Three-Dimensional-Printed Electrochemical Sensor for Simultaneous Dual Monitoring of Serotonin Overflow and Circular Muscle Contraction.

Serotonin (5-HT) is a key signaling molecule within the mucosal epithelium of the intestinal wall and has been shown to be an important modulator of motility. At present, no single approach has been established for simultaneous dual measurement of 5-HT overflow and circular muscle contraction. We developed a 3D-printed carbon black/polylactic acid (PLA) electrochemical sensor, which had a geometry suitable for ex vivo measurement in the anorectum. The device was characterized for sensitivity and stability for 5-HT measurements as well as suitability for accurate tracking of anorectal contractions. The 3D-printed electrochemical sensor had a linear range in physiological concentrations of 5-HT (1-10 µM) present within the intestinal tract and a limit of detection of 540 nM. The sensor was stable for 5-HT measurement following ex vivo tissue measurements. There was a signficant correlation in the amplitude and duration of individual contractions when comparing the measurements using an isometric force transducer and 3D-printed electrochemical sensor. Finally, in the presence of 1 µM fluoxetine, the sensor was able to monitor a reduction in contractility as well as an increase in 5-HT overflow as predicted. Overall, the 3D-printed sensor has the ability to conduct dual simultaneous measurements of 5-HT overflow and contractility. This single device will have significant potential for clinical measurements of anorectum function and signaling that can direct therapeutic management of patients with bowel disorders.

Transcriptome analysis of the central nervous system of the mollusc Lymnaea stagnalis.

BACKGROUND: The freshwater snail Lymnaea stagnalis (L. stagnalis) has served as a successful model for studies in the field of Neuroscience. However, a serious drawback in the molecular analysis of the nervous system of L. stagnalis has been the lack of large-scale genomic or neuronal transcriptome information, thereby limiting the use of this unique model. RESULTS: In this study, we report 7,712 distinct EST sequences (median length: 847 nucleotides) of a normalized L. stagnalis central nervous system (CNS) cDNA library, resulting in the largest collection of L. stagnalis neuronal transcriptome data currently available. Approximately 42% of the cDNAs can be translated into more than 100 consecutive amino acids, indicating the high quality of the library. The annotated sequences contribute 12% of the predicted transcriptome size of 20,000. Surprisingly, approximately 37% of the L. stagnalis sequences only have a tBLASTx hit in the EST library of another snail species Aplysia californica (A. californica) even using a low stringency e-value cutoff at 0.01. Using the same cutoff, approximately 67% of the cDNAs have a BLAST hit in the NCBI non-redundant protein and nucleotide sequence databases (nr and nt), suggesting that one third of the sequences may be unique to L. stagnalis. Finally, using the same cutoff (0.01), more than half of the cDNA sequences (54%) do not have a hit in nematode, fruitfly or human genome data, suggesting that the L. stagnalis transcriptome is significantly different from these species as well. The cDNA sequences are enriched in the following gene ontology functional categories: protein binding, hydrolase, transferase, and catalytic enzymes. CONCLUSION: This study provides novel molecular insights into the transcriptome of an important molluscan model organism. Our findings will contribute to functional analyses in neurobiology, and comparative evolutionary biology. The L. stagnalis CNS EST database is available at http://www.Lymnaea.org/.

Investigating the potential advantages of a new design metacarpophalangeal joint.

This paper investigates a new metacarpophalangeal joint design aimed at treating patients with moderate to severe forms of arthritis affecting the index, long, ring, and little fingers. Current small joint arthroplasty designs, including those for the metacarpophalangeal joint of the hand, have had limited success owing to mechanical failures and can be divided into two main families: single-piece elastomer implants and surface articulating implants. The design proposed in this study involves combining the principles of a surface weight-bearing articulating implant with those of a spanning elastomeric implant. The design consists of metacarpal and proximal phalangeal articulating housings and a central flexible spanning elastomer rod that maintains the alignment of the metacarpal and proximal phalangeal components. A preliminary finite element analysis was used to investigate the stresses in the design, the wear of the articulating bearing surfaces for different material combinations, and the stresses between the central elastomer rod and the articulating housings. This preliminary analysis shows that the design should withstand the probable loading conditions experienced within the human body and that the wear rates of the articulating surfaces and the central elastomer are acceptable.

