A simple 1H (12C/13C) filtered experiment to quantify and trace isotope enrichment in complex environmental and biological samples.

Nuclear magnetic resonance (NMR) based 13C tracing has broad applications across medical and environmental research. As many biological and environmental samples are heterogeneous, they experience considerable spectral overlap and relatively low signal. Here a 1D 1H-12C/13C is introduced that uses "in-phase/opposite-phase" encoding to simultaneously detect and discriminate both protons attached to 12C and 13C at full 1H sensitivity in every scan. Unlike traditional approaches that focus on the 12C/13C satellite ratios in a 1H spectrum, this approach creates separate sub-spectra for the 12C and 13C bound protons. These spectra can be used for both quantitative and qualitative analysis of complex samples with significant spectral overlap. Due to the presence of the 13C dipole, faster relaxation of the 1H-13C pairs results in slight underestimation compared to the 1H-12C pairs. However, this is easily compensated for, by collecting an additional reference spectrum, from which the absolute percentage of 13C can be calculated by difference. When combined with the result, 12C and 13C percent enrichment in both 1H-12C and 1H-13C fractions are obtained. As the approach uses isotope filtered 1H NMR for detection, it retains nearly the same sensitivity as a standard 1H spectrum. Here, a proof-of-concept is performed using simple mixtures of 12C and 13C glucose, followed by suspended algal cells with varying 12C /13C ratios representing a complex mixture. The results consistently return 12C/13C ratios that deviate less than 1 % on average from the expected. Finally, the sequence was used to monitor and quantify 13C% enrichment in Daphnia magna neonates which were fed a 13C diet over 1 week. The approach helped reveal how the organisms utilized the 12C lipids they are born with vs. the 13C lipids they assimilate from their diet during growth. Given the experiments simplicity, versatility, and sensitivity, we anticipate it should find broad application in a wide range of tracer studies, such as fluxomics, with applications spanning various disciplines.

Development of Low-Magnetic Susceptibility Microcoils via 5-Axis Machining for Analysis of Biological and Environmental Samples.

In environmental research, it is critical to understand how toxins impact invertebrate eggs and egg banks, which, due to their tiny size, are very challenging to study by conventional nuclear magnetic resonance (NMR) spectroscopy. Microcoil technology has been extensively utilized to enhance the mass-sensitivity of NMR. In a previous study, 5-axis computer numerical control (CNC) micromilling (shown to be a viable alternative to traditional microcoil production methods) was used to create a prototype copper slotted-tube resonator (STR). Despite the excellent limit of detection (LOD) of the resonator, the quality of the line shape was very poor due to the magnetic susceptibility of the copper resonator itself. This is best solved using magnetic susceptibility-matched materials. In this study, approaches are investigated that improve the susceptibility while retaining the versatility of coil milling. One method involves machining STRs from various copper/aluminum alloys, while the other involves machining ones from an aluminum 2011 alloy and electroplating them with copper. In all cases, combining copper and aluminum to produce resonators resulted in improved line shape and SNR compared to pure copper resonators due to their reduced magnetic susceptibility. However, the copper-plated aluminum resonators showed optimal performance from the devices tested. The enhanced LOD of these STRs allowed for the first 1H-13C heteronuclear multiple quantum coherence (HMQC) of a single intact 13C-labeled Daphnia magna egg (∼4 µg total biomass). This is a key step toward future screening programs that aim to elucidate the toxic processes in aquatic eggs.

Cutting without a Knife: A Slice-Selective 2D 1H-13C HSQC NMR Sequence for the Analysis of Inhomogeneous Samples.

