Dye Release Experiments Research Articles

The Water-Following Performance of Various Lagrangian Surface Drifters Measured in a Dye Release Experiment

Abstract Many different surface drifter designs have been developed recently to track near-surface ocean currents, but the degree to which these drifters slip through the water because of mechanisms associated with the wind is poorly known. In the 2020 Tracer Release Experiment (TReX), 19 drifters of eight different designs, both commercially available and home-built, were simultaneously released with a patch of rhodamine dye. The dye rapidly spread vertically through the mixed layer but also more slowly dispersed horizontally. Although winds were light, drifters moved downwind from the dye patch at speeds of 3–17 cm s−1 (0.6%–4% of wind speed) depending on the design type. Measurements were made of wind and ocean conditions, and these were incorporated into a boundary layer model at the air–sea interface to estimate complete velocity profiles above and below the surface. Then, a steady-state drag model is used with these profiles to successfully predict drifter slip. Drogued drifters (those with a subsurface drag element) can be affected by Eulerian shear in the upper 0.5 m of the water column, as well as the Stokes drift, but undrogued drifters are in addition greatly affected by direct wind drag, and possibly by resonant interactions with waves. The dye, cycling vertically in the mixed layer, is largely unaffected by all of these factors; therefore, even “perfect” surface drifters do not move with a mixed layer tracer. Significance Statement Surface drifters are used by oceanographers to measure ocean surface currents. However, different designs also slip downwind through the water at rates that are poorly known but are typically around a few percent of the wind speed. In 2020 we simultaneously deployed drifters of eight different designs along with rhodamine dye in a field experiment to see how well the different designs track the water. Here we independently and successfully model drifter slippage for the different designs. Slip factors are then estimated for a range of wind and ocean conditions.

Functionalization strategies to facilitate multi-depth, multi-molecule modifications of nanostructured oxides for triggered release applications

Metal-oxide surfaces have successfully been modified to elicit surface hydrophobicity via facile application of self-assembled monolayers (SAMs) using carbohydrate molecules comprising phosphonic acid groups. In this work, we shed light on the role of the functionalization technique used for surface modification of zirconia nanotubes (ZrNT) and compare the extent of achievable hydrophobic effect as a function of application technique, immersion in bulk solution (BI) and micro-contact printing (μCP), for different molecular chain lengths. Qualitative and quantitative evaluation of ZrNT surfaces modified via μCP and BI in SAM solutions was performed combining static water-contact angle measurements, XPS and ToF-SIMS depth-profiling to provide insights into effective multi-functional depth modifications. Deviating modification depths of the nanostructured oxide could be achieved with these methods. The use of sequential functionalization strategies via BI and μCP bears a ‘proof of principle’ to create nanostructures that are able to demonstrate both multi-molecule and multi-depth modifications. Multifunctional ZrNTs, modified with two molecules in selected depths, were obtained by the combination of both modification methods and evaluated via dye release experiments aimed to simulate drug release behavior. The dispensation of loaded dye was investigated in various solvents. While no dye could be detected in aqueous environment, a complete release of the dye was achieved in ethanol, caused by a degradation of the μCP-ODPA layer. Our results indicate that the release of the cargo of multi-functional oxide nanostructures can be triggered, e.g., by alteration of the chemical environment.

Bacterial Dye Release Measures in Response to Antimicrobial Peptides.

Assessment of bacterial dye release following exposure to antimicrobial peptides (AMPs) provides a detailed understanding regarding their interaction with the inner and outer membrane of bacteria, and the leak of bacterial intracellular materials. This underpins the overall antimicrobial mechanism of these membrane-active peptides. DiSC3(5) is a membrane potential sensitive dye and can characterize the changes in bacterial membrane potential following exposure to AMPs (see Note 1). SYTOX Green is a nucleic acid stain that enters the cell upon loss of membrane integrity after exposure to AMPs and binds to DNA. SYTO9 is another nucleic acid stain, whereas propidium iodide (PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. Both of these stains are widely used to monitor the viability of bacteria following exposure to AMPs. This chapter describes the methods of using these as bacterial dye release experiments for assessment of the antimicrobial mechanism of AMPs.

