Mechanical properties of hydrated electrospun polycaprolactone (PCL) nanofibers.

Polycaprolactone (PCL) nanofibers are a promising material for biomedical applications due to their biocompatibility, slow degradation rate, and thermal stability. We electrospun PCL fibers onto a striated substrate with 12 µm wide ridges and grooves and determined their mechanical properties in an aqueous solution with a combined atomic force/inverted optical microscopy technique. Fiber diameters, D, ranged from 27 to 280 nm. The hydrated PCL fibers had an extensibility (breaking strain), εmax, of 137%. The Young's modulus, E, and tensile strength, σT, showed a strong dependence on fiber diameter, D; decreasing steeply with increasing diameter, following empirical equations E(D)=(4.3∙103∙e-D51nm+1.1∙102) MPa and σT(D)=(2.6∙103∙e-D55nm+0.6∙102) MPa. Incremental stress-strain measurements were employed to investigate the viscoelastic behavior of these fibers. The fibers exhibited stress relaxation with a fast and slow relaxation time of 3.7 ± 1.2 s and 23 ± 8 s and these experiments also allowed the determination of the elastic and viscous moduli. Cyclic stress-strain curves were used to determine that the elastic limit of the fibers, εelastic, is between 19% and 36%. These curves were also used to determine that these fibers showed small energy losses (<20%) at small strains (ε < 10%), and over 50% energy loss at large strains (ε > 50%), asymptotically approaching 61%, as Eloss=61%·(1-e-0.04*ε). Our work is the first mechanical characterization of hydrated electrospun PCL nanofibers; all previous experiments were performed on dry PCL fibers, to which we will compare our data.

Mice expressing nonpolymerizable fibrinogen have reduced arterial and venous thrombosis with preserved hemostasis.

ABSTRACT: Elevated circulating fibrinogen levels correlate with increased risk for both cardiovascular and venous thromboembolic diseases. In vitro studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. Here, we analyzed the impact of nonpolymerizable fibrinogen on arterial and venous thrombosis as well as hemostasis in vivo using FgaEK mice that express normal levels of a fibrinogen that cannot be cleaved by thrombin. In a model of carotid artery thrombosis, FgaWT/EK and FgaEK/EK mice were protected from occlusion with 4% ferric chloride (FeCl3) challenges compared with wild-type (FgaWT/WT) mice, but this protection was lost, with injuries driven by higher concentrations of FeCl3. In contrast, fibrinogen-deficient (Fga-/-) mice showed no evidence of occlusion, even with high-concentration FeCl3 challenge. Fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content were similar in FgaWT/WT, FgaWT/EK, and FgaEK/EK mice, consistent with preserved fibrinogen-platelet interactions that support arterial thrombosis with severe challenge. In an inferior vena cava stasis model of venous thrombosis, FgaEK/EK mice had near complete protection from thrombus formation. FgaWT/EK mice also displayed reduced thrombus incidence and a significant reduction in thrombus mass relative to FgaWT/WT mice after inferior vena cava stasis, suggesting that partial expression of nonpolymerizable fibrinogen was sufficient for conferring protection. Notably, FgaWT/EK and FgaEK/EK mice had preserved hemostasis in multiple models as well as normal wound healing times after skin incision, unlike Fga-/- mice that displayed significant bleeding and delayed healing. These findings indicate that a nonpolymerizable fibrinogen variant can significantly suppress occlusive thrombosis while preserving hemostatic potential in vivo.

What is the diameter of a fibrin fiber?

