Topology profile for a glutamate receptor: Three transmembrane domains and a channel-lining reentrant membrane loop

We investigated the transmembrane topology of the GluR3 subunit that was translated in rabbit reticulocytes supplemented with microsomal membranes. A prolactin reporter epitope was fused to GluR3 at six locations, bracketing each of the proposed transmembrane domains. The sidedness of the epitope in the microsomal membrane was then assessed by proteinase K sensitivity. The N terminus and the entire region between M3 and M4 was extracellular, and the C terminus was intracellular by this method. Four native N-linked glycosylation sites in the amino terminus and one introduced site between M3 and M4 were utilized, confirming the extracellular location of these regions. Epitopes inserted upstream and downstream of M2 were protease sensitive and thus intracellular. Our results support a topological model for glutamate receptor subunits that consists of three transmembrane domains, M1, M3, and M4, and another domain, the proposed channel-lining M2, which forms a reentrant membrane segment with both ends facing the cytoplasm

The involvement of the piriform cortex in non-lesional temporal lobe epilepsy: an uncommon component of the epileptogenic network

The piriform cortex is recognized as highly epileptogenic in rodents, yet its electrophysiological role in human epilepsy remains understudied. Recent surgical outcomes have suggested potential benefits in resecting the piriform cortex for cases of medial temporal lobe epilepsy. However, little is known about its electrophysiological activity in human epilepsy. This case-series study aimed to explore the electrophysiological role of the piriform cortex within the epileptogenic network among patients with suspected temporal lobe epilepsy. Participants were recruited from Emory University Hospital or Children's Healthcare of Atlanta, with non-lesional frontotemporal or temporal lobe hypotheses, undergoing stereoelectroencephalographic studies. Specifically, focus was placed on patients with one or more electrode contacts in the piriform cortex. Primary objectives included determining piriform cortex involvement within the electrophysiologically defined epileptogenic network and assessing the effects of electrical stimulation. Twenty-two patients were included in the study. Notably, only one patient exhibited piriform cortex involvement at seizure onset, associated with an olfactory aura. Two patients showed early piriform cortex involvement, while others displayed late or no involvement. Electrical stimulation of the piriform cortex induced after-discharges in three patients and replicated a habitual seizure in one. These findings present a contrast to surgical outcome studies, suggesting that the piriform cortex may not typically play a significant role in the epileptogenic network among patients with non-lesional temporal lobe epilepsy

Transcriptional Regulation of the GluR2 Gene: Neural-Specific Expression, Multiple Promoters, and Regulatory Elements

To understand how neurons control the expression of the AMPA receptor subunit GluR2, we cloned the 5' proximal region of the rat gene and investigated GluR2 promoter activity by transient transfection. RNase protection and primer extension of rat brain mRNA revealed multiple transcription initiation sites from -340 to -481 bases upstream of the GluR2 AUG codon. The relative use of 5' start sites was different in cortex and cerebellum, indicating complexity of GluR2 transcript expression among different sets of neurons. When GluR2 promoter activity was investigated by plasmid transfection into cultured cortical neurons, cortical glia, and C6 glioma cells, the promoter construct with the strongest activity, per transfected cell, was 29.4-fold (+/- 3.7) more active in neurons than in non-neural cells. Immunostaining of cortical cultures showed that >97% of the luciferase-positive cells also expressed the neuronal marker MAP-2. Evaluation of internal deletion and substitution mutations identified a functional repressor element I RE1-like silencer and functional Sp1 and nuclear respiratory factor-1 (NRF-1) elements within a GC-rich proximal GluR2 promoter region. The GluR2 silencer reduced promoter activity in glia and non-neuronal cell lines by two- to threefold, was without effect in cortical neurons, and could bind the RE1-silencing transcription factor (REST) because cotransfection of REST into neurons reduced GluR2 promoter activity in a silencer-dependent manner. Substitution of the GluR2 silencer by the homologous NaII RE1 silencer further reduced GluR2 promoter activity in non-neuronal cells by 30-47%. Maximal positive GluR2 promoter activity required both Sp1 and NRF-1 cis elements and an interelement nucleotide bridge sequence. These results indicate that GluR2 transcription initiates from multiple sites, is highly neuronal selective, and is regulated by three regulatory elements in the 5' proximal promoter region

A Dual-Readout F2 Assay That Combines Fluorescence Resonance Energy Transfer and Fluorescence Polarization for Monitoring Bimolecular Interactions

Forster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS. Here we report the design and development of a dual-readout HTS assay that combines two assay technologies into one system using the Mcl-1 and Noxa BH3 peptide interaction as a model system. In this system, both FP and FRET signals were simultaneously monitored from one reaction, which is termed -Dual-Readout F2 assay- with F2 for FP and FRET. This dual-readout technology has been optimized in a 1,536-well ultra-HTS format for the discovery of Mcl-1 protein inhibitors and achieved a robust performance. This F2 assay was further validated by screening a library of 102,255 compounds. As two assay platforms are utilized for the same target simultaneously, hit information is enriched without increasing the screening cost. This strategy can be generally extended to other FP-based assays and is expected to enrich primary HTS information and enhance the hit quality of HTS campaigns.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/90469/1/adt-2E2010-2E0292.pd

cMix: Mixing with Minimal Real-Time Asymmetric Cryptographic Operations

We introduce cMix, a new approach to anonymous communications. Through a precomputation, the core cMix protocol eliminates all expensive realtime public-key operations --- at the senders, recipients and mixnodes --- thereby decreasing real-time cryptographic latency and lowering computational costs for clients. The core real-time phase performs only a few fast modular multiplications. In these times of surveillance and extensive profiling there is a great need for an anonymous communication system that resists global attackers. One widely recognized solution to the challenge of traffic analysis is a mixnet, which anonymizes a batch of messages by sending the batch through a fixed cascade of mixnodes. Mixnets can offer excellent privacy guarantees, including unlinkability of sender and receiver, and resistance to many traffic-analysis attacks that undermine many other approaches including onion routing. Existing mixnet designs, however, suffer from high latency in part because of the need for real-time public-key operations. Precomputation greatly improves the real-time performance of cMix, while its fixed cascade of mixnodes yields the strong anonymity guarantees of mixnets. cMix is unique in not requiring any real-time public-key operations by users. Consequently, cMix is the first mixing suitable for low latency chat for lightweight devices. Our presentation includes a specification of cMix, security arguments, anonymity analysis, and a performance comparison with selected other approaches. We also give benchmarks from our prototype