High-Accuracy Prediction of Stabilizing Surface Mutations to the Three-Helix Bundle, UBA(1), with EmCAST.

The accurate modeling of energetic contributions to protein structure is a fundamental challenge in computational approaches to protein analysis and design. We describe a general computational method, EmCAST (empirical Cα stabilization), to score and optimize the sequence to the structure in proteins. The method relies on an empirical potential derived from the database of the Cα dihedral angle preferences for all possible four-residue sequences, using the data available in the Protein Data Bank. Our method produces stability predictions that naturally correlate one-to-one with the experimental results for solvent-exposed mutation sites. EmCAST predicted four mutations that increased the stability of a three-helix bundle, UBA(1), from 2.4 to 4.8 kcal/mol by optimizing residues in both helices and turns. For a set of eight variants, the predicted and experimental stabilizations correlate very well (R2 = 0.97) with a slope near 1 and with a 0.16 kcal/mol standard error for EmCAST predictions. Tests against literature data for the stability effects of surface-exposed mutations show that EmCAST outperforms the existing stability prediction methods. UBA(1) variants were crystallized to verify and analyze their structures at an atomic resolution. Thermodynamic and kinetic folding experiments were performed to determine the magnitude and mechanism of stabilization. Our method has the potential to enable the rapid, rational optimization of natural proteins, expand the analysis of the sequence/structure relationship, and supplement the existing protein design strategies.

c-di-GMP regulates activity of the PlzA RNA chaperone from the Lyme disease spirochete.

PlzA is a c-di-GMP-binding protein crucial for adaptation of the Lyme disease spirochete Borrelia (Borreliella) burgdorferi during its enzootic life cycle. Unliganded apo-PlzA is important for vertebrate infection, while liganded holo-PlzA is important for survival in the tick; however, the biological function of PlzA has remained enigmatic. Here, we report that PlzA has RNA chaperone activity that is inhibited by c-di-GMP binding. Holo- and apo-PlzA bind RNA and accelerate RNA annealing, while only apo-PlzA can strand displace and unwind double-stranded RNA. Guided by the crystal structure of PlzA, we identified several key aromatic amino acids protruding from the N- and C-terminal domains that are required for RNA-binding and unwinding activity. Our findings illuminate c-di-GMP as a switch controlling the RNA chaperone activity of PlzA, and we propose that complex RNA-mediated modulatory mechanisms allow PlzA to regulate gene expression during both the vector and host phases of the B. burgdorferi life cycle.

Residual Structure in the Denatured State of the Fast-Folding UBA(1) Domain from the Human DNA Excision Repair Protein HHR23A.

The structure of the first ubiquitin-associated domain from HHR23A, UBA(1), was determined by X-ray crystallography at a 1.60 Å resolution, and its stability, folding kinetics, and residual structure under denaturing conditions have been investigated. The concentration dependence of thermal denaturation and size-exclusion chromatography indicate that UBA(1) is monomeric. Guanidine hydrochloride (GdnHCl) denaturation experiments reveal that the unfolding free energy, ΔGu°'(H2O), of UBA(1) is 2.4 kcal mol-1. Stopped-flow folding kinetics indicates sub-millisecond folding with only proline isomerization phases detectable at 25 °C. The full folding kinetics are observable at 4 °C, yielding a folding rate constant, kf, in the absence of a denaturant of 13,000 s-1 and a Tanford ß-value of 0.80, consistent with a compact transition state. Evaluation of the secondary structure via circular dichroism shows that the residual helical structure in the denatured state is replaced by polyproline II structure as the GdnHCl concentration increases. Analysis of NMR secondary chemical shifts for backbone 15NH, 13CO, and 13Cα atoms between 4 and 7 M GdnHCl shows three islands of residual helical secondary structure that align in sequence with the three native-state helices. Extrapolation of the NMR data to 0 M GdnHCl demonstrates that helical structure would populate to 17-33% in the denatured state under folding conditions. Comparison with NMR data for a peptide corresponding to helix 1 indicates that this helix is stabilized by transient tertiary interactions in the denatured state of UBA(1). The high helical content in the denatured state, which is enhanced by transient tertiary interactions, suggests a diffusion-collision folding mechanism.

