Benchmarking TriadAb using targets from the second antibody modeling assessment.

Computational modeling and design of antibodies has become an integral part of today's research and development in antibody therapeutics. Here we describe the Triad Antibody Homology Modeling (TriadAb) package, a functionality of the Triad protein design platform that predicts the structure of any heavy and light chain sequences of an antibody Fv domain using template-based modeling. To gauge the performance of TriadAb, we benchmarked against the results of the Second Antibody Modeling Assessment (AMA-II). On average, TriadAb produced main-chain carbonyl root-mean-square deviations between models and experimentally determined structures at 1.10 Å, 1.45 Å, 1.41 Å, 3.04 Å, 1.47 Å, 1.27 Å, 1.63 Å in the framework and the six complementarity-determining regions (H1, H2, H3, L1, L2, L3), respectively. The inaugural results are comparable to those reported in AMA-II, corroborating with our internal bench-based experiences that models generated using TriadAb are sufficiently accurate and useful for antibody engineering using the sequence design capabilities provided by Triad.

Systematic Assessment of Prosthesis Stiffness on User Biomechanics Using the Lower Leg Trajectory Error Framework and Its Implication for the Design and Evaluation of Ankle-Foot Prostheses.

Advances in understanding the effects the mechanical characteristics of prosthetic feet on user biomechanics have enabled passive prostheses to improve the walking pattern of people with lower limb amputation. However, there is no consensus on the design methodology and criteria required to maximize specific user outcomes and fully restore their mobility. The Lower Leg Trajectory Error (LLTE) framework is a novel design methodology based on the replication of lower leg dynamics. The LLTE value evaluates how closely a prosthetic foot replicates a target walking pattern. Designing a prosthesis that minimizes the LLTE value, optimizes its mechanical function to enable users to best replicate the target lower leg trajectory. Here, we conducted a systematic sensitivity investigation of LLTE-optimized prostheses. Five people with unilateral transtibial amputation walked overground at self-selected speeds using five prototype energy storage and return feet with varying LLTE values. The prototypes' LLTE values were varied by changing the stiffness of the participant's LLTE-optimized design by 60%, 80%, 120%, and 167%. Users most closely replicated the target able-bodied walking pattern with the LLTE-optimized stiffness, experimentally demonstrating that the predicted optimum was a true optimum. Additionally, the predicted LLTE values were correlated to the user's ability to replicate the target walking pattern, user preferences, and clinical outcomes including roll-over geometries, trunk sway, prosthetic energy return, and peak push-off power. This study further validates the use of the LLTE framework as a predictive and quantitative tool for designing and evaluating prosthetic feet.

Biomechanical evaluation over level ground walking of user-specific prosthetic feet designed using the lower leg trajectory error framework.

The walking pattern and comfort of a person with lower limb amputation are determined by the prosthetic foot's diverse set of mechanical characteristics. However, most design methodologies are iterative and focus on individual parameters, preventing a holistic design of prosthetic feet for a user's body size and walking preferences. Here we refined and evaluated the lower leg trajectory error (LLTE) framework, a novel quantitative and predictive design methodology that optimizes the mechanical function of a user's prosthesis to encourage gait dynamics that match their body size and desired walking pattern. Five people with unilateral below-knee amputation walked over-ground at self-selected speeds using an LLTE-optimized foot made of Nylon 6/6, their daily-use foot, and a standardized commercial energy storage and return (ESR) foot. Using the LLTE feet, target able-bodied kinematics and kinetics were replicated to within 5.2% and 13.9%, respectively, 13.5% closer than with the commercial ESR foot. Additionally, energy return and center of mass propulsion work were 46% and 34% greater compared to the other two prostheses, which could lead to reduced walking effort. Similarly, peak limb loading and flexion moment on the intact leg were reduced by an average of 13.1%, lowering risk of long-term injuries. LLTE-feet were preferred over the commercial ESR foot across all users and preferred over the daily-use feet by two participants. These results suggest that the LLTE framework could be used to design customized, high performance ESR prostheses using low-cost Nylon 6/6 material. More studies with large sample size are warranted for further verification.

