Live-Cell Cardiac-Specific High-Throughput Screening Platform for Drug-Like Molecules that Enhance Ca2+ Transport.

We engineered a concatenated fluorescent biosensor and dual-wavelength fluorescence lifetime (FLT) detection, to perform high-throughput screening (HTS) in living cells for discovery of potential heart-failure drugs. Heart failure is correlated with insufficient activity of the sarcoplasmic reticulum Ca-pump (SERCA2a), often due to excessive inhibition by phospholamban (PLB), a small transmembrane protein. We sought to discover small molecules that restore SERCA2a activity by disrupting this inhibitory interaction between PLB and SERCA2a. Our approach was to fluorescently tag the two proteins and measure fluorescence resonance energy transfer (FRET) to detect changes in binding or structure of the complex. To optimize sensitivity to these changes, we engineered a biosensor that concatenates the two fluorescently labeled proteins on a single polypeptide chain. This SERCA2a-PLB FRET biosensor construct is functionally active and effective for HTS. By implementing 2-wavelength FLT detection at extremely high speed during primary HTS, we culled fluorescent compounds as false-positive Hits. In pilot screens, we identified Hits that alter the SERCA2a-PLB interaction, and a newly developed secondary calcium uptake assay revealed both activators and inhibitors of Ca-transport. We are implementing this approach for large-scale screens to discover new drug-like modulators of SERCA2a-PLB interactions for heart failure therapeutic development.

Trajectory-Based Simulation of EPR Spectra: Models of Rotational Motion for Spin Labels on Proteins.

Direct time-domain simulation of continuous-wave (CW) electron paramagnetic resonance (EPR) spectra from molecular dynamics (MD) trajectories has become increasingly popular, especially for proteins labeled with nitroxide spin labels. Due to the time-consuming nature of simulating adequately long MD trajectories, two approximate methods have been developed to reduce the MD-trajectory length required for modeling EPR spectra: hindered Brownian diffusion (HBD) and hidden Markov models (HMMs). Here, we assess the accuracy of these two approximate methods relative to direct simulations from MD trajectories for three spin-labeled protein systems (a simple helical peptide, a soluble protein, and a membrane protein) and two nitroxide spin labels with differing mobilities (R1 and 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC)). We find that the HMMs generally outperform HBD. Although R1 dynamics partially resembles hindered Brownian diffusion, HMMs accommodate the multiple dynamic time scales for the transitions between rotameric states of R1 that cannot be captured accurately by a HBD model. The MD trajectories of the TOAC-labeled proteins show that its dynamics closely resembles slow multisite exchange between twist-boat and chair ring puckering states. This motion is modeled well by HMM but not by HBD. All MD-trajectory data processing, stochastic trajectory simulations, and CW EPR spectral simulations are implemented in EasySpin, a free software package for MATLAB.

A bifunctional spin label reports the structural topology of phospholamban in magnetically-aligned bicelles.

We have applied a bifunctional spin label and EPR spectroscopy to determine membrane protein structural topology in magnetically-aligned bicelles, using monomeric phospholamban (PLB) as a model system. Bicelles are a powerful tool for studying membrane proteins by NMR and EPR spectroscopies, where magnetic alignment yields topological constraints by resolving the anisotropic spectral properties of nuclear and electron spins. However, EPR bicelle studies are often hindered by the rotational mobility of monofunctional Cys-linked spin labels, which obscures their orientation relative to the protein backbone. The rigid and stereospecific TOAC label provides high orientational sensitivity but must be introduced via solid-phase peptide synthesis, precluding its use in large proteins. Here we show that a bifunctional methanethiosulfonate spin label attaches rigidly and stereospecifically to Cys residues at i and i+4 positions along PLB's transmembrane helix, thus providing orientational resolution similar to that of TOAC, while being applicable to larger membrane proteins for which synthesis is impractical. Computational modeling and comparison with NMR data shows that these EPR experiments provide accurate information about helix tilt relative to the membrane normal, thus establishing a robust method for determining structural topology in large membrane proteins with a substantial advantage in sensitivity over NMR.

Stem Cell Therapy in Injured Vocal Folds: A Three-Month Xenograft Analysis of Human Embryonic Stem Cells.

