Investigation Of Dopamine Dynamics In Bdnf+/- Mice Using In Vivo Microdialysis And Electrochemical Analysis Of Purine And Monoamine Molecules Using A Boron-Doped Diamond Electrode

The goal of the first study was to determine if a reduction in brain-derived neurotrophic factor (BDNF) levels in female mice lead to a dysregulation in their dopaminergic system. Through a series of in vivo microdialysis and slice voltammetry experiments, we have discerned that female BDNF+/- mice are hyperdopaminergic similar to their male BDNF+/- counterparts. The in vivo microdialysis method zero-net flux highlighted that female BDNF+/- mice had increased extracellular dopamine (DA) levels, while stimulated regional release by high potassium potentiated DA release from vesicular mediated depolarization. Using the complementary technique of fast scan cyclic voltammetry, electrical stimulation evoked greater release in the female BDNF+/- mice, while uptake was not different from female wildtype mice. When the psychostimulant methamphetamine was administered, female BDNF+/- mice had potentiated DA release compared to their wildtype counterparts. Taken together, the DA release impairments in female mice appears to result in a hyperdopaminergic phenotype with no concomitant alterations in DA uptake. The aim of the second study was to characterize how lifelong reductions in BDNF affect the striatal dopaminergic system in aged BDNF+/- mice. As BDNF+/- mice aged from 3 to 18 months, their striatal dopamine dynamics, as measured by microdialysis and slice voltammetry, `normalized\u27 with respect to time. Aged BDNF+/- mice (18 months) had elevated levels of striatal DA metabolites and decreased phasic versus tonic release of DA with time. DA levels in BDNF+/- mice were age-dependent such that low BDNF levels in early adulthood, as previously reported, led to a hyperdopaminergic state while DA dynamics in the aged BDNF+/- mice `normalized\u27 with no overt alterations in either behavior or neurochemistry. In the third study we developed a method using a commercially available BDD working electrode for detecting neurotransmitters from two different families with large oxidation potential differences, DA and adenosine (Ado). Hydrodynamic voltammograms were constructed for DA and Ado, and the optimal potential for detection of DA and Ado was determined to be +740 mV and +1200 mV versus a palladium reference electrode, respectively. A working potential of +840 mV was chosen and the detection range achieved with the BDD electrode for DA and Ado was from low nanomolar to high millimolar levels. To determine the practical function of the BDD electrode, tissue content was analyzed for seven monoamines and two purine molecules, which were resolved in a single run in less than 28 min. Our results demonstrate that the BDD electrode is sensitive and robust enough to detect monoamine and purine molecules from frontal cortex and striatal mouse samples

Drone-based Water Sampling and Characterization of Three Freshwater Harmful Algal Blooms in the United States

Freshwater harmful algal blooms (HABs), caused mostly by toxic cyanobacteria, produce a range of cyanotoxins that threaten the health of humans and domestic animals. Climate conditions and anthropogenic influences such as agricultural run-off can alter the onset and intensity of HABs. Little is known about the distribution and spread of freshwater HABs. Current sampling protocols in some lakes involve teams of researchers that collect samples by hand from a boat and/or from the shoreline. Water samples can be collected from the surface, from discrete-depth collections, and/or from depth-integrated intervals. These collections are often restricted to certain months of the year, and generally are only performed at a limited number of collection sites. In lakes with active HABs, surface samples are generally sufficient for HAB water quality assessments. We used a unique DrOne Water Sampling SystEm (DOWSE) to collect water samples from the surface of three different HABs in Ohio (Grand Lake St Marys, GLSM and Lake Erie) and Virginia (Lake Anna), United States in 2019. The DOWSE consisted of a 3D-printed sampling device tethered to a drone (uncrewed aerial system, or UAS), and was used to collect surface water samples at different distances (10–100 m) from the shore or from an anchored boat. One hundred and eighty water samples (40 at GLSM, 20 at Lake Erie, and 120 at Lake Anna) were collected and analyzed from 18 drone flights. Our methods included testing for cyanotoxins, phycocyanin, and nutrients from surface water samples. Mean concentrations of microcystins (MCs) in drone water samples were 15.00, 1.92, and 0.02 ppb for GLSM, Lake Erie, and Lake Anna, respectively. Lake Anna had low levels of anatoxin in nearly all (111/120) of the drone water samples. Mean concentrations of phycocyanin in drone water samples were 687, 38, and 62 ppb for GLSM, Lake Erie, and Lake Anna, respectively. High levels of total phosphorus were observed in the drone water samples from GLSM (mean of 0.34 mg/L) and Lake Erie (mean of 0.12 mg/L). Lake Anna had the highest variability of total phosphorus with concentrations that ranged from 0.01 mg/L to 0.21 mg/L, with a mean of 0.06 mg/L. Nitrate levels varied greatly across sites, inverse with bloom biomass, ranging from below detection to 3.64 mg/L, with highest mean values in Lake Erie followed by GLSM and Lake Anna, respectively. Drones offer a rapid, targeted collection of water samples from virtually anywhere on a lake with an active HAB without the need for a boat which can disturb the surrounding water. Drones are, however, limited in their ability to operate during inclement weather such as rain and heavy winds. Collectively, our results highlight numerous opportunities for drone-based water sampling technologies to track, predict, and respond to HABs in the future

