Tritium and Carbon-14 Contamination Reshaping the Microbial Community Structure, Metabolic Network, and Element Cycle in the Seawater Environment.

The potential ecological risks caused by entering radioactive wastewater containing tritium and carbon-14 into the sea require careful evaluation. This study simulated seawater's tritium and carbon-14 pollution and analyzed the effects on the seawater and sediment microenvironments. Tritium and carbon-14 pollution primarily altered nitrogen and phosphorus metabolism in the seawater environment. Analysis by 16S rRNA sequencing showed changes in the relative abundance of microorganisms involved in carbon, nitrogen, and phosphorus metabolism and organic matter degradation in response to tritium and carbon-14 exposure. Metabonomics and metagenomic analysis showed that tritium and carbon-14 exposure interfered with gene expression involving nucleotide and amino acid metabolites, in agreement with the results seen for microbial community structure. Tritium and carbon-14 exposure also modulated the abundance of functional genes involved in carbohydrate, phosphorus, sulfur, and nitrogen metabolic pathways in sediments. Tritium and carbon-14 pollution in seawater adversely affected microbial diversity, metabolic processes, and the abundance of nutrient-cycling genes. These results provide valuable information for further evaluating the risks of tritium and carbon-14 in marine environments.

Bioremediation of copper in sediments from a constructed wetland ex situ with the novel bacterium Cupriavidus basilensis SRS.

The H-02 constructed wetland was designed to remove metals (primarily copper and zinc) to treat building process water and storm water runoff from multiple sources associated with the Tritium Facility at the DOE-Savannah River Site, Aiken, SC. The concentration of Cu and Zn in the sediments has increased over the lifetime of the wetland and is a concern. A bioremediation option was investigated at the laboratory scale utilizing a newly isolated bacterium of the copper metabolizing genus Cupriavidus isolated from Tim's Branch Creek, a second-order stream that eventually serves as a tributary to the Savannah River, contaminated with uranium and other metals including copper, nickel, and mercury. Cupriavidus basilensis SRS is a rod-shaped, gram-negative bacterium which has been shown to have predatory tendencies. The isolate displayed resistance to the antibiotics ofloxacin, tetracycline, ciprofloxacin, select fungi, as well as Cu2+ and Zn2+. Subsequent ribosomal sequencing demonstrated a 100% confidence for placement in the genus Cupriavidus and a 99.014% match to the C. basilensis type strain. When H-02 wetland samples were inoculated with Cupriavidus basilensis SRS samples showed significant (p < 0.05) decrease in Cu2+ concentrations and variability in Zn2+ concentrations. Over the 72-h incubation there were no significant changes in the inoculate densities (106-108 cells/ML) indicating Cupriavidus basilensis SRS resiliency in this environment. This research expands our understanding of the Cupriavidus genus and demonstrates the potential for Cupriavidus basilensis SRS to bioremediate sites impacted with heavy metals, most notably copper.

Isolation and Characterization of 1-Hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic Acid, a Metabolite in Bacterial Transformation of Abscisic Acid.

We report the discovery of a new abscisic acid (ABA) metabolite, found in the course of a mass spectrometric study of ABA metabolism by the rhizosphere bacterium Rhodococcus sp. P1Y. Analogue of (+)-ABA, enriched in tritium in the cyclohexene moiety, was fed in bacterial cells, and extracts containing radioactive metabolites were purified and analyzed to determine their structure. We obtained mass spectral fragmentation patterns and nuclear magnetic resonance spectra of a new metabolite of ABA identified as 1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexene-1-acetic acid, which we named rhodococcal acid (RA) and characterized using several other techniques. This metabolite is the second bacterial ABA degradation product in addition to dehydrovomifoliol that we described earlier. Taken together, these data reveal an unknown ABA catabolic pathway that begins with side chain disassembly, as opposed to the conversion of the cyclohexene moiety in plants. The role of ABA-utilizing bacteria in interactions with other microorganisms and plants is also discussed.

Transport characteristics of 5-aminosalicylic acid into colonic epithelium: Involvement of sodium-coupled monocarboxylate transporter SMCT1-mediated transport system.

