David Schaefer

Elastic properties of the cell wall of Aspergillus nidulans studied with atomic force microscopy

Currently, little is known about the mechanical properties of filamentous fungal hyphae. To study this topic, atomic force microscopy (AFM) was used to measure cell wall mechanical properties of the model fungus Aspergillus nidulans. Wild type and a mutant strain (ΔcsmA), lacking one of the chitin synthase genes, were grown in shake flasks. Hyphae were immobilized on polylysine‐coated coverslips and AFM force‐displacement curves were collected. When grown in complete medium, wild‐type hyphae had a cell wall spring constant of 0.29 ± 0.02 N/m. When wild‐type and mutant hyphae were grown in the same medium with added KCl (0.6 M), hyphae were significantly less rigid with spring constants of 0.17 ± 0.01 and 0.18 ± 0.02 N/m, respectively. Electron microscopy was used to measure the cell wall thickness and hyphal radius. By use of finite element analysis (FEMLAB v 3.0, Burlington, MA) to simulate AFM indentation, the elastic modulus of wild‐type hyphae grown in complete medium was determined to be 110 ± 10 MPa. This decreased to 64 ± 4 MPa for hyphae grown in 0.6 M KCl, implying growth medium osmotic conditions have significant effects on cell wall elasticity. Mutant hyphae grown in KCl‐supplemented medium were found to have an elastic modulus of 67 ± 6 MPa. These values are comparable with other microbial systems (e.g., yeast and bacteria). It was also found that under these growth conditions axial variation in elastic modulus along fungal hyphae was small. To determine the relationship between composition and mechanical properties, cell wall composition was measured by anion‐exchange liquid chromatography and pulsed electrochemical detection. Results show similar composition between wild‐type and mutant strains. Together, these data imply differences in mechanical properties may be dependent on varying molecular structure of hyphal cell walls as opposed to wall composition.

Electron-Phonon Coupling in Two-Dimensional Silicene and Germanene

Following the work in graphene, we report a first-principles study of electron-phonon coupling (EPC) in low-buckled (LB) monolayer silicene and germanene. Despite of the similar honeycomb atomic arrangement and linear band dispersion, the EPC matrix-element squares of the - Eg and K-A1 modes in silicene are only about 50% of those in graphene. However, the smaller Fermi velocity in silicene compensates this reduction by providing a larger joint electronic density of states near the Dirac point. We predict that Kohn anomalies associated with these two optical modes are significant in silicene. In addition, the EPC-induced frequency shift and linewidth of the Raman-active - Eg mode in silicene are calculated as a function of doping. The results are comparable to those in graphene, indicating a similar non-adiabatic dynamical origin. In contrast, the EPC in germanene is found to be much reduced.

Assessment of Elasticity and Topography of Aspergillus nidulans Spores via Atomic Force Microscopy

ABSTRACT Previous studies have described both surface morphology and adhesive properties of fungal spores, but little information is currently available on their mechanical properties. In this study, atomic force microscopy (AFM) was used to investigate both surface topography and micromechanical properties of Aspergillus nidulans spores. To assess the influence of proteins covering the spore surface, wild-type spores were compared with spores from isogenic rodA+ and rodA− strains. Tapping-mode AFM images of wild-type and rodA+ spores in air showed characteristic “rodlet” protein structures covering a granular spore surface. In comparison, rodA− spores were rodlet free but showed a granular surface structure similar to that of the wild-type and rodA+ spores. Rodlets were removed from rodA+ spores by sonication, uncovering the underlying granular layer. Both rodlet-covered and rodlet-free spores were subjected to nanoindentation measurements, conducted in air, which showed the stiffnesses to be 110 ± 10, 120 ± 10, and 300 ± 20 N/m and the elastic moduli to be 6.6 ± 0.4, 7.0 ± 0.7, and 22 ± 2 GPa for wild-type, rodA+ and rodA− spores, respectively. These results imply the rodlet layer is significantly softer than the underlying portion of the cell wall.

Characterization of amyloidogenesis of hen egg lysozyme in concentrated ethanol solution

We show that hen egg white lysozyme [HEWL] reproducibly forms amyloid fibrils in 80% ethanol at 22 degrees C with constant agitation. Fibril formation occurs over a time course of approximately 30 days, displays polymerization nucleation kinetics, and demonstrates a marked decrease in alpha-helical structure. Seeding with as little as 0.05% v/v of fibrils cleaved into smaller seed fragments by sonication abolishes the lag phase. Thioflavin T assays confirm the amyloid nature of the fibrils. Atomic force microscopy reveals unbranched amyloid fibrils with lengths varying between 1 and 3 microm and heights ranging from 6-12 nm. The formation of amyloid fibrils in predominantly organic solvents demonstrates that the basic principles guiding fibril formation arise from interactions of the peptide backbone rather than from interactions between the amino acid side chains.