Breath Acetone Sensing Based on Single-Walled Carbon Nanotube-Titanium Dioxide Hybrids Enabled by a Custom-Built Dehumidifier.

Acetone is a metabolic byproduct found in the exhaled breath and can be measured to monitor the metabolic degree of ketosis. In this state, the body uses free fatty acids as its main source of fuel because there is limited access to glucose. Monitoring ketosis is important for type I diabetes patients to prevent ketoacidosis, a potentially fatal condition, and individuals adjusting to a low-carbohydrate diet. Here, we demonstrate that a chemiresistor fabricated from oxidized single-walled carbon nanotubes functionalized with titanium dioxide (SWCNT@TiO2) can be used to detect acetone in dried breath samples. Initially, due to the high cross sensitivity of the acetone sensor to water vapor, the acetone sensor was unable to detect acetone in humid gas samples. To resolve this cross-sensitivity issue, a dehumidifier was designed and fabricated to dehydrate the breath samples. Sensor response to the acetone in dried breath samples from three volunteers was shown to be linearly correlated with the two other ketone bodies, acetoacetic acid in urine and ß-hydroxybutyric acid in the blood. The breath sampling and analysis methodology had a calculated acetone detection limit of 1.6 ppm and capable of detecting up to at least 100 ppm of acetone, which is the dynamic range of breath acetone for someone with ketosis. Finally, the application of the sensor as a breath acetone detector was studied by incorporating the sensor into a handheld prototype breathalyzer.

Tetrahydrocannabinol Detection Using Semiconductor-Enriched Single-Walled Carbon Nanotube Chemiresistors.

Semiconductor-enriched single-walled carbon nanotubes (s-SWCNTs) have potential for application as a chemiresistor for the detection of breath compounds, including tetrahydrocannabinol (THC), the main psychoactive compound found in the marijuana plant. Herein we show that chemiresistor devices fabricated from s-SWCNT ink using dielectrophoresis can be incorporated into a hand-held breathalyzer with sensitivity toward THC generated from a bubbler containing analytical standard in ethanol and a heated sample evaporator that releases compounds from steel wool. The steel wool was used to capture THC from exhaled marijuana smoke. The generation of the THC from the bubbler and heated breath sample chamber was confirmed using ultraviolet-visible absorption spectroscopy and mass spectrometry, respectively. Enhanced selectivity toward THC over more volatile breath components such as CO2, water, ethanol, methanol, and acetone was achieved by delaying the sensor reading to allow for the desorption of these compounds from the chemiresistor surface. Additionally, machine learning algorithms were utilized to improve the selective detection of THC with better accuracy at increasing quantities of THC delivered to the chemiresistor.

Automatic Early-Onset Free Flap Failure Detection for Implantable Biomedical Devices.

OBJECTIVE: Up to 10% of free flap cases are compromised, and without prompt intervention, amputation and even death can occur. Hourly monitoring improves salvage rates, but the gold standard for monitoring requires experienced personnel to operate and suffers from high false-positive rates as high as 31% that result in costly and unnecessary surgeries. In this paper, we investigate free flap patency monitoring using automatic hardware-only classification systems that eliminate the need for experienced personnel. The expected flow ranges of the antegrade and retrograde veins for breast reconstruction are studied using a syringe pump to create the laminar flow seen in veins. METHODS: Feature data extracted from the Doppler blood flow signals are analyzed for sensitivity, specificity, and false-positive rates. Hardware is built to perform the classification automatically in real-time and output a decision at the end of the observation period. RESULTS: Experimental results using the hardware-only classifier for a 50 ms window size show high sensitivity (96.75%), specificity (90.20%), and low false-positive rate (9.803%). The experimental and theoretical classification results show close agreement. CONCLUSION: This work indicates that automatic hardware-only classifiers can eliminate the need for experienced personnel to monitor free flap patency. SIGNIFICANCE: The hardware-only classification is amenable to a monolithic implementation and future studies should study a totally implantable wirelessly-powered blood flow classifier. The high classifier performance in a short window period indicates that duty-cycled powering can be used to extend the safe operational depth of an implant. This is particularly relevant for the difficult buried free flap applications.

