Bovine adrenocortical microsomal hemeproteins P-45017 alpha and P-450C-21. Isolation, partial characterization, and comparison to P-450SCC.

A scheme is presented for the simultaneous isolation of P-45017n, and P-450c-21 from bovine adrenocortical microsomes and for their purification to specific contents of 19-20 nmol of P-450/mg of protein. Although cholate solubilized only 75% of the total amount of P- 450 and 36% of the NADPH-cytochrome C (P-450) reductase present in bovine adrenocortical microsomes as compared to Triton N-101, it produced a less complex mixture of microsomal proteins from which most of the P-450 could be removed by a 0-25% cut with polyethylene glycol. The P-450 fraction was subsequently resolved into individual P-450s by column chromatography on w-aminooctyl-Sepharose; these were then purified to homogeneity by a series of final chromatography steps including DEAE-, CM-, and w-aminooctyl-Sepharose

Search-Space Reduction via Essential Vertices

We investigate preprocessing for vertex-subset problems on graphs. While the notion of kernelization, originating in parameterized complexity theory, is a formalization of provably effective preprocessing aimed at reducing the total instance size, our focus is on finding a non-empty vertex set that belongs to an optimal solution. This decreases the size of the remaining part of the solution which still has to be found, and therefore shrinks the search space of fixed-parameter tractable algorithms for parameterizations based on the solution size. We introduce the notion of a c-essential vertex as one that is contained in all c-approximate solutions. For several classic combinatorial problems such as Odd Cycle Transversal and Directed Feedback Vertex Set, we show that under mild conditions a polynomial-time preprocessing algorithm can find a subset of an optimal solution that contains all 2-essential vertices, by exploiting packing/covering duality. This leads to FPT algorithms to solve these problems where the exponential term in the running time depends only on the number of non-essential vertices in the solution

Best Estimate Method vs Evaluation Method: a comparison of two techniques in evaluating seismic analysis and design

The concept of how two techniques, Best Estimate Method and Evaluation Method, may be applied to the traditional seismic analysis and design of a nuclear power plant is introduced. Only the four links of the seismic analysis and design methodology chain (SMC) - seismic input, soil-structure interaction, major structural response, and subsystem response - are considered. The objective is to evaluate the compounding of conservatisms in the seismic analysis and design of nuclear power plants, to provide guidance for judgments in the SMC, and to concentrate the evaluation on that part of the seismic analysis and design which is familiar to the engineering community. An example applies the effects of three-dimensional excitations on a model of a nuclear power plant structure. The example demonstrates how conservatisms accrue by coupling two links in the SMC and comparing those results to the effects of one link alone. The utility of employing the Best Estimate Method vs the Evaluation Method is also demonstrated

Multi-locus approaches for the measurement of selection on correlated genetic loci

The study of ecological speciation is inherently linked to the study of selection. Methods for estimating phenotypic selection within a generation based on associations between trait values and fitness (e.g., survival) of individuals are established. These methods attempt to disentangle selection acting directly on a trait from indirect selection caused by correlations with other traits via multivariate statistical approaches (i.e., inference of selection gradients). The estimation of selection on genotypic or genomic variation could also benefit from disentangling direct and indirect selection on genetic loci. However, achieving this goal is difficult with genomic data because the number of potentially correlated genetic loci (p) is very large relative to the number of individuals sampled (n). In other words, the number of model parameters exceeds the number of observations (p ≫ n). We present simulations examining the utility of whole genome regression approaches (i.e., Bayesian sparse linear mixed models) for quantifying direct selection in cases where p ≫ n. Such models have been used for genome-wide association mapping and are common in artificial breeding. Our results show they hold promise for studies of natural selection in the wild, and thus of ecological speciation. But we also demonstrate important limitations to the approach and discuss study designs required for more robust inferences

Correlation between compartmental tenofovir concentrations and an ex vivo rectal biopsy model of tissue infectibility in the RMP-02/MTN-006 phase 1 study

Design: Phase 1, randomized, two-site (US), double-blind, placebo-controlled study of sexually-abstinent males and females

