Limited Number Of Structures Research Articles

Deep learning permits imaging of multiple structures with the same fluorophores

Fluorescence microscopy, which employs fluorescent tags to label and observe cellular structures and their dynamics, is a powerful tool for life sciences. However, due to the spectral overlap between different dyes, a limited number of structures can be separately labeled and imaged for live-cell applications. In addition, the conventional sequential channel imaging procedure is quite time consuming, as it needs to switch either different lasers or filters. Here, we propose a novel double-structure network (DBSN) that consists of multiple connected models, which can extract six distinct subcellular structures from three raw images with only two separate fluorescent labels. DBSN combines the intensity-balance model to compensate for uneven fluorescent labels for different structures and the structure-separation model to extract multiple different structures with the same fluorescent labels. Therefore, DBSN breaks the bottleneck of the existing technologies and holds immense potential applications in the field of cell biology.

When will RNA get its AlphaFold moment?

The protein structure prediction problem has been solved for many types of proteins by AlphaFold. Recently, there has been considerable excitement to build off the success of AlphaFold and predict the 3D structures of RNAs. RNA prediction methods use a variety of techniques, from physics-based to machine learning approaches. We believe that there are challenges preventing the successful development of deep learning-based methods like AlphaFold for RNA in the short term. Broadly speaking, the challenges are the limited number of structures and alignments making data-hungry deep learning methods unlikely to succeed. Additionally, there are several issues with the existing structure and sequence data, as they are often of insufficient quality, highly biasedand missing key information. Here, we discuss these challenges in detail and suggest some steps to remedy the situation. We believe that it is possible to create an accurate RNA structure prediction method, but it will require solving several data quality and volume issues, usage of data beyond simple sequence alignments, or the development of new less data-hungry machine learning methods.

Discovery of a Hidden Trypanosoma cruzi Spermidine Synthase Binding Site and Inhibitors through In Silico, In Vitro, and X-ray Crystallography.

In drug discovery research, the selection of promising binding sites and understanding the binding mode of compounds are crucial fundamental studies. The current understanding of the proteins-ligand binding model extends beyond the simple lock and key model to include the induced-fit model, which alters the conformation to match the shape of the ligand, and the pre-existing equilibrium model, selectively binding structures with high binding affinity from a diverse ensemble of proteins. Although methods for detecting target protein binding sites and virtual screening techniques using docking simulation are well-established, with numerous studies reported, they only consider a very limited number of structures in the diverse ensemble of proteins, as these methods are applied to a single structure. Molecular dynamics (MD) simulation is a method for predicting protein dynamics and can detect potential ensembles of protein binding sites and hidden sites unobservable in a single-point structure. In this study, to demonstrate the utility of virtual screening with protein dynamics, MD simulations were performed on Trypanosoma cruzi spermidine synthase to obtain an ensemble of dominant binding sites with a high probability of existence. The structure of the binding site obtained through MD simulation revealed pockets in addition to the active site that was present in the initial structure. Using the obtained binding site structures, virtual screening of 4.8 million compounds by docking simulation, in vitro assays, and X-ray analysis was conducted, successfully identifying two hit compounds.

Rethinking textbook grammar introduction

This article describes the construction of a Swedish grammar textbook based on learnability and teachability within the processability theory (PT) paradigm. The article discusses the difficulties and choices encountered when applying the morphologic and syntactic structures defined for second-language Swedish in a learnable order. The main concerns raised in the literature, such as the limited number of structures studied within PT and the heterogeneity in the classroom, are also discussed. The textbook emphasises the distinction between processing and automatising (correct use), as it entails different types of exercises. It is argued in the article that explicit instruction on forms that are already processed may be the wrong priority. It is also argued that even though the idea of acquisitional stages is not accepted by everyone, developmental readiness does not interfere with other factors, such as frequency of input, due to the distinction between learning objectives and general input.

Dosimetric Evaluation of Simplified Knowledge-Based Plan with an Extensive Stepping Validation Approach in Volumetric-Modulated Arc Therapy-Stereotactic Body Radiotherapy for Lung Cancer.