We have previously shown that human embryonic stem cell (hESC) therapy to injured rabbit vocal folds (VFs) induces human tissue generation with regained VF vibratory capacity. The aims of this study were to test the sustainability of such effect and to what extent derivatives of the transplanted hESCs are propagated in the VFs. The VFs of 14 New Zealand rabbits were injured by a localized resection. HESCs were transplanted to 22 VFs which were analyzed for persistence of hESCs after six weeks and after three months. At three months, the VFs were also analyzed for viscoelasticity, measured as dynamic viscosity and elastic modulus, for the lamina propria (Lp) thickness and relative content of collagen type I. Three months after hESC cell therapy, the dynamic viscosity and elastic modulus of the hESC treated VFs were similar to normal controls and lower than untreated VFs (p ≤ 0.011). A normalized VF architecture, reduction in collagen type I, and Lp thickness were found compared with untreated VFs (p ≤ 0.031). At three months, no derivatives of hESCs were detected. HESCs transplanted to injured rabbit VFs restored the vibratory characteristics of the VFs, with maintained restored function for three months without remaining hESCs or derivatives.

Bifunctional Spin Labeling of Muscle Proteins: Accurate Rotational Dynamics, Orientation, and Distance by EPR.

While EPR allows for the characterization of protein structure and function due to its exquisite sensitivity to spin label dynamics, orientation, and distance, these measurements are often limited in sensitivity due to the use of labels that are attached via flexible monofunctional bonds, incurring additional disorder and nanosecond dynamics. In this chapter, we present methods for using a bifunctional spin label (BSL) to measure muscle protein structure and dynamics. We demonstrate that bifunctional attachment eliminates nanosecond internal rotation of the spin label, thereby allowing the accurate measurement of protein backbone rotational dynamics, including microsecond-to-millisecond motions by saturation transfer EPR. BSL also allows for accurate determination of helix orientation and disorder in mechanically and magnetically aligned systems, due to the label's stereospecific attachment. Similarly, labeling with a pair of BSL greatly enhances the resolution and accuracy of distance measurements measured by double electron-electron resonance (DEER). Finally, when BSL is applied to a protein with high helical content in an assembly with high orientational order (e.g., muscle fiber or membrane), two-probe DEER experiments can be combined with single-probe EPR experiments on an oriented sample in a process we call BEER, which has the potential for ab initio high-resolution structure determination.

FRET-based trilateration of probes bound within functional ryanodine receptors.

To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca(2+) channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. DPc10 is identical to the 2460-2495 segment within the cardiac muscle RyR isoform (RyR2) central domain. DPc10 binding to RyR2 results in a pathologically elevated Ca(2+) leak by destabilizing key interactions between the RyR2 N-terminal and central domains (unzipping). To localize the DPc10 binding site within RyR2, we measured FRET between five single-cysteine variants of the FK506-binding protein (FKBP) labeled with a donor probe, and DPc10 labeled with an acceptor probe (A-DPc10). Effective donor positions were calculated from simulated-annealing constrained by both the RyR cryo-EM map and the FKBP atomic structure docked to the RyR. FRET to A-DPc10 was measured in permeabilized cardiomyocytes via confocal microscopy, converted to distances, and used to trilaterate the acceptor locus within RyR. Additional FRET measurements between donor-labeled calmodulin and A-DPc10 were used to constrain the trilaterations. Results locate the DPc10 probe within RyR domain 3, ?35 Å from the previously docked N-terminal domain crystal structure. This multiscale approach may be useful in mapping other RyR sites of mechanistic interest within FRET range of FKBP.

Predicting cannabis abuse screening test (CAST) scores: a recursive partitioning analysis using survey data from Czech Republic, Italy, the Netherlands and Sweden.

