There are several advantages that functional near-infrared spectroscopy (fNIRS) presents in the study of the neural control of human movement. It is relatively flexible with respect to participant positioning and allows for some head movements during tasks. Additionally, it is inexpensive, light weight, and portable, with very few contraindications to its use. This presents a unique opportunity to study functional brain activity during motor tasks in individuals who are typically developing, as well as those with movement disorders, such as cerebral palsy. An additional consideration when studying movement disorders, however, is the quality of actual movements performed and the potential for additional, unintended movements. Therefore, concurrent monitoring of both blood flow changes in the brain and actual movements of the body during testing is required for appropriate interpretation of fNIRS results. Here, we show a protocol for the combination of fNIRS with muscle and kinematic monitoring during motor tasks. We explore gait, a unilateral multi-joint movement (cycling), and two unilateral single-joint movements (isolated ankle dorsiflexion, and isolated hand squeezing). The techniques presented can be useful in studying both typical and atypical motor control, and can be modified to investigate a broad range of tasks and scientific questions.
27 Related JoVE Articles!
Nanomechanics of Drug-target Interactions and Antibacterial Resistance Detection
Institutions: University College London.
The cantilever sensor, which acts as a transducer of reactions between model bacterial cell wall matrix immobilized on its surface and antibiotic drugs in solution, has shown considerable potential in biochemical sensing applications with unprecedented sensitivity and specificity1-5
. The drug-target interactions generate surface stress, causing the cantilever to bend, and the signal can be analyzed optically when it is illuminated by a laser. The change in surface stress measured with nano-scale precision allows disruptions of the biomechanics of model bacterial cell wall targets to be tracked in real time. Despite offering considerable advantages, multiple cantilever sensor arrays have never been applied in quantifying drug-target binding interactions.
Here, we report on the use of silicon multiple cantilever arrays coated with alkanethiol self-assembled monolayers mimicking bacterial cell wall matrix to quantitatively study antibiotic binding interactions. To understand the impact of vancomycin on the mechanics of bacterial cell wall structures1,6,7
. We developed a new model1
which proposes that cantilever bending can be described by two independent factors; i) namely a chemical factor, which is given by a classical Langmuir adsorption isotherm, from which we calculate the thermodynamic equilibrium dissociation constant (Kd
) and ii) a geometrical factor, essentially a measure of how bacterial peptide receptors are distributed on the cantilever surface. The surface distribution of peptide receptors (p
) is used to investigate the dependence of geometry and ligand loading. It is shown that a threshold value of p ~
10% is critical to sensing applications. Below which there is no detectable bending signal while above this value, the bending signal increases almost linearly, revealing that stress is a product of a local chemical binding factor and a geometrical factor combined by the mechanical connectivity of reacted regions and provides a new paradigm for design of powerful agents to combat superbug infections.
Immunology, Issue 80, Engineering, Technology, Diagnostic Techniques and Procedures, Early Diagnosis, Bacterial Infections and Mycoses, Lipids, Amino Acids, Peptides, and Proteins, Chemical Actions and Uses, Diagnosis, Therapeutics, Surface stress, vancomycin, mucopeptides, cantilever sensor
Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction
Institutions: Rutgers University, Koç University, New York University, Fairfield University.
We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the "cloud" and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in cabinet; two such cabinets (16 environments) may be controlled by one computer.
Behavior, Issue 84, genetics, cognitive mechanisms, behavioral screening, learning, memory, timing
Utility of Dissociated Intrinsic Hand Muscle Atrophy in the Diagnosis of Amyotrophic Lateral Sclerosis
Institutions: Westmead Hospital, University of Sydney, Australia.
