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PubMed2006

Fragmentation of singly protonated peptides via a combination of infrared and collisional activation.

The coupling of matrix-assisted laser desorption/ionization (MALDI) to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) provides an exceptionally capable platform for peptide analysis, but an important limitation of this approach is the difficulty in obtaining informative tandem mass spectra (MS/MS) of singly protonated peptides. This difficulty is especially pronounced with peptide ions containing basic amino acid residues (for example, tryptic peptides). While such ions can be fragmented in some instrument configurations, most FTICR instruments have comparatively little facility for high-energy fragmentation. Here, a novel MS/MS approach implemented with MALDI-FTICR-MS and specifically intended for enhanced fragmentation of singly protonated peptides is described. The method involves infrared irradiation in concert with the simultaneous application of sustained off-resonance irradiation collision-induced dissociation (SORI-CID). This form of MS/MS, described as a combination of infrared and collisional activation (CIRCA), is shown to provide a greater capacity for dissociation of singly charged model peptide ions as compared to infrared multiphoton dissociation (IRMPD) or SORI-CID alone. Overall, the CIRCA approach is demonstrated to be a feasible technique for accessing useful fragmentation pathways of singly charged peptides, including those harboring basic amino acid residues--a crucial feature in the context of proteomics.

Amino Acids, Basic↗

PubMed2006

Magnetic bead-based solid phase for selective extraction of genomic DNA.

Magnetic bead-based solid phases are widely used for the separation of nucleic acids from complex mixtures. The challenge to selectively separate specific DNA molecules (via complementary hybridization) in a single step is the selection of a linker between the capture probe and the solid support that can be exposed to high temperatures in the presence of a high salt media. This article presents a general platform for the fabrication of a magnetic bead-based selective solid phase that can be used for subtractive hybridization or sequence capture applications. Phosphorus dendrimers are used for the first time as linkers in a magnetic bead-based selective solid phase for capture of genomic DNA. Aside from providing a high loading capacity, they render a stable bond between the capture probe and the surface under the high temperature and salt conditions required for denaturation and capture to proceed in a single step. The thermal stability of the solid phase under these conditions is first demonstrated by hybridizing a Cy3-labeled target. The selective capture of DNA targets in a single step is then demonstrated by subtractive hybridization of fragmented human genomic DNA. The specificity and selectivity of the solid phase are demonstrated by the recovery of adenovirus serotype 4 DNA spiked into the human DNA target. The effect of steric and electrostatic constraints was also investigated by using dendrimers of different generations that vary in their size and the number of branches. The results demonstrate that this platform can be used for single-step subtractive hybridization applications with better performance over the conventional two-step method using streptavidin-coated magnetic beads.

Adenoviridae↗

PubMed2006

Optically transparent Au{111} substrates: flat gold nanoparticle platforms for high-resolution scanning tunneling microscopy.

We demonstrate a new type of Au{111} substrate that is both atomically flat and optically transparent, which consists of solution-grown flat gold nanoparticles (FGNPs) deposited on indium tin oxide (ITO)-coated glass. We show that FGNPs are atomically flat single-crystal plates with large {111} faces that expose only 2-4 atomic layers. These FGNPs are excellent platforms for alkanethiol self-assembled monolayers (SAMs) and for high-resolution scanning tunneling microscopy (STM). Our supported FGNPs are also low-cost Au{111} substrates, employing only basic wet chemical techniques in preparation. This approach should be broadly applicable to other types of substrates for scanning probe microscopies.

Journal Article↗

PubMed2002

Larval electroreceptors in the epidermis of mormyrid fish: I. Tuberous organs of type A and B.

Two types of larval electroreceptors, type A and B, are described in the epidermis of the head of larvae of three mormyrid species, Campylomormyrus cassaicus, Mormyrus rume proboscirostris and Pollimyrus isidori, bred in captivity. In each of these electroreceptor organs, a single sensory cell is found inside an intraepidermal cavity, sitting on a platform of accessory cells. The cavity is filled with microvilli originating both from the sensory cell and from the epidermal covering cells lining the intraepidermal cavity. These two types of tuberous larval electroreceptors differ in their distribution in the epidermis of the head, in the composition of their accessory cells, and by their innervation. The innervation found in type B organs is similar to that already described for electroreceptors of adult mormyrids. The sensorineural junction is composed of primary afferent terminal boutons, which contact the base of the sensory cell. Opposite each terminal bouton, a ribbon-like synaptic bar surrounded by vesicles is found in the cytoplasm of the sensory cell. In contrast, the base of the sensory cell in type A larval electroreceptors is not contacted by nervous terminal boutons, but instead forms closed appositions with specialized prolongations of accessory cells of the platform. The base of the sensory cell presents membrane evaginations, with hemispheric synaptic structures and few synaptic vesicles. These two types of electroreceptor organs degenerate at the time of the degeneration of the larval electric organ and the functional differentiation of the adult electric organ. The functional role of two tuberous electroreceptor types is examined.