Changes in murine anorectum signaling across the life course.

BACKGROUND: Increasing age is associated with an increase in the incidence of chronic constipation and fecal impaction. The contribution of the natural aging process to these conditions is not fully understood. This study examined the effects of increasing age on the function of the murine anorectum. METHODS: The effects of increasing age on cholinergic, nitrergic, and purinergic signaling pathways in the murine anorectum were examined using classical organ bath assays to examine tissue function and electrochemical sensing to determine age-related changes in nitric oxide and acetylcholine release. KEY RESULTS: Nitrergic relaxation increased between 3 and 6 months, peaked at 12 months and declined in the 18 and 24 months groups. These changes were in part explained by an age-related decrease in nitric oxide (NO) release. Cholinergic signaling was maintained with age by an increase in acetylcholine (ACh) release and a compensatory decrease in cholinesterase activity. Age-related changes in purinergic relaxation were qualitatively similar to nitrergic relaxation although the relaxations were much smaller. Increasing age did not alter the response of the anorectum smooth muscle to exogenously applied ACh, ATP, sodium nitroprusside or KCl. Similarly, there was no change in basal tension developed by the anorectum. CONCLUSIONS AND INFERENCES: The decrease in nitrergic signaling with increasing age may contribute to the age-related fecal impaction and constipation previously described in this model by partially obstructing defecation.

Electrochemical sensor for the detection of multiple reactive oxygen and nitrogen species from ageing central nervous system homogenates.

Reactive oxygen and nitrogen species (ROS/RNS) have been widely implicated in the ageing process and various approaches exist for monitoring these species in biological tissues. These approaches at present are limited to monitoring either a single pro-oxidant species or total pro-oxidant levels and therefore provide limited insight into the range of pro-oxidant species and their relative proportions in the ageing process. We have utilised a sensor that allows us to simultaneously monitor hydrogen peroxide, peroxynitrite, nitric oxide and nitrite. Using CNS homogenates from the pond snail, Lymnaea, we were able to show that levels of these ROS/RNS increased between young and old CNS homogenates and were different in various aged CNS regions.

X-ray transport and radiation response assessment (XTRRA) experiments at the National Ignition Facility.

Our team has developed an experimental platform to evaluate the x-ray-generated stress and impulse in materials. Experimental activities include x-ray source development, design of the sample mounting hardware and sensors interfaced to the National Ignition Facility's diagnostics insertion system, and system integration into the facility. This paper focuses on the X-ray Transport and Radiation Response Assessment (XTRRA) test cassettes built for these experiments. The test cassette is designed to position six samples at three predetermined distances from the source, each known to within ±1% accuracy. Built-in calorimeters give in situ measurements of the x-ray environment along the sample lines of sight. The measured accuracy of sample responses as well as planned modifications to the XTRRA cassette is discussed.

Synthesis and loss of activity of glucose-6-phosphate dehydrogenase in dividing plant cells.

1. Using the technique of density-labelling with deuterium oxide, evidence has been obtained for the de novo synthesis of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADPH+ 1-oxidoreductase, EC 1.1.1.49), during the culture of synchronously growing plant cells. 2. The entire increase in enzyme activity during the early cell cycles in this material can be accounted for by the appearance of an enzyme species with increased buoyand density. 3. A method is described for resolving overlapping distribution profiles after density centrifugation, which allows estimation of the amount of each species present at different times, and calculation of the loss of activity of the light species present from the start of culture. 4. Loss of activity of glucose-6-phosphate dehydrogenase in normal growing conditions in the presence of 2,4-dichlorophenoxyacetic acid is very much faster than in conditions which do not lead to cell division: in the absence of 2,4-dichlorophenoxyacetic acid, or in the presence of the inhibitor of RNA synthesis, 6-methylpurine.

Manipulating secondary metabolism in cultured plant cells.

