Christopher E. Bradburne

Characterization of Neutrophilic Fe(II)-Oxidizing Bacteria Isolated from the Rhizosphere of Wetland Plants and Description of Ferritrophicum radicicola gen. nov. sp. nov., and Sideroxydans paludicola sp. nov.

Iron deposits (Fe plaque) on wetland plant roots contain abundant microbial populations, including Fe(II)-oxidizing bacteria (FeOB) that have not been cultured previously. In this study, 4 strains of Fe plaque-associated FeOB were isolated from 4 species of wetland plants. All 4 isolates grew in tight association with Fe-oxides, but did not form any identifiable Fe-oxide structures. All strains were obligate lithotrophic Fe(II)-oxidizers that were microaerobic, and were unable to use other inorganic or organic energy sources. One strain, BrT, was shown to fix 14 CO 2 at a rate consistent with its requirement for total cell carbon. The doubling times for the strains varied between 9.5 and 15.8 hours. The fatty acid methyl ester (FAME) profiles of 2 strains, BrT and CCJ, revealed that 16:0, 15:1 isoG, and 14:0 were dominant fatty acids. Phylogenetic analysis of the 16S rRNA gene indicated that all the strains were Betaproteobacteria. Two of the strains, BrT and Br-1 belong to a new species, Sideroxydans paludicola; a third strain, LD-1, is related to Sideroxydans lithotrophicus, a recently described species of FeOB. The fourth isolate, Ferritrophicum radicicola, represented a new genus in a new order of Betaproteobacteria, the Ferritrophicales. There are no other cultured isolates in this order. A small subunit rRNA gene-based, cultivation-independent analysis of Typha latifolia collected from a wetland revealed terminal restriction fragment profiles (tRFLP) consistent with the presence of these bacteria in the rhizosphere. These novel organisms likely play an important role in Fe(II) oxidation kinetics and Fe cycling within many terrestrial and freshwater environments.

Spatiotemporal Multicolor Labeling of Individual Cells Using Peptide-Functionalized Quantum Dots and Mixed Delivery Techniques

Multicolor fluorescent labeling of both intra- and extracellular structures is a powerful technique for simultaneous monitoring of multiple complex biochemical processes. This approach remains extremely challenging, however, as it often necessitates the combinatorial use of numerous targeting probes (e.g., antibodies), multistep bioconjugation chemistries, different delivery strategies (e.g., electroporation or transfection reagents), cellular fixation coupled with membrane permeabilization, and complex spectral deconvolution. Here, we present a nanoparticle-based fluorescence labeling strategy for the multicolor labeling of distinct subcellular compartments within live cells without the need for antibody conjugation or cellular fixation/permeabilization. This multipronged approach incorporates an array of delivery strategies, which localize semiconductor quantum dots (QDs) to various subcellular structures. QD uptake is implemented in a spaciotemporal manner by staggering the delivery of QD-peptide composites and exploiting various innate (peptide-mediated endocytosis, peptide-membrane interaction, polymer-based transfection) along with physical (microinjection) cellular delivery modalities to live cells growing in culture over a 4 day period. Imaging of the different intracellular labels is simplified by the unique photophysical characteristics of the QDs in combination with Förster resonance energy transfer sensitization, which allow for multiple spectral windows to be accessed with one excitation wavelength. Using this overall approach, QDs were targeted to both early and late endosomes, the cellular cytosol, and the plasma membrane in live cells, ultimately allowing for simultaneous five-color fluorescent imaging.

Quantum dots: a powerful tool for understanding the intricacies of nanoparticle-mediated drug delivery.

Nanoparticle-mediated drug delivery (NMDD) is an emerging research area that seeks to address many of the pharmacokinetic issues encountered with traditional systemically administered drug therapies. Although the field is still in its infancy, recent research has already highlighted the potential for improved drug delivery and targeted therapeutics; however, the real promise lies in combining drug therapy with diagnostic imaging, nucleic acid delivery/gene therapy and/or biosensing applications all in one engineered nanoparticle vector. In this review, the authors discuss the unique contributions that luminescent semiconductor nanocrystals or quantum dots (QDs) offer for NMDD, how they can function as a powerful nanoscale platform to understand this process at its most basic level, and even provide drug-related properties in certain circumstances. Selected examples from the current literature are utilized to describe both their potential and the contributions they have already made towards the design and implementation of NMDD vectors. Important related issues such as QD biofunctionalization and toxicity are also discussed. The paper concludes with a perspective of how this field can be expected to develop in the future.