5-Aminosalicylic acid (5-ASA) is conventionally used as a first line drug for inflammatory bowel disease (IBD). Because 5-ASA is well absorbed in the small intestine, very high dose of 5-ASA is required to deliver it to the large intestine which is a target site. Interestingly, 5-ASA is reported to be transported into the large intestine as well as the small intestine via unknown transport system. In a heterologous expression system using Xenopus oocytes, sodium-coupled monocarboxylate transporter 1 (SMCT1) has been reported to accept 5-ASA as a substrate. Although SMCT1 is found to be expressed in the large intestine, it is unknown whether SMCT1 is responsible for 5-ASA absorption from the large intestine or not. Here we determined the transport characteristics of 5-ASA in the isolated everted sac prepared from mouse large intestine. Na+-dependent uptake of [3H]nicotinate, a substrate for SMCT1, in mouse colon was competitively inhibited by 5-ASA with IC50 value of 2.8 mM. In addition to nicotinate, 5-ASA uptake in mouse colonic mucosa was Na+-dependent and saturable with Michaelis constant (Km) of 2.4 mM. Na+-activation kinetics revealed that the Na+-to-5-ASA stoichiometry was 2:1 and concentration of Na+ necessary for half-maximal transport (K0.5Na) was 36.1 mM. Na+-dependent 5-ASA uptake was competitively inhibited by nicotinate with an inhibitory constant (Ki) of 2.1 mM was comparable to the Km value of Na+-dependent nicotinate uptake (0.99 mM). Furthermore, ibuprofen, a selective SMCT1 inhibitor, was found to have a significantly inhibitory effect on the Na+-dependent 5-ASA uptake in mouse colon (IC50 = 0.19 mM). Taken collectively, these results indicated that SMCT1 in the mouse colonic mucosa is responsible for Na+-dependent 5-ASA uptake.

Tritiodefluorination of alkyl C-F groups.

A straightforward methodology of fluorine substitution by tritium/deuterium is reported. The described method is selective towards the F─C (sp3 ) group and leaves both the aromatic F─C (sp2 ) and F2 ─C (sp3 ) moieties unaffected. Alkylfluorides, readily synthesized from appropriate alcohols by treatment with diethylaminosulfur trifluoride (DAST) reagent in an overall yield up to 76%, undergoes activation with the boron-based Lewis acid B(C6 F5 )3 , and stoichiometric in situ reduction with a tritide/deuteride reagent-the [TMP2(3) H][2(3) HB(C6 F5 )3 ] system of frustrated Lewis pair. This methodology provides an isolated yield of up to 93% of regio-specifically labeled small organic compounds with superior 2 H-enrichment of over 95%. The specific activity of prepared 1-(2-[3 H]-ethyl)naphthalene was determined at 29.0 Ci/mmol. The site selectivity of the Lewis acid/ [TMP2(3) H][2(3) HB(C6 F5 )3 ] approach is orthogonal to currently used methods and allows for isotopic labeling of complementary positions in molecules. Reported labeling methodology proceeds well at ultra-mild reaction conditions (220 mbar of T2 ), allowing very low consumption of the radioactive source (4.2 Ci/156 GBq), and producing limited amount of radioactive waste.

Cellular responses in rainbow trout (Oncorhynchus mykiss) reared in tritiated water and/or fed organically bound tritium.

For the evaluation of biological effects of low level chronic tritium exposure in fish, rainbow trout (Oncorhynchus mykiss) were exposed to tritiated water and/or fed a diet spiked with tritiated essential amino acids. Differences in fatty acid composition were noted following the tritium exposures. In addition, changes in response to a subsequent acute high dose of gamma radiation delivered in vitro were noted when evaluating DNA repair activity and fatty acid composition.

Betulinic acid, derived from the desert lavender Hyptis emoryi, attenuates paclitaxel-, HIV-, and nerve injury-associated peripheral sensory neuropathy via block of N- and T-type calcium channels.

