Biomedical subjects

James N Turner

Publications and source records attributed to James N Turner.

16 recordsLinked to original sources

PubMed2007

Functionalized hydrogel surfaces for the patterning of multiple biomolecules.

Patterning of multiple proteins and enzymes onto biocompatible surfaces can provide multiple signals to control cell attachment and growth. Acrylamide-based hydrogels were photo-polymerized in the presence of streptavidin-acrylamide, resulting in planar gel surfaces functionalized with the streptavidin protein. This surface was capable of binding biotin-labeled biomolecules. The proteins fibronectin and laminin, the enzyme alkaline phosphatase, and the photo-protein R-phycoerythrin were patterned using soft lithographic techniques. Polydimethylsiloxane stamps were used to transfer biotinylated proteins onto streptavidin-conjugated hydrogel surfaces. Stamped biomolecules were spatially resolved to feature sizes of 10 mum. Fluorescence measurements were used to assess protein transfer and enzyme functionality on modified surfaces. Our results demonstrate that hydrogel surfaces can be patterned with multiple proteins and enzymes, with retention of biological and catalytic activity. These surfaces are biocompatible and provide cues for cell attachment and growth. (c) 2006 Wiley Periodicals, Inc. J Biomed Mater Res 2007.

Animals↗

PubMed2006

Low-density neuronal networks cultured using patterned poly-l-lysine on microelectrode arrays.

Synaptic activity recorded from low-density networks of cultured rat hippocampal neurons was monitored using microelectrode arrays (MEAs). Neuronal networks were patterned with poly-l-lysine (PLL) using microcontact printing (microCP). Polydimethysiloxane (PDMS) stamps were fabricated with relief structures resulting in patterns of 2 microm-wide lines for directing process growth and 20 microm-diameter circles for cell soma attachment. These circles were aligned to electrode sites. Different densities of neurons were plated in order to assess the minimal neuron density required for development of an active network. Spontaneous activity was observed at 10-14 days in networks using neuron densities as low as 200 cells/mm(2). Immunocytochemistry demonstrated the distribution of dendrites along the lines and the location of foci of the presynaptic protein, synaptophysin, on neuron somas and dendrites. Scanning electron microscopy demonstrated that single fluorescent tracks contained multiple processes. Evoked responses of selected portions of the networks were produced by stimulation of specific electrode sites. In addition, the neuronal excitability of the network was increased by the bath application of high K(+) (10-12 mM). Application of DNQX, an AMPA antagonist, blocked all spontaneous activity, suggesting that the activity is excitatory and mediated through glutamate receptors.

Action Potentials↗

PubMed2006

Biological functionalization and surface micropatterning of polyacrylamide hydrogels.

Hydrogels are useful for linking proteins to solid surfaces because their hydrophilic nature and porous structure help them to maintain these labile molecules in the native functional state. We have developed a method for creating surface-patterned, biofunctionalized hydrogels on glass or silicon, using polyacrylamide and the disulfide-containing polyacrylamide crosslinker, bis(acryloyl)cystamine. Treatment with a reducing agent created reactive sulfhydryl (-SH) groups throughout these hydrogels that were readily conjugated to iodoacetyl biotin and streptavidin (SA). Immobilization efficiency was approximately 1-2% of the total potential binding capacity of the hydrogel. Porosity of the hydrogel was not a limiting factor for SA immobilization, as determined using fluorescence confocal microscopy. Rather, steric hindrance due to the binding of SA decreased the effective porosity near the surface of the hydrogel, restricting access to the rest of the gel. Using microcontact printing, we indirectly patterned SA on the surface of the hydrogel, generating well-resolved feature sizes of 2 microm in width. Through repeated rounds of microcontact printing, multiple, adjacent protein patterns were generated on the surface of the hydrogel. Biotinylated immune complexes and lipid vesicles readily bound to SA-functionalized hydrogels, demonstrating the feasibility of using this hydrogel system to generate complex biofunctionalized surfaces.