Purpose:We investigated the performance of the simplified knowledge-based plans (KBPs) in stereotactic body radiotherapy (SBRT) with volumetric-modulated arc therapy (VMAT) for lung cancer.Materials and Methods:For 50 cases who underwent SBRT, only three structures were registered into knowledge-based model: total lung, spinal cord, and planning target volume. We performed single auto-optimization on VMAT plans in two steps: 19 cases used for the model training (closed-loop validation) and 16 new cases outside of training set (open-loop validation) for TrueBeam (TB) and Halcyon (Hal) linacs. The dosimetric parameters were compared between clinical plans (CLPs) and KBPs: CLPclosed, KBPclosed-TB and KBPclosed-Hal in closed-loop validation, CLPopen, KBPopen-TB and KBPopen-Hal in open-loop validation.Results:All organs at risk were comparable between CLPs and KBPs except for contralateral lung: V5 of KBPs was approximately 3%–7% higher than that of CLPs. V20 of total lung for KBPs showed comparable to CLPs; CLPclosed vs. KBPclosed-TB and CLPclosed vs. KBPclosed-Hal: 4.36% ± 2.87% vs. 3.54% ± 1.95% and 4.36 ± 2.87% vs. 3.54% ± 1.94% (P = 0.54 and 0.54); CLPopen vs. KBPopen-TB and CLPopen vs. KBPopen-Hal: 4.18% ± 1.57% vs. 3.55% ± 1.27% and 4.18% ± 1.57% vs. 3.67% ± 1.26% (P = 0.19 and 0.27). CI95 of KBPs with both linacs was superior to that of the CLP in closed-loop validation: CLPclosed vs. KBPclosed-TB vs. KBPclosed-Hal: 1.32% ± 0.12% vs. 1.18% ± 0.09% vs. 1.17% ± 0.06% (P < 0.01); and open-loop validation: CLPopen vs. KBPopen-TB vs. KBPopen-Hal: 1.22% ± 0.09% vs. 1.14% ± 0.04% vs. 1.16% ± 0.05% (P ≤ 0.01).Conclusions:The simplified KBPs with limited number of structures and without planner intervention were clinically acceptable in the dosimetric parameters for lung VMAT-SBRT planning.

PH-Triggered Self-Assembly of Cellulose Oligomers with Gelatin into a Double-Network Hydrogel.

Multicomponent systems for self-assembled molecular gels provide huge opportunities to generate collective or new functions that are not inherent in individual single-component gels. However, gelation tends to require careful and complicated procedures, because, among a myriad of kinetically trapped structures related to the degree of mixing of multiple components over a wide range of scales, from molecular level to macroscopic scale, a limited number of structures that exhibit the desired function need to be constructed. This study presents a simple method for the construction of double-network (DN) hydrogels with improved stiffness composed of crystalline cellulose oligomers and gelatin. The pH-triggered self-assembly of cellulose oligomers leads to the formation of robust networks composed of crystalline nanofibers in the presence of dissolved gelatin, followed by cooling to allow for the formation of soft gelatin networks. The resultant DN hydrogels exhibit improved stiffness; the improvement in gel stiffness with double networking is comparable to that of previously reported DN hydrogels produced via a time-consuming enzymatic reaction.

Nanotechnology-Based Materials Applied in Curtain Wall System: A Case Study from Turkey

