Whole body vibration accelerates the functional recovery of motor nerve components in sciatic nerve-crush injury model rats.

This study aimed to investigate the effect of whole body vibration (WBV) on the sensory and motor nerve components with sciatic nerve injury model rats. Surgery was performed on 21 female Wister rats (6-8 weeks) under intraperitoneal anesthesia. The nerve-crush injuries for the left sciatic nerve were inflicted using a Sugita aneurysm clip. The sciatic nerve model rats were randomly divided into two groups (n=9; control group, n=12; WBV group). The rats in the WBV group walked in the cage with a vibratory stimulus (frequency 50 Hz, 20 min/day, 5 times/wk), while those in the control group walked in the cage without any vibratory stimulus. We used heat stimulation-induced sensory threshold and lumbar magnetic stimulation-induced motor-evoked potentials (MEPs) to measure the sensory and motor nerve components, respectively. Further, morphological measurements, bilateral hind-limb dimension, bilateral gastrocnemius dimension, and weight were evaluated. Consequently, there were no significant differences in the sensory threshold at the injury side between the control and WBV groups. However, at 4 and 6 weeks postoperatively, MEPs latencies in the WBV group were significantly shorter than those in the control group. Furthermore, both sides of the hind-limb dimension at 6 weeks postoperatively, the left side of the gastrocnemius dimension, and both sides of the gastrocnemius weight significantly increased. In conclusion, WBV especially accelerates the functional recovery of motor nerve components in sciatic nerve-crush injury model rats.

Clonal array profiling of scFv-displaying phages for high-throughput discovery of affinity-matured antibody mutants.

"Antibody-breeding" approach potentially generates therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Therein, antibody fragments (e.g., single-chain Fv fragments; scFvs) with a variety of mutations are displayed on bacteriophage to generate diverse phage-antibody libraries. Rare clones with improved functions are then selected via panning against immobilized or tagged target antigens. However, this selection process often ended unsuccessful, mainly due to the biased propagation of phage-antibody clones and the competition with a large excess of undesirable clones with weaker affinities. To break radically from such panning-inherent problems, we developed a novel method, clonal array profiling of scFv-displaying phages (CAP), in which colonies of the initial bacterial libraries are examined one-by-one in microwells. Progenies of scFv-displaying phages generated are, if show sufficient affinity to target antigen, captured in the microwell via pre-coated antigen and detected using a luciferase-fused anti-phage scFv. The advantage of CAP was evidenced by its application with a small error-prone-PCR-based library (~ 105 colonies) of anti-cortisol scFvs. Only two operations, each surveying only ~ 3% of the library (9,400 colonies), provided five mutants showing 32-63-fold improved Ka values (> 1010 M-1), compared with the wild-type scFv (Ka = 3.8 × 108 M-1), none of which could be recovered via conventional panning procedures operated for the entire library.

Quantitative Analysis of Velopharyngeal Movement by Applying Principal Component Analysis to Range Images Produced by a Three-Dimensional Endoscope.

Objectives: The purpose of this study was to develop a new technique for analyzing velopharyngeal movement and to investigate its utility. Materials and Methods: Velopharyngeal motion of 20 normal individuals was analyzed. A three-dimensional (3D) endoscope was inserted into the oral cavity, and the movement of the soft palate was measured using an exclusive fixation device. Range images of the soft palate were produced during phonation of the Japanese vowel /a/, and virtual grids were then overlaid on these images. Principal component analyses were applied to the 3D coordinates of the intersections of the virtual grids. The centers of gravity of the virtual grids were calculated, and the magnitude of the shift of the grid intersections during phonation was calculated. Results: The first and the second principal component scores were responsible for the upper posterior direction and the upper direction, respectively. The average magnitude of the shift of the center of gravity was 4.75 mm in males and 4.33 mm in females. Conclusions: Quantitative analysis of velopharyngeal movement was achieved by a method of applying principal component analysis (PCA) to the range images obtained from a 3D endoscope. There was no sex difference in velopharyngeal movement.

Quantitative analysis of velopharyngeal movement using a stereoendoscope: accuracy and reliability of range images.

