Functions of Phenylalanine Residues within the β-Barrel Stem of the Anthrax Toxin Pore

Background: A key step of anthrax toxin action involves the formation of a protein-translocating pore within the endosomal membrane by the Protective Antigen (PA) moiety. Formation of this transmembrane pore by PA involves interaction of the seven 2b2–2b3 loops of the heptameric precursor to generate a 14-strand transmembrane b barrel. Methodology/Principal Findings: We examined the effects on pore formation, protein translocation, and cytotoxicity, of mutating two phenylalanines, F313 and F314, that lie at the tip the b barrel, and a third one, F324, that lies part way up the barrel. Conclusions/Significance: Our results show that the function of these phenylalanine residues is to mediate membrane insertion and formation of stable transmembrane channels. Unlike F427, a key luminal residue in the cap of the pore, F313, F314, and F324 do not directly affect protein translocation through the pore. Our findings add to our knowledge of structure-function relationships of a key virulence factor of the anthrax bacillus

Domain-wall motion induced by spin transfer torque delivered by helicity-dependent femtosecond laser

In magnetic wires with perpendicular anisotropy, moving domain with only current or only circularly polarized light requires a high power. Here, we propose to reduce it by using both short current pulses and femtosecond laser pulses simultaneously. The wires were made out of perpendicularly magnetized film of Pt/Co/Ni/Co/Pt. The displacement of the domain wall is found to be dependent on the laser helicity. Based on a quantitative analysis of the current-induced domain wall motion, the spin orbit torque contribution can be neglected when compared to the spin transfer torque contribution. The effective field of the spin transfer torque is extracted from the pulsed field domain wall measurements. Finally, our result can be described using the Fatuzzo-Labrune model and considering the effective field due to the polarized laser beam, the effective field due to spin transfer torque, and the Gaussian temperature distribution of the laser spot.Comment: 14 pages, 4 figure

Electroporation-Induced Electrosensitization

BACKGROUND: Electroporation is a method of disrupting the integrity of cell membrane by electric pulses (EPs). Electrical modeling is widely employed to explain and study electroporation, but even most advanced models show limited predictive power. No studies have accounted for the biological consequences of electroporation as a factor that alters the cell's susceptibility to forthcoming EPs. METHODOLOGY/PRINCIPAL FINDINGS: We focused first on the role of EP rate for membrane permeabilization and lethal effects in mammalian cells. The rate was varied from 0.001 to 2,000 Hz while keeping other parameters constant (2 to 3,750 pulses of 60-ns to 9-µs duration, 1.8 to 13.3 kV/cm). The efficiency of all EP treatments was minimal at high rates and started to increase gradually when the rate decreased below a certain value. Although this value ranged widely (0.1-500 Hz), it always corresponded to the overall treatment duration near 10 s. We further found that longer exposures were more efficient irrespective of the EP rate, and that splitting a high-rate EP train in two fractions with 1-5 min delay enhanced the effects severalfold. CONCLUSIONS/SIGNIFICANCE: For varied experimental conditions, EPs triggered a delayed and gradual sensitization to EPs. When a portion of a multi-pulse exposure was delivered to already sensitized cells, the overall effect markedly increased. Because of the sensitization, the lethality in EP-treated cells could be increased from 0 to 90% simply by increasing the exposure duration, or the exposure dose could be reduced twofold without reducing the effect. Many applications of electroporation can benefit from accounting for sensitization, by organizing the exposure either to maximize sensitization (e.g., for sterilization) or, for other applications, to completely or partially avoid it. In particular, harmful side effects of electroporation-based therapies (electrochemotherapy, gene therapies, tumor ablation) include convulsions, pain, heart fibrillation, and thermal damage. Sensitization can potentially be employed to reduce these side effects while preserving or increasing therapeutic efficiency

A Theoretical Analysis of the Feasibility of a Singularity-Induced Micro-Electroporation System

Electroporation, the permeabilization of the cell membrane lipid bilayer due to a pulsed electric field, has important implications in the biotechnology, medicine, and food industries. Traditional macro and micro-electroporation devices have facing electrodes, and require significant potential differences to induce electroporation. The goal of this theoretical study is to investigate the feasibility of singularity-induced micro-electroporation; an electroporation configuration aimed at minimizing the potential differences required to induce electroporation by separating adjacent electrodes with a nanometer-scale insulator. In particular, this study aims to understand the effect of (1) insulator thickness and (2) electrode kinetics on electric field distributions in the singularity-induced micro-electroporation configuration. A non-dimensional primary current distribution model of the micro-electroporation channel shows that while increasing insulator thickness results in smaller electric field magnitudes, electroporation can still be performed with insulators thick enough to be made with microfabrication techniques. Furthermore, a secondary current distribution model of the singularity-induced micro-electroporation configuration with inert platinum electrodes and water electrolyte indicates that electrode kinetics do not inhibit charge transfer to the extent that prohibitively large potential differences are required to perform electroporation. These results indicate that singularity-induced micro-electroporation could be used to develop an electroporation system that consumes minimal power, making it suitable for remote applications such as the sterilization of water and other liquids