Purpose Application of nanotechnology in construction sector brings innovation to building materials like self-cleaning and antifog. These features make it possible to shorten the maintenance and repair periods of building materials, and thereby reduce maintenance costs. The development of environmentally friendly building materials become very important to reduce air pollution and adverse effects. The competitiveness of companies in the construction sector is based on technological developments. Now, it is possible to reduce costs and respond to various customer demands owing to new technologies. Nanotechnology affects many sectors as a technology that contributes significantly to the development of the properties of materials, and it has also found its place in the construction sector. The purpose of this study is to examine the use of nanomaterials used in the construction sector in the context of glass and curtain wall systems. Also, the aims of this paper are to reveal the advantages of nanomaterials by examining the differences between the traditional and the nanotechnology applications used in glass and curtain wall systems, to remark nanomaterial applications in construction sector, and finally to expand awareness in this regard. Design/Methodology/Approach This study consisted in reviewing existing nanomaterials in the curtain wall system applications through projects, and conducting a survey. A preliminary letter and a 14-question survey were prepared. This study covers aluminum composite and glass curtain wall suppliers which were contacted by email. Face to face interviews were made with those who could not be reached by e-mail. The survey that was formed by the authors was sent to 68 participants working in the aluminum composite and glass curtain wall companies operating in the construction sector. Findings The findings of the study show that there are a limited number of structures using nanomaterials in Turkey. While these materials are used in many different parts of the structures, the most preferred in the outer shell design of the buildings (curtain wall systems).The construction sector should be considered as one of the critical sectors where nanotechnology can be practiced. Research Limitations/Implications The paper contains the normal limitations associated with the survey method including potential non-response bias. Practical Implications Potential of nanotechnology applications is growing in the construction field. There are large numbers of nanotechnology applications in the sector. However, construction sector is a traditional and cost oriented industry, rather than a technology focused sector. High cost of nanotechnology-based materials is a major obstacle to applications of nanotechnology in the construction sector. Even though some steps have been taken in the field of nanotechnology, the sector should be supported with incentives by government and research institutions. Social Implications The construction sector is the largest sector that causes environmental problems through construction, operation and maintenance of buildings. Due to its potential contribution to sustainability, it makes nanotechnology one of the most promising technologies in the green building area. Nanotechnology has much to offer the construction industry as it moves towards a more sustainable future. The effects of nanotechnological materials on the environment and human health are not clearly defined yet. More studies needed about that. Originality/Value Performing such a study is considered to be crucial for construction sector of developed and developing countries, so that the construction sector also avails benefits from this revolutionary technology.

Home dialysis : what are the barriers . A French nephrologist survey

Home dialysis, which includes Peritoneal Dialysis and Home Hemodialysis, provides lots of profit to patients suffering of Chronic Kidney Disease, especially in terms of comfort, life quality and autonomy. However, its use is marginal in France, with an inhomogenous distributaion according to geographical regions. We conducted a French national survey of nephrologists to assess the barriers to the development of home dialysis. After analyzing the responses of the 230 participating nephrologists, the main obstacles to the development of the two techniques were identified and classified according to their reporting rate. The major obstacles that emerge from the survey are : the lack of information among the general public, a lack of acknowledgement of nurses specializing in these techniques, the limited number of structures that practice dialysis at home, and information difficulties among patient about dialysis techniques. The specific peritoneal dialysis-related difficulties reported are : difficulties in management of follow-up care and rehabilitation, the fear of insufficient purification and the difficulties related to the dialysis catheter. Concerning home hemodialysis, the barriers concern fear of autopunction and the need for a third party. This study helps to identify the representations of nephrologists on the major obstacles to the development of home dialysis to develop lines of thought for its promotion, both in terms of training, institutional acknowledgement, and the necessary regulatory evolution.

Modeling pKas of unfolded proteins to probe structural models of unfolded state

Modeling unfolded states of proteins has implications for protein folding and stability. Since in unfolded state proteins adopt multiple conformations, any experimentally measured quantity is ensemble averaged, therefore the computed quantity should be ensemble averaged as well. Here, we investigate the possibility that one can model an unfolded state ensemble with the coil model approach, algorithm such as “flexible-meccano” [Ozenne V et al., Flexible-meccano: A tool for the generation of explicit ensemle descriptions of intrinsically disordered proteins and their associated experimental observables, Bioinformatics 28:1463–1470, 2012], developed to generate structures for intrinsically disordered proteins. We probe such a possibility by using generated structures to calculate pKas of titratable groups and compare with experimental data. It is demonstrated that even with a small number of representative structures of unfolded state, the average calculated pKas are in very good agreement with experimentally measured pKas. Also, predictions are made for titratable groups for which there is no experimental data available. This suggests that the coil model approach is suitable for generating 3D structures of unfolded state of proteins. To make the approach suitable for large-scale modeling, which requires limited number of structures, we ranked the structures according to their solvent accessible surface area (SASA). It is shown that in the majority of cases, the top structures with smallest SASA are enough to represent unfolded state.