Horizontal and vertical spreading of dye in the coastal ocean of the northern Mid-Atlantic bight

The paper investigates the horizontal and vertical spreading of a neutrally-buoyant chemical tracer (rhodamine) that was released into the surface mixed layer in the coastal ocean. Rhodamine concentrations from the dye release experiment were combined with observations of water column properties and velocity measurements from the high-frequency radar (HFR) and ship-based ADCP in order to investigate and interpret the downward penetration of dye from the mixed layer into the stratified part of the water column and to quantify the horizontal diffusivity associated with the lateral dye spreading. Of the total amount of dye encountered during the 33-h long survey, 99% was observed within the mixed layer, suggesting that the downward penetration of dye from the mixed layer into the pycnocline is a slow process that occurs on the time scale of at least multiple days. The observed elevated dye concentrations below the mixed layer were always co-located with the elevated concentrations within the mixed layer. However, the distribution of dye below the mixed layer was patchy, and the below-mixed-layer dye concentrations were poorly correlated with those within the mixed layer. Combined with the absence of strong vertical shear of horizontal velocity across the base of the mixed layer, this suggests that the observed dye could have been ejected from the mixed layer earlier and at a different location, and then advected to the observed location by the horizontal currents below the mixed layer. The vertical diffusivity in the mixed layer is estimated to be approximately 7x10−4m2s, about two orders of magnitude larger than the diffusivity across the base of the mixed layer, which is 5x10−6m2s. Lateral spread of dye was found to be well represented by the HFR velocities (with roughly 2-km resolution) with the added small-scale turbulent diffusivity of 5 m2s.Plain Language Summary.The top part of the water column, called the surface mixed layer, is well mixed to a nearly uniform density. Below lays the pycnocline where density changes rapidly with depth. The exchange of water and properties between the mixed layer and pycnocline is an important but poorly understood oceanographic process and is the main subject of the current observational study. Specifically, this paper describes and interprets the initial 36 h of the vertical and horizontal spreading of a dye plume, released into the surface mixed layer of the coastal ocean south of Martha's Vineyard, MA. Our results indicated that the ejection and penetration of dye down from the mixed layer is a slow process that occurs over a time scales longer than multiple days. The episodic nature of the downward dye ejection from the mixed layer and the resulting patchy distribution of the subsurface dye is another important finding from this work, which has far-reaching implications for the parameterization of these small-scale vertical exchange processes in numerical models. The vertical diffusivity within the mixed layer (7x10−4m2s) is estimated to be about two orders of magnitude larger than the diffusivity across the base of the mixed layer (5x10−6m2s). Finally, the lateral spreading of the dye plume was found to be well represented by advection by the high-frequency-radar-based velocities with the added small-scale turbulent diffusivity of 5 m2s – the value that is in line with our prior estimates for this coastal region, but is larger than many prior estimates from different regions of the ocean.

Determination of Optimal Diffusion Coefficients in Lake Zirahuén through a Local Inverse Problem

In this study, optimal diffusion coefficients for Lake Zirahuén, Mexico, were found under particular conditions based on images taken with a drone of a dye release experiment. First, the dye patch concentration was discretized using image processing tools, and it was then approximated by an ellipse, finding the optimal major and minor axes. The inverse problem was implemented by comparing these observational data with the concentration obtained numerically from the 2D advection–diffusion equation, varying the diffusion tensor. When the tensor was isotropic, values of K11=K22≈0.003 m2/s were found; when nonequal coefficients were considered, it was found that K11≈0.005 m2/s and K22≈0.002 m2/s, and the cross-term K12 influenced the results of the orientation of the ellipse. It is important to mention that, with this simple technique, the parameter estimation had consequences of great importance as the value for the diffusion coefficient was bounded significantly under particular conditions for this site of study.

