Landscape phage, phage display, stripped phage, biosensors, detection, affinity reagent, nanotechnology, Salmonella typhimurium, Bacillus anthracis

Filamentous phage, such as fd used in this study, are thread-shaped bacterial viruses. Their outer coat is a tube formed by thousands equal copies of the major coat protein pVIII. We constructed libraries of random peptides fused to all pVIII domains and selected phages that act as probes specific for a panel of test antigens and biological threat agents. Because the viral carrier is infective, phage borne bio-selective probes can be cloned individually and propagated indefinitely without needs of their chemical synthesis or reconstructing. We demonstrated the feasibility of using landscape phages and their stripped fusion proteins as new bioselective materials that combine unique characteristics of affinity reagents and self assembling membrane proteins. Biorecognition layers fabricated from phage-derived probes bind biological agents and generate detectable signals. The performance of phage-derived materials as biorecognition films was illustrated by detection of streptavidin-coated beads, Bacillus anthracis spores and Salmonella typhimurium cells. With further refinement, the phage-derived analytical platforms for detecting and monitoring of numerous threat agents may be developed, since the biodetector films may be obtained from landscape phages selected against any bacteria, virus or toxin. As elements of field-use detectors, they are superior to antibodies, since they are inexpensive, highly specific and strong binders, resistant to high temperatures and environmental stresses.Comment: Submitted on behalf of TIMA Editions (http://irevues.inist.fr/tima-editions

Alamethicin adsorption to a planar lipid bilayer.

The effect of alamethicin and its derivatives on the voltage-dependent capacitance of phosphatidylethanolamine (squalane) membranes was measured using two different methods: lock-in detection and voltage pulse. Alamethicin and its derivatives modulate the voltage-dependent capacitance at voltages lower than the voltage at which alamethicin-induced conductance is detected. The magnitude and sign of this alamethicin-induced capacitance change depends on the aqueous alamethicin concentration and the kind of alamethicin used. Our experimental data can be interpreted as a potential-dependent pseudocapacitance associated with adsorbed alamethicin. Pseudocapacitance is expressed as a function of alamethicin charge, its concentration in the bathing solution and the applied electric field. The theory describes the dependence of the capacitance on applied voltage and alamethicin concentration. When alamethicin is neutral the theory predicts no change of the voltage-dependent capacitance with either sign of applied voltage. Experimental data are consistent with the model in which alamethicin molecules interact with each other while being adsorbed to the membrane surface. The energy of this interaction depends on the alamethicin concentration

Membrane surface-charge titration probed by gramicidin A channel conductance.

We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the channel conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also influenced by lipid-dependent structural factors

Engineered metal nanoparticles in the sub-nanomolar levels kill cancer cells

Vitaly Vodyanoy,1 Yasmine Daniels,2 Oleg Pustovyy,1 William A MacCrehan,2 Shin Muramoto,2 Gheorghe Stan21Department of Anatomy, Physiology and Pharmacology, Auburn University College of Veterinary Medicine, Auburn, AL, 2Material Measurement Laboratory, National Institute of Standards and Technology, Gaithersburg, MA, USA Background: Small metal nanoparticles obtained from animal blood were observed to be toxic to cultured cancer cells, whereas noncancerous cells were much less affected. In this work, engineered zinc and copper metal nanoparticles were produced from bulk metal rods by an underwater high-voltage discharge method. The metal nanoparticles were characterized by atomic force microscopy and X-ray photoelectron spectroscopy. The metal nanoparticles, with estimated diameters of 1 nm–2 nm, were determined to be more than 85% nonoxidized. A cell viability assay and high-resolution light microscopy showed that exposure of RG2, cultured rat brain glioma cancer cells, to the zinc and copper nanoparticles resulted in cell morphological changes, including decreased cell adherence, shrinking/rounding, nuclear condensation, and budding from cell bodies. The metal-induced cell injuries were similar to the effects of staurosporine, an active apoptotic reagent. The viability experiments conducted for zinc and copper yielded values of dissociation constants of 0.22±0.08 nmol/L (standard error [SE]) and 0.12±0.02 nmol/L (SE), respectively. The noncancerous astrocytes were not affected at the same conditions. Because metal nanoparticles were lethal to the cancer cells at sub-nanomolar concentrations, they are potentially important as nanomedicine.Purpose: Lethal concentrations of synthetic metal nanoparticles reported in the literature are a few orders of magnitude higher than the natural, blood-isolated metal nanoparticles; therefore, in this work, engineered metal nanoparticles were examined to mimic the properties of endogenous metal nanoparticles.Materials and methods: RG2, rat brain glioma cells CTX TNA2 brain rat astrocytes, obtained from the American Type Culture Collection, high-voltage discharge, atomic force microscope, X-ray photoelectron spectroscopy, high-resolution light microscopy, zeta potential measurements, and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay were used in this work.Results: Engineered zinc and copper metal nanoparticles of size 1 nm–2 nm were lethal to cultured RG2 glioma cancer cells. Cell death was confirmed by MTT assay, showing that the relative viability of RG2 glioma cells is reduced in a dose-dependent manner at sub-nanomolar concentrations of the nanoparticles. The noncancerous astrocytes were not affected at the same conditions.Conclusion: The engineered and characterized zinc and copper nanoparticles are potentially significant as biomedicine.Keywords: nanoparticles, XPS, atomic force, glioma cancer cell, zinc, coppe

Probability of alamethicin conductance states varies with nonlamellar tendency of bilayer phospholipids.

With few exceptions, membrane lipids are usually regarded as a kind of filler or passive solvent for membrane proteins. Yet, cells exquisitely control membrane composition. Many phospholipids found in plasma membrane bilayers favor packing into inverted hexagonal bulk phases. It was suggested that the strain of forcing such lipids into a bilayer may affect membrane protein function, such as the operation of transmembrane channels. To investigate this, we have inserted the peptide alamethicin into bilayer membranes composed of lipids of empirically determined inverted hexagonal phase "spontaneous radii" Ro, which will have expectably different degrees of strain when forced into bilayer form. We observe a correlation between measured Ro and the relative probabilities of different conductance states. States of higher conductance are more probable in dioleoylphosphatidylethanolamine, the lipid of highest curvature, 1/Ro, than in dioleoylphosphatidylcholine, the lipid of lowest curvature

Cell volume measurement using scanning ion conductance microscopy.

We report a novel scanning ion conductance microscopy (SICM) technique for assessing the volume of living cells, which allows quantitative, high-resolution characterization of dynamic changes in cell volume while retaining the cell functionality. The technique can measure a wide range of volumes from 10(-19) to 10(-9) liter. The cell volume, as well as the volume of small cellular structures such as lamelopodia, dendrites, processes, or microvilli, can be measured with the 2.5 x 10(-20) liter resolution. The sample does not require any preliminary preparation before cell volume measurement. Both cell volume and surface characteristics can be simultaneously and continuously assessed during relatively long experiments. The SICM method can also be used for rapid estimation of the changes in cell volume. These are important when monitoring the cell responses to different physiological stimuli