, to investigate in vivo electrophysiological parameters by intracardiac electrical stimulation and to quantify heart rate turbulence after ischemia/reperfusion or ventricular pacing in TASK-1(+/+) and TASK-1(-/-) mice. Methods: Rate corrected QT intervals (QTc) were recorded in conscious mice by telemetry and in surface ECGs following administration of various anesthetics (tribromoethanol (Avertin (R)), pentobarbital and isoflurane). TASK-1(+/+) and TASK-1(-/-) mice were characterized by programmed electrical stimulation using an intracardiac octapolar catheter. The baroreceptor reflex was analyzed by heart rate turbulence (turbulence onset and slope) after ischemia/reperfusion and by stimulated premature ventricular contractions

A detailed clinical and molecular survey of subjects with nonsyndromic USH2A retinopathy reveals an allelic hierarchy of disease-causing variants.

Defects in USH2A cause both isolated retinal disease and Usher syndrome (ie, retinal disease and deafness). To gain insights into isolated/nonsyndromic USH2A retinopathy, we screened USH2A in 186 probands with recessive retinal disease and no hearing complaint in childhood (discovery cohort) and in 84 probands with recessive retinal disease (replication cohort). Detailed phenotyping, including retinal imaging and audiological assessment, was performed in individuals with two likely disease-causing USH2A variants. Further genetic testing, including screening for a deep-intronic disease-causing variant and large deletions/duplications, was performed in those with one likely disease-causing change. Overall, 23 of 186 probands (discovery cohort) were found to harbour two likely disease-causing variants in USH2A. Some of these variants were predominantly associated with nonsyndromic retinal degeneration ('retinal disease-specific'); these included the common c.2276 G>T, p.(Cys759Phe) mutation and five additional variants: c.2802 T>G, p.(Cys934Trp); c.10073 G>A, p.(Cys3358Tyr); c.11156 G>A, p.(Arg3719His); c.12295-3 T>A; and c.12575 G>A, p.(Arg4192His). An allelic hierarchy was observed in the discovery cohort and confirmed in the replication cohort. In nonsyndromic USH2A disease, retinopathy was consistent with retinitis pigmentosa and the audiological phenotype was variable. USH2A retinopathy is a common cause of nonsyndromic recessive retinal degeneration and has a different mutational spectrum to that observed in Usher syndrome. The following model is proposed: the presence of at least one 'retinal disease-specific' USH2A allele in a patient with USH2A-related disease results in the preservation of normal hearing. Careful genotype-phenotype studies such as this will become increasingly important, especially now that high-throughput sequencing is widely used in the clinical setting.European Journal of Human Genetics advance online publication, 4 February 2015; doi:10.1038/ejhg.2014.283

Increased Motor-Impairing Effects of the Neuroactive Steroid Pregnanolone in Mice with Targeted Inactivation of the GABA(A) Receptor gamma 2 Subunit in the Cerebellum

Endogenous neurosteroids and neuroactive steroids have potent and widespread actions on the brain via inhibitory GABA(A) receptors. In recombinant receptors and genetic mouse models their actions depend on the alpha, beta, and delta subunits of the receptor, especially on those that form extrasynaptic GABA(A) receptors responsible for non-synaptic (tonic) inhibition, but they also act on synaptically enriched gamma 2 subunit containing receptors and even on alpha beta binary receptors. Here we tested whether behavioral sensitivity to the neuroactive steroid agonist 5 beta-pregnan-3 alpha-ol-20-one is altered in genetically engineered mouse models that have deficient GABA(A) receptor mediated synaptic inhibition in selected neuronal populations. Mouse lines with the GABA(A) receptor gamma 2 subunit gene selectively deleted either in parvalbumin-containing cells (including cerebellar Purkinje cells), cerebellar granule cells, or just in cerebellar Purkinje cells were trained on the accelerated rotating rod and then tested for motor impairment after cumulative intraperitoneal dosing of 5 beta-pregnan-3 alpha-ol-20-one. Motor impairing effects of 5 beta-pregnan-3 alpha-ol-20-one were strongly increased in all three mouse models in which gamma 2 subunit-dependent synaptic GABA(A) responses in cerebellar neurons were genetically abolished. Furthermore, rescue of postsynaptic GABA(A) receptors in Purkinje cells normalized the effect of the steroid. Anxiolytic/explorative effects of the steroid in elevated plus maze and light:dark exploration tests in mice with Purkinje cell gamma 2 subunit inactivation were similar to those in control mice. The results suggest that, when the deletion of gamma 2 subunit has removed synaptic GABA(A) receptors from the specific cerebellar neuronal populations, the effects of neuroactive steroids solely on extrasynaptic alpha beta or alpha beta delta receptors lead to enhanced changes in the cerebellum-generated behavior.Peer reviewe

Organism-sediment interactions govern post-hypoxia recovery of ecosystem functioning