Nuclear magnetic resonance (NMR) is a powerful technique with applications ranging from small molecule structure elucidation to metabolomics studies of living organisms. Typically, solution-state NMR requires a homogeneous liquid, and the whole sample is analyzed as a single entity. While adequate for homogeneous samples, such an approach is limited if the composition varies as would be the case in samples that are naturally heterogeneous or layered. In complex samples such as living organisms, magnetic susceptibility distortions lead to broad 1H line shapes, and thus, the additional spectral dispersion afforded by 2D heteronuclear experiments is often required for metabolite discrimination. Here, a novel, slice-selective 2D, 1H-13C heteronuclear single quantum coherence (HSQC) sequence was developed that exclusively employs shaped pulses such that only spins in the desired volume are perturbed. In turn, this permits multiple volumes in the tube to be studied during a single relaxation delay, increasing sensitivity and throughput. The approach is first demonstrated on standards and then used to isolate specific sample/sensor elements from a microcoil array and finally study slices within a living earthworm, allowing metabolite changes to be discerned with feeding. Overall, slice-selective NMR is demonstrated to have significant potential for the study of layered and other inhomogeneous samples of varying complexity. In particular, its ability to select subelements is an important step toward developing microcoil receive-only arrays to study environmental toxicity in tiny eggs, cells, and neonates, whereas localization in larger living species could help better correlate toxin-induced biochemical responses to the physical localities or organs involved.

Exploring the Potential of Broadband Complementary Metal Oxide Semiconductor Micro-Coil Nuclear Magnetic Resonance for Environmental Research.

With sensitivity being the Achilles' heel of nuclear magnetic resonance (NMR), the superior mass sensitivity offered by micro-coils can be an excellent choice for tiny, mass limited samples such as eggs and small organisms. Recently, complementary metal oxide semiconductor (CMOS)-based micro-coil transceivers have been reported and demonstrate excellent mass sensitivity. However, the ability of broadband CMOS micro-coils to study heteronuclei has yet to be investigated, and here their potential is explored within the lens of environmental research. Eleven nuclei including 7Li, 19F, 31P and, 205Tl were studied and detection limits in the low to mid picomole range were found for an extended experiment. Further, two environmentally relevant samples (a sprouting broccoli seed and a D. magna egg) were successfully studied using the CMOS micro-coil system. 13C NMR was used to help resolve broad signals in the 1H spectrum of the 13C enriched broccoli seed, and steady state free precession was used to improve the signal-to-noise ratio by a factor of six. 19F NMR was used to track fluorinated contaminants in a single D. magna egg, showing potential for studying egg-pollutant interactions. Overall, CMOS micro-coil NMR demonstrates significant promise in environmental research, especially when the future potential to scale to multiple coil arrays (greatly improving throughput) is considered.

Integrated Digital Microfluidics NMR Spectroscopy: A Key Step toward Automated In Vivo Metabolomics.

Toxicity testing is currently undergoing a paradigm shift from examining apical end points such as death, to monitoring sub-lethal toxicity in vivo. In vivo nuclear magnetic resonance (NMR) spectroscopy is a key platform in this endeavor. A proof-of-principle study is presented which directly interfaces NMR with digital microfluidics (DMF). DMF is a "lab on a chip" method allowing for the movement, mixing, splitting, and dispensing of µL-sized droplets. The goal is for DMF to supply oxygenated water to keep the organisms alive while NMR detects metabolomic changes. Here, both vertical and horizontal NMR coil configurations are compared. While a horizontal configuration is ideal for DMF, NMR performance was found to be sub-par and instead, a vertical-optimized single-sided stripline showed most promise. In this configuration, three organisms were monitored in vivo using 1H-13C 2D NMR. Without support from DMF droplet exchange, the organisms quickly showed signs of anoxic stress; however, with droplet exchange, this was completely suppressed. The results demonstrate that DMF can be used to maintain living organisms and holds potential for automated exposures in future. However, due to numerous limitations of vertically orientated DMF, along with space limitations in standard bore NMR spectrometers, we recommend future development be performed using a horizontal (MRI style) magnet which would eliminate practically all the drawbacks identified here.

