A Morphometric Analysis of Platelet Dense Granules of Patients with Unexplained Bleeding: A New Entity of Delta-Microgranular Storage Pool Deficiency.

One thousand and eighty patients, having prolonged bleeding times, frequent epistaxis, menorrhagia or easy bruising or other bleeding manifestations, and excluding those with von Willebrand's disease, were evaluated for platelet dense granule deficiency. The mean diameter of platelet dense granules was determined for all patients using image analysis. Four hundred and ninety-nine had "classic" dense (delta) granule storage pool deficiency (δ-SPD). Five hundred and eighty-one individuals (53.8%) were found to have a normal mean number of dense granules, but for some of these patients, the dense granules were smaller than for the controls. Of the patients having a normal number of dense granules, 165 (28.4%) were found to have significantly smaller granules than the platelets obtained from the control subjects. Their average granule diameter was 123.35 ± 0.86 nm, that is more than three standard deviations below the mean of the control data. Total δ-granule storage pool volumes (TDGV)/platelet were calculated using these measurements. Individuals with δ-SPD had half the number of granules (2.25 ± 0.04 DG/PL) and storage pool volume (3.88 ± 1.06 × 106 nm3) when compared to our control data (4.64 ± 0.11 DG/PL; 10.79 × 106 nm3 ± 0.42). Individuals having a bleeding history but a normal average of small dense granules had a calculated storage pool volume statistically different than controls and essentially the same storage pool volume as patients with δ-SPD. We have identified a sub-classification of δ-SPD that we have defined as micro-granular storage pool deficiency (δ-MGSPD).

Pulse electrodeposited manganese oxide on carbon fibers as electrodes for high capacity supercapacitors.

Arrays of manganese dioxide (MnO2), a pseudocapacitive material, have been deposited on the carbon fibers (CF) of a carbon woven fabric by electrodeposition from a solution containing manganese sulfate and sulfuric acid using galvanostatic square waves. The thickness of the MnO2 was varied by increasing/decreasing the time of deposition, and the electrochemical performance of the MnO2 has been analyzed. The CF serves as a substrate material with high surface area, good electrical conductivity and excellent mechanical strength. The electrochemical properties of the resultant electrode were examined by cyclic voltammogram (CV), galvanostatic charge/discharge, and electrochemical impedance spectroscopy in a three-electrode system. From the specific capacitance calculations obtained from CV and charge-discharge, a high specific capacitance of 769 F g-1 @ 5 mV s-1 (low weight electrode) has been achieved. The maximum area capacitance, estimated from the charge-discharge curves, was 790 mF cm-2 @ 5 mV s-1. The high-performance is attributed to the double layer capacitance of the CFs combined with the pseudocapacitive nature of MnO2, the large surface area, and high degree of ordering of the ultrathin MnO2.

Initial performance studies of a wearable brain positron emission tomography camera based on autonomous thin-film digital Geiger avalanche photodiode arrays.

Using analytical and Monte Carlo modeling, we explored performance of a lightweight wearable helmet-shaped brain positron emission tomography (PET), or BET camera, based on thin-film digital Geiger avalanche photodiode arrays with Lutetium-yttrium oxyorthosilicate (LYSO) or [Formula: see text] scintillators for imaging in vivo human brain function of freely moving and acting subjects. We investigated a spherical cap BET and cylindrical brain PET (CYL) geometries with 250-mm diameter. We also considered a clinical whole-body (WB) LYSO PET/CT scanner. The simulated energy resolutions were 10.8% (LYSO) and 3.3% ([Formula: see text]), and the coincidence window was set at 2 ns. The brain was simulated as a water sphere of uniform F-18 activity with a radius of 100 mm. We found that BET achieved [Formula: see text] better noise equivalent count (NEC) performance relative to the CYL and [Formula: see text] than WB. For 10-mm-thick [Formula: see text] equivalent mass systems, LYSO (7-mm thick) had [Formula: see text] higher NEC than [Formula: see text]. We found that [Formula: see text] scintillator crystals achieved [Formula: see text] full-width-half-maximum spatial resolution without parallax errors. Additionally, our simulations showed that LYSO generally outperformed [Formula: see text] for NEC unless the timing resolution for [Formula: see text] was considerably smaller than that presently used for LYSO, i.e., well below 300 ps.

Neurodegeneration by activated microglia across a nanofiltration membrane.

