Incomplete Incorporation of Tandem Subunits in Recombinant Neuronal Nicotinic Receptors

Tandem constructs are increasingly being used to restrict the composition of recombinant multimeric channels. It is therefore important to assess not only whether such approaches give functional channels, but also whether such channels completely incorporate the subunit tandems. We have addressed this question for neuronal nicotinic acetylcholine receptors, using a channel mutation as a reporter for subunit incorporation. We prepared tandem constructs of nicotinic receptors by linking α (α2–α4, α6) and β (β2, β4) subunits by a short linker of eight glutamine residues. Robust functional expression in oocytes was observed for several tandems (β4_α2, β4_α3, β4_α4, and β2_α4) when coexpressed with the corresponding β monomer subunit. All tandems expressed when injected alone, except for β4_α3, which produced functional channels only together with β4 monomer and was chosen for further characterization. These channels produced from β4_α3 tandem constructs plus β4 monomer were identical with receptors expressed from monomer α3 and β4 constructs in acetylcholine sensitivity and in the number of α and β subunits incorporated in the channel gate. However, separately mutating the β subunit in either the monomer or the tandem revealed that tandem-expressed channels are heterogeneous. Only a proportion of these channels contained as expected two copies of β subunits from the tandem and one from the β monomer construct, whereas the rest incorporated two or three β monomers. Such inaccuracies in concatameric receptor assembly would not have been apparent with a standard functional characterization of the receptor. Extensive validation is needed for tandem-expressed receptors in the nicotinic superfamily

NGPaaS framework for enriched and customized virtual network functions-as-a-service

This paper describes how the novel Next Generation Platform-as-a-Service (NGPaaS) framework can facilitate major benefits for Network Operators and Vertical Service Providers (VSPs) who wish to leverage Virtual Network Functions-as-a-Service (VNFaaS) capabilities. Network Operators can benefit by providing an "on demand" PaaS with required features for the VSPs, thus generating new revenue streams but with low operational overhead due to the high degree of automation. VSPs can benefit from the PaaS-oriented approach, by being able to flexibly on-board new VNF types and "value-added" service capabilities like monitoring, healing and profiling, to deliver customized service blueprints to meet the needs of their end customers. The paper outlines the design of an early prototype, built on the Open-CORD platform and using industry-standard Virtualised Network Functions (VNFs)

Human α3β4 Neuronal Nicotinic Receptors Show Different Stoichiometry if They Are Expressed in Xenopus Oocytes or Mammalian HEK293 Cells

The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the α3 and β4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the α5 and/or β2 subunits. We compared the properties of human α3β4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and α:β subunit ratio.Two distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the α subunit, as reported for α4β2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9' of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike α4β2 channels, for α3β4 receptors the putative two-α form is the predominant one in oocytes (at 1:1 α:β cRNA ratio). This two-α form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-α form is the predominant one in HEK cells transfected with a 1:1 α:β DNA ratio or in oocytes at 9:1 α:β RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-α form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-α form gave rise to short bursts (14 ms) of high conductance (39 pS).Like other neuronal nicotinic receptors, the α3β4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected

The Next Generation Platform as A Service: Composition and Deployment of Platforms and Services

The emergence of widespread cloudification and virtualisation promises increased flexibility, scalability, and programmability for the deployment of services by Vertical Service Providers (VSPs). This cloudification also improves service and network management, reducing the Capital and Operational Expenses (CAPEX, OPEX). A truly cloud-native approach is essential, since 5G will provide a diverse range of services - many requiring stringent performance guarantees while maximising flexibility and agility despite the technological diversity. This paper proposes a workflow based on the principles of build-to-order, Build-Ship-Run, and automation; following the Next Generation Platform as a Service (NGPaaS) vision. Through the concept of Reusable Functional Blocks (RFBs), an enhancement to Virtual Network Functions, this methodology allows a VSP to deploy and manage platforms and services, agnostic to the underlying technologies, protocols, and APIs. To validate the proposed workflow, a use case is also presented herein, which illustrates both the deployment of the underlying platform by the Telco operator and of the services that run on top of it. In this use case, the NGPaaS operator facilitates a VSP to provide Virtual Network Function as a Service (VNFaaS) capabilities for its end customers

Mendelian randomisation implicates hyperlipidaemia as a risk factor for colorectal cancer.

While elevated blood cholesterol has been associated with an increased risk of colorectal cancer (CRC) in observational studies, causality is uncertain. Here we apply a Mendelian randomisation (MR) analysis to examine the potential causal relationship between lipid traits and CRC risk. We used single nucleotide polymorphisms (SNPs) associated with blood levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) as instrumental variables (IV). We calculated MR estimates for each risk factor with CRC using SNP-CRC associations from 9,254 cases and 18,386 controls. Genetically predicted higher TC was associated with an elevated risk of CRC (odds ratios (OR) per unit SD increase = 1.46, 95% confidence interval [CI]: 1.20-1.79, P=1.68x10−4). The pooled ORs for LDL, HDL, and TG were 1.05 (95% CI: 0.92-1.18, P=0.49), 0.94 (95% CI: 0.84-1.05, P= 0.27), and 0.98 (95% CI: 0.85-1.12, P=0.75) respectively. A genetic risk score for 3-hydoxy-3-methylglutaryl-coenzyme A reductase (HMGCR) to mimic the effects of statin therapy was associated with a reduced CRC risk (OR=0.69, 95% CI: 0.49-0.99, P=0.046). This study supports a causal relationship between higher levels of TC with CRC risk, and a further rationale for implementing public health strategies to reduce the prevalence of hyperlipidaemia. This article is protected by copyright. All rights reserved

Temporal properties of human information processing: Tests of discrete versus continuous models,

Cognitive psychologists have characterized the temporal properties of human information processing in terms of discrete and continuous models. Discrete models postulate that component mental processes transmit a finite number of intermittent outputs (quanta) of information over time, whereas continuous models postulate that information is transmitted in a gradual fashion. These postulates may be tested by using an adaptive response-priming procedure and analysis of reaction-time mixture distributions. Three experiments based on this procedure and analysis are reported. The experiments involved varying the temporal interval between the onsets of a prime stimulus and a subsequent test stimulus to which a response had to be made. Reaction time was measured as a function of the duration of the priming interval and the type of prime stimulus. Discrete models predict that manipulations of the priming interval should yield a family of reaction-time mixture distributions formed from a finite number of underlying basis distributions, corresponding to distinct preparatory states. Continuous models make a different prediction. Goodness-of-fit tests between these predictions and the data supported either the discrete or the continuous models, depending on the nature of the stimuli and responses being used. When there were only two alternative responses and the stimulus-response mapping was a compatible one, discrete models with two or three states of preparation fit the results best. For larger response sets with an incompatible stimulus-response mapping, a continuous model fit some of the data better. These results are relevant to the interpretation of reaction-time data in a variety of contexts and to the analysis of speed-accuracy trade-offs in mental processes.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25558/1/0000100.pd