BioAssay Research Database (BARD): chemical biology and probe-development enabled by structured metadata and result types.

BARD, the BioAssay Research Database (https://bard.nih.gov/) is a public database and suite of tools developed to provide access to bioassay data produced by the NIH Molecular Libraries Program (MLP). Data from 631 MLP projects were migrated to a new structured vocabulary designed to capture bioassay data in a formalized manner, with particular emphasis placed on the description of assay protocols. New data can be submitted to BARD with a user-friendly set of tools that assist in the creation of appropriately formatted datasets and assay definitions. Data published through the BARD application program interface (API) can be accessed by researchers using web-based query tools or a desktop client. Third-party developers wishing to create new tools can use the API to produce stand-alone tools or new plug-ins that can be integrated into BARD. The entire BARD suite of tools therefore supports three classes of researcher: those who wish to publish data, those who wish to mine data for testable hypotheses, and those in the developer community who wish to build tools that leverage this carefully curated chemical biology resource.

Compton suppression method and epithermal NAA in the determination of nutrients and heavy metals in Nigerian food and beverages.

We used in this study Compton suppression method and epithermal neutron activation analysis to determine the concentration of nutrients and heavy metals in Nigerian food and beverages. The work was performed at the University of Texas TRIGA Reactor by short, medium, and long irradiation protocols, using thermal flux of 1.4x10(12)n cm(-2)s(-1) and epithermal flux of 1.4x10(11)n cm(-2)s(-1). Application of Compton suppression method has reduced interferences from Compton scattered photons thereby allowing easy evaluation of Na, Cl, Ca, Cu, Mn, Mg, Co, Cr, Rb, Fe, and Se. The epithermal NAA method has enabled determination of Cd, As, Ba, Sr, Br, I, and V with little turn-around time. Quality Control and Quality Assurance of the method was tested by analyzing four Standard Reference Materials (non-fat powdered milk, apple leaves, citrus leaves, and peach leaves) obtained from National Institute for Standards and Technology. Our results show that sorghum, millet, and maize have high values of Zn, Mn, Fe, low values of Cd, As, and Se. Powdered milks, rice, beans, and soybeans were found to have moderate amounts of all the elements. Tobacco recorded high content of Cd, Mn, and As, whereas tea, tsobo leaves, Baobab leaves, and okro seed have more As values than others. However, biscuits, macaroni, spaghetti, and noodles show lower concentrations of all the elements. The distribution of these nutrients and heavy metals in these food and beverages shows the need to fortify biscuits and pastas with micro and macro-nutrients and reduce the use of tobacco, tea, tsobo leaves, Baobab leaves, and Okro seed to avoid intake of heavy elements.

Netrin-1 promotes thalamic axon growth and is required for proper development of the thalamocortical projection.

The thalamocortical axon (TCA) projection originates in dorsal thalamus, conveys sensory input to the neocortex, and has a critical role in cortical development. We show that the secreted axon guidance molecule netrin-1 acts in vitro as an attractant and growth promoter for dorsal thalamic axons and is required for the proper development of the TCA projection in vivo. As TCAs approach the hypothalamus, they turn laterally into the ventral telencephalon and extend toward the cortex through a population of netrin-1-expressing cells. DCC and neogenin, receptors implicated in mediating the attractant effects of netrin-1, are expressed in dorsal thalamus, whereas unc5h2 and unc5h3, netrin-1 receptors implicated in repulsion, are not. In vitro, dorsal thalamic axons show biased growth toward a source of netrin-1, which can be abolished by netrin-1-blocking antibodies. Netrin-1 also enhances overall axon outgrowth from explants of dorsal thalamus. The biased growth of dorsal thalamic axons toward the internal capsule zone of ventral telencephalic explants is attenuated, but not significantly, by netrin-1-blocking antibodies, suggesting that it releases another attractant activity for TCAs in addition to netrin-1. Analyses of netrin-1 -/- mice reveal that the TCA projection through the ventral telencephalon is disorganized, their pathway is abnormally restricted, and fewer dorsal thalamic axons reach cortex. These findings demonstrate that netrin-1 promotes the growth of TCAs through the ventral telencephalon and cooperates with other guidance cues to control their pathfinding from dorsal thalamus to cortex.