Background: Altered fibrin fiber structure is linked to pathologic states, including coronary heart disease, ischemic stroke, and atherosclerosis. However, several different techniques are commonly utilized for studying fibrin structures, and comparison of results obtained using different techniques can be challenging due to lack of standardization. Objectives: This study provides a path toward standardization by comparing fibrin fiber diameters for a range of physiologic fibrinogen and thrombin concentrations using multiple different complementary experimental methods. Methods: We determined fiber diameter using scanning electron microscopy (SEM), superresolution (stochastic optical reconstruction microscopy) fluorescence microscopy, and 4 commonly utilized turbidimetric approaches to determine the congruence between the results and the conditions under which each should be used. Results: We found that diameter values obtained using SEM and superresolution imaging agree within 10% for nearly all conditions tested. We also found that when a wavelength range of 500 to 800 nm was used for measurements and accounting for the wavelength dependence of the refractive index and specific refractive index increment, diameters obtained using the corrected Yeromonahos turbidimetric approach agree with SEM within 20% for most conditions. Conclusion: We performed a systematic, multitechnique survey assessing fibrin fiber diameters under a range of biochemical conditions. The similarity in the diameter values obtained using SEM and superresolution imaging suggests that drying and fixation during SEM sample preparation do not dramatically alter fiber cross-sections. Congruence, under certain conditions, between diameter values obtained using SEM, superresolution fluorescence imaging, and turbidimetry demonstrates the feasibility of a fibrin diameter standardization project.

The Mechanical Properties of Blended Fibrinogen:Polycaprolactone (PCL) Nanofibers.

Electrospinning is a process to produce versatile nanoscale fibers. In this process, synthetic and natural polymers can be combined to produce novel, blended materials with a range of physical, chemical, and biological properties. We electrospun biocompatible, blended fibrinogen:polycaprolactone (PCL) nanofibers with diameters ranging from 40 nm to 600 nm, at 25:75 and 75:25 blend ratios and determined their mechanical properties using a combined atomic force/optical microscopy technique. Fiber extensibility (breaking strain), elastic limit, and stress relaxation times depended on blend ratios but not fiber diameter. As the fibrinogen:PCL ratio increased from 25:75 to 75:25, extensibility decreased from 120% to 63% and elastic limit decreased from a range between 18% and 40% to a range between 12% and 27%. Stiffness-related properties, including the Young's modulus, rupture stress, and the total and relaxed, elastic moduli (Kelvin model), strongly depended on fiber diameter. For diameters less than 150 nm, these stiffness-related quantities varied approximately as D-2; above 300 nm the diameter dependence leveled off. 50 nm fibers were five-ten times stiffer than 300 nm fibers. These findings indicate that fiber diameter, in addition to fiber material, critically affects nanofiber properties. Drawing on previously published data, a summary of the mechanical properties for fibrinogen:PCL nanofibers with ratios of 100:0, 75:25, 50:50, 25:75 and 0:100 is provided.

The Applicability of Current Turbidimetric Approaches for Analyzing Fibrin Fibers and Other Filamentous Networks.

Turbidimetry is an experimental technique often used to study the structure of filamentous networks. To extract structural properties such as filament diameter from turbidimetric data, simplifications to light scattering theory must be employed. In this work, we evaluate the applicability of three commonly utilized turbidimetric analysis approaches, each using slightly different simplifications. We make a specific application towards analyzing fibrin fibers, which form the structural scaffold of blood clots, but the results are generalizable. Numerical simulations were utilized to assess the applicability of each approach across a range of fiber lengths and diameters. Simulation results indicated that all three turbidimetric approaches commonly underestimate fiber diameter, and that the "Carr-Hermans" approach, utilizing wavelengths in the range of 500−800 nm, provided <10% error for the largest number of diameter/length combinations. These theoretical results were confirmed, under select conditions, via the comparison of fiber diameters extracted from experimental turbidimetric data, with diameters obtained using super-resolution microscopy.

Development of Transient Recombinant Expression and Affinity Chromatography Systems for Human Fibrinogen.

Fibrin forms the structural scaffold of blood clots and has great potential for biomaterial applications. Creating recombinant expression systems of fibrinogen, fibrin's soluble precursor, would advance the ability to construct mutational libraries that would enable structure-function studies of fibrinogen and expand the utility of fibrin as a biomaterial. Despite these needs, recombinant fibrinogen expression systems, thus far, have relied on the time-consuming creation of stable cell lines. Here we present tests of a transient fibrinogen expression system that can rapidly generate yields of 8-12 mg/L using suspension HEK Expi293TM cells. We report results from two different plasmid systems encoding the fibrinogen cDNAs and two different transfection reagents. In addition, we describe a novel, affinity-based approach to purifying fibrinogen from complex media such as human plasma. We show that using a high-affinity peptide which mimics fibrin's knob 'A' sequence enables the purification of 50-75% of fibrinogen present in plasma. Having robust expression and purification systems of fibrinogen will enable future studies of basic fibrin(ogen) biology, while paving the way for the ubiquitous use of fibrin as a biomaterial.

