Gait analysis of the hind limb in Labrador Retrievers with and without cranial cruciate ligament disease

Cranial cruciate ligament (CCL) deficiency is the leading cause of lameness affecting the stifle joints of large breed dogs, especially Labrador Retrievers. Although CCL disease has been studied extensively, its exact pathogenesis and the primary cause leading to CCL rupture remain controversial. However, weakening secondary to repetitive microtrauma is currently believed to cause the majority of CCL instabilities diagnosed in dogs. Techniques of gait analysis have become the most productive tools to investigate normal and pathological gait in human and veterinary subjects. The inverse dynamics analysis approach models the limb as a series of connected linkages and integrates morphometric data to yield information about the net joint moment, patterns of muscle power and joint reaction forces. The results of these studies have greatly advanced our understanding of the pathogenesis of joint diseases in humans. A muscular imbalance between the hamstring and quadriceps muscles has been suggested as a cause for anterior cruciate ligament rupture in female athletes. Based on these findings, neuromuscular training programs leading to a relative risk reduction of up to 80% has been designed. In spite of the cost and morbidity associated with CCL disease and its management, very few studies have focused on the inverse dynamics gait analysis of this condition in dogs. The general goals of this research were (1) to further define gait mechanism in Labrador Retrievers with and without CCL-deficiency, (2) to identify individual dogs that are susceptible to CCL disease, and (3) to characterize their gait. The mass, location of the center of mass (COM), and mass moment of inertia of hind limb segments were calculated using a noninvasive method based on computerized tomography of normal and CCL-deficient Labrador Retrievers. Regression models were developed to determine predictive equations to estimate body segment parameters on the basis of simple morphometric measurements, providing a basis for nonterminal studies of inverse dynamics of the hind limbs in Labrador Retrievers. Kinematic, ground reaction forces (GRF) and morphometric data were combined in an inverse dynamics approach to compute hock, stifle and hip net moments, powers and joint reaction forces (JRF) while trotting in normal, CCL-deficient or sound contralateral limbs. Reductions in joint moment, power, and loads observed in CCL-deficient limbs were interpreted as modifications adopted to reduce or avoid painful mobilization of the injured stifle joint. Lameness resulting from CCL disease affected predominantly reaction forces during the braking phase and the extension during push-off. Kinetics also identified a greater joint moment and power of the contralateral limbs compared with normal, particularly of the stifle extensor muscles group, which may correlate with the lameness observed, but also with the predisposition of contralateral limbs to CCL deficiency in dogs. For the first time, surface EMG patterns of major hind limb muscles during trotting gait of healthy Labrador Retrievers were characterized and compared with kinetic and kinematic data of the stifle joint. The use of surface EMG highlighted the co-contraction patterns of the muscles around the stifle joint, which were documented during transition periods between flexion and extension of the joint, but also during the flexion observed in the weight bearing phase. Identification of possible differences in EMG activation characteristics between healthy patients and dogs with or predisposed to orthopedic and neurological disease may help understanding the neuromuscular abnormality and gait mechanics of such disorders in the future. Conformation parameters, obtained from femoral and tibial radiographs, hind limb CT images, and dual-energy X-ray absorptiometry, of hind limbs predisposed to CCL deficiency were compared with the conformation parameters from hind limbs at low risk. A combination of tibial plateau angle and femoral anteversion angle measured on radiographs was determined optimal for discriminating predisposed and non-predisposed limbs for CCL disease in Labrador Retrievers using a receiver operating characteristic curve analysis method. In the future, the tibial plateau angle (TPA) and femoral anteversion angle (FAA) may be used to screen dogs suspected of being susceptible to CCL disease. Last, kinematics and kinetics across the hock, stifle and hip joints in Labrador Retrievers presumed to be at low risk based on their radiographic TPA and FAA were compared to gait data from dogs presumed to be predisposed to CCL disease for overground and treadmill trotting gait. For overground trials, extensor moment at the hock and energy generated around the hock and stifle joints were increased in predisposed limbs compared to non predisposed limbs. For treadmill trials, dogs qualified as predisposed to CCL disease held their stifle at a greater degree of flexion, extended their hock less, and generated more energy around the stifle joints while trotting on a treadmill compared with dogs at low risk. This characterization of the gait mechanics of Labrador Retrievers at low risk or predisposed to CCL disease may help developing and monitoring preventive exercise programs to decrease gastrocnemius dominance and strengthened the hamstring muscle group.