Slit inhibition of retinal axon growth and its role in retinal axon pathfinding and innervation patterns in the diencephalon.

We have analyzed the role of the Slit family of repellent axon guidance molecules in the patterning of the axonal projections of retinal ganglion cells (RGCs) within the embryonic rat diencephalon and whether the slits can account for a repellent activity for retinal axons released by hypothalamus and epithalamus. At the time RGC axons extend over the diencephalon, slit1 and slit2 are expressed in hypothalamus and epithalamus but not in the lateral part of dorsal thalamus, a retinal target. slit3 expression is low or undetectable. The Slit receptors robo2, and to a limited extent robo1, are expressed in the RGC layer, as are slit1 and slit2. In collagen gels, axon outgrowth from rat retinal explants is biased away from slit2-transfected 293T cells, and the number and length of axons are decreased on the explant side facing the cells. In addition, in the presence of Slit2, overall axon outgrowth is decreased, and bundles of retinal axons are more tightly fasciculated. This action of Slit2 as a growth inhibitor of retinal axons and the expression patterns of slit1 and slit2 correlate with the fasciculation and innervation patterns of RGC axons within the diencephalon and implicate the Slits as components of the axon repellent activity associated with the hypothalamus and epithalamus. Our findings suggest that in vivo the Slits control RGC axon pathfinding and targeting within the diencephalon by regulating their fasciculation, preventing them or their branches from invading nontarget tissues, and steering them toward their most distal target, the superior colliculus.

Attenuation of brain hyperbaric oxygen toxicity by fasting is not related to ketosis.

The effect of 24 h of fasting and changes in blood glucose and beta-hydroxybutyrate (BHB) level on latency to seizures in hyperbaric oxygen (HBO2) was studied. Conscious, unrestrained rats implanted with cortical electroencephalogram electrodes were exposed to 0.5 MPa (gauge pressure) O2 until seizures were observed. Fasting for 24 h significantly (P < 0.01) decreased blood glucose (from 8.6 +/- 0.9 in fed to 6.9 +/- 0.7 mM in the fasted group), increased blood BHB (0.07 +/- 0.02 mM to 0.38 +/- 0.10 mM, respectively), and prolonged the latency to seizures compared with normally fed animals (21.0 +/- 9.8 vs. 34.6 +/- 17.7 min, P < 0.05). Injection of the ketone precursor 1,3-butanediol (BD) to the fed animals increased blood BHB level to 0.72 +/- 0.32; however, seizure latency remained the same as in fed animals. Restoration of blood glucose in fasted animals to the same level as in the fed group did not reverse the protection achieved by fast; instead it increased the latency to seizures. The results indicate that the protection against HBO2 seizures by fasting in short starvation is not related to the increase in circulating ketone bodies or decrease in blood glucose.

Thalamocortical axons are influenced by chemorepellent and chemoattractant activities localized to decision points along their path.

Thalamocortical axons (TCAs), which originate in dorsal thalamus, project ventrally in diencephalon and then dorsolaterally in ventral telencephalon to their target, the neocortex. To elucidate potentially key decision points in TCA pathfinding and hence the possible localization of guidance cues, we used DiI-tracing to describe the initial trajectory of TCAs in mice. DiI-labeled TCAs extend ventrally on the lateral surface of ventral thalamus. Rather than continuing this trajectory onto the lateral surface of the hypothalamus, TCAs make a sharp lateral turn into ventral telencephalon. This behavior suggests that the hypothalamus is repulsive and the ventral telencephalon attractive for TCAs. In support of this hypothesis, we find that axon outgrowth from explants of dorsal thalamus is biased away from hypothalamus and toward ventral telencephalon when cocultured at a distance in collagen gels. The in vivo DiI analysis also reveals a broad cluster of retrogradely labeled neurons in the medial part of ventral telencephalon positioned within or adjacent to the thalamocortical pathway prior to or at the time TCAs are extending through it. The axons of these neurons extend into or through dorsal thalamus and appear to be coincident with the oppositely extending TCAs. These findings suggest that multiple cues guide TCAs along their pathway from dorsal thalamus to neocortex: TCAs may fasciculate on the axons of ventral telencephalic neurons as they extend through ventral thalamus and the medial part of ventral telencephalon, and chemorepellent and chemoattractant activities expressed by hypothalamus and ventral telencephalon, respectively, may cooperate to promote the turning of TCAs away from hypothalamus and into ventral telencephalon.