A multi-compartment single and multiple dose pharmacokinetic comparison of rectally applied tenofovir 1% gel and oral tenofovir disoproxil fumarate

This Phase 1, randomized, two-site (United States), double-blind, placebo-controlled study enrolled 18 sexually abstinent men and women. All received a single 300-mg dose of oral tenofovir disoproxil fumarate (TDF) and were then randomized 2:1 to receive single and then seven daily rectal exposures of vaginally-formulated tenofovir (TFV) 1% gel or a hydroxyethyl cellulose (HEC) placebo gel. Blood, colonic biopsies and rectal and vaginal mucosal fluids were collected after the single oral TDF, the single topical TFV gel dose, and after 7 days of topical TFV gel dosing for extracellular analysis of TFV and intracellular analysis of the active metabolite tenofovir diphosphate (TFVdp) in peripheral blood mononuclear cells (PBMCs) and isolated mucosal mononuclear cells (MMC), including CD4+ and CD4- cell subsets. With a single rectal dose, TFV plasma concentrations were 24-33 fold lower and half-life was 5 h shorter compared to a single oral dose (p = 0.02). TFVdp concentrations were also undetectable in PBMCs with rectal dosing. Rectal tissue exposure to both TFV and TFVdp was 2 to 4-log10 higher after a single rectal dose compared to a single oral dose, and after 7 daily doses, TFVdp accumulated 4.5 fold in tissue. TFVdp in rectal tissue homogenate was predictive (residual standard error, RSE = 0.47) of tissue MMC intracellular TFVdp concentration, with the CD4+ cells having a 2-fold higher TFVdp concentration than CD4- cells. TFV concentrations from rectal sponges was a modest surrogate indicator for both rectal tissue TFV and TFVdp (RSE = 0.67, 0.66, respectively) and plasma TFV (RSE = 0.38). TFV penetrates into the vaginal cavity after oral and rectal dosing, with rectal dosing leading to higher vaginal TFV concentrations (p<0.01)

MTN-001: Randomized Pharmacokinetic Cross-Over Study Comparing Tenofovir Vaginal Gel and Oral Tablets in Vaginal Tissue and Other Compartments

Background: Oral and vaginal preparations of tenofovir as pre-exposure prophylaxis (PrEP) for human immunodeficiency virus (HIV) infection have demonstrated variable efficacy in men and women prompting assessment of variation in drug concentration as an explanation. Knowledge of tenofovir concentration and its active form, tenofovir diphosphate, at the putative vaginal and rectal site of action and its relationship to concentrations at multiple other anatomic locations may provide key information for both interpreting PrEP study outcomes and planning future PrEP drug development. Objective: MTN-001 was designed to directly compare oral to vaginal steady-state tenofovir pharmacokinetics in blood, vaginal tissue, and vaginal and rectal fluid in a paired cross-over design. Methods and Findings: We enrolled 144 HIV-uninfected women at 4 US and 3 African clinical research sites in an open label, 3-period crossover study of three different daily tenofovir regimens, each for 6 weeks (oral 300 mg tenofovir disoproxil fumarate, vaginal 1% tenofovir gel [40 mg], or both). Serum concentrations after vaginal dosing were 56-fold lower than after oral dosing (p<0.001). Vaginal tissue tenofovir diphosphate was quantifiable in ≥90% of women with vaginal dosing and only 19% of women with oral dosing. Vaginal tissue tenofovir diphosphate was ≥130-fold higher with vaginal compared to oral dosing (p<0.001). Rectal fluid tenofovir concentrations in vaginal dosing periods were higher than concentrations measured in the oral only dosing period (p<0.03). Conclusions: Compared to oral dosing, vaginal dosing achieved much lower serum concentrations and much higher vaginal tissue concentrations. Even allowing for 100-fold concentration differences due to poor adherence or less frequent prescribed dosing, vaginal dosing of tenofovir should provide higher active site concentrations and theoretically greater PrEP efficacy than oral dosing; randomized topical dosing PrEP trials to the contrary indicates that factors beyond tenofovir's antiviral effect substantially influence PrEP efficacy. Trial Registration: ClinicalTrials.gov NCT00592124