The Federal Pain Research Strategy recommended development of nonopioid analgesics as a top priority in its strategic plan to address the significant public health crisis and individual burden of chronic pain faced by >100 million Americans. Motivated by this challenge, a natural product extracts library was screened and identified a plant extract that targets activity of voltage-gated calcium channels. This profile is of interest as a potential treatment for neuropathic pain. The active extract derived from the desert lavender plant native to southwestern United States, when subjected to bioassay-guided fractionation, afforded 3 compounds identified as pentacyclic triterpenoids, betulinic acid (BA), oleanolic acid, and ursolic acid. Betulinic acid inhibited depolarization-evoked calcium influx in dorsal root ganglion (DRG) neurons predominantly through targeting low-voltage-gated (Cav3 or T-type) and CaV2.2 (N-type) calcium channels. Voltage-clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after BA exposure. Betulinic acid inhibited spontaneous excitatory postsynaptic currents and depolarization-evoked release of calcitonin gene-related peptide from lumbar spinal cord slices. Notably, BA did not engage human mu, delta, or kappa opioid receptors. Intrathecal administration of BA reversed mechanical allodynia in rat models of chemotherapy-induced peripheral neuropathy and HIV-associated peripheral sensory neuropathy as well as a mouse model of partial sciatic nerve ligation without effects on locomotion. The broad-spectrum biological and medicinal properties reported, including anti-HIV and anticancer activities of BA and its derivatives, position this plant-derived small molecule natural product as a potential nonopioid therapy for management of chronic pain.

Long-Term Neurobehavioral Consequences of a Single Ketamine Neonatal Exposure in Rats: Effects on Cellular Viability and Glutamate Transport in Frontal Cortex and Hippocampus.

The neonatal exposure to general anesthetics has been associated with neuronal apoptosis and dendritic spines morphologic changes in the developing brain. Ketamine, a noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, is widely used in pediatric patients to induce general anesthesia, analgesia, and perioperative sedation. In the present study, we investigated short- and long-term effects of a single ketamine (20 mg/kg, s.c.) neonatal exposure at postnatal day 7 in rats on the hippocampal and frontal cortical cellular viability. Additionally, putative neurochemical alterations and neurobehavioral impairments were evaluated in the adulthood. Ketamine neonatal administration selectively decreased cellular viability in the hippocampus, but not in the frontal cortex, 24 h after the treatment. Interestingly, a single ketamine neonatal exposure prevented the vulnerability to glutamate-induced neurotoxicity in the frontal cortex of adult rats. No short- or long-term damage to cellular membranes, as an indicative of cell death, was observed in hippocampal or cortical slices. However, ketamine induced a long-term increase in hippocampal glutamate uptake. Regarding behavioral analysis, neonatal ketamine exposure did not alter locomotor activity and anxiety-related parameters evaluated in the open-field test. However, ketamine administration disrupted the hippocampal-dependent object recognition ability of adult rats, while improved the motor coordination addressed on the rotarod. These findings indicate that a single neonatal ketamine exposure induces a short-term reduction in the hippocampal, but not in cortical, cellular viability, and long-term alterations in hippocampal glutamate transport, improvement on motor performance, and short-term recognition memory impairment.

Inhibitory effects of luteolin­4'­O­ß­D­glucopyranoside on P2Y12 and thromboxane A2 receptor­mediated amplification of platelet activation in vitro.

Platelet activation and subsequent accumulation at sites of vascular injury are central to thrombus formation, which is considered to be a trigger of several cardiovascular diseases. Callicarpa nudiflora (C. nudiflora) Hook is a traditional Chinese medicinal herb for promoting blood circulation by removing blood stasis. In our previous study, several compounds extracted from this herb, including luteolin­4'­O­ß­D­glucopyranoside (LGP), were revealed to exert inhibitory effects on adenosine diphosphate (ADP)­induced platelet aggregation. The aim of present study was to confirm these antiplatelet effects and elucidate the potential mechanisms. Using a platelet­aggregation assay, it was revealed that LGP significantly inhibited platelet aggregation induced by ADP, U46619 and arachidonic acid. It was also found that LGP exhibited marked inhibitory effects on the activation of αIIbß3 integrin, the secretion of serotonin from granules, and the synthesis of thromboxane A2. In addition, the results showed that LGP suppressed Ras homolog family member A and phosphoinositide 3­kinase/Akt/glycogen synthase kinase 3ß signal transduction. Data from a radiolabeled ligand­binding assay indicated that LGP exhibited apparent competing effects on thromboxane receptor (TP) and P2Y12 receptors. In conclusion, the data presented here demonstrated that LGP, a natural compound from C. nudiflora Hook, inhibited the development of platelet aggregation and amplification of platelet activation. These inhibitory effects may be associated with its dual­receptor inhibition on P2Y12 and TP receptors.