Effect of MK-801 on seizures induced by exposure to hyperbaric oxygen: comparison with AP-7.

The effect of the noncompetitive N-methyl-d-aspartate (NMDA)-receptor antagonist MK-801 on seizures induced by hyperbaric oxygen in relation to changes in cerebral blood flow (CBF) was investigated. Rats were injected with MK-801 (0.005-8 mg/kg) 30 min before exposure to 100% O2 at 5 atm (gauge pressure). MK-801 administration resulted in a biphasic response in seizure latency. Doses of 0.1-4 mg/kg significantly decreased time to EEG and motor seizures, while 8 mg/kg had no effect on seizure latency. MK-801 had no effect on seizure duration. In a dose range 0.1-8 mg/kg MK-801 increased CBF in awake animals, which might be responsible for the decreased seizure latency. The gradual increase in seizure latency with increasing MK-801 doses suggests involvement of an additional factor probably related to the drug's anticonvulsive effect. Unlike MK-801, a competitive NMDA receptor antagonist, AP-7, at a dose 250 mg/kg had no effect on latency to seizures or CBF.

Role of cerebral blood flow in seizures from hyperbaric oxygen exposure.

Hyperbaric O2 exposure causes seizures by an unknown mechanism. Cerebral blood flow (CBF) may affect seizure latency, although no studies have demonstrated a direct relationship. Awake rats (male, Sprague-Dawley, 350-450 g), instrumented for measuring electroencephalographic activity (EEG) and CBF (laser-Doppler flowmetry), were exposed to 100% O2 at 4 or 5 atm (gauge pressure) until EEG seizures. Compression with O2 caused vasoconstriction to about 70% of control flow that was maintained for various times. CBF then suddenly, but transiently, increased at a time that was reliably related to seizure latency (r=0.8, p<0.01). Additional animals were treated with agents that have diverse pharmacology and their effects on CBF and latency were measured. Glutamate receptor antagonists MK-801 (1 or 4 mg/kg) and ketamine (20-100 mg/kg) significantly increased CBF by 60-80% and decreased seizure latency from about 17+/-8 min (+/-S.D.) in controls to 5+/-1 and 6+/-2 min, respectively. In opposite, a nitric oxide synthase (NOS) inhibitor, N-nitro-L-arginine (NNA)(25 mg/kg) decreased CBF by about 25% and increased time to seizure to 60+/-16 min. If these effects occur in humans, non-invasive measurement of CBF could potentially improve the safety and reliability of hyperbaric O2 usage in clinical and diving applications. It also appears that the effect of drugs on seizure latency can be explained, at least in part, by their effect on CBF.

Retinal axon guidance by region-specific cues in diencephalon.

Retinal axons show region-specific patterning along the dorsal-ventral axis of diencephalon: retinal axons grow in a compact bundle over hypothalamus, dramatically splay out over thalamus, and circumvent epithalamus as they continue toward the dorsal midbrain. In vitro, retinal axons are repulsed by substrate-bound and soluble activities in hypothalamus and epithalamus, but invade thalamus. The repulsion is mimicked by a soluble floor plate activity. Tenascin and neurocan, extracellular matrix molecules that inhibit retinal axon growth in vitro, are enriched in hypothalamus and epithalamus. Within thalamus, a stimulatory activity is specifically upregulated in target nuclei at the time that retinal axons invade them. These findings suggest that region-specific, axon repulsive and stimulatory activities control retinal axon patterning in the embryonic diencephalon.

