Developing an Analytical Method for Separating and Quantifying RNA Generated in In Vitro Transcription Reactions

The generation of run-off transcripts from in vitro transcription reactions is a useful technique in the study of transcription regulation. There are currently limited ways in which these run-off transcripts can be analyzed, and few that are truly quantitative. Our aim is to establish a method using ion-pair reverse phase high performance liquid chromatography (IP RP HPLC) to analyze RNA transcripts from in vitro transcription reactions. We were able to demonstrate that we could recapitulate the separation of DNA based on size using our IP RP HPLC method. The application of this method was also able to effectively separate RNA based on size in the size range of 281 – 1908 bp. Using a simple in vitro transcription system with T7 RNA polymerase and a linear DNA template with a single promoter, we showed detection of the RNA run-off transcript in the size range demonstrated. We would like to apply this method to more complex in vitro transcription systems and demonstrate the ability to quantify RNA using IP RP HPLC

Quantification of DNA Products Using Ion-Pair Reverse Phase Liquid Chromatography

The transcription of DNA via RNA polymerases is a fundamental process in cellular systems. In eukaryotic cells, we observe transcription in the nucleus (via genomic DNA) as well as in the mitochondria (via mitochondrial DNA). There are many tools available to investigate nuclear transcription; however, few tools exist to study mitochondrial transcription even though the mitochondrial DNA encodes several essential proteins. Recently an in vitro transcription system using purified mitochondrial transcription proteins, including the mitochondrial RNA polymerase, and linear mitochondrial DNA templates has been developed. Quantitative analysis of the DNA templates can be done via ion-pair reverse-phase high performance liquid chromatography (IP-RP HPLC), a high-resolution technique in separating DNA based on size. Using IP-RP HPLC our aim is to assess the lower limits of separation, and our quantification method is based on measuring peak area and the peak height

Non-linear hydrodynamic instability and turbulence in eccentric astrophysical discs with vertical structure

Non-linear evolution of the parametric instability of inertial waves inherent to eccentric discs is studied by way of a new local numerical model. Mode coupling of tidal deformation with the disc eccentricity is known to produce exponentially growing eccentricities at certain mean-motion resonances. However, the details of an efficient saturation mechanism balancing this growth still are not fully understood. This paper develops a local numerical model for an eccentric quasi-axisymmetric shearing box which generalizes the often-used Cartesian shearing box model. The numerical method is an overall second-order well-balanced finite volume method which maintains the stratified and oscillatory steady-state solution by construction. This implementation is employed to study the non-linear outcome of the parametric instability in eccentric discs with vertical structure. Stratification is found to constrain the perturbation energy near the mid-plane and localize the effective region of inertial wave breaking that sources turbulence. A saturated marginally sonic turbulent state results from the non-linear breaking of inertial waves and is subsequently unstable to large-scale axisymmetric zonal flow structures. This resulting limit-cycle behaviour reduces access to the eccentric energy source and prevents substantial transport of angular momentum radially through the disc. Still, the saturation of this parametric instability of inertial waves is shown to damp eccentricity on a time-scale of a thousand orbital periods. It may thus be a promising mechanism for intermittently regaining balance with the exponential growth of eccentricity from the eccentric Lindblad resonances and may also help explain the occurrence of ’bursty’ dynamics such as the superhump phenomenon

Nonlinear hydrodynamic instability and turbulence in eccentric astrophysical discs

