Long-term effects of amphetamine neurotoxicity on tyrosine hydroxylase mRNA and protein in aged rats

ABSTRACT Four injections (intraperitoneal) of 3 mg/kg amphetamine (2 hr apart) produced pronounced hyperthermia and sustained decreases in dopamine levels and tyrosine hydroxylase (TH) protein levels in the striatum of 15-month-old male rats. A partial recovery of striatal dopamine levels was observed at 4 months after amphetamine. In contrast, TH mRNA and TH protein levels in the midbrain were unaffected at all time points tested up to 4 months after amphetamine treatment. The number of THimmunopositive cells in the midbrain was also unchanged at 4 months after amphetamine, even though the number of THpositive axons in the striatum remained dramatically decreased at this time point. Interestingly, TH-immunopositive cell bodies were observed 4 months after amphetamine in the lateral caudate/putamen, defined anteriorly by the genu of the corpus collosum and posteriorly by the junction of the anterior commissures; these striatal TH-positive cells were not observed in saline-or amphetamine-treated rats that did not become hyperthermic. In addition, low levels (orders of magnitude lower than that present in the midbrain) of TH mRNA were detected using reverse transcription-polymerase chain reaction in the striatum of these amphetamine-treated rats. Our results suggest that even though there is a partial recovery of striatal dopamine levels, which occurs within 4 months after amphetamine treatment, this recovery is not associated with increased TH gene expression in the midbrain. Furthermore, new THpositive cells are generated in the striatum at this 4-month time point

Species Association of Hepatitis B Virus (HBV) in Non-Human Apes; Evidence for Recombination between Gorilla and Chimpanzee Variants

Hepatitis B virus (HBV) infections are widely distributed in humans, infecting approximately one third of the world's population. HBV variants have also been detected and genetically characterised from Old World apes; Gorilla gorilla (gorilla), Pan troglodytes (chimpanzee), Pongo pygmaeus (orang-utan), Nomascus nastusus and Hylobates pileatus (gibbons) and from the New World monkey, Lagothrix lagotricha (woolly monkey). To investigate species-specificity and potential for cross species transmission of HBV between sympatric species of apes (such as gorillas and chimpanzees in Central Africa) or between humans and chimpanzees or gorillas, variants of HBV infecting captive wild-born non-human primates were genetically characterised. 9 of 62 chimpanzees (11.3%) and two from 11 gorillas (18%) were HBV-infected (15% combined frequency), while other Old world monkey species were negative. Complete genome sequences were obtained from six of the infected chimpanzee and both gorillas; those from P. t .ellioti grouped with previously characterised variants from this subspecies. However, variants recovered from P. t. troglodytes HBV variants also grouped within this clade, indicative of transmission between sub-species, forming a paraphyletic clade. The two gorilla viruses were phylogenetically distinct from chimpanzee and human variants although one showed evidence for a recombination event with a P.t.e.-derived HBV variant in the partial X and core gene region. Both of these observations provide evidence for circulation of HBV between different species and sub-species of non-human primates, a conclusion that differs from the hypothesis if of strict host specificity of HBV genotypes

THE JOURNAL OF PHARMACOLOGY ANt) EXPERIMENTAL THERAPEUTICS K Channel and Adenylate Cyclase Involvement in Regulation of Ca2 evoked Release of [3H]Dopamine from Synaptosomes'

ABSTRACT In superfused striatal synaptosomes, previously unexposed to Ca2 during isolation and superfusion

An Iterative Leave-One-Out Approach to Outlier Detection in RNA-Seq Data.