Cytotoxicity of Quantum Dots Used for In Vitro Cellular Labeling: Role of QD Surface Ligand, Delivery Modality, Cell Type, and Direct Comparison to Organic Fluorophores

Interest in taking advantage of the unique spectral properties of semiconductor quantum dots (QDs) has driven their widespread use in biological applications such as in vitro cellular labeling/imaging and sensing. Despite their demonstrated utility, concerns over the potential toxic effects of QD core materials on cellular proliferation and homeostasis have persisted, leaving in question the suitability of QDs as alternatives for more traditional fluorescent materials (e.g., organic dyes, fluorescent proteins) for in vitro cellular applications. Surprisingly, direct comparative studies examining the cytotoxic potential of QDs versus these more traditional cellular labeling fluorophores remain limited. Here, using CdSe/ZnS (core/shell) QDs as a prototypical assay material, we present a comprehensive study in which we characterize the influence of QD dose (concentration and incubation time), QD surface capping ligand, and delivery modality (peptide or cationic amphiphile transfection reagent) on cellular viability in three human cell lines representing various morphological lineages (epithelial, endothelial, monocytic). We further compare the effects of QD cellular labeling on cellular proliferation relative to those associated with a panel of traditionally employed organic cell labeling fluorophores that span a broad spectral range. Our results demonstrate the important role played by QD dose, capping ligand structure, and delivery agent in modulating cellular toxicity. Further, the results show that at the concentrations and time regimes required for robust QD-based cellular labeling, the impact of our in-house synthesized QD materials on cellular proliferation is comparable to that of six commercial cell labeling fluorophores. Cumulatively, our results demonstrate that the proper tuning of QD dose, surface ligand, and delivery modality can provide robust in vitro cell labeling reagents that exhibit minimal impact on cellular viability.

Sensors for detecting biological agents

Biological agents including viruses, bacteria, and other naturally occurring pathogenic organisms, along with the toxins they produce, are considered far harder to detect and defend against than chemical agents. Here we provide an overview of the predominant molecular sensing technologies for the detection of these agents. This includes biosensing strategies based upon use of antibodies, genomic analysis, biochemical testing, other recognition interactions, and cellular-based responses. We survey some popular sensing approaches, illustrate them with current examples showing how they have been applied, and discuss their intrinsic benefits and potential liabilities. Lastly, within the context of security applications, some approaches for integrating sensing technologies into field-portable devices are discussed.

Transcriptional and apoptotic responses of THP-1 cells to challenge with toxigenic, and non-toxigenic Bacillus anthracis

BackgroundBacillus anthracis secretes several virulence factors targeting different host organs and cell types during inhalational anthrax infection. The bacterial expression of a key virulence factor, lethal toxin (LeTx) is closely tied to another factor, edema toxin (EdTx). Both are transcribed on the same virulence plasmid (pXO1) and both have been the subject of much individual study. Their combined effect during virulent anthrax likely modulates both the global transcriptional and the phenotypic response of macrophages and phagocytes. In fact, responses brought about by the toxins may be different than each of their individual effects.ResultsHere we report the transcriptional and apoptotic responses of the macrophage-like phagocytic cell line THP-1 exposed to B. anthracis Sterne (pXO1+) spores, and B. anthracis Δ Sterne (pXO1-) spores. These cells are resistant to LeTx-induced cytolysis, a phenotype seen in macrophages from several mouse strains which are sensitive to toxigenic anthrax infection. Our results indicate that the pXO1-containing strain induces higher pro-inflammatory transcriptional responses during the first 4 hours of interaction with bacterium, evident in the upregulation of several genes relevant to Nf-κB, phosphatases, prostaglandins, and TNF-α, along with decreases in expression levels of genes for mitochondrial components. Both bacterial strains induce apoptosis, but in the toxigenic strain-challenged cells, apoptosis is delayed.ConclusionThis delay in apoptosis occurs despite the much higher level of TNF-α secretion induced by the toxigenic-strain challenge. Interestingly, CFLAR, an important apoptotic inhibitor which blocks apoptosis induced by large amounts of extracellular TNF-α, is upregulated significantly during toxigenic-strain infection, but not at all during non-toxigenic-strain infection, indicating that it may play a role in blocking or delaying TNF-α-mediated apoptosis. The suppression of apoptosis by the toxigenic anthrax strain is consistent with the notion that apoptosis itself may represent a protective host cell response.

Assessment of methods and analysis of outcomes for comprehensive optimization of nucleofection

