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High-definition transcranial direct current stimulation (HD-tDCS) has recently been developed as a noninvasive brain stimulation approach that increases the accuracy of current delivery to the brain by using arrays of smaller 'high-definition' electrodes, instead of the larger pad-electrodes of conventional tDCS. Targeting is achieved by energizing electrodes placed in predetermined configurations. One of these is the 4x1-ring configuration. In this approach, a center ring electrode (anode or cathode) overlying the target cortical region is surrounded by four return electrodes, which help circumscribe the area of stimulation. Delivery of 4x1-ring HD-tDCS is capable of inducing significant neurophysiological and clinical effects in both healthy subjects and patients.

Furthermore, its tolerability is supported by studies using intensities as high as 2.0 milliamperes for up to twenty minutes. Even though 4x1 HD-tDCS is simple to perform, correct electrode positioning is important in order to accurately stimulate target cortical regions and exert its neuromodulatory effects.

The use of electrodes and hardware that have specifically been tested for HD-tDCS is critical for safety and tolerability. Given that most published studies on 4x1 HD-tDCS have targeted the primary motor cortex (M1), particularly for pain-related outcomes, the purpose of this article is to systematically describe its use for M1 stimulation, as well as the considerations to be taken for safe and effective stimulation. However, the methods outlined here can be adapted for other HD-tDCS configurations and cortical targets. A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system.

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These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid.

Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

Improved methods for assessing bulbar impairment are necessary for expediting diagnosis of bulbar dysfunction in ALS, for predicting disease progression across speech subsystems, and for addressing the critical need for sensitive outcome measures for ongoing experimental treatment trials. To address this need, we are obtaining longitudinal profiles of bulbar impairment in 100 individuals based on a comprehensive instrumentation-based assessment that yield objective measures. Using instrumental approaches to quantify speech-related behaviors is very important in a field that has primarily relied on subjective, auditory-perceptual forms of speech assessment 1. Our assessment protocol measures performance across all of the speech subsystems, which include respiratory, phonatory (laryngeal), resonatory (velopharyngeal), and articulatory. The articulatory subsystem is divided into the facial components (jaw and lip), and the tongue. Prior research has suggested that each speech subsystem responds differently to neurological diseases such as ALS. The current protocol is designed to test the performance of each speech subsystem as independently from other subsystems as possible.

The speech subsystems are evaluated in the context of more global changes to speech performance. These speech system level variables include speaking rate and intelligibility of speech. The protocol requires specialized instrumentation, and commercial and custom software. The respiratory, phonatory, and resonatory subsystems are evaluated using pressure-flow (aerodynamic) and acoustic methods. The articulatory subsystem is assessed using 3D motion tracking techniques.

The objective measures that are used to quantify bulbar impairment have been well established in the speech literature and show sensitivity to changes in bulbar function with disease progression. The result of the assessment is a comprehensive, across-subsystem performance profile for each participant. The profile, when compared to the same measures obtained from healthy controls, is used for diagnostic purposes. Currently, we are testing the sensitivity and specificity of these measures for diagnosis of ALS and for predicting the rate of disease progression. In the long term, the more refined endophenotype of bulbar ALS derived from this work is expected to strengthen future efforts to identify the genetic loci of ALS and improve diagnostic and treatment specificity of the disease as a whole.

The objective assessment that is demonstrated in this video may be used to assess a broad range of speech motor impairments, including those related to stroke, traumatic brain injury, multiple sclerosis, and Parkinson disease. Rat tail tendons (RTTs) are a common biological model used in experimental in vitro studies in the fields of tendon physiology and tendinopathy. Working with those tissues is challenging because they are very fragile, and until now there was no rigorously detailed protocol for their isolation. Faced with these challenges, we have developed methods and instruments to facilitate manipulation of RTTs and control tissue viability, sterility and integrity. This article describes the experimental procedures used to prepare RTTs for biomechanical and mechanobiological studies.

Our work is divided into four main steps: extraction, cross-sectional area measurement, rinsing and loading into the bioreactor chamber. At each step, all procedures, materials and manipulations are presented in detail so that they can be easily reproduced.