Evaluation of double-tuned single-sided planar microcoils for the analysis of small 13 C enriched biological samples using 1 H-13 C 2D heteronuclear correlation NMR spectroscopy.

Microcoils provide a cost-effective approach to improve detection limits for mass-limited samples. Single-sided planar microcoils are advantageous in comparison to volume coils, in that the sample can simply be placed on top. However, the considerable drawback is that the RF field that is produced by the coil decreases with distance from the coil surface, which potentially limits more complex multi-pulse NMR pulse sequences. Unfortunately, 1 H NMR alone is not very informative for intact biological samples due to line broadening caused by magnetic susceptibility distortions, and 1 H-13 C 2D NMR correlations are required to provide the additional spectral dispersion for metabolic assignments in vivo or in situ. To our knowledge, double-tuned single-sided microcoils have not been applied for the 2D 1 H-13 C analysis of intact 13 C enriched biological samples. Questions include the following: Can 1 H-13 C 2D NMR be performed on single-sided planar microcoils? If so, do they still hold sensitivity advantages over conventional 5 mm NMR technology for mass limited samples? Here, 2D 1 H-13 C HSQC, HMQC, and HETCOR variants were compared and then applied to 13 C enriched broccoli seeds and Daphnia magna (water fleas). Compared to 5 mm NMR probes, the microcoils showed a sixfold improvement in mass sensitivity (albeit only for a small localized region) and allowed for the identification of metabolites in a single intact D. magna for the first time. Single-sided planar microcoils show practical benefit for 1 H-13 C NMR of intact biological samples, if localized information within ~0.7 mm of the 1 mm I.D. planar microcoil surface is of specific interest.

5-Axis CNC Micromilling for Rapid, Cheap, and Background-Free NMR Microcoils.

The superior mass sensitivity of microcoil technology in nuclear magnetic resonance (NMR) spectroscopy provides potential for the analysis of extremely small-mass-limited samples such as eggs, cells, and tiny organisms. For optimal performance and efficiency, the size of the microcoil should be tailored to the size of the mass-limited sample of interest, which can be costly as mass-limited samples come in many shapes and sizes. Therefore, rapid and economic microcoil production methods are needed. One method with great potential is 5-axis computer numerical control (CNC) micromilling, commonly used in the jewelry industry. Most CNC milling machines are designed to process larger objects and commonly have a precision of >25 µm (making the machining of common spiral microcoils, for example, impossible). Here, a 5-axis MiRA6 CNC milling machine, specifically designed for the jewelry industry, with a 0.3 µm precision was used to produce working planar microcoils, microstrips, and novel microsensor designs, with some tested on the NMR in less than 24 h after the start of the design process. Sample wells could be built into the microsensor and could be machined at the same time as the sensors themselves, in some cases leaving a sheet of Teflon as thin as 10 µm between the sample and the sensor. This provides the freedom to produce a wide array of designs and demonstrates 5-axis CNC micromilling as a versatile tool for the rapid prototyping of NMR microsensors. This approach allowed the experimental optimization of a prototype microstrip for the analysis of two intact adult Daphnia magna organisms. In addition, a 3D volume slotted-tube resonator was produced that allowed for 2D 1H-13C NMR of D. magna neonates and exhibited 1H sensitivity (nLODω600 = 1.49 nmol s1/2) close to that of double strip lines, which themselves offer the best compromise between concentration and mass sensitivity published to date.

The ion channel TRPM7 is required for B cell lymphopoiesis.

The transient receptor potential (TRP) family is a large family of widely expressed ion channels that regulate the intracellular concentration of ions and metals and respond to various chemical and physical stimuli. TRP subfamily M member 7 (TRPM7) is unusual in that it contains both an ion channel and a kinase domain. TRPM7 is a divalent cation channel with preference for Ca2+ and Mg2+ It is required for the survival of DT40 cells, a B cell line; however, deletion of TRPM7 in T cells does not impair their development. We found that expression of TRPM7 was required for B cell development in mice. Mice that lacked TRPM7 in B cells failed to generate peripheral B cells because of a developmental block at the pro-B cell stage. The loss of TRPM7 kinase activity alone did not affect the proportion of peripheral mature B cells or the development of B cells in the bone marrow. However, supplementation with a high concentration of extracellular Mg2+ partially rescued the development of TRPM7-deficient B cells in vitro. Thus, our findings identify a critical role for TRPM7 ion channel activity in B cell development.