Microglia have been implicated in the pathogenesis of several neurodegenerative diseases, but their precise role remains elusive. Although neuron loss in the presence of lipopolysaccharide-stimulated microglia has been well documented, a novel coculture paradigm was developed as a new approach to assess the diffusible, soluble mediators of neurodegeneration. Isolated microglia were plated on membrane inserts that were coated with a layer of cellulose acetate. The cellulose acetate-coated membranes have nanofiltration properties, in that only molecules with masses less than 350 Da can pass through. Products released from activated microglia that were separated from primary ventral mesencephalon cells beneath the nanofiltering membrane were able to kill the dopamine neurons. Microglial cytokines cannot diffuse through this separating membrane. Addition of a nitric oxide synthase inhibitor prevented the loss of the dopamine neurons. These data describe a novel coculture system for studying diffusible factors and further support nitric oxide production as an important mediator in microglia-induced neuron death.

Isolation of tumor cells using size and deformation.

The isolation and analysis of circulating tumor cells (CTCs) from blood are the subject of intense research. Although tests to detect metastasis on a molecular level are available, progress has been hampered by a lack of tumor-specific markers and predictable DNA abnormalities. The main challenge in this endeavor is the small number of available cells of interest, 1-2 per mL in whole blood. We have designed a micromachined device to fractionate whole blood using physical means to enrich for and/or isolate rare cells from peripheral circulation. It has arrays of four successively narrower channels, each consisting of a two-dimensional array of columns. Current devices have channels ranging in width from 20 to 5 microm, and in depth from 20 to 5 microm. Several optimizations resulting in the fabrication of a total of 10 derivative devices have been carried out; only two types are used in this study. Both have increasingly narrower gap widths between the columns along the flow axis with 20, 15, 10, and 5 microm spacing all on one device. The first 20 microm wide segment disperses the cell suspension and creates an evenly distributed flow over the entire device, whereas the others were designed to retain increasingly smaller cells. The channel depth is constant across the entire device, the first type was 10 microm deep and the second type is 20 microm deep. When cells from each of eight tumor cell lines were loaded into the device, all cancerous cells were isolated. In mixing experiments using human whole blood, we were able to fractionate cancer cells without interference from the blood cells. Additionally, either intact cells, or DNA, could be extracted for molecular analysis. The ultimate goal of this work is to characterize the cells on the molecular level to provide non-invasive methods to monitor patients, stage disease, and assess treatment efficacy. Furthermore, this work will use gene expression profiles to gain insights into metastasis.

Release of nerve growth factor from HEMA hydrogel-coated substrates and its effect on the differentiation of neural cells.

Local pharmacological intervention may be needed to ensure the long-term performance of neural prosthetic devices because of insertion-related neuron loss and reactive cell responses that form compact sheaths, leading to decreased device performance. We propose that local delivery of neurotrophins would enhance neuron survival and promote neuron sprouting toward device electrodes, thus providing improved electrode-neuron communication and device performance for recording and stimulating CNS activity. In this study, three different types of poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels were developed and assessed for storage capacity and release rates of the neurotrophin, nerve growth factor (NGF). Additionally, a method was developed for routine coating of microfabricated neuroprosthetic devices with the different pHEMA hydrogels. Biological responses to hydrogel-delivered NGF from the devices were measured using primary cell cultures of dorsal root ganglion (DRG) neurons. Neuron process growth was used to assess biological responses to released NGF. When targeted media concentrations were the same, responses to bath-applied NGF and NGF released from pHEMA hydrogels were not significantly different. When NGF was released from lysine-conjugated pHEMA hydrogels, a significant increase in process growth was observed. Our studies demonstrate that pHEMA coatings can be used on neural devices consistent with the needs for local neurotrophin delivery in the brain.

Light-emitting diodes are better illumination sources for biological microscopy than conventional sources.

Light-emitting diodes (LEDs) can be easily and inexpensively integrated into modern light microscopes. There are numerous advantages of LEDs as illumination sources; most notably, they provide brightness and spectral control. We demonstrate that for transmitted light imaging, an LED can replace the traditional tungsten filament bulb while offering longer life; no color temperature change with intensity change; reduced emission in the infrared region, which is important for live cell imaging; and reduced cost of ownership. We show a direct substitution of the typical tungsten bulb with a commercially available LED and demonstrated the color stability by imaging a histology section over a wide range of light intensities. For fluorescent imaging, where the typical illumination sources are mercury or xenon lamps, we demonstrate that LEDs offer advantages of providing a longer lifespan, having a more constant intensity output over time, more homogeneous illumination, and significantly lower photon dose. Our LED equipped system was used to image and deconvolve dual fluorescently labeled cells, as well as image cells undergoing mitosis expressing green fluorescent protein-histone 2B complex. The timing of the stages of mitosis is well established as an indicator of cell viability.

Improved detection of branching points in algorithms for automated neuron tracing from 3D confocal images.