Knee Swing Phase Flexion Resistance Affects Several Key Features of Leg Swing Important to Safe Transfemoral Prosthetic Gait.

We systematically investigate in-vivo the effect of increasing prosthetic knee flexion damping on key features of the swing phase of individuals with transfemoral amputation during walking. Five experienced prosthesis users walked using a prototype device in a motion capture laboratory. A range of interchangeable hydraulic rotary dampers was used to progressively modify swing phase flexion resistance in isolation. Toe clearance (TC; vertical distance toe to floor), effective leg length (ELL; distance hip to toe), and knee flexion angle during swing phase were computed, alongside the sensitivities of vertical toe position to angular displacements at the hip, knee and ankle. Key features of these profiles were compared across 5 damping conditions. With higher damping, knee extension occurred earlier in swing phase, promoting greater symmetry. However, with implications for toe catch, minimum TC reduced, and minimum TC and maximum ELL occurred earlier; temporally closer to mid-swing, when the limb must pass the stance limb. Further, TC became less sensitive to changes in hip flexion, suggesting a lesser ability to control toe clearance without employing proximal or contralateral compensations. There is a trade-off between key features related to gait safety when selecting an appropriate resistance for a mechanical prosthetic knee. In addition to highlighting broader implications surrounding swing phase damping selection for the optimization of mechanical knees, this work reveals design considerations that may be of utility in the formulation of control strategies for computerized devices.

Experimental Demonstration of the Lower Leg Trajectory Error Framework Using Physiological Data as Inputs.

While many studies have attempted to characterize the mechanical behavior of passive prosthetic feet to understand their influence on amputee gait, the relationship between mechanical design and biomechanical performance has not yet been fully articulated from a fundamental physics perspective. A novel framework, called lower leg trajectory error (LLTE) framework, presents a means of quantitatively optimizing the constitutive model of prosthetic feet to match a reference kinematic and kinetic dataset. This framework can be used to predict the required stiffness and geometry of a prosthesis to yield a desired biomechanical response. A passive prototype foot with adjustable ankle stiffness was tested by a unilateral transtibial amputee to evaluate this framework. The foot condition with LLTE-optimal ankle stiffness enabled the user to replicate the physiological target dataset within 16% root-mean-square (RMS) error. Specifically, the measured kinematic variables matched the target kinematics within 4% RMS error. Testing a range of ankle stiffness conditions from 1.5 to 24.4 N·m/deg with the same user indicated that conditions with lower LLTE values deviated the least from the target kinematic data. Across all conditions, the framework predicted the horizontal/vertical position, and angular orientation of the lower leg during midstance within 1.0 cm, 0.3 cm, and 1.5 deg, respectively. This initial testing suggests that prosthetic feet designed with low LLTE values could offer benefits to users. The LLTE framework is agnostic to specific foot designs and kinematic/kinetic user targets, and could be used to design and customize prosthetic feet.

Whole Exome Sequencing reveals NOTCH1 mutations in anaplastic large cell lymphoma and points to Notch both as a key pathway and a potential therapeutic target.

Patients diagnosed with Anaplastic Large Cell Lymphoma (ALCL) are still treated with toxic multi-agent chemotherapy and as many as 25-50% of patients relapse. To understand disease pathology and to uncover novel targets for therapy, Whole-Exome Sequencing (WES) of Anaplastic Lymphoma Kinase (ALK)+ ALCL was performed as well as Gene-Set Enrichment Analysis. This revealed that the T-cell receptor (TCR) and Notch pathways were the most enriched in mutations. In particular, variant T349P of NOTCH1, which confers a growth advantage to cells in which it is expressed, was detected in 12% of ALK+ and ALK- ALCL patient samples. Furthermore, we demonstrate that NPM-ALK promotes NOTCH1 expression through binding of STAT3 upstream of NOTCH1. Moreover, inhibition of NOTCH1 with γ-secretase inhibitors (GSIs) or silencing by shRNA leads to apoptosis; co-treatment in vitro with the ALK inhibitor Crizotinib led to additive/synergistic anti-tumour activity suggesting this may be an appropriate combination therapy for future use in the circumvention of ALK inhibitor resistance. Indeed, Crizotinib-resistant and sensitive ALCL were equally sensitive to GSIs. In conclusion, we show a variant in the extracellular domain of NOTCH1 that provides a growth advantage to cells and confirm the suitability of the Notch pathway as a second-line druggable target in ALK+ ALCL.