Moreover, the specific instruments developed are presented: a manipulation plate used to segregate RTTs, an optic micrometer to position the tissue during the cross-sectional area measurement and an anchoring system to attach the RTTs onto a bioreactor. Finally, we describe the results obtained after multiple tests to validate our methods. The viability, sterility and integrity evaluations demonstrate that our procedures are sufficiently rigorous for manipulations of fragile tissues such as rat tail tendons. Reference scanners are used in dental medicine to verify a lot of procedures. The main interest is to verify impression methods as they serve as a base for dental restorations.

The current limitation of many reference scanners is the lack of accuracy scanning large objects like full dental arches, or the limited possibility to assess detailed tooth surfaces. A new reference scanner, based on focus variation scanning technique, was evaluated with regards to highest local and general accuracy. A specific scanning protocol was tested to scan original tooth surface from dental impressions. Also, different model materials were verified. The results showed a high scanning accuracy of the reference scanner with a mean deviation of 5.3 ± 1.1 µm for trueness and 1.6 ± 0.6 µm for precision in case of full arch scans.

Current dental impression methods showed much higher deviations (trueness: 20.4 ± 2.2 µm, precision: 12.5 ± 2.5 µm) than the internal scanning accuracy of the reference scanner. Smaller objects like single tooth surface can be scanned with an even higher accuracy, enabling the system to assess erosive and abrasive tooth surface loss.

The reference scanner can be used to measure differences for a lot of dental research fields. The different magnification levels combined with a high local and general accuracy can be used to assess changes of single teeth or restorations up to full arch changes. Precise measurement of neurological and neuropsychological impairment and disability in multiple sclerosis is challenging. We report a new test, the Multiple Sclerosis Performance Test (MSPT), which represents a new approach to quantifying MS related disability.

The MSPT takes advantage of advances in computer technology, information technology, biomechanics, and clinical measurement science. The resulting MSPT represents a computer-based platform for precise, valid measurement of MS severity. Based on, but extending the Multiple Sclerosis Functional Composite (MSFC), the MSPT provides precise, quantitative data on walking speed, balance, manual dexterity, visual function, and cognitive processing speed.

The MSPT was tested by 51 MS patients and 49 healthy controls (HC). MSPT scores were highly reproducible, correlated strongly with technician-administered test scores, discriminated MS from HC and severe from mild MS, and correlated with patient reported outcomes. Measures of reliability, sensitivity, and clinical meaning for MSPT scores were favorable compared with technician-based testing.

The MSPT is a potentially transformative approach for collecting MS disability outcome data for patient care and research. Because the testing is computer-based, test performance can be analyzed in traditional or novel ways and data can be directly entered into research or clinical databases. The MSPT could be widely disseminated to clinicians in practice settings who are not connected to clinical trial performance sites or who are practicing in rural settings, drastically improving access to clinical trials for clinicians and patients. The MSPT could be adapted to out of clinic settings, like the patient’s home, thereby providing more meaningful real world data. The MSPT represents a new paradigm for neuroperformance testing.

This method could have the same transformative effect on clinical care and research in MS as standardized computer-adapted testing has had in the education field, with clear potential to accelerate progress in clinical care and research. Diffusion tensor imaging (DTI) techniques provide information on the microstructural processes of the cerebral white matter (WM) in vivo. The present applications are designed to investigate differences of WM involvement patterns in different brain diseases, especially neurodegenerative disorders, by use of different DTI analyses in comparison with matched controls. DTI data analysis is performed in a variate fashion, i.e. Voxelwise comparison of regional diffusion direction-based metrics such as fractional anisotropy (FA), together with fiber tracking (FT) accompanied by tractwise fractional anisotropy statistics (TFAS) at the group level in order to identify differences in FA along WM structures, aiming at the definition of regional patterns of WM alterations at the group level. Transformation into a stereotaxic standard space is a prerequisite for group studies and requires thorough data processing to preserve directional inter-dependencies.

