The Ferric-Superoxo Intermediate of the TxtE Nitration Pathway Resists Reduction, Facilitating Its Reaction with Nitric Oxide.

TxtE is a cytochrome P450 (CYP) homologue that mediates the nitric oxide (NO)-dependent direct nitration of l-tryptophan (Trp) to form 4-nitro-l-tryptophan (4-NO2-Trp). A recent report showed evidence that TxtE activity requires NO to react with a ferric-superoxo intermediate. Given this minimal mechanism, it is not clear how TxtE avoids Trp hydroxylation, a mechanism that also traverses the ferric-superoxo intermediate. To provide insight into canonical CYP intermediates that TxtE can access, electron coupling efficiencies to form 4-NO2-Trp under single- or limited-turnover conditions were measured and compared to steady-state efficiencies. As previously reported, Trp nitration by TxtE is supported by the engineered self-sufficient variant, TB14, as well as by reduced putidaredoxin. Ferrous (FeII) TxtE exhibits excellent electron coupling (70%), which is 50-fold higher than that observed under turnover conditions. In addition, two- or four-electron reduced TB14 exhibits electron coupling (∼6%) that is 2-fold higher than that of one-electron reduced TB14 (3%). The combined results suggest (1) autoxidation is the sole TxtE uncoupling pathway and (2) the TxtE ferric-superoxo intermediate cannot be reduced by these electron transfer partners. The latter conclusion is further supported by ultraviolet-visible absorption spectral time courses showing neither spectral nor kinetic evidence for reduction of the ferric-superoxo intermediate. We conclude that resistance of the ferric-superoxo intermediate to reduction is a key feature of TxtE that increases the lifetime of the intermediate and enables its reaction with NO and efficient nitration activity.

A universal and label-free impedimetric biosensing platform for discrimination of single nucleotide substitutions in long nucleic acid strands.

We report a label-free universal biosensing platform for highly selective detection of long nucleic acid strands. The sensor consists of an electrode-immobilized universal stem-loop (USL) probe and two adaptor strands that form a 4J structure in the presence of a specific DNA/RNA analyte. The sensor was characterized by electrochemical impedance spectroscopy (EIS) using K3[Fe(CN)6]/K4[Fe(CN)6] redox couple in solution. An increase in charge transfer resistance (RCT) was observed upon 4J structure formation, the value of which depends on the analyte length. Cyclic voltammetry (CV) was used to further characterize the sensor and monitor the electrochemical reaction in conjunction with thickness measurements of the mixed DNA monolayer obtained using spectroscopic ellipsometry. In addition, the electron transfer was calculated at the electrode/electrolyte interface using a rotating disk electrode. Limits of detection in the femtomolar range were achieved for nucleic acid targets of different lengths (22 nt, 60 nt, 200 nt). The sensor produced only a background signal in the presence of single base mismatched analytes, even in hundred times excess in concentration. This label-free and highly selective biosensing platform is versatile and can be used for universal detection of nucleic acids of varied lengths which could revolutionize point of care diagnostics for applications such as bacterial or cancer screening.

Estrogen effects on high-affinity choline uptake in primary cultures of rat basal forebrain.

Basal forebrain cholinergic neurons (BFCNs) degenerate in aging and Alzheimer's disease. It has been proposed that estrogen can affect the survival and function of BFCNs. This study characterized primary rat BFCN cultures and investigated the effect of estrogen on high-affinity choline uptake (HACU). BFCNs were identified by immunoreactivity to the vesicular acetylcholine transporter (VAChT) and represented up to 5% of total cells. HACU was measured in living BFCN cultures and differentiated from low-affinity choline uptake by hemicholinium-3 (HC-3) inhibition. A HC-3 concentration curve showed that 0.3 muM HC-3, but not higher concentrations that inhibit LACU, could distinguish the two transport activities. 17-beta-Estradiol treatment increased HACU in some culture preparations that contained non-neuronal cells. Elimination of dividing cells using antimitotic treatments resulted in a lack of estrogen effects on HACU. These results suggest that estrogen may have indirect effects on BFCNs that are mediated through non-neuronal cells.

Controlling pattern formation in nanoparticle assemblies via directed solvent dewetting.

We have achieved highly localized control of pattern formation in two-dimensional nanoparticle assemblies by direct modification of solvent dewetting dynamics. A striking dependence of nanoparticle organization on the size of atomic force microscope-generated surface heterogeneities is observed and reproduced in numerical simulations. Nanoscale features induce a rupture of the solvent-nanoparticle film, causing the local flow of solvent to carry nanoparticles into confinement. Microscale heterogeneities instead slow the evaporation of the solvent, producing a remarkably abrupt interface between different nanoparticle patterns.

A genetic algorithm approach to probing the evolution of self-organized nanostructured systems.

