Closed-loop automated reaching apparatus (CLARA) for interrogating complex motor behaviors.

Objective.Closed-loop neuromodulation technology is a rapidly expanding category of therapeutics for a broad range of indications. Development of these innovative neurological devices requires high-throughput systems for closed-loop stimulation of model organisms, while monitoring physiological signals and complex, naturalistic behaviors. To address this need, we developed CLARA, a closed-loop automated reaching apparatus.Approach.Using breakthroughs in computer vision, CLARA integrates fully-automated, markerless kinematic tracking of multiple features to classify animal behavior and precisely deliver neural stimulation based on behavioral outcomes. CLARA is compatible with advanced neurophysiological tools, enabling the testing of neurostimulation devices and identification of novel neurological biomarkers.Results.The CLARA system tracks unconstrained skilled reach behavior in 3D at 150 Hz without physical markers. The system fully automates trial initiation and pellet delivery and is capable of accurately delivering stimulation in response to trial outcome with short latency. Kinematic data from the CLARA system provided novel insights into the dynamics of reach consistency over the course of learning, suggesting that learning selectively improves reach failures but does not alter the kinematics of successful reaches. Additionally, using the closed-loop capabilities of CLARA, we demonstrate that vagus nerve stimulation (VNS) improves skilled reach performance and increases reach trajectory consistency in healthy animals.Significance.The CLARA system is the first mouse behavior apparatus that uses markerless pose tracking to provide real-time closed-loop stimulation in response to the outcome of an unconstrained motor task. Additionally, we demonstrate that the CLARA system was essential for our investigating the role of closed-loop VNS stimulation on motor performance in healthy animals. This approach has high translational relevance for developing neurostimulation technology based on complex human behavior.

Validation of an adipose-liver human-on-a-chip model of NAFLD for preclinical therapeutic efficacy evaluation.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and strongly correlates with the growing incidence of obesity and type II diabetes. We have developed a human-on-a-chip model composed of human hepatocytes and adipose tissue chambers capable of modeling the metabolic factors that contribute to liver disease development and progression, and evaluation of the therapeutic metformin. This model uses a serum-free, recirculating medium tailored to represent different human metabolic conditions over a 14-day period. The system validated the indirect influence of adipocyte physiology on hepatocytes that modeled important aspects of NAFLD progression, including insulin resistant biomarkers, differential adipokine signaling in different media and increased TNF-α-induced steatosis observed only in the two-tissue model. This model provides a simple but unique platform to evaluate aspects of an individual factor's contribution to NAFLD development and mechanisms as well as evaluate preclinical drug efficacy and reassess human dosing regimens.

Thermal conductivity and its relation to atomic structure for symmetrical tilt grain boundaries in silicon.

We perform a systematic study of thermal resistance/conductance of tilt grain boundaries (GBs) in Si using classical molecular dynamics. The GBs studied are naturally divided into three groups according to the structural units forming the GB core. We find that, within each group, the GB thermal conductivity strongly correlates with the excess GB energy. All three groups predict nearly the same GB conductivity extrapolated to the high-energy limit. This limiting value is close to the thermal conductivity of amorphous Si, suggesting similar heat transport mechanisms. While the lattice thermal conductivity decreases with temperature, the GB conductivity slightly increases. However, at high temperatures it turns over and starts decreasing if the GB structure undergoes a premelting transformation. Analysis of vibrational spectra of GBs resolved along different directions sheds light on the mechanisms of their thermal resistance. The existence of alternating tensile and compressive atomic environments in the GB core gives rise to localized vibrational modes, frequency gaps creating acoustic mismatch with lattice phonons, and anharmonic vibrations of loosely-bound atoms residing in open atomic environments.

La práctica oftalmológica en México del siglo xviii / The practice of ophthalmology in Mexico in the 18th century

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Nickel nanoparticles set a new record of strength.

Material objects with micrometer or nanometer dimensions can exhibit much higher strength than macroscopic objects, but this strength rarely approaches the maximum theoretical strength of the material. Here, we demonstrate that faceted single-crystalline nickel (Ni) nanoparticles exhibit an ultrahigh compressive strength (up to 34 GPa) unprecedented for metallic materials. This strength matches the available estimates of Ni theoretical strength. Three factors are responsible for this record-high strength: the large Ni shear modulus, the smooth edges and corners of the nanoparticles, and the thin oxide layer on the particle surface. This finding is supported by molecular dynamics simulations that closely mimic the experimental conditions, which show that the mechanical failure of the strongest particles is triggered by homogeneous nucleation of dislocation loops inside the particle. The nucleation of a stable loop is preceded by multiple nucleation attempts accompanied by unusually large local atomic displacements caused by thermal fluctuations.

Functional analysis of human intrafusal fiber innervation by human γ-motoneurons.