Effects of Glycon and Temperature on Self-Assembly Behaviors of α-Galactosyl Ceramide in Water.

α-Galactosyl ceramide (GalCer) is an anticancer glycolipid consisting of d-galactose and phytosphingosine-based ceramide. Although the amphiphilic structure of GalCer is expected to form self-associates in water, the self-assembly behaviors of GalCer and its derivatives have not been systematically investigated at this moment in spite of its great importance. The evaluation of morphologies and properties of the associates should open new insights into glycolipid chemistry such as the application of GalCer derivatives to a nanocarrier and the elucidation of the detailed pharmacological mechanism of GalCer. Herein, we show the synthesis of the aglycon fragment (Aglycon) of GalCer and the self-assembly behaviors of both GalCer and Aglycon in water. The critical aggregation concentrations of Aglycon and GalCer were determined using UV-vis spectral measurements at various concentrations. The transmission electron microscopy observations of the aqueous sample solutions indicated that the solution of GalCer includes vesicles, while that of Aglycon comprises giant micelles in the absence of vesicles. The vesicle formation in the solution of GalCer was also confirmed by Triton X-100-triggered dye-release experiments. To reveal the effects of glycon on the self-assembly behaviors in detail, we performed the measurements of dynamic light scattering, temperature-dependence of turbidity, differential scanning calorimetry, and wide-angle X-ray diffraction. The results clarify that the glycon moiety of GalCer has a significant role in the formation inhibition of second associates and the plasticization of the hydrophobe. This work will shed light on the other natural glycosides to evaluate the self-assembly behaviors for supramolecular and pharmacological applications in the near future.

Dispersion in the Open Ocean Seasonal Pycnocline at Scales of 1–10 km and 1–6 days

AbstractResults are presented from two dye release experiments conducted in the seasonal thermocline of the Sargasso Sea, one in a region of low horizontal strain rate (~10−6 s−1), the second in a region of intermediate horizontal strain rate (~10−5 s−1). Both experiments lasted ~6 days, covering spatial scales of 1–10 and 1–50 km for the low and intermediate strain rate regimes, respectively. Diapycnal diffusivities estimated from the two experiments were κz = (2–5) × 10−6 m2 s−1, while isopycnal diffusivities were κH = (0.2–3) m2 s−1, with the range in κH being less a reflection of site-to-site variability, and more due to uncertainties in the background strain rate acting on the patch combined with uncertain time dependence. The Site I (low strain) experiment exhibited minimal stretching, elongating to approximately 10 km over 6 days while maintaining a width of ~5 km, and with a notable vertical tilt in the meridional direction. By contrast, the Site II (intermediate strain) experiment exhibited significant stretching, elongating to more than 50 km in length and advecting more than 150 km while still maintaining a width of order 3–5 km. Early surveys from both experiments showed patchy distributions indicative of small-scale stirring at scales of order a few hundred meters. Later surveys show relatively smooth, coherent distributions with only occasional patchiness, suggestive of a diffusive rather than stirring process at the scales of the now larger patches. Together the two experiments provide important clues as to the rates and underlying processes driving diapycnal and isopycnal mixing at these scales.

A Global Glacial Ocean State Estimate Constrained by Upper-Ocean Temperature Proxies