Development and validation of analytical methods for ultra-trace beryllium in biological matrices.

Beryllium (Be) is still not well understood from a toxicological point of view, and studies that involve the determination of different Be compounds species in tissues need to be conducted. In this paper we describe the development and validation of reliable methods for the detection of ultra-trace levels of Be in various biological matrices. Blood and tissues (liver, lung, spleen, and kidney) were used in this study. The samples were digested with a mixture of nitric and perchloric acids for Be and BeAl and an addition of sulfuric acid was made for BeO. The solutions were analyzed by inductively coupled plasma mass spectrometry with (6)Li as internal standard. The detection limits are in the order of 0.02 ng/g for tissue and 0.03 ng/mL for blood, and were compared to existing reference methods. To our knowledge, this is the first study that assesses dissolution of the different Be compounds in biological matrices, while also undergoing a rigorous optimization and complete validation. This method has proven that it is reliable, among the most sensitive available in the literature, and that it can be used in trace toxicological studies for Be.

Urinary levels, tissue concentrations and lung inflammation after nose-only exposure to three different chemical forms of beryllium.

This study aimed to determine the toxicity and toxicokinetic of three Be chemical species A total of 120 mice (four groups of 30) were nose-only exposed. The first group was used as a control while the three others were exposed to 250 microg m(-3) of fine particles of three different Be species (Be metal, Be-F; Be oxide, BeO-F; Be aluminium, BeAl-F). Exposure lasted over three consecutive weeks, five days per week and 6 h per day. Blood and several tissues were collected one week after exposure. Urines were collected before the beginning of exposure, at the end of every week of exposure and one week after exposure. Results showed that urine concentrations were different from one Be species to another and that excretion continued after the end of exposure. Except for BeO-F, where Be urine concentrations were stable during the three weeks of exposure, concentrations of Be-F and BeAl-F reached a peak after the first week. According to particle size, BeO-F obtained the highest theoretical pulmonary deposition rate, which partially led to the highest Be lung concentration. This group also presented the lowest urine concentration but that did not lead to more severe lung inflammation. Moreover, even if BeAl-F obtained the lowest percentage theoretical pulmonary deposition, it showed the highest Be urinary concentration, the lowest Be lung concentration and the lowest lung toxicity. In this specific case, a high Be concentration in urine did not reflect a high exposure or a severe toxic effect.

Effect of a psychoneurotherapy on brain electromagnetic tomography in individuals with major depressive disorder.

Recent advances in power spectral analysis of electroencephalography (EEG) signals and brain-computer interface (BCI) technology may significantly contribute to the development of psychoneurotherapies. The goal of this study was to measure the effect of a psychoneurotherapy on brain source generators of abnormal EEG activity in individuals with major depressive disorder (MDD). Thirty participants with unipolar MDD were recruited in the community. The proposed psychoneurotherapy was developed based on the relationship between the localization of abnormal EEG activity and depressive symptomatology. Brain electromagnetic abnormalities in MDD were identified with low resolution brain electromagnetic tomography (LORETA) and a normative EEG database. Localization of brain changes after treatment was assessed through the standardized version of LORETA (sLORETA). Before treatment, excessive high-beta (18-30 Hz) activity was noted in several brain regions located in the fronto-temporal regions. After treatment, only participants who successfully normalized EEG activity in cortico-limbic/paralimbic regions could be considered in clinical remission. In these regions, significant correlations were found between the percentage of change of depressive symptoms and the percentage of reduction in high-beta activity. These results suggest that the normalization of high-beta activity in cortico-limbic/paralimbic regions can be associated with a significant reduction of depressive symptoms.

