Inferring models of bacterial dynamics toward point sources

Experiments have shown that bacteria can be sensitive to small variations in chemoattractant (CA) concentrations. Motivated by these findings, our focus here is on a regime rarely studied in experiments: bacteria tracking point CA sources (such as food patches or even prey). In tracking point sources, the CA detected by bacteria may show very large spatiotemporal fluctuations which vary with distance from the source. We present a general statistical model to describe how bacteria locate point sources of food on the basis of stochastic event detection, rather than CA gradient information. We show how all model parameters can be directly inferred from single cell tracking data even in the limit of high detection noise. Once parameterized, our model recapitulates bacterial behavior around point sources such as the "volcano effect". In addition, while the search by bacteria for point sources such as prey may appear random, our model identifies key statistical signatures of a targeted search for a point source given any arbitrary source configuration

ANALYSIS OF MULTICHANNEL SIGNALS USING A CHANNEL SIMULATOR

poster abstractsIn biological systems ion channels can be thought of as opening and clos-ing pores that allow the flow of ion through a membrane. Ion channels are responsible for intracellular communication and keeping an osmotic equilib-rium across cell membranes. Measurements of ion channel activities that generate multichannel events (the opening of more than one channel at a time) are important to understand, but difficult to analyze. In many cases the multichannel events are discarded instead of used in the analysis. The availability of a channel signal simulator offers an excellent opportunity to develop and test statistical models for analysis of multichannel signals. We have generated single channel traces for various open probabilities and then digitally superimposed these signals to obtain multichannel events. We then applied our simplified analysis to these multichannel traces to calculate sin-gle channel parameters such as the average ON and OFF times and their statistical distribution of the ON times. The average ON time as well as the ON time distribution matched the single channel input mean open time. The importance of our findings is that our proposed analysis will be able to use the statistical distribution of ON times, in addition to the traditional “dwell time” (or average ON time) parameter, to characterize ion channels

Direct affinity of dopamine to lipid membranes investigated by Nuclear Magnetic Resonance spectroscopy

Dopamine, a naturally occurring neurotransmitter, plays an important role in the brain’s reward system and acts on sensory receptors in the brain. Neurotransmitters are contained in lipid membraned vesicles and are released by exocytosis. All neurotransmitters interact with transport and receptor proteins in glial cells, on neuronal dendrites, and at the axonal button, and also must interact with membrane lipids. However, the extent of direct interaction between lipid membranes in the absence of receptors and transport proteins has not been extensively investigated. In this report, we use UV and NMR spectroscopy to determine the affinity and the orientation of dopamine interacting with lipid vesicles made of either phosphatidylcholine (PC) or phosphatidylserine (PS) lipids which are primary lipid components of synaptic vesicles. We quantify the interaction of dopamine's aromatic ring with lipid membranes using our newly developed method that involves reference spectra in hydrophobic environments. Our measurements show that dopamine interacts with lipid membranes primarily through the aromatic side opposite to the hydroxyl groups, with this aromatic side penetrating deeper into the hydrophobic region of the membrane. Since dopamine's activity involves its release into extracellular space, we have used our method to also investigate dopamine's release from lipid vesicles. We find that dopamine trapped inside PC and PS vesicles is released into the external solution despite its affinity to membranes. This result suggests that dopamine's interaction with lipid membranes is complex and involves both binding as well as permeation through lipid bilayers, a combination that could be an effective trigger for apoptosis of dopamine-generating cells

Hydrophobic Effects on Tyrosyl Ring 1H Chemical Shifts in Peptides

poster abstractHydrophobic environmental effects on tyrosine are measurable by 1H NMR spectroscopy and can allow us to detect interactions between peptides and lipid membranes. We first investigated the effects of hydrophobic environments on the 1H chemical shifts of tyrosine ring protons by using varying concentrations of isopropanol to mimic and calibrate the effects of hydrophobicity. Compared with this calibration, we then measured the interaction of tyrosine-containing peptides with sonicated unilamellar vesicles of phosholipids such as phosphatidylcholine and phosphatidylserine that are commonly found in biological membranes