Strength, deformability and toughness of uncrosslinked fibrin fibers from theoretical reconstruction of stress-strain curves.

Structural mechanisms underlying the mechanical properties of fibrin fibers are elusive. We combined tensile testing of uncrosslinked fibrin polymers in vitro and in silico to explore their material properties. The experimental stress (σ) - strain (ε) curves for fibrin fibers are characterized by elastic deformations with a weaker elastic response for ε<160% due to unraveling of αC tethers and straightening of fibrin protofibrils, and a stronger response for ε>160% owing to unfolding of the coiled coils and γ nodules in fibrin monomers. Fiber rupture for strains ε>212% is due to dissociation of the knob-hole bonds and rupture of D:D interfaces. We developed the Fluctuating Bilinear Spring model to interpret the σ-ε profiles in terms of the free energy for protofibril alignment ΔG0 = 10.1-11.5 kBT, Young's moduli for protofibril alignment Yu = 1.9-3.2 MPa and stretching Ya = 5.7-9.7 MPa, strain scale ε˜≈ 12-40% for fiber rupture, and protofibril cooperativity m= 3.6-8. We applied the model to characterize the fiber strength σcr≈ 12-13 MPa, deformability εcr≈ 222%, and rupture toughness U≈ 9 MJ/m3, and to resolve thermodynamic state functions, 96.9 GJ/mol entropy change for protofibril alignment (at room temperature) and 113.6 GJ/mol enthalpy change for protofibril stretching, which add up to 210.5 GJ/mol free-energy change. Fiber elongation is associated with protofibril dehydration and sliding mechanism to create an ordered protofibril array. Fibrin fibers behave like a hydrogel; protofibril dehydration and water expulsion account for ∼94-98% of the total free-energy changes for fiber elongation and rupture. STATEMENT OF SIGNIFICANCE: Structural mechanisms underlying the mechanical properties of fibrin fibers, major components of blood clots and obstructive thrombi, are elusive. We performed tensile testing of uncrosslinked fibrin polymers in vitro and in silico to explore their material properties. Fluctuating Bilinear Spring theory was developed to interpret the stress-strain profiles in terms of the energy for protofibril alignment, elastic moduli for protofibril alignment and stretching, and strain scale for fiber rupture, and to probe the limits of fiber strength, extensibility and toughness. Fibrin fibers behave like a hydrogel. Fiber elongation is defined by the protofibril dehydration and sliding. Structural rearrangements in water matrix control fiber elasticity. These results contribute to fundamental understanding of blood clot breakage that underlies thrombotic embolization.

Automated Fiber Diameter and Porosity Measurements of Plasma Clots in Scanning Electron Microscopy Images.

Scanning Electron Microscopy (SEM) is a powerful, high-resolution imaging technique widely used to analyze the structure of fibrin networks. Currently, structural features, such as fiber diameter, length, density, and porosity, are mostly analyzed manually, which is tedious and may introduce user bias. A reliable, automated structural image analysis method would mitigate these drawbacks. We evaluated the performance of DiameterJ (an ImageJ plug-in) for analyzing fibrin fiber diameter by comparing automated DiameterJ outputs with manual diameter measurements in four SEM data sets with different imaging parameters. We also investigated correlations between biophysical fibrin clot properties and diameter, and between clot permeability and DiameterJ-determined clot porosity. Several of the 24 DiameterJ algorithms returned diameter values that highly correlated with and closely matched the values of the manual measurements. However, optimal performance was dependent on the pixel size of the images-best results were obtained for images with a pixel size of 8-10 nm (13-16 pixels/fiber). Larger or smaller pixels resulted in an over- or underestimation of diameter values, respectively. The correlation between clot permeability and DiameterJ-determined clot porosity was modest, likely because it is difficult to establish the correct image depth of field in this analysis. In conclusion, several DiameterJ algorithms (M6, M5, T3) perform well for diameter determination from SEM images, given the appropriate imaging conditions (13-16 pixels/fiber). Determining fibrin clot porosity via DiameterJ is challenging.

