Block of Kcnk3 by protons. Evidence that 2-P-domain potassium channel subunits function as homodimers.

KCNK subunits have two pore-forming P domains and four predicted transmembrane segments. To assess the number of subunits in each pore, we studied external proton block of Kcnk3, a subunit prominent in rodent heart and brain. Consistent with a pore-blocking mechanism, inhibition was dependent on voltage, potassium concentration, and a histidine in the first P domain (P1H). Thus, at pH 6.8 with 20 mm potassium half the current passed by P1H channels was blocked (apparently via two sites approximately 10% into the electrical field) whereas channels with an asparagine substitution (P1N) were fully active. Furthermore, pore blockade by barium was sensitive to pH in P1H but not P1N channels. Although linking two Kcnk3 subunits in tandem to produce P1H-P1H and P1N-P1N channels bearing four P domains did not alter these attributes, the mixed tandems P1H-P1N and P1N-P1H were half-blocked at pH approximately 6.4, apparently via a single site. This implicates a dimeric structure for Kcnk3 channels with two (and only two) P1 domains in each pore and argues that P2 domains also contribute to pore formation

Proton block and voltage gating are potassium-dependent in the cardiac leak channel Kcnk3.

Potassium leak conductances were recently revealed to exist as independent molecular entities. Here, the genomic structure, cardiac localization, and biophysical properties of a murine example are considered. Kcnk3 subunits have two pore-forming P domains and unique functional attributes. At steady state, Kcnk3 channels behave like open, potassium-selective, transmembrane holes that are inhibited by physiological levels of proton. With voltage steps, Kcnk3 channels open and close in two phases, one appears to be immediate and one is time-dependent (tau = approximately 5 ms). Both proton block and gating are potassium-sensitive; this produces an anomalous increase in outward flux as external potassium levels rise because of decreased proton block. Single Kcnk3 channels open across the physiological voltage range; hence they are "leak" conductances; however, they open only briefly and rarely even after exposure to agents that activate other potassium channels

Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus.

Usher 1 patients are born profoundly deaf and then develop retinal degeneration. Thus they are readily identified before the onset of retinal degeneration, making gene therapy a viable strategy to prevent their blindness. Here, we have investigated the use of adeno-associated viruses (AAVs) for the delivery of the Usher 1B gene, MYO7A, to retinal cells in cell culture and in Myo7a-null mice. MYO7A cDNA, under control of a smCBA promoter, was packaged in single AAV2 and AAV5 vectors and as two overlapping halves in dual AAV2 vectors. The 7.9-kb smCBA-MYO7A exceeds the capacity of an AAV vector; packaging of such oversized constructs into single AAV vectors may involve fragmentation of the gene. Nevertheless, the AAV2 and AAV5 single vector preparations successfully transduced photoreceptor and retinal pigment epithelium cells, resulting in functional, full-length MYO7A protein and correction of mutant phenotypes, suggesting successful homologous recombination of gene fragments. With discrete, conventional-sized dual AAV2 vectors, full-length MYO7A was detected, but the level of protein expression was variable, and only a minority of cells showed phenotype correction. Our results show that MYO7A therapy with AAV2 or AAV5 single vectors is efficacious; however, the dual AAV2 approach proved to be less effective

Role of glucose as a modulator of anabolic and catabolic gene expression in normal and osteoarthritic human chondrocytes

Cartilage matrix homeostasis involves a dynamic balance between numerous signals that modulate chondrocyte functions. This study aimed at elucidating the role of the extracellular glucose concentration in modulating anabolic and catabolic gene expression in normal and osteoarthritic (OA) human chondrocytes and its ability to modify the gene expression responses induced by pro-anabolic stimuli, namely Transforming Growth Factor-β (TGF). For this, we analyzed by real time RT-PCR the expression of articular cartilage matrix-specific and non-specific genes, namely collagen types II and I, respectively. The expression of the matrix metalloproteinases (MMPs)-1 and -13, which plays a major role in cartilage degradation in arthritic conditions, and of their tissue inhibitors (TIMP) was also measured. The results showed that exposure to high glucose (30 mM) increased the mRNA levels of both MMPs in OA chondrocytes, whereas in normal ones only MMP-1 increased. Collagen II mRNA was similarly increased in normal and OA chondrocytes, but the increase lasted longer in the later. Exposure to high glucose for 24 h prevented TGF-induced downregulation of MMP-13 gene expression in normal and OA chondrocytes, while the inhibitory effect of TGF on MMP-1 expression was only partially reduced. Other responses were not significantly modified. In conclusion, exposure of human chondrocytes to high glucose, as occurs in vivo in diabetes mellitus patients and in vitro for the production of engineered cartilage, favors the chondrocyte catabolic program. This may promote articular cartilage degradation, facilitating OA development and/or progression, as well as compromise the quality and consequent in vivo efficacy of tissue engineered cartilage