Automated tracing of neuronal processes from 3D confocal microscopy images is essential for quantitative neuroanatomy and neuronal assays. Two basic approaches are described in the literature-one based on skeletonization and another based on sequential tracing along neuronal processes. This article presents algorithms for improving the rate of detection, and the accuracy of estimating the location and process angles at branching points for the latter class of algorithms. The problem of simultaneously detecting branch points and estimating their measurements is formulated as a generalized likelihood ratio test defined on a spatial neighborhood of each candidate point, in which likelihoods were computed using a ridge detection approach. The average detection rate increased from from 37 to 86%. The average error in locating the branch points decreased from 2.6 to 2.1 voxels in 3D images. The generalized hypothesis test improves the rate of detection of branching points, and the accuracy of location estimates, enabling a more complete extraction of neuroanatomy and more accurate counting of branch points in neuronal assays. More accurate branch point morphometry is valuable for image registration and change analysis.

Applications of hydrogels for neural cell engineering.

Hydrogels, three-dimensional networks of hydrophilic polymers, are currently being investigated for use in a variety of tissue-engineering applications. Hydrogel materials generally exhibit a number of properties including permeability to oxygen and nutrients, which make these materials attractive for use in biological applications. However, in order for such polymers to be successfully integrated into biological systems, these constructs must be engineered so as to mimic native biological interfaces. In this paper we discuss the use of acrylate-based polymers and their derivatives, for use in biomedical applications, including drug delivery, tissue engineering, cell encapsulation, and as templates for directed cell growth, attachment and proliferation.

Electrical stimulation-induced cell clustering in cultured neural networks.

Planar microelectrode arrays (MEAs) are widely used to record electrical activity from neural networks. However, only a small number of functional recording sites frequently show electrical activity. One contributing factor may be that neurons in vitro receive insufficient synaptic input to develop into fully functional networks. In this study, electrical stimulation was applied to neurons mimicking synaptic input. Various stimulation paradigms were examined. Stimulation amplitude and frequency were tailored to prevent cell death. Two effects of stimulation were observed when 3-week-old cultures were stimulated: (1) clusters of neural cells were observed adjacent to stimulating electrodes and (2) an increase in spontaneous neuronal activity was recorded at stimulating electrodes. Immunocytochemical analysis indicates that stimulation may cause both new neuron process growth as well as astrocyte activation. These data indicate that electrical stimulation can be used as a tool to modify neural networks at specific electrode sites and promote electrical activity.

Biochip for separating fetal cells from maternal circulation.

Isolation of fetal cells from maternal circulation is the subject of intense research to eliminate the need for currently used invasive prenatal diagnosis procedures. Fetal cells can be isolated using magnetic-activated cell sorting or fluorescence-activated cell sorting, however no technique to specifically isolate and use fetal cells for genetic diagnosis has reached routine clinical practice. This paper demonstrates the use of a micromachined device to separate fetal cells from maternal circulation based on differences in size and deformation characteristics. Nucleated fetal red blood cells range in diameter from 9 to 12 microm can deform and pass through a channel as small as 2.5 microm wide and 5 microm deep. Although the white blood cells range in diameter from 10 to 20 microm, they cannot deform and are retained by the 2.5 microm wide and 5 microm deep channels under our experimental conditions. Fetal cells were isolated from cord blood and DNA analysis confirmed their fetal origin with ruled out maternal contamination.

Directed cell growth on protein-functionalized hydrogel surfaces.

Biochemical surface modification has been used to direct cell attachment and growth on a biocompatible gel surface. Acrylamide-based hydrogels were photo-polymerized in the presence of an acroyl-streptavidin monomer to create planar, functionalized surfaces capable of binding biotin-labelled proteins. Soft protein lithography (microcontact printing) of proteins was used to transfer the biotinylated extracellular matrix proteins, fibronectin and laminin, and the laminin peptide biotin-IKVAV, onto modified surfaces. As a biological assay, we plated LRM55 astroglioma and primary rat hippocampal neurons on patterned hydrogels. We found both cell types to selectively adhere to areas patterned with biotin-conjugated proteins. Fluorescence and bright-field modes of microscopy were used to assess cell attachment and cell morphology on modified surfaces. LRM55 cells were found to attach to protein-stamped regions of the hydrogel only. Neurons exhibited significant neurite extension after 72h in vitro, and remained viable on protein-stamped areas for more than 4 weeks. Patterned neurons developed functionally active synapses, as measured by uptake of the dye FM1-43FX. Results from this study suggest that hydrogel surfaces can be patterned with multiple proteins to direct cell growth and attachment.

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.

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.

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.

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.

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.

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.

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.

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.