Acrylic Resins↗

PubMed2006

BCI Meeting 2005--workshop on signals and recording methods.

This paper describes the highlights of presentations and discussions during the Third International BCI Meeting in a workshop that evaluated potential brain-computer interface (BCI) signals and currently available recording methods. It defined the main potential user populations and their needs, addressed the relative advantages and disadvantages of noninvasive and implanted (i.e., invasive) methodologies, considered ethical issues, and focused on the challenges involved in translating BCI systems from the laboratory to widespread clinical use. The workshop stressed the critical importance of developing useful applications that establish the practical value of BCI technology.

Algorithms↗

PubMed2005

On-chip micro-biosensor for the detection of human CD4(+) cells based on AC impedance and optical analysis.

The current study was undertaken to fabricate a small micro-electrode on-chip to rapidly detect and quantify human CD4(+) cells in a minimal volume of blood through impedance measurements made with simple electronics that could be battery operated implemented in a hand held device. The micro-electrode surface was non-covalently modified sequentially by incubation with solutions of protein G', human albumin, monoclonal mouse anti-human CD4, and mouse IgG. The anti-human CD4 antibody served as the recognition and capture molecule for CD4(+) cells present in human blood. The binding of these biomolecules to the micro-electrodes was verified by impedance and cyclic voltammetry measurements. An increase in impedance was detected for each layer of protein adsorbed onto the micro-electrode surface. This process was shown to be highly repeatable. Increased impedance was measured when CD4(+) cells were captured on the micro-electrode, and the impedance also increased as the number of captured cells increased. Fluorescence microscopy of captured cells immunolabeled with anti-human CD4, CD8, and CD19 antibodies, and the nuclear label DAPI, confirmed that only CD4(+) cells were captured. The results were highly dependent on the specimen preparation method used. We conclude that the on-chip capture system can efficiently quantify the number of CD4(+) cells.

Biosensing Techniques↗

PubMed2005

Development and characterization of on-chip biopolymer membranes.

In lab-on-a-chip applications, filtration is currently performed prior to sample loading or through pre-cast membranes adhered to the substrate. These membranes cannot be patterned to micrometer resolution, and their adhesion may be incompatible with the fabrication process or may introduce contaminants. We have developed an on-chip separation process using a biocompatible polymer that can be patterned and has controllable molecular rejection properties. We spun cast cellulose acetate (CA) membranes directly onto silicon wafers. Characterization of the molecular flux across the membrane showed that molecular weight and charge are major factors contributing to the membranes' rejection characteristics. Altering casting conditions such as polymer concentration in the casting solution and the quenching-bath composition and/or temperature allowed control of the molecular weight cut-off (MWCO). Three MWCOs; 300, 350, and 700 Da have been achieved for non-linear molecules. Molecular shape is also very important as much higher molecular weight single-stranded DNA was electrophoresed across the membranes while heme with a similar negative charge density was rejected. This was due to DNA's small molecular cross section. This is an important result because heme inhibits polymerase chain reactions (PCR) reducing the detection and characterization of DNA from blood samples.

Base Sequence↗

PubMed2005

Automated image analysis methods for 3-D quantification of the neurovascular unit from multichannel confocal microscope images.

BACKGROUND: There is a need for integrative and quantitative methods to investigate the structural and functional relations among elements of complex systems, such as the neurovascular unit (NVU), that involve multiple cell types, microvasculatures, and various genomic/proteomic/ionic functional entities. METHODS: Vascular casting and selective labeling enabled simultaneous three-dimensional imaging of the microvasculature, cell nuclei, and cytoplasmic stains. Multidimensional segmentation was achieved by (i) bleed-through removal and attenuation correction; (ii) independent segmentation and morphometry for each corrected channel; and (iii) spatially associative feature computation across channels. The combined measurements enabled cell classification based on nuclear morphometry, cytoplasmic signals, and distance from vascular elements. Specific spatial relations among the NVU elements could be quantified. RESULTS: A software system combining nuclear and vessel segmentation codes and associative features was constructed and validated. Biological variability contributed to misidentified nuclei (9.3%), undersegmentation of nuclei (3.7%), hypersegmentation of nuclei (14%), and missed nuclei (4.7%). Microvessel segmentation errors occurred rarely, mainly due to nonuniform lumen staining. CONCLUSIONS: Associative features across fluorescence channels, in combination with standard features, enable integrative structural and functional analysis of the NVU. By labeling additional structural and functional entities, this method can be scaled up to larger-scale systems biology studies that integrate spatial and molecular information.

