William R. LaCourse

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.

Pulsed electrochemical detection of thiocompounds following microchromatographic separations

Abstract Pulsed electrochemical detection (PED) following microchromatographic separations has been applied to the determination of thiocompounds (e.g., methionine, cystamine, glutathione, glutathione disulfide) under typical reversed-phase conditions. Both reduced and oxidized thiol moieties are detected with high selectivity and sensitivity without the need of derivatization. The anodic responses of model thiol redox couples are shown to be based upon oxide-catalyzed anodic oxygen transfer reactions at noble metal electrodes. The mechanism of detection and optimization of PED waveform parameters are studied using cyclic and pulsed voltammetry at rotated disk electrodes. In a comparative study of pulsed amperometric detection (PAD) and integrated pulsed amperometric detection (IPAD), IPAD is determined to be better suited to manage the large oxide-formation (background) currents, which accompany the Faradaic signal of the analyte. IPAD results in more stable baselines, eliminates oxide-induced artifacts, and yields lower limits of detection. The higher frequency waveforms (1 Hz) needed to define the narrow peaks of microchromatographic separations and the fast scan rates (1000 mV s−1) of the IPAD waveform require the use of microelectrodes (≤ 1 mm), which exhibit reduced capacitance effects. Mass detection limits for thiocompounds are typically 0.2–3 pmole for microbore chromatography and 0.1-0.3 pmole for capillary liquid chromatography. The high selectivity of PED for thiocompounds reduces sample preparation and produces simpler chromatograms of complex mixtures, such as liver extracts.

Genetic analysis of acd6-1 reveals complex defense networks and leads to identification of novel defense genes in Arabidopsis

Pathogen infection leads to the activation of defense signaling networks in plants. To study these networks and the relationships between their components, we introduced various defense mutations into acd6-1, a constitutive gain-of-function Arabidopsis mutant that is highly disease resistant. acd6-1 plants show spontaneous cell death, reduced stature, and accumulate high levels of camalexin (an anti-fungal compound) and salicylic acid (SA; a signaling molecule). Disruption of several defense genes revealed that in acd6-1, SA levels/signaling were positively correlated with the degree of disease resistance and defense gene expression. Salicylic acid also modulates the severity of cell death. However, accumulation of camalexin in acd6-1 is largely unaffected by reducing the level of SA. In addition, acd6-1 shows ethylene- and jasmonic acid-mediated signaling that is antagonized and therefore masked by the presence of SA. Mutant analysis revealed a new relationship between the signaling components NPR1 and PAD4 and also indicated that multiple defense pathways were required for phenotypes conferred by acd6-1. In addition, our data confirmed that the size of acd6-1 was inversely correlated with SA levels/signaling. We exploited this unique feature of acd6-1 to identify two genes disrupted in acd6-1 suppressor (sup) mutants: one encodes a known SA biosynthetic component (SID2) and the other encodes an uncharacterized putative metalloprotease (At5g20660). Taken together, acd6-1 is a powerful tool not only for dissecting defense regulatory networks but also for discovering novel defense genes.

Pulsed electrochemical detection of sulfur-containing antibiotics following high performance liquid chromatography

Pulsed electrochemical detection (PED) following reversed-phase chromatography has been applied to the direct detection of sulfur-containing antibiotics, specifically, penicillins, cephalosporins, and lincomycin. The compounds are detected sensitively and selectively without the need for derivatization. Integrated pulsed amperometric detection (IPAD) yields limits of detection lower than UV detection for these compounds. Detection limits using an optimized IPAD waveform are typically 10 ppb or less. The high selectivity of PED for thiocompounds reduces sample preparation. This work is applied to the determination of penicillin and related analogues in various pharmaceutical formulations/preparations, including a chicken feed.

Pulsed electrochemical detection of thiols and disulfides following capillary electrophoresis.

Pulsed electrochemical detection (PED) following capillary electrophoresis (CE) has been applied to the direct detection of thiocompounds. Both reduced and oxidized thiol moieties are detected without the need of derivatization. Thiocompounds can be detected over a wide range of pH conditions (i.e., pH 0-14), and except for pH, their response is relatively unperturbed by buffer composition. Integrated pulsed amperometric detection (IPAD) results in more stable baselines, eliminates oxide-induced artifacts, and yields lower limits of detection than other PED waveforms. Mass detection limits using optimized IPAD waveforms are typically 2 pg (5 fmol) or less. The high selectivity of PED for thiocompounds reduces sample preparation and produces simpler electropherograms of complex samples containing these biologically significant compounds.