Animal Structures↗

PubMed1997

Posttraining estradiol injections enhance memory in ovariectomized rats: cholinergic blockade and synergism.

The present experiments examined acute posttraining estrogenic influences on memory in ovariectomized rats. In experiment 1 rats received a single 8-trial (30-s ITI) training session with a submerged escape platform located in the same quadrant of a circular water maze on all trials. Following trial 8, rats received a posttraining intraperitoneal injection of either an estradiol-cyclodextrin inclusion complex (0.1, 0.2, or 0.4 mg/kg) or saline. On a retention test session 24 h later, the escape latencies of rats given injections of estradiol (0.2 mg/kg) were significantly lower than those of saline-treated rats, indicating an enhancement of memory. Injections of estradiol delayed 2 h posttraining did not affect retention, demonstrating a time-dependent effect of estradiol on memory storage processes. In experiment 2a, posttraining injections of the cholinergic muscarinic receptor antagonist scopolamine (0.4 mg/kg) impaired memory in ovariectomized rats. In experiment 2b, the memory-enhancing effect of estradiol (0.2 mg/kg) was blocked by concurrent posttraining administration of a subeffective dose (0.1 mg/kg) of scopolamine, suggesting an interaction between estradiol and muscarinic cholinergic systems in memory modulation. In experiment 3a, posttraining injections of the cholinergic muscarinic receptor agonist oxotremorine (0.2 mg/kg) enhanced memory in ovariectomized rats. In experiment 3b, concurrent posttraining injection a subeffective dose of estradiol (0.1 mg/kg) and a subeffective dose of oxotremorine (0.1 mg/kg) enhanced memory, indicating a synergistic effect of estradiol and muscarinic receptor activation on memory.

Animals↗

PubMed2004

A model of cerebellum stabilized and scheduled hybrid long-loop control of upright balance.

A recurrent integrator proportional integral derivative (PID) model that has been used to account for cerebrocerebellar stabilization and scaling of transcortical proprioceptive feedback in the control of horizontal planar arm movements has been augmented with long-loop force feedback and gainscheduling to describe the control of human upright balance. The cerebellar component of the controller is represented by two sets of gains that each provide linear scaling of same-joint and interjoint long-loop stretch responses between ankle, knee, and hip. The cerebral component of the model includes a single set of same-joint linear force feedback gains. Responses to platform translations of a three-segment body model operating under this hybrid proprioception and force-based long-loop control were simulated. With low-velocity platform disturbances, "ankle-strategy"-type postural recovery kinematics and electromyogram (EMG) patterns were generated using the first set of cerebeller control gains. With faster disturbances, balance was maintained by including the second set of gains cerebellar control gains that yielded "mixed ankle-hip strategy"-type kinematics and EMG patterns. The addition of small amounts of simulated muscular coactivation improved the fit to certain human datasets. It is proposed that the cerebellum switches control gainsets as a function of sensed body kinematic state. Reduction of cerebellar gains with a compensatory increase in muscular stiffness yielded posture recovery with abnormal motions consistent with those found in cerebellar disease. The model demonstrates that stabilized hybrid long-loop feedback with scheduling of linear gains may afford realistic balance control in the absence of explicit internal dynamics models and suggests that the cerebellum and cerebral cortex may contribute to balance control by such a mechanism.

Biomechanical Phenomena↗

PubMed2003

Repetitive acute pain in infancy increases anxiety but does not alter spatial learning ability in juvenile mice.

