PATHOGENS IN QUAILS OF THE TRANS-PECOS ECOREGION OF TEXAS.

Texas quail populations have declined over the past few decades. While habitat loss has been identified as the primary cause, it has been speculated that pathogens may also play a role in this decline. To help address this, we collected scaled quail, Callipepla squamata, Gambel's quail, Callipepla gambelii, and Montezuma quail, Cyrtonyx montezumae, from across the Trans-Pecos ecoregion of Texas via hunter-harvest. Quail samples were then necropsied to document pathogens not previously recorded in the host species. Pathogens were submitted to the Texas A&M University Veterinary Medicine Diagnostic Lab (TVMDL), where parasite identification and histopathological analyses were conducted. From this, we identified several parasites that had never been documented in the quails of the Trans-Pecos ecoregion of Texas. This study was the first to document Mycobacterium sp. and Sarcocystis sp. in scaled quail, Subulura sp. and Physaloptera sp. in Montezuma quail, and Oxyspirura petrowi and Aulonocephalus pennula in a Texas Gambel's quail.

Bioinspired Synthesis of Pinoxaden Metabolites Using a Site-Selective C-H Oxidation Strategy.

A bioinspired synthesis of Pinoxaden metabolites 2-5 is described herein. A site-selective C-H oxidation strategy validated by density functional theory (DFT) calculations was devised for preparing metabolites 2-4. Oxidation of the benzylic C-H bond in tertiary alcohol 7 using K2S2O8 and catalytic AgNO3 formed the desired metabolite 2 that enabled access to metabolites 3 and 4 in a single step. Unlike most metal/persulfate-catalyzed transformations reported for the C-C and C-O bond formation reactions wherein the metal acts as a catalyst, we propose that Ag(I)/K2S2O8 plays the role of an initiator in the oxidation of intermediate 7 to 2. Metabolite 2 was subjected to a ruthenium tetroxide-mediated C-H oxidation to form metabolites 3 and 4 as a mixture that were purified to isolate pure standards of these metabolites. Metabolite 5 was synthesized from readily available advanced intermediate 9 via a House-Meinwald-type rearrangement in one step using a base.

First Documentation of Nematode Dispharynx sp. in Scaled Quail.

Quail populations in the United States have been declining for several decades, and the role that parasites may be playing in this decline is not well understood. The goal of this study was to document novel parasites that inhabited the scaled quail, Callipepla squamata, of the Trans-Pecos ecoregion of Texas. To do this, quail were collected by hunter-harvest, night-netting, and funnel-trapping and were necropsied in the laboratory to determine the parasites they hosted. After analyzing 386 birds, we identified Dispharynx sp. in one of the samples. This specimen is the first to be officially documented in scaled quail.

Low temperature assembly of functional 3D DNA-PNA-protein complexes.

Proteins have evolved to carry out nearly all the work required of living organisms within complex inter- and intracellular environments. However, systematically investigating the range of interactions experienced by a protein that influence its function remains challenging. DNA nanostructures are emerging as a convenient method to arrange a broad range of guest molecules. However, flexible methods are needed for arranging proteins in more biologically relevant 3D geometries under mild conditions that preserve protein function. Here we demonstrate how peptide nucleic acid (PNA) can be used to control the assembly of cytochrome c (12.5 kDa, pI 10.5) and azurin (13.9 kDa, pI 5.7) proteins into separate 3D DNA nanocages, in a process that maintains protein function. Toehold-mediated DNA strand displacement is introduced as a method to purify PNA-protein conjugates. The PNA-proteins were assembled within 2 min at room temperature and within 4 min at 11 °C, and hybridize with even greater efficiency than PNA conjugated to a short peptide. Gel electrophoresis and steady state and time-resolved fluorescence spectroscopy were used to investigate the effect of protein surface charge on its interaction with the negatively charged DNA nanocage. These data were used to generate a model of the DNA-PNA-protein complexes that show the negatively charged azurin protein repelled away from the DNA nanocage while the positively charged cytochrome c protein remains within and closely interacts with the DNA nanocage. When conjugated to PNA and incorporated into the DNA nanocage, the cytochrome c secondary structure and catalytic activity were maintained, and its redox potential was reduced modestly by 20 mV possibly due to neutralization of some positive surface charges. This work demonstrates a flexible new approach for using 3D nucleic acid (PNA-DNA) nanostructures to control the assembly of functional proteins, and facilitates further investigation of protein interactions as well as engineer more elaborate 3D protein complexes.

Purification and assembly of thermostable Cy5 labeled γ-PNAs into a 3D DNA nanocage.

