Glucagon-like peptide-1 receptor agonist, exendin-4, reduces reinstatement of heroin-seeking behavior in rats.

Opioid use disorder (OUD) causes the death of nearly 130 Americans daily. It is evident that new avenues for treatment are needed. To this end, studies have reported that 'satiety' agents such as the glucagon-like peptide-1 receptor (GLP-1R) agonist, exendin-4 (Ex-4), decreases responding for addictive drugs such as cocaine, nicotine, alcohol, and oxycodone, but no work has been done with heroin. In this study, we used a reward devaluation model in which rats avoid ingesting a saccharin solution that predicts drug availability to test the effects of 2.4 µg/kg Ex-4 on responding for a natural reward cue (i.e., saccharin) and on cue- and drug-induced heroin seeking. The results showed that treatment with Ex-4 during the 16-day abstinence period and on the test day decreased cue-induced heroin seeking. Drug-induced heroin seeking also was reduced by Ex-4, but only when using a 1 h, but not a 6 h, pretreatment time. Treatment with Ex-4 did not alter intake of the saccharin cue when the drug was on board, but a history of treatment with Ex-4 increased acceptance of the saccharin cue in later extinction trials. Finally, treatment with Ex-4 did not alter body weight, but was associated with increased Orexin 1 receptor (OX1) mRNA expression in the nucleus accumbens shell. Taken together, these findings are the first to show that treatment with a GLP-1R agonist can reduce both cue-induced seeking and drug-induced reinstatement of heroin seeking. As such, a GLP-1R agonist may serve as an effective treatment for OUD in humans.

Impact of the NaV1.8 variant, A1073V, on post-sigmoidectomy pain and electrophysiological function in rat sympathetic neurons.

NaV1.8 channels play a crucial role in regulating the action potential in nociceptive neurons. A single nucleotide polymorphism in the human NaV1.8 gene SCN10A, A1073V (rs6795970, G>A), has been linked to the diminution of mechanical pain sensation as well as cardiac conduction abnormalities. Furthermore, studies have suggested that this polymorphism may result in a "loss-of-function" phenotype. In the present study, we performed genomic analysis of A1073V polymorphism presence in a cohort of patients undergoing sigmoid colectomy who provided information regarding perioperative pain and analgesic use. Homozygous carriers reported significantly reduced severity in postoperative abdominal pain compared with heterozygous and wild-type carriers. Homozygotes also trended toward using less analgesic/opiates during the postoperative period. We also heterologously expressed the wild-type and A1073V variant in rat superior cervical ganglion neurons. Electrophysiological testing demonstrated that the mutant NaV1.8 channels activated at more depolarized potentials compared with wild-type channels. Our study revealed that postoperative abdominal pain is diminished in homozygous carriers of A1073V and that this is likely due to reduced transmission of action potentials in nociceptive neurons. Our findings reinforce the importance of NaV1.8 and the A1073V polymorphism to pain perception. This information could be used to develop new predictive tools to optimize patient pain experience and analgesic use in the perioperative setting.NEW & NOTEWORTHY We present evidence that in a cohort of patients undergoing sigmoid colectomy, those homozygous for the NaV1.8 polymorphism (rs6795970) reported significantly lower abdominal pain scores than individuals with the homozygous wild-type or heterozygous genotype. In vitro electrophysiological recordings also suggest that the mutant NaV1.8 channel activates at more depolarizing potentials than the wild-type Na+ channel, characteristic of hypoactivity. This is the first report linking the rs6795970 mutation with postoperative abdominal pain in humans.

Functional characterization of the S41Y (C2755A) polymorphism of tryptophan hydroxylase 2.