The split hand
phenomenon refers to predominant wasting of thenar muscles and is an early and specific feature of amyotrophic lateral sclerosis (ALS). A novel split hand index (SI) was developed to quantify the split hand phenomenon, and its diagnostic utility was assessed in ALS patients. The split hand index was derived by dividing the product of the compound muscle action potential (CMAP) amplitude recorded over the abductor pollicis brevis and first dorsal interosseous muscles by the CMAP amplitude recorded over the abductor digiti minimi muscle. In order to assess the diagnostic utility of the split hand index, ALS patients were prospectively assessed and their results were compared to neuromuscular disorder patients. The split hand index was significantly reduced in ALS when compared to neuromuscular disorder patients (P<0.0001). Limb-onset ALS patients exhibited the greatest reduction in the split hand index, and a value of 5.2 or less reliably differentiated ALS from other neuromuscular disorders. Consequently, the split hand index appears to be a novel diagnostic biomarker for ALS, perhaps facilitating an earlier diagnosis.
Medicine, Issue 85, Amyotrophic Lateral Sclerosis (ALS), dissociated muscle atrophy, hypothenar muscles, motor neuron disease, split-hand index, thenar muscles
A Proboscis Extension Response Protocol for Investigating Behavioral Plasticity in Insects: Application to Basic, Biomedical, and Agricultural Research
Institutions: Arizona State University.
Insects modify their responses to stimuli through experience of associating those stimuli with events important for survival (e.g.
, food, mates, threats). There are several behavioral mechanisms through which an insect learns salient associations and relates them to these events. It is important to understand this behavioral plasticity for programs aimed toward assisting insects that are beneficial for agriculture. This understanding can also be used for discovering solutions to biomedical and agricultural problems created by insects that act as disease vectors and pests. The Proboscis Extension Response (PER) conditioning protocol was developed for honey bees (Apis mellifera
) over 50 years ago to study how they perceive and learn about floral odors, which signal the nectar and pollen resources a colony needs for survival. The PER procedure provides a robust and easy-to-employ framework for studying several different ecologically relevant mechanisms of behavioral plasticity. It is easily adaptable for use with several other insect species and other behavioral reflexes. These protocols can be readily employed in conjunction with various means for monitoring neural activity in the CNS via electrophysiology or bioimaging, or for manipulating targeted neuromodulatory pathways. It is a robust assay for rapidly detecting sub-lethal effects on behavior caused by environmental stressors, toxins or pesticides.
We show how the PER protocol is straightforward to implement using two procedures. One is suitable as a laboratory exercise for students or for quick assays of the effect of an experimental treatment. The other provides more thorough control of variables, which is important for studies of behavioral conditioning. We show how several measures for the behavioral response ranging from binary yes/no to more continuous variable like latency and duration of proboscis extension can be used to test hypotheses. And, we discuss some pitfalls that researchers commonly encounter when they use the procedure for the first time.
Neuroscience, Issue 91, PER, conditioning, honey bee, olfaction, olfactory processing, learning, memory, toxin assay
Barnes Maze Testing Strategies with Small and Large Rodent Models
Institutions: University of Missouri, Food and Drug Administration.
Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.
bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.
distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.
random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.
shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.
Behavior, Issue 84, spatial navigation, rats, Peromyscus, mice, intra- and extra-maze cues, learning, memory, latency, search strategy, escape motivation
The Multiple Sclerosis Performance Test (MSPT): An iPad-Based Disability Assessment Tool
Institutions: Cleveland Clinic Foundation, Cleveland Clinic Foundation, Cleveland Clinic Foundation, Cleveland Clinic Foundation.
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.
Medicine, Issue 88, Multiple Sclerosis, Multiple Sclerosis Functional Composite, computer-based testing, 25-foot walk test, 9-hole peg test, Symbol Digit Modalities Test, Low Contrast Visual Acuity, Clinical Outcome Measure
Scalable Nanohelices for Predictive Studies and Enhanced 3D Visualization
Institutions: University of California Merced, University of California Merced.