Drone-based water sampling and characterization of three freshwater harmful algal blooms in the United States

Freshwater harmful algal blooms (HABs), caused mostly by toxic cyanobacteria, produce a range of cyanotoxins that threaten the health of humans and domestic animals. Climate conditions and anthropogenic influences such as agricultural run-off can alter the onset and intensity of HABs. Little is known about the distribution and spread of freshwater HABs. Current sampling protocols in some lakes involve teams of researchers that collect samples by hand from a boat and/or from the shoreline. Water samples can be collected from the surface, from discrete-depth collections, and/or from depth-integrated intervals. These collections are often restricted to certain months of the year, and generally are only performed at a limited number of collection sites. In lakes with active HABs, surface samples are generally sufficient for HAB water quality assessments. We used a unique DrOne Water Sampling SystEm (DOWSE) to collect water samples from the surface of three different HABs in Ohio (Grand Lake St Marys, GLSM and Lake Erie) and Virginia (Lake Anna), United States in 2019. The DOWSE consisted of a 3D-printed sampling device tethered to a drone (uncrewed aerial system, or UAS), and was used to collect surface water samples at different distances (10–100 m) from the shore or from an anchored boat. One hundred and eighty water samples (40 at GLSM, 20 at Lake Erie, and 120 at Lake Anna) were collected and analyzed from 18 drone flights. Our methods included testing for cyanotoxins, phycocyanin, and nutrients from surface water samples. Mean concentrations of microcystins (MCs) in drone water samples were 15.00, 1.92, and 0.02 ppb for GLSM, Lake Erie, and Lake Anna, respectively. Lake Anna had low levels of anatoxin in nearly all (111/120) of the drone water samples. Mean concentrations of phycocyanin in drone water samples were 687, 38, and 62 ppb for GLSM, Lake Erie, and Lake Anna, respectively. High levels of total phosphorus were observed in the drone water samples from GLSM (mean of 0.34 mg/L) and Lake Erie (mean of 0.12 mg/L). Lake Anna had the highest variability of total phosphorus with concentrations that ranged from 0.01 mg/L to 0.21 mg/L, with a mean of 0.06 mg/L. Nitrate levels varied greatly across sites, inverse with bloom biomass, ranging from below detection to 3.64 mg/L, with highest mean values in Lake Erie followed by GLSM and Lake Anna, respectively. Drones offer a rapid, targeted collection of water samples from virtually anywhere on a lake with an active HAB without the need for a boat which can disturb the surrounding water. Drones are, however, limited in their ability to operate during inclement weather such as rain and heavy winds. Collectively, our results highlight numerous opportunities for drone-based water sampling technologies to track, predict, and respond to HABs in the future

Comparative Analysis of Microcystin Prevalence in Michigan Lakes by Online Concentration LC/MS/MS and ELISA