We assessed the long-term behavioural effects of a single acute or repetitive inflammatory pain experienced during infancy. Groups of male and female CD1 mice were subjected to either an acute single pain, i.e. a tail clip or sham pain at P8, or acute repetitive pain in the form of needle pricks or sham pain from P8 to P14. All of the subjects were tested in the elevated plus maze at P30 and in the Morris Water Maze from P31 to P38. Mice in the acute single pain and sham groups did not differ on measures of anxiety in the plus maze. Mice in the repetitive pain group demonstrated significantly more anxious behaviours than controls in the elevated plus maze as they spent less time in the open arms, made fewer open arm entries, displayed fewer head dips and showed more stretch attend postures. There were no effects of single or repetitive pain treatments in the latency to find the hidden platform in the Morris Water Maze. Overall, these data suggest that acute repetitive pain experienced during infancy may increase anxiety later in life but it does not influence spatial learning as measured in the Morris Water Maze. The origin of the anxiogenic profile shown in the acute repetitive pain mice may be a result of changes in neural circuitry or context dependent learning and is currently under further investigation with this paradigm.

Acute Disease↗

PubMed1991

Effects of short-lasting inactivations of the ventral hippocampus and medial septum on long-term and short-term acquisition of spatial information in rats.

This study was aimed at testing the effects of a reversible inactivation of the hippocampal formation on long-term and short-term acquisition of spatial information. Rats chronically equipped with either bilateral cannulae into the ventral hippocampus or a single cannula into the medial septum had to locate, in a circular platform with 18 holes on the periphery, the unique hole leading to a hidden shelter in order to avoid bright light. In Expt. 1, following 16 days of training (1 trial/day, 24 h ITI) without physical intervention, the location of the correct hole was changed on both Days 17 and 23, and the rats were either sham-injected or injected with lidocaine. Both hippocampally and septally lidocaine-injected rats relearned the new location at a rate similar to corresponding sham-injected animals. In Expt. 2, a massed-trial version of the task was used, in which the rats had to learn a new hole location on each daily session (3 trials, ITI = 1 min). Animals were sham-injected or lidocaine-injected on alternate sessions. While sham-injected rats improved in orientational accuracy over successive trials, both hippocampally and septally lidocaine-injected rats failed to display any between-trial improvement. The impairment displayed by lidocaine-injected rats when their hippocampus was inactivated confirms the role of the hippocampus in short-term spatial memory (Expt. 2). In contrast, short-lasting inactivation of the hippocampus did not prevent long-term spatial learning (Expt. 1). These results suggest that the hippocampus could process information 'off-line' in the delay between temporally discontiguous learning trials, and show that short-term and long-term spatial learning rely on distinct neurobiological mechanisms.

Animals↗

PubMed2004

Development of a microfabricated cytometry platform for characterization and sorting of individual leukocytes.

Organizing leukocytes into high-density arrays makes these cells amenable to rapid optical characterization and subsequent sorting, pointing to clinical and basic science applications. The present paper describes development of a cytometry platform for creating high-density leukocyte arrays and demonstrates retrieval of single cells from the array. Poly(ethylene glycol)(PEG) photolithography was employed to fabricate arrays of microwells composed of PEG hydrogel walls and glass attachment pads 20 microm x 20 microm and 15 microm x 15 microm in size. PEG micropatterned glass surfaces were further modified with cell-adhesive ligands, poly-L-lysine, anti-CD5 and anti-CD19 antibodies, in order to engineer specific cell-surface interactions within the individual wells. Localization of the fluorescently-labeled proteins in the glass attachment pads of PEG microwells was visualized by fluorescence microscopy. Glass slides micropatterned with PEG and cell-adhesive ligands were exposed to T-lymphocytes for 30 min. These anchorage-independent cells became selectively captured in the ligand-modified microwells forming high-density cell arrays. Cell occupancy in the microwells was found to be antibody-dependent, reaching 94.6 +/- 2.3% for microwells decorated with T-cell specific anti-CD5 antibodies. Laser capture microdissection (LCM) was investigated as a method for sorting cells from the array and retrieval of single selected cells was demonstrated.

B-Lymphocytes↗

PubMed1990

A new single-stage step test for the clinical assessment of maximal oxygen consumption.

Assessments of cardiorespiratory fitness are important in any health-assessment program or routine involving variables that may be influenced by an individual's level of physical fitness or ability to sustain an exercise program. Recently, a new single-stage step test was introduced that uses a height-adjustable platform to normalize the height of stepping for individuals of varying stature. This computer program is designed to simplify the use of this test and to facilitate the implementation in clinical environments where equipment or time is limited, yet accuracy and validity are essential.

Adolescent↗

PubMed2026

Complete genome sequence of the Anaplasma phagocytophilum clinical isolate NCH-1.

Anaplasma phagocytophilum is an obligate intracellular gram-negative bacterium and etiologic agent of human granulocytic anaplasmosis. A. phagocytophilum genomic sequencing has historically been performed via short-read platforms. Our optimized bacterial isolation protocol combined with Nanopore sequencing produced a single, closed 1,481,805 bp circular A. phagocytophilum strain NCH-1 chromosome.