Vapor Annealing and Colloid Lithography: An Effective Tool To Control Spatial Resolution of Surface Modification.

Colloid lithography represents a simple and efficient method for creation of a large-scale template for subsequent surface patterning, deposition of regular metal nanostructures, or periodical surface structures. However, this method is significantly restricted by its ability to create only a limited number of structures with confined geometry and symmetry features. To overcome this limitation, different techniques, such as plasma treatment or tilting angle metal deposition, have been proposed. In this paper, an alternative method based on the vapor annealing of ordered single polystyrene (PS) microspheres layer, followed by the surface grafting with arenediazonium tosylates is proposed. Application of vapor treatment before surface grafting allows effective control of the area screened by PS microspheres. Pristine and vapor-annealed microsphere arrays on the gold substrate were electrochemically modified using ADTs. Subsequent removal of the PS microsphere mask enabled to prepare well-defined nanostructures with controllable surface features. In particular, prepared periodic arrangements were achieved by the grafting of OFGs to the empty interspaces between nanopore arrays. The process of sample preparation was controlled, and the properties of prepared structures were characterized by various techniques, including atomic force microscopy (AFM), conductive AFM, scanning electron microscopy energy-dispersive X-ray spectrometry, Raman spectroscopy, and voltammetry.

Trait-Related Cortical-Subcortical Dissociation in Bipolar Disorder: Analysis of Network Degree Centrality.

Bipolar disorder is a systemic brain disorder. Accumulated evidence suggested that cortical-subcortical imbalance could be a trait-related pathogenic factor of bipolar disorder. Degree centrality, a robust index of focal connectivity in which the number of direct connections from one node to all nodes is counted, has not previously been studied in bipolar disorder as a whole. Resting state functional magnetic resonance imaging was performed on 52 patients with DSM-IV bipolar I disorder and 70 healthy controls recruited between September 2009 and July 2014. Degree centrality was calculated within cerebral gray matter for each subject and compared between patients with bipolar disorder and healthy controls. Hub distributions of both groups were explored. Effects of medication exposure and mood state on degree centrality, as well as cortical-subcortical degree centrality correlations, were explored. Compared to healthy controls, patients with bipolar disorder exhibited significant decrease in degree centrality in cortical regions, including the middle temporal pole, inferior temporal gyrus, and ventral prefrontal cortex, but showed significant increase in degree centrality mainly in subcortical regions, including caudate, thalamus, parahippocampal gyrus, hippocampi, anterior cingulate, insula, and amygdala, and a small portion of cortical regions, such as superior and middle frontal gyrus (P < .05, corrected). Spatial distributions of the 2 groups were very similar. No significant effects of medication exposure or mood state on degree centrality were found. Patients with bipolar disorder also showed significant decrease in cortical-subcortical degree centrality correlation (P = .003). These findings further contribute to the mounting evidence of cortical-subcortical dissociation in bipolar disorder pathophysiology. In addition, this study supports the continued development and implementation of graph-based techniques to enhance our understanding of the underlying neural mechanisms in mental disorders such as bipolar disorder, which are increasingly viewed as systemic brain disorders rather than disorders arising from disruption within a single structure or a limited number of structures. Due to the heterogeneity of our sample, as well as the small sample size of each medication and mood state subgroups, further investigation is needed to support our findings.

