Investigation of 89Zr-Siderophores as Molecular Imaging Agents for Positron Emission Tomography Imaging of Bacterial Infections

Siderophores are small molecules synthesized by bacteria to harvest Fe3+ from their environment. In infection scenarios, their production can increase infection virulence by increasing the ability of bacteria to obtain Fe3+ and therefore grow more rapidly. The selective uptake of siderophores in vivo in multi-bacteria environments indicates that this class of molecules has a potential use as selective imaging agents. In this work, DFO-NCS and a library of trihydroxamate siderophores were evaluated as vehicles to deliver 89Zr selectively to bacteria for Positron Emission Tomography (PET) imaging of bacterial infections. Productive work with radiometals involves thorough knowledge of the element’s chemistry as well as the sources and detrimental effects of any contaminating metal ions present with the radiometal in the reaction mixture. As a case study to determine the factors likely to interfere with the complexation of any given radiometal, the quality control assay used to determine effective specific activity (ESA) of 64Cu was intensely examined. The purpose of this study was to identify sources of cold metal contaminants in the 64Cu production process and to identify which of those metals interfere with the binding of 64Cu to the TETA chelator. The TETA titration method for determining 64Cu ESA has relative standard deviations of 27.6% and 40.3% for repeatability and reproducibility respectively and the chelator TETA is selective for picomolar amounts of Cu2+ in the presence of low millimolar concentrations of Zn2+ and Ni2+. When the 89Zr-DFO-NCS complex was tested against a panel of cell types, the uptake by human cells (SKBR3), Staphylococcus aureus cells, and Pseudomonas aeruginosa cells was significantly different (pS. aureus. The Zr chemistry and bacterial uptake behavior of a library of trihydroxamate siderophores was then evaluated and compared to that of DFO-NCS. The uptake of 89Zr-DFO-NCS and a siderophore library member (89Zr-V-129) were tested in a murine lung infection model (P. aeruginosa, PA M57-15) and the lung uptake of 89Zr-V-129 was found to be significantly higher in infected mice (p=0.012035) than in control mice. The uptake of 89Zr-DFO-NCS did not differ significantly between control and infected mice (p=0.831). 89Zr-siderophores have been shown to possess potential to be selective, specific PET tracers for imaging bacterial infections in vivo and their utility for infection imaging should be more thoroughly explored

Design and Experimental Validation of Continuous DNA Separation using ITP-Based Microfluidic Device

We have demonstrated a chip-based microfluidic device suitable for rapid and high-throughput DNA separation and purification for downstream analysis. The glass-based device incorporates a transverse free-flow isotachophoresis (tFF-ITP) that overcomes the volume limitations of CE approaches, and allows continuous sample processing in contrast to batch-mode solid-phase extraction (SPE). The device is able to focus DNA at flow rates up to 100 uL/min, and sample conductivity up to 2 mS/cm. Downstream of the chip, 30-40% of DNA from the input sample is recovered as a result of ITP focusing, and preliminary results indicate that this design is able to purify DNA from contaminating species, particularly those that inhibit PCR

\u3ci\u3eDrosophila\u3c/i\u3e Muller F Elements Maintain a Distinct Set of Genomic Properties Over 40 Million Years of Evolution

The Muller F element (4.2 Mb, ~80 protein-coding genes) is an unusual autosome of Drosophila melanogaster; it is mostly heterochromatic with a low recombination rate. To investigate how these properties impact the evolution of repeats and genes, we manually improved the sequence and annotated the genes on the D. erecta, D. mojavensis, and D. grimshawi F elements and euchromatic domains from the Muller D element. We find that F elements have greater transposon density (25–50%) than euchromatic reference regions (3–11%). Among the F elements, D. grimshawi has the lowest transposon density (particularly DINE-1: 2% vs. 11–27%). F element genes have larger coding spans, more coding exons, larger introns, and lower codon bias. Comparison of the Effective Number of Codons with the Codon Adaptation Index shows that, in contrast to the other species, codon bias in D. grimshawi F element genes can be attributed primarily to selection instead of mutational biases, suggesting that density and types of transposons affect the degree of local heterochromatin formation. F element genes have lower estimated DNA melting temperatures than D element genes, potentially facilitating transcription through heterochromatin. Most F element genes (~90%) have remained on that element, but the F element has smaller syntenic blocks than genome averages (3.4–3.6 vs. 8.4–8.8 genes per block), indicating greater rates of inversion despite lower rates of recombination. Overall, the F element has maintained characteristics that are distinct from other autosomes in the Drosophila lineage, illuminating the constraints imposed by a heterochromatic milieu