OBJECTIVE: We developed a novel method of producing accurate range images of the velopharynx using a three-dimensional (3D) endoscope to obtain detailed measurements of velopharyngeal movements. The purpose of the present study was to determine the relationship between the distance from the endoscope to an object, elucidate the measurement accuracy along the temporal axes, and determine the degree of blurring when using a jig to fix the endoscope. METHODS: An endoscopic measuring system was developed in which a pattern projection system was incorporated into a commercially available 3D endoscope. After correcting the distortion of the camera images, range images were produced using pattern projection to achieve stereo matching. Graph paper was used to measure the appropriate distance from the camera to an object, the mesial buccal cusp of the right maxillary first molar was measured to clarify the range image stability, and an electric actuator was used to evaluate the measurement accuracy along the temporal axes. RESULTS: The measurement error was substantial when the distance from the camera to the subject was >6.5 cm. The standard error of the 3D coordinate value produced from 30 frames was within 0.1 mm (range, 0.01-0.08 mm). The measurement error of the temporal axes was 9.16% in the horizontal direction and 9.27% in the vertical direction. CONCLUSION: The optimal distance from the camera to an object is <6.5 cm. The present endoscopic measuring system can provide stable range images of the velopharynx when using an appropriate fixation method and enables quantitative analysis of velopharyngeal movements.

A case of squamous cell carcinoma arising from branchial cleft cyst.

Squamous cell carcinoma throughout the epithelium of a lateral cervical cyst is considered extremely rare. This report describes an additional case of this very rare clinical condition. A 70-year-old man presented with a well-defined, immobile, painless mass in the left neck that was excised with a diagnosis of branchial cyst. Histologic findings of the excised specimen were lateral branchial cyst with high-grade dysplasia and carcinoma of the squamous epithelial lining. The patient was followed for more than 2 years 10 months and no evidence of recurrence or other cancer has been found.

Insulator-based dielectrophoresis with ß-galactosidase in nanostructured devices.

Insulator-based dielectrophoresis (iDEP) has been explored as a powerful analytical technique in recent years. Unlike with larger entities such as cells, bacteria or organelles, the mechanism of iDEP transport of proteins remains little explored. In this work, we extended the pool of proteins investigated with iDEP in nanostructured devices with ß-galactosidase. Our work indicates that ß-galactosidase shows concentration due to negative DEP which we compare to DEP response of immunoglobulin G (IgG) encapsulated in micelles also showing negative DEP. Experimental observations are further compared with numerical simulations to elucidate the influence of electrokinetic transport and the magnitude of DEP mobility. Numerical simulations suggest that the DEP mobility calculated using the classical model underestimates the actual contribution of DEP on the experimentally monitored concentration effect of proteins. Moreover, we observed a unique voltage dependent ß-galactosidase concentration which we attribute to an additional factor influencing the protein concentration at the nanoconstrictions, namely ion concentration polarization. Our work aids in understanding factors influencing protein iDEP transport which is required for the future development of protein preconcentration or separation methods based on iDEP.

Gender differences in posed smiles using principal component analysis.

OBJECTIVES: The purpose of this study was to clarify gender differences in posed smiles using principal component analysis (PCA). MATERIALS AND METHODS: Fourteen adult volunteers, 7 males and 7 females, were enrolled. Using the motion analyzing system we developed, range images and 5 × 5 virtual grids were produced across the whole sequence while the volunteers were asked to smile. Two sets of all intersections of the virtual grids captured while the subject was smiling were regarded as PCA variables. Discriminate analysis was then applied to compare the males and females. RESULTS: The first and second principal component scores (PCSs) were plotted on the x-axis and y-axis, respectively. The center of gravity of the PCSs is shown by the plus on the x-axis and minus on the y-axis for the males and by the minus on the x-axis and the plus on the y-axis for the females. Discriminate analyses of the PCSs revealed a correct gender classification rate of 74.4% for posed smiles. CONCLUSIONS: While the sample size is too small to extrapolate from these results, we can conclude that PCA can be used to identify gender differences while smiling.

Different therapeutic mechanisms of rigid and semi-rigid mandibular repositioning devices in obstructive sleep apnea syndrome.

To clarify the mechanisms of rigid and semi-rigid mandibular repositioning devices (MRDs) in obstructive sleep apnea syndrome (OSAS), seven and 13 patients received rigid and semi-rigid MRDs, respectively. Each patient underwent polysomnographic and computed tomographic examinations at the initial consultation and after symptom improvement. Three-dimensional models of the upper airway (hard palate level to epiglottic base) were reconstructed by image processing software (Mimics version 14.2) to measure airway morphology. The mean age and body mass index were 58.1 years and 24.8 kg/m(2), respectively, in the rigid MRD group and 57.9 years and 23.2 kg/m(2), respectively, in the semi-rigid MRD group. The apnea-hypopnea index significantly improved (P < 0.05, Wilcoxon signed-rank test) from 22.0 to 8.9 and 20.5 to 11.5 events per hour of sleep in the respective groups. The cross-sectional areas measured at the epiglottic tip (from 2.0 to 2.6 cm(2)) and hard palate (from 2.6 to 3.3 cm(2)) levels also increased in the respective groups (P < 0.05). However, airway volume, cross-sectional area measured at the uvular tip level, and anteroposterior and transverse diameters of the airway were not significantly different. In conclusion, both types of MRDs improve respiratory status, but they affect different parts of the airway.

