Phytocannabinoids Reduce Seizures in Larval Zebrafish and Affect Endocannabinoid Gene Expression.

Cannabis has demonstrated anticonvulsant properties, and about thirty percent of epileptic patients do not have satisfactory seizure management with standard treatment and could potentially benefit from cannabis-based intervention. Here, we report the use of cannabinoids to treat pentylenetetrazol (PTZ)-induced convulsions in a zebrafish model, their effect on gene expression, and a simple assay for assessing their uptake in zebrafish tissues. Using an optimized behavioral assay, we show that cannabidiol (CBD) and cannabichromene (CBC) and cannabinol (CBN) are effective at reducing seizures at low doses, with little evidence of sedation, and our novel HPLC assay indicates that CBC is effective with the lowest accumulation in larval tissues. All cannabinoids tested were effective at higher concentrations. Pharmacological manipulation of potential receptors demonstrates that Gpr55 partially mediates the anticonvulsant effects of CBD. Treatment of zebrafish larvae with endocannabinoids, such as 2-arachidonoylglycerol (2-AG) and anandamide (AEA), altered larvae movement, and the expression of genes that regulate their metabolism was affected by phytocannabinoid treatment, highlighting the possibility that changes to endocannabinoid levels may represent one facet of the anticonvulsant effect of phytocannabinoids.

Development and application of a thin-film molecularly imprinted polymer for the measurement of mycophenolic acid in human plasma.

BACKGROUND: Mycophenolic acid (MPA) is used to suppress the immune response following organ transplantation; however, complex pharmacokinetic behavior and a large interpersonal variability necessitate therapeutic drug monitoring. To overcome the limitations of current sample preparation techniques, we present a novel thin-film molecularly imprinted polymer (TF-MIP) extraction device as part of a simple, sensitive, and fast method for analysis of MPA from human plasma. METHODS: Mycophenolic acid is extracted from plasma using a tailor-made TF-MIP that is subsequently desorbed into an organic solvent system compatible with mass spectrometry. The MIP yielded higher recovery of MPA relative to a corresponding non-imprinted polymer. The method allows for the determination of MPA in 45 min including analysis time and can be scaled for high throughput to process as many as 96 samples per hour. RESULTS: The method gave an LOD of 0.3 ng mL-1 and was linear from 5 to 250 ng mL-1 . Patient plasma samples (35 µL) were diluted using charcoal-stripped pooled plasma to a final extraction volume of 700 µL; when MPA in patient plasma is high, this ratio can easily be adjusted to ensure samples are within the method linear range. Intra- and inter-day variability were 13.8% and 4.3% (at 15 ng mL-1 ) and 13.5% and 11.0% (at 85 ng mL-1 ), respectively (n = 3); inter-device variability was 9.6% (n = 10). CONCLUSIONS: Low inter-device variability makes these devices suitable for single use in a clinical setting, and the fast and robust method is suitable for therapeutic drug monitoring, where throughput and time-to-result are critical.

Purification of Functional Gene Transfer Agents Using Two-Step Preparative Monolithic Chromatography.

Background: Gene transfer agents (GTAs) are phage-like particles that transfer cellular genomic DNA between cells. A hurdle faced in studying GTA function and interactions with cells is the difficulty in obtaining pure and functional GTAs from cultures. Materials and Methods: We used a novel two-step method for purification of GTAs from R. capsulatus by monolithic chromatography. Results: Our efficient and simple process had advantages compared to previous approaches. The purified GTAs retained gene transfer activity and the packaged DNA could be used for further studies. Conclusions: This method is applicable to GTAs produced by other species and small phages, and could be useful for therapeutic applications.

Micro-gel thin film molecularly imprinted polymer coating for extraction of organophosphorus pesticides from water and beverage samples.

