Using zeta-potential measurements to quantify peptide partition to lipid membranes

© The Author(s) 2011. This article is published with open access at Springerlink.com.Open Access: This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.Many cellular phenomena occur on the biomembranes. There are plenty of molecules (natural or xenobiotics) that interact directly or partially with the cell membrane. Biomolecules, such as several peptides (e.g., antimicrobial peptides) and proteins, exert their effects at the cell membrane level. This feature makes necessary investigating their interactions with lipids to clarify their mechanisms of action and side effects necessary. The determination of molecular lipid/water partition constants (Kp) is frequently used to quantify the extension of the interaction. The determination of this parameter has been achieved by using different methodologies, such as UV-Vis absorption spectrophotometry, fluorescence spectroscopy and ζ-potential measurements. In this work, we derived and tested a mathematical model to determine the Kp from ζ-potential data. The values obtained with this method were compared with those obtained by fluorescence spectroscopy, which is a regular technique used to quantify the interaction of intrinsically fluorescent peptides with selected biomembrane model systems. Two antimicrobial peptides (BP100 and pepR) were evaluated by this new method. The results obtained by this new methodology show that ζ-potential is a powerful technique to quantify peptide/lipid interactions of a wide variety of charged molecules, overcoming some of the limitations inherent to other techniques, such as the need for fluorescent labeling.This work was partially supported by project PTDC/QUI/ 69937/2006 from Fundação para a Ciência e Tecnologia-Ministério da Ciência, Tecnologia e Ensino Superior (FCT-MCTES, Portugal), and by Fundação Calouste Gulbenkian (Portugal). JMF and MMD also thank FCT-MCTES for grants IMM/BT/37-2010 and SFRH/BD/41750/2007, respectively

A Study of Problem Solving Using Blocks Vehicle in a STEAM Course for Lower Elementary Levels

STEAM education is currently one of the most important parts of the elementary school curriculum. If STEAM learning can cultivate good problem-solving ability, it will also help improve judgment and thinking abilities. Several voices in the literature have argued for cooperative learning in STEAM courses. Although the effectiveness of course learning often is evaluated using course feedback forms, there is comparatively little emphasis on whether a course succeeds in realizing cooperative learning. For a course involving self-propelled toy-brick cars, there is little research on the application of low-grade pupils. Therefore, based on the integration of STEAM courses into self-propelled toy-brick car learning, this study applied two learning strategies of cooperative learning and individual learning to low-grade pupils in the second grade in elementary schools. After completing the course problem-solving ability indicators were measured and analyzed using the problem-solving ability test. The results show that the mean score of the experimental group in the problem-solving ability test was higher than that of the control group. In the problem-solving ability test, the scores of the two groups were also significantly different, which suggests that cooperative learning is more effective than individual learning strategies.acceptedVersio

Characterization of highly stable liposomal and immunoliposomal formulations of vincristine and vinblastine

Liposome and immunoliposome formulations of two vinca alkaloids, vincristine and vinblastine, were prepared using intraliposomal triethylammonium sucroseoctasulfate and examined for their ability to stabilize the drug for targeted drug delivery in vivo. The pharmacokinetics of both the encapsulated drug (vincristine or vinblastine) and liposomal carrier were examined in Sprague Dawley rats, and the in vivo drug release rates determined. Anti-HER2 immunoliposomal vincristine was prepared from a human anti-HER2/neu scFv and studied for targeted cytotoxic activity in cell culture, and antitumor efficacy in vivo. Nanoliposome formulations of vincristine and vinblastine demonstrated similar pharmacokinetic profiles for the liposomal carrier, but increased clearance for liposome encapsulated vinblastine (t 1/2 = 9.7 h) relative to vincristine (t 1/2 = 18.5 h). Immunoliposome formulations of vincristine targeted to HER2 using an anti-HER2 scFv antibody fragment displayed a marked enhancement in cytotoxicity when compared to non-targeted liposomal vincristine control; 63- or 253-fold for BT474 and SKBR3 breast cancer cells, respectively. Target-specific activity was also demonstrated in HER2-overexpressing human tumor xenografts, where the HER2-targeted formulation was significantly more efficacious than either free vincristine or non-targeted liposomal vincristine. These results demonstrate that active targeting of solid tumors with liposomal formulations of vincristine is possible when the resulting immunoliposomes are sufficiently stabilized

Lack of efficacy of Doxil® in TNF-α-based isolated limb perfusion in sarcoma-bearing rats

textabstractHere we show that Doxil® has minimal antitumour activity in the isolated limb perfusion (ILP) setting and its activity was not enhanced by the addition of tumour necrosis factor (TNF). Doxil® accumulation in tumour tissue was low and also not augmented by TNF. In contrast, activity of free conventional doxorubicin was enhanced by TNF. We conclude that application of Doxil® in a TNF-based ILP is not a useful alternative to free conventional doxorubicin or melphalan

High-order chromatin architecture determines the landscape of chromosomal alterations in cancer

The rapid growth of cancer genome structural information provides an opportunity for a better understanding of the mutational mechanisms of genomic alterations in cancer and the forces of selection that act upon them. Here we test the evidence for two major forces, spatial chromosome structure and purifying (or negative) selection, that shape the landscape of somatic copy-number alterations (SCNAs) in cancer1. Using a maximum likelihood framework we compare SCNA maps and three-dimensional genome architecture as determined by genome-wide chromosome conformation capture (HiC) and described by the proposed fractal-globule (FG) model2. This analysis provides evidence that the distribution of chromosomal alterations in cancer is spatially related to three-dimensional genomic architecture and additionally suggests that purifying selection as well as positive selection shapes the landscape of SCNAs during somatic evolution of cancer cells