BackgroundNucleofection is an emerging technology for delivery of nucleic acids into both the cytoplasm and nucleus of eukaryotic cells with high efficiency. This makes it an ideal technology for gene delivery and siRNA applications. A 96-well format has recently been made available for high-throughput nucleofection, however conditions must be optimized for delivery into each specific cell type. Screening each 96-well plate can be expensive, and descriptions of methods and outcomes to determine the best conditions are lacking in the literature. Here we employ simple methods, including cell counting, microscopy, viability and cytotoxicity assays to describe the minimal experimental methods required to optimize nucleofection conditions for a given cell line.MethodsWe comprehensively measured and analyzed the outcomes of the 96-well nucleofection of pmaxGFP plasmids encoding green fluorescent protein (GFP) into the A-549 human lung epithelial cell line. Fluorescent microscopy and a plate reader were used to respectively observe and quantify green fluorescence in both whole and lysed cells. Cell viability was determined by direct counting/permeability assays, and by both absorbance and fluorescence-based plate reader cytotoxicity assays. Finally, an optimal nucleofection condition was used to deliver siRNA and gene specific knock-down was demonstrated.ResultsGFP fluorescence among conditions ranged from non-existent to bright, based upon the fluorescent microscopy and plate reader results. Correlation between direct counting of cells and plate-based cytotoxicity assays were from R = .81 to R = .88, depending on the assay. Correlation between the GFP fluorescence of lysed and unlysed cells was high, ranging from R = .91 to R = .97. Finally, delivery of a pooled sample of siRNAs targeting the gene relA using an optimized nucleofection condition resulted in a 70–95% knock down of the gene over 48 h with 90–97% cell viability.ConclusionOur results show the optimal 96-well nucleofection conditions for the widely-used human cell line, A-549. We describe simple, effective methods for determining optimal conditions with high confidence, providing a useful road map for other laboratories planning optimization of specific cell lines or primary cells. Our analysis of outcomes suggests the need to only measure unlysed, whole-cell fluorescence and cell metabolic activity using a plate reader cytotoxicity assay to determine the best conditions for 96-well nucleofection.

Delivery of quantum dot bioconjugates to the cellular cytosol: release from the endolysosomal system

To realize their full potential as intracellular imaging and sensing reagents, robust and efficient methods for the targeted cellular delivery of luminescent semiconductor quantum dots (QDs) must be developed. We have previously shown that QDs decorated with histidine-terminated polyarginine cell-penetrating peptides (CPP) are rapidly and specifically internalized via endocytosis by several mammalian cell lines with no cytotoxicity. Here we demonstrate the long-term intracellular stability and fate of these QD-peptide conjugates. We found that the QD-peptide conjugates remain sequestered within endolysosomal vesicles for up to three days after delivery. However, the CPP appeared to remain stably associated with the QD within these acidic vesicles over this time period. Hence, we explored a number of techniques to either actively deliver QDs directly to the cytosol or to facilitate the endosomal release of endocytosed QDs to the cytosol. Active methods (e.g., electroporation) delivered only modest amounts of QDs to the cytosol that appeared to form aggregates. Delivery of QDs using polymer-based transfection reagents resulted primarily in the endosomal sequestration of the QDs, although one commercial polymer tested delivered QDs to the cytosol but only after several days in culture and with a considerable degree of polymer-induced toxicity. Finally, a modular, amphiphilic peptide containing functionalities designed for cell penetration and vesicular membrane interaction demonstrated the ability to deliver QDs in a well-dispersed manner to the cytosol. This peptide mediated rapid QD uptake followed by a slower efficient endosomal release of the QDs to the cytosol that peaked at 48 hours post-delivery. Importantly, this QD-peptide conjugate elicited minimal cytotoxicity in two cell lines tested. A more detailed understanding of the mechanism of the peptides uptake and endosomal escape attributes will lead to the design of further QD conjugates for targeted imaging and sensing applications.

Specific cellular delivery and intracellular fate of quantum dot-peptide and quantum dot-polymer nanoassemblies

Luminescent semiconductor quantum dots (QDs) possess several unique optical and spectroscopic properties that are of great interest and promise in biology. These properties suggest that QDs will be integral to the development of the next generation of biosensors capable of detecting molecular processes in both living and fixed cells. We are developing robust and facile delivery schemes for the selective intracellular delivery of QD-based nanoassemblies. These schemes are based upon the self-assembly and subsequent cellular uptake of QD-peptide and QD-polymer bioconjugates. The QD-peptide structures are generated by the self-assembly of the peptide onto CdSe-ZnS core-shell QDs via metal ion coordination between the peptides polyhistidine motif and the Zn-rich QD shell. The polymer-based QD assemblies are formed via the electrostatic interaction of aqueous cationic liposomes with available carboxylate moieties on the QD surface ligands. Cellular delivery experiments utilizing both delivery schemes will be presented. The advantages and disadvantages of each approach will be discussed, including the intracellular fate and stability of the QD-nanoassemblies.

Controlling the intracellular fate of nano-bioconjugates: pathways for realizing nanoparticle-mediated theranostics

For nanomaterials to realize their full potential in disease diagnosis and drug delivery applications, one must be able to exert fine control over their cellular delivery, localization and long-term fate in biological systems. Our laboratory has been active in developing methodologies for the controlled and site-specific delivery of a range of nanomaterials (e.g., quantum dots, colloidal gold, nematic liquid crystals) for cellular labeling, imaging and sensing. This talk will highlight several examples from these efforts and will demonstrate the use of peptide- and protein-mediated facilitated delivery of nanomaterials to discrete cellular locations including the endocytic pathway, the plasma membrane and the cellular cytosol. The implications of the ability to exert fine control over nanomaterial constructs in biological settings will be discussed with a particular focus on their use in nanoparticle-based theranostics.