Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “patch”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 577 records · Page 32
All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

Quality control and crop characterization framework for multi-temporal UAV LiDAR data over mechanized agricultural fields

Recent developments in remote sensing are enabling automatic, high resolution, and non-destructive survey of agriculture fields, providing the key basis for advancing plant breeding. Among the used remote sensing modalities, LiDAR has attracted wide attention for its ability to directly provide accurate 3D information. Despite the increasing utilization of LiDAR technology in phenotyping, there is still a lack of effective quality control strategies, in particular, quality control of LiDAR data collected on a multi-temporal basis. This study proposes a targetless framework for multi-temporal LiDAR data quality control and crop characterization in mechanized agricultural fields. Features extracted from the fields – terrain patches and row/alley locations – are utilized for evaluating the vertical and planimetric relative accuracy of the point clouds. Row/alley locations in the field are automatically identified from the point clouds based on the assumption that higher point density and/or higher elevation correspond to plant locations. The performance of the proposed quality control strategies is evaluated using multi-temporal datasets collected in agricultural fields of different sizes, orientation, crops, and growth stages. The result shows that the net vertical and planimetric discrepancies between multi-temporal point clouds are ±3 cm and ±8 cm, respectively. While the former reflects the actual accuracy of the point clouds, the latter is a combined effect of the LiDAR point cloud accuracy, rasterization artifacts, crop type, growth pattern, and wind condition during data acquisition. In terms of row and alley detection, the result shows that the proposed strategy achieves high performance and can deal with different planting orientation, crop types, growth stages, canopy cover, and planting density. In conclusion, this study presents a quality control framework for multi-temporal LiDAR data. Finally, the row and alley detection leads to automated extraction of plots, and hence facilitates the use of remotely sensed data for automated phenotyping.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

An ecologically-constrained deep learning model for tropical leaf phenology monitoring using PlanetScope satellites

In tropical forests, leaf phenology signals leaf-on/off status and exhibits considerable variability across scales from a single tree-crown to the entire forest ecosystem. Such phenology signals importantly regulate large-scale biogeochemical cycles and regional climate. PlanetScope CubeSats data with a 3-m resolution and near-daily global coverage provide an unprecedented opportunity to monitor both fine- and ecosystem-scale phenology variability along large environmental gradients. However, a scalable method that accurately characterizes leaf phenology from PlanetScope with biophysically meaningful metrics remains lacking. Here we developed an index-guided, ecologically constrained autoencoder (IG-ECAE) method to automatically derive a deciduousness metric (i.e. percentage of tree canopies with leaf-off status within an image pixel) from PlanetScope. The IG-ECAE first estimated the reflectance spectra of leafy/leafless canopies based on their spectral indices characteristics, then used the derived reflectance spectra to guide an autoencoder deep learning method with additional ecological constraints to refine the reflectance spectra, and finally used linear spectral unmixing to estimate the relative abundance of leafless canopies (or deciduousness) per PlanetScope image pixel. We tested the IG-ECAE method at 16 tropical forest sites spanning multiple continents and a large precipitation gradient (1470-2819 mm year -1 ). Among these sites, we evaluated the PlanetScope-derived deciduousness with corresponding measures derived from WorldView-2 (n=9 sites) and local phenocams (n=9 sites). Our results show that PlanetScope-derived deciduousness agrees: 1) with that derived from WorldView-2 at the patch level (90m×90m) with r 2 =0.89 across all sites; and 2) with that derived from phenocams to quantify ecosystem-scale seasonality with r 2 ranging from 0.62-0.96. These results demonstrate the effectiveness and scalability of IG-ECAE in characterizing the wide variability in deciduousness across scales from pixels to forest ecosystems, and from a single date to the full annual cycle, indicating the potential for using high-resolution satellites to track the large-scale phenological patterns and response of tropical forests to climate change.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2023

The stability of FeH x and hydrogen transport at Earth’s core mantle boundary

Iron hydride in Earth’s interior can be formed by the reaction between hydrous minerals (water) and iron. Studying iron hydride improves our understanding of hydrogen transportation in Earth’s interior. Our high-pressure experiments found that face-centered cubic (fcc) FeH x (x≤1) is stable up to 165 GPa, and our ab initio molecular dynamics simulations predicted that fcc FeH x transforms to a superionic state under lower mantle conditions. In the superionic state, H-ions in fcc FeH become highly diffusive-like fluids with a high diffusion coefficient of ~3.7 × 10 -4 cm 2 s -1 , which is comparable to that in the liquid Fe-H phase. The densities and melting temperatures of fcc FeH x were systematically calculated. Similar to superionic ice, the extra entropy of diffusive H-ions increases the melting temperature of fcc FeH. Further, the wide stability field of fcc FeH enables hydrogen transport into the outer core to create a potential hydrogen reservoir in Earth’s interior, leaving oxygen-rich patches (ORP) above the core mantle boundary (CMB).