Human mammary epithelial cells in a mature, stratified epithelial layer flatten and stiffen compared to single and confluent cells.

BACKGROUND: The epithelium forms a protective barrier against external biological, chemical and physical insults. So far, AFM-based, micro-mechanical measurements have only been performed on single cells and confluent cells, but not yet on cells in mature layers. METHODS: Using a combination of atomic force, fluorescence and confocal microscopy, we determined the changes in stiffness, morphology and actin distribution of human mammary epithelial cells (HMECs) as they transition from single cells to confluency to a mature layer. RESULTS: Single HMECs have a tall, round (planoconvex) morphology, have actin stress fibers at the base, have diffuse cortical actin, and have a stiffness of 1 kPa. Confluent HMECs start to become flatter, basal actin stress fibers start to disappear, and actin accumulates laterally where cells abut. Overall stiffness is still 1 kPa with two-fold higher stiffness in the abutting regions. As HMECs mature and form multilayered structures, cells on apical surfaces become flatter (apically more level), wider, and seven times stiffer (mean, 7 kPa) than single and confluent cells. The main drivers of these changes are actin filaments, as cells show strong actin accumulation in the regions where cells adjoin, and in the apical regions. CONCLUSIONS: HMECs stiffen, flatten and redistribute actin upon transiting from single cells to mature, confluent layers. GENERAL SIGNIFICANCE: Our findings advance the understanding of breast ductal morphogenesis and mechanical homeostasis.

Mechanical Properties of Electrospun, Blended Fibrinogen: PCL Nanofibers.

Electrospun nanofibers manufactured from biocompatible materials are used in numerous bioengineering applications, such as tissue engineering, creating organoids or dressings, and drug delivery. In many of these applications, the morphological and mechanical properties of the single fiber affect their function. We used a combined atomic force microscope (AFM)/optical microscope technique to determine the mechanical properties of nanofibers that were electrospun from a 50:50 fibrinogen:PCL (poly-ε-caprolactone) blend. Both of these materials are widely available and biocompatible. Fibers were spun onto a striated substrate with 6 µm wide grooves, anchored with epoxy on the ridges and pulled with the AFM probe. The fibers showed significant strain softening, as the modulus decreased from an initial value of 1700 MPa (5-10% strain) to 110 MPa (>40% strain). Despite this extreme strain softening, these fibers were very extensible, with a breaking strain of 100%. The fibers exhibited high energy loss (up to 70%) and strains larger than 5% permanently deformed the fibers. These fibers displayed the stress-strain curves of a ductile material. We provide a comparison of the mechanical properties of these blended fibers with other electrospun and natural nanofibers. This work expands a growing library of mechanically characterized, electrospun materials for biomedical applications.

Interpretation and Validation of Maximum Absorbance Data Obtained from Turbidimetry Analysis of Plasma Clots.

Turbidimetry is used to characterize fibrin clot properties. In purified systems, maximum absorbance (MA) directly relates to fibrin fiber cross-sectional area. However, in plasma samples there are discrepancies in the relationships between MA and fibrinogen concentration, fiber diameter, other clot properties, and cardiovascular disease outcomes, which complicate data interpretation. This study aims to advance understanding of MA of plasma clots through testing how well it relates to fundamental dependence on fibrinogen concentration and fiber diameter as predicted by light scattering theory, other clot properties and lifestyle, and biochemical variables. Plasma samples from 30 apparently healthy individuals with a fibrinogen concentration from 2.4 to 6.4 g/L were included. We performed turbidimetry, permeability, scanning electron microscopy, and rheometry on in vitro formed plasma clots. MA correlated more strongly with fibrinogen concentration (r = 0.65; p < 0.001) than with fiber diameter (r = 0.47; p = 0.01), which combined explained only 46% of the MA variance. Of additional variables measured, only low-density lipoprotein cholesterol correlated with MA (r = 0.46; p = 0.01) and clot lysis (r = 0.62; p < 0.0001) but not with fiber diameter or fibrinogen concentration. MA correlated with clot lysis time (r = 0.59; p = 0.001), storage modulus (r = 0.61; p = 0.001), and loss modulus (r = 0.59; p = 0.001), and negatively with clot permeability (r = -0.60; p = 0.001) also after adjustment for fibrinogen concentration and fiber diameter. Increased MA is indicative of a prothrombotic clot phenotype irrespective of fibrinogen concentration. MA is more indicative of overall clot density than of fiber diameter. Other plasma components can alter internal fiber density without altering fiber diameter and should be considered when interpreting MA of plasma samples.