Human TPH2 (hTPH2) catalyzes the rate-limiting step in CNS serotonin biosynthesis. We characterized a single-nucleotide polymorphism (C2755A) in the hTPH2 gene that substitutes tyrosine for serine at position 41 in the regulatory domain of the enzyme. This polymorphism is associated with bipolar disorder and peripartum depression in a Chinese population. Recombinant h TPH2 human proteins were expressed in bacteria and also stably expressed in PC12 cells. Following bacterial expression and purification, the tyrosine for serine substitution at position 41 (S41Y) polymorphic enzyme displayed increased Vmax with unchanged Km values. By contrast, enzyme stability was decreased in vitro from 32 min to 4 min (37 °C) for the S41Y enzyme (as compared to the wild-type enzyme). The S41Y polymorphism decreased cyclic AMP-dependent protein kinase A-mediated phosphorylation ~ 50% relative to wild-type hTPH2, suggesting that the S41Y mutation may disrupt the post-translational regulation of this enzyme. Transfected PC12 cells expressed hTPH2 mRNA, active protein, and synthesized and released serotonin. Paradoxically, while S41Y-transfected PC12 cells expressed higher levels of hTPH2 than wild type, they synthesized less serotonin. These findings suggest a modified regulation of the S41Y gene variant leading to altered regulation and reduced neurotransmitter synthesis that may contribute to association of the polymorphism with bipolar disorder and depression. We report the functional implications of a polymorphic human tryptophan hydroxylase-2 gene associated with depression and bipolar disorder. The polymorphic enzyme (serine-41 converted to tyrosine) has increased activity, but decreased enzyme stability and serotonin production. Moreover, cyclic AMP-dependent protein kinase (PKA)-mediated phosphorylation of the mutant enzyme is decreased suggesting modified regulation of the S41Y variant leading to altered serotonin.

Complex molecular regulation of tyrosine hydroxylase.

Tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, is strictly controlled by several interrelated regulatory mechanisms. Enzyme synthesis is controlled by epigenetic factors, transcription factors, and mRNA levels. Enzyme activity is regulated by end-product feedback inhibition. Phosphorylation of the enzyme is catalyzed by several protein kinases and dephosphorylation is mediated by two protein phosphatases that establish a sensitive process for regulating enzyme activity on a minute-to-minute basis. Interactions between tyrosine hydroxylase and other proteins introduce additional layers to the already tightly controlled production of catecholamines. Tyrosine hydroxylase degradation by the ubiquitin-proteasome coupled pathway represents yet another mechanism of regulation. Here, we revisit the myriad mechanisms that regulate tyrosine hydroxylase expression and activity and highlight their physiological importance in the control of catecholamine biosynthesis.

Mesenchymal Stem Cell Identification After Delayed Cord Clamping.

We sought to determine the feasibility of identifying and quantifying mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) after delayed cord clamping in preterm and term births. We obtained 3 mL of UCB at various gestational ages after delayed cord clamping. UCB separated by density gradient centrifugation within 4 h of delivery was passed through magnetic bead micro-columns to exclude the CD34 + cell population. The samples were incubated with fluorescent-tagged mesenchymal cell marker antibodies CD 29, CD44, CD73, CD105, and hematopoietic cell marker CD45. The cell populations were analyzed by flow cytometry. Viable cells were assessed with 7-aminoactinomycin-D. The results were expressed in median (minimum to maximum) MSCs and compared between preterm and term samples. A total of 12 UCB samples (32-40 weeks) were obtained, 10 of which demonstrated MSCs, accounting for 0.0174% (0-14.7%) of the viable UCB mononuclear cells. MSCs comprised 0.148% (0.0006-1.59%) and 0.116% (0-14.7%) of the viable UCB mononuclear cells in the term (n = 5), 38.4 ± 1.3 weeks, and preterm (n = 7) samples, 34.6 ± 1.1, respectively, p = 0.17. There was an overall median of 96 (0-39,574) MSCs. There was no difference in the median numbers of MSCs identified between term and preterm UCB samples, 3384 (23-6042) and 36 (0-39,574), respectively, p = 0.12. Mesenchymal stem cells were identified and quantified in 5 of 7 preterm and all 5 term UCB 3-mL samples obtained after delayed cord clamping.