58 GEOSCIENCES↗
DOE OSTI2018

Thermal impact of adhesive-mounted rooftop PV on underlying roof shingles

Adhesive mounting of residential rooftop photovoltaics (PV) is an alternative to traditional rack mounting that reduces installation costs. Adhesive mounting is fast, simple and reduces the need for skilled labor. In our novel design that further reduces the installation costs, a lightweight (glassless and frameless) PV module is directly adhered to a shingled roof using an adhesive tape, creating a <5 mm air gap between the PV back-panel and the roof shingle surface. Although the gap is sufficient for moisture and rainwater transport under the PV panel, potential heat buildup under the module may adversely impact the long-term durability of the shingles. Heat buildup may also increase the heat flux through the roof, resulting in an overall increase in building cooling loads. This study investigates the thermal behavior of the roof under an adhered PV system. Two identical test huts with dark shingle-covered roofs were located in the hot, desert climate of Albuquerque, NM. Adhesively-mounted lightweight PV modules were installed on the south-facing roof of one of the test huts (PV hut), with the other one serving as a reference hut. During the summer season, the asphalt roof shingles under the PV modules experienced a 13 °C reduction in daytime peak temperature compared with the exposed shingles. No evidence of heat buildup under the PV module was observed. It was also found that the temperature of shingles underneath the adhesive was up to 6 °C higher than for shingles underneath the gap space at the daily peak time. Thin but ventilated air gap between the PV back-panel and the roof shingles helped remove the heat, while the adhesive pads (patches) served as thermal bridges between the PV module and the roof. Daily peak heat flow through the attic ceiling was almost 49% lower in the PV hut compared to the reference hut. These results show no evidence of an adverse thermal impact of the adhesive-mounted PV system on the roofing materials, while demonstrating a potential for a notable reduction in space conditioning energy requirements.

14 SOLAR ENERGY↗
DOE OSTI2025

Tomographic assessment of peening for metallurgical bonding in cold spray

It is widely asserted that peening densifies cold spray coatings and improves particle–particle and particle–substrate bonding, yet systematic studies analyzing the evolution of bonding due to peening particle strikes are scarce. We explore peening at the level of individual particle impacts, by creating two-particle Cu-on-Cu stacks using laser-induced particle impact testing. Exploring impact velocities that range from the onset of bonding at the “critical velocity”, V cr , to its peak near 1.4⋅V cr , we perform full 3D tomography of the particle stacks to characterize the bonding state at the particle-substrate interface. Direct labeling of non-bonded patches and the total particle-substrate interface permits quantification and comparison of bonding in single impacts with bonding after subsequent impacts to reveal the origins of the peening effect. The data reveal a surprising finding: although a second impact directly atop the first particle increases the area of the particle-substrate interface (~40% area increase), there is no improvement in metallurgical bonding. Analysis of these data suggest that not only area expansion (and associated oxide rarefication), but also stress-induced extrusion of metal at that interface needs to occur to achieve metal-on-metal contact to see an enhancement in bonding. Furthermore, our analysis suggests that the subsequent impacting particle needs to have ~70% more kinetic energy to produce additional bonding; this implies that particle and velocity distributions within a spray may be helpful, and that peening may rely on unaligned impacts.