Molecularly imprinted polymers (MIPs) have become an important class of materials for selective and efficient adsorption of target analytes. Despite versatility of MIPs for fabrication in numerous formats, these materials have been primarily reported as solid phase extraction packing materials. An effective thin film MIP prepared on stainless steel substrate is reported here for high throughput enrichment of organophosphorus pesticides (OPPs) from water and beverage samples followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The key factors controlling performance as well as best practices for optimized fabrication of thin film MIPs are presented. A pseudo-phase diagram is introduced to evaluate and predict the effect of the ratio of porogen (solvent, 1-octanol) volume to relative crosslinker mass on the desired polymer features (i.e., porosity, surface area, capacity, and selectivity). At low porogen ratios, a macroporous polymer with insignificant selectivity is formed, whereas at high porogen ratios a micro-gel polymer with superior selectivity towards targets is obtained. The porosity and morphology determined with nitrogen adsorption and scanning electron microscopy were attributed to specific regions in the pseudo-phase diagram. Other factors influencing selectivity and stability of the polymer, such as type of the template and its ratios with monomer (methacrylic acid) and crosslinker (ethylene glycol dimethacrylate) were optimized. The prepared thin film MIPs were characterized using adsorption isotherms and adsorption kinetics, and evaluated for matrix effects (high humic acid content) and cross-reactivity in presence of other pesticides and pharmaceuticals. The optimized method provided limits of quantitation (LOQs) ranged from 0.002 to 0.02 ng mL-1 in water and from 0.095 to 0.48 ng g-1 in apple juice. Regarding inter-device variability (CV∼10% without normalization), excellent linearity (R2 > 0.99), satisfactory accuracies (90-110%) and precisions (<15%) were obtained.

Thin film molecularly imprinted polymer (TF-MIP), a selective and single-use extraction device for high-throughput analysis of biological samples.

Enhancing selectivity, reducing matrix effects and increasing analytical throughput have been the main objectives in the development of biological sample preparation techniques. A thin film molecularly imprinted polymer (MIP) is employed for extraction and analysis of tricyclic antidepressants (TCAs) as a model class of compounds in human plasma for the first time to reach the abovementioned goals. The thin film MIPs prepared on a metal substrate can be used directly for extraction from biological matrices with no sample manipulation steps and no pre-conditioning. This method was validated with good linearity (R2 > 0.99 in 1.0-500.0 ng mL-1 range), excellent accuracy (90% -110%) and precision (RSD % value less than 15%) in pooled human plasma samples (N = 3). The limits of quantitation (LOQ) for TCAs in plasma samples were between 1.0-5.0 ng mL-1 which are lower than the therapeutic ranges of these drugs. Kinetic and isotherm studies showed the superior performance of MIP sorbent compared to a non-imprinted polymer (NIP) sorbent in extracting TCAs from a bovine serum albumin (BSA) solution. The optimized and validated method for pooled human plasma was utilized for monitoring the concentration of TCAs in three patient samples who had been prescribed TCAs. These selective single-use thin film extraction devices are promising for efficient and fast procedures for analyzing biological samples.

A Two-Component System Acquired by Horizontal Gene Transfer Modulates Gene Transfer and Motility via Cyclic Dimeric GMP.

Bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) is an important intracellular signaling molecule that affects diverse physiological processes in bacteria. The intracellular levels of c-di-GMP are controlled by proteins acting as diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes that synthesize and degrade c-di-GMP, respectively. In the alphaproteobacterium Rhodobacter capsulatus, flagellar motility and gene exchange via production of the gene transfer agent RcGTA are regulated by c-di-GMP. One of the R. capsulatus proteins involved in this regulation is Rcc00620, which contains an N-terminal two-component system response regulator receiver (REC) domain and C-terminal DGC and PDE domains. We demonstrate that the enzymatic activity of Rcc00620 is regulated through the phosphorylation status of its REC domain, which is controlled by a cognate histidine kinase protein, Rcc00621. In this system, the phosphorylated form of Rcc00620 is active as a PDE enzyme and stimulates gene transfer and motility. In addition, we discovered that the rcc00620 and rcc00621 genes are present in only one lineage within the genus Rhodobacter and were acquired via horizontal gene transfer from a distantly related alphaproteobacterium in the order Sphingomonadales. Therefore, a horizontally acquired regulatory system regulates gene transfer in the recipient organism.

Cyclic di-GMP-Mediated Regulation of Gene Transfer and Motility in Rhodobacter capsulatus.