The present applications show optimized technical approaches for this preservation of quantitative and directional information during spatial normalization in data analyses at the group level. On this basis, FT techniques can be applied to group averaged data in order to quantify metrics information as defined by FT. Additionally, application of DTI methods, i.e. Differences in FA-maps after stereotaxic alignment, in a longitudinal analysis at an individual subject basis reveal information about the progression of neurological disorders.

Further quality improvement of DTI based results can be obtained during preprocessing by application of a controlled elimination of gradient directions with high noise levels. In summary, DTI is used to define a distinct WM pathoanatomy of different brain diseases by the combination of whole brain-based and tract-based DTI analysis. HIV-1 drug resistance has the potential to seriously compromise the effectiveness and impact of antiretroviral therapy (ART). As ART programs in sub-Saharan Africa continue to expand, individuals on ART should be closely monitored for the emergence of drug resistance. Surveillance of transmitted drug resistance to track transmission of viral strains already resistant to ART is also critical. Unfortunately, drug resistance testing is still not readily accessible in resource limited settings, because genotyping is expensive and requires sophisticated laboratory and data management infrastructure.

An open access genotypic drug resistance monitoring method to manage individuals and assess transmitted drug resistance is described. The method uses free open source software for the interpretation of drug resistance patterns and the generation of individual patient reports. The genotyping protocol has an amplification rate of greater than 95% for plasma samples with a viral load >1,000 HIV-1 RNA copies/ml. The sensitivity decreases significantly for viral loads.

We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion. Arcgis License File Crack Mixtape. Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes.

The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo.

Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians. Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself. The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Many biological and clinical studies require the longitudinal study and analysis of morphology and function with cellular level resolution. Traditionally, multiple experiments are run in parallel, with individual samples removed from the study at sequential time points for evaluation by light microscopy.

Several intravital techniques have been developed, with confocal, multiphoton, and second harmonic microscopy all demonstrating their ability to be used for imaging in situ 1. With these systems, however, the required infrastructure is complex and expensive, involving scanning laser systems and complex light sources. Here we present a protocol for the design and assembly of a high-resolution microendoscope which can be built in a day using off-the-shelf components for under US$5,000. The platform offers flexibility in terms of image resolution, field-of-view, and operating wavelength, and we describe how these parameters can be easily modified to meet the specific needs of the end user. We and others have explored the use of the high-resolution microendoscope (HRME) in in vitro cell culture 2-5, in excised 6 and living animal tissues 2,5, and in human tissues in vivo 2,7. Users have reported the use of several different fluorescent contrast agents, including proflavine 2-4, benzoporphyrin-derivative monoacid ring A (BPD-MA) 5, and fluoroscein 6,7, all of which have received full, or investigational approval from the FDA for use in human subjects. High-resolution microendoscopy, in the form described here, may appeal to a wide range of researchers working in the basic and clinical sciences.

The technique offers an effective and economical approach which complements traditional benchtop microscopy, by enabling the user to perform high-resolution, longitudinal imaging in situ. The scaled subprofile model (SSM) 1-4 is a multivariate PCA-based algorithm that identifies major sources of variation in patient and control group brain image data while rejecting lesser components ( Figure 1).

Applied directly to voxel-by-voxel covariance data of steady-state multimodality images, an entire group image set can be reduced to a few significant linearly independent covariance patterns and corresponding subject scores. Each pattern, termed a group invariant subprofile (GIS), is an orthogonal principal component that represents a spatially distributed network of functionally interrelated brain regions.

Large global mean scalar effects that can obscure smaller network-specific contributions are removed by the inherent logarithmic conversion and mean centering of the data 2,5,6. Subjects express each of these patterns to a variable degree represented by a simple scalar score that can correlate with independent clinical or psychometric descriptors 7,8. Using logistic regression analysis of subject scores ( i.e.