INTRODUCTION: Cannabis is Europe's most commonly used illicit drug. Some users do not develop dependence or other problems, whereas others do. Many factors are associated with the occurrence of cannabis-related disorders. This makes it difficult to identify key risk factors and markers to profile at-risk cannabis users using traditional hypothesis-driven approaches. Therefore, the use of a data-mining technique called binary recursive partitioning is demonstrated in this study by creating a classification tree to profile at-risk users. METHODS: 59 variables on cannabis use and drug market experiences were extracted from an internet-based survey dataset collected in four European countries (Czech Republic, Italy, Netherlands and Sweden), n = 2617. These 59 potential predictors of problematic cannabis use were used to partition individual respondents into subgroups with low and high risk of having a cannabis use disorder, based on their responses on the Cannabis Abuse Screening Test. Both a generic model for the four countries combined and four country-specific models were constructed. RESULTS: Of the 59 variables included in the first analysis step, only three variables were required to construct a generic partitioning model to classify high risk cannabis users with 65-73% accuracy. Based on the generic model for the four countries combined, the highest risk for cannabis use disorder is seen in participants reporting a cannabis use on more than 200 days in the last 12 months. In comparison to the generic model, the country-specific models led to modest, non-significant improvements in classification accuracy, with an exception for Italy (p = 0.01). CONCLUSION: Using recursive partitioning, it is feasible to construct classification trees based on only a few variables with acceptable performance to classify cannabis users into groups with low or high risk of meeting criteria for cannabis use disorder. The number of cannabis use days in the last 12 months is the most relevant variable. The identified variables may be considered for use in future screeners for cannabis use disorders.

Phosphorylated phospholamban stabilizes a compact conformation of the cardiac calcium-ATPase.

The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with ß-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture.

N-terminal and central segments of the type 1 ryanodine receptor mediate its interaction with FK506-binding proteins.

We used site-directed labeling of the type 1 ryanodine receptor (RyR1) and fluorescence resonance energy transfer (FRET) measurements to map RyR1 sequence elements forming the binding site of the 12-kDa binding protein for the immunosuppressant drug, FK506. This protein, FKBP12, promotes the RyR1 closed state, thereby inhibiting Ca(2+) leakage in resting muscle. Although FKBP12 function is well established, its binding determinants within the RyR1 protein sequence remain unresolved. To identify these sequence determinants using FRET, we created five single-Cys FKBP variants labeled with Alexa Fluor 488 (denoted D-FKBP) and then targeted these D-FKBPs to full-length RyR1 constructs containing decahistidine (His10) "tags" placed within N-terminal (amino acid residues 76-619) or central (residues 2157-2777) regions of RyR1. The FRET acceptor Cy3NTA bound specifically and saturably to these His tags, allowing distance analysis of FRET measured from each D-FKBP variant to Cy3NTA bound to each His tag. Results indicate that D-FKBP binds proximal to both N-terminal and central domains of RyR1, thus suggesting that the FKBP binding site is composed of determinants from both regions. These findings further imply that the RyR1 N-terminal and central domains are proximal to one another, a core premise of the domain-switch hypothesis of RyR function. We observed FRET from GFP fused at position 620 within the N-terminal domain to central domain His-tagged sites, thus further supporting this hypothesis. Taken together, these results support the conclusion that N-terminal and central domain elements are closely apposed near the FKBP binding site within the RyR1 three-dimensional structure.

Nucleotide activation of the Ca-ATPase.

We have used fluorescence spectroscopy, molecular modeling, and limited proteolysis to examine structural dynamics of the sarcoplasmic reticulum Ca-ATPase (SERCA). The Ca-ATPase in sarcoplasmic reticulum vesicles from fast twitch muscle (SERCA1a isoform) was selectively labeled with fluorescein isothiocyanate (FITC), a probe that specifically reacts with Lys-515 in the nucleotide-binding site. Conformation-specific proteolysis demonstrated that FITC labeling does not induce closure of the cytoplasmic headpiece, thereby assigning FITC-SERCA as a nucleotide-free enzyme. We used enzyme reverse mode to synthesize FITC monophosphate (FMP) on SERCA, producing a phosphorylated pseudosubstrate tethered to the nucleotide-binding site of a Ca(2+)-free enzyme (E2 state to prevent FMP hydrolysis). Conformation-specific proteolysis demonstrated that FMP formation induces SERCA headpiece closure similar to ATP binding, presumably due to the high energy phosphoryl group on the fluorescent probe (ATP·E2 analog). Subnanosecond-resolved detection of fluorescence lifetime, anisotropy, and quenching was used to characterize FMP-SERCA (ATP·E2 state) versus FITC-SERCA in Ca(2+)-free, Ca(2+)-bound, and actively cycling phosphoenzyme states (E2, E1, and EP). Time-resolved spectroscopy revealed that FMP-SERCA exhibits increased probe dynamics but decreased probe accessibility compared with FITC-SERCA, indicating that ATP exhibits enhanced dynamics within a closed cytoplasmic headpiece. Molecular modeling was used to calculate the solvent-accessible surface area of FITC and FMP bound to SERCA crystal structures, revealing a positive correlation of solvent-accessible surface area with quenching but not anisotropy. Thus, headpiece closure is coupled to substrate binding but not active site dynamics. We propose that dynamics in the nucleotide-binding site of SERCA is important for Ca(2+) binding (distal allostery) and phosphoenzyme formation (direct activation).