Plasma diagnostics and ITO film deposition by RF-assisted closed-field dual magnetron system

Deposition of indium tin oxide (ITO) among various transparent conductive materials on flexible organic substrates has been intensively investigated among academics and industrials for a whole new array of imaginative optoelectronic products. One critical challenge coming with the organic materials is their poor thermal endurances, considering that the process currently used to produce industry-standard ITO usually involves relatively high substrate temperature in excess of 200°C and post-annealing. A lower processing temperature is thus demanded, among other desires of high deposition rate, large substrate area, good uniformity, and high quality of the deposited materials. For this purpose, we developed an RF-assisted closed-field dual magnetron sputtering system. The “prototype” system consists of a 3-inch unbalanced dual magnetron operated at a closed-field configuration. An RF coil was fabricated and placed between the two magnetron cathodes to initiate a secondary plasma. The concept is to increase the ionization faction with the RF enhancement and utilize the ion energy instead of thermal energy to facilitate the ITO film growth. The closed-field unbalanced magnetrons create a plasma in the intervening region rather than confine it near the target, thus achieving a large-area processing capability. An RF-compensated Langmuir probe was used to characterize and compare the plasmas in mirrored balanced and closed-field unbalanced magnetron configurations. The spatial distributions of the electron density ne and electron temperature Te were measured. The density profiles reflect the shapes of the plasma. Rather than intensively concentrated to the targets/cathodes in the balanced magnetrons, the plasma is more dispersive in the closed-field mode with a twice higher electron density in the substrate region. The RF assistance significantly enhances ne by one or two orders of magnitude higher. The effect of various other parameters, such as pressure, on the plasma was also studied. The ionization fractions of the sputtered atoms were measured using a gridded energy analyzer (GEA) combined with a quartz crystal microbalance (QCM). The presence of the RF plasma effectively increases the ITO ionization fraction to around 80% in both the balanced and closed-field unbalanced configurations. The ionization fraction also varies with pressure, maximizing at 5-10 mTorr. The study of the ionization not only facilitates understanding the plasma behaviors in the RF-assisted magnetron sputtering, but also provides a criterion for optimizing the film deposition process. ITO films were deposited on both glass and plastic (PET) substrates in the 3-inch RF-assisted closed-field magnetrons. The electrical resistivity and optical transmission transparency of the ITO films were measured. Appropriate RF assistance was shown to dramatically reduce the electrical resistivity. An ITO film with a resistivity of 1.2×10-3 Ω-cm and a visible light transmittance of 91% was obtained with a 225 W RF enhancement, while the substrate temperature was monitored as below 110°C. X-ray photoelectron spectroscopy (XPS) was employed to confirm the ITO film stoichiometry. The surface morphology of the ITO films and its effect on the film properties were studied using atomic force microscopy (AFM). The prototype of RF-assisted closed-field magnetron was further extended to a larger rectangular shaped dual magnetron in a flat panel display manufacturing system. Similar improvement of the ITO film conductivities by the auxiliary RF was observed on the large-area PET substrates. Meanwhile, significant deposition rates of 25-42 nm/min were achieved.

Single-molecule fluorescence resonance energy transfer study of SNARE-mediated membrane fusion

This is a comprehensive study of protein-mediated membrane fusion through single-molecule fluorescence resonance energy transfer (smFRET). Membrane fusion is one of the important cellular processes by which two initially distinct lipid bilayers merge their hydrophobic cores, resulting in one interconnected structure. For example, exocytosis, fertilization of an egg by a sperm and communication between neurons are a few among many processes that rely on some form of fusion. Proteins called soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) play a central role in fusion processes which is also regulated by many accessory proteins, such as synaptotagmin, complexin and Munc18. By a new lipid mixing method at the single-vesicle level, we are able to accurately detect different stages of SNARE-mediated membrane fusion including docking, hemi and full fusion via FRET value of single donor/acceptor vesicle pair. Through this single-vesicle lipid mixing assay, we discovered the vesicle aggregation induced by C2AB/Ca2+, the dual function of complexin, and the fusion promotion role of Munc18/SNARE-core binding mode. While this new method provides the information regarding the extent of the ensemble lipid mixing, the fusion pore opening between two vesicular cavities and the interaction between proteins cannot be detected. In order to overcome these limitations, we then developed a single-vesicle content mixing method to reveal the key factor of pore expansion by detecting the FRET change of dual-labeled DNA probes encapsulated in vesicles. Through our single-vesicle content mixing assay, we found the fusion pore expansion role of yeast SNAREs as well as neuronal SNAREs plus synaptotagmin 1.