Animals↗

PubMed2005

Upstream migration of Xylella fastidiosa via pilus-driven twitching motility.

Xylella fastidiosa is a xylem-limited nonflagellated bacterium that causes economically important diseases of plants by developing biofilms that block xylem sap flow. How the bacterium is translocated downward in the host plant's vascular system against the direction of the transpiration stream has long been a puzzling phenomenon. Using microfabricated chambers designed to mimic some of the features of xylem vessels, we discovered that X. fastidiosa migrates via type IV-pilus-mediated twitching motility at speeds up to 5 mum min(-1) against a rapidly flowing medium (20,000 mum min(-1)). Electron microscopy revealed that there are two length classes of pili, long type IV pili (1.0 to 5.8 mum) and short type I pili (0.4 to 1.0 mum). We further demonstrated that two knockout mutants (pilB and pilQ mutants) that are deficient in type IV pili do not twitch and are inhibited from colonizing upstream vascular regions in planta. In addition, mutants with insertions in pilB or pilQ (possessing type I pili only) express enhanced biofilm formation, whereas a mutant with an insertion in fimA (possessing only type IV pili) is biofilm deficient.

Bacterial Proteins↗

PubMed2004

Extracellular recordings from patterned neuronal networks using planar microelectrode arrays.

Neuronal cell networks have been reconstructed on planar microelectrode arrays (MEAs) from dissociated hippocampal pyramidal neurons. Microcontact printing (microCP) and a photoresist-liftoff method were used to selectively localize poly-L-lysine (PLL) on the surface of MEAs. Haptotaxis led to the organization of the neurons into networks localized adjacent to microelectrodes. Various grids of PLL with 2-25-microm-wide lines spaced by 50-200 microm with 15-25-microm nodes at intersection points were used to guide cell body attachment and neurite outgrowth. Bursting activity with spike amplitude attenuation was observed, and multichannel recordings detected instances of coincident firing activity. Finally, we present here an extracellular recording from a approximately 2 microm bundle of guided neurites.

Action Potentials↗

PubMed2004

Model neural prostheses with integrated microfluidics: a potential intervention strategy for controlling reactive cell and tissue responses.

Model silicon intracortical probes with microfluidic channels were fabricated and tested to examine the feasibility of using diffusion-mediated delivery to deliver therapeutic agents into the volume of tissue exhibiting reactive responses to implanted devices. Three-dimensional probe structures with microfluidic channels were fabricated using surface micromachining and deep reactive ion etching (DRIE) techniques. In vitro functional tests of devices were performed using fluorescence microscopy to record the transient release of Texas Red labeled transferrin (TR-transferrin) and dextran (TR-dextran) from the microchannels into 1% w/v agarose gel. In vivo performance was characterized by inserting devices loaded with TR-transferrin into the premotor cortex of adult male rats. Brain sections were imaged using confocal microscopy. Diffusion of TR-transferrin into the extracellular space and uptake by cells up to 400 microm from the implantation site was observed in brain slices taken 1 h postinsertion. The reactive tissue volume, as indicated by the presence of phosphorylated mitogen-activated protein kinases (MAPKs), was characterized using immunohistochemistry and confocal microscopy. The reactive tissue volume extended 600, 800, and 400 microm radially from the implantation site at 1 h, 24 h, and 6 weeks following insertion, respectively. These results indicate that diffusion-mediated delivery can be part of an effective intervention strategy for the treatment of reactive tissue responses around chronically implanted intracortical probes.

Animals↗

PubMed2004

Development of a rare cell fractionation device: application for cancer detection.