Totally Implantable Wireless Ultrasonic Doppler Blood Flowmeters: Toward Accurate Miniaturized Chronic Monitors.

Totally implantable wireless ultrasonic blood flowmeters provide direct-access chronic vessel monitoring in hard-to-reach places without using wired bedside monitors or imaging equipment. Although wireless implantable Doppler devices are accurate for most applications, device size and implant lifetime remain vastly underdeveloped. We review past and current approaches to miniaturization and implant lifetime extension for wireless implantable Doppler devices and propose approaches to reduce device size and maximize implant lifetime for the next generation of devices. Additionally, we review current and past approaches to accurate blood flow measurements. This review points toward relying on increased levels of monolithic customization and integration to reduce size. Meanwhile, recommendations to maximize implant lifetime should include alternative sources of power, such as transcutaneous wireless power, that stand to extend lifetime indefinitely. Coupling together the results will pave the way for ultra-miniaturized totally implantable wireless blood flow monitors for truly chronic implantation.

A System for Simple Real-Time Anastomotic Failure Detection and Wireless Blood Flow Monitoring in the Lower Limbs.

Current totally implantable wireless blood flow monitors are large and cannot operate alongside nearby monitors. To alleviate the problems with the current monitors, we developed a system to monitor blood flow wirelessly, with a simple and easily interpretable real-time output. To the best of our knowledge, the implanted electronics are the smallest in reported literature, which reduces bio-burden. Calibration was performed across realistic physiological flow ranges using a syringe pump. The device's sensors connected directly to the bilateral femoral veins of swine. For each 1 min, blood flow was monitored, then, an occlusion was introduced, and then, the occlusion was removed to resume flow. Each vein of four pigs was monitored four times, totaling 32 data collections. The implant measured 1.70 cm3 without battery/encapsulation. Across its calibrated range, including equipment tolerances, the relative error is less than ±5% above 8 mL/min and between -0.8% and +1.2% at its largest calibrated flow rate, which to the best of our knowledge is the lowest reported in the literature across the measured calibration range. The average standard deviation of the flow waveform amplitude was three times greater than that of no-flow. Establishing the relative amplitude for the flow and no-flow waveforms was found necessary, particularly for noise modulated Doppler signals. Its size and accuracy, compared with other microcontroller-equipped totally implantable monitors, make it a good candidate for future tether-free free flap monitoring studies.

The Development of a Wireless Implantable Blood Flow Monitor.

Microvascular anastomotic failure remains an uncommon but devastating problem. Although the implantable Doppler probe is helpful in flap monitoring, the devices are cumbersome, easily dislodged, and plagued by false-positive results. The authors have developed an implantable wireless Doppler monitor prototype from off-the-shelf components and tested it in a swine model. The wireless probe successfully distinguished between femoral vein flow, occlusion, and reflow, and wirelessly reported the different signals reliably. This is the first description of a wireless implantable blood flow sensor for flap monitoring. Future iterations will incorporate an integrated microchip-based Doppler system that will decrease the size to 1 mm, small enough to fit onto an anastomotic coupler.

Innovation and translation efforts in wireless medical connectivity, telemedicine and eMedicine: a story from the RFID Center of Excellence at the University of Pittsburgh.

Translational research has recently been rediscovered as one of the basic tenants of engineering. Although many people have numerous ideas of how to accomplish this successfully, the fundamental method is to provide an innovative and creative environment. The University of Pittsburgh has been accomplishing this goal though a variety of methodologies. The contents of this paper are exemplary of what can be achieved though the interaction of students, staff, faculty and, in one example, high school teachers. While the projects completed within the groups involved in this paper have spanned other areas, the focus of this paper is on the biomedical devices, that is, towards improving and maintaining health in a variety of areas. The spirit of the translational research is discovery, invention, intellectual property protection, and the creation of value through the spinning off of companies while providing better health care and creating jobs. All but one of these projects involve wireless radio frequency (RF) energy for delivery. The remaining device can be wirelessly connected for data collection.