Artifacts associated with the measurement of oxidized DNA bases.

In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, the situation is confusing because variations that may be as large as two orders of magnitude have been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings clearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and -32P--postlabeling may lead to a significant overestimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 10(4) normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7, 8-dihydroadenine,5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolysate prior to the derivatization reaction prevents artefactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2;-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropriate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cell gel electrophoresis (comet) assay

The Advertisement Value of Transformational & Informational Appeal on Company Facebook Pages

The advertisement value of Facebook is an under-developed area of social media research. Transformational and informational advertising appeal has yet to be studied as it relates to social media. This paper utilizes established classification and measurement scales from marketing literature to classify companies and their Facebook posts and measures the advertisement value of these posts. The study uses a sample of 100 companies from the 2015 Fortune 500 list. Results indicate that posts with transformational appeal are more engaging to the consumer than informational appeal; however, posts with informational appeal have greater advertisement value for the company. The results also indicate there is no relationship between type of company and type of appeal used by companies

Palaeoceanographic implications of new and revised bio-chronostratigraphic constraints from the Profitis Ilias Unit (Rhodes, Greece)

Middle Oxfordian-early Kimmeridgian radiolaria, extracted from the top of radiolarites of Profitis Ilias unit (Rhodes island), suggest that the latter are essentially Middle Jurassic in age and the overlying siliceous shales Late Jurassic. The previously identified Calpionellid horizon at the top of Profitis Ilias siliceous marls is now regarded as early Valanginian in age. The above chronostratigraphic constraints allow tentative correlations to be made between Profitis Ilias and Pindos-Olonos sedimentary units. Finally, the palaeoceanographic significance of the studied series in Rhodes and potentially similar pelagic sequences in the Marmaris area of Turkey are discussed

Methamphetamine Induces TET1- and TET3-Dependent DNA Hydroxymethylation of Crh and Avp Genes in the Rat Nucleus Accumbens

Methamphetamine (METH) addiction is a biopsychosocial disorder that is accompanied by multiple relapses even after prolonged abstinence, suggesting the possibilities of long-lasting maladaptive epigenetic changes in the brain. Here, we show that METH administration produced time-dependent increases in the expression of corticotropin-releasing hormone (Crh/Crf), arginine vasopressin (Avp), and cocaine- and amphetamine-regulated transcript prepropeptide (Cartpt) mRNAs in the rat nucleus accumbens (NAc). Chromatin immunoprecipitation (ChIP) assays revealed that METH increased the abundance of phosphorylated CREB (pCREB) at the promoter of Cartpt but not at Avp or Crh DNA sequences. In contrast, METH produced DNA hypomethylation at sites near the Crh transcription start site (TSS) and at intragenic Avp sequences. METH also increased DNA hydroxymethylation at the Crh TSS and at intragenic Avp sites. In addition, METH increased the protein expression of ten-eleven-translocation enzymes that catalyze DNA hydroxymethylation. Importantly, METH increased TET1 binding at the Crh promoter and increased TET3 binding at Avp intragenic regions. We further tested the role of TET enzymes in METH-induced changes in gene expression by using the TET inhibitor, 1,5-isoquinolinediol (IQD), and found that IQD blocked METH-induced increases in Crh and Avp mRNA expression. Together, these results indicate that METH produced changes in neuropeptide transcription by both activation of the cAMP/CREB pathway and stimulation of TET-dependent DNA hydroxymethylation. These results provide molecular evidence for epigenetic controls of METH-induced changes in the expression of neuropeptides.Fil: Jayanthi, Subramaniam. National Institutes of Health; Estados UnidosFil: Gonzalez, Betina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: McCoy, Michael T.. National Institutes of Health; Estados UnidosFil: Ladenheim, Bruce. National Institutes of Health; Estados UnidosFil: Bisagno, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Farmacológicas. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Investigaciones Farmacológicas; ArgentinaFil: Cadet, Jean Lud. National Institutes of Health; Estados Unido

Methamphetamine Induces Dopamine D1 Receptor-Dependent Endoplasmic Reticulum Stress-Related Molecular Events in the Rat Striatum