The discrete data structure and large sequencing depth of RNA sequencing (RNA-seq) experiments can often generate outlier read counts in one or more RNA samples within a homogeneous group. Thus, how to identify and manage outlier observations in RNA-seq data is an emerging topic of interest. One of the main objectives in these research efforts is to develop statistical methodology that effectively balances the impact of outlier observations and achieves maximal power for statistical testing. To reach that goal, strengthening the accuracy of outlier detection is an important precursor. Current outlier detection algorithms for RNA-seq data are executed within a testing framework and may be sensitive to sparse data and heavy-tailed distributions. Therefore, we propose a univariate algorithm that utilizes a probabilistic approach to measure the deviation between an observation and the distribution generating the remaining data and implement it within in an iterative leave-one-out design strategy. Analyses of real and simulated RNA-seq data show that the proposed methodology has higher outlier detection rates for both non-normalized and normalized negative binomial distributed data

Multi-class computational evolution: development, benchmark evaluation and application to RNA-Seq biomarker discovery

Abstract Background A computational evolution system (CES) is a knowledge discovery engine that can identify subtle, synergistic relationships in large datasets. Pareto optimization allows CESs to balance accuracy with model complexity when evolving classifiers. Using Pareto optimization, a CES is able to identify a very small number of features while maintaining high classification accuracy. A CES can be designed for various types of data, and the user can exploit expert knowledge about the classification problem in order to improve discrimination between classes. These characteristics give CES an advantage over other classification and feature selection algorithms, particularly when the goal is to identify a small number of highly relevant, non-redundant biomarkers. Previously, CESs have been developed only for binary class datasets. In this study, we developed a multi-class CES. Results The multi-class CES was compared to three common feature selection and classification algorithms: support vector machine (SVM), random k-nearest neighbor (RKNN), and random forest (RF). The algorithms were evaluated on three distinct multi-class RNA sequencing datasets. The comparison criteria were run-time, classification accuracy, number of selected features, and stability of selected feature set (as measured by the Tanimoto distance). The performance of each algorithm was data-dependent. CES performed best on the dataset with the smallest sample size, indicating that CES has a unique advantage since the accuracy of most classification methods suffer when sample size is small. Conclusion The multi-class extension of CES increases the appeal of its application to complex, multi-class datasets in order to identify important biomarkers and features

Plasma Levels of Parent Compound and Metabolites after Doses of Either d-Fenfluramine or d-3,4-Methylenedioxymethamphetamine (MDMA) that Produce Long-Term Serotonergic Alterations

Plasma levels of parent compounds and metabolites were determined in adult rhesus monkeys after doses of either 5 mg/kg d-fenfluramine (FEN) or 10 mg/kg d-3, 4-methylenedioxymethamphetamine (MDMA) i.m. twice daily for four consecutive days. These treatment regimens have been previously shown to produce long-term serotonin (5-HT) depletions. Peak plasma levels of 2.0±0.4 μM FEN were reached within 40 min after the first dose of FEN, and then declined rapidly, while peak plasma levels (0.4±0.1 μM) of the metabolite norfenfluramine (NFEN) were not reached until 6 h after dosing. After the seventh (next to last) dose of FEN, peak plasma levels of FEN were 35% greater than after the first dose while peak NFEN-levels were 500% greater. The t 1/2 for FEN was 2.6±0.3 h after the first dose and 3.2±0.2 h after the seventh. The estimated t 1/2 for NFEN was more than 37.6±20.5 h. Peak plasma levels of 9.5±2.5 μM MDMA were reached within 20 min after the first dose of MDMA, and then declined rapidly, while peak plasma levels (0.9±0.2 μM) of the metabolite 3,4-methylenedioxyamphetamine (MDA) were not reached until 3–6 h after dosing. After the seventh (next to last) dose of MDMA, peak plasma levels of MDMA were 30% greater than the first dose while peak MDA levels were elevated over 200%. The t 1/2 for MDMA was 2.8±0.4 h after the first and 3.9±1.1 h after the seventh dose. The estimated t 1/2 for MDA was about 8.3±1.0 h. Variability in plasma levels of MDMA and MDA between subjects was much greater than that for FEN and NFEN. This variability in MDMA and MDA exposure levels may have lead to variability in the subsequent disruption of some behaviors seen in these same subjects. There were 80% reductions in the plasma membrane-associated 5-HT transporters 6 months after either the FEN or MDMA dosing regimen indicating that both treatments produced long-term serotonergic effects

Identification of whole blood mRNA and microRNA biomarkers of tissue damage and immune function resulting from amphetamine exposure or heat stroke in adult male rats.