Investigation of neuromuscular deficits and diseases such as SMA, as well as for next generation prosthetics, utilizing in vitro phenotypic models would benefit from the development of a functional neuromuscular reflex arc. The neuromuscular reflex arc is the system that integrates the proprioceptive information for muscle length and activity (sensory afferent), to modify motoneuron output to achieve graded muscle contraction (actuation efferent). The sensory portion of the arc is composed of proprioceptive sensory neurons and the muscle spindle, which is embedded in the muscle tissue and composed of intrafusal fibers. The gamma motoneurons (γ-MNs) that innervate these fibers regulate the intrafusal fiber's stretch so that they retain proper tension and sensitivity during muscle contraction or relaxation. This mechanism is in place to maintain the sensitivity of proprioception during dynamic muscle activity and to prevent muscular damage. In this study, a co-culture system was developed for innervation of intrafusal fibers by human γ-MNs and demonstrated by morphological and immunocytochemical analysis, then validated by functional electrophysiological evaluation. This human-based fusimotor model and its incorporation into the reflex arc allows for a more accurate recapitulation of neuromuscular function for applications in disease investigations, drug discovery, prosthetic design and neuropathic pain investigations.

Re-shaping NHS orthodontic provision - the North Wales experience.

The orthodontic service provision within North Wales, in common with many areas of the United Kingdom, was experiencing increasing waiting times for assessment and treatment. Reasons for this included an increasing population, patient demand and fixed NHS contracted orthodontic provision. In addition to these universal challenges, the geography of North Wales contributed to difficulties in accessing care. It was felt that with a reshaping of the orthodontic services there was potential to enhance the quality of orthodontic care available to patients and deliver prudent NHS orthodontic services. Three distinct, but inter-related steps, were identified to progress the reshaping of the service with the intended outcome of achieving an improved co-ordinated service. Initially, this involved the re-commissioning of the primary care specialist service through a formal retendering process. Following this, a standardised orthodontic referral form was developed, to be used for all orthodontic referrals regardless of whether their destination was a primary or secondary care provider. Finally, a formal accreditation process for all non-specialist dentists who were undertaking NHS orthodontic treatment was developed and implemented. The successful outcome of this process was only possible because of the close working partnership between the North Wales Orthodontic Managed Clinical Network (OMCN) and Betsi Cadwaladr University Health Board.

Energy spectrum of a Langevin oscillator.

We derive analytical solutions for the autocorrelation and cross-correlation functions of the kinetic, potential, and total energy of a Langevin oscillator. These functions are presented in both the time and frequency domains and validated by independent numerical simulations. The results are applied to address the long-standing issue of temperature fluctuations in canonical systems.

Mechanistic investigation of adult myotube response to exercise and drug treatment in vitro using a multiplexed functional assay system.

The ability to accurately measure skeletal muscle functional performance at the single-cell level would be advantageous for exercise physiology studies and disease modeling applications. To that end, this study characterizes the functional response of individual skeletal muscle myotubes derived from adult rodent tissue to creatine treatment and chronic exercise. The observed improvements to functional performance in response to these treatments appear to correlate with alterations in hypertrophic and mitochondrial biogenesis pathways, supporting previously published in vivo and in vitro data, which highlights the role of these pathways in augmenting skeletal muscle output. The developed system represents a multiplexed functional in vitro assay capable of long-term assessment of contractile cellular outputs in real-time that is compatible with concomitant molecular biology analysis. Adoption of this system in drug toxicity and efficacy studies would improve understanding of compound activity on physical cellular outputs and provide more streamlined and predictive data for future preclinical analyses.

A multiplexed chip-based assay system for investigating the functional development of human skeletal myotubes in vitro.

This report details the development of a non-invasive in vitro assay system for investigating the functional maturation and performance of human skeletal myotubes. Data is presented demonstrating the survival and differentiation of human myotubes on microscale silicon cantilevers in a defined, serum-free system. These cultures can be stimulated electrically and the resulting contraction quantified using modified atomic force microscopy technology. This system provides a higher degree of sensitivity for investigating contractile waveforms than video-based analysis, and represents the first system capable of measuring the contractile activity of individual human muscle myotubes in a reliable, high-throughput and non-invasive manner. The development of such a technique is critical for the advancement of body-on-a-chip platforms toward application in pre-clinical drug development screens.

Correlation of embryonic skeletal muscle myotube physical characteristics with contractile force generation on an atomic force microscope-based bio-microelectromechanical systems device.