Temporal and spatial temperature measurement in insulator-based dielectrophoretic devices.

Insulator-based dielectrophoresis is a relatively new analytical technique with a large potential for a number of applications, such as sorting, separation, purification, fractionation, and preconcentration. The application of insulator-based dielectrophoresis (iDEP) for biological samples, however, requires the precise control of the microenvironment with temporal and spatial resolution. Temperature variations during an iDEP experiment are a critical aspect in iDEP since Joule heating could lead to various detrimental effects hampering reproducibility. Additionally, Joule heating can potentially induce thermal flow and more importantly can degrade biomolecules and other biological species. Here, we investigate temperature variations in iDEP devices experimentally employing the thermosensitive dye Rhodamin B (RhB) and compare the measured results with numerical simulations. We performed the temperature measurement experiments at a relevant buffer conductivity range commonly used for iDEP applications under applied electric potentials. To this aim, we employed an in-channel measurement method and an alternative method employing a thin film located slightly below the iDEP channel. We found that the temperature does not deviate significantly from room temperature at 100 µS/cm up to 3000 V applied such as in protein iDEP experiments. At a conductivity of 300 µS/cm, such as previously used for mitochondria iDEP experiments at 3000 V, the temperature never exceeds 34 °C. This observation suggests that temperature effects for iDEP of proteins and mitochondria under these conditions are marginal. However, at larger conductivities (1 mS/cm) and only at 3000 V applied, temperature increases were significant, reaching a regime in which degradation is likely to occur. Moreover, the thin layer method resulted in lower temperature enhancement which was also confirmed with numerical simulations. We thus conclude that the thin film method is preferable providing closer agreement with numerical simulations and further since it does not depend on the iDEP channel material. Overall, our study provides a thorough comparison of two experimental techniques for direct temperature measurement, which can be adapted to a variety of iDEP applications in the future. The good agreement between simulation and experiment will also allow one to assess temperature variations for iDEP devices prior to experiments.

Three-dimensional intra-rater and inter-rater reliability during a posed smile using a video-based motion analyzing system.

OBJECTIVES: The purpose of this study was to determine the three-dimensional reproducibility of lip movement during a posed smile using a video-based motion analyzing system. MATERIALS AND METHODS: In six adult volunteers (4 males and 2 females), the lip motions during a posed smile were recorded six times. Using our recently-developed motion analyzing system, range images were produced across the whole sequence during the posed smile. Virtual grids of 5 × 5 were fitted onto the surfaces, and the three-dimensional coordinates of the intersections of these grids were then computed. The magnitude of the shift of the intersections during smiling was calculated and summed in each area. Intraclass correlation coefficients (ICC), ICC (1,1) for intra-rater reliability and ICC (2,1) for inter-rater reliability were calculated. The number of repeated measurements necessary for an ICC level beyond 0.8 was determined using the formula of Spearman-Brown. RESULTS: The ICC (1,1) and ICC (2,1) ranged from 0.71 to 0.83 and from 0.77 to 0.99, respectively. The number of repeated measurements necessary for an ICC beyond 0.8 was 2. CONCLUSIONS: From the present study, both the three-dimensional intra-rater and inter-rater reliabilities during a posed smile were considered to be relatively high, and enough reliability could be expected by calculating the average of the values measured two times. However, the sample size was very small, this could not be generalized simplistically.

Range image of the velopharynx produced using a 3-D endoscope with pattern projection.

OBJECTIVES/HYPOTHESIS: To measure movements of the velopharynx in detail, a novel method of producing range images of the velopharynx was developed using a 3-D endoscope. The purpose of this paper is to introduce this system and to clarify its accuracy. STUDY DESIGN: The standard errors of repeated measurements, intraclass correlation coefficients (ICC), ICC(1,1) for intrarater reliability, and ICC(2,1) for interrater reliability were measured using a phantom of the nasopharynx. METHODS: An endoscopic measuring system was developed in which a pattern projection system was incorporated into a commercially available 3-D endoscope. Right and left images of the endoscope were integrated into one video file and digitized at a horizontal and vertical resolution of 640 × 480 pixels, as odd and even scanning lines, respectively. After separation of the video file into right and left images by interlace interpolation, correcting the distortion of the camera images, rectifying, and stereo-matching range images of the velopharynx were produced. The distances between two points, which were marked at an interval of 5 mm on the uvula and the pharynx of the phantom, were measured. RESULTS: The standard errors of repeated measurements were 0.02 horizontally and 0.01 vertically. The ICC(1,1) and ICC(2,1) were 0.83 and 0.94, and both correlation coefficients were considered to be "almost perfect." CONCLUSION: The present endoscopic measuring system provided relatively accurate and reliable range images of the velopharynx, and enabled quantitative analysis of movements of the velopharynx.