Graded and lamina-specific distributions of ligands of EphB receptor tyrosine kinases in the developing retinotectal system.

Molecular gradients have been postulated to control the topographic mapping of retinal axons in their central targets. Based initially on their expression patterns, and more recently on functional studies, members of the EphA subfamily of receptor tyrosine kinases and their ephrin-A ligands have been implicated in the guidance of retinal axons along the anterior-posterior axis of the chick optic tectum. The report that a receptor of the EphB subfamily, EphB2/Cek5/Nuk/Sek3, is expressed in a high ventral to low dorsal gradient in the developing chick retina and is present on ganglion cell axons suggests that it may be involved in the mapping of retinal axons along the corresponding dorsal-ventral axis of the tectum. To address this issue, we have determined the expression and distribution of ephrin-B1/LERK-2/Cek5-L and ephrin-B2/LERK-5/Htk-L/ELF-2, ligands for EphB2, in the developing chick retinotectal system using riboprobes, immunocytochemistry, and receptor affinity probes. Both ephrin-B1 and ephrin-B2 transcripts are expressed in a high dorsal to low ventral gradient in the developing retina, complementary to the distribution of EphB2. Ephrin-B1 and ephrin-B2 proteins are predominantly found in the developing plexiform layers, suggesting a role in the development of intraretinal connections. Neither protein is detected on ganglion cell axons. In tectum, ephrin-B1 transcripts are expressed in a high dorsal to low ventral gradient in the neuroepithelium and the protein is present along the processes of radial glia and is concentrated at their endfeet in the stratum opticum, at the time retinal axons are growing through it. This distribution of ephrin-B1 suggests that it influences retinal axon mapping along the dorsal-ventral tectal axis and may also be involved in intratectal development. In contrast, ephrin-B2 transcripts and protein are localized to the deeper retinorecipient laminae in the tectum at the time retinal axons begin to arborize in them, suggesting that this ligand may influence the laminar patterning of retinal axon terminations.

Maturation-dependent upregulation of growth-promoting molecules in developing cortical plate controls thalamic and cortical neurite growth.

We have tested the hypothesis that maturation-dependent changes in the cortical plate affect the spatiotemporal growth patterns of developing thalamocortical and corticocortical axonal projections. Given a choice between alternating lanes of embryonic (E18-19) and neonatal (P0-1) rat cortical plate membranes, embryonic (E18-19) thalamic and cortical neurites prefer to extend on neonatal membranes. Thalamic and cortical explants do extend neurites on uniform carpets of E19 cortical plate membranes, but the outgrowth is consistently greater on uniform carpets of P1 cortical plate membranes. These experiments demonstrate a maturation-dependent enhancement in the ability of cortical plate to support neurite growth from thalamic and cortical explants. In contrast, retinal and cerebellar neurites, which do not grow into cortex in vivo, generally grew poorly on these membranes, suggesting a degree of specificity to the neurite growth response. Immunohistochemical analysis of developing cortex suggests that several extracellular matrix (ECM) and cell adhesion molecules are upregulated in cortical plate. However, immunocharacterization of membrane carpets for these same ECM and cell adhesion molecules suggests that the growth preferences of thalamic and cortical neurites in vitro are predominantly influenced by membrane-anchored, rather than ECM, molecules. Western analysis of E19 and P1 cortical plate membranes supports this conclusion, and indicates that the membrane-anchored cell adhesion molecules L1 and N-CAM are more abundant in the P1 cortical plate membrane preparation. Experiments in which cortical plate membranes were treated to remove molecules sensitive to phosphatidylinositol (PI)-specific phospholipase C demonstrate that neurite growth promoters present in E19 cortical plate membranes are predominantly PI linked, whereas those present in P1 membranes are predominantly non-PI linked. These findings indicate that the neurite growth preferences are mediated, at least in part, by an upregulation of neurite growth-promoting molecules in developing cortical plate that are not PI linked. Taken together, these findings suggest that a maturation-dependent upregulation of neurite growth-promoting molecules on cortical plate cells controls the invasion of the cortical plate by thalamocortical and corticocortical axons.