Bonding↗
DOE OSTI2014

A novel assessment of nefazodone-induced hERG inhibition by electrophysiological and stereochemical method

Nefazodone was used widely as an antidepressant until it was withdrawn from the U.S. market in 2004 due to hepatotoxicity. We have investigated methods to predict various toxic effects of drug candidates to reduce the failure rate of drug discovery. An electrophysiological method was used to assess the cardiotoxicity of drug candidates. Small molecules, including withdrawn drugs, were evaluated using a patch-clamp method to establish a database of hERG inhibition. Nefazodone inhibited hERG channel activity in our system. However, nefazodone-induced hERG inhibition indicated only a theoretical risk of cardiotoxicity. Nefazodone inhibited the hERG channel in a concentration-dependent manner with an IC{sub 50} of 45.3 nM in HEK-293 cells. Nefazodone accelerated both the recovery from inactivation and its onset. Nefazodone also accelerated steady-state inactivation, although it did not modify the voltage-dependent character. Alanine mutants of hERG S6 and pore region residues were used to identify the nefazodone-binding site on hERG. The hERG S6 point mutants Y652A and F656A largely abolished the inhibition by nefazodone. The pore region mutant S624A mildly reduced the inhibition by nefazodone but T623A had little effect. A docking study showed that the aromatic rings of nefazodone interact with Y652 and F656 via π–π interactions, while an amine interacted with the S624 residue in the pore region. In conclusion, Y652 and F656 in the S6 domain play critical roles in nefazodone binding. - Highlights: • Nefazodone inhibits hERG channels with an IC{sub 50} of 45.3 nM in HEK-293 cells. • Nefazodone blocks hERG channels by binding to the open channels. • Y652 and F656 are important for binding of nefazodone. • The aromatic rings of nefazodone interact with Y652 and F656 via π–π interactions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

A novel dihydropyridine with 3-aryl meta-hydroxyl substitution blocks L-type calcium channels in rat cardiomyocytes

Rationale: Dihydropyridines are widely used for the treatment of several cardiac diseases due to their blocking activity on L-type Ca{sup 2+} channels and their renowned antioxidant properties. Methods: We synthesized six novel dihydropyridine molecules and performed docking studies on the binding site of the L-type Ca{sup 2+} channel. We used biochemical techniques on isolated adult rat cardiomyocytes to assess the efficacy of these molecules on their Ca{sup 2+} channel-blocking activity and antioxidant properties. The Ca{sup 2+} channel-blocking activity was evaluated by confocal microscopy on fluo-3AM loaded cardiomyocytes, as well as using patch clamp experiments. Antioxidant properties were evaluated by flow cytometry using the ROS sensitive dye 1,2,3 DHR. Results: Our docking studies show that a novel compound with 3-OH substitution inserts into the active binding site of the L-type Ca{sup 2+} channel previously described for nitrendipine. In biochemical assays, the novel meta-OH group in the aryl in C4 showed a high blocking effect on L-type Ca{sup 2+} channel as opposed to para-substituted compounds. In the tests we performed, none of the molecules showed antioxidant properties. Conclusions: Only substitutions in C2, C3 and C5 of the aryl ring render dihydropyridine compounds with the capacity of blocking LTCC. Based on our docking studies, we postulate that the antioxidant activity requires a larger group than the meta-OH substitution in C2, C3 or C5 of the dihydropyridine ring. - Highlights: • Dihydropyridine (DHP) molecules are widely used in cardiovascular disease. • DHPs block Ca{sup 2+} entry through LTCC—some DHPs have antioxidant activity as well. • We synthesized 6 new DHPs and tested their Ca{sup 2+} blocking and antioxidant activities. • 3-Aryl meta-hydroxyl substitution strongly increases their Ca{sup 2+} blocking activity. • 3-Aryl meta-hydroxyl substitution did not affect the antioxidant properties.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Predicting changes in cardiac myocyte contractility during early drug discovery with in vitro assays

Cardiovascular-related adverse drug effects are a major concern for the pharmaceutical industry. Activity of an investigational drug at the L-type calcium channel could manifest in a number of ways, including changes in cardiac contractility. The aim of this study was to define which of the two assay technologies – radioligand-binding or automated electrophysiology – was most predictive of contractility effects in an in vitro myocyte contractility assay. The activity of reference and proprietary compounds at the L-type calcium channel was measured by radioligand-binding assays, conventional patch-clamp, automated electrophysiology, and by measurement of contractility in canine isolated cardiac myocytes. Activity in the radioligand-binding assay at the L-type Ca channel phenylalkylamine binding site was most predictive of an inotropic effect in the canine cardiac myocyte assay. The sensitivity was 73%, specificity 83% and predictivity 78%. The radioligand-binding assay may be run at a single test concentration and potency estimated. The least predictive assay was automated electrophysiology which showed a significant bias when compared with other assay formats. Given the importance of the L-type calcium channel, not just in cardiac function, but also in other organ systems, a screening strategy emerges whereby single concentration ligand-binding can be performed early in the discovery process with sufficient predictivity, throughput and turnaround time to influence chemical design and address a significant safety-related liability, at relatively low cost. - Highlights: • The L-type calcium channel is a significant safety liability during drug discovery. • Radioligand-binding to the L-type calcium channel can be measured in vitro. • The assay can be run at a single test concentration as part of a screening cascade. • This measurement is highly predictive of changes in cardiac myocyte contractility.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2015