Effects of Carbon Nanotubes in Barrier Epithelial Cells via Effects on Lipid Bilayers

poster abstractCarbon nanotubes (CNTs) are one of many nanoparticles (NP) which are being developed as part of the burgeoning nanotechnology. The tubes have similar physical properties to known toxic materials, such as asbestos; yet there is a lack of evidence showing that they may be hazardous to humans, specifically to our barrier epithelial cells. We measured the effects of CNTs on human airway epithelial cells (Calu-3 cell line) using electrophysiology. This is a technique which measures transepithelial electrical resistance (TEER), a measure of monolayer integrity; and short circuit current (SCC) a measure of net ion transport across the cell. Exposed cells showed significant decreases in TEER when incubated for 48 hours with physiologically relevant concentrations of 4μg/cm2 - 0.4ng/cm2 of multi-wall (MW) and 4μg/cm2 - 0.04ng/cm2 single-wall (SW) CNT. TEER is a measure of barrier function which is important in cells that maintain separate compartments in the body. The impaired barrier function, despite sustained cell viability, led us to investigate the mechanism by which the CNT were interacting with the cell when applied topically. Model lipid membranes connected to an ion channel amplifier, Planar Bilayer Workstation (BLM), were used. Membranes were formed using the neutral diphytanoylphosphatidylcholine (DPhPC) and negatively charged diphytanoyl phosphatidylserine (DPhPS) lipids. CNTs caused random, transient currents ranging from 0pA to 6479pA to traverse the membrane. In the presence of Gramicidin A, an ion channel reporter protein, the tubes induced increased gramicidin channel formation in the membrane to saturation level and then membrane lysis. This CNT- lipid interaction indicated that short MWCNTs permits unregulated ion movement across the lipid membrane. Disruption in the selective permeability of the plasmalemma may impact the tissue’s barrier function

Fluorescence Measurements of Aromatic Amino Acids in the Presence of Lipid Membranes

Amphiphilic peptides are capable of finding their way to, and occasionally through, cellular membranes using a mechanism that includes specific amino acid sequences. Physical measurements of amino acid-lipid interactions are of interest for a quantitative description of peptide affinities to biological membranes. In this study, we investigate small peptide-lipid interactions using the fluorescence of the aromatic amino acids tyrosine (Tyr), tryptophan (Trp) and phenylalanine (Phe). Reference spectra in deuterated isopropanol solutions are obtained to mimic hydrophobic environments and are used to quantify the interaction of Lys-Tyr-Lys, Trp-Gly, and Gly-Phe with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and palmitoyl-oleoyl phosphatidylserine (POPS) lipid membranes. These fluorescence data complement previously reported UV absorption data and have the advantage of eliminating background and scatter from solution. Together with NMR data, these results can be used to more fully characterize lipid-aromatic amino residue interactions

Effect of carbon nanotubes on transepithelial resistance in barrier epithelial cells

poster abstractThe burgeoning of the nanotechnology industry has revolutionized engineering, medicine and the fashion industry amongst many other technologies. The arrays of products that are synthesized from nanomaterials include high definition TV and computer screens, artificial organs, as well as antibacterial food containers. With these novel applications there is a paralleled concern of the negative implications of nano-material contamination in our environment and food chains. We are interested in carbon nanotubes because they are the most abundantly occurring nanoparticles that are found in the workplace. We have recently conducted studies in barrier epithelial cells to show that long single-wall and multi-wall carbon nanotubes (CNTs) caused a decrease in the transepithelial electrical resistance, a measure of the barrier function ,of renal principal cells at very low concentrations (0.4 ng/cm2- 4 g/cm2). These results suggested that nanoparticles may also cause an effect on other barrier epithelial cells such as those lining the human digestive and respiratory tracks. After 48 hours of CNT exposure, to airway and colon cell lines, Calu-3 and t84 respectively, the calculated resistances were approximately half of the control monolayers’, indicating that the barrier function of the tissue had been compromised, while the cellular monolayer remained intact. We sought to determine the mechanism of action of the nanoparticles, by investigating the interaction of CNTs with model lipid membranes using a bilayer clamp amplifier. Measurements showed that the presence of nanoparticles caused transient disruptions in lipid membranes made of phosphatidylcholine lipids. Nanotubes also caused transient interruptions in the current allowed by the ion channel reporter (gramicidin A). These results began to elucidate the mode of action of the particles and indicated that it is important to develop a complete understanding of how nanoparticles interact with cells if we are to safeguard against changes that these materials will cause in vivo