Isolation and characterization of plasma membrane-associated cortical granules from sea urchin eggs

Cortical granules, which are specialized secretory organelles found in ova of many organisms, have been isolated from the eggs of the sea urchins Arbacia punctulata and Strongylocentrtus pupuratus by a simple, rapid procedure. Electron micropscope examination of cortical granules prepared by this procedure reveals that they are tightly attached to large segments of the plasma membrane and its associated vitelline layer. Further evidence that he cortical granules were associated with these cell surface layers was obtained by (125)I-labeling techniques. The cortical granule preparations were found to be rich in proteoesterase, which was purified 32-fold over that detected in a crude homogenate. Similarly, the specific radioactivity of a (125)I-labeled, surface glycoprotein was increased 40-fold. These facts, coupled with electron microscope observations, indicate the isolation procedure yields a preparation in which both the cortical granules and the plasma membrane-vitelline layer are purified to the same extent. Gel electrophoresis of the membrane-associated cortical granule preparation reveals the presence of at least eight polypeptides. The major polypeptide, which is a glycotprotein of apparent mol wt of 100,000, contains most of the radioactivity introduced by (125)I-labeling of the intact eggs. Lysis of the cortical granules is observed under hypotonic conditions, or under isotonic conditions if Ca(2+) ion is present. When lysis is under isotonic conditions is induced by addition of Ca(2+) ion, the electron-dense contents of the granules remain insoluble. In contrast, hypotonic lysis results in release of the contents of the granule in a soluble form. However, in both cases the (125)I-labeled glycoprotein remains insoluble, presumably because it is a component of either the plasma membrane or the vitelline layer. All these findings indicate that, using this purified preparation, it should be possible to carry out in vitro studies to better define some of the initial, surface-related events observed in vivo upon fertilization.     

Programmatic Cost Evaluation of Nontargeted Opt-Out Rapid HIV Screening in the Emergency Department

Background

The Centers for Disease Control and Prevention recommends nontargeted opt-out HIV screening in healthcare settings. Cost effectiveness is critical when considering potential screening methods. Our goal was to compare programmatic costs of nontargeted opt-out rapid HIV screening with physician-directed diagnostic rapid HIV testing in an urban emergency department (ED) as part of the Denver ED HIV Opt-Out Trial.

Methods

This was a prospective cohort study nested in a larger quasi-experiment. Over 16 months, nontargeted rapid HIV screening (intervention) and diagnostic rapid HIV testing (control) were alternated in 4-month time blocks. During the intervention phase, patients were offered HIV testing using an opt-out approach during registration; during the control phase, physicians used a diagnostic approach to offer HIV testing to patients. Each method was fully integrated into ED operations. Direct program costs were determined using the perspective of the ED. Time-motion methodology was used to estimate personnel activity costs. Costs per patient newly-diagnosed with HIV infection by intervention phase, and incremental cost effectiveness ratios were calculated.

Results

During the intervention phase, 28,043 eligible patients were included, 6,933 (25%) completed testing, and 15 (0.2%, 95% CI: 0.1%–0.4%) were newly-diagnosed with HIV infection. During the control phase, 29,925 eligible patients were included, 243 (0.8%) completed testing, and 4 (1.7%, 95% CI: 0.4%–4.2%) were newly-diagnosed with HIV infection. Total annualized costs for nontargeted screening were $148,997, whereas total annualized costs for diagnostic HIV testing were $31,355. The average costs per HIV diagnosis were $9,932 and $7,839, respectively. Nontargeted HIV screening identified 11 more HIV infections at an incremental cost of $10,693 per additional infection.

Conclusions

Compared to diagnostic testing, nontargeted HIV screening was more costly but identified more HIV infections. More effective and less costly testing strategies may be required to improve the identification of patients with undiagnosed HIV infection in the ED.     

Automated Bayesian model development for frequency detection in biological time series

Background

The ability to perform quantitative studies using isotope tracers and metabolic flux analysis (MFA) is critical for detecting pathway bottlenecks and elucidating network regulation in biological systems, especially those that have been engineered to alter their native metabolic capacities. Mathematically, MFA models are traditionally formulated using separate state variables for reaction fluxes and isotopomer abundances. Analysis of isotope labeling experiments using this set of variables results in a non-convex optimization problem that suffers from both implementation complexity and convergence problems.

Results

This article addresses the mathematical and computational formulation of ¹³C MFA models using a new set of variables referred to as fluxomers. These composite variables combine both fluxes and isotopomer abundances, which results in a simply-posed formulation and an improved error model that is insensitive to isotopomer measurement normalization. A powerful fluxomer iterative algorithm (FIA) is developed and applied to solve the MFA optimization problem. For moderate-sized networks, the algorithm is shown to outperform the commonly used 13CFLUX cumomer-based algorithm and the more recently introduced OpenFLUX software that relies upon an elementary metabolite unit (EMU) network decomposition, both in terms of convergence time and output variability.