The calmodulin inhibitor CGS 9343B inhibits voltage-dependent K{sup +} channels in rabbit coronary arterial smooth muscle cells

We investigated the effects of the calmodulin inhibitor CGS 9343B on voltage-dependent K{sup +} (Kv) channels using whole-cell patch clamp technique in freshly isolated rabbit coronary arterial smooth muscle cells. CGS 9343B inhibited Kv currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC{sub 50}) value of 0.81 μM. The decay rate of Kv channel inactivation was accelerated by CGS 9343B. The rate constants of association and dissociation for CGS 9343B were 2.77 ± 0.04 μM{sup −1} s{sup −1} and 2.55 ± 1.50 s{sup −1}, respectively. CGS 9343B did not affect the steady-state activation curve, but shifted the inactivation curve toward to a more negative potential. Train pulses (1 or 2 Hz) application progressively increased the CGS 9343B-induced Kv channel inhibition. In addition, the inactivation recovery time constant was increased in the presence of CGS 9343B, suggesting that CGS 9343B-induced inhibition of Kv channel was use-dependent. Another calmodulin inhibitor, W-13, did not affect Kv currents, and did not change the inhibitory effect of CGS 9343B on Kv current. Our results demonstrated that CGS 9343B inhibited Kv currents in a state-, time-, and use-dependent manner, independent of calmodulin inhibition. - Highlights: • We investigated the effects of CGS 9394B on Kv channels. • CGS 9394B inhibited Kv current in a state-, time-, and use-dependent manner. • Caution is required when using CGS 9394B in vascular function studies.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Evaluation of nefazodone-induced cardiotoxicity in human induced pluripotent stem cell-derived cardiomyocytes

The recent establishment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), which express the major cardiac ion channels and recapitulate spontaneous mechanical and electrical activities, may provide a possible solution for the lack of in vitro human-based cardiotoxicity testing models. Cardiotoxicity induced by the antidepressant nefazodone was previously revealed to cause an acquired QT prolongation by hERG channel blockade. To elucidate the cellular mechanisms underlying the cardiotoxicity of nefazodone beyond hERG, its effects on cardiac action potentials (APs) and ion channels were investigated using hiPSC-CMs with whole-cell patch clamp techniques. In a proof of principle study, we examined the effects of cardioactive channel blockers on the electrophysiological profile of hiPSC-CMs in advance of the evaluation of nefazodone. Nefazodone dose-dependently prolonged the AP duration at 90% (APD{sub 90}) and 50% (APD{sub 50}) repolarization, reduced the maximum upstroke velocity (dV/dt{sub max}) and induced early after depolarizations. Voltage-clamp studies of hiPSC-CMs revealed that nefazodone inhibited various voltage-gated ion channel currents including I{sub Kr}, I{sub Ks}, I{sub Na}, and I{sub Ca}. Among them, I{sub Kr} and I{sub Na} showed relatively higher sensitivity to nefazodone, consistent with the changes in the AP parameters. In summary, hiPSC-CMs enabled an integrated approach to evaluate the complex interactions of nefazodone with cardiac ion channels. These results suggest that hiPSC-CMs can be an effective model for detecting drug-induced arrhythmogenicity beyond the current standard assay of heterologously expressed hERG K{sup +} channels. - Highlights: • Nefazodone prolonged APD and decreased upstroke velocity of APs in hiPSC-CMs. • Nefazodone inhibited cardiac ion channels, especially I{sub Kr} and I{sub Na}, in hiPSC-CMs. • Nefazodone-induced AP changes are mainly the result of I{sub Kr} and I{sub Na} inhibition. • hiPSC-CMs are sensitive model to validate nefazodone-induced cardiotoxicity. • hiPSC-CMs provide an integrated approach for evaluating mechanism of drug actions.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Inhibition by ketamine and amphetamine analogs of the neurogenic nitrergic vasodilations in porcine basilar arteries