Methamphetamine (METH) is an illicit toxic psychostimulant which is widely abused. Its toxic effects depend on the release of excessive levels of dopamine (DA) that activates striatal DA receptors. Inhibition of DA-mediated neurotransmission by the DA D1 receptor antagonist, SCH23390, protects against METH-induced neuronal apoptosis. The initial purpose of the present study was to investigate, using microarray analyses, the influence of SCH23390 on transcriptional responses in the rat striatum caused by a single METH injection at 2 and 4 hours after drug administration. We identified 545 out of a total of 22,227 genes as METH-responsive. These include genes which are involved in apoptotic pathways, endoplasmic reticulum (ER) stress, and in transcription regulation, among others. Of these, a total of 172 genes showed SCH23390-induced inhibition of METH-mediated changes. Among these SCH23390-responsive genes were several genes that are regulated during ER stress, namely ATF3, HSP27, Hmox1, HSP40, and CHOP/Gadd153. The secondary goal of the study was to investigate the role of DA D1 receptor stimulation on the expression of genes that participate in ER stress-mediated molecular events. We thus used quantitative PCR to confirm changes in the METH-responsive ER genes identified by the microarray analyses. We also measured the expression of these genes and of ATF4, ATF6, BiP/GRP78, and of GADD34 over a more extended time course. SCH23390 attenuated or blocked METH-induced increases in the expression of the majority of these genes. Western blot analysis revealed METH-induced increases in the expression of the antioxidant protein, Hmox1, which lasted for about 24 hours after the METH injection. Additionally, METH caused DA D1 receptor-dependent transit of the Hmox1 regulator protein, Nrf2, from cytosolic into nuclear fractions where the protein exerts its regulatory functions. When taken together, these findings indicate that SCH23390 can provide protection against neuronal apoptosis by inhibiting METH-mediated DA D1 receptor-mediated ER stress in the rat striatum. Our data also suggest that METH-induced toxicity might be a useful model to dissect molecular mechanisms involved in ER stress-dependent events in the rodent brain

Compulsive methamphetamine taking in the presence of punishment is associated with increased oxytocin expression in the nucleus accumbens of rats

Methamphetamine addiction is mimicked in rats that self-administer the drug. However, these self-administration (SA) models do not include adverse consequences that are necessary to reach a diagnosis of addiction in humans. Herein, we measured genome-wide transcriptional consequences of methamphetamine SA and footshocks in the rat brain. We trained rats to self-administer methamphetamine for 20 days. Thereafter, lever-presses for methamphetamine were punished by mild footshocks for 5 days. Response-contingent punishment significantly reduced methamphetamine taking in some rats (shock-sensitive, SS) but not in others (shock-resistant, SR). Rats also underwent extinction test at one day and 30 days after the last shock session. Rats were euthanized one day after the second extinction test and the nucleus accumbens (NAc) and dorsal striatum were collected to measure gene expression with microarray analysis. In the NAc, there were changes in the expression of 13 genes in the SRvsControl and 9 genes in the SRvsSS comparison. In the striatum, there were 9 (6 up, 3 down) affected genes in the SRvsSS comparison. Among the upregulated genes was oxytocin in the NAc and CARTpt in the striatum of SR rats. These observations support a regional role of neuropeptides in the brain after a long withdrawal interval when animals show incubation of methamphetamine craving

Chronic Methamphetamine Administration Causes Differential Regulation of Transcription Factors in the Rat Midbrain

Methamphetamine (METH) is an addictive and neurotoxic psychostimulant widely abused in the USA and throughout the world. When administered in large doses, METH can cause depletion of striatal dopamine terminals, with preservation of midbrain dopaminergic neurons. Because alterations in the expression of transcription factors that regulate the development of dopaminergic neurons might be involved in protecting these neurons after toxic insults, we tested the possibility that their expression might be affected by toxic doses of METH in the adult brain. Male Sprague-Dawley rats pretreated with saline or increasing doses of METH were challenged with toxic doses of the drug and euthanized two weeks later. Animals that received toxic METH challenges showed decreases in dopamine levels and reductions in tyrosine hydroxylase protein concentration in the striatum. METH pretreatment protected against loss of striatal dopamine and tyrosine hydroxylase. In contrast, METH challenges caused decreases in dopamine transporters in both saline- and METH-pretreated animals. Interestingly, METH challenges elicited increases in dopamine transporter mRNA levels in the midbrain in the presence but not in the absence of METH pretreatment. Moreover, toxic METH doses caused decreases in the expression of the dopamine developmental factors, Shh, Lmx1b, and Nurr1, but not in the levels of Otx2 and Pitx3, in saline-pretreated rats. METH pretreatment followed by METH challenges also decreased Nurr1 but increased Otx2 and Pitx3 expression in the midbrain. These findings suggest that, in adult animals, toxic doses of METH can differentially influence the expression of transcription factors involved in the developmental regulation of dopamine neurons. The combined increases in Otx2 and Pitx3 expression after METH preconditioning might represent, in part, some of the mechanisms that served to protect against METH-induced striatal dopamine depletion observed after METH preconditioning