Pattern expression values), linear coefficients can be derived to combine multiple principal components into single disease-related spatial covariance patterns, i.e. Composite networks with improved discrimination of patients from healthy control subjects 5,6. Cross-validation within the derivation set can be performed using bootstrap resampling techniques 9. Forward validation is easily confirmed by direct score evaluation of the derived patterns in prospective datasets 10. Once validated, disease-related patterns can be used to score individual patients with respect to a fixed reference sample, often the set of healthy subjects that was used (with the disease group) in the original pattern derivation 11. These standardized values can in turn be used to assist in differential diagnosis 12,13 and to assess disease progression and treatment effects at the network level 7,14-16.

We present an example of the application of this methodology to FDG PET data of Parkinson's Disease patients and normal controls using our in-house software to derive a characteristic covariance pattern biomarker of disease. It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility.

The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software.

Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3.

Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature.

Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance 6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain 8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system. In cardiac myocytes a complex network of membrane tubules - the transverse-axial tubule system (TATS) - controls deep intracellular signaling functions. While the outer surface membrane and associated TATS membrane components appear to be continuous, there are substantial differences in lipid and protein content.

In ventricular myocytes (VMs), certain TATS components are highly abundant contributing to rectilinear tubule networks and regular branching 3D architectures. It is thought that peripheral TATS components propagate action potentials from the cell surface to thousands of remote intracellular sarcoendoplasmic reticulum (SER) membrane contact domains, thereby activating intracellular Ca 2+ release units (CRUs).

In contrast to VMs, the organization and functional role of TATS membranes in atrial myocytes (AMs) is significantly different and much less understood. Taken together, quantitative structural characterization of TATS membrane networks in healthy and diseased myocytes is an essential prerequisite towards better understanding of functional plasticity and pathophysiological reorganization. Here, we present a strategic combination of protocols for direct quantitative analysis of TATS membrane networks in living VMs and AMs. For this, we accompany primary cell isolations of mouse VMs and/or AMs with critical quality control steps and direct membrane staining protocols for fluorescence imaging of TATS membranes. Using an optimized workflow for confocal or superresolution TATS image processing, binarized and skeletonized data are generated for quantitative analysis of the TATS network and its components. Unlike previously published indirect regional aggregate image analysis strategies, our protocols enable direct characterization of specific components and derive complex physiological properties of TATS membrane networks in living myocytes with high throughput and open access software tools.

In summary, the combined protocol strategy can be readily applied for quantitative TATS network studies during physiological myocyte adaptation or disease changes, comparison of different cardiac or skeletal muscle cell types, phenotyping of transgenic models, and pharmacological or therapeutic interventions. The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality.

We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.

To disseminate these methods for broader use we present Protein WISDOM (a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design.

Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods. Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies.

All these characteristics need to be considered when deciding on which approach to take for segmentation. The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets. Asdm Demo Mode Installer more.

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts.

The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (α-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity.

All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual's oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods.

We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual's ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity. Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases.

Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.

Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P.

Gingivalis accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention. We describe an indoor, portable, standardized course that can be used to evaluate obstacle avoidance in persons who have ultralow vision.

Six sighted controls and 36 completely blind but otherwise healthy adult male (n=29) and female (n=13) subjects (age range 19-85 years), were enrolled in one of three studies involving testing of the BrainPort sensory substitution device. Subjects were asked to navigate the course prior to, and after, BrainPort training. They completed a total of 837 course runs in two different locations. Means and standard deviations were calculated across control types, courses, lights, and visits. We used a linear mixed effects model to compare different categories in the PPWS (percent preferred walking speed) and error percent data to show that the course iterations were properly designed.

The course is relatively inexpensive, simple to administer, and has been shown to be a feasible way to test mobility function. Data analysis demonstrates that for the outcome of percent error as well as for percentage preferred walking speed, that each of the three courses is different, and that within each level, each of the three iterations are equal. This allows for randomization of the courses during administration. Abbreviations: preferred walking speed (PWS) course speed (CS) percentage preferred walking speed (PPWS). Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.

Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success.

Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18% raffinose and 3% skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains. Here we demonstrate a newly developed I•Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (>60%) and rapid progressive motility (>45%) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains.

Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I•Cryo kit and later recovered by IVF. Preclinical models of restenosis are essential to unravel the pathophysiological processes that lead to in-stent restenosis and to optimize existing and future drug-eluting stents. A variety of antibodies and transgenic and knockout strains are available in rats. Consequently, a model for in-stent restenosis in the rat would be convenient for pathobiological and pathophysiological studies. In this video, we present the full procedure and pit-falls of a rat stent model suitable for high throughput stent research. We will show the surgical procedure of stent deployment, and the assessment of in-stent restenosis using the most elegant technique of OCT (Optical Coherence Tomography).

This technique provides high accuracy in assessing plaque CSAs (cross section areas) and correlates well with histological sections, which require special and time consuming embedding and sectioning techniques. OCT imaging further allows longitudinal monitoring of the development of in-stent restenosis within the same animal compared to one-time snapshots using histology. Baculoviruses are widely used both as protein expression vectors and as insect pest control agents. This video shows how lepidopteran larvae can be infected with polyhedra by droplet feeding and diet plug-based bioassays. This accompanying Springer Protocols section provides an overview of the baculovirus lifecycle and use of baculoviruses as insecticidal agents, including discussion of the pros and cons for use of baculoviruses as insecticides, and progress made in genetic enhancement of baculoviruses for improved insecticidal efficacy.

What is Visualize? JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library. How does it work? We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos. Video X seems to be unrelated to Abstract Y. In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult.

In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.

Methods In 1996 and 1997, we measured thyrotropin in stored serum samples collected from 25,216 pregnant women between January 1987 and March 1990. We then located 47 women with serum thyrotropin concentrations at or above the 99.7th percentile of the values for all the pregnant women, 15 women with values between the 98th and 99.6th percentiles, inclusive, in combination with low thyroxine levels, and 124 matched women with normal values. Their seven-to-nine-year-old children, none of whom had hypothyroidism as newborns, underwent 15 tests relating to intelligence, attention, language, reading ability, school performance, and visual–motor performance. Results The children of the 62 women with high serum thyrotropin concentrations performed slightly less well on all 15 tests. Their full-scale IQ scores on the Wechsler Intelligence Scale for Children, third edition, averaged 4 points lower than those of the children of the 124 matched control women (P=0.06); 15 percent had scores of 85 or less, as compared with 5 percent of the matched control children.

Of the 62 women with thyroid deficiency, 48 were not treated for the condition during the pregnancy under study. The full-scale IQ scores of their children averaged 7 points lower than those of the 124 matched control children (P=0.005); 19 percent had scores of 85 or less. Eleven years after the pregnancy under study, 64 percent of the untreated women and 4 percent of the matched control women had confirmed hypothyroidism. The link between hypothyroidism caused by iodine deficiency during pregnancy and mental retardation in the offspring has been recognized for nearly 100 years.

Iodine deficiency is associated with thyroid deficiency in both mother and fetus, a situation that makes it impossible to determine whether the mental retardation of the fetus is due to maternal hypothyroidism or both maternal and fetal hypothyroidism. In developed countries, chronic autoimmune thyroiditis is the most common cause of hypothyroidism among women in their childbearing years. Antibodies responsible for compromising maternal thyroid function can cross the placenta and, in some instances, compromise fetal and neonatal thyroid function. One such antibody, the thyrotropin-receptor–blocking antibody, has been implicated in cases of transient congenital hypothyroidism that were identified by screening programs for newborns. In 1969, Man and Jones suggested that mild maternal hypothyroidism alone was associated with lower IQ levels in the offspring; their study involved a cohort of 1349 children, and measurements of serum butanol extractable iodine were used to distinguish between euthyroidism and hypothyroidism in women. A study by Matsuura and Konishi in 1990 documented that fetal brain development is adversely affected when both the mother and fetus have hypothyroidism caused by chronic autoimmune thyroiditis.