Spring-like materials are ubiquitous in nature and of interest in nanotechnology for energy harvesting, hydrogen storage, and biological sensing applications. For predictive simulations, it has become increasingly important to be able to model the structure of nanohelices accurately. To study the effect of local structure on the properties of these complex geometries one must develop realistic models. To date, software packages are rather limited in creating atomistic helical models. This work focuses on producing atomistic models of silica glass (SiO2
) nanoribbons and nanosprings for molecular dynamics (MD) simulations. Using an MD model of “bulk” silica glass, two computational procedures to precisely create the shape of nanoribbons and nanosprings are presented. The first method employs the AWK programming language and open-source software to effectively carve various shapes of silica nanoribbons from the initial bulk model, using desired dimensions and parametric equations to define a helix. With this method, accurate atomistic silica nanoribbons can be generated for a range of pitch values and dimensions. The second method involves a more robust code which allows flexibility in modeling nanohelical structures. This approach utilizes a C++ code particularly written to implement pre-screening methods as well as the mathematical equations for a helix, resulting in greater precision and efficiency when creating nanospring models. Using these codes, well-defined and scalable nanoribbons and nanosprings suited for atomistic simulations can be effectively created. An added value in both open-source codes is that they can be adapted to reproduce different helical structures, independent of material. In addition, a MATLAB graphical user interface (GUI) is used to enhance learning through visualization and interaction for a general user with the atomistic helical structures. One application of these methods is the recent study of nanohelices via MD simulations for mechanical energy harvesting purposes.
Physics, Issue 93, Helical atomistic models; open-source coding; graphical user interface; visualization software; molecular dynamics simulations; graphical processing unit accelerated simulations.
Magnetic Tweezers for the Measurement of Twist and Torque
Institutions: Delft University of Technology.
Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a “conventional” magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the “conventional” magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.
Bioengineering, Issue 87, magnetic tweezers, magnetic torque tweezers, freely-orbiting magnetic tweezers, twist, torque, DNA, single-molecule techniques
Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma
Institutions: Southern Illinois University School of Medicine.
A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2
. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5
. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6
. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7
. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9
. Bevacizumab however failed to prolong overall survival in a recent phase III trial26
. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians’ choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12
, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.
Medicine, Issue 92, Tumor Treating Fields, TTF System, TTF Therapy, Recurrent Glioblastoma, Bevacizumab, Brain Tumor
From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data
Institutions: Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory, Lawrence Berkeley National Laboratory.
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.
Bioengineering, Issue 90, 3D electron microscopy, feature extraction, segmentation, image analysis, reconstruction, manual tracing, thresholding
A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Institutions: University of Notre Dame.
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ
hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
Developmental Biology, Issue 93, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites
Institutions: Royal Military College of Canada, Queen's University.
The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g.
carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.
Environmental Sciences, Issue 93, biochar, characterization, carbon sequestration, remediation, International Biochar Initiative (IBI), soil amendment
Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?
Institutions: Cedars-Sinai Medical Center.
Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.
Chemistry, Issue 88, Cancer treatment, personalized medicine, polymalic acid, nanodrug, biopolymer, targeting, host compatibility, biodegradability
Localization and Relative Quantification of Carbon Nanotubes in Cells with Multispectral Imaging Flow Cytometry
Institutions: CNRS/Université Paris Diderot, CNRS/Université Paris Diderot, CNRS/Institut de Biologie Moléculaire et Cellulaire.
Carbon-based nanomaterials, like carbon nanotubes (CNTs), belong to this type of nanoparticles which are very difficult to discriminate from carbon-rich cell structures and de facto
there is still no quantitative method to assess their distribution at cell and tissue levels. What we propose here is an innovative method allowing the detection and quantification of CNTs in cells using a multispectral imaging flow cytometer (ImageStream, Amnis). This newly developed device integrates both a high-throughput of cells and high resolution imaging, providing thus images for each cell directly in flow and therefore statistically relevant image analysis. Each cell image is acquired on bright-field (BF), dark-field (DF), and fluorescent channels, giving access respectively to the level and the distribution of light absorption, light scattered and fluorescence for each cell. The analysis consists then in a pixel-by-pixel comparison of each image, of the 7,000-10,000 cells acquired for each condition of the experiment. Localization and quantification of CNTs is made possible thanks to some particular intrinsic properties of CNTs: strong light absorbance and scattering; indeed CNTs appear as strongly absorbed dark spots on BF and bright spots on DF with a precise colocalization.