Gene transfer agents (GTAs) are bacteriophage-like particles produced by several bacterial and archaeal lineages that contain small pieces of the producing cells' genomes that can be transferred to other cells in a process similar to transduction. One well-studied GTA is RcGTA, produced by the alphaproteobacterium Rhodobacter capsulatus RcGTA gene expression is regulated by several cellular regulatory systems, including the CckA-ChpT-CtrA phosphorelay. The transcription of multiple other regulator-encoding genes is affected by the response regulator CtrA, including genes encoding putative enzymes involved in the synthesis and hydrolysis of the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP). To investigate whether c-di-GMP signaling plays a role in RcGTA production, we disrupted the CtrA-affected genes potentially involved in this process. We found that disruption of four of these genes affected RcGTA gene expression and production. We performed site-directed mutagenesis of key catalytic residues in the GGDEF and EAL domains responsible for diguanylate cyclase (DGC) and c-di-GMP phosphodiesterase (PDE) activities and analyzed the functions of the wild-type and mutant proteins. We also measured RcGTA production in R. capsulatus strains where intracellular levels of c-di-GMP were altered by the expression of either a heterologous DGC or a heterologous PDE. This adds c-di-GMP signaling to the collection of cellular regulatory systems controlling gene transfer in this bacterium. Furthermore, the heterologous gene expression and the four gene disruptions had similar effects on R. capsulatus flagellar motility as found for gene transfer, and we conclude that c-di-GMP inhibits both RcGTA production and flagellar motility in R. capsulatusIMPORTANCE Gene transfer agents (GTAs) are virus-like particles that move cellular DNA between cells. In the alphaproteobacterium Rhodobacter capsulatus, GTA production is affected by the activities of multiple cellular regulatory systems, to which we have now added signaling via the second messenger dinucleotide molecule bis-(3'-5')-cyclic dimeric GMP (c-di-GMP). Similar to the CtrA phosphorelay, c-di-GMP also affects R. capsulatus flagellar motility in addition to GTA production, with lower levels of intracellular c-di-GMP favoring increased flagellar motility and gene transfer. These findings further illustrate the interconnection of GTA production with global systems of regulation in R. capsulatus, providing additional support for the notion that the production of GTAs has been maintained in this and related bacteria because it provides a benefit to the producing organisms.

Optimized Oxidative Stress Protocols for Low-microliter Volumes of Mammalian Plasma.

Small blood volumes commonly obtained from small mammals during field studies are only sufficient for a single biochemical assay. In this study, we used blood collected from a population of wild eastern chipmunks (Tamias striatus) and developed modified methods to improve analytical selectivity and sensitivity required for measuring markers of oxidative stress using small blood volumes. Specifically, we proposed a modified malondialdehyde (MDA) analysis protocol by high performance liquid chromatography (HPLC) and also optimized both the uric acid independent ferric reducing antioxidant power (FRAP) and hypochlorous acid shock capacity (HASC) assays. We present methods in which a total volume of less than 60 µl of plasma is required to obtain a comprehensive portrait of an individual's oxidative profile.

Development of small blood volume assays for the measurement of oxidative stress markers in mammals.

Measuring oxidative stress has become increasingly valuable in ecological studies, especially when different markers are measured on the same individual. However, many of the current methods lack sensitivity for analysis of low blood volume samples, which represent a challenge for longitudinal field studies of small organisms. Small blood volumes can usually only be analysed by using a single assay, therefore providing limited information on individual's oxidative profile. In this study, we used blood collected from a population of wild eastern chipmunks (Tamias striatus) and modified methods presented in the literature to improve analytical selectivity and sensitivity required for small blood volumes. Specifically, we proposed a modified malondialdehyde (MDA) analysis protocol by HPLC and also optimized both the uric acid independent ferric reducing antioxidant power (FRAP) and hypochlorous acid shock capacity (HASC) assays. Development of the three modified methods was achieved with a sensitivity and repeatability that meets standards of field ecology while allowing measurement of all three assays in duplicate using less than 60 µL of plasma. Availability of these tests using small blood volumes will provide ecologists with a more comprehensive portrait of an individual's oxidative profile and a better understanding of its determinants and interactions with the environment.