Eric Slimko

Selective Electrical Silencing of Mammalian Neurons In Vitro by the Use of Invertebrate Ligand-Gated Chloride Channels

Selectively reducing the excitability of specific neurons will (1) allow for the creation of animal models of human neurological disorders and (2) provide insight into the global function of specific sets of neurons. We focus on a combined genetic and pharmacological approach to silence neurons electrically. We express invertebrate ivermectin (IVM)-sensitive chloride channels (Caenorhabditis elegans GluCl α and β) with a Sindbis virus and then activate these channels with IVM to produce inhibition via a Cl− conductance. We constructed a three-cistron Sindbis virus that expresses the α and β subunits of a glutamate-gated chloride channel (GluCl) along with the green fluorescent protein (EGFP) marker. Expression of the C. elegans channel does not affect the normal spike activity or GABA/glutamate postsynaptic currents of cultured embryonic day 18 hippocampal neurons. At concentrations as low as 5 nm, IVM activates a Cl− current large enough to silence infected neurons effectively. This conductance reverses in 8 hr. These low concentrations of IVM do not potentiate GABA responses. Comparable results are observed with plasmid transfection of yellow fluorescent protein-tagged (EYFP) GluCl α and cyan fluorescent protein-tagged (ECFP) GluCl β. The present study provides an in vitromodel mimicking conditions that can be obtained in transgenic mice and in viral-mediated gene therapy. These experiments demonstrate the feasibility of using invertebrate ligand-activated Cl− channels as an approach to modulate excitability.

Codon optimization of Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels.

Organisms use synonymous codons in a highly non-random fashion. These codon usage biases sometimes frustrate attempts to express high levels of exogenous genes in hosts of widely divergent species. The Caenorhabditis elegans GluClalpha1 and GluClbeta genes form a functional glutamate and ivermectin-gated chloride channel when expressed in Xenopus oocytes, but expression is weak in mammalian cells. We have constructed synthetic genes that retain the amino acid sequence of the wild-type GluCl channel proteins, but use codons that are optimal for mammalian cell expression. We have tagged the native and codon-optimized GluCl cDNAs with enhanced yellow fluorescent protein (EYFP, GluClalpha1 subunit) and enhanced cyan fluorescent protein (EFCP, GluClbeta subunit), expressed the channels in E18 rat hippocampal neurons and measured the relative expression levels of the two genes with fluorescence microscopy as well as with electrophysiology. Codon optimization provides a 6- to 9-fold increase in expression, allowing the conclusions that the ivermectin-gated channel has an EC(50) of 1.2 nM and a Hill coefficient of 1.9. We also confirm that the Y182F mutation in the codon-optimized beta subunit results in a heteromeric channel that retains the response to ivermectin while reducing the response to 100 microM glutamate by 7-fold. The engineered GluCl channel is the first codon-optimized membrane protein expressed in mammalian cells and may be useful for selectively silencing specific neuronal populations in vivo.

Selective elimination of glutamate activation and introduction of fluorescent proteins into a Caenorhabditis elegans chloride channel

Glutamate‐gated chloride (GluCl) channels from invertebrates can be activated by ivermectin (IVM) to produce electrical silencing in mammalian neurons. To improve this GluCl/IVM strategy, we sought to mutate the Caenorhabditis elegans GluCl channels so that they become insensitive to glutamate but retain their sensitivity to IVM. Based on structure–function studies of nicotinic acetylcholine receptor superfamily members, we tested in oocytes 19 point mutants at 16 residues in the β‐subunit likely to be involved in the response to glutamate. Y182F reduces the glutamate response by greater than six‐fold, with little change to IVM responses, when coexpressed with wild‐type (WT) GluCl α. For GluCl αβ(Y182F), the EC50 and Hill coefficient for glutamate are similar to those of WT, indicating that the mutant decreases the efficacy of glutamate, but not the potency. Also, fluorescent proteins (enhanced green fluorescent protein, enhanced yellow fluorescent protein, enhanced cyan fluorescent protein; XFP) were inserted into the M3–M4 loop of the GluCl α, β and β(Y182F). We found no significant functional difference between these XFP‐tagged receptors and WT receptors. The modified GluCl channel, without glutamate sensitivity but with a fluorescent tag, may be more useful in GluCl silencing strategies.