Novel clinically relevant antibiotic resistance genes associated with sewage sludge and industrial waste streams revealed by functional metagenomic screening.

A growing body of evidence indicates that anthropogenic activities can result in increased prevalence of antimicrobial resistance genes (ARGs) in bacteria in natural environments. Many environmental studies have used next-generation sequencing methods to sequence the metagenome. However, this approach is limited as it does not identify divergent uncharacterized genes or demonstrate activity. Characterization of ARGs in environmental metagenomes is important for understanding the evolution and dissemination of resistance, as there are several examples of clinically important resistance genes originating in environmental species. The current study employed a functional metagenomic approach to detect genes encoding resistance to extended spectrum ß-lactams (ESBLs) and carbapenems in sewage sludge, sludge amended soil, quaternary ammonium compound (QAC) impacted reed bed sediment and less impacted long term curated grassland soil. ESBL and carbapenemase genes were detected in sewage sludge, sludge amended soils and QAC impacted soil with varying degrees of homology to clinically important ß-lactamase genes. The flanking regions were sequenced to identify potential host background and genetic context. Novel ß-lactamase genes were found in Gram negative bacteria, with one gene adjacent to an insertion sequence ISPme1, suggesting a recent mobilization event and/ the potential for future transfer. Sewage sludge and quaternary ammonium compound (QAC) rich industrial effluent appear to disseminate and/or select for ESBL genes which were not detected in long term curated grassland soils. This work confirms the natural environment as a reservoir of novel and mobilizable resistance genes, which may pose a threat to human and animal health.

A Novel Framework for Quantitatively Connecting the Mechanical Design of Passive Prosthetic Feet to Lower Leg Trajectory.

This paper presents a novel framework that quantitatively connects the mechanical design of a prosthetic foot to its anticipated biomechanical performance. The framework uses kinetic inputs (ground reaction forces and center of pressure) to predict kinematic outputs of the lower leg segment by knowing the geometry and stiffness of the foot. The error between the predicted and target kinematics is evaluated using a root-mean-square error function called the Lower Leg Trajectory Error (LLTE). Using physiological kinetic inputs and kinematic targets, three model foot architectures were optimized to minimize the LLTE. The resulting predicted lower leg kinematics were compared to those of the same foot architectures optimized for physiological roll-over geometry. The feet with minimized LLTE had lower leg kinematics closer to physiological than those optimized for roll-over geometry. A prosthetic foot that exactly mimics physiological roll-over geometry may result in gait kinematics that differ greatly from physiological, as roll-over geometry omits information about the foot-ground contact constraint, lower leg orientation, and temporal progression of a step. The LLTE-based framework is agnostic to specific foot designs provided their constitutive behavior can be characterized, and it can accept alternate inputs and targets depending on what performance and clinical objectives are desired.

Modeling the future of irrigation: A parametric description of pressure compensating drip irrigation emitter performance.