Development of Zinc Chelating Resin Polymer Beads for the Removal of Cell-Free Hemoglobin.

Red blood cell (RBC) hemolysis is one of the most common storage lesions in packed RBCs (pRBC). Older units of pRBCs, especially those > 21 days old, have increasing levels of hemolysis leading to increased oxidative stress and premature platelet activation. This effect can mostly be attributed to the increase of cell-free hemoglobin (Hb). Therefore, removal of cell-free Hb from pRBCs prior to transfusion could mitigate these deleterious effects. We propose a new method for the removal of Hb from pRBCs using zinc beads. Prepared Hb solutions and pRBCs were treated with zinc beads using two different protocols. UV-Vis spectrophotometry was used to determine Hb concentrations, before and after treatment. Experiments were run in triplicate and paired t tests were used to determine significant differences between groups. Zinc beads removed on average 94% of cell-free Hb within 15 min and 78% Hb from pRBCs (p < 0.0001), demonstrating a maximum binding capacity ~ 66.2 ± 0.7 mg Hb/mL beads. No differences in RBC morphology or deformability were observed after treatment. This study demonstrates the feasibility of using zinc beads for the rapid and targeted removal of Hb from pRBC units. Further investigation is needed to scale this method for large volume removal.

Erythrocytic bioactivation of nitrite and its potentiation by far-red light.

BACKGROUND: Nitrite is reduced by heme-proteins and molybdenum-containing enzymes to form the important signaling molecule nitric oxide (NO), mediating NO signaling. Substantial evidence suggests that deoxygenated hemoglobin within red blood cells (RBCs) is the main erythrocytic protein responsible for mediating nitrite-dependent NO signaling. In other work, infrared and far red light have been shown to have therapeutic potential that some attribute to production of NO. Here we explore whether a combination of nitrite and far red light treatment has an additive effect in NO-dependent processes, and whether this effect is mediated by RBCs. METHODS AND RESULTS: Using photoacoustic imaging in a rat model as a function of varying inspired oxygen, we found that far red light (660 nm, five min. exposure) and nitrite feeding (three weeks in drinking water at 100 mg/L) each separately increased tissue oxygenation and vessel diameter, and the combined treatment was additive. We also employed inhibition of human platelet activation measured by flow cytometry to assess RBC-dependent nitrite bioactivation and found that far red light dramatically potentiates platelet inhibition by nitrite. Blocking RBC-surface thiols abrogated these effects of nitrite and far-red light. RBC-dependent production of NO was also shown to be enhanced by far red light using a chemiluminescence-based nitric oxide analyzer. In addition, RBC-dependent bioactivation of nitrite led to prolonged lag times for clotting in platelet poor plasma that was enhanced by exposure to far red light. CONCLUSIONS: Our results suggest that nitrite leads to the formation of a photolabile RBC surface thiol-bound species such as an S-nitrosothiol or heme-nitrosyl (NO-bound heme) for which far red light enhances NO signaling. These findings expand our understanding of RBC-mediated NO production from nitrite. This pathway of NO production may have therapeutic potential in several applications including thrombosis, and, thus, warrants further study.

A simple and robust approach to reducing contact resistance in organic transistors.