PNA is hybrid molecule ideally suited for bridging the functional landscape of polypeptides with the structural diversity that can be engineered with DNA nanostructures. However, PNA can be more challenging to work with in aqueous solvents due to its hydrophobic nature. A solution phase method using strain promoted, copper free click chemistry was developed to conjugate the fluorescent dye Cy5 to 2 bifunctional PNA strands as a first step toward building cyclic PNA-polypeptides that can be arranged within 3D DNA nanoscaffolds. A 3D DNA nanocage was designed with binding sites for the 2 fluorescently labeled PNA strands in close proximity to mimic protein active sites. Denaturing polyacrylamide gel electrophoresis (PAGE) is introduced as an efficient method for purifying charged, dye-labeled PNA conjugates from large excesses of unreacted dye and unreacted, neutral PNA. Elution from the gel in water was monitored by fluorescence and found to be more efficient for the more soluble PNA strand. Native PAGE shows that both PNA strands hybridize to their intended binding sites within the DNA nanocage. Förster resonance energy transfer (FRET) with a Cy3 labeled DNA nanocage was used to determine the dissociation temperature of one PNA-Cy5 conjugate to be near 50°C. Steady-state and time resolved fluorescence was used to investigate the dye orientation and interactions within the various complexes. Bifunctional, thermostable PNA molecules are intriguing candidates for controlling the assembly and orientation of peptides within small DNA nanocages for mimicking protein catalytic sites.

Purification and assembly of thermostable Cy5 labeled γ-PNAs into a 3D DNA nanocage.

PNA is hybrid molecule ideally suited for bridging the functional landscape of polypeptides with the structural diversity that can be engineered with DNA nanostructures. However, PNA can be more challenging to work with in aqueous solvents due to its hydrophobic nature. A solution phase method using strain promoted, copper free click chemistry was developed to conjugate the fluorescent dye Cy5 to 2 bifunctional PNA strands as a first step toward building cyclic PNA-polypeptides that can be arranged within 3D DNA nanoscaffolds. A 3D DNA nanocage was designed with binding sites for the 2 fluorescently labeled PNA strands in close proximity to mimic protein active sites. Denaturing polyacrylamide gel electrophoresis (PAGE) is introduced as an efficient method for purifying charged, dye-labeled NA conjugates from large excesses of unreacted dye and unreacted, neutral PNA. Elution from the gel in water was monitored by fluorescence and found to be more efficient for the more soluble PNA strand. Native PAGE shows that both PNA strands hybridize to their intended binding sites within the DNA nanocage. Förster resonance energy transfer (FRET) with a Cy3 labeled DNA nanocage was used to determine the dissociation temperature of one PNA-Cy5 conjugate to be near 50C. Steady-state and time resolved fluorescence was used to investigate the dye orientation and interactions within the various complexes. Bifunctional, thermostable PNA molecules are intriguing candidates for controlling the assembly and orientation of peptides within small DNA nanocages for mimicking protein catalytic sites.

Biofilm formation in cochlear implants with cochlear drug delivery channels in an in vitro model.

BACKGROUND: Cochlear implant (CI) drug delivery (DD) may improve electrophysiological outcomes, but it may also increase the risk of suppurative complications. The aim of this study was to evaluate the development of bacterial biofilms on DD ports when subjected to varying types of penetration. METHODS: Silastic models were constructed to represent CIs with a DD channel, with an intact port, a widely opened port, a noncoring needle penetrating the port, and a noncoring needle removed from the port. CIs were exposed to a culture of a biofilm-forming strain of Staphylococcus aureus for 5 days. Biofilm formation was assessed with quantitative bacterial counts (after eliminating planktonic bacteria) and scanning electron microscopy. RESULTS: Bacterial counts were significantly higher in CIs with widely fenestrated ports than all other port conditions (P = 0.0003). CONCLUSIONS: Biofilm formation may be minimized on CIs with DD by using fine, noncoring needles and limiting the duration of port penetration.

Biofilm formation in an in vitro model of cochlear implants with removable magnets.

BACKGROUND: Cochlear implant (CI) recesses, such as the removable magnet pocket, appear to harbor more biofilm than smooth surfaces. The aim of this study was to examine the impact of removable magnets on biofilm formation in an in vitro model. METHODS: Silastic models were constructed to represent CIs with and without a magnet pocket and with and without a titanium blank in the pocket. CIs were exposed to a culture of a biofilm forming strain of Staphylococcus aureus. Adherence of planktonic bacteria and biofilm formation were assessed with quantitative bacterial counts and scanning electron microscopy. RESULTS: Adherent bacterial counts were significantly higher in CI models with an empty magnet pocket (P = 0.0097). Biofilm formation was significantly lower in CI models without a magnet pocket (P = 0.0121). CONCLUSIONS: CI magnet pockets harbor bacteria that can increase biofilm development in an in vitro model.