Anaplasma phagocytophilum↗

PubMed2006

Model-projected mechanistic bases for sex differences in growth hormone regulation in humans.

Models of physiological systems facilitate rational experimental design, inference, and prediction. A recent construct of regulated growth hormone (GH) secretion interlinks the actions of GH-releasing hormone (GHRH), somatostatin (SRIF), and GH secretagogues (GHS) with GH feedback in the rat (Farhy LS, Veldhuis JD. Am J Physiol Regul Integr Comp Physiol 288: R1649-R1663, 2005). In contrast, no comparable formalism exists to explicate GH dynamics in any other species. The present analyses explore whether a unifying model structure can represent species- and sex-defined distinctions in the human and rodent. The consensus principle that GHRH and GHS synergize in vivo but not in vitro was explicable by assuming that GHS 1) evokes GHRH release from the brain, 2) opposes inhibition by SRIF both in the hypothalamus and on the pituitary gland, and 3) stimulates pituitary GH release directly and additively with GHRH. The gender-selective principle that GH pulses are larger and more irregular in women than men was conferrable by way of 4) higher GHRH potency and 5) greater GHS efficacy. The overall construct predicts GHRH/GHS synergy in the human only in the presence of SRIF when the brain-pituitary nexus is intact, larger and more irregular GH pulses in women, and observed gender differences in feedback by GH and the single and paired actions of GHRH, GHS, and SRIF. The proposed model platform should enhance the framing and interpretation of novel clinical hypotheses and create a basis for interspecies generalization of GH-axis regulation.

Animals↗

PubMed1975

Naloxone-induced jumping in morphine dependent mice: stimulus control and motivation.

When naloxone-precipitated abstinence occurs in morphine-dependent mice, they exhibit stereotyped jumping. The number of jumps has been used as an abstinence measure and the motivation for them described as 'escape'. Studies were designed to assess stimulus control and motivation here. Groups of ten dependent and ten saline mice were singly tested in both light and dark conditions in each of five covered cylinders (2-23 in high). Jumps were recorded automatically for 10 min after 10 mg/kg naloxone. At 2 in, which permitted tactile contact with the cover, no jumps occurred in the light or the dark; at 5-11 in, a high incidence of jumping occurred in both the light and the dark; at 23 in, too high for the animal to reach, relatively few jumps occurred in the light but many occurred in the dark. In another study, similar groups were singly tested in the light after naloxone on each of five circular platforms (18 in high, 3-48 in diameter). As the diameter increased, the number of mice jumping to the floor showed a linear decrease concomitant with an increase in latency. This suggests that jumping is controlled by visual and tactile cues; mice do not jump when escape is prevented by a cover which can be touched or a height which cannot be reached; but do jump when such cues are eliminated by intermediate heights or darkness. Latency varies with size of immediate explorable environment.

Animals↗

PubMed2006

Multilayer image grid reconstruction technology: four-dimensional interactive image reconstruction of microsurgical neuroanatomic dissections.

OBJECTIVE: Cadaveric dissection is the gold standard for training physicians in various surgical specialties. However, limitations in acquiring and storing sufficient cadaveric material, recent pressures in training opportunities, and progress in digital image technology have led to advances in virtual or artificial visual means to augment surgical training. For training neurosurgeons, the appearance of reality is still crucial for learning anatomic structures and procedures. We developed a four-dimensional (including time) multilayer digital image reconstruction technology (MIGRT) that allows users to manipulate a "volumetric" set of photographic image data from exquisite cadaveric intracranial dissections and to navigate through stages of neurosurgical procedures as the dissection progresses. METHODS: A robotic microscope with two digital cameras was used to capture dissection images, usually in stereoscopic mode. A grid space was created to define positions at which images are captured. Images were acquired from identical angles at the same grid coordinates but at different stages of various dissections. RESULTS: Image data are reconstructed according to the sequence of acquisition into a multilayer image grid system by the MIGRT software. The single interactive, four-dimensional montage is viewable a on common computer platform. CONCLUSION: MIGRT uniquely focuses on capturing anatomic content that preserves natural appearances, including procedure, texture, and color, which is far superior and preferable to images and a reconstructed image environment based on artificial or animated concepts. MIGRT shows time-dependent changes in procedures, provides depth perception by stereoscopy or unique sequential motion, and allows simultaneous interactivity at each step of the procedure.