Sizing and Margins Assessment of Mars Science Laboratory Aeroshell Thermal Protection System

The methodology employed for the thermal design and margins assessment of the Mars Science Laboratory aeroshell thermal protection system is reviewed. A new thermal margins policy was developed in the course of this work that provides additional rigor over previous methods. Because of a late change of thermal protection materials from the heritage super lightweight ablator 561V to phenolic impregnated carbon ablator, the design of the heat shield followed a nontraditional path in which the flight thickness was selected based on a mass (rather than thermal) limit. The material switch was followed by detailed thermal analyses that demonstrated that the baselined thickness was sufficient to provide adequate thermal protection to the vehicle without violating design requirements during a 3-sigma worst-case entry condition. The backshell material thickness was also finalized before the thermal sizing was completed, and the resulting analysis showed that there was more than sufficient material on the backshell....

SMAP L-Band Microwave Radiometer: Instrument Design and First Year on Orbit

The Soil Moisture Active–Passive (SMAP) L-band microwave radiometer is a conical scanning instrument designed to measure soil moisture with 4% volumetric accuracy at 40-km spatial resolution. SMAP is NASA’s first Earth Systematic Mission developed in response to its first Earth science decadal survey. Here, the design is reviewed and the results of its first year on orbit are presented. Unique features of the radiometer include a large 6-m rotating reflector, fully polarimetric radiometer receiver with internal calibration, and radio-frequency interference detection and filtering hardware. The radiometer electronics are thermally controlled to achieve good radiometric stability. Analyses of on-orbit results indicate that the electrical and thermal characteristics of the electronics and internal calibration sources are very stable and promote excellent gain stability. Radiometer NEDT < 1 K for 17-ms samples. The gain spectrum exhibits low noise at frequencies >1 MHz and 1/f noise rising at longer time scales fully captured by the internal calibration scheme. Results from sky observations and global swath imagery of all four Stokes antenna temperatures indicate that the instrument is operating as expected.

Design and performance of Astromesh reflector onboard Soil Moisture Active Passive spacecraft

The Soil Moisture Active Passive1 (SMAP) instrument includes a conically scanning 6-m diameter deployable Astromesh reflector and feedhorn that rotates relative to a de-spun spacecraft at 14.6 RPM. This is the first application of a spinning Astromesh reflector. This paper describes the design and performance of the Reflector/Boom Assembly (RBA) under multiple constraints and requirements that are inherent to a spinning large flexible reflector/structure. The deployed RBA has stringent mass property control and knowledge requirements, structural natural frequency separation requirements, and all other typical ones including the antenna performance. Finally the validation of the design on the ground by analysis/test and its difficulties due to gravity are discussed.

Preliminary Design of the Cruise, Entry, Descent, and Landing Mechanical Subsystem for MSL

Mars Science Laboratory is a scientific mission to the surface of Mars that would include a rover with 10 science instruments. In order to accomplish this mission, the rover must be transported from Earth to the Martian surface. The mechanical hardware that transports the rover is developed by the cruise, entry, descent, and landing (CEDL) mechanical subsystem team. This mechanical hardware includes the cruise stage structure, the aeroshell subsystem, the parachute deceleration subsystem, the descent stage structure, the bridle & umbilical device, and the pyro and separation devices that allow for the numerous separation events that occur in the EDL sequence. The key challenges for this system lie in the complex configuration (both geometric and subsystem accommodations), multiple unique load cases, and numerous separation events. This paper will describe the preliminary design and key challenges of each of these mechanical assemblies that comprise the CEDL mechanical subsystem.

MSL heatshield development: From failure to success

Mars Science Laboratory is a scientific mission that places a 900 kg rover with 10 science instruments on the surface of Mars. 12A key component for landing on the surface of Mars is the aeroshell, which decelerates the entry vehicle to just 1% of its initial kinetic energy at atmospheric entry. The forward portion of the aeroshell is the heatshield which must be designed to withstand the pressure, heating, and shear stresses it experiences during the atmospheric deceleration process. The original exterior material chosen for the heatshield, SLA-561V, was the material used on all previous Mars aeroshells but required new qualification testing. Unfortunately, the material failed the more extreme environment 3 months after the aeroshell and project Critical Design Reviews. A rapid decision was required to switch to another material under test by the Orion program call Phenolic Impregnated Carbon Ablator (PICA). With 18 months left to design, fabricate, test, and deliver this new heatshield for the MSL 2009 launch opportunity, the PICA heatshield team had to rapidly respond to the challenge and work in a manner that ensured success. This paper discusses the lessons learned from the original SLA-561V design implementation and the process used to ensure the success of the PICA design implementation.