Rigorous analysis of muscle function in in vitro systems is needed for both acute and chronic biomedical applications. Forces generated by skeletal myotubes on bio-microelectromechanical cantilevers were calculated using a modified version of Stoney's thin-film equation and finite element analysis (FEA), then analyzed for regression to physical parameters. The Stoney's equation results closely matched the more intensive FEA and the force correlated to cross-sectional area (CSA). Normalizing force to measured CSA significantly improved the statistical sensitivity and now allows for close comparison of in vitro data to in vivo measurements for applications in exercise physiology, robotics, and modeling neuromuscular diseases.

JAK inhibitors suppress t(8;21) fusion protein-induced leukemia.

Oncogenic mutations in components of the JAK/STAT pathway, including those in cytokine receptors and JAKs, lead to increased activity of downstream signaling and are frequently found in leukemia and other hematological disorders. Thus, small-molecule inhibitors of this pathway have been the focus of targeted therapy in these hematological diseases. We previously showed that t(8;21) fusion protein acute myeloid leukemia (AML)1-ETO and its alternatively spliced variant AML1-ETO9a (AE9a) enhance the JAK/STAT pathway via downregulation of CD45, a negative regulator of this pathway. To investigate the therapeutic potential of targeting JAK/STAT in t(8;21) leukemia, we examined the effects of a JAK2-selective inhibitor TG101209 and a JAK1/2-selective inhibitor INCB18424 on t(8;21) leukemia cells. TG101209 and INCB18424 inhibited proliferation and promoted apoptosis of these cells. Furthermore, TG101209 treatment in AE9a leukemia mice reduced tumor burden and significantly prolonged survival. TG101209 also significantly impaired the leukemia-initiating potential of AE9a leukemia cells in secondary recipient mice. These results demonstrate the potential therapeutic efficacy of JAK inhibitors in treating t(8;21) AML.

A functional system for high-content screening of neuromuscular junctions in vitro.

High-content phenotypic screening systems are the logical extension of the current efficient, yet low information content, pre-clinical screens for drug discovery. A physiologically accurate in vitro neuromuscular junction (NMJ) screening system would therefore be of tremendous benefit to the study of peripheral neuropathies as well as for basic and applied neuromuscular research. To date, no fully-defined, selective assay system has been developed which would allow investigators to determine the functional output of cultured muscle fibers (myotubes) when stimulated via the NMJ in real time for both acute and chronic applications. Here we present the development of such a phenotypic screening model, along with evidence of NMJ formation and motoneuron initiated neuromuscular transmission in an automated system. Myotubes assembled on silicon cantilevers allowed for measurement of substrate deflection in response to contraction and provided the basis for monitoring the effect of controlled motoneuron stimulation on the contractile behavior. The effect was blocked by treatment with D-tubocurarine, confirming NMJ functionality in this highly multiplexed assay system.

An optimization-based study of equivalent circuit models for representing recordings at the neuron-electrode interface.

Extracellular neuroelectronic interfacing is an emerging field with important applications in the fields of neural prosthetics, biological computation, and biosensors. Traditionally, neuron-electrode interfaces have been modeled as linear point or area contact equivalent circuits but it is now being increasingly realized that such models cannot explain the shapes and magnitudes of the observed extracellular signals. Here, results were compared and contrasted from an unprecedented optimization-based study of the point contact models for an extracellular "on-cell" neuron-patch electrode and a planar neuron-microelectrode interface. Concurrent electrophysiological recordings from a single neuron simultaneously interfaced to three distinct electrodes (intracellular, "on-cell" patch, and planar microelectrode) allowed novel insights into the mechanism of signal transduction at the neuron-electrode interface. After a systematic isolation of the nonlinear neuronal contribution to the extracellular signal, a consistent underestimation of the simulated suprathreshold extracellular signals compared to the experimentally recorded signals was observed. This conclusively demonstrated that the dynamics of the interfacial medium contribute nonlinearly to the process of signal transduction at the neuron-electrode interface. Further, an examination of the optimized model parameters for the experimental extracellular recordings from sub- and suprathreshold stimulations of the neuron-electrode junctions revealed that ionic transport at the "on-cell" neuron-patch electrode is dominated by diffusion whereas at the neuron-microelectrode interface the electric double layer (EDL) effects dominate. Based on this study, the limitations of the equivalent circuit models in their failure to account for the nonlinear EDL and ionic electrodiffusion effects occurring during signal transduction at the neuron-electrode interfaces are discussed.

Design optimization of liquid-phase flow patterns for microfabricated lung on a chip.