A REVIEW OF NATURAL RADIONUCLIDES IN CANADIAN DRINKING WATER (1975-16).

Historical results of natural radioactivity in drinking water were reviewed for a total of 21 communities across Canada from 1975 to 2016. Analyses for 226Ra, 210Pb and total uranium were carried out on monthly composite samples of drinking water from selected Canadian municipalities. Generally speaking, levels of 226Ra, 210Pb and total uranium were found to be low compared to national and international standards for drinking water quality. Because levels were low, federal monitoring programs were discontinued in most communities in 1986 except for Regina, Elliot Lake and Port Hope. The population-weighted average levels for these three communities, using data from the most recent 5 years, are <1 mBq/L for 226Ra, <5 mBq/L for 210Pb and <0.4 µg/L for total uranium. The average effective dose resulting from drinking water intake at these levels would be <4.3 µSv per year.

Relationship between receptor occupancy and the antinociceptive effect of mu opioid receptor agonists in male rats.

The analgesic mechanisms of mu opioid receptor (MOR) agonists, including receptor occupancy at the site of action, are not completely understood. The aims of the present study were to evaluate: (i) receptor occupancy in the rat brain after administration of MOR agonists; (ii) the relationship between occupancy and the antinociceptive effect. Morphine (2 or 4 mg/kg) or oxycodone (1 or 3 mg/kg) was subcutaneously administered to rats. The antinociceptive effect of these drugs was measured by the hot-plate test. MOR occupancy in the thalamus was assessed by conducting an ex vivo receptor binding assay using [3H] [D-Ala2, N-MePhe4, Gly-ol]-enkephalin, followed by autoradiographic analysis. Both drugs produced antinociception in a dose-dependent manner, and these effects disappeared after the time point at which the maximal effect was elicited. Thalamic MOR occupancy was observed in a dose-dependent manner at the time point at which maximal antinociception was elicited, and relatively low occupancy was observed when the antinociceptive effect was decreasing. Good correlation between thalamic MOR occupancy and the antinociceptive effect was observed. These findings provide direct evidence for the receptor occupancy of MOR agonists at the site of action and its relationship with the analgesic effect.

Characterization of the binding site for d-deprenyl in human inflamed synovial membrane.

AIMS: d-Deprenyl when used as a positron emission tomography tracer visualizes peripheral inflammation. The major aim of the current study was to identify and investigate the properties of the binding target for d-deprenyl in synovial membrane explants from arthritic patients. MAIN METHODS: Thirty patients diagnosed with arthritis or osteoarthritis were enrolled into the study. Homologous and competitive radioligand binding assays utilizing [3H]d-deprenyl were performed to investigate the biochemical characteristics of the binding site and assess differences in the binding profile in synovial membranes exhibiting varying levels of inflammation. KEY FINDINGS: The [3H]d-deprenyl binding assay confirmed the existence of a single, saturable population of membrane-bound protein binding sites in synovial membrane homogenates. The macroscopically determined level of inflammation correlated with an increase in [3H]d-deprenyl binding affinity, without significant alterations in binding site density. Selective monoamine oxidase B inhibitor, selegiline competed for the same site as [3H]d-deprenyl, but failed to differentiate the samples with regard to their inflammation grade. A monoamine oxidase A inhibitor, pirlindole mesylate showed only weak displacement of [3H]d-deprenyl binding. No significant alterations in monoamine oxidase B expression was detected, thus it was not confirmed whether it could serve as a marker for ongoing inflammation. SIGNIFICANCE: Our study was the first to show the biochemical characteristics of the [3H]d-deprenyl binding site in inflamed human synovium. We confirmed that d-deprenyl could differentiate between patients with varying severity of synovitis in the knee joint by binding to a protein target distinct from monoamine oxidase B.

Consequences of perinatal hypoxia in developing brain: Changes in GABA transporter functioning in cortical, hippocampal and thalamic rat nerve terminals.