The abuse of ketamine and amphetamine analogs is associated with incidence of hypertension and strokes involving activation of sympathetic activities. Large cerebral arteries at the base of the brain from several species receive dense sympathetic innervation which upon activation causes parasympathetic-nitrergic vasodilation with increased regional blood flow via axo-axonal interaction mechanism, serving as a protective mechanism to meet O{sub 2} demand in an acutely stressful situation. The present study was designed to examine effects of ketamine and amphetamine analogs on axo-axonal interaction-mediated neurogenic nitrergic vasodilation in porcine basilar arteries using techniques of blood-vessel myography, patch clamp and two-electrode voltage clamp, and calcium imaging. In U46619-contracted basilar arterial rings, nicotine (100 μM) and electrical depolarization of nitrergic nerves by transmural nerve stimulation (TNS, 8 Hz) elicited neurogenic nitrergic vasodilations. Ketamine and amphetamine analogs concentration-dependently inhibited nicotine-induced parasympathetic-nitrergic vasodilation without affecting that induced by TNS, nitroprusside or isoproterenol. Ketamine and amphetamine analogs also concentration-dependently blocked nicotine-induced inward currents in Xenopus oocytes expressing α3β2-nicotinic acetylcholine receptors (nAChRs), and nicotine-induced inward currents as well as calcium influxes in rat superior cervical ganglion neurons. The potency in inhibiting both inward-currents and calcium influxes is ketamine > methamphetamine > hydroxyamphetamine. These results indicate that ketamine and amphetamine analogs, by blocking nAChRs located on cerebral perivascular sympathetic nerves, reduce nicotine-induced, axo-axonal interaction mechanism-mediated neurogenic dilation of the basilar arteries. Chronic abuse of these drugs, therefore, may interfere with normal sympathetic-parasympathetic interaction mechanism resulting in diminished neurogenic vasodilation and, possibly, normal blood flow in the brainstem. - Highlights: • Ketamine/amphetamines inhibit nicotine-induced cerebral neurogenic vasdilation. • Ketamine/amphetamines block cerebral perivascular sympathetic nAChR-mediated current. • The inhibitory potency is ketamine > D-amphetamine > methamphetamine > OH-amphetamine.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Assessment of drug-induced arrhythmic risk using limit cycle and autocorrelation analysis of human iPSC-cardiomyocyte contractility

Cardiac safety assays incorporating label-free detection of human stem-cell derived cardiomyocyte contractility provide human relevance and medium throughput screening to assess compound-induced cardiotoxicity. In an effort to provide quantitative analysis of the large kinetic datasets resulting from these real-time studies, we applied bioinformatic approaches based on nonlinear dynamical system analysis, including limit cycle analysis and autocorrelation function, to systematically assess beat irregularity. The algorithms were integrated into a software program to seamlessly generate results for 96-well impedance-based data. Our approach was validated by analyzing dose- and time-dependent changes in beat patterns induced by known proarrhythmic compounds and screening a cardiotoxicity library to rank order compounds based on their proarrhythmic potential. We demonstrate a strong correlation for dose-dependent beat irregularity monitored by electrical impedance and quantified by autocorrelation analysis to traditional manual patch clamp potency values for hERG blockers. In addition, our platform identifies non-hERG blockers known to cause clinical arrhythmia. Our method provides a novel suite of medium-throughput quantitative tools for assessing compound effects on cardiac contractility and predicting compounds with potential proarrhythmia and may be applied to in vitro paradigms for pre-clinical cardiac safety evaluation. - Highlights: • Impedance-based monitoring of human iPSC-derived cardiomyocyte contractility • Limit cycle analysis of impedance data identifies aberrant oscillation patterns. • Nonlinear autocorrelation function quantifies beat irregularity. • Identification of hERG and non-hERG inhibitors with known risk of arrhythmia • Automated software processes limit cycle and autocorrelation analyses of 96w data.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Using DR52c/Ni{sup 2+} mimotope tetramers to detect Ni{sup 2+} reactive CD4{sup +} T cells in patients with joint replacement failure