Brain activity in near-death experiencers during a meditative state.

AIM: To measure brain activity in near-death experiencers during a meditative state. METHODS: In two separate experiments, brain activity was measured with functional magnetic resonance imaging (fMRI) and electroencephalography (EEG) during a Meditation condition and a Control condition. In the Meditation condition, participants were asked to mentally visualize and emotionally connect with the "being of light" allegedly encountered during their "near-death experience". In the Control condition, participants were instructed to mentally visualize the light emitted by a lamp. RESULTS: In the fMRI experiment, significant loci of activation were found during the Meditation condition (compared to the Control condition) in the right brainstem, right lateral orbitofrontal cortex, right medial prefrontal cortex, right superior parietal lobule, left superior occipital gyrus, left anterior temporal pole, left inferior temporal gyrus, left anterior insula, left parahippocampal gyrus and left substantia nigra. In the EEG experiment, electrode sites showed greater theta power in the Meditation condition relative to the Control condition at FP1, F7, F3, T5, P3, O1, FP2, F4, F8, P4, Fz, Cz and Pz. In addition, higher alpha power was detected at FP1, F7, T3 and FP2, whereas higher gamma power was found at FP2, F7, T4 and T5. CONCLUSIONS: The results indicate that the meditative state was associated with marked hemodynamic and neuroelectric changes in brain regions known to be involved either in positive emotions, visual mental imagery, attention or spiritual experiences.

The neural basis of unconditional love.

Functional neuroimaging studies have shown that romantic love and maternal love are mediated by regions specific to each, as well as overlapping regions in the brain's reward system. Nothing is known yet regarding the neural underpinnings of unconditional love. The main goal of this functional magnetic resonance imaging study was to identify the brain regions supporting this form of love. Participants were scanned during a control condition and an experimental condition. In the control condition, participants were instructed to simply look at a series of pictures depicting individuals with intellectual disabilities. In the experimental condition, participants were instructed to feel unconditional love towards the individuals depicted in a series of similar pictures. Significant loci of activation were found, in the experimental condition compared with the control condition, in the middle insula, superior parietal lobule, right periaqueductal gray, right globus pallidus (medial), right caudate nucleus (dorsal head), left ventral tegmental area and left rostro-dorsal anterior cingulate cortex. These results suggest that unconditional love is mediated by a distinct neural network relative to that mediating other emotions. This network contains cerebral structures known to be involved in romantic love or maternal love. Some of these structures represent key components of the brain's reward system.

EEG activity in Carmelite nuns during a mystical experience.

Mystical experiences relate to a fundamental dimension of human existence. These experiences, which are characterized by a sense of union with God, are commonly reported across all cultures. To date, no electroencephalography (EEG) study has been conducted to identify the neuroelectrical correlates of such experiences. The main objective of this study was to measure EEG spectral power and coherence in 14 Carmelite nuns during a mystical experience. EEG activity was recorded from 19 scalp locations during a resting state, a control condition and a mystical condition. In the mystical condition compared to control condition, electrode sites showed greater theta power at F3, C3, P3, Fz, Cz and Pz, and greater gamma1 power was detected at T4 and P4. Higher delta/beta ratio, theta/alpha ratio and theta/beta ratio were found for several electrode sites. In addition, FP1-C3 pair of electrodes displayed greater coherence for theta band while F4-P4, F4-T6, F8-T6 and C4-P4 pairs of electrodes showed greater coherence for alpha band. These results indicate that mystical experiences are mediated by marked changes in EEG power and coherence. These changes implicate several cortical areas of the brain in both hemispheres.

In vivo measurements of brain trapping of C-labelled alpha-methyl-L-tryptophan during acute changes in mood states.