Perinatal hypoxia leads to behavioral abnormalities, cognitive disabilities, and epilepsy resulting from alterations in neurodevelopment, maturation and construction of the network. Considering a particular role of γ-aminobutyric acid (GABA) for an immature brain, we analysed transporter-mediated [3H]GABA uptake in the cortical, hippocampal and thalamic nerve terminals isolated from rats of different age in the control and after perinatal hypoxia. The state of hypoxia was induced by exposure of rats at the age of 10 postnatal days (pd) (that corresponds approximately to the time of birth in humans) to a respiratory medium with low O2 content (4% O2 and 96%N2) for 12min (up to the initiation of clonico-tonic seizures). Here, we found that the initial rate of [3Н]GABA uptake was higher in the young rats (pd 17-19) as compared to the older ones (pd 24-26, 38-40 and 66-73) in both control and hypoxia groups. It decreased abruptly by 50% in the thalamus and by 25% in the cortex for the period from pd 17-19 to pd 66-73. In the hippocampus, a decrease in the rate during the same time interval was 25%. Exposure to hypoxia had no effect on the intensity of [3Н]GABA uptake by the cortical and thalamic nerve terminals, but caused a significant age-dependent attenuation (by 35%) of the uptake intensity in the hippocampal ones. Significant age-dependent hypoxia-independent decrease in [3Н]GABA uptake with step-like dynamics of changes was shown in the thalamus and cortex. Gradual age-dependent hypoxia-dependent decrease in [3Н]GABA uptake was revealed in the hippocampus, and so a particular vulnerability of the latest structure to hypoxia as compared to the cortex and thalamus was revealed.

PEG modification of Amorfrutin B from Amorpha fructicosa increases gastric absorption, circulation half-life and glucose uptake by T3T-L1 adipocytes.

Through a simple PEG-conjugation of the natural product Amorfrutin B, we enhanced its pharmacokinetic profile. The PEGylated molecule displayed significantly improved gastrointestinal absorption (p<0.05) and had a longer systemic circulation life (p<0.05). Oral glucose tolerance study showed PEGylated Amorfrutin B displayed longer protection against oral glucose load compared to Amorfrutin B (p<0.05). It also showed significant improvement in glucose uptake in-vitro by T3T-L1 adipocytes (p<0.05). The PEGylated molecule also showed reduced propensity of crossing the blood brain barrier and accumulating in the brain (p<0.05). It also showed reduced accumulation in the adipose tissue. Preliminary liver and kidney toxicity screening showed no significant alteration in liver or kidney function of Amorfrutin B or its PEGylated form. In conclusion, PEG modification can be an attractive strategy to reduce lipophilicity and enhance pharmacokinetic properties of natural products, derived from traditional medicine.

4-phenylselenyl-7-chloroquinoline, a novel multitarget compound with anxiolytic activity: Contribution of the glutamatergic system.

A growing body of evidence demonstrates that quinoline compounds have attracted much attention in the field of drug development. Accordingly, 4-phenylselenyl-7-chloroquinoline (4-PSQ) is a new quinoline derivative containing selenium, which showed a potential antioxidant, antinociceptive and anti-inflammatory effect. The present study was undertaken to evaluate the anxiolytic-like properties of 4-PSQ. Mice were orally pretreated with 4-PSQ (5-50 mg/kg) or vehicle, 30 min prior to the elevated plus-maze (EPM), light-dark (LDT) or open field (OFT) tests. A time-response curve was carried out by administration of 4-PSQ (50 mg/kg) at different times before the EPM test. The involvement of glutamate uptake/release and Na+, K+-ATPase activity in the anxiolytic-like effect was investigated in cerebral cortices. In addition, the effectiveness of acute treatment with 4-PSQ was evaluated in a model of kainate (KA)-induced anxiety-related behavior. Finally, acute toxicity of this compound was investigated. 4-PSQ produced an anxiolytic-like action, both in EPM and LDT. In OFT, 4-PSQ did not affect locomotor and exploratory activities. 4-PSQ anxiolytic-like effect started at 0.5 h and remained significant up to 72 h after administration. Treatment with 4-PSQ reduced [3H] glutamate uptake, but the [3H] glutamate release and Na+, K+-ATPase activity were not altered. KA-induced anxiety-related behavior was protected by 4-PSQ pretreatment. Additionally, 4-PSQ exposure did not alter urea levels, aspartate (AST) and alanine aminotrasferase (ALT) activities in plasma. Parameters of oxidative stress in brain and liver of mice were not modified by 4-PSQ. Taken together these data demonstrated that the anxiolytic-like effect caused by 4-PSQ seems to be mediated by involvement of the glutamatergic system.