Simultaneous Monitoring of Peptide Aggregate Distributions, Structure, and Kinetics Using Amide Hydrogen Exchange: Application to Aβ(1–40) Fibrillogenesis

Increasing evidence indicates that soluble aggregates of amyloid beta protein (Aβ) are neurotoxic. However, difficulty in isolating these unstable, dynamic species impedes studies of Aβ and other aggregating peptides and proteins. In this study, hydrogen–deuterium exchange (HX) detected by mass spectrometry (MS) was used to measure Aβ(1‐40) aggregate distributions without purification or modification that might alter the aggregate structure or distribution. Different peaks in the mass spectra were assigned to monomer, low molecular weight oligomer, intermediate, and fibril based on HX labeling behavior and complementary assays. After 1 h labeling, the intermediates incorporated approximately ten more deuterons relative to fibrils, indicating a more solvent exposed structure of such intermediates. HX‐MS also showed that the intermediate species dissociated much more slowly to monomer than did the very low molecular weight oligomers that were formed at very early times in Aβ aggregation. Atomic force microscopy (AFM) measurements revealed the intermediates were roughly spherical with relatively homogenous diameters of 30–50 nm. Quantitative analysis of the HX mass spectra showed that the amount of intermediate species was correlated with Aβ toxicity patterns reported in a previous study under the same conditions. This study also demonstrates the potential of the HX‐MS approach to characterizing complex, multi‐component oligomer distributions of aggregating peptides and proteins. Biotechnol. Bioeng. 2008;100: 1214–1227.

Plasmon-induced magnetization of metallic nanostructures

In this paper we report on the observation of novel and highly unusual magnetic state of light. It appears that in small holes light quanta behave as small magnets so that light propagation through such holes may be affected by magnetic field. When arrays of such holes are made, magnetic light of the individual holes forms novel and highly unusual two-dimensional magnetic light material. Magnetic light may soon become a great new tool for quantum communication and computing.Zero-point energy of surface plasmon modes of mesoscopic metal samples in an external magnetic field has been considered. The magnetic response of plasmon vacuum has shown to be diamagnetic. In thin films this surface vacuum diamagnetism is at least of the same order of magnitude as the magnetism of the bulk electrons. Thus, a novel type of magnetism shown by thin metallic samples, which is complementary to the well known Pauli paramagnetic and Landau diamagnetic contributions to the magnetism of the electron gas has been demonstrated.

Characterization of surface modification in atomic force microscope-induced nanolithography of oxygen deficient La0.67Ba0.33MnO3−δ thin films

We report our studies of the nanolithographic surface modifications induced by an Atomic Force Microscope (AFM) in epitaxial thin films of oxygen deficient Lanthanum Barium Manganese Oxide (La0.67Ba0.33MnO3−δ). The pattern characteristics depend on the tip voltage, tip polarity, voltage duration, tip force, and humidity. We have used Electron Energy Dispersive X-Ray Spectroscopy (EDS) to analyze the chemical changes associated with the surface modifications produced with a negatively biased AFM tip. A significant increase in the oxygen stoichiometry for the patterned regions relative to the pristine film surface is observed. The results also indicate changes in the cation stoichiometry, specifically a decrease in the Lanthanum and Manganese concentrations and an increase in the Barium concentration in the patterned regions.

Possible mechanisms in atomic force microscope-induced nano-oxidation lithography in epitaxial La0.67Ba0.33MnO3-δ thin films

Atomic force microscope (AFM) induced nanolithography has been successfully utilized on perovskite manganite thin films by several groups to create nanoscale patterns for various fundamental mesoscopic-scale transport studies. However, the chemical and physical processes involved have not been understood. This work presents possible microscopic mechanisms for AFM induced nanolithography in La2/3Ba1/3MnO3-δ films induced by an AFM tip, which is negatively biased with respect to the sample in a humid environment. A self-consistent conceptual framework, which accounts for the previously reported observations of changes in the nanomodified regions such as volume increases, selective acid etching, as well as changes in the chemical composition detected by energy dispersive spectroscopy, is reported. Microscopic mechanisms delineated in this work are based on the following: existence of known compounds composed of the available elements (La, Ba, Mn,O, and H) resulting in equal or higher formal oxidation states,...

Chapter 4 – Aspects of Particle Adhesion and Removal

This chapter describes the forces, interactions, and factors that control the adhesion and cohesion of particles. It also discusses how the adhesion of particles to surfaces is commonly determined. Forces of nature are generally categorized as one of four types: strong interactions, weak interactions, electromagnetic interactions, and gravitational interactions. Which of these are important in a given instance depends on the size of the particles and the separations between the particles or between the particles and the substrates. Particles adhere to surfaces because of both the existence of attractive forces between the particles and the substrates and the mechanical responses of these materials to these attractive forces. Specifically, the interactions between a particle and a contacting substrate cause stresses in both materials. These stresses cause the materials to deform and it is the combination of the deformation and the strength of the interactions that determines how strongly a particle is bonded to a substrate. This discussion concludes that the choice of the means of cleaning must consider the particle-substrate interactions as well as the process speeds.

Effect of Rapamycin on Filamentous Fungal Cell Walls

Filamentous fungi are widely used in the bioprocess industry to produce a variety of products resulting in billion dollar returns. Often times in industrial bioprocesses, fungi experience nutrient limitation, and we hypothesize that autophagy, a nutrient starvation response, is induced. We further hypothesize that autophagy leads to changes in the mechanical properties of filamentous fungal cell walls, resulting in thinner, weaker and stiffer walls. We test this hypothesis here, using rapamycin (an immunosuppressant drug) to gratuitously induce autophagy in the model fungi Aspergillus nidulans. Atomic force microscopy (AFM) is used to assess the mechanical properties of the cell wall, electron microscopy is used to assess wall thickness and a novel fragmentation assay is used to determine relative tensile strength of the culture. We will report on these studies and how they support our hypotheses.