Recent Advances in Small Clusters and Polymetallic Assemblies Based on Transition Metals and Dithiocarboxylate Zwitterions Derived from N-Heterocyclic Carbenes

N-Heterocyclic carbenes (NHCs) react readily with carbon disulfide to form stable, crystalline, zwitterionic adducts. In this review, we outline the various strategies that were applied to synthesize transition metal clusters based on such NHC·CS2 ligands. We also briefly survey their most salient structural features and catalytic properties. Because this research field is still in its infancy, only a limited number of structures containing metals from Group 7 (Mn and Re) and 8 (Fe and Ru) have been described so far. Homo- and heterobimetallic manganese and rhenium carbonyl clusters with the generic formula [MM′(CO)6(μ-κ2-S,S′-κ3-S,C,S′-S2C·NHC)] (MM′ = Mn2, MnRe, or Re2) were most thoroughly investigated. Isolated examples of dinuclear iron and tetranuclear ruthenium compounds, in which the dithiocarboxylate unit underwent fragmentation, were also reported. Other related polymetallic entities recently elaborated through the use of azolium-2-dithiocarboxylate zwitterions and Group 11 metals (Cu and Au) include copper-based coordination polymers, gold nanoparticles, and self-assembled monolayers.

Predictive Self-Assembly of Complex Crystals via Building Block Design

A primary challenge for the development of bulk, scalable, and high yield materials with interesting properties is the limited number of structures that can be obtained via self-assembly of nano and micrometer sized particles. Systematic and extensive computational studies of hard polyhedral particles have demonstrated that anisotropy of the building blocks can be a viable route for increasing variability of assembled patterns [1, 2, 3]. Interestingly, the types of structures assembled from this method were shown to be predictable from information contained already in the dense fluid, prior to crystallization. In this talk, the role of such local structures for self-assembly will be rationalized and we will demonstrate how this information can be used as a strategy for design of crystalline and quasicrystalline patterns for both symmetric and asymmetric particles.

Mechatronic Approaches for Functional Structural Synthesis of Mechanical Systems of Industrial Robots

Abstract The necessity of new approaches for structural synthesis of mechanical systems, which directly identify a limited number of structures that carry a potential for solving the technical problems and meet the specific requirements for the design of mechanical systems, mainly for specialised robots, is specified. The identified five types of kinematic chains (primary, parallel, secondary, additional and subsidiary), with different functionality, allow the synthesis of manipulators structures according to the defined goal tasks and specific functional requirements for hybrid systems, in particular specialised industrial robots. Two mechatronic approaches for functional structural synthesis of mechanical systems of industrial robots, where the main manipulation mechanism is a path generator, were introduced. A limited number of structures that meets the set of objectives and technical requirements to design mechanisms, is directly determined. Emphasis is placed on the tasks of passive (mechanical) control of manipulation systems associated with specialized robotics. Two mechatronic approaches for functional structural synthesis of mechanical systems of industrial robots, where the main manipulation mechanism is a motion generator, are introduced. A limited number of structures is directly defined that meets the set of technical objectives and requirements for the designed mechanisms. Emphasis is placed on the tasks of passive control of the manipulation systems associated with the specialized robotics.

Imaging Review of the Temporal Bone: Part I. Anatomy and Inflammatory and Neoplastic Processes

From a clinical-radiologic standpoint, there are a limited number of structures and disease entities in the temporal bone with which one must be familiar in order to proficiently interpret a computed tomographic or magnetic resonance imaging study of the temporal bone. It is helpful to examine the region in an organized and systematic fashion, going through the same checklist of key structures each time. This is the first of a two-part review that provides a practical approach to understanding temporal bone anatomy, localizing a pathologic process with a focus on inflammatory and neoplastic processes, identifying pertinent positives and negatives, and formulating a differential diagnosis.