In such cases, transient neonatal hypothyroidism is present. In an earlier, population-based survey of 2000 pregnancies, we measured serum thyrotropin concentrations during the second trimester. The concentrations were high (above 6 mU per liter) in 49 of the women, of whom 6 (3 per 1000) had low serum free thyroxine concentrations. If a lowering of the IQ levels of the offspring were to occur sufficiently often in association with this degree and frequency of maternal thyroid deficiency, then systematically determining the thyroid status of all women before or very early in pregnancy might be justified. The aim of the current study was to determine whether undetected or inadequately treated maternal thyroid deficiency during pregnancy is associated with lower IQ scores in the offspring in the absence of neonatal hypothyroidism. Methods The Foundation for Blood Research administers a statewide, second-trimester prenatal serum screening program for open neural-tube defects and Down's syndrome in Maine. Aliquots of serum that remain after screening are routinely coded and stored at –20°C.

Outcome information is available through a contract with the state's Bureau of Vital Records. The current study cohort was limited to women with viable singleton pregnancies, who were screened between January 1987 and March 1990, and their infants whose birth weight was at least 1500 g. The serum from the mothers was shipped to the New England Newborn Screening Program in Boston, where serum thyrotropin was measured. Samples from 25,216 women were analyzed in five batches over a two-year period. Selection of Women with Hypothyroidism and Control Subjects We recruited women with hypothyroidism during pregnancy, as determined by a high serum thyrotropin concentration, without regard to treatment status, and we tested their children between March 1996 and December 1997. We contacted 55 of the 75 pregnant women with serum thyrotropin concentrations at or above the 99.7th percentile of the values for all the pregnant women; 47 (85 percent) agreed to participate. In 2 of the 75 pregnancies, the women were enrolled through a previous pregnancy.

Of the 18 women not contacted, 3 had moved to another state, 1 had died, and for 1, the child was a ward of the state. The remaining 13 were not contacted because of limited funds. At the urging of a grant review panel, we recruited 18 more women to represent a range of milder cases, defined by a serum thyrotropin concentration between the 98th and 99.6th percentiles, inclusive, and a serum thyroxine concentration below 7.75 μg per deciliter (99.7 nmol per liter). To identify this second subgroup, we measured serum thyroxine concentrations in 247 pregnant women with serum thyrotropin concentrations between the 98th and 99.7th percentiles.

Fifteen of the 18 women identified (83 percent) agreed to participate. After recruitment, we measured thyroxine, free thyroxine, and antithyroid peroxidase antibodies in the original serum samples from all women in the study. For each woman with hypothyroidism, we identified potential control subjects who had serum thyrotropin concentrations below the 98th percentile and who were matched according to the following criteria: mother's age at delivery (within one year), number of years of education of the mother (within one year), gestational age at the time of sampling (same completed week), duration of storage of serum sample (within one month), and sex of the child. From this group, two women were randomly selected and recruited for each woman with hypothyroidism. Additional matched control women were available from the same list, if one initially declined participation. The protocols for the additional assays and the follow-up study were approved by the institutional review board at the Foundation for Blood Research. Enrollment began with a telephone call to the woman and a letter describing the study.

Then an appointment was arranged, at which informed consent was requested and, if consent was granted, testing was performed on the child. The neuropsychological test results were provided to the family within one month after the child's testing was completed. At the end of the study, we contacted the women with hypothyroidism and the matched control women again to determine whether hypothyroidism had been clinically diagnosed since the pregnancy in those who had not received a diagnosis of hypothyroidism at the time of pregnancy. The contact was initially by a letter, which also included information about the thyrotropin concentrations in the stored serum samples. The letter was followed by a telephone call, during which a questionnaire was administered and a blood test for measuring thyrotropin was offered. For those who agreed to be tested, blood spots were collected on filter paper by a finger prick. Study Procedures We collected standardized information about socioeconomic status and medical history from all women enrolled in the study, using the Four Factor Index of Social Status (the Hollingshead score).

Each woman and her husband or partner were assigned an education code ranging from 1 (corresponding to less than seven years of schooling) to 7 (corresponding to graduate or professional training) and an occupation code ranging from 1 (e.g., “farm worker”) to 9 (e.g., “higher executive”). Each couple's individual education scores were multiplied by 3, the occupation scores were multiplied by 5, and the two values were then added together. The final score was the average of the scores of the woman and her partner. When one partner was not employed, the final score was taken to be the score of the employed partner. The woman was also asked whether her child had repeated a grade and about her child's school performance, including whether the child had had learning problems or other difficulties in school. Neuropsychological testing of the women's children included assessment of intelligence, attention, language, reading ability, school performance, and visual–motor performance.