The A328 V/E (rs2887147) polymorphisms in human tryptophan hydroxylase 2 compromise enzyme activity.

Human tryptophan hydroxylase 2 (hTPH2) is the rate-limiting enzyme for serotonin biosynthesis in the brain. A number of naturally-occurring single nucleotide polymorphisms (SNPs) have been reported for hTPH2. We investigated the activity and kinetic characteristics of the most common missense polymorphism rs2887147 (A328 V/E; 0.92% allelic frequency for the two different reported SNPs at the same site) using bacterially expressed hTPH2. The recombinant full-length enzyme A328E had no measurable enzyme activity, but A328V displayed decreased enzyme activity (Vmax). A328V also displayed substrate inhibition and decreased stability compared to the wild-type enzyme. By contrast, in constructs lacking the N-terminal 150 amino acid regulatory domain, the A328V substitution had no effect; that is, there was no substrate inhibition, enzyme stabilities (for wild-type and A328V) were dramatically increased, and Vmax values were not different (while the A328E variant remained inactive). These findings, in combination with molecular modeling, suggest that substitutions at A328 affect catalytic activity by altering the conformational freedom of the regulatory domain. The reduced activity and substrate inhibition resulting from these polymorphisms may ultimately reduce serotonin synthesis and contribute to behavioral perturbations, emotional stress, and eating disorders.

Cannabinoids as Potential Cancer Therapeutics: The Concentration Conundrum.

Background: Studies have reported that cannabinoids, in particular Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), significantly reduce cancer cell viability in vitro. Unfortunately, treatment conditions vary significantly across reports. In particular, a majority of reports utilize conditions with reduced serum concentrations (0-3%) that may compromise the growth of the cells themselves, as well as the observed results. Objectives: This study was designed to test the hypothesis that, based on their known protein binding characteristics, cannabinoids would be less effective in the presence of fetal bovine serum (FBS). Moreover, we wished to determine if the treatments served to be cytotoxic or cytostatic under these conditions. Methods: Six cancer cell lines, representing two independent lines of three different types of cancer (glioblastoma, melanoma, and colorectal cancer [CRC]), were treated with 10 µM pure Δ9-THC, CBD, KM-233, and HU-331 for 48 h (in the presence or absence of FBS). Cell viability was measured with the MTT assay. Dose-response curves were then generated comparing the potencies of the four cannabinoids under the same conditions. Results: We found that serum-free medium alone produces cell cycle arrest for CRC cells and slows cell growth for the other cancer types. The antineoplastic effects of three of the four cannabinoids (Δ9-THC, CBD, and KM-233) increase when serum is omitted from the media. In addition, dose-response curves for these drugs demonstrated lower IC50 values for serum-free media compared with the media with 10% serum in all cell lines. The fourth compound, HU-331, was equally effective under both conditions. A further confound we observed is that omission of serum produces dramatic binding of Δ9-THC and CBD to plastic. Conclusions: Treatment of cancer cells in the absence of FBS appears to enhance the potency of cannabinoids. However, omission of FBS itself compromises cell growth and represents a less physiological condition. Given the knowledge that cannabinoids are 90-95% protein bound and have well-known affinities for plastic, it may be ill-advised to treat cells under conditions where the cells are not growing optimally and where known concentrations cannot be assumed (i.e., FBS-free conditions).

A retrospective chart review to identify perinatal factors associated with food allergies.