We present a new methodology, based on a combination of genetic algorithms and image morphometry, for matching the outcome of a Monte Carlo simulation to experimental observations of a far-from-equilibrium nanosystem. The Monte Carlo model used simulates a colloidal solution of nanoparticles drying on a solid substrate and has previously been shown to produce patterns very similar to those observed experimentally. Our approach enables the broad parameter space associated with simulated nanoparticle self-organization to be searched effectively for a given experimental target morphology.

Charge transport in cellular nanoparticle networks: meandering through nanoscale mazes.

The transport of electrons through topologically complex two-dimensional Au nanoparticle networks has been investigated using a combination of low temperature (4.5 K) direct current I(V) measurements and numerical simulations. Intricate, spatially correlated nanostructured networks were formed via spin-casting. The topological complexity of the nanoparticle assemblies produces I(V) curves associated with nonlinearity exponents, zeta approximately 4.0. Simulations based on tunneling transport in sparse and inhomogeneous planar networks are used to elucidate the influence of topology on the value of zeta.

Effectiveness of statins in reducing the rate of severe sepsis: a retrospective evaluation.

STUDY OBJECTIVES: To determine whether use of 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) is associated with a reduced rate of severe sepsis, and to further characterize the effect of statins on the frequency of organ dysfunction in patients with severe sepsis. DESIGN: Retrospective cohort study. SETTING: University-associated teaching hospital. PATIENTS: Fifty-three patients admitted with sepsis; 16 were receiving statins and 37 were not receiving statins (controls) before admission. MEASUREMENTS AND MAIN RESULTS: Patients were identified by the International Classification of Diseases, Ninth Revision, Clinical Modification codes. Patient demographics, vital signs, and laboratory values were collected from their electronic medical records. The primary end point was rate of severe sepsis, defined in accordance with guidelines from the American College of Chest Physicians and the Society of Critical Care Medicine. Secondary end points were in-hospital mortality rate and rate of five categories of organ dysfunction (cardiovascular, renal, pulmonary, hematologic, and metabolic). Preadmission statin therapy, compared with no statin therapy, was associated with a 30% lower rate of severe sepsis (56% vs 86%, p<0.02). In-hospital mortality was not significantly different between groups (38% vs 49%, p=0.33); however, the rate of cardiovascular dysfunction, defined as hypotension requiring vasopressor therapy, was significantly lower in the statin group (38% vs 73%, p<0.02). No significant differences in the other organ dysfunction categories were noted between groups. CONCLUSION: Statins appear to prevent sepsis from becoming severe, most notably through prevention of sepsis-induced hypotension. This potential role for statins in the prevention and treatment of severe sepsis should be further evaluated in a randomized controlled trial.

Aspirin resistance: an evaluation of current evidence and measurement methods.

Aspirin resistance is a poorly characterized phenomenon, whereby certain patients do not benefit from the antithrombotic effect of aspirin. The frequency of aspirin resistance is unknown, but estimates range from 5-60%. The mechanism of aspirin resistance also is unknown; proposed mechanisms are poor patient compliance, poor aspirin absorption, increased isoprostane activity, platelet hypersensitivity to agonists, increased cyclooxygenase-2 activity, a cyclooxygenase-1 polymorphism, and the platelet alloantigen 2 polymorphism of platelet glycoprotein IIIa. Aspirin resistance appears to be dose related in some patients and therefore may be overcome with high doses. Evidence indicates that aspirin resistance is a dynamic state, with significant intrapatient variability in aspirin sensitivity with time. To date, a sensitive and specific assay of aspirin effect that reliably predicts treatment failure has not emerged. However, several commercially available products are being marketed for this purpose without convincing clinical data. Despite a wealth of literature on the topic, aspirin resistance remains an enigma. Further investigation is needed regarding strategies to identify and treat patients resistant to aspirin.

Potential in vitro interaction between tenecteplase and unfractionated heparin.

STUDY OBJECTIVE: To explore the potential of a direct drug interaction between unfractionated heparin (UFH) and tenecteplase that lowers the pharmacologic propensity of UFH to prolong the activated partial thromboplastin time (aPTT). DESIGN: In vitro experiment. SETTING: Texas Tech University School of Pharmacy, with sample analysis performed at an independent, contract laboratory. Samples. Blood samples collected from healthy volunteers. INTERVENTION: Three separate in vitro experiments were conducted to explore the relative influence of various thrombolytic agents with and without UFH on aPTT prolongation. In each experiment, blood from healthy volunteers (12 for each experiment) was treated with different concentrations and combinations of tenecteplase and UFH. MEASUREMENTS AND MAIN RESULTS: When the effects of tenecteplase plus UFH versus UFH alone on aPTT prolongation were compared, each experiment demonstrated attenuation of aPTT with the combination versus UFH alone. In contrast, findings for other thrombolytic agents combined with UFH demonstrate elevation of the aPTT compared with UFH alone. CONCLUSION: The results indicate a possible drug interaction between tenecteplase and UFH, with tenecteplase attenuating the intensity of anticoagulation of UFH in vitro. Further investigation into this possible interaction is warranted in the clinical setting.