Metabotropic glutamate receptor 2/3 density and its relation to the hippocampal neuropathology in a model of temporal lobe epilepsy in rats.

Dysregulation in the glutamatergic function is considered a major contributor to hyperexcitatory neuronal networks in mesial temporal lobe epilepsy (MTLE). Studies in animal models of MTLE have shown positive outcomes of augmenting group 2-metabotropic receptor functions that can regulate neuronal excitability from extrasynaptic locations. To assist in efficient translation of these findings to the clinical settings, we aimed to characterise the expression of mGluR2/3 receptors in the brain areas relevant to MTLE. mGluR2/3 density was determined by autoradiographic techniques using [3H]-LY341495 at various cross-sectional timepoints following kainic acid-induced status epilepticus (KASE) covering the acute, latent and chronic phases of epilepsy pathogenesis. We found a significant reduction in the mGluR density in the CA1 and temporal cortex during the acute (2day) timepoint after SE in KASE rats whereas a reduced receptor density was only found in temporal cortex during the latent period (7day). During the late latent phase (14day), a generalised increase in the receptor density was found in widely distributed brain areas of KASE rats. Finally, in the chronic periods (day 42 and 84) a significant decrease was seen in the stratum lacunosum moleculare in the KASE rats. Moreover, mGluR2/3 density in the CA1 regions strongly correlated with the neuronal cell scores in the hippocampal regions. Our findings suggest a time dependent evolving pattern of mGluR2/3 density during the pathogenesis of MTLE and provide insights for utilising this data for in vivo imaging to predict the specific timepoints and responsiveness to the therapy targeting mGluR2/3.

Membrane fluidity does not explain how solvents act on the middle-ear reflex.

Some volatile aromatic solvents have similar or opposite effects to anesthetics in the central nervous system. Like for anesthetics, the mechanisms of action involved are currently the subject of debate. This paper presents an in vivo study to determine whether direct binding or effects on membrane fluidity best explain how solvents counterbalance anesthesia's depression of the middle-ear reflex (MER). Rats were anesthetized with a mixture of ketamine and xylazine while also exposed to solvent vapors (toluene, ethylbenzene, or one of the three xylene isomers) and the amplitude of their MER was monitored. The depth of anesthesia was standardized based on the magnitude of the contraction of the muscles involved in the MER, determined by measuring cubic distortion product oto-acoustic emissions (DPOAEs) while triggering the bilateral reflex with contralateral acoustic stimulation. The effects of the aromatic solvents were quantified based on variations in the amplitude of the DPOAEs. The amplitude of the alteration to the MER measured in anesthetized rats did not correlate with solvent lipophilocity (as indicated by logKow values). Results obtained with the three xylene isomers indicated that the positions of two methyl groups around the benzene ring played a determinant role in solvent/neuronal cell interaction. Additionally, Solid-state Nuclear Magnetic Resonance (NMR) spectra for brain microsomes confirmed that brain lipid fluidity was unaffected by solvent exposure, even after three days (6h/day) at an extremely high concentration (3000ppm). Therefore, aromatic solvents appear to act directly on the neuroreceptors involved in the acoustic reflex circuit, rather than on membrane fluidity. The affinity of this interaction is determined by stereospecific parameters rather than lipophilocity.

Development of a Novel High-Density [3H]Hypoxanthine Scintillation Proximity Assay To Assess Plasmodium falciparum Growth.

