Theoretical Principles of In Vitro Selection Using Combinatorial Nucleic Acid Libraries

A new paradigm for drug discovery and biological research has developed from technologies that integrate combinatorial chemistry with rounds of selection and amplification, a technique called in vitro selection or systematic evolution of ligands by exponential enrichment (SELEX). This overview unit discusses nucleic acid libraries that can be used, affinity probability distributions, an equilibrium model for SELEX, and optimal conditions including concentrations and signalâ toâ noise ratios.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/143632/1/cpnc0901.pd

Effect of N3-Methyladenine and an Isosteric Stable Analogue on DNA Polymerization

N3-methyladenine (3-mA) is a cytotoxic lesion formed by the reaction of DNA with many methylating agents, including antineoplastic drugs, environmental agents and endogenously generated compounds. The toxicity of 3-mA has been attributed to its ability to block DNA polymerization. Using Me-lex, a compound that selectively and efficiently reacts with DNA to afford 3-mA, we have observed in yeast a mutational hotspot at the 5′-terminus of an A4 tract. In order to explore the potential role of sequence-dependent DNA polymerase bypass of 3-mA, we developed an in vitro system to prepare 3-mA modified substrates using Me-lex. We detail the effects of 3-mA, its stable isostere analogue, 3-methyl-3-deazaadenine, 3-deazaadenine and an THF abasic site on DNA polymerization within an A4 sequence. The methyl group on 3-mA and 3-methyl-3-deazaadenine has a pronounced inhibitory effect on DNA polymerization. There was no sequence selectivity for the bypass of any of the lesions, except for the abasic site, which was most efficiently by-passed when it was on the 5′-terminus of the A4 tract. The results indicate that the weak mutational pattern induced by Me-lex may result form the depurination of 3-mA to an abasic site that is bypassed in a sequence dependent context

A study of 7-deaza-2′-deoxyguanosine–2′-deoxycytidine base pairing in DNA

The incorporation of 7-deazaguanine modifications into DNA is frequently used to probe protein recognition of H-bonding information in the major groove of DNA. While it is generally assumed that 7-deazaguanine forms a normal Watson–Crick base pair with cytosine, detailed thermodynamic and structural analyses of this modification have not been reported. The replacement of the 7-N atom on guanine with a C–H, alters the electronic properties of the heterocycle and eliminates a major groove cation-binding site that could affect the organization of salts and water in the major groove. We report herein the characterization of synthetic DNA oligomers containing 7-deazaguanine using a variety of complementary approaches: UV thermal melting, differential scanning calorimetry (DSC), circular dichroism (CD), chemical probing and NMR. The results indicate that the incorporation of a 7-deazaguanine modification has a significant effect on the dynamic structure of the DNA at the flanking residue. This appears to be mediated by changes in hydration and cation organization

Magnetic Energy and Helicity Budgets in the Active-Region Solar Corona. I. Linear Force-Free Approximation

We self-consistently derive the magnetic energy and relative magnetic helicity budgets of a three-dimensional linear force-free magnetic structure rooted in a lower boundary plane. For the potential magnetic energy we derive a general expression that gives results practically equivalent to those of the magnetic Virial theorem. All magnetic energy and helicity budgets are formulated in terms of surface integrals applied to the lower boundary, thus avoiding computationally intensive three-dimensional magnetic field extrapolations. We analytically and numerically connect our derivations with classical expressions for the magnetic energy and helicity, thus presenting a so-far lacking unified treatment of the energy/helicity budgets in the constant-alpha approximation. Applying our derivations to photospheric vector magnetograms of an eruptive and a noneruptive solar active regions, we find that the most profound quantitative difference between these regions lies in the estimated free magnetic energy and relative magnetic helicity budgets. If this result is verified with a large number of active regions, it will advance our understanding of solar eruptive phenomena. We also find that the constant-alpha approximation gives rise to large uncertainties in the calculation of the free magnetic energy and the relative magnetic helicity. Therefore, care must be exercised when this approximation is applied to photospheric magnetic field observations. Despite its shortcomings, the constant-alpha approximation is adopted here because this study will form the basis of a comprehensive nonlinear force-free description of the energetics and helicity in the active-region solar corona, which is our ultimate objective.Comment: 44 pages, 8 figures, 2 tables. The Astrophysical Journal, in pres