The nonlinear evolution of the parametric instability of inertial waves inherent to eccentric discs is studied by way of a new local numerical model. Mode coupling of tidal deformation with the disc eccentricity is known to produce exponentially growing eccentricities at mean-motion resonances, most prominently via the 3:1 eccentric Lindblad resonance in circumstellar binaries. However, the details of an efficient saturation mechanism for this growth are still not fully understood. Linear theory for the parametric instability of inertial waves in eccentric discs has previously been studied by Barker and Ogilvie (2014), the nonlinear evolution of which dictates the transport rates and may help saturate this unmitigated eccentricity growth. This thesis develops a generalised numerical model for an eccentric quasi-axisymmetric shearing box based on the theoretical model developed by Ogilvie and Barker (2014) which itself generalises the often-used local cartesian shearing box model. The numerical method is an overall 2nd order well-balanced finite volume method which maintains the stratified and oscillatory steady state solution to machine precision. This implementation is employed to study the nonlinear outcome of this parametric instability in eccentric discs with vertical structure. Stratification is found to constrain the perturbation energy near the midplane, and localises the effective region of the secondary instability. A saturated marginally sonic turbulent state is then maintained by the breaking of inertial waves cascading energy into smaller scales. The resulting turbulence is quite inefficient at transporting angular momentum through the disc, as might be expected from a restricted axisymmetric instability. Still, the saturation of this parametric instability of inertial waves is shown to damp the eccentricity on the timescale of 50 to 100 orbital periods, and so is a promising mechanism for balancing the exponential growth of eccentricity from the eccentric Lindblad resonances

Cytochromeâ P450â Induced Ordering of Microsomal Membranes Modulates Affinity for Drugs

Although membrane environment is known to boost drug metabolism by mammalian cytochromeâ P450s, the factors that stabilize the structural folding and enhance protein function are unclear. In this study, we use peptideâ based lipid nanodiscs to â trapâ the lipid boundaries of microsomal cytochromeâ P450 2B4. We report the first evidence that CYP2B4 is able to induce the formation of raft domains in a biomimetic compound of the endoplasmic reticulum. NMR experiments were used to identify and quantitatively determine the lipids present in nanodiscs. A combination of biophysical experiments and molecular dynamics simulations revealed a sphingomyelin binding region in CYP2B4. The proteinâ induced lipid raft formation increased the thermal stability of P450 and dramatically altered ligand binding kinetics of the hydrophilic ligand BHT. These results unveil membrane/protein dynamics that contribute to the delicate mechanism of redox catalysis in lipid membrane.Redoxkatalyse in der Lipidmembran: Eine neue Anwendung von Peptidnanoscheiben zeigt, dass Cytochrom P450 2B4 die Bildung von Lipidâ Raftâ Domänen in einer biomimetischen Verbindung des endoplasmatischen Retikulums (ER) induzieren kann. Die proteininduzierten Lipidflöà e steigern die thermische Stabilität von Cytochrom P450 und modulieren die Ligandenbindungskinetik des hydrophilen BHTâ Liganden.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/142938/1/ange201713167.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142938/2/ange201713167_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/142938/3/ange201713167-sup-0001-misc_information.pd

Clinically Relevant Interactions between Newer Antidepressants and Second-Generation Antipsychotics

INTRODUCTION: Combinations of newer antidepressants and second-generation antipsychotics (SGAs) are frequently used by clinicians. Pharmacokinetic drug interaction (PK DI) and poorly understood pharmacodynamic (PD) drug interaction (PD DI) can occur between them. AREAS COVERED: This paper comprehensively reviews PD DI and PK DI studies. EXPERT OPINION: More PK DI studies are needed to better establish dose correction factors after adding fluoxetine and paroxetine to aripiprazole, iloperidone and risperidone. Further PK DI studies and case reports are also needed to better establish the need for dose correction factors after adding i) fluoxetine to clozapine, lurasidone, quetiapine and olanzapine; ii) paroxetine to olanzapine; iii) fluvoxamine to asenapine, aripiprazole, iloperidone, lurasidone, olanzapine, quetiapine and risperidone; iv) high sertraline doses to aripiprazole, clozapine, iloperidone and risperidone: v) bupropion and duloxetine to aripiprazole, clozapine, iloperidone and risperidone; and vi) asenapine to paroxetine and venlafaxine. Possible beneficial PD DI effects occur after adding SGAs to newer antidepressants for treatment-resistant major depressive and obsessive-compulsive disorders. The lack of studies combining newer antidepressants and SGAs in psychotic depression is worrisome. PD DIs between newer antidepressants and SGAs may be more likely for mirtazapine and bupropion. Adding selective serotonin reuptake inhibitors and SGAs may increase QTc interval and may very rarely contribute to torsades de pointes

In vitro Effects of Four Native Brazilian Medicinal Plants in CYP3A4 mRNA Gene Expression, Glutathione Levels, and P-Glycoprotein Activity.