This methodology could have a considerable impact on studies about interactions between nanomaterials and cells given that this protocol is applicable for a large range of nanomaterials, insofar as they are capable of absorbing (and/or scattering) strongly enough the light.
Bioengineering, Issue 82, bioengineering, imaging flow cytometry, Carbon Nanotubes, bio-nano-interactions, cellular uptake, cell trafficking
Cargo Loading onto Kinesin Powered Molecular Shuttles
Institutions: University of Florida, Columbia University.
Cells have evolved sophisticated molecular machinery, such as kinesin motor proteins and microtubule filaments, to support active intracellular transport of cargo. While kinesins tail domain binds to a variety of cargoes, kinesins head domains utilize the chemical energy stored in ATP molecules to step along the microtubule lattice. The long, stiff microtubules serve as tracks for long-distance intracellular transport.
These motors and filaments can also be employed in microfabricated synthetic environments as components of molecular shuttles 1
. In a frequently used design, kinesin motors are anchored to the track surface through their tails, and functionalized microtubules serve as cargo carrying elements, which are propelled by these motors. These shuttles can be loaded with cargo by utilizing the strong and selective binding between biotin and streptavidin. The key components (biotinylated tubulin, streptavidin, and biotinylated cargo) are commercially available.
Building on the classic inverted motility assay 2
, the construction of molecular shuttles is detailed here. Kinesin motor proteins are adsorbed to a surface precoated with casein; microtubules are polymerized from biotinylated tubulin, adhered to the kinesin and subsequently coated with rhodamine-labeled streptavidin. The ATP concentration is maintained at subsaturating concentration to achieve a microtubule gliding velocity optimal for loading cargo 3
. Finally, biotinylated fluorescein-labeled nanospheres are added as cargo. Nanospheres attach to microtubules as a result of collisions between gliding microtubules and nanospheres adhering to the surface.
The protocol can be readily modified to load a variety of cargoes such as biotinylated DNA4
, quantum dots 5
or a wide variety of antigens via biotinylated antibodies 4-6
Cellular Biology, Issue 45, motility assay, microtubules, kinesin, motor protein, molecular shuttle, nanobiotechnology
Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays
Institutions: University of North Carolina at Chapel Hill.
Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels. One of its many advantages is a lower detection limit compared to other methods of gene expression profiling while using smaller amounts of input for each assay. Automated qPCR setup has improved this field by allowing for greater reproducibility. Its convenient and rapid setup allows for high-throughput experiments, enabling the profiling of many different genes simultaneously in each experiment. This method along with internal plate controls also reduces experimental variables common to other techniques.
We recently developed a qPCR assay for profiling of pre-microRNAs (pre-miRNAs) using a set of 186 primer pairs. MicroRNAs have emerged as a novel class of small, non-coding RNAs with the ability to regulate many mRNA targets at the post-transcriptional level. These small RNAs are first transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) transcript, which is then cleaved into the precursor miRNA (pre-miRNA). Pre-miRNAs are exported to the cytoplasm where Dicer cleaves the hairpin loop to yield mature miRNAs. Increases in miRNA levels can be observed at both the precursor and mature miRNA levels and profiling of both of these forms can be useful. There are several commercially available assays for mature miRNAs; however, their high cost may deter researchers from this profiling technique. Here, we discuss a cost-effective, reliable, SYBR-based qPCR method of profiling pre-miRNAs. Changes in pre-miRNA levels often reflect mature miRNA changes and can be a useful indicator of mature miRNA expression. However, simultaneous profiling of both pre-miRNAs and mature miRNAs may be optimal as they can contribute nonredundant information and provide insight into microRNA processing. Furthermore, the technique described here can be expanded to encompass the profiling of other library sets for specific pathways or pathogens.