BACKGROUND: Gut flora are important immunomodulators that may be disrupted in individuals with atopic conditions. Probiotic bacteria have been suggested as therapeutic modalities to mitigate or prevent food allergic manifestations. We wished to investigate whether perinatal factors known to disrupt gut flora increase the risk of IgE-mediated food allergies. METHODS: Birth records obtained from 192 healthy children and 99 children diagnosed with food allergies were reviewed retrospectively. Data pertaining to delivery method, perinatal antibiotic exposure, neonatal nursery environment, and maternal variables were recorded. Logistic regression analysis was used to assess the association between variables of interest and subsequent food allergy diagnosis. RESULTS: Retrospective investigation did not find perinatal antibiotics, NICU admission, or cesarean section to be associated with increased risk of food allergy diagnosis. However, associations between food allergy diagnosis and male gender (66 vs. 33; p=0.02) were apparent in this cohort. Additionally, increasing maternal age at delivery was significantly associated with food allergy diagnosis during childhood (OR, 1.05; 95% CI, 1.017 to 1.105; p=0.005). CONCLUSIONS: Gut flora are potent immunomodulators, but their overall contribution to immune maturation remains to be elucidated. Additional understanding of the interplay between immunologic, genetic, and environmental factors underlying food allergy development need to be clarified before probiotic therapeutic interventions can routinely be recommended for prevention or mitigation of food allergies. Such interventions may be well-suited in male infants and in infants born to older mothers.

Neural regeneration therapies for Alzheimer's and Parkinson's disease-related disorders.

Neurodegenerative diseases are devastating mental illnesses without a cure. Alzheimer's disease (AD) characterized by memory loss, multiple cognitive impairments, and changes in personality and behavior. Although tremendous progress has made in understanding the basic biology in disease processes in AD and PD, we still do not have early detectable biomarkers for these diseases. Just in the United States alone, federal and nonfederal funding agencies have spent billions of dollars on clinical trials aimed at finding drugs, but we still do not have a drug or an agent that can slow the AD or PD disease process. One primary reason for this disappointing result may be that the clinical trials enroll patients with AD or PD at advances stages. Although many drugs and agents are tested preclinical and are promising, in human clinical trials, they are mostly ineffective in slowing disease progression. One therapy that has been promising is 'stem cell therapy' based on cell culture and pre-clinical studies. In the few clinical studies that have investigated therapies in clinical trials with AD and PD patients at stage I. The therapies, such as stem cell transplantation - appear to delay the symptoms in AD and PD. The purpose of this article is to describe clinical trials using 1) stem cell transplantation methods in AD and PD mouse models and 2) regenerative medicine in AD and PD mouse models, and 3) the current status of investigating preclinical stem cell transplantation in patients with AD and PD.

Cannabidiol (CBD) Oil Does Not Display an Entourage Effect in Reducing Cancer Cell Viability in vitro.

INTRODUCTION: Several studies have found that cannabinoids, particularly delta-9-tetrahydrocannabinol and cannabidiol (CBD), have the ability to reduce cancer cell viability. An ongoing debate regarding the use of medical Cannabis revolves around the effectiveness of pure compounds versus intact plant material for treatment. Proponents for the use of intact plant material or botanical extracts argue that there is a synergistic effect between the different cannabinoids, terpenoids, and flavonoids; this is commonly referred to as the "entourage effect." Our study was designed to test the validity of the proposed entourage effect in a narrow application using a cancer cell viability model. MATERIALS AND METHODS: Six cancer cell lines, from 3 different types of human cancer were treated with 10 µM pure CBD or 10 µM CBD from hemp (Cannabis sativa) oil (obtained from 3 different commercial sources) for 48 h, and cell viability was measured with the MTS assay. Dose-response curves were then performed to compare the potencies of pure CBD to CBD oils. CBD concentrations were independently confirmed in the commercial oils, and cannabinoid and terpene composition were also compared. RESULTS: CBD (10 µM) was able to reduce cell viability in 3 of the 6 cell lines tested, and this was found to be cell line specific and not specific to select cancers. None of the CBD oils tested were able to reduce viability to a greater extent than that of pure CBD. Additionally, dose-response curves found lower IC50 values for pure CBD compared to the most potent CBD oil tested. Interestingly, some oils actually appeared to protect cancer cells from the effects of CBD. CONCLUSIONS: We found that pure CBD was as potent or more potent at reducing cancer cell viability as the most potent oil tested, suggesting that there is no "entourage" effect under these specific in vitro conditions.