Conclusions

Substantial improvements in convergence time and statistical quality of results can be achieved by applying fluxomer variables and the FIA algorithm to compute best-fit solutions to MFA models. We expect that the fluxomer formulation will provide a more suitable basis for future algorithms that analyze very large scale networks and design optimal isotope labeling experiments.     

Identification and characterization of 17 microsatellite primers for the tick,Ixodes ricinus,using enriched genomic libraries

Ixodes ricinus is the main vector for important infectious diseases in both humans and in animals. Microsatellite loci were isolated from a dinucleotide‐enriched library made from I. ricinus sampled in Norway. Seventeen polymorphic microsatellites were further characterized among 24 individuals sampled from an island in the Oslofjord region. The number of observed alleles ranged from two to 17 and the observed heterozygosities between 0.10 and 0.83. Analysis of family materials gives evidence of non‐Mendelian inheritance of several of the characterized loci, among which most could be explained by presence of null alleles.     

First Record of Teratopactus nodicollis (Coleoptera: Curculionidae) in Dry Bean (Phaseolus vulgaris)

Observations on the bioecology and damage of Teratopactus nodicollis Boheman on Phaseolus vulgaris were carried out on field samples by assessing the number of larvae and root damage in 40?ha of a dry bean field from the Federal District, Brazil (16°4??28.41???W; 47°30??21.13???S). Larvae caused the greatest damage at the stage of germination, emergence, and primary leaves, producing 50?% stand reduction. Most larvae pupated in August and September, and adult emergence occurred in middle October. Some larvae were infected with the fungus Metarhizium spp., a biological agent that would be naturally controlling this insect.     

Motif D of Viral RNA-Dependent RNA Polymerases Determines Efficiency and Fidelity of Nucleotide Addition

Fast, accurate nucleotide incorporation by polymerases facilitates expression and maintenance of genomes. Many polymerases use conformational dynamics of a conserved α helix to permit efficient nucleotide addition only when the correct nucleotide substrate is bound. This α helix is missing in structures of RNA-dependent RNA polymerases (RdRps) and RTs. Here, we use solution-state nuclear magnetic resonance to demonstrate that the conformation of conserved structural motif D of an RdRp is?linked to the nature (correct versus incorrect) of the bound nucleotide and the protonation state of a conserved, motif-D lysine. Structural data also reveal the inability of motif D to achieve its optimal conformation after incorporation of an incorrect nucleotide. Functional data are consistent with the conformational change of motif D becoming rate limiting during and after nucleotide misincorporation. We conclude that motif D of RdRps and, by inference, RTs is the functional equivalent to the fidelity helix of other polymerases.     

Determinants of RNA-dependent RNA polymerase (in)fidelity revealed by kinetic analysis of the polymerase encoded by a foot-and-mouth disease virus mutant with reduced sensitivity to ribavirin

A mutant poliovirus (PV) encoding a change in its polymerase (3Dpol) at a site remote from the catalytic center (G64S) confers reduced sensitivity to ribavirin and forms a restricted quasispecies, because G64S 3Dpol is a high-fidelity enzyme. A foot-and-mouth disease virus (FMDV) mutant that encodes a change in the polymerase catalytic site (M296I) exhibits reduced sensitivity to ribavirin without restricting the viral quasispecies. In order to resolve this apparent paradox, we have established a minimal kinetic mechanism for nucleotide addition by wild-type (WT) FMDV 3Dpol that permits a direct comparison to PV 3Dpol as well as to FMDV 3Dpol derivatives. Rate constants for correct nucleotide addition were on par with those of PV 3Dpol, but apparent binding constants for correct nucleotides were higher than those observed for PV 3Dpol. The A-to-G transition frequency was calculated to be 1/20,000, which is quite similar to that calculated for PV 3Dpol. The analysis of FMDV M296I 3Dpol revealed a decrease in the calculated ribavirin incorporation frequency (1/8,000) relative to that (1/4,000) observed for the WT enzyme. Unexpectedly, the A-to-G transition frequency was higher (1/8,000) than that observed for the WT enzyme. Therefore, FMDV selected a polymerase that increases the frequency of the misincorporation of natural nucleotides while specifically decreasing the frequency of the incorporation of ribavirin nucleotide. These studies provide a mechanistic framework for understanding FMDV 3Dpol structure-function relationships, provide the first direct analysis of the fidelity of FMDV 3Dpol in vitro, identify the β9-α11 loop as a (in)fidelity determinant, and demonstrate that not all ribavirin-resistant mutants will encode high-fidelity polymerases.