Fatigue assessment of structures on the Docklands Light Railway, London, UK

The Docklands Light Railway in London, UK has been in service for some 25 years and has now been upgraded for the second time to enable it to use longer trains at higher frequencies. The structures had to be assessed and, when necessary, strengthened. The assessment would normally have been done using a similar approach to that which would be used for a new design. However, this showed up a significant need for strengthening purely for fatigue. It was therefore decided to strain gauge some structures and the results showed significantly smaller ranges than predicted. Rather than use the results directly, they were used to develop a more realistic assessment approach. This was partly because of the need to apply results for a limited number of structures subjected to two-car trains, to a larger number of structures subjected to three-car trains. Comparison with the strain gauge readings suggested the adopted approach was still very conservative but major savings were still made compared with the strengthening that would have been required by the conventional approach. Two of the structures had more specific and localised problems. Here it was possible to strain gauge all the critical elements and more direct use of the results was possible.

The impact response of graded foam sandwich structures

Low velocity impact tests have been undertaken on sandwich structures based on cores fabricated by bonding foams of different densities together. Here, a range of linear PVC, crosslinked PVC and PEI foams were bonded together to produce a three layer core. Carbon fibre skins were then bonded to the core and the structures were loaded by a drop-weight impact carriage with a hemispherical head. It has been observed that the majority of the panels failed in a through-thickness shearing mode, leaving a clear cylindrical hole in the multi-layered core. A limited number of structures also exhibited cone cracking on the exit surface, due to failure in a mixed tensile/shear mode. The impact response of the graded sandwich structures was modelled by finite element analysis and the predicted load–displacement responses and failure modes compared. Agreement between the FE model and the experimental data was good across the range of structures investigated, with the model accurately predicting the impact responses and failure characteristics observed within the panels. It has also been shown that graded core structures can out-perform their monolithic counterparts. Finally when normalised by their unit cost, significant differences in the perforation resistances of the structures have been observed.

The A 1 receptor agonist R-Pia reduces the imbalance between cerebral glucose metabolism and blood flow during status epilepticus: Could this mechanism be involved with neuroprotection?

It is well known that the uncoupling between local cerebral glucose utilization (LCGU) and local cerebral blood flow (LCBF), i.e. decrease in LCBF rates with high LCGU, is frequently associated with seizure-induced neuronal damage. This study was performed to assess if the neuroprotective effect of the adenosinergic A 1 receptor agonist R-N-phenylisopropyladenosine (R-Pia) injected prior to pilocarpine is able to reduce the uncoupling between LCGU and LCBF during status epilepticus (SE). Four groups of rats were studied: Saline, Pilo, R-Pia + Saline and R-Pia + Pilo. For LCGU and LCBF studies, rats were subjected to autoradiography using [ 14C]-2-deoxyglucose and [ 14C]-iodoantypirine, respectively. Radioligands were injected 4 h after SE onset. Neuronal loss was evaluated by Fluorojade-B (FJB) at two time points after SE onset (24 h and 7 days). The results showed a significant increase in LCGU in almost all brain regions studied in the Pilo and R-Pia + Pilo groups compared to controls. However, in R-Pia pretreated rats, the uncoupling between LCGU and LCBF was moderated in a limited number of structures as substantia nigra pars reticulata and hippocampal formation rather in favor of hyperperfusion. Significant increases in LCBF were observed in the entorhinal cortex, thalamic nuclei, mammillary body, red nucleus, zona incerta, pontine nucleus and visual cortex. The neuroprotective effect of R-Pia assessed by FJB showed a lower density of degenerating cells in the hippocampal formation, piriform cortex and basolateral amygdala. In conclusion our data shows that the neuroprotective effect of R-Pia was accompanied by a compensatory metabolic input in brain areas involved with seizures generation.

Erratum: “The Neural Nanoprobe: Physically Decoupling the Neural Recording Site From the Headstage” [Journal of Medical Devices, 2009, 3(2), p. 027524