Fast and reliable workflows are needed to quantitate microcystins (MCs), a ubiquitous class of hepatotoxic cyanotoxins, so that the impact of human and environmental exposure is assessed quickly and minimized. Our goal was to develop a high-throughput online concentration liquid chromatography tandem mass spectrometry (LC/MS/MS) workflow to quantitate the 12 commercially available MCs and nodularin in surface and drinking waters. The method run time was 8.5 min with detection limits in the low ng/L range and minimum reporting levels between 5 and 10 ng/L. This workflow was benchmarked by determining the prevalence of MCs and comparing the Adda-ELISA quantitation to our new workflow from 122 samples representing 31 waterbodies throughout Michigan. The frequency of MC occurrence was MC-LA > LR > RR > D-Asp3-LR > YR > HilR > WR > D-Asp3-RR > HtyR > LY = LW = LF, while MC-RR had the highest concentrations. MCs were detected in 33 samples and 13 of these samples had more than 20% of their total MC concentration from MCs not present in US Environmental Protection Agency (US EPA) Method 544. Furthermore, seasonal deviations between the LC/MS/MS and Adda-ELISA data suggest Adda-ELISA cross-reacts with MC degradation products. This workflow provides less than 24-h turnaround for quantification and also identified key differences between LC/MS/MS and ELISA quantitation that should be investigated further

Dhb Microcystins Discovered in USA Using an Online Concentration LC–MS/MS Platform

Based on current structural and statistical calculations, thousands of microcystins (MCs) can exist; yet, to date, only 246 MCs have been identified and only 12 commercial MC standards are available. Standard mass spectrometry workflows for known and unknown MCs need to be developed and validated for basic and applied harmful algal bloom research to advance. Our investigation focuses on samples taken in the spring of 2018 from an impoundment fed by Oser and Bischoff Reservoirs, Indiana, United States of America (USA). The dominant cyanobacterium found during sampling was Planktothrix agardhii. The goal of our study was to identify and quantify the MCs in the impoundment sample using chemical derivatization and mass spectrometry. Modifying these techniques to use online concentration liquid chromatography tandem mass spectrometry (LC–MS/MS), two untargeted MCs have been identified, [d-Asp3, Dhb7]-MC-LR and tentative [Dhb7]-MC-YR. [Dhb7]-MC-YR is not yet reported in the literature to date, and this was the first reported incidence of Dhb MCs in the United States. Furthermore, it was discovered that the commercially available [d-Asp3]-MC-RR standard was [d-Asp3, Dhb7]-MC-RR. This study highlights a workflow utilizing online concentration LC–MS/MS, high-resolution MS (HRMS), and chemical derivatization to identify isobaric MCs

Identification and Quantification of Degradation Products of Microcystins using High-Resolution UHPLC-Orbitrap-MS

Sharmila I. Thenuwara1, Jyotshana Gautam2, Samuel Simpson1, Johnna Birbeck3, Judy A. Westrick3, Jason F. Huntley2, Dragan Isailovic1 1Department of Chemistry and Biochemistry, University of Toledo, Toledo, OH 43606 2Department of Medical Microbiology and Immunology, University of Toledo College of Medicine and Life Sciences, Toledo, OH 43614 3Lumigen Instrumentation Center, Department of Chemistry, Wayne State University, Detroit, MI 48202 Microcystins (MCs) are heptapeptides produced by freshwater harmful algal blooms (HABs). Exposure to hepatotoxic MCs is a threat to humans and animals. Although conventional municipal water treatment processes can treat MC contaminated water, biodegradation of cyanotoxins using indigenous bacteria is cost-effective and environmentally friendly. We previously isolated and characterized five bacterial isolates from Lake Erie that degraded MC-LR into non-toxic fragments (Thees et al. 2018). Herein, degradation of MCs that are abundant in Lake Erie HABs, MC-LR and MC-RR, is investigated qualitatively and quantitatively using high-resolution LC-MS. UHPLC-Selected ion monitoring (SIM)-Orbitrap-MS analysis revealed two peaks at different retention times corresponding to m/z of a tetrapeptide degradation product. Fragmentation spectra of both peaks showed characteristic ADDA fragment with m/z 135.08. From the MS/MS spectrum, it was concluded that the peak with the shorter retention time than the substrate is linear tetrapeptide. These results indicate that the MC biodegradation mechanisms in Lake Erie bacteria may be distinct from those in other MC degrading bacteria. The enzymatic pathways and MC breakdown products are being investigated further

Discovery and Structural Elucidation of Novel Microcystins Using MS and MSn with Python Code