Injection of human mesenchymal stem cells improves healing of vocal folds after scar excision--a xenograft analysis.

OBJECTIVES: Using a xenograft model the aim was to analyze if injection of human mesenchymal stem cells (hMSC) into the rabbit vocal fold (VF), after excision of an established scar, can improve the functional healing of the VF. STUDY DESIGN: Prospective design with an experimental xenograft model. METHODS: The VFs of 12 New Zealand rabbits were injured by a bilateral localized resection. After 9 weeks the scar after the resection was excised and hMSC were injected into the VFs. After another 10 weeks 10 VFs were dissected and stained for histology. Lamina propria thickness and relative content of collagen type I were measured. Viscoelasticity of 14 VFs at phonatory frequencies was quantified by a simple-shear rheometer. The hMSC survival was determined using a human DNA specific reference probe, that is, FISH analysis. RESULTS: The viscoelastic measurements, that is, dynamic viscosity and elastic shear modulus for the hMSC-treated VFs, were found to be similar to those of normal controls and were significantly lower than those of untreated controls (P < .05). A significant reduction in lamina propria thickness was also shown for the hMSC treated VFs compared with the untreated VFs (P < .05). This histologic finding corresponded with the viscoelastic results. No hMSC survived 10 weeks after the injection. CONCLUSIONS: Human mesenchymal stem cells injected into the rabbit VF following the excision of a chronic scar, were found to enhance the functional healing of the VF with reduced lamina propria thickness and restored viscoelastic shear properties.

Injection of human mesenchymal stem cells improves healing of scarred vocal folds: analysis using a xenograft model.

OBJECTIVES/HYPOTHESIS: The aims were to analyze if improved histological and viscoelastic properties seen after injection of human mesenchymal stem cells (hMSCs) in scarred vocal folds (VFs) of rabbits are sustainable and if the injected hMSCs survive 3 months in the VFs. STUDY DESIGN: Experimental xenograft model. METHODS: Eighteen VFs of 11 New Zealand white rabbits were scarred by a bilateral localized resection. After 3 months the animals were sacrificed. Twelve VFs were dissected and stained for histology, lamina propria thickness, and relative collagen type I analyses. The hMSCs survival was analyzed using a human DNA-specific reference probe, that is, fluorescence in situ hybridization staining. Viscoelasticity, measured as the dynamic viscosity and elastic modulus, was analyzed in a parallel-plate rheometer for 10 VFs. RESULTS: The dynamic viscosity and elastic modulus of hMSC-treated VFs were similar to that of normal controls and significantly improved compared to untreated controls (P < .05). A reduction in lamina propria thickness and relative collagen type 1 content were also shown for the hMSC-treated VFs compared to the untreated VFs (P < .05). The histological pictures corresponded well to the viscoelastic results. No hMSCs survived. CONCLUSIONS: Human mesenchymal stem cells injected into a scarred vocal fold of rabbit enhance healing of the vocal fold with reduced lamina propria thickness and collagen type I content and restore the viscoelastic function.

Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR.

We present a structurally dynamic model for nucleotide- and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in Dictyostelium myosin II. The actin-binding cleft is a solvent-filled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin-spin interactions, as measured by continuous wave EPR for distances of 0.7-2 nm or pulsed EPR (double electron-electron resonance) for distances of 1.7-6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex.