T cell mediated hypersensitivity to nickel (Ni{sup 2+}) is one of the most common causes of allergic contact dermatitis. Ni{sup 2+} sensitization may also contribute to the failure of Ni{sup 2+} containing joint implants, and revision to non-Ni{sup 2+} containing hardware can be costly and debilitating. Previously, we identified Ni{sup 2+} mimotope peptides, which are reactive to a CD4{sup +} T cell clone, ANi2.3 (Vα1, Vβ17), isolated from a Ni{sup 2+} hypersensitive patient with contact dermatitis. This T cell is restricted to the major histocompatibility complex class II (MHCII) molecule, Human Leukocyte Antigen (HLA)-DR52c (DRA, DRB3*0301). However, it is not known if Ni{sup 2+} induced T cell responses in sensitized joint replacement failure patients are similar to subjects with Ni{sup 2+} induced contact dermatitis. Here, we generated DR52c/Ni{sup 2+} mimotope tetramers, and used them to test if the same Ni{sup 2+} T cell activation mechanism could be generalized to Ni{sup 2+} sensitized patients with associated joint implant failure. We confirmed the specificity of these tetramers by staining of ANi2.3 T cell transfectomas. The DR52c/Ni{sup 2+} mimotope tetramer detected Ni{sup 2+} reactive CD4{sup +} T cells in the peripheral blood mononuclear cells (PBMC) of patients identified as Ni{sup 2+} sensitized by patch testing and a positive Ni{sup 2+} LPT. When HLA-typed by a DR52 specific antibody, three out of four patients were DR52 positive. In one patient, Ni{sup 2+} stimulation induced the expansion of Vβ17 positive CD4{sup +} T cells from 0.8% to 13.3%. We found that the percentage of DR52 positivity and Vβ17 usage in Ni{sup 2+} sensitized joint failure patients are similar to Ni sensitized skin allergy patients. Ni{sup 2+} independent mimotope tetramers may be a useful tool to identify the Ni{sup 2+} reactive CD4{sup +} T cells. - Highlight: • Nickel hypersensitive patients with implant failure are dominantly DR52 positive. • These patients may have similar TCR beta chain usage to that of contact dermatitis. • The nickel mimotope tetramers are a tool to identify the nickel reactive CD4{sup +} T cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2017

Modulation of the heart's electrical properties by the anticonvulsant drug retigabine

Retigabine, currently used as antiepileptic drug, has a wide range of potential medical uses. Administration of the drug in patients can lead to QT interval prolongation in the electrocardiogram and to cardiac arrhythmias in rare cases. This suggests that the drug may perturb the electrical properties of the heart, and the underlying mechanisms were investigated here. Effects of retigabine on currents through human cardiac ion channels, heterologously expressed in tsA-201 cells, were studied in whole-cell patch-clamp experiments. In addition, the drug's impact on the cardiac action potential was tested. This was done using ventricular cardiomyocytes isolated from Langendorff-perfused guinea pig hearts and cardiomyocytes derived from human induced pluripotent stem cells. Further, to unravel potential indirect effects of retigabine on the heart which might involve the autonomic nervous system, membrane potential and noradrenaline release from sympathetic ganglionic neurons were measured in the absence and presence of the drug. Retigabine significantly inhibited currents through hKv11.1 potassium, hNav1.5 sodium, as well as hCav1.2 calcium channels, but only in supra-therapeutic concentrations. In a similar concentration range, the drug shortened the action potential in both guinea pig and human cardiomyocytes. Therapeutic concentrations of retigabine, on the other hand, were sufficient to inhibit the activity of sympathetic ganglionic neurons. We conclude that retigabine- induced QT interval prolongation, and the reported cases of cardiac arrhythmias after application of the drug in a typical daily dose range, cannot be explained by a direct modulatory effect on cardiac ion channels. They are rather mediated by indirect actions at the level of the autonomic nervous system. - Highlights: • We study effects of anticonvulsant retigabine on the heart's electrical properties. • Retigabine does not modulate cardiac ion channels in therapeutic concentrations. • The activity of sympathetic ganglionic neurons is inhibited by retigabine. • Ganglionic block may generate QT interval prolongation and cardiac arrhythmias.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

A systematic strategy for estimating hERG block potency and its implications in a new cardiac safety paradigm