We use the method of least squares with Lagrange multipliers to fit an ocean general circulation model to the Multiproxy Approach for the Reconstruction of the Glacial Ocean Surface (MARGO) estimate of near sea surface temperature (NSST) at the Last Glacial Maximum (LGM; circa 23–19 thousand years ago). Compared to a modern simulation, the resulting global, last-glacial ocean state estimate, which fits the MARGO data within uncertainties in a free-running coupled ocean–sea ice simulation, has global-mean NSSTs that are 2°C lower and greater sea ice extent in all seasons in both the Northern and Southern Hemispheres. Increased brine rejection by sea ice formation in the Southern Ocean contributes to a stronger abyssal stratification set principally by salinity, qualitatively consistent with pore fluid measurements. The upper cell of the glacial Atlantic overturning circulation is deeper and stronger. Dye release experiments show similar distributions of Southern Ocean source waters in the glacial and modern western Atlantic, suggesting that LGM NSST data do not require a major reorganization of abyssal water masses. Outstanding challenges in reconstructing LGM ocean conditions include reducing effects from model biases and finding computationally efficient ways to incorporate abyssal tracers in global circulation inversions. Progress will be aided by the development of coupled ocean–atmosphere–ice inverse models, by improving high-latitude model processes that connect the upper and abyssal oceans, and by the collection of additional paleoclimate observations.

Behavioural modification of local hydrodynamics by asteroids enhances reproductive success

The reproduction of apex species, such as sea stars, is important for sustaining many marine ecosystems. Many sea star species reproduce externally, introducing gametes in the turbulent benthic boundary layer. Sea stars often aggregate and adopt characteristic behaviour, such as arched posturing, while spawning. Here we quantify, for the first time, the hydrodynamic advantages of postural changes and the extent to which they enhance the efficiency of external reproduction. Hydrodynamic and fertilisation kinetic theoretical modelling were used to provide context and comparison. The arched posture was clearly important in the downstream advection of gametes. Digital particle image velocimetry, acoustic doppler velocimetry and dye release experiments indicated reduced wake and lower shear stresses downstream of arched sea stars, which increased downstream transport of gametes compared to those in the flat position. In all cases, sperm concentration decay rates of two orders-of-magnitude over distances <20cm were inferred from fluorometry, confirming the requirement for close aggregation. The level of turbulence and hence downstream gamete dilution was increased by greater current speeds and a rougher seabed. Both an arched posture and hydrodynamic conditions may improve external reproduction efficiency, with behavioural mechanisms providing the primary contribution.

Synthesis and characterisation of robust emulsion-templated silica microcapsules

Robust silica microcapsules were synthesised using an emulsion template via a seeded growth strategy. Multiple additions of the silica precursor tetraethyl orthosilicate (TEOS) were observed to result in a number of physical and property changes of the capsule shells as compared to a single coating. Scanning electron microscopy indicated a morphological transition from a smooth to a roughened surface. Improved cargo retention and consolidation of the pore structure of the silica shells were observed using dye release experiments and nitrogen porosimetry respectively. In comparison to a typical hollow silica shell synthesis procedure, this one-pot loading and synthesis allows the simple production of robust capsules that are capable of sustained release, using mild conditions and reagents.

Bis-clickable Mesoporous Silica Nanoparticles: Straightforward Preparation of Light-Actuated Nanomachines for Controlled Drug Delivery with Active Targeting.

Bis(clickable) mesoporous silica nanospheres (ca. 100 nm) were obtained by the co-condensation of TEOS with variable amounts (2-5 % each) of two clickable organosilanes in the presence of CTAB. Such nanoparticles could be easily functionalized with two independent functions using the copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction to transform them into nanomachines bearing cancer cell targeting ligands with the ability to deliver drugs on-demand. The active targeting was made possible after anchoring folic acid by CuAAC click reaction, whereas the controlled delivery was performed by clicked azobenzene fragments. Indeed, the azobenzene groups are able to obstruct the pores of the nanoparticles in the dark whereas upon irradiation in the UV or in the blue range, their trans-to-cis photoisomerization provokes disorder in the pores, enabling the delivery of the cargo molecules. The on-command delivery was proven in solution by dye release experiments, and in vitro by doxorubicin delivery. The added value of the folic acid ligand was clearly evidenced by the difference of cell killing induced by doxorubicin-loaded nanoparticles under blue irradiation, depending on whether the particles featured the clicked folic acid ligand or not.