Microcystins (MCs) are cyclic heptapeptide hepatotoxins with a vast structural diversity (\u3e300 congeners). However, many more congeners are theoretically possible, and a workflow was developed for putative identification of novel MCs using liquid chromatography (LC) coupled to high-resolution mass spectrometry (HRMS) and MSn with Python code. Water samples collected from Lake Erie were sonicated and filtered, and solid-phase extraction was performed. A portion of the water sample was reacted with mercaptoethanol using the method developed by Miles et al. for structural elucidation. Extracted MCs were analyzed using UHPLC coupled to an Orbitrap Fusion instrument for HRMS. Collision-induced dissociation (CID) and higher-energy CID (HCD) were used for MSn analyses. A total of 33 MCs were found in lake water samples, including two unknown MCs. Two Python codes were developed to elucidate the structures of MCs. Code 1 was written to generate a list of masses of theoretically possible MC congeners. Code 2 was written to compare the experimentally obtained masses of MC fragment ions to the theoretical fragment masses. Using Codes 1 and 2, the two unknown MCs with m/z values of 526.7980 (z=+2) and 1025.5302 (z=+1) were putatively identified as MC-HarR and MC-E(OMe)R, respectively

Quantification of microcystin production and biodegradation rates in the western basin of Lake Erie

Cyanobacterial biomass forecasts currently cannot predict the concentrations of microcystin, one of the most ubiquitous cyanotoxins that threaten human and wildlife health globally. Mechanistic insights into how microcystin production and biodegradation by heterotrophic bacteria change spatially and throughout the bloom season can aid in toxin concentration forecasts. We quantified microcystin production and biodegradation during two growth seasons in two western Lake Erie sites with different physicochemical properties commonly plagued by summer Microcystis blooms. Microcystin production rates were greater with elevated nutrients than under ambient conditions and were highest nearshore during the initial phases of the bloom, and production rates were lower in later bloom phases. We examined biodegradation rates of the most common and toxic microcystin by adding extracellular stable isotope-labeled microcystin-LR (1 μg L−1), which remained stable in the abiotic treatment (without bacteria) with minimal adsorption onto sediment, but strongly decreased in all unaltered biotic treatments, suggesting biodegradation. Greatest biodegradation rates (highest of −8.76 d−1, equivalent to the removal of 99.98% in 18 h) were observed during peak bloom conditions, while lower rates were observed with lower cyanobacteria biomass. Cell-specific nitrogen incorporation from microcystin-LR by nanoscale imaging mass spectrometry showed that a small percentage of the heterotrophic bacterial community actively degraded microcystin-LR. Microcystin production and biodegradation rates, combined with the microcystin incorporation by single cells, suggest that microcystin predictive models could be improved by incorporating toxin production and biodegradation rates, which are influenced by cyanobacterial bloom stage (early vs. late bloom), nutrient availability, and bacterial community composition.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/173130/1/lno12096_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/173130/2/lno12096.pd

Development and Application of Extraction Methods for LC-MS Quantification of Microcystins in Liver Tissue

A method was developed to extract and quantify microcystins (MCs) from mouse liver with limits of quantification (LOQs) lower than previously reported. MCs were extracted from 40-mg liver samples using 85:15 (v:v) CH3CN:H2O containing 200 mM ZnSO4 and 1% formic acid. Solid-phase extraction with a C18 cartridge was used for sample cleanup. MCs were detected and quantified using HPLC-orbitrap-MS with simultaneous MS/MS detection of the 135.08 m/z fragment from the conserved Adda amino acid for structural confirmation. The method was used to extract six MCs (MC-LR, MC-RR, MC-YR, MC-LA, MC-LF, and MC-LW) from spiked liver tissue and the MC-LR cysteine adduct (MC-LR-Cys) created by the glutathione detoxification pathway. Matrix-matched internal standard calibration curves were constructed for each MC (R2 ≥ 0.993), with LOQs between 0.25 ng per g of liver tissue (ng/g) and 0.75 ng/g for MC-LR, MC-RR, MC-YR, MC-LA, and MC-LR-Cys, and 2.5 ng/g for MC-LF and MC-LW. The protocol was applied to extract and quantify MC-LR and MC-LR-Cys from the liver of mice that had been gavaged with 50 µg or 100 µg of MC-LR per kg bodyweight and were euthanized 2 h, 4 h, or 48 h after final gavage. C57Bl/6J (wild type, control) and Leprdb/J (experiment) mice were used as a model to study non-alcoholic fatty liver disease. The Leprdb/J mice were relatively inefficient in metabolizing MC-LR into MC-LR-Cys, which is an important defense mechanism against MC-LR exposure. Trends were also observed as a function of MC-LR gavage amount and time between final MC-LR gavage and euthanasia/organ harvest