This review attempts to present an overall strategy for the production of useful secondary metabolites by cultured plant cells. After consideration of the nature and utility of secondary metabolites and the possible role of these substances to the plant, the review focuses attention on the properties of the plant cells in culture and how the cell populations and their physical and chemical environment can be manipulated to encourage the synthesis and accumulation of secondary products. Finally, consideration is given to the involvement of genetic engineering in the production of cells to perform particular metabolic tasks and how these techniques might contribute to the development of a new strategy to enable the production of useful secondary metabolites on a commercial scale. CONTENTS Summary 553 I. Introduction 553 II. Development of a strategy 555 III. Concluding remarks 565 Acknowledgements 565 References 565.

Developmental changes in Sitka spruce as indices of physiological age I. Changes in needle morphology.

There is a paucity of data regarding developmental changes in trees. Measurements of length, width, length/width ratio, perimeter/width and width/height ratios of tranverse sections, projected and total surface areas, dry weight, specific leaf area and the weight of epicuticular waxes per unit leaf area were made on needles sampled from the uppermost whorl of Sitka spruce trees aged 3, 5,10,15, 20 and 38 years. All characteristics, except needle length and the weight of epicuticular waxes, showed asymptotic changes with age that could adequately be described by the Gompertz growth function, and offer potential as indices of physiological age. Asymptotes were reached at different ages (width and the length/width ratio having the slowest rates of change and the perimeter/width ratio, specific leaf area, and the width/height ratio having the fastest) suggesting a predictable sequence of changes. The observed changes in needle morphology are interpreted as a transition from shade to sun leaves.

Developmental changes in Sitka spruce as indices of physiological age: II. Rooting of cuttings and callusing of needle explants.

Adventitious rooting ability is considered a good indicator of juvenility in trees and has often been used to assess explants subjected to treatments to alter developmental stage. However, rooting assessments are not always practical and the callusing of needles in vitro was investigated as an alternative bioassay of physiological age. Shoots were sampled from the uppermost whorl of Sitka spruce trees aged 1, 3, 4, 6, 11, 16, 21, 35 and 39 years and from the lowest whorls of 3- and 4-year-old trees. Needles were sampled for measurement of length, width, length/width ratio, projected and total surface areas, dry weight and specific leaf area. For explants of uppermost whorl origin, there were decreases in the rooting ability of cuttings and the callusing response of needles with increasing age of tree. Associated with the loss in callusing ability was an increase in the production of a black exudate. The changes with age for rooiing, callusing and exudation could each be adequately described by the Gompertz growth function which showed their rates of change to be similar. The observed changes are closely associated with changes in needle area, weight and specific leaf area. Euclidean distances (using the mean values of selected characteristics for each age class) were computed as a measure of physiological age. The shoots and needles of basal shoots are more juvenile in character than those taken from the uppermost whorls of the same trees.

Myenteric neuron numbers are maintained in aging mouse distal colon.

BACKGROUND Age-associated myenteric neuronal loss has been described in several species. In some studies,cholinergic neurons have been reported to be selectively vulnerable, whereas nitrergic neurons are spared. Aging of the mouse enteric nervous system(ENS) and the subtypes of mouse myenteric neurons that may be lost have been little studied. We therefore investigated changes in the numbers of total neurons and two neuronal subpopulations in the mouse distal colon during aging. METHODS Wholemount preparations from 3­4-, 12­13-, 18­19-, and 24­25-month-old C57BL/6 mice were double immunolabeled with HuC/D antibody to identify the total neuronal population and antisera to either calbindin or neuronal nitric oxide synthase (nNOS) to identify myenteric neuronal subpopulations. Samples were analyzed by confocal microscopy. New procedures were employed to ensure unbiased counting and to correct for changes in gut dimensions with age and stretch during sample preparation. The density of nerve fibers in the tertiary plexus was also studied. KEY RESULTS No significant change in numbers of total neurons or of either subpopulation with age was measured, but because of gut growth, the density of myenteric neurons decreased between 3­4 and 12­13 months. The density of nNOS-immunoreactive nerve fibers in the tertiary plexus increased significantly with age, up to 18­19 months. Numerous swollen processes of CB and nNOS-immunoreactive neurons were observed in 18­19- and 24­25-month-old animals. Conclusions &Inferences These results indicate that aging does not result in a loss of myenteric neurons in mouse distal colon at the ages studied, although neurodegenerative changes, which may impact on neuronal function, do occur.