Daily mood, partner support, sexual interest, and sexual activity among adolescent women

This is a post print version of the article. The official published version can be accessed from the link below.Objective: to examine day-to-day associations of coitus, sexual interest, partner emotional support, negative mood and positive mood among adolescent women. Methods: Women (ages 14 – 17 at enrollment; N=146) enrolled from one of three adolescent primary care clinics completed up to five 84-day diaries over a 27-month period. The diaries assessed partner interactions, sexual activity, substance use and mood. Partner-specific measures assessed on each day included partner emotional support (4 items; alpha = 0.94), argument with a partner (no/yes) and coitus (no/yes). Within-day measures assessed marijuana use (no/yes), Positive Mood (3-items; alpha = 0. 86); Negative Mood (3-items; alpha = 0.82) and Sexual Interest (1-item). Lagged measures of mood and sexual activity were included in multivariate models to control for recent mood and sexual behavior effects on current day mood and coitus. Two main analyses were conducted: coitus as a predictor of positive and negative mood; and the role of positive and negative mood as predictors of coitus. Analyses were conducted by multivariate mixed effect regression and mixed effect logistic regression models. Results: Data represent 28,376 days from 146 participants. The average number of diary days was 194 days per participant. Sexual activity was reported on 8.3% of days, with condoms used for 27.0% of these coital events. Marijuana was used on 11% of days. Significant predictors of positive mood on a given day included partner support, marijuana use, and coitus. Negative mood was associated with having an argument with a partner and with prior day coitus. Predictors of coitus on a given day included age (Odds ratio = 1.22), increased coital frequency in previous week (OR = 1.49), coitus on the previous day (1.21), increased same-day sexual interest (OR = 2.8) and decreased same-day negative mood (OR = 0.92). Conclusions: The data demonstrate complex associations of sexual interest, mood, partner interactions and sexual activity

Relationship between DNA methylation and mutational patterns induced by a sequence selective minor groove methylating agent.

Me-lex, a methyl sulfonate ester appended to a neutral N-methylpyrrolecarboxamide-based dipeptide, was synthesized to preferentially generate N3-methyladenine (3-MeA) adducts which are expected to be cytotoxic rather than mutagenic DNA lesions. In the present study, the sequence specificity for DNA alkylation by Me-lex was determined in the p53 cDNA through the conversion of the adducted sites into single strand breaks and sequencing gel analysis. In order to establish the mutagenic and lethal properties of Me-lex lesions, a yeast expression vector harboring the human wild-type p53 cDNA was treated in vitro with Me-lex, and transfected into a yeast strain containing the ADE2 gene regulated by a p53-responsive promoter. The results showed that: 1) more than 99% of the lesions induced by Me-lex are 3-MeA; 2) the co-addition of distamycin quantitatively inhibited methylation at all minor groove sites; 3) Me-lex selectively methylated A's that are in, or immediately adjacent to, the lex equilibrium binding sites; 4) all but 6 of the 33 independent mutations were base pair substitutions, the majority of which (17/33; 52%) were AT-targeted; 5) AT --TA transversions were the predominant mutations observed (13/33; 39%); 6) 13 out of 33 (39%) independent mutations involved a single lex-binding site encompassing positions A600-602 and 9 occurred at position 602 which is a real Me-lex mutation hotspot (n = 9, p10(-6), Poisson's normal distribution). A hypothetical model for the interpretation of mutational events at this site is proposed. The present work is the first report on mutational properties of Me-lex. Our results suggest that 3-MeA is not only a cytotoxic but also a premutagenic lesion which exerts this unexpected property in a strict sequence-dependent manner

Influences of base excision repair defects on the lethality and mutagenicity induced by Me-lex, a sequence-selective N3-adenine methylating agent.