Brain↗

PubMed2006

Progressive stent technologies: new approaches for the treatment of cardiovascular diseases.

The CYPHER (Cordis, Johnson & Johnson) sirolimus-eluting stent and the TAXU (Boston Scientific) paclitaxel-eluting stent have been extensively evaluated and have been proven to be significant novel tools for the treatment of coronary artery disease. Several sirolimus derivatives have already emerged, receiving CE Mark approval. However, in the future, it is likely that drugs presently under investigation will address additional mechanisms associated with neointimal formation, either as single agents or in combination with antiproliferative compounds. Concurrently, alterations on stent platform design (helicoidal, open-closed cell), coatings (biodegradable, bioabsorbable, nanoporous) and polymers are being explored.

Angioplasty, Balloon, Coronary↗

PubMed2006

Expression profiling of drug response--from genes to pathways.

Understanding individual response to a drug-what determines its efficacy and tolerability-is the major bottleneck in current drug development and clinical trials. Intracellular response and metabolism, for example through cytochrome P-450 enzymes, may either enhance or decrease the effect of different drugs, dependent on the genetic variant. Microarrays offer the potential to screen the genetic composition of the individual patient. However, experiments are "noisy" and must be accompanied by solid and robust data analysis. Furthermore, recent research aims at the combination of high-throughput data with methods of mathematical modeling, enabling problem-oriented assistance in the drug discovery process. This article will discuss state-of-the-art DNA array technology platforms and the basic elements of data analysis and bioinformatics research in drug discovery. Enhancing single-gene analysis, we will present a new method for interpreting gene expression changes in the context of entire pathways. Furthermore, we will introduce the concept of systems biology as a new paradigm for drug development and highlight our recent research-the development of a modeling and simulation platform for biomedical applications. We discuss the potentials of systems biology for modeling the drug response of the individual patient.

Animals↗

PubMed2004

Novel fluorescence-based approaches for the study of biogenic amine transporter localization, activity, and regulation.

Pre-synaptic norepinephrine (NE) and dopamine (DA) transporters (NET and DAT) terminate catecholamine synaptic transmission through reuptake of released neurotransmitter. Recent studies reveal that NET and DAT are tightly regulated by receptor and second messenger-linked signaling pathways. Common approaches for studying these transporters involve use of radiolabeled substrates or antagonists, methods possessing limited spatial resolution and that bear limited opportunities for repeated monitoring of living preparations. To circumvent these issues, we have explored two novel assay platforms that permit temporally resolved quantitation of transport activity and transporter protein localization. To monitor the binding and transport function of NET and DAT in real-time, we have investigated the uptake of the fluorescent organic compound 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (ASP+). We have extended our previous single cell level application of this substrate to monitor transport activity via high-throughput assay platforms. Compared to radiotracer uptake methods, acquisition of ASP+ fluorescence is non-isotopic and allows for continuous, repeated transport measurements on both transfected and native preparations. Secondly, we have extended our application of small-molecule-conjugated fluorescent CdSe/ZnS nanocrystals, or quantum dots (Qdots), to utilize antibody and peptide ligands that can identify surface expressed transporters, receptors and other membrane proteins in living cell systems. Unlike typical organic fluorophores, Qdots are highly resistant to bleaching and can be conjugated to multiple ligands. They can also be illuminated by conventional light sources, yet produce narrow, gaussian emission spectra compatible with multiple target visualization (multiplexing). Together, these approaches offer novel opportunities to investigate changes in transporter function and distribution in real-time with superior spatial and temporal resolution.

Antibodies↗

PubMed2006

Self-assembled single-crystal silicon circuits on plastic.

We demonstrate the use of self-assembly for the integration of freestanding micrometer-scale components, including single-crystal, silicon field-effect transistors (FETs) and diffusion resistors, onto flexible plastic substrates. Preferential self-assembly of multiple microcomponent types onto a common platform is achieved through complementary shape recognition and aided by capillary, fluidic, and gravitational forces. We outline a microfabrication process that yields single-crystal, silicon FETs in a freestanding, powder-like collection for use with self-assembly. Demonstrations of self-assembled FETs on plastic include logic inverters and measured electron mobility of 592 cm2/V-s. Finally, we extend the self-assembly process to substrates each containing 10,000 binding sites and realize 97% self-assembly yield within 25 min for 100-microm-sized elements. High-yield self-assembly of micrometer-scale functional devices as outlined here provides a powerful approach for production of macroelectronic systems.

Journal Article↗