Protein dielectrophoresis: advances, challenges, and applications.

Protein dielectrophoresis (DEP) has the potential to play an important role as a manipulation, fractionation, preconcentration, and separation method in bioanalysis and as manipulation tool for nanotechnological applications. The first demonstrations of protein DEP have been reported almost 20 years ago. Since then various experimental realizations to manipulate proteins by DEP as well as more targeted applications employing protein DEP have been demonstrated. This review summarizes the experimental studies in the field of protein DEP trapping and focusing as well as specific applications in separation, molecular patterning, on bioprobes and biosensors. While a comprehensive theoretical model describing protein DEP is still lacking we also attempt to provide an overview of the factors influencing protein DEP and relate to currently available theoretical models. We further point out the variations in experimental conditions used in the past to study the somewhat 20 proteins as well as the implications of protein molecular structure to the DEP response.

Tuning direct current streaming dielectrophoresis of proteins.

Dielectrophoresis (DEP) of biomolecules has large potential to serve as a novel selectivity parameter for bioanalytical methods such as (pre)concentration, fractionation, and separation. However, in contrast to well-characterized biological cells and (nano)particles, the mechanism of protein DEP is poorly understood, limiting bioanalytical applications for proteins. Here, we demonstrate a detailed investigation of factors influencing DEP of diagnostically relevant immunoglobulin G (IgG) molecules using insulator-based DEP (iDEP) under DC conditions. We found that the pH range in which concentration of IgG due to streaming iDEP occurs without aggregate formation matches the pH range suitable for immunoreactions. Numerical simulations of the electrokinetic factors pertaining to DEP streaming in this range further suggested that the protein charge and electroosmotic flow significantly influence iDEP streaming. These predictions are in accordance with the experimentally observed pH-dependent iDEP streaming profiles as well as the determined IgG molecular properties. Moreover, we observed a transition in the streaming behavior caused by a change from positive to negative DEP induced through micelle formation for the first time experimentally, which is in excellent qualitative agreement with numerical simulations. Our study thus relates molecular immunoglobulin properties to observed iDEP, which will be useful for the future development of protein (pre)concentration or separation methods based on DEP.

Quantification of pH gradients and implications in insulator-based dielectrophoresis of biomolecules.

Direct current (DC) insulator-based dielectrophoretic (iDEP) microdevices have the potential to replace traditional alternating current dielectrophoretic devices for many cellular and biomolecular separation applications. The use of large DC fields suggest that electrode reactions and ion transport mechanisms can become important and impact ion distributions in the nanoliters of fluid in iDEP microchannels. This work tracked natural pH gradient formation in a 100 µm wide, 1 cm-long microchannel under applicable iDEP protein manipulation conditions. Using fluorescence microscopy with the pH-sensitive dye FITC Isomer I and the pH-insensitive dye TRITC as a reference, pH was observed to drop drastically in the microchannels within 1 min in a 3000 V/cm electric field; pH drops were observed in the range of 6-10 min within a 100 V/cm electric field and varied based on the buffer conductivity. To address concerns of dye transport impacting intensity data, electrokinetic mobilities of FITC were carefully examined and found to be (i) toward the anode and (ii) 1 to 2 orders of magnitude smaller than H⁺ transport which is responsible for pH drops from the anode toward the cathode. COMSOL simulations of ion transport showed qualitative agreement with experimental results. The results indicate that pH changes are severe enough and rapid enough to influence the net charge of a protein or cause aggregation during iDEP experiments. The results also elucidate reasonable time periods over which the phosphate buffering capacity can counter increases in H⁺ and OH⁻ for unperturbed iDEP manipulations.

Immunoglobulin G and bovine serum albumin streaming dielectrophoresis in a microfluidic device.

Dielectrophoresis (DEP) has demonstrated to be a versatile tool to manipulate micro- and nanoparticles with applications for positioning, separation and fractionation. Recent developments of DEP have also shown that DEP can be used for the manipulation of biomolecules, such as DNA. Here, we focus on the manipulation of proteins using insulator-based dielectrophoresis (iDEP). We designed suitable post arrays in a microfluidic channel and use numerical simulations to calculate the electric field distribution as well as concentration of proteins according to a convection-diffusion model for both negative and positive DEP. Experimentally, we find DEP trapping of mainly protein aggregates in phosphate buffer. However, when adding a charged zwitterionic detergent, we observed DEP streamlining of immunoglobulin G (IgG) and bovine serum albumin (BSA). Our experimental observations are in excellent agreement with numerical simulations and indicate positive DEP behavior of IgG and BSA under the employed experimental conditions. Our results demonstrate DEP streaming of proteins in an iDEP device for the first time and indicate the potential of protein DEP for separation and fractionation.