In the original manuscript, the fourth author, A. G. Erdman, was inadvertently omitted. The complete author list and accompanying abstract are reprinted below.K. M. Al-AshmounyElectrical Engineering and Computer Science, University of MichiganC. BoldtDepartment of Neuroscience, University of MinnesotaJ. E. FergusonDepartment of Biomedical Engineering, University of MinnesotaA. G. ErdmanMechanical Engineering, University of MinnesotaA. D. RedishDepartment of Neuroscience, University of MinnesotaE. YoonElectrical Engineering and Computer Science, University of MichiganThe ability to record neural ensembles from awake, behaving animals is one of the most important and successful components of the neuroscience experimental toolbox. However, even the most advanced modern systems have limitations due to the physical coupling of the recording site with the headstage. These systems can only record from a limited number of structures at any one time and have particular difficulty recording large ensembles from animals with thin skulls (e.g., mice, songbirds). Current systems cannot record from fragile structures (spinal cord, peripheral nerves and ganglia) during behavior because the wire electrodes would shred the fragile nerves as the animal moves. We propose the concept of a neural nanoprobe that is physically decoupled from a separately implanted waystation. Because the nanoprobes are not connected to the waystation by physical wires, multiple nanoprobes could be placed in multiple neural structures, all transmitting to a single, separate waystation. Because the nanoprobes effectively float in the cellular matrix, they are safe to put in fragile structures. The waystation does not need to be implanted in the fragile structures; it only needs to be electrically coupled to them. The first step to the realization of this device is a low-power, high-fidelity method for communicating between the nanoprobe and the waystation. In this abstract, we report a successful test proving the viability of using the brain itself as the conducting medium through which the nanoprobe and waystation can communicate. Initial tests show that neural signals from multiple transmission sites can be sent to a single, separated receiver. We first identified the current-loss of sine-waves transmitted through live (anesthetized) brain tissue. We found negligible current-loss across frequencies ranging from 100 kHz–50 MHz across distances as much as 15 mm. As these frequencies are larger than any known frequencies used by neural signals, they are unlikely to interfere with neural function. We next measured the ability to transmit and receive pre-recorded neural signals (sampled at 20 kHz), using prerecorded signals to determine the fidelity of transmission. The two different signals were transmitted, received, and successfully demodulated with high-fidelity, even with transmission currents as low as 2 μA. Both the transmitters and the receiver each had their own battery power supply to ensure that they used separate, floating grounds. Finally, to ensure that the intra-brain communication signals did not interfere with neural activity, we recorded extra-cellular potentials before, during, and after the test. No changes were observed in spike shape, spike frequency, bursting, or other cellular properties, demonstrating the safety of this technique. Supported by a grant from the Institute for Engineering in Medicine (U Minnesota) and training grant support from T90-DK070106. Corresponding author; email: redish@umn.edu

Rearrangements in the Relative Orientation of Cytoplasmic Domains Induced by a Membrane-anchored Protein Mediate Modulations in Kv Channel Gating

Interdomain interactions between intracellular N and C termini have been described for various K(+) channels, including the voltage-gated Kv2.1, and suggested to affect channel gating. However, no channel regulatory protein directly affecting N/C interactions has been demonstrated. Most Kv2.1 channel interactions with regulatory factors occur at its C terminus. The vesicular SNARE that is also present at a high concentration in the neuronal plasma membrane, VAMP2, is the only protein documented to affect Kv2.1 gating by binding to its N terminus. As its binding target has been mapped near a site implicated in Kv2.1 N/C interactions, we hypothesized that VAMP2 binding to the N terminus requires concomitant conformational changes in the C terminus, which wraps around the N terminus from the outside, to give VAMP2 access. Here, we first determined that the Kv2.1 N terminus, although crucial, is not sufficient to convey functional interaction with VAMP2, and that, concomitant to its binding to the "docking loop" at the Kv2.1 N terminus, VAMP2 binds to the proximal part of the Kv2.1 C terminus, C1a. Next, using computational biology approaches (ab initio modeling, docking, and molecular dynamics simulations) supported by molecular biology, biochemical, electrophysiological, and fluorescence resonance energy transfer analyses, we mapped the interaction sites on both VAMP2 and Kv2.1 and found that this interaction is accompanied by rearrangements in the relative orientation of Kv2.1 cytoplasmic domains. We propose that VAMP2 modulates Kv2.1 inactivation by interfering with the interaction between the docking loop and C1a, a mechanism for gating regulation that may pertain also to other Kv channels.