Drip irrigation is a means of distributing the exact amount of water a plant needs by dripping water directly onto the root zone. It can produce up to 90% more crops than rain-fed irrigation, and reduce water consumption by 70% compared to conventional flood irrigation. Drip irrigation may enable millions of poor farmers to rise out of poverty by growing more and higher value crops, while not contributing to overconsumption of water. Achieving this impact will require broadening the engineering knowledge required to design new, low-cost, low-power drip irrigation technology, particularly for poor, off-grid communities in developing countries. For more than 50 years, pressure compensating (PC) drip emitters-which can maintain a constant flow rate under variations in pressure, to ensure uniform water distribution on a field-have been designed and optimized empirically. This study presents a parametric model that describes the fluid and solid mechanics that govern the behavior of a common PC emitter architecture, which uses a flexible diaphragm to limit flow. The model was validated by testing nine prototypes with geometric variations, all of which matched predicted performance to within R2 = 0.85. This parametric model will enable irrigation engineers to design new drip emitters with attributes that improve performance and lower cost, which will promote the use of drip irrigation throughout the world.

The Effects of the Inertial Properties of Above-Knee Prostheses on Optimal Stiffness, Damping, and Engagement Parameters of Passive Prosthetic Knees.

Our research aims to design low-cost, high-performance, passive prosthetic knees for developing countries. In this study, we determine optimal stiffness, damping, and engagement parameters for a low-cost, passive prosthetic knee that consists of simple mechanical elements and may enable users to walk with the normative kinematics of able-bodied humans. Knee joint power was analyzed to divide gait into energy-based phases and select mechanical components for each phase. The behavior of each component was described with a polynomial function, and the coefficients and polynomial order of each function were optimized to reproduce the knee moments required for normative kinematics of able-bodied humans. Sensitivity of coefficients to prosthesis mass was also investigated. The knee moments required for prosthesis users to walk with able-bodied normative kinematics were accurately reproduced with a mechanical system consisting of a linear spring, two constant-friction dampers, and three clutches (R2=0.90 for a typical prosthetic leg). Alterations in upper leg, lower leg, and foot mass had a large influence on optimal coefficients, changing damping coefficients by up to 180%. Critical results are reported through parametric illustrations that can be used by designers of prostheses to select optimal components for a prosthetic knee based on the inertial properties of the amputee and his or her prosthetic leg.

The Effects of Prosthesis Inertial Properties on Prosthetic Knee Moment and Hip Energetics Required to Achieve Able-Bodied Kinematics.

There is a major need in the developing world for a low-cost prosthetic knee that enables users to walk with able-bodied kinematics and low energy expenditure. To efficiently design such a knee, the relationship between the inertial properties of a prosthetic leg and joint kinetics and energetics must be determined. In this paper, using inverse dynamics, the theoretical effects of varying the inertial properties of an above-knee prosthesis on the prosthetic knee moment, hip power, and absolute hip work required for walking with able-bodied kinematics were quantified. The effects of independently varying mass and moment of inertia of the prosthesis, as well as independently varying the masses of each prosthesis segment, were also compared. Decreasing prosthesis mass to 25% of physiological leg mass increased peak late-stance knee moment by 43% and decreased peak swing knee moment by 76%. In addition, it reduced peak stance hip power by 26%, average swing hip power by 76%, and absolute hip work by 22%. Decreasing upper leg mass to 25% of its physiological value reduced absolute hip work by just 2%, whereas decreasing lower leg and foot mass reduced work by up to 22%, with foot mass having the greater effect. Results are reported in the form of parametric illustrations that can be utilized by researchers, designers, and prosthetists. The methods and outcomes presented have the potential to improve prosthetic knee component selection, facilitate able-bodied kinematics, and reduce energy expenditure for users of low-cost, passive knees in developing countries, as well as for users of advanced active knees in developed countries.

Waste water effluent contributes to the dissemination of CTX-M-15 in the natural environment.

