ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations

Torsion pendulum facility for direct force measurements of LISA GRS related disturbances

A four mass torsion pendulum facility for testing of the LISA GRS is under development in Trento. With a LISA-like test mass suspended off-axis with respect to the pendulum fiber, the facility allows for a direct measurement of surface force disturbances arising in the GRS. We present here results with a prototype pendulum integrated with very large-gap sensors, which allows an estimate of the intrinsic pendulum noise floor in the absence of sensor related force noise. The apparatus has shown a torque noise near to its mechanical thermal noise limit, and would allow to place upper limits on GRS related disturbances with a best sensitivity of 300 fN/Hz^(1/2) at 1mHz, a factor 50 from the LISA goal. Also, we discuss the characterization of the gravity gradient noise, one environmental noise source that could limit the apparatus performances, and report on the status of development of the facility.Comment: Submitted to Proceedings of the 6th International LISA Symposium, AIP Conference Proceedings 200

Use of surrogate endpoints in healthcare policy : proposal for consistent adoption of a validation framework

We propose a three step framework for the evaluation of surrogate endpoints for health policy decisions on health technologies

Combined sanger and ngs sequence analysis of the myostatin gene (mstn) in the Camelus dromedarius species

Different mutations have been identified in the myostatin gene (MSTN), some of which are responsible for protein inactivation and double muscling phenotype in mammals. So far, no extensive polymorphism survey has been carried out in Camelus dromedarius. We therefore performed a sequence analysis, adopting a combined strategy involving Sanger and next generation sequencing (NGS). Notably, 3.6 kb of the MSTN locus were Sanger sequenced in a population dataset including samples from Algeria (10), Tunisia (5), Egypt (9), Mauritania (5), Sudan (5) and Saudi Arabia (9). A further wholegenome dataset, including 7 C. dromedarius from Pakistan (1), Kenya (1), Saudi Arabia (3), Canary Islands (1) and Oman (1) were sequenced using the Illumina Hi-Seq 2000 technique at an average 15-fold coverage. Whole-genome NGS sequence data from 9 C. bactrianus and 7 C. ferus samples were also available for comparison. Overall, only four polymorphisms were detected, all of them were observed in intronic regions, corresponding to an average presence of one SNP per 1200 bps. Ten fixed sites were observed when comparing C. dromedarius MSTN sequences with those from C. bactrianus and C. ferus. The apparent low sequence diversity observed at the MSTN locus may reflect the peculiar evolutionary history of this species, with purifying selection and drift phenomena as the most likely acting forces.(Résumé d'auteur

Upper limits to surface force disturbances on LISA proof masses and the possibility of observing galactic binaries

We report on the measurement of parasitic surface force noise on a hollow replica of a LISA (Laser Interferometer Space Antenna for the observation of gravitational waves) proof mass surrounded by a faithful representation of its in flight surroundings, namely the capacitive sensor used to detect proof-mass motion. Parasitic forces are detected through the corresponding torque exerted on the proof mass and measured with a torsion pendulum in the frequency range 0.1 30 mHz. The sensor electrodes, electrode housing and associated readout electronics have the same nominal design as for the flight hardware, including 4 mm gaps around the proof mass along the sensitive laser interferometry axis. We show that the measured upper limit for surface forces would allow detection of a number of galactic binaries signals with signal to noise ratio up to approximately 40 for 1 year integration. We also discuss how the flight test under development, LISA Pathfinder, will substantially improve this limit, approaching the performance required for LISA.Comment: 3 Figures. Submitted to Physical Review Letter

The Dusty Starburst Nucleus of M33

We have thoroughly characterized the ultraviolet to near-infrared (0.15 - 2.2 micron) spectral energy distribution (SED) of the central parsec of the M33 nucleus through new infrared photometry and optical/near-infrared spectroscopy, combined with ultraviolet/optical observations from the literature and the HST archive. The SED shows evidence for a significant level of attenuation, which we model through a Monte Carlo radiative transfer code as a shell of clumpy Milky Way-type dust (tau_V ~ 2 +/- 1). The discovery of Milky Way-type dust (with a strong 2175 A bump) internal to the M33 nucleus is different from previous work which has found SMC-like dust (no bump) near starburst regions. The amount by which dust can be processed may be related to the mass and age of the starburst as well as the extent to which the dust can shield itself. Our starburst models include the effects of this dust and can fit the SED if the nucleus was the site of a moderate (~10^8 L_sun at 10 Myrs) episode of coeval star formation about 70 Myrs ago. This result is quite different from previous studies which resorted to multiple stellar populations (between 2 and 7) attenuated by either no or very little internal dust. The M33 nuclear starburst is remarkably similar to an older version (70 Myr versus 10 Myr) of the ultra-compact starburst in the center of the Milky Way.Comment: 29 pages, 9 embedded figures, ApJ, in pres

Digital detection of exosomes by interferometric imaging

Exosomes, which are membranous nanovesicles, are actively released by cells and have been attributed to roles in cell-cell communication, cancer metastasis, and early disease diagnostics. The small size (30–100 nm) along with low refractive index contrast of exosomes makes direct characterization and phenotypical classification very difficult. In this work we present a method based on Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows multiplexed phenotyping and digital counting of various populations of individual exosomes (>50 nm) captured on a microarray-based solid phase chip. We demonstrate these characterization concepts using purified exosomes from a HEK 293 cell culture. As a demonstration of clinical utility, we characterize exosomes directly from human cerebrospinal fluid (hCSF). Our interferometric imaging method could capture, from a very small hCSF volume (20 uL), nanoparticles that have a size compatible with exosomes, using antibodies directed against tetraspanins. With this unprecedented capability, we foresee revolutionary implications in the clinical field with improvements in diagnosis and stratification of patients affected by different disorders.This work was supported by Regione Lombardia and Fondazione Cariplo through POR-FESR, project MINER (ID 46875467); Italian Ministry of Health, Ricerca Corrente. This work was partially supported by The Scientific and Technological Research Council of Turkey (grant #113E643). (Regione Lombardia; 46875467 - Fondazione Cariplo through POR-FESR, project MINER; Italian Ministry of Health, Ricerca Corrente; 113E643 - Scientific and Technological Research Council of Turkey)Published versio