U-dual fluxes and Generalized Geometry

We perform a systematic analysis of generic string flux compactifications, making use of Exceptional Generalized Geometry (EGG) as an organizing principle. In particular, we establish the precise map between fluxes, gaugings of maximal 4d supergravity and EGG, identifying the complete set of gaugings that admit an uplift to 10d heterotic or type IIB supegravity backgrounds. Our results reveal a rich structure, involving new deformations of 10d supergravity backgrounds, such as the RR counterparts of the $\beta$-deformation. These new deformations are expected to provide the natural extension of the $\beta$-deformation to full-fledged F-theory backgrounds. Our analysis also provides some clues on the 10d origin of some of the particularly less understood gaugings of 4d supergravity. Finally, we derive the explicit expression for the effective superpotential in arbitrary N = 1 heterotic or type IIB orientifold compactifications, for all the allowed fluxes.Comment: 58 pages, 6 table

Fake supersymmetry versus Hamilton-Jacobi

We explain when the first-order Hamilton-Jacobi equations for black holes (and domain walls) in (gauged) supergravity, reduce to the usual first-order equations derived from a fake superpotential. This turns out to be equivalent to the vanishing of a newly found constant of motion and we illustrate this with various examples. We show that fake supersymmetry is a necessary condition for having physically sensible extremal black hole solutions. We furthermore observe that small black holes become scaling solutions near the horizon. When combined with fake supersymmetry, this leads to a precise extension of the attractor mechanism to small black holes: The attractor solution is such that the scalars move on specific curves, determined by the black hole charges, that are purely geodesic, although there is a non-zero potential.Comment: 20 pages, v2: Typos corrected, references adde

Moduli Stabilization and Cosmology of Type IIB on SU(2)-Structure Orientifolds

We consider type IIB flux compactifications on six-dimensional SU(2)-structure manifolds with O5- and O7-planes. These six-dimensional spaces allow not only for F_3 and H_3 fluxes but also for F_1 and F_5 fluxes. We derive the four-dimensional N=1 scalar potential for such compactifications and present one explicit example of a fully stabilized AdS vacuum with large volume and small string coupling. We then discuss cosmological aspects of these compactifications and derive several no-go theorems that forbid dS vacua and slow-roll inflation under certain conditions. We also study concrete examples of cosets and twisted tori and find that our no-go theorems forbid dS vacua and slow-roll inflation in all but one of them. For the latter we find a dS critical point with \epsilon numerically zero. However, the point has two tachyons and eta-parameter \eta \approx -3.1.Comment: 35 pages + appendices, LaTeX2e; v2: numerical dS extremum added, typos corrected, references adde

Black holes in supergravity and integrability

Stationary black holes of massless supergravity theories are described by certain geodesic curves on the target space that is obtained after dimensional reduction over time. When the target space is a symmetric coset space we make use of the group-theoretical structure to prove that the second order geodesic equations are integrable in the sense of Liouville, by explicitly constructing the correct amount of Hamiltonians in involution. This implies that the Hamilton-Jacobi formalism can be applied, which proves that all such black hole solutions, including non-extremal solutions, possess a description in terms of a (fake) superpotential. Furthermore, we improve the existing integration method by the construction of a Lax integration algorithm that integrates the second order equations in one step instead of the usual two step procedure. We illustrate this technology with a specific example.Comment: 44 pages, small typos correcte

Application of Nanotechnology in the Agro-Food Sector

Nanotechnology is an emerging field of research that has been widely applied in different scientific and engineering areas. The agro-food sector is not an exception, which considers its applicability in several areas of major interest for both consumers and producers. This review considers major concepts related to nanostructures and nano-based instruments used in the food sector, as well as their applications in agro-food products. Food safety through the use of nanosensors for pathogen detection, smart packaging, and valorisation of food products by nanoencapsulation/nanodelivery of food ingredients (e.g. flavours) are examples of important areas of nanotechnology. Consumers' apprehension regarding food stability and safety issues is also considered