Aaron P. Mosier

Inhibition of biofilm formation, quorum sensing and infection in Pseudomonas aeruginosa by natural products-inspired organosulfur compounds.

Using a microplate-based screening assay, the effects on Pseudomonas aeruginosa PAO1 biofilm formation of several S-substituted cysteine sulfoxides and their corresponding disulfide derivatives were evaluated. From our library of compounds, S-phenyl-L-cysteine sulfoxide and its breakdown product, diphenyl disulfide, significantly reduced the amount of biofilm formation by P. aeruginosa at levels equivalent to the active concentration of 4-nitropyridine-N-oxide (NPO) (1 mM). Unlike NPO, which is an established inhibitor of bacterial biofilms, our active compounds did not reduce planktonic cell growth and only affected biofilm formation. When used in a Drosophila-based infection model, both S-phenyl-L-cysteine sulfoxide and diphenyl disulfide significantly reduced the P. aeruginosa recovered 18 h post infection (relative to the control), and were non-lethal to the fly hosts. The possibility that the observed biofilm inhibitory effects were related to quorum sensing inhibition (QSI) was investigated using Escherichia coli-based reporters expressing P. aeruginosa lasR or rhIR response proteins, as well as an endogenous P. aeruginosa reporter from the lasI/lasR QS system. Inhibition of quorum sensing by S-phenyl-L-cysteine sulfoxide was observed in all of the reporter systems tested, whereas diphenyl disulfide did not exhibit QSI in either of the E. coli reporters, and showed very limited inhibition in the P. aeruginosa reporter. Since both compounds inhibit biofilm formation but do not show similar QSI activity, it is concluded that they may be functioning by different pathways. The hypothesis that biofilm inhibition by the two active compounds discovered in this work occurs through QSI is discussed.

The regulation of focal adhesion complex formation and salivary gland epithelial cell organization by nanofibrous PLGA scaffolds

Nanofiber scaffolds have been useful for engineering tissues derived from mesenchymal cells, but few studies have investigated their applicability for epithelial cell-derived tissues. In this study, we generated nanofiber (250 nm) or microfiber (1200 nm) scaffolds via electrospinning from the polymer, poly-l-lactic-co-glycolic acid (PLGA). Cell-scaffold contacts were visualized using fluorescent immunocytochemistry and laser scanning confocal microscopy. Focal adhesion (FA) proteins, such as phosphorylated FAK (Tyr397), paxillin (Tyr118), talin and vinculin were localized to FA complexes in adult cells grown on planar surfaces but were reduced and diffusely localized in cells grown on nanofiber surfaces, similar to the pattern observed in adult mouse salivary gland tissues. Significant differences in epithelial cell morphology and cell clustering were also observed and quantified, using image segmentation and computational cell-graph analyses. No statistically significant differences in scaffold stiffness between planar PLGA film controls compared to nanofibers scaffolds were detected using nanoindentation with atomic force microscopy, indicating that scaffold topography rather than mechanical properties accounts for changes in cell attachments and cell structure. Finally, PLGA nanofiber scaffolds could support the spontaneous self-organization and branching of dissociated embryonic salivary gland cells. Nanofiber scaffolds may therefore have applicability in the future for engineering an artificial salivary gland.

Development of antifouling surfaces to reduce bacterial attachment

It is well documented that bacterial adhesion to surfaces is mediated by the physical and chemical properties of the substrate, as well as the surface characteristics of the organism. Topographical features that limit cell–surface interactions have been shown to reduce surface colonization and biofilm formation. In this study, bacterial attachment to medically relevant materials was evaluated. Our data show that Escherichia coli attachment to glass, silicone, and titanium surfaces was most affected by the surface energy of these materials, as determined by water contact angle. The inherent roughness of the surface, however, was not correlated with cell attachment density. To study the effect of engineered surface roughness on bacterial attachment, topographical features, including arrays of holes and repeating lines/trenches, were formed from silicon wafers and then used as a template to imprint silicone-based polydimethylsiloxane (PDMS). Patterned silicone surfaces were then used in static and microfluidic flow-based experiments to evaluate cellular settlement and attachment. Cell attachment was observed to be strongly dependent upon the topographical features under both static and microfluidic flow conditions. The highest attachment density was observed on flat, un-patterned surfaces, while linear patterned surfaces showed greatly reduced cell attachment. Moreover, surfaces consisting of arrays of holes further reduced cell attachment as compared to linear patterns. These results demonstrate that the size, spacing, and shape of surface features play a significant role in cell–surface attachment and provide insight for the design of surfaces with antifouling properties.

A novel microfluidic device for the in situ optical and mechanical analysis of bacterial biofilms.