Biochemistry, Issue 46, pre-microRNAs, qPCR, profiling, Tecan Freedom Evo, robot
Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro
Institutions: The University of Chicago Medical Center, The University of Chicago Medical Center.
Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro
using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro
preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers.
In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo
counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure
neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic
SD immune signaling.
Neuroscience, Issue 52, innate immunity, hormesis, microglia, T-cells, hippocampus, slice culture, gene expression, laser dissection microscopy, real-time qPCR, interferon-gamma
Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique
Institutions: University of North Carolina School of Medicine, University of North Carolina .
Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4
. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications.
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5
(Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10
Bioengineering, Issue 55, Nanoparticles, nanomedicine, drug delivery, polymeric micelles, polymeric nanoparticles, diblock co-polymers, nanoplatform, nanoparticle molecular imaging, polymer conjugation.
Gold Nanostar Synthesis with a Silver Seed Mediated Growth Method
Institutions: The University of Texas at San Antonio, Centro de Investigaciones en Optica A. C., The University of Texas at San Antonio.
The physical, chemical and optical properties of nano-scale colloids depend on their material composition, size and shape 1-5
. There is a great interest in using nano-colloids for photo-thermal ablation, drug delivery and many other biomedical applications 6
. Gold is particularly used because of its low toxicity 7-9
. A property of metal nano-colloids is that they can have a strong surface plasmon resonance 10
. The peak of the surface plasmon resonance mode depends on the structure and composition of the metal nano-colloids. Since the surface plasmon resonance mode is stimulated with light there is a need to have the peak absorbance in the near infrared where biological tissue transmissivity is maximal 11, 12
We present a method to synthesize star shaped colloidal gold, also known as star shaped nanoparticles 13-15
or nanostars 16
. This method is based on a solution containing silver seeds that are used as the nucleating agent for anisotropic growth of gold colloids 17-22
. Scanning electron microscopy (SEM) analysis of the resulting gold colloid showed that 70 % of the nanostructures were nanostars. The other 30 % of the particles were amorphous clusters of decahedra and rhomboids. The absorbance peak of the nanostars was detected to be in the near infrared (840 nm). Thus, our method produces gold nanostars suitable for biomedical applications, particularly for photo-thermal ablation.
Bioengineering, Issue 59, thermal ablation, surface plasmon resonance, nanoparticle, nanotechnology, silver seeds
In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage
Institutions: Baylor College of Medicine , Michael E. DeBakey Veterans Affairs Medical Center, Baylor College of Medicine .
Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6
. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9
. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10
. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12
. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12
, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4
blunt-ended break (DNase I-type breaks)11,12
, if T4 DNA ligase is present in the solution 13,14
. When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4
blunt-ended breaks, the ligase reacts with 5'PO4
DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4
blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.
Bioengineering, Issue 59, molecular machine, bio-nanotechnology, 5'OH DNA breaks, 5'PO4 DNA breaks, apoptosis labeling, in situ detection, vaccinia topoisomerase I, DNA breaks, green nanotechnology
Perceptual and Category Processing of the Uncanny Valley Hypothesis' Dimension of Human Likeness: Some Methodological Issues
Institutions: University of Zurich.