BACKGROUND: Little is known about the specific contribution of serotonin (5-HT) to the neurobiology of emotion and mood in healthy people. In an exploratory study, we sought to investigate the effect of rapid and sustained changes of emotional state on the trapping of 11C-labelled alpha-methyl-L-tryptophan (11C-alphaMtrp) used as a proxy of 5-HT synthesis, using positron emission tomography (PET). METHOD: In a within-subject repeated-measure design, participants recalled autobiographical memories to self-induce sadness, happiness and a neutral emotional state during scanning to measure brain trapping of 11C-alphaMtrp. Three separate scan acquisitions, counterbalanced for order across subjects, took place at the McConnell Brain Imaging Center, Montréal. RESULTS: Whole brain analysis revealed positive and negative correlations between experienced levels of emotions and 11C-alphaMtrp trapping in the right anterior cingulate cortex. CONCLUSION: These findings point to a mechanism whereby state-related changes in a proxy of 5-HT synthesis underscore aspects of the self-regulation of normal mood.

Neural correlates of a mystical experience in Carmelite nuns.

The main goal of this functional magnetic resonance imaging (fMRI) study was to identify the neural correlates of a mystical experience. The brain activity of Carmelite nuns was measured while they were subjectively in a state of union with God. This state was associated with significant loci of activation in the right medial orbitofrontal cortex, right middle temporal cortex, right inferior and superior parietal lobules, right caudate, left medial prefrontal cortex, left anterior cingulate cortex, left inferior parietal lobule, left insula, left caudate, and left brainstem. Other loci of activation were seen in the extra-striate visual cortex. These results suggest that mystical experiences are mediated by several brain regions and systems.

Dysfunction in the neural circuitry of emotional self-regulation in major depressive disorder.

An inability to self-regulate negative emotions appears to play a pivotal role in the genesis of major depressive disorder. This inability may be related to a dysfunction of the neural circuitry underlying emotional self-regulation. This functional magnetic resonance imaging study was conducted to test this hypothesis. Depressed individuals and controls were scanned while they attempted to voluntarily down-regulate sad feelings. The degree of difficulty experienced during down-regulation of sadness was higher in depressed individuals. Furthermore, there was greater activation in the right dorsal anterior cingulate cortex, right anterior temporal pole, right amygdala, and right insula in depressed individuals. These results suggest that emotional dysregulation in major depressive disorder is related to a disturbance in the neural circuitry of emotional self-regulation.

alpha-[11C]Methyl-L-tryptophan trapping in the orbital and ventral medial prefrontal cortex of suicide attempters.

Low serotonin neurotransmission is thought to increase vulnerability to suicidal behavior. To test this hypothesis, we measured brain regional serotonin synthesis, as indexed by PET and alpha-[(11)C]methyl-L-tryptophan trapping, in 10 patients who had made a high-lethality suicide attempt and 16 healthy controls. Compared to healthy controls, suicide attempters had reduced normalized alpha-[(11)C]methyl-L-tryptophan trapping in orbital and ventromedial prefrontal cortex. alpha-[(11)C]Methyl-L-tryptophan trapping in these regions correlated negatively with suicide intent. Low serotonin synthesis in the prefrontal cortex might lower the threshold for suicidal behavior.

Separate neural circuits for primary emotions? Brain activity during self-induced sadness and happiness in professional actors.

The question of whether distinct or similar neural substrates underlie primary emotions has not been resolved yet. To address this issue, we used fMRI to scan professional actors during self-induced states of sadness and happiness. Results demonstrated that, relative to an emotionally Neutral state, both the Sad and the Happy states were associated with significant loci of activation, bilaterally, in the orbitofrontal cortex, and in the left medial prefrontal cortex, left ventrolateral prefrontal cortex, left anterior temporal pole, and right pons. These loci of activation were localized distinctly within these regions, that is, in different sub-regions. These results suggest that sadness and happiness may be associated with similar brain regions but distinct sub-regions and neural circuits.