Dispersal and dilution of wastewater from an ocean outfall at Davis Station, Antarctica, and resulting environmental contamination

The Antarctic Treaty permits the discharge of wastewater into Antarctic marine waters providing that conditions exist for initial dilution and rapid dispersal. We investigated the dilution and dispersal of macerated wastewater around Australia's Davis Station in East Antarctica and examined sediments for evidence of contaminants. Methods used to examine hydrodynamic conditions included current meters, dye release experiments and measurement of sewage-associated microbial markers and surfactants in the water column. We measured marine sediments for metals, nutrients, PBDEs, hydrocarbons and faecal sterols. We propose that if there is adequate dilution and dispersal there would be no significant difference in contaminant concentrations in sediments around the outfall compared to distant control sites. Currents were strongly correlated with prevailing wind conditions. Modelling indicated that diffusivity of wastewater had the greatest effect on dilution factors and that neither discharge rates nor local currents had as much effect. During summer conditions of open water, wastewater is likely to be constrained in a narrow plume close to the coast. Concentrations of sewage bacteria were high around the outfall and detected up to 1.5 km away, along with dye. There were significant differences in sediment concentrations of metals, PBDEs, hydrocarbons, nutrients and faecal sterols between sites within 2 km of the outfall and control sites. We conclude that dilution and dispersal conditions at the Davis outfall are insufficient to prevent the accumulation of contaminants in local sediments and that microbial hazards posed by wastewater are an environmental risk to local wildlife.

Hydrological connectivity in river deltas: The first‐order importance of channel‐island exchange

AbstractDeltaic systems are composed of distributary channels and interdistributary islands. While previous work has focused either on the channels or on the islands, here we study the hydrological exchange between channels and islands and point at its important role in delta morphology and ecology. We focus our analysis on Wax Lake Delta in coastal Louisiana (USA) and characterize the surface water component of hydrological connectivity through measurements of water discharge and hydraulic tracer propagation. We find that deltaic islands are zones of significant water flux as 23–54% of the incoming distributary channel flux enters the islands. A calculation of the travel times through a channel‐island complex shows travel times through the islands to be at least 3 times their channel counterparts. A dye release experiment also indicates that travel times in islands are much longer that those within channels as dye remained in the island for the 3.8 day duration of the experiment. Additionally, islands are more sensitive than channels to environmental forces such as tides, which cause flow reversal and thus can increase travel times through the islands. Our work defines the “hydrological network” of a river delta to include not only the distributary channel network but also the interdistributary islands, quantifies the implications of channel‐island hydrological connectivity to travel times through the system, and discusses the relevance of our findings to channel mouth dynamics at the delta front and the potential for denitrification in coastal systems.

Horizontal dispersion in shelf seas: High resolution modelling as an aid to sparse sampling

The ability of a hydrodynamic model to reproduce the results of a dye release experiment conducted in a wide shelf sea environment was investigated with the help of the Massachusetts Institute of Technology general circulation model (MITgcm). In the field experiment a fluorescent tracer, Rhodamine WT, was injected into the seasonal pycnocline, and its evolution was tracked for two days using a towed undulating vehicle equipped with a fluorometer and a CTD. With a 50m horizontal resolution grid, and with three different forcings initialized in the model (viz: tides, stationary current, and wind stress on the free surface), it was possible to replicate the dye patch evolution quite accurately. The mechanisms responsible for the enhancement of horizontal dispersion were investigated on the basis of the model results. It was found that enhancement of the dye dispersion was controlled by vertically sheared currents that, in combination with vertical diapycnal mixing, led to a substantial increase in the “effective” horizontal mixing. The values of “effective” horizontal mixing found from the model runs were in good agreement with those obtained from in-situ data, and the probable degree to which the observational techniques undersampled the dye patch was revealed.