Selective regeneration of photoreceptors in goldfish retina.

Previous work has shown that the neural retina in adult goldfish can regenerate. Following retinal damage elicited by surgical or cytotoxic lesions, missing neurons are replaced by foci of proliferating neuroepithelial cells, which previous studies have suggested are derived from rod precursors. In the intact retina, rod precursors proliferate but produce only new rods. The regenerative responses observed previously have involved replacement of neurons in all retinal layers; selective regeneration of specific neuronal types (except for rod photoreceptors) has not been reported. In the experiments described here, we specifically destroyed either cones alone or cones and rods with an argon laser, and we found that both types of photoreceptors regenerated within a few weeks. The amount of cone regeneration varied in proportion to the degree of rod loss. This is the first demonstration of selective regeneration of a specific class of neuron (i.e., cones) in a region of central nervous tissue where developmental production of that class of neuron has ceased. Selective regeneration may be limited to photoreceptors, however, because when dopaminergic neurons in the inner retina were ablated with intraocular injections of 6-hydroxydopamine, in combination with laser lesions that destroyed photoreceptors, the dopaminergic neurons did not regenerate, but the photoreceptors did. These data support previous studies which showed that substantial cell loss is required to trigger regeneration of inner retinal neurons, including dopaminergic neurons. New observations here bring into question the presumption that rod precursors are the only source of neuronal progenitors during the regenerative response. Finally, a model is presented which suggests a possible mechanism for regulating the phenotypic fate of retinal progenitor cells during retinal regeneration.

Increased red cell osmotic fragility and hematocrit after hyperbaric O2 exposure are related to acidosis.

Exposure to hyperbaric O2 (0.6 MPa) until convulsion occurred resulted in increased red blood cell osmotic fragility, increased hematocrit, and acidosis. Correction of the mixed metabolic and respiratory acidosis in arterial blood samples completely restored osmotic fragility and lowered hematocrit by 20%. Rats exposed to intermittent hyperbaric O2 (repeated cycles of 7 minutes of O2 and 7 minutes of air) tolerated significantly more O2 time than those exposed continuously. Intermittently exposed animals had smaller increases in osmotic fragility and less-severe acidosis. We verified the influence of pH on osmotic fragility and hematocrit in rats made acutely acidotic and corrected in vivo. Acidosis caused by CO2 inhalation and lactic and hydrochloric acid infusion raised osmotic fragility and hematocrit; these effects were completely reversed in the animal when we restored normal acid-base status. These studies demonstrate that conditions causing acidosis, including hyperbaric O2 exposure, increase red cell fragility and size and increase hematocrit.

Continued search for the cellular signals that regulate regeneration of dopaminergic neurons in goldfish retina.

Intraocular injections of low doses (0.7-1.4 mM estimated intraocular concentration) of 6-hydroxydopamine (6OHDA) selectively destroy dopaminergic neurons in the inner nuclear layer (INL) of goldfish retina, and they never regenerate. However, injection of a higher dose of 6OHDA (2.9 mM) destroys > 30% (but not all) of the cells in both the INL and the outer nuclear layer (ONL), but within 3 weeks, neurons in both the INL (including dopaminergic neurons) and the ONL regenerate. We hypothesize that the regenerated neurons derive from mitotic rod precursors in the ONL and that damage to the surrounding micro-environment (i.e. destruction of photoreceptors) triggers the regenerative response. To directly test this hypothesis, we selectively ablated > 99% of dopaminergic neurons (with low doses of 6OHDA) and up to 55% of rod photoreceptors (with tunicamycin), and asked whether the dopaminergic neurons regenerated, as evidenced by double immunolabeling with anti-tyrosine hydroxylase and anti-bromodeoxyuridine. After 38 days, the number of bromodeoxyuridine-immunoreactive rod nuclei was increased 2.4-fold compared to controls, but no regenerated dopaminergic neurons were found. These data suggest that although the rate of rod production increases, rod precursors do not alter their normal pathway of development to replace dopaminergic neurons in the INL when damage to the ONL is limited to destruction of rods.

