The suitability of high throughput automated patch clamp for physiological applications.

Although automated patch clamp (APC) devices have been around for many years and have become an integral part of many aspects of drug discovery, high throughput instruments with gigaohm seal data quality are relatively new. Experiments where a large number of compounds are screened against ion channels are ideally suited to high throughput APC, particularly when the amount of compound available is low. Here we evaluate different APC approaches using a variety of ion channels and screening settings. We have performed a screen of 1920 compounds on GluN1/GluN2A NMDA receptors for negative allosteric modulation using both the SyncroPatch 384 and FLIPR. Additionally, we tested the effect of 36 arthropod venoms on NaV 1.9 using a single 384-well plate on the SyncroPatch 384. As an example for mutant screening, a range of acid-sensing ion channel variants were tested and the success rate increased through fluorescence-activated cell sorting (FACS) prior to APC experiments. Gigaohm seal data quality makes the 384-format accessible to recording of primary and stem cell-derived cells on the SyncroPatch 384. We show recordings in voltage and current clamp modes of stem cell-derived cardiomyocytes. In addition, the option of intracellular solution exchange enabled investigations into the effects of intracellular Ca2+ and cAMP on TRPC5 and HCN2 currents, respectively. Together, these data highlight the broad applicability and versatility of APC platforms and also outlines some limitations of the approach. KEY POINTS: High throughput automated patch clamp (APC) can be used for a variety of applications involving ion channels. Lower false positive rates were achieved using automated patch clamp versus a fluorometric imaging plate reader (FLIPR) in a high throughput compound screen against NMDA receptors.  Genetic variants and mutations can be screened on a single 384-well plate to reduce variability of experimental parameters. Intracellular solution can be perfused to investigate effects of ions and second messenger systems without the need for excised patches. Primary cells and stem cell-derived cells can be used on high throughput APC with reasonable success rates for cell capture, voltage clamp measurements and action potential recordings in current clamp mode.

Physical principles for scalable neural recording.

Simultaneously measuring the activities of all neurons in a mammalian brain at millisecond resolution is a challenge beyond the limits of existing techniques in neuroscience. Entirely new approaches may be required, motivating an analysis of the fundamental physical constraints on the problem. We outline the physical principles governing brain activity mapping using optical, electrical, magnetic resonance, and molecular modalities of neural recording. Focusing on the mouse brain, we analyze the scalability of each method, concentrating on the limitations imposed by spatiotemporal resolution, energy dissipation, and volume displacement. Based on this analysis, all existing approaches require orders of magnitude improvement in key parameters. Electrical recording is limited by the low multiplexing capacity of electrodes and their lack of intrinsic spatial resolution, optical methods are constrained by the scattering of visible light in brain tissue, magnetic resonance is hindered by the diffusion and relaxation timescales of water protons, and the implementation of molecular recording is complicated by the stochastic kinetics of enzymes. Understanding the physical limits of brain activity mapping may provide insight into opportunities for novel solutions. For example, unconventional methods for delivering electrodes may enable unprecedented numbers of recording sites, embedded optical devices could allow optical detectors to be placed within a few scattering lengths of the measured neurons, and new classes of molecularly engineered sensors might obviate cumbersome hardware architectures. We also study the physics of powering and communicating with microscale devices embedded in brain tissue and find that, while radio-frequency electromagnetic data transmission suffers from a severe power-bandwidth tradeoff, communication via infrared light or ultrasound may allow high data rates due to the possibility of spatial multiplexing. The use of embedded local recording and wireless data transmission would only be viable, however, given major improvements to the power efficiency of microelectronic devices.

Conserved glycine 33 residue in flexible domain I of hepatitis C virus core protein is critical for virus infectivity.