The discovery and development of new antimalarial drugs are becoming imperative because of the spread of resistance to current clinical treatments. The lack of robustly validated antimalarial targets and the difficulties with the building in of whole-cell activity in screening hits are hampering target-based approaches. However, phenotypic screens of structurally diverse molecule libraries are offering new opportunities for the identification of novel antimalarials. Several methodologies can be used to determine the whole-cell in vitro potencies of antimalarial hits. The [(3)H]hypoxanthine incorporation assay is considered the "gold standard" assay for measurement of the activity of antimalarial compounds against intraerythrocytic forms of Plasmodium falciparum However, the method has important limitations, as the assay is not amenable for high-throughput screening since it remains associated with the 96-well plate format. We have overcome this drawback by adapting the [(3)H]hypoxanthine incorporation method to a 384-well high-density format by coupling a homogeneous scintillation proximity assay (SPA) and thus eliminating the limiting filtration step. This SPA has been validated using a diverse set of 1,000 molecules, including both a representative set from the Tres Cantos Antimalarial Set (TCAMS) of compounds and molecules inactive against whole cells. The results were compared with those from the P. falciparum lactate dehydrogenase whole-cell assay, another method that is well established as a surrogate for parasite growth and is amenable for high-throughput screening. The results obtained demonstrate that the SPA-based [(3)H]hypoxanthine incorporation assay is a suitable design that is adaptable to high-throughput antimalarial drug screening and that maintains the features, robustness, and reliability of the standard filtration hypoxanthine incorporation method.

Variations in myo-inositol in fronto-limbic regions and clinical response to electroconvulsive therapy in major depression.

Though electroconvulsive therapy (ECT) is an established treatment for severe depression, the neurobiological factors accounting for the clinical effects of ECT are largely unknown. Myo-inositol, a neurometabolite linked with glial activity, is reported as reduced in fronto-limbic regions in patients with depression. Whether changes in myo-inositol relate to the antidepressant effects of ECT is unknown. Using magnetic resonance spectroscopy ((1)H-MRS), we measured dorsomedial anterior cingulate cortex (dmACC) and left and right hippocampal myo-inositol in 50 ECT patients (mean age: 43.78, 14 SD) and 33 controls (mean age: 39.33, 12 SD) to determine cross sectional effects of diagnosis and longitudinal effects of ECT. Patients were scanned prior to treatment, after the second ECT and at completion of the ECT index series. Controls were scanned twice at intervals corresponding to patients' baseline and end of treatment scans. Myo-inositol increased over the course of ECT in the dmACC (p = 0.042). A significant hemisphere by clinical response effect was observed for the hippocampus (p = 0.003) where decreased myo-inositol related to symptom improvement in the left hippocampus. Cross-sectional differences between patients and controls at baseline were not detected. Changes in myo-inositol observed in the dmACC in association with ECT and in the hippocampus in association with ECT-related clinical response suggest the mechanisms of ECT could include gliogenesis or a reversal of gliosis that differentially affect dorsal and ventral limbic regions. Change in dmACC myo-inositol diverged from control values with ECT suggesting compensation, while hippocampal change suggested normalization.

Effect of dietary docosahexaenoic acid (DHA) in phospholipids or triglycerides on brain DHA uptake and accretion.

Tracer studies suggest that phospholipid DHA (PL-DHA) more effectively targets the brain than triglyceride DHA (TAG-DHA), although the mechanism and whether this translates into higher brain DHA concentrations are not clear. Rats were gavaged with [U-(3)H]PL-DHA and [U-(3)H]TAG-DHA and blood sampled over 6h prior to collection of brain regions and other tissues. In another experiment, rats were supplemented for 4weeks with TAG-DHA (fish oil), PL-DHA (roe PL) or a mixture of both for comparison to a low-omega-3 diet. Brain regions and other tissues were collected, and blood was sampled weekly. DHA accretion rates were estimated using the balance method. [U-(3)H]PL-DHA rats had higher radioactivity in cerebellum, hippocampus and remainder of brain, with no differences in other tissues despite higher serum lipid radioactivity in [U-(3)H]TAG-DHA rats. TAG-DHA, PL-DHA or a mixture were equally effective at increasing brain DHA. There were no differences between DHA-supplemented groups in brain region, whole-body, or tissue DHA accretion rates except heart and serum TAG where the PL-DHA/TAG-DHA blend was higher than TAG-DHA. Apparent DHA ß-oxidation was not different between DHA-supplemented groups. This indicates that more labeled DHA enters the brain when consumed as PL; however, this may not translate into higher brain DHA concentrations.