Regeneration of dopaminergic neurons in goldfish retina.

The conditions necessary to trigger regeneration of dopaminergic neurons were investigated in the goldfish retina. Intraocular injection of 6-hydroxydopamine (6-OHDA) was used to destroy dopaminergic neurons, and neuronal regeneration was monitored by injections of the thymidine analog bromodeoxyuridine (BUdR). Regenerated dopaminergic neurons, (identified by double-labeling with anti-tyrosine hydroxylase and anti-BUdR antibodies) were found within 3 weeks after 2 injections of 0.6 mg/ml 6-OHDA (estimated intraocular concentration), but not after injection of lower doses. All retinas with regenerated dopaminergic neurons also contained other types of regenerated neurons, including cones and ganglion cells, consistent with nuclear counts which revealed non-selective cell loss (34-36%) in both the outer and inner nuclear layers after exposure to the high dose, but not lower doses of 6-OHDA. Regenerated neurons were produced by clusters of dividing neuroepithelial cells probably derived from rod precursors in the outer nuclear layer. These results demonstrate that dopaminergic neurons will not regenerate after they are selectively ablated but only as part of a developmental process that involves generation of multiple cell types.

Response of antioxidant enzymes to intermittent and continuous hyperbaric oxygen.

Rats and guinea pigs were exposed to O2 at 2.8 ATA (HBO) delivered either continuously or intermittently (repeated cycles of 10 min of 100% O2 followed by 2.5 min of air). The O2 time required to produce convulsions and death was increased significantly in both species by intermittency. To determine whether changes in brain and lung superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSHPx) correlated with the observed tolerance, enzyme activities were measured after short or long HBO exposures. For each exposure duration, one group received continuous and one intermittent HBO; O2 times were matched. HBO had marked effects on these enzymes: lung SOD increased (guinea pigs 47%, rats 88%) and CAT and GSHPx activities decreased (33%) in brain and lung. No differences were seen in lung GSHPx or brain CAT in rats or brain SOD in either species. In guinea pigs, but less so in rats, the observed changes in activity were usually modulated by intermittency. Increases in hematocrit, organ protein, and lung DNA, which may also reflect ongoing oxidative damage, were also slowed with intermittency in guinea pigs. Intermittency benefited both species by postponing gross symptoms of toxicity, but its modulation of changes in enzyme activities and other biochemical variables was more pronounced in guinea pigs than in rats, suggesting that there are additional mechanisms for tolerance.

The adolescent ballet dancer. Nutritional practices and characteristics associated with anorexia nervosa.

A questionnaire was administered to 45 female ballet dancers aged 12-21 years and 44 nonathletic female subjects aged 14-16 years. Dietary practices and nutrition beliefs of the dancers were assessed, and a comparison was made of the prevalence of anorexia nervosa characteristics among the two groups. Ballet dancers reported characteristics of anorexia nervosa significantly more often than did controls. Characteristics used to differentiate between the groups were underweight (p less than 0.05), distorted body image (p less than 0.005), amenorrhea (p less than 0.02), and binge eating (p less than 0.005). Nutrition practices and beliefs among adolescent ballet dancers included frequent use of weight reduction strategies such as fasting, binging, and selective food restriction. Supplements were used by 60% of the dancers, primarily a multivitamin or vitamin C supplement. Fluids or carbohydrates wer not viewed as an important aid to performance by the majority of dancers. This study indicates the need for improved education for ballet dancers, coaches, trainers, and parents, including information on nutrition needs and the management of behaviors associated with anorexia nervosa.