Hepatitis C virus core protein forms the viral nucleocapsid and plays a critical role in the formation of infectious particles. In this study, we demonstrate that the highly conserved residue G33, located within domain 1 of the core protein, is important for the production of cell culture-infectious virus (HCVcc). Alanine substitution at this position in the JFH1 genome did not alter viral RNA replication but reduced infectivity by ∼2 logs. Virus production by this core mutant could be rescued by compensatory mutations located immediately upstream and downstream of the original G33A mutation. The examination of the helix-loop-helix motif observed in the core protein structure (residues 15 to 41; Protein Data Bank entry 1CWX) indicated that the residues G33 and F24 are in close contact with each other, and that the G33A mutation induces a steric clash with F24. Molecular simulations revealed that the compensatory mutations increase the helix-loop-helix flexibility, allowing rescue of the core active conformation required for efficient virus production. Taken together, these data highlight the plasticity of core domain 1 conformation and illustrate the relationship between its structural tolerance to mutations and virus infectivity.

Requirement of cellular DDX3 for hepatitis C virus replication is unrelated to its interaction with the viral core protein.

The cellular DEAD-box protein DDX3 was recently shown to be essential for hepatitis C virus (HCV) replication. Prior to that, we had reported that HCV core binds to DDX3 in yeast-two hybrid and transient transfection assays. Here, we confirm by co-immunoprecipitation that this interaction occurs in cells replicating the JFH1 virus. Consistent with this result, immunofluorescence staining of infected cells revealed a dramatic redistribution of cytoplasmic DDX3 by core protein to the virus assembly sites around lipid droplets. Given this close association of DDX3 with core and lipid droplets, and its involvement in virus replication, we investigated the importance of this host factor in the virus life cycle. Mutagenesis studies located a single amino acid in the N-terminal domain of JFH1 core that when changed to alanine significantly abrogated this interaction. Surprisingly, this mutation did not alter infectious virus production and RNA replication, indicating that the core-DDX3 interaction is dispensable in the HCV life cycle. Consistent with previous studies, siRNA-led knockdown of DDX3 lowered virus production and RNA replication levels of both WT JFH1 and the mutant virus unable to bind DDX3. Thus, our study shows for the first time that the requirement of DDX3 for HCV replication is unrelated to its interaction with the viral core protein.

Inhibition of Dugbe nairovirus replication by human MxA protein.

Sensitivity to the interferon-induced protein, MxA, has previously been demonstrated for viruses belonging to the Orthobunyavirus, Hantavirus and Phlebovirus genera of the Bunyaviridae family. We have extended these findings to a member of the fourth and remaining genus containing viruses that infect man and other animals, the nairovirus Dugbe virus (DUGV). Indirect immunofluorescence experiments using VA9 cells (Vero cells permanently transfected with MxA cDNA) revealed strongly reduced DUGV antigen expression, suggesting that MxA inhibited DUGV replication. Western and Northern blot analyses showed significantly lower DUGV nucleocapsid (N) protein expression and DUGV genomic RNA, respectively, in the presence of MxA. Viral titres were also reduced by more than two orders of magnitude in VA9 cells compared with control VN36 cells. This finding may have application to nairovirus therapeutics.

Dugbe nairovirus S segment: correction of published sequence and comparison of five isolates.

The sequence of the S (small) RNA segment of the ArD 44313 isolate of Dugbe nairovirus (DUG) has been redetermined, and a number of apparent errors in the previously reported sequence (V. K. Ward, A. C. Marriott, A. A. El-Ghorr, and P. A. Nuttall, 1990, Virology 175, 518-524) were revealed. Our results indicate that the S RNA is 1716 nucleotides (nt) in length and contains one large open reading frame spanning 1449 nt. This can encode a 483 amino acid polypeptide, M(r) 53.9 kDa, corresponding to the viral nucleocapsid protein N. The DUG N protein is thus similar in length to the N proteins of Hazara (HAZ) and Crimean-Congo haemorrhagic fever (CCHF) nairoviruses, which are 485 and 482 amino acids in length, respectively. S segment RNA sequences were also determined for DUG isolates IbAr 1792, IbH 11480, ArD 16095, and KT 281/75; only the KT 281/75 sequence differed markedly from that of ArD 44313. Phylogenetic trees were constructed for these nairovirus S segment sequences.