Viable methods for bacterial biofilm remediation require a fundamental understanding of biofilm mechanical properties and their dependence on dynamic environmental conditions. Mechanical test data, such as elasticity or adhesion, can be used to perform physical modelling of biofilm behaviour, thus enabling the development of novel remediation strategies. To achieve real-time, dynamic measurements of these properties, a novel microfluidic flowcell device has been designed and fabricated for in situ analysis using atomic force microscopy (AFM). The flowcell consists of microfluidic channels for biofilm establishment that are then converted into an open architecture, laminar flow channel for AFM measurement in a liquid environment. Finite element analysis (FEA) was used to profile fluid conditions within the flowcell during biofilm establishment. Force-mode AFM was used to measure the elastic properties of mature Pseudomonas aeruginosa PAO1 biofilms as well as polyacrylamide hydrogels. Elastic moduli ranging from 0.58 to 2.61kPa were determined for the mature biofilm, which fall within the range of moduli previously reported by optical, rheometric, and microindentation techniques. These results demonstrate the validity of the microfluidic flowcell system as an effective platform for future investigations of biofilm mechanical and morphological response to dynamic environmental conditions.

Analysis of Bacterial Surface Interactions Using Microfluidic Systems

Modern microbiological research has increasingly focused on the interactions between bacterial cells and the surfaces that they inhabit. To this end, microfluidic devices have played a large role in enabling research of cell-surface interactions, especially surface attachment and biofilm formation. This review provides background on microfluidic devices and their use in biological systems, as well specific examples from current literature. Methods to observe and interrogate cells within microfluidic devices are described, as well as the analytical techniques that are used to collect these data.

Microfluidic Platform for the Elastic Characterization of Mouse Submandibular Glands by Atomic Force Microscopy

The ability to characterize the microscale mechanical properties of biological materials has the potential for great utility in the field of tissue engineering. The development and morphogenesis of mammalian tissues are known to be guided in part by mechanical stimuli received from the local environment, and tissues frequently develop to match the physical characteristics (i.e., elasticity) of their environment. Quantification of these material properties at the microscale may provide valuable information to guide researchers. Presented here is a microfluidic platform for the non-destructive ex vivo microscale mechanical characterization of mammalian tissue samples by atomic force microscopy (AFM). The device was designed to physically hold a tissue sample in a dynamically controllable fluid environment while allowing access by an AFM probe operating in force spectroscopy mode to perform mechanical testing. Results of measurements performed on mouse submandibular gland samples demonstrate the ability of the analysis platform to quantify sample elasticity at the microscale, and observe chemically-induced changes in elasticity.

Differential effects of silver nanoparticles on DNA damage and DNA repair gene expression in Ogg1-deficient and wild type mice

Abstract Due to extensive use in consumer goods, it is important to understand the genotoxicity of silver nanoparticles (AgNPs) and identify susceptible populations. 8-Oxoguanine DNA glycosylase 1 (OGG1) excises 8-oxo-7,8-dihydro-2-deoxyguanine (8-oxoG), a pro-mutagenic lesion induced by oxidative stress. To understand whether defects in OGG1 is a possible genetic factor increasing an individual’s susceptibly to AgNPs, we determined DNA damage, genome rearrangements, and expression of DNA repair genes in Ogg1-deficient and wild type mice exposed orally to 4 mg/kg of citrate-coated AgNPs over a period of 7 d. DNA damage was examined at 3 and 7 d of exposure and 7 and 14 d post-exposure. AgNPs induced 8-oxoG, double strand breaks (DSBs), chromosomal damage, and DNA deletions in both genotypes. However, 8-oxoG was induced earlier in Ogg1-deficient mice and 8-oxoG levels were higher after 7-d treatment and persisted longer after exposure termination. AgNPs downregulated DNA glycosylases Ogg1, Neil1, and Neil2 in wild type mice, but upregulated Myh, Neil1, and Neil2 glycosylases in Ogg1-deficient mice. Neil1 and Neil2 can repair 8-oxoG. Thus, AgNP-mediated downregulation of DNA glycosylases in wild type mice may contribute to genotoxicity, while upregulation thereof in Ogg1-deficient mice could serve as an adaptive response to AgNP-induced DNA damage. However, our data show that Ogg1 is indispensable for the efficient repair of AgNP-induced damage. In summary, citrate-coated AgNPs are genotoxic in both genotypes and Ogg1 deficiency exacerbates the effect. These data suggest that humans with genetic polymorphisms and mutations in OGG1 may have increased susceptibility to AgNP-mediated DNA damage.

Novel approaches to biosensing and nano-biological interactions

Nanotechnology has recently been applied to a wide range of biological systems. In particular, there is a current push to examine the interface between the biological world and micro/nano-scale systems. Our research in this field has led to the development of novel strategies for spatial patterning of biomolecules, electrical and optical biosensing, nanomaterial delivery systems, single-cell manipulation, and the study of cellular interactions with nano-structured surfaces. Current work on these topics will be presented, including work on novel, semiconductor-based DNA detection methods and mechanical, atomic force microscopy (AFM)-based characterization of bacterial biofilms in threedimensional microfluidic systems.

Bacterial biofilms for sequestration of Cu +2 from CMP wastewater

The process of chemical mechanical planarization (CMP) employed by the semiconductor industry creates large quantities of wastewater containing toxic levels of copper. Use of biomass-based filtration may offer a less expensive and more sustainable means of treating wastewater prior to disposal. We screened a library of microorganisms for ability to bind Cu2+ from solution and form biofilm within packed bed filtration columns. Filter beds supporting L. casei/P. pastoris biofilms were found to exhibit a two-phase adsorption behavior during flow-through binding experiments. Bound Cu2+ was able to be recovered via acid wash, and the biomass quickly regenerated and could be reused.