Due to its minor groove selectivity, Me-lex preferentially generates N3-methyladenine (3-MeA) adducts in double-stranded DNA. We undertook a genetic approach in yeast to establish the influence of base excision repair (BER) defects on the processing of Me-lex lesions on plasmid DNA that harbors the p53 cDNA as target. We constructed a panel of isogenic strains containing a reporter gene to test p53 function and the following gene deletions: deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. When compared with the wild-type strain, a decrease in survival was observed in deltamag1, deltaapn1apn2, and deltaapn1apn2mag1. The Me-lex-induced mutation frequency increased in the following order: wild typedeltamag1deltaapn1apn2 = deltaapn1apn2mag1. A total of 77 mutants (23 in wild type, 31 in deltamag1, and 23 in deltaapn1apn2) were sequenced. Eighty-one independent mutations (24 in wild type, 34 in deltamag1, and 23 in deltaapn1apn2) were detected. The majority of base pair substitutions were AT-targeted in all strains (14/23, 61% in wild type; 20/34, 59%, in deltamag1; and 14/23, 61%, in deltaapn1apn2). The Mag1 deletion was associated with a significant decrease of GCAT transitions when compared with both the wild-type and the AP endonuclease mutants. This is the first time that the impact of Mag1 and/or AP endonuclease defects on the mutational spectra caused by 3-MeA has been determined. The results suggest that 3-MeA is critical for Me-lex cytotoxicity and that its mutagenicity is slightly elevated in the absence of Mag1 glycosylase activity but significantly higher in the absence of AP endonuclease activity

Improved gastrointestinal profile with diroximel fumarate is associated with a positive impact on quality of life compared with dimethyl fumarate: Results from the randomized, double-blind, phase III EVOLVE-MS-2 study

BACKGROUND: Diroximel fumarate (DRF) is a novel oral fumarate approved for relapsing forms of multiple sclerosis (MS). DRF demonstrated significantly improved gastrointestinal (GI) tolerability METHODS: A RESULTS: In total, 504 patients (DRF, CONCLUSIONS: The improved GI tolerability with DRF translated into clinically meaningful benefits to QoL, as patients experienced less impact on daily life and work and required less concomitant symptomatic medication use. TRIAL REGISTRATION: [ClinicalTrials.gov identifier: NCT03093324]

Effects of esomeprazole treatment for gastroesophageal reflux disease on quality of life in 12- to 17-year-old adolescents: an international health outcomes study

<p>Abstract</p> <p>Background</p> <p>Although gastroesophageal reflux disease (GERD) is common in adolescents, the burden of GERD on health-related quality of life (HRQOL) in adolescents has not been previously evaluated. Therefore, the objective of the study was to examine the effect of GERD on HRQOL in adolescents.</p> <p>Methods</p> <p>This international, 31-site, 8-week safety study randomized adolescents, aged 12 to 17 years inclusive, with GERD to receive esomeprazole 20 or 40 mg once daily. The Quality of Life in Reflux and Dyspepsia questionnaire (QOLRAD), previously validated in adults, consists of 25 questions grouped into 5 domains: emotional distress, sleep disturbance, food/drink problems, physical/social functioning, and vitality. The QOLRAD was administered at the baseline and week-8 (final) visits.</p> <p>Results</p> <p>Of the 149 patients randomized, 134 completed the QOLRAD at baseline and final visits and were eligible for analysis of their HRQOL data. Baseline QOLRAD scores indicated GERD had a negative effect on the HRQOL of these adolescents, especially in the domains of vitality and emotional distress, and problems with food/drink. At the final visit, mean scores for all 5 QOLRAD domains improved significantly (<it>P </it>< .0001); change of scores (ie, delta) for all domains met or exceeded the adult QOLRAD minimal clinically significant difference standard of 0.5 units.</p> <p>Conclusion</p> <p>GERD had a negative effect on QOL in adolescents. After esomeprazole treatment, statistically and clinically significant improvements occurred in all domains of the QOLRAD for these adolescents.</p> <p>Trial Registration</p> <p>D9614C00098; ClinicalTrials.gov Identifier NCT00241501</p