Injection of embryonic stem cells into scarred rabbit vocal folds enhances healing and improves viscoelasticity: short-term results.

OBJECTIVES: Scarring caused by trauma, postcancer treatment, or inflammation in the vocal folds is associated with stiffness of the lamina propria and results in severe voice problems. Currently there is no effective treatment. Human embryonic stem cells (hESC) have been recognized as providing a potential resource for cell transplantations, but in the undifferentiated state, they are generally not considered for therapeutic use due to risk of inadvertent development. This study assesses the functional potential of hESC to prevent or diminish scarring and improve viscoelasticity following grafting into scarred rabbit vocal folds. STUDY DESIGN: hESC were injected into 22 scarred vocal folds of New Zealand rabbits. After 1 month, the vocal folds were dissected and analyzed for persistence of hESC by fluorescence in situ hybridization using a human specific probe, and for differentiation by evaluation in hematoxylin-eosin-stained tissues. Parallel-plate rheometry was used to evaluate the functional effects, i.e., viscoelastic properties, after treatment with hESC. RESULTS: The results revealed significantly improved viscoelasticity in the hESC-treated vs. non-treated vocal folds. An average of 5.1% engraftment of human cells was found 1 month after hESC injection. In the hESC-injected folds, development compatible with cartilage, muscle and epithelia in close proximity or inter-mixed with the appropriate native rabbit tissue was detected in combination with less scarring and improved viscoelasticity. CONCLUSIONS: The histology and location of the surviving hESC-derived cells strongly indicate that the functional improvement was caused by the injected cells, which were regenerating scarred tissue. The findings point toward a strong impact from the host microenvironment, resulting in a regional specific in vivo hESC differentiation and regeneration of three types of tissue in scarred vocal folds of adult rabbits.

Silicone oil content in ophthalmic viscosurgical devices.

PURPOSE: To examine the silicone oil content in 5 brands of ophthalmic viscoelastic devices (OVDs). SETTING: Department of Ophthalmology, Central Hospital of Vasteras, Vasteras, Sweden. METHODS: Phacoemulsification with intraocular lens (IOL) implantation was performed in 250 patients. Five brands of OVD were used, each one in 50 procedures. From each brand, 5 separate batches, each consisting of 10 syringes, were used. The 250 samples from identical batches were sent for spectrophotometric analysis, and 250 samples were used during surgery. RESULTS: The silicone oil content varied significantly between the OVD brands. CONCLUSIONS: Silicone oil is a common contaminant in many OVDs. Ophthalmic viscosurgical devices with relatively low silicone oil content are available.

Mechanisms of shock wave induced endothelial cell injury.

BACKGROUND AND OBJECTIVES: Medical procedures, for example, laser angioplasty and extracorporeal lithotripsy as well as high-energy trauma expose human tissues to shock waves (SWs) that may cause tissue injury. The mechanisms for this injury, often affecting blood vessel walls, are poorly understood. Here we sought to assess the role of two suggested factors, viz., cavitation or reactive oxygen species (ROS). STUDY DESIGN/MATERIALS AND METHODS: A laser driven flyer-plate model was used to expose human umbilical cord vein endothelial cell (HUVEC) monolayers to SWs or to SWs plus cavitation (SWC). Cell injury was quantified with morphometry, trypan blue staining, and release of (51)Cr from labeled HUVECs. RESULTS: HUVECs, exposed to SWs only, could not be distinguished from controls in morphological appearance or ability to exclude trypan blue. Yet, release of (51)Cr, indicated a significant cell injury (P < 0.05). HUVEC cultures exposed to SWC, exhibited cell detachment and cell membrane damage detectable with trypan blue. Release of (51)Cr was fourfold compared to SW samples (P < 0.01). Signs of cell injury were evident at 15 minutes and did not change over the next 4 hours. No protective effects of ROS scavengers were demonstrated. CONCLUSIONS: Independent of ROS, SWC generated an immediate cell injury, which can explain, for example, vessel wall perturbation described in relation to SW treatments and trauma.