Erythrina mulungu Benth. (Fabaceae), Cordia verbenacea A. DC. (Boraginaceae), Solanum paniculatum L. (Solanaceae) and Lippia sidoides Cham. (Verbenaceae) are medicinal plant species native to Brazil shortlisted by the Brazilian National Health System for future clinical use. However, nothing is known about their effects in metabolic and transporter proteins, which could potentially lead to herb-drug interactions (HDI). In this work, we assess non-toxic concentrations (100 μg/mL) of the plant infusions for their in vitro ability to modulate CYP3A4 mRNA gene expression and intracellular glutathione levels in HepG2 cells, as well as P-glycoprotein (P-gp) activity in vincristine-resistant Caco-2 cells (Caco-2 VCR). Their mechanisms of action were further studied by measuring the activation of human pregnane X receptor (hPXR) in transiently co-transfected HeLa cells and the inhibition of γ-glutamyl transferase (GGT) in HepG2 cells. Our results show that P-gp activity was not affected in any case and that only Solanum paniculatum was able to significantly change CYP3A4 mRNA gene expression (twofold decrease, p < 0.05), this being correlated with an antagonist effect upon hPXR (EC50 = 0.38 mg/mL). Total intracellular glutathione levels were significantly depleted by exposure to Solanum paniculatum (-44%, p < 0.001), Lippia sidoides (-12%, p < 0.05) and Cordia verbenacea (-47%, p < 0.001). The latter plant extract was able to decrease GGT activity (-48%, p < 0.01). In conclusion, this preclinical study shows that the administration of some of these herbal medicines may be able to cause disturbances to metabolic mechanisms in vitro. Although Erythrina mulungu appears safe in our tests, active pharmacovigilance is recommended for the other three species, especially in the case of Solanum paniculatum

Identification, Replication, and Functional Fine-Mapping of Expression Quantitative Trait Loci in Primary Human Liver Tissue

The discovery of expression quantitative trait loci (“eQTLs”) can help to unravel genetic contributions to complex traits. We identified genetic determinants of human liver gene expression variation using two independent collections of primary tissue profiled with Agilent (n = 206) and Illumina (n = 60) expression arrays and Illumina SNP genotyping (550K), and we also incorporated data from a published study (n = 266). We found that ∼30% of SNP-expression correlations in one study failed to replicate in either of the others, even at thresholds yielding high reproducibility in simulations, and we quantified numerous factors affecting reproducibility. Our data suggest that drug exposure, clinical descriptors, and unknown factors associated with tissue ascertainment and analysis have substantial effects on gene expression and that controlling for hidden confounding variables significantly increases replication rate. Furthermore, we found that reproducible eQTL SNPs were heavily enriched near gene starts and ends, and subsequently resequenced the promoters and 3′UTRs for 14 genes and tested the identified haplotypes using luciferase assays. For three genes, significant haplotype-specific in vitro functional differences correlated directly with expression levels, suggesting that many bona fide eQTLs result from functional variants that can be mechanistically isolated in a high-throughput fashion. Finally, given our study design, we were able to discover and validate hundreds of liver eQTLs. Many of these relate directly to complex traits for which liver-specific analyses are likely to be relevant, and we identified dozens of potential connections with disease-associated loci. These included previously characterized eQTL contributors to diabetes, drug response, and lipid levels, and they suggest novel candidates such as a role for NOD2 expression in leprosy risk and C2orf43 in prostate cancer. In general, the work presented here will be valuable for future efforts to precisely identify and functionally characterize genetic contributions to a variety of complex traits