Addressing Health Literacy in Patient Decision Aids:An Update from the International Patient Decision Aid Standards

BACKGROUND: There is increasing recognition of the importance of addressing health literacy in patient decision aid (PtDA) development. PURPOSE: An updated review as part of IPDAS 2.0 examined the extent to which PtDAs are designed to meet the needs of low health literacy/disadvantaged populations. DATA SOURCES: Reference list of Cochrane review of randomised controlled trials (RCTs) of PtDAs (2014, 2017 and upcoming 2021 versions). STUDY SELECTION: RCTs that assessed the impact of PtDAs on low health literacy or other disadvantaged groups (i.e. ≥50% participants from disadvantaged groups and/or subgroup analysis in disadvantaged group/s). DATA EXTRACTION: Two researchers independently extracted data into a standardized form including PtDA development and evaluation details. We searched online repositories and emailed authors to access PtDAs to verify reading level, understandability and actionability. DATA SYNTHESIS: Twenty-five out of 213 RCTs met inclusion criteria illustrating that only 12% of studies addressed the needs of low health literacy or other disadvantaged groups. Reading age was calculated in 8/25 studies (33%), which is recommended in previous IPDAS guidelines. We accessed and independently assessed 11 PtDAs. None were written at 6(th) grade level or below. Ten PtDAs met the recommended threshold for understandability, but only 5 met the recommended threshold for actionability. We also conducted a post-hoc subgroup meta-analysis and found that knowledge improvements after receiving a PtDA were greater in studies that reported using strategies to reduce cognitive demand in PtDA development compared to studies that did not (Chi(2)=14.11, p=0.0002, I(2)=92.9%). LIMITATIONS: We were unable to access 13 out of 24 PtDAs. CONCLUSIONS: Greater attention to health literacy and disadvantaged populations is needed in the field of PtDAs to ensure equity in decision support

Decontamination of MDA Reagents for Single Cell Whole Genome Amplification

Single cell genomics is a powerful and increasingly popular tool for studying the genetic make-up of uncultured microbes. A key challenge for successful single cell sequencing and analysis is the removal of exogenous DNA from whole genome amplification reagents. We found that UV irradiation of the multiple displacement amplification (MDA) reagents, including the Phi29 polymerase and random hexamer primers, effectively eliminates the amplification of contaminating DNA. The methodology is quick, simple, and highly effective, thus significantly improving whole genome amplification from single cells

Methamphetamine Self-Administration Is Associated with Persistent Biochemical Alterations in Striatal and Cortical Dopaminergic Terminals in the Rat

Methamphetamine (meth) is an illicit psychostimulant that is abused throughout the world. Repeated passive injections of the drug given in a single day or over a few days cause significant and long-term depletion of dopamine and serotonin in the mammalian brain. Because meth self-administration may better mimic some aspects of human drug-taking behaviors, we examined to what extent this pattern of drug treatment might also result in damage to monoaminergic systems in the brain. Rats were allowed to intravenously self-administer meth (yoked control rats received vehicle) 15 hours per day for 8 days before being euthanized at either 24 hours or at 7 and 14 days after cessation of drug taking. Meth self-administration by the rats was associated with a progressive escalation of daily drug intake to 14 mg/kg per day. Animals that self-administered meth exhibited dose-dependent decreases in striatal dopamine levels during the period of observation. In addition, there were significant reductions in the levels of striatal dopamine transporter and tyrosine hydroxylase proteins. There were also significant decreases in the levels of dopamine, dopamine transporter, and tyrosine hydroxylase in the cortex. In contrast, meth self-administration caused only transient decreases in norepinephrine and serotonin levels in the two brain regions, with these values returning to normal at seven days after cessation of drug taking. Importantly, meth self-administration was associated with significant dose-dependent increases in glial fibrillary acidic protein in both striatum and cortex, with these changes being of greater magnitude in the striatum. These results suggest that meth self-administration by rats is associated with long-term biochemical changes that are reminiscent of those observed in post-mortem brain tissues of chronic meth abusers