The V81M variant of tyrosine hydroxylase is associated with more severe freezing of gait in Parkinson's disease.

INTRODUCTION: Many of the symptoms and signs of Parkinson's disease (PD) arise from the death of midbrain dopamine neurons that utilize tyrosine hydroxylase (TH) as the rate-limiting enzyme in catecholamine biosynthesis. METHODS: We investigated whether the presence of a common TH polymorphism affects the clinical outcomes in 101 PD subjects. We further examined the effect of this polymorphism on the purified recombinant enzyme. RESULTS: PD subjects homozygous for the common V81M polymorphism, have higher overall freezing of gait scores after controlling for disease duration, although this polymorphism does not associate with the occurrence of PD or FOG. In vitro functional assays on pure recombinant wild type TH and V81M TH revealed that the Km of the mutant enzyme for tyrosine was twice that of the wild-type. This polymorphism, however, did not change the stability of the enzyme, nor did it affect the Vmax or Km for the co-substrate BH4. CONCLUSION: The data suggest that presence of a homozygous V81M polymorphism is associated with more severe FOG, possibly due to lower catecholamine synthetic capacity. Further studies are warranted to investigate the role of subtle changes in catecholamine availability in the development of FOG.

Identification and characterization of victorin sensitivity in Arabidopsis thaliana.

Cochliobolus victoriae is a necrotrophic fungus that produces a host-selective toxin called victorin. Victorin is considered to be host selective because it has been known to affect only certain allohexaploid oat cultivars containing the dominant Vb gene. Oat cultivars containing Vb are also the only genotypes susceptible to C. victoriae. Assays were developed to screen the "nonhost" plant of C. victoriae, Arabidopsis thaliana, for victorin sensitivity. Sensitivity to victorin was identified in six of 433 bulk populations of Arabidopsis. In crosses of Col-4 (victorin-insensitive) x victorin-sensitive Arabidopsis ecotypes, victorin sensitivity segregated as a single dominant locus, as it does in oats. This Arabidopsis locus was designated LOV, for locus orchestrating victorin effects. Allelism tests indicate that LOV loci are allelic or closely linked in all six victorin-sensitive ecotypes identified. LOV was localized to the north arm of Arabidopsis thaliana chromosome I. The victorin-sensitive Arabidopsis line LOV1 but not the victorin-insensitive line Col-4 was susceptible to C. victoriae infection. Consequently, the LOV gene appears to be a genetically dominant, disease susceptibility gene.

TPH2 in the ventral tegmental area of the male rat brain.

This study surveyed the distribution of tryptophan hydroxylase 2 (TPH2) mRNA, protein, and enzymatic activity throughout the male Sprague-Dawley rat brain. TPH2 is the genetic isoform of TPH that catalyzes the rate-limiting step in serotonin biosynthesis within the central nervous system. Although cell bodies of serotonergic neurons are located mainly in the raphe, serotonin-containing axons innervate many regions of the brain. In the present study, we assessed the levels of mRNA, protein expression, and enzyme activity of TPH2 in the rat raphe, ventral tegmental area (VTA), substantia nigra, hippocampus, cerebellum, dorsal striatum, nucleus accumbens, amygdala, and medial prefrontal cortex to more fully understand the distribution of this enzyme throughout the central nervous system. The pineal gland was used as a control tissue that expresses TPH1 (the peripheral enzyme), but not TPH2. As expected, the raphe showed the highest brain TPH2 activity and protein expression. In the contrast to other reports, however, the VTA followed the raphe as the region with the second-highest amount of TPH2 activity, mRNA and protein expression. There were significantly lower TPH activities and levels of TPH2 protein in the other regions. In addition, TPH2 immunocytochemistry demonstrated the presence of TPH-positive cell bodies within the VTA. The results of this study indicate that TPH2 and serotonergic signaling may play an important role in the mesolimbic/mesocortical reward pathway.