Hypoxia represents one of the major causes of biodiversity and ecosystem functioning loss for coastal waters. Since eutrophication-induced hypoxic events are becoming increasingly frequent and intense, understanding the response of ecosystems to hypoxia is of primary importance to understand and predict the stability of ecosystem functioning. Such ecological stability may greatly depend on the recovery patterns of communities and the return time of the system properties associated to these patterns. Here, we have examined how the reassembly of a benthic community contributed to the recovery of ecosystem functioning following experimentally-induced hypoxia in a tidal flat. We demonstrate that organism-sediment interactions that depend on organism size and relate to mobility traits and sediment reworking capacities are generally more important than recovering species richness to set the return time of the measured sediment processes and properties. Specifically, increasing macrofauna bioturbation potential during community reassembly significantly contributed to the recovery of sediment processes and properties such as denitrification, bedload sediment transport, primary production and deep pore water ammonium concentration. Such bioturbation potential was due to the replacement of the small-sized organisms that recolonised at early stages by large-sized bioturbating organisms, which had a disproportionately stronger influence on sediment. This study suggests that the complete recovery of organism-sediment interactions is a necessary condition for ecosystem functioning recovery, and that such process requires long periods after disturbance due to the slow growth of juveniles into adult stages involved in these interactions. Consequently, repeated episodes of disturbance at intervals smaller than the time needed for the system to fully recover organism-sediment interactions may greatly impair the resilience of ecosystem functioning.

Genetic landscape of 6089 inherited retinal dystrophies affected cases in Spain and their therapeutic and extended epidemiological implications

Inherited retinal diseases (IRDs), defined by dysfunction or progressive loss of photoreceptors, are disorders characterized by elevated heterogeneity, both at the clinical and genetic levels. Our main goal was to address the genetic landscape of IRD in the largest cohort of Spanish patients reported to date. A retrospective hospital-based cross-sectional study was carried out on 6089 IRD affected individuals (from 4403 unrelated families), referred for genetic testing from all the Spanish autonomous communities. Clinical, demographic and familiar data were collected from each patient, including family pedigree, age of appearance of visual symptoms, presence of any systemic findings and geographical origin. Genetic studies were performed to the 3951 families with available DNA using different molecular techniques. Overall, 53.2% (2100/3951) of the studied families were genetically characterized, and 1549 different likely causative variants in 142 genes were identified. The most common phenotype encountered is retinitis pigmentosa (RP) (55.6% of families, 2447/4403). The most recurrently mutated genes were PRPH2, ABCA4 and RS1 in autosomal dominant (AD), autosomal recessive (AR) and X-linked (XL) NON-RP cases, respectively; RHO, USH2A and RPGR in AD, AR and XL for non-syndromic RP; and USH2A and MYO7A in syndromic IRD. Pathogenic variants c.3386G > T (p.Arg1129Leu) in ABCA4 and c.2276G > T (p.Cys759Phe) in USH2A were the most frequent variants identified. Our study provides the general landscape for IRD in Spain, reporting the largest cohort ever presented. Our results have important implications for genetic diagnosis, counselling and new therapeutic strategies to both the Spanish population and other related populations.This work was supported by the Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Health (FIS; PI16/00425 and PI19/00321), Centro de Investigación Biomédica en Red Enfermedades Raras (CIBERER, 06/07/0036), IIS-FJD BioBank (PT13/0010/0012), Comunidad de Madrid (CAM, RAREGenomics Project, B2017/BMD-3721), European Regional Development Fund (FEDER), the Organización Nacional de Ciegos Españoles (ONCE), Fundación Ramón Areces, Fundación Conchita Rábago and the University Chair UAM-IIS-FJD of Genomic Medicine. Irene Perea-Romero is supported by a PhD fellowship from the predoctoral Program from ISCIII (FI17/00192). Ionut F. Iancu is supported by a grant from the Comunidad de Madrid (CAM, PEJ-2017-AI/BMD7256). Marta del Pozo-Valero is supported by a PhD grant from the Fundación Conchita Rábago. Berta Almoguera is supported by a Juan Rodes program from ISCIII (JR17/00020). Pablo Minguez is supported by a Miguel Servet program from ISCIII (CP16/00116). Marta Corton is supported by a Miguel Servet program from ISCIII (CPII17/00006). The funders played no role in study design, data collection, data analysis, manuscript preparation and/or publication decisions

A Single Acidic Residue Can Guide Binding Site Selection but Does Not Govern QacR Cationic-Drug Affinity

Structures of the multidrug-binding repressor protein QacR with monovalent and bivalent cationic drugs revealed that the carboxylate side-chains of E90 and E120 were proximal to the positively charged nitrogens of the ligands ethidium, malachite green and rhodamine 6G, and therefore may contribute to drug neutralization and binding affinity. Here, we report structural, biochemical and in vivo effects of substituting these glutamate residues. Unexpectedly, substitutions had little impact on ligand affinity or in vivo induction capabilities. Structures of QacR(E90Q) and QacR(E120Q) with ethidium or malachite green took similar global conformations that differed significantly from all previously described QacR-drug complexes but still prohibited binding to cognate DNA. Strikingly, the QacR(E90Q)-rhodamine 6G complex revealed two mutually exclusive rhodamine 6G binding sites. Despite multiple structural changes, all drug binding was essentially isoenergetic. Thus, these data strongly suggest that rather than contributing significantly to ligand binding affinity, the role of acidic residues lining the QacR multidrug-binding pocket is primarily to attract and guide cationic drugs to the “best available” positions within the pocket that elicit QacR induction

