FRET-Based Quantum Dot Immunoassay for Rapid and Sensitive Detection of Aspergillus amstelodami

In this study, a fluorescence resonance energy transfer (FRET)-based quantum dot (QD) immunoassay for detection and identification of Aspergillus amstelodami was developed. Biosensors were formed by conjugating QDs to IgG antibodies and incubating with quencher-labeled analytes; QD energy was transferred to the quencher species through FRET, resulting in diminished fluorescence from the QD donor. During a detection event, quencher-labeled analytes are displaced by higher affinity target analytes, creating a detectable fluorescence signal increase from the QD donor. Conjugation and the resulting antibody:QD ratios were characterized with UV-Vis spectroscopy and QuantiT protein assay. The sensitivity of initial fluorescence experiments was compromised by inherent autofluorescence of mold spores, which produced low signal-to-noise and inconsistent readings. Therefore, excitation wavelength, QD, and quencher were adjusted to provide optimal signal-to-noise over spore background. Affinities of anti-Aspergillus antibody for different mold species were estimated with sandwich immunoassays, which identified A. fumigatus and A. amstelodami for use as quencher-labeled- and target-analytes, respectively. The optimized displacement immunoassay detected A. amstelodami concentrations as low as 103 spores/mL in five minutes or less. Additionally, baseline fluorescence was produced in the presence of 105 CFU/mL heat-killed E. coli O157:H7, demonstrating high specificity. This sensing modality may be useful for identification and detection of other biological threat agents, pending identification of suitable antibodies. Overall, these FRET-based QD-antibody biosensors represent a significant advancement in detection capabilities, offering sensitive and reliable detection of targets with applications in areas from biological terrorism defense to clinical analysis

Solid oxide fuel cell reactor analysis and optimisation through a novel multi-scale modelling strategy

The simulation of a solid oxide fuel cell (SOFC) that incorporates a detailed user-developed model was performed within the commercial flowsheet simulator Aspen Plus. It allows modification of the SOFC's governing equations, as well as the configuration of the cell's fuel-air flow pattern at the flowsheet level. Initially, the dynamic behaviour of single compartment of a cell was examined with a 0D model, which became the building block for more complex SOFC configurations. Secondly, a sensitivity analysis was performed at the channel (1D) scale for different flow patterns. Thirdly, the effect of fuel and air flow rates on the predominant distributed variables of a cell was tested on a 2D assembly. Finally, an optimisation study was carried out on the 2D cell, leading to a robust, optimal air distribution profile that minimises the internal temperature gradient. This work forms the foundation of future stack and system scale studies

Elucidating the Molecular Basis of Protein and Polymer Display in Gram-Positive Bacteria for Novel Antibiotic Development

The emergence of multi-drug resistant bacteria has prompted novel antibiotic development by targeting non-essential pathways, such as virulence factor production and display during cell wall biosynthesis. Within Gram-positive bacteria, sortase transpeptidases covalently attach proteins to the cell wall or assemble pili using class A-F enzymes. Interestingly, class E sortases display proteins via recognition of a non-canonical LAXTG motif. We have determined the first crystal structure of a class E sortase, the 1.93 � resolution structure of SrtE1 from Streptomyces coelicolor. The SrtE1 enzyme possesses structurally distinct β3/β4 and β6/β7 active site loops that contact the LAXTG substrate. Furthermore, molecular dynamics studies have identified a conserved tyrosine residue that likely confers substrate specificity for class E sortases. A second anti-virulence target, the TarA glycosyltransferase (GT), is highly conserved among Gram-positive bacteria and produces surface-anchored wall teichoic acid (WTA) polymers. The WTA biosynthetic mechanism involving TarA and other membrane-associated, enzymes is poorly understood due to a lack of structural characterization. We have determined the 2.0 � resolution crystal structure of the TarA enzyme from Thermobacter italicus, which adopts a structurally novel protein fold, termed class GT-E, and represents the first structurally characterized member of the WecB-TagA-CpsF GT family. Sequence conservation mapping onto experimentally observed TarA oligomer structures has identified putative functional residues and suggests formation of a competent active site through oligomerization, which will guide studies of substrate binding and catalysis. Furthermore, we describe two target-specific, cell-based assays for the discovery of sortase and TarA inhibitors. The first assay monitors sortase-dependent growth inhibition of wild-type and sortase-deficient Actinomyces oris strains in the presence of small molecule inhibitors. A pilot screen of 1280 compounds returned a hit rate of 0.3%, which has prompted large-scale high-throughput screening. The second assay utilizes a TarA-dependent morphological transition of a mutant B. subtilis strain complemented with the TarA enzyme from S. aureus (TarA+) to replace the endogenous enzyme activity. The drastic rod-shape to spherical morphological transition provides a robust HTS platform with a Z-prime score of 0.76. Ultimately, these structural and cell-based studies will promote the development of anti-virulence inhibitors to combat bacterial resistance

Elucidating the Molecular Basis of Protein and Polymer Display in Gram-Positive Bacteria for Novel Antibiotic Development

The emergence of multi-drug resistant bacteria has prompted novel antibiotic development by targeting non-essential pathways, such as virulence factor production and display during cell wall biosynthesis. Within Gram-positive bacteria, sortase transpeptidases covalently attach proteins to the cell wall or assemble pili using class A-F enzymes. Interestingly, class E sortases display proteins via recognition of a non-canonical LAXTG motif. We have determined the first crystal structure of a class E sortase, the 1.93 � resolution structure of SrtE1 from Streptomyces coelicolor. The SrtE1 enzyme possesses structurally distinct β3/β4 and β6/β7 active site loops that contact the LAXTG substrate. Furthermore, molecular dynamics studies have identified a conserved tyrosine residue that likely confers substrate specificity for class E sortases. A second anti-virulence target, the TarA glycosyltransferase (GT), is highly conserved among Gram-positive bacteria and produces surface-anchored wall teichoic acid (WTA) polymers. The WTA biosynthetic mechanism involving TarA and other membrane-associated, enzymes is poorly understood due to a lack of structural characterization. We have determined the 2.0 � resolution crystal structure of the TarA enzyme from Thermobacter italicus, which adopts a structurally novel protein fold, termed class GT-E, and represents the first structurally characterized member of the WecB-TagA-CpsF GT family. Sequence conservation mapping onto experimentally observed TarA oligomer structures has identified putative functional residues and suggests formation of a competent active site through oligomerization, which will guide studies of substrate binding and catalysis. Furthermore, we describe two target-specific, cell-based assays for the discovery of sortase and TarA inhibitors. The first assay monitors sortase-dependent growth inhibition of wild-type and sortase-deficient Actinomyces oris strains in the presence of small molecule inhibitors. A pilot screen of 1280 compounds returned a hit rate of 0.3%, which has prompted large-scale high-throughput screening. The second assay utilizes a TarA-dependent morphological transition of a mutant B. subtilis strain complemented with the TarA enzyme from S. aureus (TarA+) to replace the endogenous enzyme activity. The drastic rod-shape to spherical morphological transition provides a robust HTS platform with a Z-prime score of 0.76. Ultimately, these structural and cell-based studies will promote the development of anti-virulence inhibitors to combat bacterial resistance