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

Selection signatures of fat tail in sheep

The investigation of the genes with a role in lipid metabolism enjoy considerable scientific and commercial interest because of the strong correlations between fat deposition and the risk of cardiovascular disease. The fat tail characteristic of sheep is the adaptive response to harsh environment, and beyond representing a valuable energy reserve for facing future climate changes provides clues for elucidating the physiology of fat deposition. Studies on various sheep populations detected fat-tail signatures on chromosomes 2, 3, 5, 6, 7 and 13. Fat-tailed sheep represent about 25% of the world\u2019s sheep population, and the genes with a role in this phenotype are likely not the same for every breed, since the wild ancestor of sheep had a thin tail, and the fat tail was selected by humans in longstanding husbandry practices in different regions. In the present work, a genome-wide scan using ~50,000 Single Nucleotide Polymorphisms was performed to identify selection signatures for the f at tail in the Barbaresca sheep, an Italian breed originating from North Africa. Fst values of differentiation, and \u3c72 test of significance of allele frequency were calculated, for each marker, between the Barbaresca and each of 13 Italian thin-tailed breeds. Strong signals of selection were detected for all 13 breeds on chromosome 6, in a region encoding the SLIT homolog 2 gene, this gene acting as a molecular guidance cue in cellular migration. The signature on chromosome 7 was very strong only in some of the breeds used for comparison: the detected signal was located in proximity of the Vertnin gene, a candidate for variation in vertebral number, and was already revealed in Iranian and Mediterranean fat-tailed breeds, but not in the Chinese sheep, so confirming the complexity of the fat-tail phenotype, which is associated in some breeds to long and pendulous tail, while, in other breeds, to the short tail

The influence of phosphatidylserine localisation and lipid phase on membrane remodelling by the ESCRT-II/ESCRT-III complex

The endosomal sorting complex required for transport (ESCRT) organises in supramolecular structures on the surface of lipid bilayers to drive membrane invagination and scission of intraluminal vesicles (ILVs), a process also controlled by membrane mechanics. However, ESCRT association with the membrane is also mediated by electrostatic interactions with anionic phospholipids. Phospholipid distribution within natural biomembranes is inhomogeneous due to, for example, the formation of lipid rafts and curvature-driven lipid sorting. Here, we have used phase-separated giant unilamellar vesicles (GUVs) to investigate the link between phosphatidylserine (PS)-rich lipid domains and ESCRT activity. We employ GUVs composed of phase separating lipid mixtures, where unsaturated DOPS and saturated DPPS lipids are incorporated individually or simultaneously to enhance PS localisation in liquid disordered (Ld) and/or liquid ordered (Lo) domains, respectively. PS partitioning between the coexisting phases is confirmed by a fluorescent Annexin V probe. Ultimately, we find that ILV generation promoted by ESCRTs is significantly enhanced when PS lipids localise within Ld domains. However, the ILVs that form are rich in Lo lipids. We interpret this surprising observation as preferential recruitment of the Lo phase beneath the ESCRT complex due to its increased rigidity, where the Ld phase is favoured in the neck of the resultant buds to facilitate the high membrane curvature in these regions of the membrane during the ILV formation process. Ld domains offer lower resistance to membrane bending, demonstrating a mechanism by which the composition and mechanics of membranes can be coupled to regulate the location and efficiency of ESCRT activity

Demographic genetics of the endangered Amiata donkey breed

The demogenetic structure of the Amiata donkey, an endangered breed from Central Italy, was investigated using information from pedigrees. Genealogical data of 602 donkeys reared in Tuscany were recorded in a database and analysed by the computer package ENDOG. Population size increased from 89 subjects in 1995 to 503 (129 males and 374 females) in 2005. Animals were distributed among 152 herds, but the effective number of herds was 21, suggesting that a small number of herds provided stallions for the entire breed. The maximum number of traced generation was 4, the mean maximum generation was 1.14, the mean com- plete generation was 0.53, and the mean equivalent generation was 0.78. The average relatedness coeffi- cient (AR) in the 503 alive animals was 0.94% while the mean F was 0.29% so the effective population size was 172.41. Among 24 animals with a 4-generation history, 3 (12.5%) were 25% inbred. Although the incompleteness of genealogical information did not permit accurate inference of the current values of popu- lation genetic parameters, the present work represents a first step towards an efficient management of the breed

Health technology assessment of medical devices: a survey of non-European union agencies.

PublishedJournal ArticleResearch Support, Non-U.S. Gov'tThis is the final version of the article. Available from Cambridge University Press via the DOI in this record.OBJECTIVES: The aim of this study was to review and compare current health technology assessment (HTA) activities for medical devices across non-European Union HTA agencies. METHODS: HTA activities for medical devices were evaluated from three perspectives: organizational structure, processes, and methods. Agencies were primarily selected upon membership of existing HTA networks. The data collection was performed in two stages: stage 1-agency Web-site assessment using a standardized questionnaire, followed by review and validation of the collected data by a representative of the agency; and stage 2-semi-structured telephone interviews with key informants of a sub-sample of agencies. RESULTS: In total, thirty-six HTA agencies across twenty non-EU countries assessing medical devices were included. Twenty-seven of thirty-six (75 percent) agencies were judged at stage 1 to have adopted HTA-specific approaches for medical devices (MD-specific agencies) that were largely organizational or procedural. There appeared to be few differences in the organization, process and methods between MD-specific and non-MD-specific agencies. Although the majority (69 percent) of both categories of agency had specific methods guidance or policy for evidence submission, only one MD-specific agency had developed methodological guidelines specific to medical devices. In stage 2, many MD-specific agencies cited insufficient resources (budget, skilled employees), lack of coordination (between regulator and reimbursement bodies), and the inability to generalize findings from evidence synthesis to be key challenges in the HTA of medical devices. CONCLUSIONS: The lack of evidence for differentiation in scientific methods for HTA of devices raises the question of whether HTA needs to develop new methods for medical devices but rather adapt existing methodological approaches. In contrast, organizational and/or procedural adaptation of existing HTA agency frameworks to accommodate medical devices appear relatively commonplace.This study was supported by a research grant from the European Community’s Seventh Framework Program (FP7 - HEALTH Grant Agreement no. 305694). The sponsor had no role in the study design, collection and analysis of data, writing of the report, or submission of the paper for publication. The authors wish to thank all interviewees and agencies’ assessment forms verifiers for their invaluable contribution to the completion of this study