IL-21 signaling is essential for optimal host resistance against Mycobacterium tuberculosis infection

IL-21 is produced predominantly by activated CD4(+) T cells and has pleiotropic effects on immunity via the IL-21 receptor (IL-21R), a member of the common gamma chain (gamma(c)) cytokine receptor family. We show that IL-21 signaling plays a crucial role in T cell responses during Mycobacterium tuberculosis infection by augmenting CD8(+) T cell priming, promoting T cell accumulation in the lungs, and enhancing T cell cytokine production. In the absence of IL-21 signaling, more CD4(+) and CD8(+) T cells in chronically infected mice express the T cell inhibitory molecules PD-1 and TIM-3. We correlate these immune alterations with increased susceptibility of IL-21R(-/-) mice, which have increased lung bacterial burden and earlier mortality compared to WT mice. Finally, to causally link the immune defects with host susceptibility, we use an adoptive transfer model to show that IL-21R(-/-) T cells transfer less protection than WT T cells. These results prove that IL-21 signaling has an intrinsic role in promoting the protective capacity of T cells. Thus, the net effect of IL-21 signaling is to enhance host resistance to M. tuberculosis. These data position IL-21 as a candidate biomarker of resistance to tuberculosis.This work was supported by National Institutes of Health Grants R21 AI100766, R01 AI106725, and P01 AI073748

Membrane Partitioning: “Classical” and “Nonclassical” Hydrophobic Effects

The free energy of transfer of nonpolar solutes from water to lipid bilayers is often dominated by a large negative enthalpy rather than the large positive entropy expected from the hydrophobic effect. This common observation has led to the idea that membrane partitioning is driven by the “nonclassical” hydrophobic effect. We examined this phenomenon by characterizing the partitioning of the well-studied peptide melittin using isothermal titration calorimetry (ITC) and circular dichroism (CD). We studied the temperature dependence of the entropic (−TΔS) and enthalpic (ΔH) components of free energy (ΔG) of partitioning of melittin into lipid membranes made of various mixtures of zwitterionic and anionic lipids. We found significant variations of the entropic and enthalpic components with temperature, lipid composition and vesicle size but only small changes in ΔG (entropy–enthalpy compensation). The heat capacity associated with partitioning had a large negative value of about −0.5 kcal mol−1 K−1. This hallmark of the hydrophobic effect was found to be independent of lipid composition. The measured heat capacity values were used to calculate the hydrophobic-effect free energy ΔGhΦ, which we found to dominate melittin partitioning regardless of lipid composition. In the case of anionic membranes, additional free energy comes from coulombic attraction, which is characterized by a small effective peptide charge due to the lack of additivity of hydrophobic and electrostatic interactions in membrane interfaces [Ladokhin and White J Mol Biol 309:543–552, 2001]. Our results suggest that there is no need for a special effect—the nonclassical hydrophobic effect—to describe partitioning into lipid bilayers

Firsthand Experience and The Subsequent Role of Reflected Knowledge in Cultivating Trust in Global Collaboration

While scholars contend that firsthand experience - time spent onsite observing the people, places, and norms of a distant locale - is crucial in globally distributed collaboration, how such experience actually affects interpersonal dynamics is poorly understood. Based on 47 semistructured interviews and 140 survey responses in a global chemical company, this paper explores the effects of firsthand experience on intersite trust. We find firsthand experience leads not just to direct knowledge of the other, but also knowledge of the self as seen through the eyes of the other - what we call “reflected knowledge”. Reflected and direct knowledge, in turn, affect trust through identification, adaptation, and reduced misunderstandings

Spatial Orientation in Japanese Quails (Coturnix coturnix japonica)

Finding a given location can be based on a variety of strategies, for example on the estimation of spatial relations between landmarks, called spatial orientation. In galliform birds, spatial orientation has been demonstrated convincingly in very young domestic chicks. We wanted to know whether adult Japanese quails (Coturnix coturnix japonica) without food deprivation are also able to use spatial orientation. The quails had to learn the relation of a food location with four conspicuous landmarks which were placed in the corners of a square shaped arena. They were trained to find mealworms in three adjacent food cups in a circle of 20 such cups. The rewarded feeders were located during training between the same two landmarks each of which showed a distinct pattern. When the birds had learned the task, all landmarks were displaced clockwise by 90 degrees. When tested in the new situation, all birds redirected their choices with respect to the landmark shift. In subsequent tests, however, the previously correct position was also chosen. According to our results, quails are using conspicuous landmarks as a first choice for orientation. The orientation towards the previously rewarded location, however, indicates that the neuronal representation of space which is used by the birds also includes more fine grain, less conspicuous cues, which are probably also taken into account in uncertain situations. We also presume that the rare orientation towards never rewarded feeders may be due to a foraging strategy instead of being mistakes

Search for rare quark-annihilation decays, B --> Ds(*) Phi

We report on searches for B- --> Ds- Phi and B- --> Ds*- Phi. In the context of the Standard Model, these decays are expected to be highly suppressed since they proceed through annihilation of the b and u-bar quarks in the B- meson. Our results are based on 234 million Upsilon(4S) --> B Bbar decays collected with the BABAR detector at SLAC. We find no evidence for these decays, and we set Bayesian 90% confidence level upper limits on the branching fractions BF(B- --> Ds- Phi) Ds*- Phi)<1.2x10^(-5). These results are consistent with Standard Model expectations.Comment: 8 pages, 3 postscript figues, submitted to Phys. Rev. D (Rapid Communications