Mori's Uncanny Valley Hypothesis1,2
proposes that the perception of humanlike characters such as robots and, by extension, avatars (computer-generated characters) can evoke negative or positive affect (valence) depending on the object's degree of visual and behavioral realism along a dimension of human likeness
) (Figure 1
). But studies of affective valence of subjective responses to variously realistic non-human characters have produced inconsistent findings 3, 4, 5, 6
. One of a number of reasons for this is that human likeness is not perceived as the hypothesis assumes. While the DHL can be defined following Mori's description as a smooth linear change in the degree of physical humanlike similarity, subjective perception of objects along the DHL can be understood in terms of the psychological effects of categorical perception (CP) 7
. Further behavioral and neuroimaging investigations of category processing and CP along the DHL and of the potential influence of the dimension's underlying category structure on affective experience are needed. This protocol therefore focuses on the DHL and allows examination of CP. Based on the protocol presented in the video as an example, issues surrounding the methodology in the protocol and the use in "uncanny" research of stimuli drawn from morph continua to represent the DHL are discussed in the article that accompanies the video. The use of neuroimaging and morph stimuli to represent the DHL in order to disentangle brain regions neurally responsive to physical human-like similarity from those responsive to category change and category processing is briefly illustrated.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Psychology, Neuropsychology, uncanny valley, functional magnetic resonance imaging, fMRI, categorical perception, virtual reality, avatar, human likeness, Mori, uncanny valley hypothesis, perception, magnetic resonance imaging, MRI, imaging, clinical techniques
How to Detect Amygdala Activity with Magnetoencephalography using Source Imaging
Institutions: University of Wisconsin-Milwaukee, Montreal Neurological Institute, McGill University, Medical College of Wisconsin .
In trace fear conditioning a conditional stimulus (CS) predicts the occurrence of the unconditional stimulus (UCS), which is presented after a brief stimulus free period (trace interval)1
. Because the CS and UCS do not co-occur temporally, the subject must maintain a representation of that CS during the trace interval. In humans, this type of learning requires awareness of the stimulus contingencies in order to bridge the trace interval2-4
. However when a face is used as a CS, subjects can implicitly learn to fear the face even in the absence of explicit awareness*. This suggests that there may be additional neural mechanisms capable of maintaining certain types of "biologically-relevant" stimuli during a brief trace interval. Given that the amygdala is involved in trace conditioning, and is sensitive to faces, it is possible that this structure can maintain a representation of a face CS during a brief trace interval.
It is challenging to understand how the brain can associate an unperceived face with an aversive outcome, even though the two stimuli are separated in time. Furthermore investigations of this phenomenon are made difficult by two specific challenges. First, it is difficult to manipulate the subject's awareness of the visual stimuli. One common way to manipulate visual awareness is to use backward masking. In backward masking, a target stimulus is briefly presented (< 30 msec) and immediately followed by a presentation of an overlapping masking stimulus5
. The presentation of the mask renders the target invisible6-8
. Second, masking requires very rapid and precise timing making it difficult to investigate neural responses evoked by masked stimuli using many common approaches. Blood-oxygenation level dependent (BOLD) responses resolve at a timescale too slow for this type of methodology, and real time recording techniques like electroencephalography (EEG) and magnetoencephalography (MEG) have difficulties recovering signal from deep sources.
However, there have been recent advances in the methods used to localize the neural sources of the MEG signal9-11
. By collecting high-resolution MRI images of the subject's brain, it is possible to create a source model based on individual neural anatomy. Using this model to "image" the sources of the MEG signal, it is possible to recover signal from deep subcortical structures, like the amygdala and the hippocampus*.
Behavior, Issue 76, Neuroscience, Neurobiology, Molecular Biology, Medicine, Physiology, Anatomy, Psychology, Amygdala, Magnetoencephalography, Fear, awareness, masking, source imaging, conditional stimulus, unconditional stimulus, hippocampus, brain, magnetic resonance imaging, MRI, fMRI, imaging, clinical techniques
Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns
Institutions: University of Calgary , University of Calgary .
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.
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.
Medicine, Issue 78, Anatomy, Physiology, Cancer Biology, angular spread, architectural distortion, breast cancer, Computer-Assisted Diagnosis, computer-aided diagnosis (CAD), entropy, fractional Brownian motion, fractal dimension, Gabor filters, Image Processing, Medical Informatics, node map, oriented texture, Pattern Recognition, phase portraits, prior mammograms, spectral analysis
Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model
Institutions: University Hospital Zurich, EMPA Swiss Federal Laboratories for Materials Testing and Research, University of Bern.
Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1
. Therefore, the ex vivo
dual recirculating human placental perfusion, developed by Panigel et al.
in 1967 2
and continuously modified by Schneider et al.
in 1972 3
, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo
dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo
human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.
Biomedical Engineering, Issue 76, Medicine, Bioengineering, Anatomy, Physiology, Molecular Biology, Biochemistry, Biophysics, Pharmacology, Obstetrics, Nanotechnology, Placenta, Pharmacokinetics, Nanomedicine, humans, ex vivo perfusion, perfusion, biological barrier, xenobiotics, nanomaterials, clinical model
Fabricating Nanogaps by Nanoskiving
Institutions: University of Groningen.
There are several methods of fabricating nanogaps with controlled spacings, but the precise control over the sub-nanometer spacing between two electrodes-and generating them in practical quantities-is still challenging. The preparation of nanogap electrodes using nanoskiving, which is a form of edge lithography, is a fast, simple and powerful technique. This method is an entirely mechanical process which does not include any photo- or electron-beam lithographic steps and does not require any special equipment or infrastructure such as clean rooms. Nanoskiving is used to fabricate electrically addressable nanogaps with control over all three dimensions; the smallest dimension of these structures is defined by the thickness of the sacrificial layer (Al or Ag) or self-assembled monolayers. These wires can be manually positioned by transporting them on drops of water and are directly electrically-addressable; no further lithography is required to connect them to an electrometer.
Chemistry, Issue 75, Materials Science, Chemical Engineering, Electrical Engineering, Physics, Nanotechnology, nanodevices (electronic), Nanoskiving, nanogaps, nanofabrication, molecular electronics, nanowires, fabrication, etching, ultramicrotome, scanning electron microscopy, SEM
Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer
Institutions: Scientific Instruments.
Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF · (OD260-OD320)· NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1
Protein concentration results can be expressed as μg/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.
Molecular Biology, Issue 48, Nucleic acid quantitation, protein quantitation, micro-volume analysis, label quantitation
Bioelectric Analyses of an Osseointegrated Intelligent Implant Design System for Amputees
Institutions: Department of Veteran Affairs, University of Utah, University of Utah, University of Utah, University of Utah.
The projected number of American amputees is expected to rise to 3.6 million by 2050. Many of these individuals depend on artificial limbs to perform routine activities, but prosthetic suspensions using traditional socket technology can prove to be cumbersome and uncomfortable for a person with limb loss. Moreover, for those with high proximal amputations, limited residual limb length may prevent exoprosthesis attachment all together. Osseointegrated implant technology is a novel operative procedure which allows firm skeletal attachment between the host bone and an implant. Preliminary results in European amputees with osseointegrated implants have shown improved clinical outcomes by allowing direct transfer of loads to the bone-implant interface. Despite the apparent advantages of osseointegration over socket technology, the current rehabilitation procedures require long periods of restrictive load bearing prior which may be reduced with expedited skeletal attachment via electrical stimulation. The goal of the osseointegrated intelligent implant design (OIID) system is to make the implant part of an electrical system to accelerate skeletal attachment and help prevent periprosthetic infection. To determine optimal electrode size and placement, we initiated proof of concept with computational modeling of the electric fields and current densities that arise during electrical stimulation of amputee residual limbs. In order to provide insure patient safety, subjects with retrospective computed tomography scans were selected and three dimensional reconstructions were created using customized software programs to ensure anatomical accuracy (Seg3D and SCIRun) in an IRB and HIPAA approved study. These software packages supported the development of patient specific models and allowed for interactive manipulation of electrode position and size. Preliminary results indicate that electric fields and current densities can be generated at the implant interface to achieve the homogenous electric field distributions required to induce osteoblast migration, enhance skeletal fixation and may help prevent periprosthetic infections. Based on the electrode configurations experimented with in the model, an external two band configuration will be advocated in the future.
Medicine, Issue 29, Osseointegration, Electrical Stimulation, Osteogenesis, Amputation, Percutaneous