Efficient injection of charge carriers from the contacts into the semiconductor layer is crucial for achieving high-performance organic devices. The potential drop necessary to accomplish this process yields a resistance associated with the contacts, namely the contact resistance. A large contact resistance can limit the operation of devices and even lead to inaccuracies in the extraction of the device parameters. Here, we demonstrate a simple and efficient strategy for reducing the contact resistance in organic thin-film transistors by more than an order of magnitude by creating high work function domains at the surface of the injecting electrodes to promote channels of enhanced injection. We find that the method is effective for both organic small molecule and polymer semiconductors, where we achieved a contact resistance as low as 200 Ωcm and device charge carrier mobilities as high as 20 cm2V-1s-1, independent of the applied gate voltage.

Analysis of single, cisplatin-induced DNA bends by atomic force microscopy and simulations.

Bent DNA, or DNA that is locally more flexible, is a recognition motif for many DNA binding proteins. These DNA conformational properties can thus influence many cellular processes, such as replication, transcription, and DNA repair. The importance of these DNA conformational properties is juxtaposed to the experimental difficulty to accurately determine small bends, locally more flexible DNA, or a combination of both (bends with increased flexibility). In essence, many current bulk methods use average quantities, such as the average end-to-end distance, to extract DNA conformational properties; they cannot access the additional information that is contained in the end-to-end distance distributions. We developed a method that exploits this additional information to determine DNA conformational parameters. The method is based on matching end-to-end distance distributions obtained experimentally by atomic force microscopy imaging to distributions obtained from simulations. We applied this method to investigate cisplatin GG biadducts. We found that cisplatin induces a bend angle of 36° and softens the DNA locally around the bend.

Nonuniform Internal Structure of Fibrin Fibers: Protein Density and Bond Density Strongly Decrease with Increasing Diameter.

The major structural component of a blood clot is a meshwork of fibrin fibers. It has long been thought that the internal structure of fibrin fibers is homogeneous; that is, the protein density and the bond density between protofibrils are uniform and do not depend on fiber diameter. We performed experiments to investigate the internal structure of fibrin fibers. We formed fibrin fibers with fluorescently labeled fibrinogen and determined the light intensity of a fiber, I, as a function of fiber diameter, D. The intensity and, thus, the total number of fibrin molecules in a cross-section scaled as D1.4. This means that the protein density (fibrin per cross-sectional area), ρp , is not homogeneous but instead strongly decreases with fiber diameter as D-0.6. Thinner fibers are denser than thicker fibers. We also determined Young's modulus, Y, as a function of fiber diameter. Y decreased strongly with increasing D; Y scaled as D-1.5. This implies that the bond density, ρb , also scales as D-1.5. Thinner fibers are stiffer than thicker fibers. Our data suggest that fibrin fibers have a dense, well-connected core and a sparse, loosely connected periphery. In contrast, electrospun fibrinogen fibers, used as a control, have a homogeneous cross-section.

Stretching single fibrin fibers hampers their lysis.

Blood clots, whose main structural component is a mesh of microscopic fibrin fibers, experience mechanical strain from blood flow, clot retraction and interactions with platelets and other cells. We developed a transparent, striated and highly stretchable substrate made from fugitive glue (a styrenic block copolymer) to investigate how mechanical strain affects lysis of single, suspended fibrin fibers. In this suspended fiber assay, lysis manifested itself by fiber elongation, thickening (disassembly), fraying and collapse. Stretching single fibrin fibers significantly hampered their lysis. This effect was seen in uncrosslinked and crosslinked fibers. Crosslinking (without stretching) also hampered single fiber lysis. Our data suggest that strain is a novel mechanosensitive factor that regulates blood clot dissolution (fibrinolysis) at the single fiber level. At the molecular level of single fibrin molecules, strain may distort, or hinder access to, plasmin cleavage sites and thereby hamper lysis. STATEMENT OF SIGNIFICANCE: Fibrin fibers are the major structural component of a blood clot. We developed a highly stretchable substrate made from fugitive glue and a suspended fibrin fiber lysis assay to investigate the effect of stretching on single fibrin fibers lysis. The key findings from our experiments are: 1) Fibers thicken and elongate upon lysis; 2) stretching strongly reduces lysis; 3) this effect is more pronounced for uncrosslinked fibers; and 4) stretching fibers has a similar effect on reducing lysis as crosslinking fibers. At the molecular level, strain may distort plasmin cleavage sites, or restrict access to those sites. Our results suggest that strain may be a novel mechanobiological factor that regulates fibrinolysis.