Variable temperature ultra-nanoindentation system: Elevated and cryogenic temperature measurements

One of the primary motivations for development of instrumented indentation was to measure the mechanical properties of thin films. Characterization of thin film mechanical properties as a function of temperature is of immense industrial and scientific interest. The major bottlenecks in variable temperature measurements have been thermal drift, signal stability (noise) and oxidation of/condensation on the surface. Thermal drift is a measurement artifact that arises due to thermal expansion/contraction of indenter tip and loading column. This gets superimposed on the mechanical behavior data precluding accurate extraction of mechanical properties of the sample at elevated/cryogenic temperatures [1]. Reliable load-displacement measurements up to 700 °C have additional technical requirements including a differential displacement measurement system, independent tip and sample heating and active thermal management of the system as well as answers to scientific questions like the temperature in the contact area or the tip wear. It is then mandatory to have a suitable device for exploring such scientific limits to technical goals and understanding nanoscale high temperature deformation and fracture. Such a device must be able to maintain the thermal drift below 0.1 nm/sec, and should be implemented in a robust system which minimizes noise (electrical, vibrational, thermal, etc..), with a continuous correction based on active top- referencing. A novel vacuum nanoindentation system that can perform reliable load-displacement measurements over a wide temperature range (-150 to 700 °C) will be presented. This system is based on the Ultra Nanoindentation Tester (UNHT [2], [3]) that utilizes an active surface referencing technique comprising of two independent axes, one for surface referencing and another for indentation. This results in negligible compliance of the system and very low thermal drift rates. Vacuum is essential to prevent sample/tip oxidation at elevated temperatures and condensation at cryogenic temperatures. The sample, indenter and reference tip are heated separately and the surface temperatures matched establishing an Infrared bath to obtain drift rates as low as 5nm/min at 700 °C. Instrumentation development, system characterization, experimental protocol, operational refinements and thermal drift characteristics at various temperatures will be presented. The system was validated by performing extensive testing on calibration materials like fused silica and single crystal aluminum. Case studies on elevated temperature properties of P91 and 316L steels and low temperature properties of nanocrystalline nickel and copper will be presented. Finally, the current status and future roadmap for variable temperature nanoindentation testing will be discussed

Genetic variability of three local cattle breeds (Calvana, Pontremolese, Garfagnina) by STR analysis

AbstractThe dramatic size contraction of local cattle breeds due to replacement with cosmopolite improved breeds highlights the need for native genetic resources conservation. In 1985, the Anagraphic Register of local cattle breeds and small-size ethnic groups was established by the Italian Ministry of Agriculture and Forestry. Calvana, Pontremolese and Garfagnina are among the included breeds. They are all native from Tuscany. Present breeding area covers the provinces of Firenze, Prato, Pistoia and Siena for the Calvana breed (around 280 heads), while it is restricted to the province of Lucca for both Garfagnina (around 180 heads) and Pontremolese. This latter breed consists, nowadays, of less than 40 heads, while being around 15000 in 1940s.The characterization of the genetic structure and variability via molecular markers could provide useful information for breed management and conservation. In the present study, a total of 149 animals, evenly distributed among the three breeds, were genetically char..

Effect of research impact on emerging camel husbandry, welfare and social-related awareness

The lack of applied scientific research on camels, despite them being recognized as production animals, compels the reorganization of emerging camel breeding systems with the aim of achieving successful camel welfare management strategies all over the world. Relevant and properly-framed research widely impacts dissemination of scientific contents and drives public willingness to enhance ethically acceptable conditions for domestic animals. Consumer perception of this livestock industry will improve and high-quality products will be obtained. This paper draws on bibliometric indicators as promoting factors for camel-related research advances, tracing historical scientific publications indexed in ScienceDirect directory from 1880–2019. Camel as a species did not affect Journal Citation Reports (JCR) impact (p > 0.05) despite the journal, author number, corresponding author origin, discipline and publication year affecting it (p < 0.001). Countries with traditionally well-established camel farming are also responsible for the papers with the highest academic impact. However, camel research advances may have only locally and partially influenced welfare related laws, so intentional harming acts and basic needs neglect may persist in these species. A sustainable camel industry requires those involved in camel research to influence business stakeholders and animal welfare advocacies by highlighting the benefits of camel wellbeing promotion, co-innovation partnership establishment and urgent enhancement of policy reform

Small optic suspensions for Advanced LIGO input optics and other precision optical experiments

We report on the design and performance of small optic suspensions developed to suppress seismic motion of out-of-cavity optics in the Input Optics subsystem of the Advanced LIGO interferometric gravitational wave detector. These compact single stage suspensions provide isolation in all six degrees of freedom of the optic, local sensing and actuation in three of them, and passive damping for the other three