OBJECTIVES: Multidrug-resistant Enterobacteriaceae pose a significant threat to public health. We aimed to study the impact of sewage treatment effluent on antibiotic resistance reservoirs in a river. METHODS: River sediment samples were taken from downstream and upstream of a waste water treatment plant (WWTP) in 2009 and 2011. Third-generation cephalosporin (3GC)-resistant Enterobacteriaceae were enumerated. PCR-based techniques were used to elucidate mechanisms of resistance, with a new two-step PCR-based assay developed to investigate bla(CTX-M-15) mobilization. Conjugation experiments and incompatibility replicon typing were used to investigate plasmid ecology. RESULTS: We report the first examples of bla(CTX-M-15) in UK river sediment; the prevalence of bla(CTX-M-15) was dramatically increased downstream of the WWTP. Ten novel genetic contexts for this gene were identified, carried in pathogens such as Escherichia coli ST131 as well as indigenous aquatic bacteria such as Aeromonas media. The bla(CTX-M-15) -gene was readily transferable to other Gram-negative bacteria. We also report the first finding of an imipenem-resistant E. coli in a UK river. CONCLUSIONS: The high diversity and host range of novel genetic contexts proves that evolution of novel combinations of resistance genes is occurring at high frequency and has to date been significantly underestimated. We have identified a worrying reservoir of highly resistant enteric bacteria in the environment that poses a threat to human and animal health.

Functional metagenomic analysis reveals rivers are a reservoir for diverse antibiotic resistance genes.

The environment harbours a significant diversity of uncultured bacteria and a potential source of novel and extant resistance genes which may recombine with clinically important bacteria disseminated into environmental reservoirs. There is evidence that pollution can select for resistance due to the aggregation of adaptive genes on mobile elements. The aim of this study was to establish the impact of waste water treatment plant (WWTP) effluent disposal to a river by using culture independent methods to study diversity of resistance genes downstream of the WWTP in comparison to upstream. Metagenomic libraries were constructed in Escherichia coli and screened for phenotypic resistance to amikacin, gentamicin, neomycin, ampicillin and ciprofloxacin. Resistance genes were identified by using transposon mutagenesis. A significant increase downstream of the WWTP was observed in the number of phenotypic resistant clones recovered in metagenomic libraries. Common ß-lactamases such as blaTEM were recovered as well as a diverse range of acetyltransferases and unusual transporter genes, with evidence for newly emerging resistance mechanisms. The similarities of the predicted proteins to known sequences suggested origins of genes from a very diverse range of bacteria. The study suggests that waste water disposal increases the reservoir of resistance mechanisms in the environment either by addition of resistance genes or by input of agents selective for resistant phenotypes.

Localized fluidization burrowing mechanics of Ensis directus.

Muscle measurements of Ensis directus, the Atlantic razor clam, indicate that the organism only has sufficient strength to burrow a few centimeters into the soil, yet razor clams burrow to over 70 cm. In this paper, we show that the animal uses the motions of its valves to locally fluidize the surrounding soil and reduce burrowing drag. Substrate deformations were measured using particle image velocimetry (PIV) in a novel visualization system that enabled us to see through the soil and watch E. directus burrow in situ. PIV data, supported by soil and fluid mechanics theory, show that contraction of the valves of E. directus locally fluidizes the surrounding soil. Particle and fluid mixtures can be modeled as a Newtonian fluid with an effective viscosity based on the local void fraction. Using these models, we demonstrate that E. directus is strong enough to reach full burrow depth in fluidized soil, but not in static soil. Furthermore, we show that the method of localized fluidization reduces the amount of energy required to reach burrow depth by an order of magnitude compared with penetrating static soil, and leads to a burrowing energy that scales linearly with depth rather than with depth squared.

MRI findings in acute Hendra virus meningoencephalitis.

AIM: To describe serial changes in brain magnetic resonance imaging (MRI) in acute human infection from two outbreaks of Hendra virus (HeV), relate these changes to disease prognosis, and compare HeV encephalitis to reported cases of Nipah virus encephalitis. MATERIALS AND METHODS: The MRI images of three human cases (two of which were fatal) of acute HeV meningoencephalitis were reviewed. RESULTS: Cortical selectivity early in the disease is evident in all three patients, while deep white matter involvement appears to be a late and possibly premorbid finding. This apparent early grey matter selectivity may be related to viral biology or ribavirin pharmacokinetics. Neuronal loss is evident at MRI, and the rate of progression of MRI abnormalities can predict the outcome of the infection. In both fatal cases, the serial changes in the MRI picture mirrored the clinical course. CONCLUSION: This is the first comprehensive report of serial MRI findings in acute human cerebral HeV infection from two outbreaks. The cortical selectivity appears to be an early finding while deep white matter involvement a late, and possibly premorbid, finding. In both fatal cases, the serial changes in MRI mirrored the clinical course.