Solution-Processed Organic and Halide Perovskite Transistors on Hydrophobic Surfaces.

Solution-processable electronic devices are highly desirable due to their low cost and compatibility with flexible substrates. However, they are often challenging to fabricate due to the hydrophobic nature of the surfaces of the constituent layers. Here, we use a protein solution to modify the surface properties and to improve the wettability of the fluoropolymer dielectric Cytop. The engineered hydrophilic surface is successfully incorporated in bottom-gate solution-deposited organic field-effect transistors (OFETs) and hybrid organic-inorganic trihalide perovskite field-effect transistors (HTP-FETs) fabricated on flexible substrates. Our analysis of the density of trapping states at the semiconductor-dielectric interface suggests that the increase in the trap density as a result of the chemical treatment is minimal. As a result, the devices exhibit good charge carrier mobilities, near-zero threshold voltages, and low electrical hysteresis.

Diffusion and Binding of Mismatch Repair Protein, MSH2, in Breast Cancer Cells at Different Stages of Neoplastic Transformation.

The interior of cells is a highly complex medium, containing numerous organelles, a matrix of different fibers and a viscous, aqueous fluid of proteins and small molecules. The interior of cells is also a highly dynamic medium, in which many components move, either by active transport or passive diffusion. The mobility and localization of proteins inside cells can provide important insights into protein function and also general cellular properties, such as viscosity. Neoplastic transformation affects numerous cellular properties, and our goal was to investigate the diffusional and binding behavior of the important mismatch repair (MMR) protein MSH2 in live human cells at various stages of neoplastic transformation. Toward this end, noncancerous, immortal, tumorigenic, and metastatic mammary epithelial cells were transfected with EGFP and EGFP-tagged MSH2. MSH2 forms two MMR proteins (MutSα and MutSß) and we assume MSH2 is in the complex MutSα, though our results are similar in either case. Unlike the MutS complexes that bind to nuclear DNA, EGFP diffuses freely. EGFP and MutSα-EGFP diffusion coefficients were determined in the cytoplasm and nucleus of each cell type using fluorescence recovery after photobleaching. Diffusion coefficients were 14-24 µm2/s for EGFP and 3-7 µm2/s for MutSα-EGFP. EGFP diffusion increased in going from noncancerous to immortal cells, indicating a decrease in viscosity, with smaller changes in subsequent stages. MutSα produces an effective diffusion coefficient that, coupled with the free EGFP diffusion measurements, can be used to extract a pure diffusion coefficient and a pseudo-equilibrium constant K*. The MutSα nuclear K* increased sixfold in the first stage of cancer and then decreased in the more advanced stages. The ratio of nuclear to cytoplasmic K*for MutSα increased almost two orders of magnitude in going from noncancerous to immortal cells, suggesting that this quantity may be a sensitive metric for recognizing the onset of cancer.

Fluorinated benzalkylsilane molecular rectifiers.

We report on the synthesis and electrical properties of nine new alkylated silane self-assembled monolayers (SAMs) - (EtO)3Si(CH2)nN = CHPhX where n = 3 or 11 and X = 4-CF3, 3,5-CF3, 3-F-4-CF3, 4-F, or 2,3,4,5,6-F, and explore their rectification behavior in relation to their molecular structure. The electrical properties of the films were examined in a metal/insulator/metal configuration, with a highly-doped silicon bottom contact and a eutectic gallium-indium liquid metal (EGaIn) top contact. The junctions exhibit high yields (>90%), a remarkable resistance to bias stress, and current rectification ratios (R) between 20 and 200 depending on the structure, degree of order, and internal dipole of each molecule. We found that the rectification ratio correlates positively with the strength of the molecular dipole moment and it is reduced with increasing molecular length.