Changes in the innervation of the mouse internal anal sphincter during aging.

BACKGROUND: The innervation of the mouse internal anal sphincter (IAS) has been little studied, and how it changes during aging has not previously been investigated. The aim of this study was therefore to characterize the distribution and density of subtypes of nerve fibers in the IAS and underlying mucosa in 3-, 12- to 13-, 18- and 24- to 25-month-old male C57BL/6 mice. METHODS: Nerve fibers were immunolabeled with antibodies against protein gene product 9.5 (PGP9.5), neuronal nitric oxide synthase (nNOS), vasoactive intestinal polypeptide (VIP), substance P (SP), calcitonin gene-related peptide (CGRP), and calretinin (CR). Immunoreactivity in nerve fibers in the circular muscle and mucosa was quantified using Image J software. KEY RESULTS: In young adult (3 month) mice, nNOS-immunoreactive (IR) nerve fibers were densely distributed in the circular muscle, but relatively few in the mucosa; VIP-IR nerve fibers were abundant in the circular muscle and common in the mucosa; SP-IR nerve fibers were common in circular muscle and mucosa; CGRP- and CR-IR nerve fibers were dense in mucosa and sparse in circular muscle. The density of PGP9.5 immunoreactivity (IRY) was not significantly reduced with age, but a significant reduction in nNOS-IRY and SP-IRY with age was found in the IAS circular muscle. Neuronal nitric oxide synthase-, VIP-, and SP-IRY in the anal mucosa were significantly reduced with age. CGRP-IRY in both circular muscle and mucosa was increased in 18-month-old animals. CONCLUSIONS & INFERENCES: The density of immunoreactivity of markers for some types of IAS nerve fibers decreases during aging, which may contribute to age-related ano-rectal dysfunction.

The magnetic recoil spectrometer for measurements of the absolute neutron spectrum at OMEGA and the NIF.

The neutron spectrum produced by deuterium-tritium (DT) inertial confinement fusion implosions contains a wealth of information about implosion performance including the DT yield, ion-temperature, and areal-density. The Magnetic Recoil Spectrometer (MRS) has been used at both the OMEGA laser facility and the National Ignition Facility (NIF) to measure the absolute neutron spectrum from 3 to 30 MeV at OMEGA and 3 to 36 MeV at the NIF. These measurements have been used to diagnose the performance of cryogenic target implosions to unprecedented accuracy. Interpretation of MRS data requires a detailed understanding of the MRS response and background. This paper describes ab initio characterization of the system involving Monte Carlo simulations of the MRS response in addition to the commission experiments for in situ calibration of the systems on OMEGA and the NIF.

National Ignition Facility neutron time-of-flight measurements (invited).

The first 3 of 18 neutron time-of-flight (nTOF) channels have been installed at the National Ignition Facility (NIF). The role of these detectors includes yield, temperature, and bang time measurements. This article focuses on nTOF data analysis and quality of results obtained for the first set of experiments to use all 192 NIF beams. Targets produced up to 2×10(10) 2.45 MeV neutrons for initial testing of the nTOF detectors. Differences in neutron scattering at the OMEGA laser facility where the detectors were calibrated and at NIF result in different response functions at the two facilities. Monte Carlo modeling shows this difference. The nTOF performance on these early experiments indicates that the nTOF system with its full complement of detectors should perform well in future measurements of yield, temperature, and bang time.

The National Ignition Facility neutron time-of-flight system and its initial performance (invited).

The National Ignition Facility (NIF) successfully completed its first inertial confinement fusion (ICF) campaign in 2009. A neutron time-of-flight (nTOF) system was part of the nuclear diagnostics used in this campaign. The nTOF technique has been used for decades on ICF facilities to infer the ion temperature of hot deuterium (D(2)) and deuterium-tritium (DT) plasmas based on the temporal Doppler broadening of the primary neutron peak. Once calibrated for absolute neutron sensitivity, the nTOF detectors can be used to measure the yield with high accuracy. The NIF nTOF system is designed to measure neutron yield and ion temperature over 11 orders of magnitude (from 10(8) to 10(19)), neutron bang time in DT implosions between 10(12) and 10(16), and to infer areal density for DT yields above 10(12). During the 2009 campaign, the three most sensitive neutron time-of-flight detectors were installed and used to measure the primary neutron yield and ion temperature from 25 high-convergence implosions using D(2) fuel. The OMEGA yield calibration of these detectors was successfully transferred to the NIF.