One of two certified psychologists performed the testing, and the project's consulting psychologist supervised the testing and rescored the tests. The staff involved in the neuropsychological testing did not know whether the children's mothers were women with hypothyroidism or control women. Intelligence was measured with use of the Wechsler Intelligence Scale for Children, third edition, the most widely used intelligence test.

This test provides a full-scale IQ score and subscale scores (range, 40 to 160) for verbal skills, performance, and freedom from distractibility. To measure hearing deficits in the children, the staff administered subtests on word discrimination and word articulation from the Test of Language Development, second edition (range of scores, 1 to 20). We used the norms for children 8 years 11 months of age, because the version for older children did not have scales for word discrimination or articulation. The Peabody Individual Achievement Test, revised (PIAT-R), was used to measure reading recognition and reading comprehension (range of scores, 55 to 145). The staff administered the Conners' Continuous Performance Test to measure sustained vigilance and attention, using a computer program that employs a go–no go paradigm (range of overall index score, 1 to 30). The Developmental Test of Visual-Motor Integration was administered to provide a standard measure of visual perception and fine motor skills (range, 55 to 145).

The grooved pegboard test was administered to assess visual–motor coordination and dexterity by measuring the time required to insert pegs with both the preferred and nonpreferred hand (for this test, it is recommended that normative data be derived from control children in the study). Assay Methods We measured serum thyrotropin using a coated-tube radioimmunoassay (Diagnostic Products, Los Angeles). Thyrotropin was measured on dried blood spots with a time-resolved immunofluorometric assay (Wallac Oy, Turku, Finland). Serum thyroxine was measured with a solid-phase radioimmunoassay or a time-resolved immunofluorometric assay (Wallac Oy); serum free thyroxine was measured with a time-resolved immunofluorometric assay. We measured serum antithyroid peroxidase antibodies using the Kalibre enzyme-linked immunosorbent assay (Kronus, San Clemente, Calif.) (normal concentration, ≤2 U per milliliter). Statistical Analysis The serum thyrotropin, thyroxine, and free thyroxine concentrations were logarithmically transformed before analysis.

We used geometric means and logarithmic standard deviations to summarize the results (after censoring seven measurements that were more than 3 SD above or below the group mean). We compared categorical variables using an exact test of significance or odds ratios, and we compared continuous variables using the Student's t-test. When necessary, the t-test was modified to allow for unequal variances.

The primary analysis was of all 62 women with hypothyroidism and all 124 control women; we preserved matching by comparing the result from the child of a woman with hypothyroidism with the average result from the two matched control children. No adjustment was made for multiple comparisons.

All statistical tests were two-sided. Results According to records from the New England Newborn Screening Program, none of the children of the 62 women with high serum thyrotropin concentrations while pregnant were identified as having transient or permanent congenital hypothyroidism. The distribution of serum thyrotropin concentrations in the 62 women with hypothyroidism and the 124 matched control women is shown in Figure 1 Distribution of Serum Thyrotropin Concentrations during Pregnancy in the 62 Women with Hypothyroidism and the 124 Matched Control Women. Open circles indicate the 14 women who were treated for hypothyroidism during the pregnancy under study. Selected percentiles are shown for the entire cohort of 25,216 pregnant women.. Fifteen of the 62 women with hypothyroidism reported that they had received a diagnosis of hypothyroidism before the pregnancy, and 14 of these 15 women were treated for hypothyroidism during that pregnancy. Two of the control women reported that they had had hypothyroidism in the distant past but were never treated.

Demographic and pregnancy-related information about the women with hypothyroidism and the control women and the remainder of the cohort from which the women were selected is shown in Table 1 Characteristics of the Study Women, the Remainder of the Cohort, and Their Children.. There were no significant differences between the women with hypothyroidism and the control women for any of the variables. Four of the variables were used for matching: number of years of education of the mother, mother's age at delivery, gestational week when the serum sample was obtained, and sex of the child.