Identification and evaluation of the Atlantic razor clam (Ensis directus) for biologically inspired subsea burrowing systems.

In this article, we identify and analyze a subsea organism to serve as a model for biologically inspired burrowing technology to be used in applications such as anchoring, installation of cables, and recovery of oil. After inspecting myriad forms of life that live on or within ocean substrates, the Atlantic razor clam, Ensis directis, stood out as an attractive basis for new burrowing technology because of its low-energy requirements associated with digging (0.21 J/cm), its speed and depth of burrrowing (∼1 cm/s and 70 cm, respectively), and its size and simplicity relative to man-made machines. As anchoring is a prime application for the technology resulting from this work, the performance of an Ensis directus-based anchoring system was compared to existing technologies. In anchoring force per embedment energy, the E. directus-based anchor beats existing technology by at least an order of magnitude. In anchoring force per weight of device, the biologically inspired system weighs less than half that of current anchors. The article concludes with a review of E. directus's digging strategy, which involves motions of its valves to locally fluidize the substrate to reduce burrowing drag and energy, and the successful adaptation of E. directus's burrowing mechanisms into an engineering system: the RoboClam burrowing robot, which, like the animal, uses localized fluidization to achieve digging energy that scales linearly with depth, rather than depth squared, for moving through static soil.

Generalized valence bond wave functions in quantum Monte Carlo.

We present a technique for using quantum Monte Carlo (QMC) to obtain high quality energy differences. We use generalized valence bond (GVB) wave functions, for an intuitive approach to capturing the important sources of static correlation, without needing to optimize the orbitals with QMC. Using our modifications to Walker branching and Jastrows, we can then reliably use diffusion quantum Monte Carlo to add in all the dynamic correlation. This simple approach is easily accurate to within a few tenths of a kcal/mol for a variety of problems, which we demonstrate for the adiabatic singlet-triplet splitting in methylene, the vertical and adiabatic singlet-triplet splitting in ethylene, 2+2 cycloaddition, and Be(2) bond breaking.

Efficient algorithm for "on-the-fly" error analysis of local or distributed serially correlated data.

We describe the Dynamic Distributable Decorrelation Algorithm (DDDA) which efficiently calculates the true statistical error of an expectation value obtained from serially correlated data "on-the-fly," as the calculation progresses. DDDA is an improvement on the Flyvbjerg-Petersen renormalization group blocking method (Flyvberg and Peterson, J Chem Phys 1989, 91, 461). This "on-the-fly" determination of statistical quantities allows dynamic termination of Monte Carlo calculations once a specified level of convergence is attained. This is highly desirable when the required precision might take days or months to compute, but cannot be accurately estimated prior to the calculation. Furthermore, DDDA allows for a parallel implementation which requires very low communication, O(log(2)N), and can also evaluate the variance of a calculation efficiently "on-the-fly." Quantum Monte Carlo calculations are presented to illustrate "on-the-fly" variance calculations for serial and massively parallel Monte Carlo calculations.

Potassium and calcium current blocking properties of the novel antiarrhythmic agent H 345/52: implications for proarrhythmic potential.