The use of finite element methods and genetic algorithms in search of an optimal fabric reinforced porous graft system.

The mechanics of arteries result from the properties of the soft tissue constituents and the interaction of the wall layers, predominantly media and adventitia. This concept was adopted in this study for the design of a tissue regenerative vascular graft. To achieve the desired structural properties of the graft, most importantly a diametric compliance of 6%/100 mmHg, finite element methods and genetic algorithms were used in an integrated approach to identify the mechanical properties of an adventitial fabric layer that were required to optimally complement an intimal/medial polyurethane layer with interconnected porosity of three different size classes. The models predicted a compliance of 16.0, 19.2, and 31.5%/100 mmHg for the non-reinforced grafts and 5.3, 5.5, and 6.0%/100 mmHg for the fabric-reinforced grafts. The latter, featuring fabrics manufactured according to the required non-linear mechanical characteristics numerically predicted, exhibited an in vitro compliance of 2.1 +/- 0.8, 3.0 +/- 2.4, and 4.0 +/- 0.7% /100 mmHg. The combination of finite element methods and genetic algorithms was shown to be able to successfully optimize the mechanical design of the composite graft. The method offers potential for the application to alternative concepts of modular vascular grafts and the incorporation of tissue ingrowth and biodegradation.

Changes in the properties of the modulatory cerebral giant cells contribute to aging in the feeding system of Lymnaea.

This study examined whether electrophysiological changes in the endogenous properties and connectivity of the modulatory serotonergic cerebral giant cells (CGCs) contributed to the age-related changes in feeding behavior of the pond snail, Lymnaea. With increasing age there was a decrease in spontaneous CGC firing rates and decreased excitability of the CGCs to both chemosensory stimulation (0.05M sucrose applied to the lips) and direct intracellular current injection. These changes could be accounted for by a decrease in the input resistance of the neuron and an increase in the amplitude and the duration of the after-hyperpolarization. Decreases were also seen in the % of CGC pairs that were electrically coupled causing asynchronous firing. Together these changes would tend to reduce the ability of the CGCs to gate and control the frequency of the feeding behavior. Part of the ability of the CGCs to gate and frequency control the feeding network is to provide a background level of excitation to the feeding motor neurons. Recordings from B1 and B4 motor neurons showed an age-related hyperpolarization of the resting membrane potential consistent with a deficit in CGC function. Increases were seen in the strength of the evoked CGC-->B1 connection, however, this increase failed to compensate for the deficits in CGC excitability. In summary, age-related changes in the properties of the CGCs were consistent with them contributing to the age-related changes in feeding behavior seen in Lymnaea.

Effects of age on feeding behavior and chemosensory processing in the pond snail, Lymnaea stagnalis.

This study used behavioral and electrophysiological techniques to examine age-related changes in the feeding behavior and chemosensory processing in the pond snail, Lymnaea stagnalis. Increasing age was associated with a 50% decrease in long-term food consumption. Analysis of short-term sucrose-evoked feeding bouts showed an age-related increase in the number of animals that failed to respond to the stimulus. Of the animals that did respond increasing age was associated with a decrease in the number of sucrose-evoked bites and a increase in the duration of the swallow phase. These changes were observed with both 0.01 and 0.05M sucrose stimuli but were not seen when 0.1M sucrose was used as the stimulus. Electrophysiological analysis of the chemosensory pathway in semi-intact lip-CNS preparations failed to demonstrate a significant change in the neuronal information entering the cerebral ganglia from the lips via the median lip nerve, but did demonstrate an age-related deficit in the neuronal output from the cerebral ganglia. This deficit was also dependent on the sucrose concentration and mirrored the concentration-dependent changes in feeding behavior. In summary, aging appeared to affect central but not peripheral processing of chemosensory information and suggests that this deficit contributes to the age-related changes in feeding behavior.