The use of the number of years of education as a matching variable was intended to control for socioeconomic status. To assess the effectiveness of this matching, we used the Hollingshead score as an additional measure. This score took into account the mother's educational level and occupation and also the father's educational level and occupation.

The mean Hollingshead score in the women with hypothyroidism was one point lower than that in the control women (P=0.43). The study children were similar in most respects to the remainder of the cohort, but more were girls, their mothers were older, a higher percentage of their mothers were married, and more of their mothers were multiparous.

The results of the measurements of thyroid function in the women with hypothyroidism and the control women during pregnancy are shown in Table 2 Measurements of Thyroid Function in the Study Women during Pregnancy.. As expected, according to the selection process, the women with hypothyroidism had higher serum thyrotropin and lower serum thyroxine concentrations. The children's neuropsychological test scores are shown in Table 3 Neuropsychological Test Scores among the Children of Women with Hypothyroidism during Pregnancy as Compared with the Children of Matched Control Women.. All the children were between seven and nine years of age when tested, and the child of a woman with hypothyroidism and his or her matched control children were tested at the same age.

The analysis preserved matching for each of the tests by expressing the relative performance between case and control children as a mean difference (the value in the case child minus the average of the values in the two control children). The case children performed less well on all the tests; 2 of the 15 differences reached statistical significance (P. Discussion The current study shows that hypothyroidism in pregnant women can adversely affect their children's subsequent performance on neuropsychological tests. Decreases in performance can occur even when the pregnant woman's hypothyroidism is mild and probably asymptomatic.

The presence of high serum concentrations of antithyroid peroxidase antibodies in 77 percent of the women with hypothyroidism indicates that chronic autoimmune thyroiditis was the most frequent cause of hypothyroidism in these women. Treating maternal hypothyroidism during pregnancy appears to be beneficial for the child, even when treatment is inadequate as determined by measurements of thyrotropin. If our findings were to be confirmed, and routine screening for hypothyroidism during pregnancy were to be instituted, what might the benefits be? The main benefit — an increase of approximately 4 points in IQ scores — would occur in the children of women with serum thyrotropin concentrations at or above the 98th percentile. A secondary benefit would be reduced morbidity for women who were systematically identified and treated.

The present study shows that a large percentage of pregnant women with high serum thyrotropin concentrations subsequently have clinically apparent hypothyroidism. Because the symptoms associated with hypothyroidism are nonspecific, the condition can be difficult to diagnose, as reflected by the five-year median time to diagnosis in the women. Before about 12 weeks' gestation, when the fetal thyroid gland becomes active, the mother is the sole source of thyroid hormones. Maternal thyroid sufficiency might therefore be most important in the first trimester. This theory is supported by a recent study in a small cohort of 220 healthy infants in which lower maternal serum free thyroxine concentrations at 12 weeks' gestation were associated with impaired psychomotor development at 10 months of age.

However, the later stages of fetal brain development involve neuronal migration and organization. Since these processes are responsible for functions measured by the neuropsychological tests used in the present study, thyroid insufficiency beyond the first trimester is also likely to have adverse effects. In rats, triiodothyronine receptors are first detected in the brain in the second trimester, and the induction by triiodothyronine of enzymes that are important in nervous-system development begins late in fetal development. The current study documents a relatively long average interval between early biochemical evidence of hypothyroidism and clinical diagnosis, a finding that suggests that ongoing maternal health problems might hinder the child's development after birth. In the absence of objective data, the most prudent policy would be to identify and treat maternal hypothyroidism as early in pregnancy as possible, keeping in mind that the need for thyroxine increases during pregnancy. We conclude that systematic screening for hypothyroidism early in pregnancy may be worthwhile, even when the degree of deficiency is mild and does not cause immediate clinical manifestations in the woman. If routine screening were to be introduced, the most conservative policy would be to perform testing at the first prenatal visit, preferably in the first trimester.

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