Isolating rare cells from biological fluids including whole blood or bone marrow is an interesting biological problem. Characterization of a few metastatic cells from cancer patients for further study is desirable for prognosis/diagnosis. Traditional methods have not proven adequate, due to the compositional complexity of blood, with its large numbers of cell types. To separate individual cells based on their mechanical characteristics, we have developed a series of massively parallel microfabricated sieving device. These devices were constructed with four successively narrower regions of channels numbering approximately 1800 per region. As cells traversed the device, they encountered each region and stopped at a gap width that prohibited passage due to their size. Cultured neuroblastoma cells, when mixed with whole blood and applied to the device, were retained in the 10-microm-wide by 20-microm-deep channels. All other cells migrated to the output. A derivative of the same device was utilized to characterize migration of whole blood. Adult white blood cells were retained at the 2.5-microm-wide by 5-microm-deep channels, while red blood cells passed through these channels. Devices designed to capture rare cells in peripheral circulation for downstream analysis will provide an important tool for diagnosis and treatment.

Cell Separation↗

PubMed2003

Automated three-dimensional tracing of neurons in confocal and brightfield images.

Automated three-dimensional (3-D) image analysis methods are presented for tracing of dye-injected neurons imaged by fluorescence confocal microscopy and HRP-stained neurons imaged by transmitted-light brightfield microscopy. An improved algorithm for adaptive 3-D skeletonization of noisy images enables the tracing. This algorithm operates by performing connectivity testing over large N x N x N voxel neighborhoods exploiting the sparseness of the structures of interest, robust surface detection that improves upon classical vacant neighbor schemes, improved handling of process ends or tips based on shape collapse prevention, and thickness-adaptive thinning. The confocal image stacks were skeletonized directly. The brightfield stacks required 3-D deconvolution. The results of skeletonization were analyzed to extract a graph representation. Topological and metric analyses can be carried out using this representation. A semiautomatic method was developed for reconnection of dendritic fragments that are disconnected due to insufficient dye penetration, an imaging deficiency, or skeletonization errors.

Animals↗

PubMed2003

Median-based robust algorithms for tracing neurons from noisy confocal microscope images.

This paper presents a method to exploit rank statistics to improve fully automatic tracing of neurons from noisy digital confocal microscope images. Previously proposed exploratory tracing (vectorization) algorithms work by recursively following the neuronal topology, guided by responses of multiple directional correlation kernels. These algorithms were found to fail when the data was of lower quality (noisier, less contrast, weak signal, or more discontinuous structures). This type of data is commonly encountered in the study of neuronal growth on microfabricated surfaces. We show that by partitioning the correlation kernels in the tracing algorithm into multiple subkernels, and using the median of their responses as the guiding criterion improves the tracing precision from 41% to 89% for low-quality data, with a 5% improvement in recall. Improved handling was observed for artifacts such as discontinuities and/or hollowness of structures. The new algorithms require slightly higher amounts of computation, but are still acceptably fast, typically consuming less than 2 seconds on a personal computer (Pentium III, 500 MHz, 128 MB). They produce labeling for all somas present in the field, and a graph-theoretic representation of all dendritic/axonal structures that can be edited. Topological and size measurements such as area, length, and tortuosity are derived readily. The efficiency, accuracy, and fully-automated nature of the proposed method makes it attractive for large-scale applications such as high-throughput assays in the pharmaceutical industry, and study of neuron growth on nano/micro-fabricated structures. A careful quantitative validation of the proposed algorithms is provided against manually derived tracing, using a performance measure that combines the precision and recall metrics.

Algorithms↗

PubMed2003

Controlling cellular reactive responses around neural prosthetic devices using peripheral and local intervention strategies.