Electrocatalytic Oxidations at Electrodeposited Bismuth (III)‐Doped Beta‐Lead Dioxide Film Electrodes

Heterogeneous rate constants for the anodic oxidation of many inorganic and organic compounds at electrolytically deposited beta-PbO{sub 2} film electrodes in 1.0M HClO{sub 4} are increased by doping the oxide with Bi(III). The rate constants at the mixed-oxide electrode rise sharply for a change from 0 to 10 mole percent (m/o) Bi(III) in the electrodeposition solution, and reach a mass transport-limited value for some compounds at 40 m/o Bi(III). Results of kinetic, voltammetric, and spectroscopic studies of these electrodes are described. In addition, the lifetime of Bi(III)-doped PbO{sub 2} film electrodes is discussed as a function of concentration, rotation speed, analyte, and applied voltage.

A platform for on-site environmental analysis of explosives using high performance liquid chromatography with UV absorbance and photo-assisted electrochemical detection

High-performance liquid chromatography with photo-assisted electrochemical detection (HPLC-PAED) is used in conjunction with ultraviolet absorbance (UV) detection for determining explosives in environmental samples. The system utilizes an on-line solid-phase extraction technique for sample pretreatment (i.e., fractionation and concentration), thus reducing the required ground water sample size from 1L to 2mL and minimizing sample handling. Limits of detection for explosives using solid-phase extraction and PAED range from 0.0007 to 0.4mug/L, well below those achieved with UV detection for several important explosives (e.g., RDX). The method has demonstrated good accuracy, precision, and recovery for all tested explosives, thus proving that the method is suitable for evaluation of explosives in ground water with competitive advantages over the U.S. Environmental Protection Agency (EPA) Method 8330. A system adaptable for the on-site environmental analysis of explosives has been developed and validated.

Competency-based reforms of the undergraduate biology curriculum: integrating the physical and biological sciences.

In response to the Association of American Medical Colleges–Howard Hughes Medical Institute report Scientific Foundations for Future Physicians, a collaborative effort by four institutions has produced an introductory physics for life sciences course that stresses competency building and helps students apply strategies from the physical sciences to solve authentic biological problems.

Experimental determination and calculations of redox potential descriptors of compounds directed against retroviral zinc fingers: Implications for rational drug design

A diverse set of electrophilic compounds that react with cysteine thiolates in retroviral nucleocapsid (NC) proteins and abolish virus infectivity has been identified. Although different in chemical composition, these compounds are all oxidizing agents that lead to the ejection of Zn(II) ions bound to conserved structural motifs (zinc fingers) present in retroviral NC proteins. The reactivity of a congeneric series of aromatic disulfides toward the NC protein of the human immunodeficiency virus type 1 (HIV‐1), NCp7, has been characterized by HPLC separation of starting reagents from reaction products. We calculated the absolute redox potentials of these compounds in the gas phase and in aqueous solvent, using a density functional theory method and a continuum solvation model. Pulsed polarography experiments were performed and showed a direct correlation between calculated and experimentally determined redox propensities. A dependence between protein reactivity and redox potential for a specific compound was shown: Reaction with NCp7 did not take place below a threshold value of redox potential. This relationship permits the distinction between active and nonactive compounds targeted against NCp7, and provides a theoretical basis for a scale of reactivity with retroviral zinc fingers. Our results indicate that electrophilic agents with adequate thiophilicity to react with retroviral NC fingers can now be designed using known or calculated electrochemical properties. This may assist in the design of antiretroviral compounds with greater specificity for NC protein. Such electrophilic agents can be used in retrovirus inactivation with the intent of preparing a whole‐killed virus vaccine formulation that exhibits unaffected surface antigenic properties.

Indirect pulsed electrochemical detection of amino acids and proteins following high performance liquid chromatography

Pulsed electrochemical detection (PED) following liquid chromatographic separation has been applied to the indirect determination of amino acids and proteins. Here, the adsorption of these analytes at noble metal electrodes is exploited to suppress the oxidation of polyols and carbohydrates under alkaline conditions to elicit an indirect response. Of the reagents tested, gluconic acid gave the best overall signal-to-noise values for the indirect detection of amino acids following high performance anion-exchange chromatography (HPAEC). Limits of detection of amino acids were found to be 2-30pmol using optimized potential-time waveforms at an Au electrode. Indirect PED provided much greater detection sensitivity toward amino acids than direct PED. Analytical sensitivity of indirect PED is a function of both the analytes ability to adsorb to the electrode surface and its molecular size, which was demonstrated by the separation and detection of bovine serum albumin, ovalbumin, and myoglobin following gel-filtration chromatography (GFC).