Dye Release Experiments with Dextran Loaded Vesicles

Dye release experiments are a widely used method to assess the interactions between membrane-active molecules and lipid membranes. Of particular interest is the ability to assess the degree of the lipid bilayer perturbation by simultaneously encapsulating dye of different sizes, such as dextrans grafted with a chromophore. In this assay, dextran linked to rhodamine or fluorescein are both encapsulated in lipid vesicles to allow quantifying the leakage of each dextran individually from a single sample. For instance, the size evaluation of the lipid pore formed by an antimicrobial peptide has been recently achieved using this protocol (Sani et al., 2013)., 染料释放实验是一种广泛使用的方法来评估膜 - 活性分子和脂质膜之间的相互作用。 特别感兴趣的是通过同时包封不同尺寸的染料（例如用发色团接枝的葡聚糖）来评估脂质双层扰动的程度的能力。 在该测定中，连接到罗丹明或荧光素的葡聚糖都封装在脂质囊泡中，以允许从单个样品单独地量化每个葡聚糖的渗漏。 例如，最近使用该方案获得了由抗微生物肽形成的脂质孔的尺寸评价（Sani等人，2013）。

Aggregation Modulators Interfere with Membrane Interactions of β2-Microglobulin Fibrils

Amyloid fibril accumulation is a pathological hallmark of several devastating disorders, including Alzheimer’s disease, prion diseases, type II diabetes, and others. Although the molecular factors responsible for amyloid pathologies have not been deciphered, interactions of misfolded proteins with cell membranes appear to play important roles in these disorders. Despite increasing evidence for the involvement of membranes in amyloid-mediated cytotoxicity, the pursuit for therapeutic strategies has focused on preventing self-assembly of the proteins comprising the amyloid plaques. Here we present an investigation of the impact of fibrillation modulators upon membrane interactions of β2-microglobulin (β2m) fibrils. The experiments reveal that polyphenols (epigallocatechin gallate, bromophenol blue, and resveratrol) and glycosaminoglycans (heparin and heparin disaccharide) differentially affect membrane interactions of β2m fibrils measured by dye-release experiments, fluorescence anisotropy of labeled lipid, and confocal and cryo-electron microscopies. Interestingly, whereas epigallocatechin gallate and heparin prevent membrane damage as judged by these assays, the other compounds tested had little, or no, effect. The results suggest a new dimension to the biological impact of fibrillation modulators that involves interference with membrane interactions of amyloid species, adding to contemporary strategies for combating amyloid diseases that focus on disruption or remodeling of amyloid aggregates.

Solute dispersion in the coastal boundary layer of southern Lake Michigan

We evaluate a three‐dimensional, nested‐grid nearshore model of Lake Michigan for its ability to describe key aspects of hydrodynamics and solute transport using data from a field study conducted in summer 2008. Velocity comparisons with observations from five bottom‐mounted ADCPs at different depths in the coastal boundary layer (CBL) show that the numerical model was able to simulate currents and flow reversals accurately within the inertial boundary layer, however model accuracy reduced close to the shoreline. Power spectra of observed and simulated velocity time series at different locations showed that the hydrodynamic model was able to describe the energy contained in the inertial scales, but over‐predicted turbulent dissipation rates. As a result, model‐predicted values of energy in the smaller, dissipation scales were lower compared to values calculated from observations in the CBL. Differences between energy contained in the observed and simulated velocity spectra increased as the shoreline is approached. Observations showed that vertical variations in the alongshore and cross‐shore velocities were dominated by inertial waves. Inaccuracies in representing energy dissipation rates and processes not explicitly described in the hydrodynamic model (e.g., anisotropy, waves) could potentially contribute to errors in describing transport in the CBL. Measurements from a continuous dye release experiment from a riverine outfall were described using a nearshore model with a mean horizontal dispersion coefficient of 5.6 m2/s. Improved representations of physical processes (such as turbulence, internal waves and wave‐current interactions) can be expected to provide better descriptions of solute transport in the CBL.