Introduction: hERG block potency is widely used to calculate a drug's safety margin against its torsadogenic potential. Previous studies are confounded by use of different patch clamp electrophysiology protocols and a lack of statistical quantification of experimental variability. Since the new cardiac safety paradigm being discussed by the International Council for Harmonisation promotes a tighter integration of nonclinical and clinical data for torsadogenic risk assessment, a more systematic approach to estimate the hERG block potency and safety margin is needed. Methods: A cross-industry study was performed to collect hERG data on 28 drugs with known torsadogenic risk using a standardized experimental protocol. A Bayesian hierarchical modeling (BHM) approach was used to assess the hERG block potency of these drugs by quantifying both the inter-site and intra-site variability. A modeling and simulation study was also done to evaluate protocol-dependent changes in hERG potency estimates. Results: A systematic approach to estimate hERG block potency is established. The impact of choosing a safety margin threshold on torsadogenic risk evaluation is explored based on the posterior distributions of hERG potency estimated by this method. The modeling and simulation results suggest any potency estimate is specific to the protocol used. Discussion: This methodology can estimate hERG block potency specific to a given voltage protocol. The relationship between safety margin thresholds and torsadogenic risk predictivity suggests the threshold should be tailored to each specific context of use, and safety margin evaluation may need to be integrated with other information to form a more comprehensive risk assessment.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2014

Functional analysis of the C-terminal region of human adenovirus E1A reveals a misidentified nuclear localization signal

The immortalizing function of the human adenovirus 5 E1A oncoprotein requires efficient localization to the nucleus. In 1987, a consensus monopartite nuclear localization sequence (NLS) was identified at the C-terminus of E1A. Since that time, various experiments have suggested that other regions of E1A influence nuclear import. In addition, a novel bipartite NLS was recently predicted at the C-terminal region of E1A in silico. In this study, we used immunofluorescence microscopy and co-immunoprecipitation analysis with importin-α to verify that full nuclear localization of E1A requires the well characterized NLS spanning residues 285–289, as well as a second basic patch situated between residues 258 and 263 ({sup 258}RVGGRRQAVECIEDLLNEPGQPLDLSCKRPRP{sup 289}). Thus, the originally described NLS located at the C-terminus of E1A is actually a bipartite signal, which had been misidentified in the existing literature as a monopartite signal, altering our understanding of one of the oldest documented NLSs. - Highlights: • Human adenovirus E1A is localized to the nucleus. • The C-terminus of E1A contains a bipartite nuclear localization signal (NLS). • This signal was previously misidentified to be a monopartite NLS. • Key basic amino acid residues within this sequence are highly conserved.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Polymer microfiber meshes facilitate cardiac differentiation of c-kit{sup +} human cardiac stem cells

Electrospun microfiber meshes have been shown to support the proliferation and differentiation of many types of stem cells, but the phenotypic fate of c-kit{sup +} human cardiac stem cells (hCSCs) have not been explored. To this end, we utilized thin (~5 µm) elastomeric meshes consisting of aligned 1.7 µm diameter poly (ester-urethane urea) microfibers as substrates to examine their effect on hCSC viability, morphology, proliferation, and differentiation relative to cells cultured on tissue culture polystyrene (TCPS). The results showed that cells on microfiber meshes displayed an elongated morphology aligned in the direction of fiber orientation, lower proliferation rates, but increased expressions of genes and proteins majorly associated with cardiomyocyte phenotype. The early (NK2 homeobox 5, Nkx2.5) and late (cardiac troponin I, cTnI) cardiomyocyte genes were significantly increased on meshes (Nkx=2.5 56.2±13.0, cTnl=2.9±0.56,) over TCPS (Nkx2.5=4.2±0.9, cTnl=1.6±0.5, n=9, p<0.05 for both groups) after differentiation. In contrast, expressions of smooth muscle markers, Gata6 and myosin heavy chain (SM-MHC), were decreased on meshes. Immunocytochemical analysis with cardiac antibody exhibited the similar pattern of above cardiac differentiation. We conclude that aligned microfiber meshes are suitable for guiding cardiac differentiation of hCSCs and may facilitate stem cell-based therapies for treatment of cardiac diseases. - Highlights: • First study to characterize c-kit{sup +} human cardiac stem cells on microfiber meshes. • Microfiber meshes seem reducing cell proliferation, but no effect on cell viability. • Microfiber meshes facilitate the elongation of human cardiac stem cells in culture. • Cardiac but not smooth muscle differentiation were enhanced on microfiber meshes. • Microfiber meshes may be used as cardiac patches in cell-based cardiac therapy.

60 APPLIED LIFE SCIENCES↗