This work extends the understanding of how toxic exposures to amphetamine (AMPH) adversely affect the immune system and lead to tissue damage. Importantly, it determines which effects of AMPH are and are not due to pronounced hyperthermia. Whole blood messenger RNA (mRNA) and whole blood and serum microRNA (miRNA) transcripts were identified in adult male Sprague-Dawley rats after exposure to toxic AMPH under normothermic conditions, AMPH when it produces pronounced hyperthermia, or environmentally-induced hyperthermia (EIH). mRNA transcripts with large increases in fold-change in treated relative to control rats and very low expression in the control group were a rich source of organ-specific transcripts in blood. When severe hyperthermia was produced by either EIH or AMPH, significant increases in circulating organ-specific transcripts for liver (Alb, Fbg, F2), pancreas (Spink1), bronchi/lungs (F3, Cyp4b1), bone marrow (Np4, RatNP-3b), and kidney (Cesl1, Slc22a8) were observed. Liver damage was suggested also by increased miR-122 levels in the serum. Increases in muscle/heart-enriched transcripts were produced by AMPH even in the absence of hyperthermia. Expression increases in immune-related transcripts, particularly Cd14 and Vcan, indicate that AMPH can activate the innate immune system in the absence of hyperthermia. Most transcripts specific for T-cells decreased 50-70% after AMPH exposure or EIH, with the noted exception of Ccr5 and Chst12. This is probably due to T-cells leaving the circulation and down-regulation of these genes. Transcript changes specific for B-cells or B-lymphoblasts in the AMPH and EIH groups ranged widely from decreasing ≈ 40% (Cd19, Cd180) to increasing 30 to 100% (Tk1, Ahsa1) to increasing ≥500% (Stip1, Ackr3). The marked increases in Ccr2, Ccr5, Pld1, and Ackr3 produced by either AMPH or EIH observed in vivo provide further insight into the initial immune system alterations that result from methamphetamine and AMPH abuse and could modify risk for HIV and other viral infections

Evaluating the Stability of RNA-Seq Transcriptome Profiles and Drug-Induced Immune-Related Expression Changes in Whole Blood.

Methods were developed to evaluate the stability of rat whole blood expression obtained from RNA sequencing (RNA-seq) and assess changes in whole blood transcriptome profiles in experiments replicated over time. Expression was measured in globin-depleted RNA extracted from the whole blood of Sprague-Dawley rats, given either saline (control) or neurotoxic doses of amphetamine (AMPH). The experiment was repeated four times (paired control and AMPH groups) over a 2-year span. The transcriptome of the control and AMPH-treated groups was evaluated on: 1) transcript levels for ribosomal protein subunits; 2) relative expression of immune-related genes; 3) stability of the control transcriptome over 2 years; and 4) stability of the effects of AMPH on immune-related genes over 2 years. All, except one, of the 70 genes that encode the 80s ribosome had levels that ranked in the top 5% of all mean expression levels. Deviations in sequencing performance led to significant changes in the ribosomal transcripts. The overall expression profile of immune-related genes and genes specific to monocytes, T-cells or B-cells were well represented and consistent within treatment groups. There were no differences between the levels of ribosomal transcripts in time-matched control and AMPH groups but significant differences in the expression of immune-related genes between control and AMPH groups. AMPH significantly increased expression of some genes related to monocytes but down-regulated those specific to T-cells. These changes were partially due to changes in the two types of leukocytes present in blood, which indicate an activation of the innate immune system by AMPH. Thus, the stability of RNA-seq whole blood transcriptome can be verified by assessing ribosomal protein subunits and immune-related gene expression. Such stability enables the pooling of samples from replicate experiments to carry out differential expression analysis with acceptable power