The high activity of 3C 454.3 in autumn 2007: Monitoring by the WEBT during the AGILE detection

The quasar-type blazar 3C 454.3 underwent a phase of high activity in summer and autumn 2007, which was intensively monitored in the radio-to-optical bands by the Whole Earth Blazar Telescope (WEBT). The gamma-ray satellite AGILE detected this source first in late July, and then in November-December 2007. In this letter we present the multifrequency data collected by the WEBT and collaborators during the second AGILE observing period, complemented by a few contemporaneous data from UVOT onboard the Swift satellite. The aim is to trace in detail the behaviour of the synchrotron emission from the blazar jet, and to investigate the contribution from the thermal emission component. Optical data from about twenty telescopes have been homogeneously calibrated and carefully assembled to construct an R-band light curve containing about 1340 data points in 42 days. This extremely well-sampled optical light curve allows us to follow the dramatic flux variability of the source in detail. In addition, we show radio-to-UV spectral energy distributions (SEDs) at different epochs, which represent different brightness levels. In the considered period, the source varied by 2.6 mag in a couple of weeks in the R band. Many episodes of fast (i.e. intranight) variability were observed, most notably on December 12, when a flux increase of about 1.1 mag in 1.5 hours was detected, followed by a steep decrease of about 1.2 mag in 1 hour. The contribution by the thermal component is difficult to assess, due to the uncertainties in the Galactic, and possibly also intrinsic, extinction in the UV band. However, polynomial fitting of radio-to-UV SEDs reveals an increasing spectral bending going towards fainter states, suggesting a UV excess likely due to the thermal emission from the accretion disc

Multifrequency variability of the blazar AO 0235+164 the WEBT campaign in 2004-2005 and long-term SED analysis

A huge multiwavelength campaign targeting the blazar AO 0235+164 was organized by the Whole Earth Blazar Telescope (WEBT) in 2003-2005 to study the variability properties of the source. Monitoring observations were carried out at cm and mm wavelengths, and in the near-IR and optical bands, while three pointings by the XMM-Newton satellite provided information on the X-ray and UV emission. We present the data acquired during the second observing season, 2004-2005, by 27 radio-to-optical telescopes. They reveal an increased near-IR and optical activity with respect to the previous season. Increased variability is also found at the higher radio frequencies, down to 15 GHz, but not at the lower ones. The radio (and optical) outburst predicted to peak around February-March 2004 on the basis of the previously observed 5-6 yr quasi-periodicity did not occur. The analysis of the optical light curves reveals now a longer characteristic time scale of 8 yr, which is also present in the radio data. The spectral energy distributions corresponding to the XMM-Newton observations performed during the WEBT campaign are compared with those pertaining to previous pointings of X-ray satellites. Bright, soft X-ray spectra can be described in terms of an extra component, which appears also when the source is faint through a hard UV spectrum and a curvature of the X-ray spectrum. Finally, there might be a correlation between the X-ray and optical bright states with a long time delay of about 5 yr, which would require a geometrical interpretation

The GASP-WEBT monitoring of 3C 454.3 during the 2008 optical-to-radio and γ-ray outburst

Since 2001, the radio quasar 3C 454.3 has undergone a period of high optical activity, culminating in the brightest optical state ever observed, during the 2004-2005 outburst. The Whole Earth Blazar Telescope (WEBT) consortium has carried out several multifrequency campaigns to follow the source behaviour. The GLAST-AGILE Support Program (GASP) was born from the WEBT to provide long-term continuous optical-to-radio monitoring of a sample of gamma-loud blazars, during the operation of the AGILE and GLAST (now known as Fermi GST) gamma-ray satellites. The main aim is to shed light on the mechanisms producing the high-energy radiation, through correlation analysis with the low-energy emission. Thus, since 2008 the monitoring task on 3C 454.3 passed from the WEBT to the GASP, while both AGILE and Fermi detected strong gamma-ray emission from the source. We present the main results obtained by the GASP at optical, mm, and radio frequencies in the 2008-2009 season, and compare them with the WEBT results from previous years. An optical outburst was observed to peak in mid July 2008, when Fermi detected the brightest gamma-ray levels. A contemporaneous mm outburst maintained its brightness for a longer time, until the cm emission also reached the maximum levels. The behaviour compared in the three bands suggests that the variable relative brightness of the different-frequency outbursts may be due to the changing orientation of a curved inhomogeneous jet. The optical light curve is very well sampled during the entire season, which is also well covered by the various AGILE and Fermi observing periods. The relevant cross-correlation studies will be very important in constraining high-energy emission models