While chronic use of indwelling micromachined neural prosthetic devices has great potential, the development of reactive responses around them results in a decrease in electrode function over time. Since the cellular events responsible for these responses may be anti-inflammatory in nature, we have tested the effectiveness of dexamethasone and cyclosporin A as potential drugs for developing intervention strategies following insertion of single-shank micromachined silicon devices. Peripheral injection of dexamethasone was effective in attenuating increased expression of glial fibrillary acidic protein and astrocyte hyperplasia observed during both initial- and sustained-reactive responses observed at one and six weeks post insertion, respectively. Peripheral injection of cyclosporin A had no positive effect. If anything, application of this drug increased the early reactive response. Effectiveness of local release of dexamethasone in rat neocortex was tested by inserting ribbons of poly (ethyl-vinyl) acetate containing 35% (w/w) dexamethasone. Initial concentrations of dexamethasone were similar to those obtained by peripheral injection. Local drug release provided continued control of cellular reactive responses during the six-week study period. These results demonstrate that peripheral delivery of dexamethasone can be used to control reactive responses and that local drug delivery by slow-release from biocompatible polymers may be a more effective method of drug intervention. Incorporating these strategies on micromachined devices may provide an intervention strategy that will insure the chronic functioning of electrodes on intracortical neuroprosthetic devices.

Animals↗

PubMed2002

Rapid automated three-dimensional tracing of neurons from confocal image stacks.

Algorithms are presented for fully automatic three-dimensional (3-D) tracing of neurons that are imaged by fluorescence confocal microscopy. Unlike previous voxel-based skeletonization methods, the present approach works by recursively following the neuronal topology, using a set of 4 x N2 directional kernels (e.g., N = 32), guided by a generalized 3-D cylinder model. This method extends our prior work on exploratory tracing of retinal vasculature to 3-D space. Since the centerlines are of primary interest, the 3-D extension can be accomplished by four rather than six sets of kernels. Additional modifications, such as dynamic adaptation of the correlation kernels, and adaptive step size estimation, were introduced for achieving robustness to photon noise, varying contrast, and apparent discontinuity and/or hollowness of structures. The end product is a labeling of all somas present, graph-theoretic representations of all dendritic/axonal structures, and image statistics such as soma volume and centroid, soma interconnectivity, the longest branch, and lengths of all graph branches originating from a soma. This method is able to work directly with unprocessed confocal images, without expensive deconvolution or other preprocessing. It is much faster that skeletonization, typically consuming less than a minute to trace a 70-MB image on a 500-MHz computer. These properties make it attractive for large-scale automated tissue studies that require rapid on-line image analysis, such as high-throughput neurobiology/angiogenesis assays, and initiatives such as the Human Brain Project.

Algorithms↗

PubMedDate not supplied

Evidence for adhesive activity of the ventrolateral flange in Giardia lamblia.

Giardia lamblia is an intestinal protozoan that inhabits the intestinal tract of man and other mammals by attaching to the mucosal surface via the contractile activity of an attachment organelle called the ventral adhesive disk. We have investigated the presence of other attachment mechanisms in G. lamblia trophozoites by using microfabricated substrates that sterically interfere with formation of the hypothesized "negative pressure" under the ventral adhesive disk that would mediate attachment to a substratum. Pillars measuring 1 microm high and 2 microm in diam. were constructed in microarrays with spacings smaller than the diameter of the ventral adhesive disk. Using high resolution field emission scanning electron microscopy, the attachment of trophozoites to the tops of pillars in the microfabricated substrates was investigated. Firm adhesion of trophozoites was observed to be mediated by direct attachment of the ventrolateral flange membrane to the tops of microfabricated pillars. Attachment to microfabricated surfaces was 16% of that observed for attachment mediated by the ventral adhesive disk (4.4 +/- 1.5 cells/100 micro2 micropillar surface vs. 25.9 +/- 3.1 cells/100 micro2 flat substrate, p < 0.0001) This is the first report of trophozoite adhesion to a substratum by a mechanism other than the direct attachment of the ventral adhesive disk, and provides experimental evidence that the ventrolateral flange may play a role in trophozoite adhesion. A hypothesis is presented describing how the adhesive nature of the ventrolateral flange might be involved in normal attachment of G. lamblia trophozoites to a substratum.

Animals↗