Propagation of undifferentiated human embryonic stem cells with nano-liposomal ceramide.

Human embryonic stem (hES) cells, located on the periphery of the colonies, express the neuroectodermal markers nestin and Tuj1, suggesting a prematurely differentiated subgroup of cells. Here, we report that ceramide, a bioactive sphingolipid, selectively eliminates hES cells differentially expressing nestin and Tuj1. In contrast, undifferentiated cells are resistant to the apoptotic effects of ceramide. Ceramide-resistant hES cells express higher levels of the messenger RNA for ceramide-metabolizing enzymes that convert ceramide into pro-mitogenic metabolites. Based on these findings, we conducted long-term studies to determine whether liposomal ceramide can be used to maintain undifferentiated hES cells free of feeder cells. We continuously cultured hES cells on matrigel for 4 months with liposomal ceramide in a feeder cell-free system. Human ES cells treated with liposomal ceramide maintained their pluripotent state as determined by in vivo and in vitro differentiation studies and contained no chromosomal abnormalities. In conclusion, our findings suggest that exposure to ceramide provides a viable strategy to prevent premature hES cell differentiation and to maintain pluripotent stem cell populations in the absence of feeder cells.

Plasma biomarkers in pediatric patients undergoing cardiopulmonary bypass.

It is critical to identify at-risk patients and minimize the deleterious effects of cardiopulmonary bypass (CPB) procedures in pediatric populations. The present study screened the plasma proteome of pediatric patients undergoing CPB procedures to identify potential clinical biomarkers related to tissue damage, inflammation, or other pathologies. Blood samples were collected at five different time points from 10 children undergoing a CPB procedure. Plasma was isolated and analyzed using two-dimensional differential in-gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry. Levels of differentially regulated proteins identified by two-dimensional differential in-gel electrophoresis, and related proteins were then measured in all time points and patients. As well, associated small molecules and ions were measured. The present study identified 13 proteins and protein isoforms altered in expression, including hemopexin, ceruloplasmin, inter-alpha inhibitor H4, and alpha-2-macroglobulin. Immunoblot analysis revealed significant decreases in each of these proteins during the CPB procedure. Significant changes in the levels of copper, iron, Hb, epinephrine, norepinephrine, and serotonin were observed. The potential markers of pathology (inflammation, oxidative stress) identified during this preliminary study may illuminate opportunities for preventative measures and/or treatments during and following CPB procedures in pediatric patients.

Functional domains of human tryptophan hydroxylase 2 (hTPH2).

Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in serotonin biosynthesis. A novel gene, termed TPH2, has recently been described. This gene is preferentially expressed in the central nervous system, while the original TPH1 is the peripheral gene. We have expressed human tryptophan hydroxylase 2 (hTPH2) and two deletion mutants (NDelta150 and NDelta150/CDelta24) using isopropyl beta-D-thiogalactopyranoside-free autoinduction in Escherichia coli. This expression system produced active wild type TPH2 with relatively low solubility. The solubility was increased for mutants lacking the NH(2)-terminal regulatory domain. The solubility of hTPH2, NDelta150, and NDelta150/CDelta24 are 6.9, 62, and 97.5%, respectively. Removal of the regulatory domain also produced a more than 6-fold increase in enzyme stability (t((1/2)) at 37 degrees C). The wild type hTPH2, like other members of the aromatic amino acid hydroxylase superfamily, exists as a homotetramer (236 kDa on size exclusion chromatography). Similarly, NDelta150 also migrates as a tetramer (168 kDa). In contrast, removal of the NH(2)-terminal domain and the COOH-terminal, putative leucine zipper tetramerization domain produces monomeric enzyme (39 kDa). Interestingly, removal of the NH(2)-terminal regulatory domain did not affect the Michaelis constants for either substrate but did increase V(max) values. These data identify the NH(2)-terminal regulatory domain as the source of hTPH2 instability and reduced solubility.