Efficacy of Combined Therapy with Amantadine, Oseltamivir, and Ribavirin In Vivo against Susceptible and Amantadine-Resistant Influenza A Viruses

The limited efficacy of existing antiviral therapies for influenza – coupled with widespread baseline antiviral resistance – highlights the urgent need for more effective therapy. We describe a triple combination antiviral drug (TCAD) regimen composed of amantadine, oseltamivir, and ribavirin that is highly efficacious at reducing mortality and weight loss in mouse models of influenza infection. TCAD therapy was superior to dual and single drug regimens in mice infected with drug-susceptible, low pathogenic A/H5N1 (A/Duck/MN/1525/81) and amantadine-resistant 2009 A/H1N1 influenza (A/California/04/09). Treatment with TCAD afforded >90% survival in mice infected with both viruses, whereas treatment with dual and single drug regimens resulted in 0% to 60% survival. Importantly, amantadine had no activity as monotherapy against the amantadine-resistant virus, but demonstrated dose-dependent protection in combination with oseltamivir and ribavirin, indicative that amantadine's activity had been restored in the context of TCAD therapy. Furthermore, TCAD therapy provided survival benefit when treatment was delayed until 72 hours post-infection, whereas oseltamivir monotherapy was not protective after 24 hours post-infection. These findings demonstrate in vivo efficacy of TCAD therapy and confirm previous reports of the synergy and broad spectrum activity of TCAD therapy against susceptible and resistant influenza strains in vitro

A limited role for unforced internal variability in 20th century warming.

The early twentieth-century warming (EW; 1910–45) and the mid-twentieth-century cooling (MC; 1950–80) have been linked to both internal variability of the climate system and changes in external radiative forcing. The degree to which either of the two factors contributed to EW and MC, or both, is still debated. Using a two-box impulse response model, we demonstrate that multidecadal ocean variability was unlikely to be the driver of observed changes in global mean surface temperature (GMST) after AD 1850. Instead, virtually all (97%–98%) of the global low-frequency variability (.30 years) can be explained by external forcing. We find similarly high percentages of explained variance for interhemispheric and land–ocean temperature evolution. Three key aspects are identified that underpin the conclusion of this new study: inhomogeneous anthropogenic aerosol forcing (AER), biases in the instrumental sea surface temperature (SST) datasets, and inadequate representation of the response to varying forcing factors. Once the spatially heterogeneous nature of AER is accounted for, the MC period is reconcilable with external drivers. SST biases and imprecise forcing responses explain the putative disagreement between models and observations during the EW period. As a consequence, Atlantic multidecadal variability (AMV) is found to be primarily controlled by external forcing too. Future attribution studies should account for these important factors when discriminating between externally forced and internally generated influences on climate. We argue that AMV must not be used as a regressor and suggest a revised AMV index instead [the North Atlantic Variability Index (NAVI)]. Our associated best estimate for the transient climate response (TCR) is 1.57 K (60.70 at the 5%–95% confidence level)

Empirical Legal Studies Before 1940: A Bibliographic Essay

The modern empirical legal studies movement has well-known antecedents in the law and society and law and economics traditions of the latter half of the 20th century. Less well known is the body of empirical research on legal phenomena from the period prior to World War II. This paper is an extensive bibliographic essay that surveys the English language empirical legal research from approximately 1940 and earlier. The essay is arranged around the themes in the research: criminal justice, civil justice (general studies of civil litigation, auto accident litigation and compensation, divorce, small claims, jurisdiction and procedure, civil juries), debt and bankruptcy, banking, appellate courts, legal needs, legal profession (including legal education), and judicial staffing and selection. Accompanying the essay is an extensive bibliography of research articles, books, and reports

Effects of F313 and F314 mutations on PA permeabilization of membranes to K⁺.

<p>For clarity, only selected mutants are shown. The double Trp (WW) and double Tyr (YY) replacements had approximately the same kinetics of K⁺ release as the WT (FF). The kinetics of release by the double Leu (LL), double Ala (AA), and double Arg (RR), as well as the F313A and F314A mutants are also shown. The double His (HH), double Asp (DD), and the buffer control results were indistinguishable from those of RR.</p

Cellular translocation assay of F313X/F314X mutants.

<p>(A) PA₈₃ was titrated, mixed with a fixed concentration of LF_N-DTA (100 pM), added to CHO-K1 cells, and incubated at 37°C for 4 hours. The level of protein synthesis was measured by detecting [³H] leucine incorporation and was expressed as fraction of that observed with PA-WT. Only WT, S1 (deletion of F313 and F314) and DNI (K397D, D425K) are shown. (B) EC₅₀ for all F313X/F314X mutants as calculated from sigmoidal fits to cytotoxicity experiments.</p

Locations of F313, F314, F324 and β strands 1–4 in the β barrel and the pore.

<p>The approximate boundaries of the membrane-spanning region are indicated by the dashed lines. The illustration of the pore structure (left), reconstructed from single-pore images obtained by electron microscopy, is modified from Figure 4 of reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006280#pone.0006280-Katayama1" target="_blank">[4]</a>. The amino acid sequence of the ® hairpin is as follows: E(302)VHGNAEVHASFFDIGGSVSAGFS(325).</p