OBJECTIVES: To study the blocking effects of H 345/52 on ionic currents of rabbit ventricular myocytes and how these features translate into a proarrhythmic potential. METHODS: The single electrode voltage clamp technique was used to study the effects of H 345/52 on the rapid component of the delayed rectifying potassium current, I(Kr), and the L-type calcium current (I(Ca)). Differential effects of H 345/52 and almokalant on APD prolongation were studied in a rabbit Purkinje fibre/ventricular muscle preparation. The temporal variability of the action potential duration (APD) and its relation to proarrhythmias was examined in Langendorff-perfused rabbit hearts administered H 345/52 or almokalant. Anaesthetised, methoxamine-sensitised rabbits were used to assess the propensity of intravenous H 345/52 and ibutilide to induce torsades de pointes (TdP). RESULTS: H 345/52 potently blocked I(Kr) (IC(50)=40 nM) without consequential use-dependency. The I(Ca) was also blocked, but at higher concentrations (IC(50)=1.3 microM). Block of I(Ca) was markedly frequency-dependent (positive) and influenced by membrane potential, such that H 345/52 was more effective following clamp steps from plateau potentials than from -80 mV. In the Purkinje fibre-ventricular muscle preparation, almokalant prolonged the Purkinje fibre APD preferentially, whereas H 345/52 homogeneously prolonged APD in both tissue types. In the perfused rabbit heart, H 345/52 (1 microM) and almokalant (0.3 microM) prolonged APD to a similar degree but increased the temporal variability of APD differently, from 3+/-0.4 ms in control hearts to 8+/-1.2 ms and to 38+/-7.5 ms (P<0.001 vs. H 345/52), respectively. Unequivocal early after-depolarisations were seen in 5/6 almokalant-perfused hearts but in no heart administered H 345/52 (P<0.05). In anaesthetised rabbits, H 345/52 (17.4 micromol/kg) or ibutilide (2.6 micromol/kg maximum), maximally lengthened the QT interval from 133+/-4.5 to 177+/-8.0 ms and from 125+/-5.1 to 166+/-9.3 ms (P<0.001, n=8). However, whereas ibutilide induced TdP in all animals at 0.06+/-0.009 micromol/kg, H 345/52 did not induce TdP (P=0.0002) at up to 17.4 micromol/kg. CONCLUSIONS: H 345/52 blocks I(Kr) with high potency and I(Ca) with somewhat lower potency and was found to delay ventricular repolarisation without substantially increasing temporal or spatial dispersion and without inducing early after-depolarisations or TdP.

Effects of the calcium sensitizer [+]-EMD 60263 and its enantiomer [-]-EMD 60264 on cardiac ionic currents of guinea pig and rat ventricular myocytes.

The thiadiazinone enantiomers [+]-EMD 60263 and [-]-EMD 60264 ((+)-5-(1-(alpha-ethylimino-3,4-dimethoxybenzyl)-1,2,3,4-tetrah ydroquinoline-6-yl)-6-methyl-3,6-dihydro-2H-1,3,4-thiadiazine-2 -on) exhibit distinct stereoselectivity for Ca2+-sensitizing action ([+]-enantiomer) and phosphodiesterase inhibition ([-]-enantiomer). However, in isolated guinea pig papillary muscle, both compounds cause an action-potential prolongation that has been related to a nonselective depression of the delayed rectifier potassium current. Because [-]-EMD 60264 did not increase force of contraction despite phosphodiesterase inhibition, we postulated that one or several additional actions may oppose the anticipated positive inotropic effect. Therefore we investigated whether other membrane currents were also affected in voltage-clamped ventricular cardiomyocytes. Both [+]-EMD 60263 and [-]-EMD 60264 reduced sodium current as well as L-type calcium current in guinea pig ventricular myocytes, but steady-state inactivation or conductance curves of I(Na) and I(Ca) were not shifted along the voltage axis. Inward rectifier and transient outward current were studied in rat myocytes, but neither current was affected. We conclude that the positive inotropic action of [+]-EMD 60263 can be explained by prevalence of the Ca2+-sensitizing effect over its inhibitory actions on Na+ and Ca2+ current, whereas the negative inotropic effect of [-]-EMD 60264 may be caused by inhibition of I(Ca) predominating over PDE inhibition.