An experimental study of the spatial structures of the instabilities of longshore currents on plane beaches

A laboratory experiment on alongshore currents is conducted for two plane beaches with slopes 1:40 and 1:100 to investigate the instability of alongshore currents. The dye release experiment is also performed synchronously in surf zone. Complicated and strongly unstable motions of alongshore currents are observed in the experiment. To examine the spatial and temporal variations of the shear instabilities of longshore clearly, dye batches are released in surf zone. The deformation of the dye patch is observed efficiently and effectively with charge coupled device (CCD) system. Some essential characteristics of the shear instability are validated from the results of image analyses of the temporal variation of the dye patch. The influences of alongshore currents, Stokes drift, large-scale vorticity and the shear instabilities on the transport of dye are analyzed using the collected images. The spatial structure of the instabilities of longshore currents is studied by analyzing collected images of the dye patch. And the phase velocity of the meandering movements is obtained through measuring the movement distances of the oscillations of dye patch in alongshore direction with time. The results suggest that the propagation speed of the shear instability is approximately 50%–75% of maximum of mean alongshore currents for irregular and regular waves. The calculated propagation speed using a linear instability analysis theory is compared with the experimental results. The comparison shows agreements between them.

Combinatorial Effects of Charge Characteristics and Hydrophobicity of Silk Fibroin on the Sorption and Release of Charged Dyes

The present study was designed to examine the influence of the charge characteristics of silk fibroin on the sorption and release of charged dyes by varying the pH values of the sorption and release media as well as types of charged dyes. Negatively charged dyes (phenol red and chromotrope 2R) and positively charged dyes (crystal violet and indoine blue) were used as the model compounds. Silk fibroin films were prepared by using a solution casting technique. The prepared films were then treated with an aqueous methanol solution or annealed with water to control their conformation. The sorption behavior of the model compounds made by the methanol-treated and water-annealed silk fibroin films was investigated. Compared to the water- annealed silk fibroin films, a higher hydrophobicity of the methanol-treated silk fibroin films caused a higher sorption of the hydrophobic dyes. The dye molecules had a fairly high affinity to the silk fibroin film, even though the dye and the matrix possessed the same charge. However, in the presence of two charged groups in a single dye molecule, the electrostatic repulsion become more dominant. Stronger interaction was observed when the charges of the film and the dye were opposite. The results of dye sorption and release experiments showed that the degree of synergism or competition between electrostatic and hydrophobic interactions directly depended on the charges and chemical structure of the dye molecules and the environmental pH conditions of the existing silk fibroin film.

Relative Spatial Positions of Tryptophan and Cationic Residues in Helical Membrane-active Peptides Determine Their Cytotoxicity

The cytotoxic activity of 10 analogs of the idealized amphipathic helical 21-mer peptide (KAAKKAA)3, where three of the Ala residues at different positions have been replaced with Trp residues, has been investigated. The peptide's cytotoxic activity was found to be markedly dependent upon the position of the Trp residues within the hydrophobic sector of an idealized α-helix. The peptides with Trp residues located opposite the cationic sector displayed no antitumor activity, whereas those peptides with two or three Trp residues located adjacent to the cationic sector exhibited high cytotoxic activity when tested against three different cancer cell lines. Dye release experiments revealed that in contrast to the peptides with Trp residues located opposite the cationic sector, the peptides with Trp residues located adjacent to the cationic sector induced a strong permeabilizing activity from liposomes composed of a mixture of zwitterionic phosphatidylcholine and negatively charged phosphatidylserine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS)) (2:1) but not from liposomes composed of zwitterionic phosphatidylcholine, POPC. Fluorescence blue shift and quenching experiments revealed that Trp residues inserted deeper into the hydrophobic environment of POPC/POPS liposomes for peptides with high cytotoxic activity. Through circular dichroism studies, a correlation between the cytotoxic activity and the α-helical propensity was established. Structural studies of one inactive and two active peptides in the presence of micelles using NMR spectroscopy showed that only the active peptides adopted highly coiled to helical structures when bound to a membrane surface.