Microreactors experience significant deviations from plug flow due to the no-slip boundary condition at the walls of the chamber. The development of stagnation zones leads to widening of the residence time distribution at the outlet of the reactor. A hybrid design optimization process that combines modeling and experiments has been utilized to minimize the width of the residence time distribution in a microreactor. The process was used to optimize the design of a microfluidic system for an in vitro model of the lung alveolus. Circular chambers to accommodate commercial membrane supported cell constructs are a particularly challenging geometry in which to achieve a uniform residence time distribution. Iterative computational fluid dynamics (CFD) simulations were performed to optimize the microfluidic structures for two different types of chambers. The residence time distributions of the optimized chambers were significantly narrower than those of non-optimized chambers, indicating that the final chambers better approximate plug flow. Qualitative and quantitative visualization experiments with dye indicators demonstrated that the CFD results accurately predicted the residence time distributions within the bioreactors. The results demonstrate that such a hybrid optimization process can be used to design microreactors that approximate plug flow for in vitro tissue engineered systems. This technique has broad application for optimization of microfluidic body-on-a-chip systems for drug and toxin studies.

Bax activation by engagement with, then release from, the BH3 binding site of Bcl-xL.

Bcl-2 homologues (such as Bcl-x(L)) promote survival in part through sequestration of "activator" BH3-only proteins (such as Puma), preventing them from directly activating Bax. It is thus assumed that inhibition of interactions between activators and Bcl-x(L) is a prerequisite for small molecules to antagonize Bcl-x(L) and induce cell death. The biological properties, described here of a terphenyl-based alpha-helical peptidomimetic inhibitor of Bcl-x(L) attest that displacement of Bax from Bcl-x(L) is also critical. Terphenyl 14 triggers Bax-dependent but Puma-independent cell death, disrupting Bax/Bcl-x(L) interactions without affecting Puma/Bcl-x(L) interactions. In cell-free assays, binding of inactive Bax to Bcl-x(L), followed by its displacement from Bcl-x(L) by terphenyl 14, produces mitochondrially permeabilizing Bax molecules. Moreover, the peptidomimetic kills yeast cells that express Bax and Bcl-x(L), and it uses Bax-binding Bcl-x(L) to induce mammalian cell death. Likewise, ectopic expression of Bax in yeast and mammalian cells enhances sensitivity to another Bcl-x(L) inhibitor, ABT-737, when Bcl-x(L) is present. Thus, the interaction of Bcl-x(L) with Bax paradoxically primes Bax at the same time it keeps Bax activity in check, and displacement of Bax from Bcl-x(L) triggers an apoptotic signal by itself. This mechanism might contribute to the clinical efficiency of Bcl-x(L) inhibitors.

Controlled exploration of structural databases: the case of farnesyl transferase inhibitors.

Among the newer and promising weapons against cancer are Farnesyl Transferase Inhibitors (FTI). Indeed it is known that the enzyme Farnesyl Transferase (FT), catalyses the prenylation of cysteine residues of several proteins associated with cancer progression, including oncogenic forms of Ras.FTI could alter tumour progression. Exploration of our corporate structural database, based on concepts of diversity and similarity, brought forward a quinazoline-2,4-dione possessing weak farnesyl transferase inhibitory properties. A systematic modulation of structural parameters allowed the elaboration of a series of analogs out of which the most potent compound (21b) exhibited an IC(50) of 19 nM on FT, an excellent cellular activity on the oncogenic H-Ras-transfected cell line Ras #1, as well as selectivity (ratio of IC(50) on parental RAT2 cells/ IC(50) on Ras#1 cells > 2000). Moreover this compound also showed encouraging "in vivo" activity. The synthesis of these new chemical entities as well as the structure activity relationships found following pharmacological testing, is described.

Nanoscale Nonlinear dynamic characterization of the neuron-electrode junction.

Extracellular recordings from neurons using microelectrode and field effect transistor arrays suffer from many problems including low signal to noise ratio, signal attenuation due to counter-ion diffusion from the bulk extracellular medium and a modification of the shape of the cell-generated potentials due to the presence of a highly dispersive dielectric medium in the cell-electrode cleft. Attempts to date to study the neuron-electrode interface have focused on point or area contact linear-equivalent-circuit models. We present here the results obtained from a 'data-true' nonlinear dynamic characterization of the neuron-electrode junction using Volterra-Wiener modeling. For the characterization, NG108-15 cells were cultured on microelectrode arrays and stimulated with broadband Gaussian white noise under voltage clamp mode. A Volterra-Wiener model was then estimated using the input signal and the extracellular signal recorded on the microelectrode. The existence of the second order Wiener kernel confirmed that the recorded extracellular signal had a nonlinear component. The verification of the estimated model was carried out by employing the intracellular action potential as an input to the Volterra-Wiener model and comparing the predicted extracellular response with the corresponding extracellular signal recorded on the microelectrode. We believe that a 'data-true' Volterra-Wiener model of the neuron-electrode junction shall not only facilitate a direct insight into the physicochemical processes taking place at the interface during signal transduction but will also allow one to evolve strategies for engineering the neuron-electrode interface using surface chemical modification of the microelectrodes.