Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.     

Dietary pyrroloquinoline quinone (PQQ) alters indicators of inflammation and mitochondrial-related metabolism in human subjects

Pyrroloquinoline quinone (PQQ) influences energy-related metabolism and neurologic functions in animals. The mechanism of action involves interactions with cell signaling pathways and mitochondrial function. However, little is known about the response to PQQ in humans. Using a crossover study design, 10 subjects (5 females, 5 males) ingested PQQ added to a fruit-flavored drink in two separate studies. In study 1, PQQ was given in a single dose (0.2 mg PQQ/kg). Multiple measurements of plasma and urine PQQ levels and changes in antioxidant potential [based on total peroxyl radical-trapping potential and thiobarbituric acid reactive product (TBAR) assays] were made throughout the period of 48 h. In study 2, PQQ was administered as a daily dose (0.3 mg PQQ/kg). After 76 h, measurements included indices of inflammation [plasma C-reactive protein, interleukin (IL)-6 levels], standard clinical indices (e.g., cholesterol, glucose, high-density lipoprotein, low-density lipoprotein, triglycerides, etc.) and ¹H-nuclear magnetic resonance estimates of urinary metabolites related in part to oxidative metabolism. The standard clinical indices were normal and not altered by PQQ supplementation. However, dietary PQQ exposure (Study 1) resulted in apparent changes in antioxidant potential based on malonaldehyde-related TBAR assessments. In Study 2, PQQ supplementation resulted in significant decreases in the levels of plasma C-reactive protein, IL-6 and urinary methylated amines such as trimethylamine N-oxide, and changes in urinary metabolites consistent with enhanced mitochondria-related functions. The data are among the first to link systemic effects of PQQ in animals to corresponding effects in humans.     

Proteomic patterns for classification of ovarian cancer and CTCL serum samples utilizing peak pairs indicative of post-translational modifications

Proteomic patterns as a potential diagnostic technology has been well established for several cancer conditions and other diseases. The use of machine learning techniques such as decision trees, neural networks, genetic algorithms, and other methods has been the basis for pattern determination. Cancer is known to involve signaling pathways that are regulated through PTM of proteins. These modifications are also detectable with high confidence using high-resolution MS. We generated data using a prOTOF mass spectrometer on two sets of patient samples: ovarian cancer and cutaneous t-cell lymphoma (CTCL) with matched normal samples for each disease. Using the knowledge of mass shifts caused by common modifications, we built models using peak pairs and compared this to a conventional technique using individual peaks. The results for each disease showed that a small number of peak pairs gave classification equal to or better than the conventional technique that used multiple individual peaks. This simple peak picking technique could be used to guide identification of important peak pairs involved in the disease process.     

Position of single amino acid substitutions in the collagen triple helix determines their effect on structure of collagen fibrils

Collagen II fibrils are a critical structural component of the extracellular matrix of cartilage providing the tissue with its unique biomechanical properties. The self-assembly of collagen molecules into fibrils is a spontaneous process that depends on site-specific binding between specific domains belonging to interacting molecules. These interactions can be altered by mutations in the COL2A1 gene found in patients with a variety of heritable cartilage disorders known as chondrodysplasias. Employing recombinant procollagen II, we studied the effects of R75C or R789C mutations on fibril formation. We determined that both R75C and R789C mutants were incorporated into collagen assemblies. The effects of the R75C and R789C substitutions on fibril formation differed significantly. The R75C substitution located in the thermolabile region of collagen II had no major effect on the fibril formation process or the morphology of fibrils. In contrast, the R789C substitution located in the thermostable region of collagen II caused profound changes in the morphology of collagen assemblies. These results provide a basis for identifying pathways leading from single amino acid substitutions in collagen II to changes in the structure of individual fibrils and in the organization of collagenous matrices.     

Polyproline II helical structure in protein unfolded states: lysine peptides revisited

The left-handed polyproline II (PPII) helix gives rise to a circular dichroism spectrum that is remarkably similar to that of unfolded proteins. This similarity has been used as the basis for the hypothesis that unfolded proteins possess considerable PPII helical content. It has long been known that homopolymers of lysine adopt the PPII helical conformation at neutral pH, presumably a result of electrostatic repulsion between side chains. It is shown here that a seven-residue lysine peptide also adopts the PPII conformation. In contrast with homopolymers of lysine, this short peptide is shown to retain PPII helical character under conditions in which side-chain charges are heavily screened or even neutralized. The most plausible explanation for these observations is that the peptide backbone favors the PPII conformation to maximize favorable interactions with solvent. These data are evidence that unfolded proteins do indeed possess PPII content, indicating that the ensemble of unfolded states is significantly smaller than is commonly assumed.     

Pheromone trap for the eastern tent caterpillar moth

The discovery that the eastern tent caterpillar Malacosoma americanum (F.) causes mare reproductive loss syndrome (MRLS), and thus has the potential to continue to result in major economic losses to the equine industry of Kentucky, has resulted in an intensive effort to identify practical means to monitor and control this defoliator, including these experiments to optimize a sex pheromone trap for this pest. A pheromone-baited delta trap with a large opening, such as InterceptST Delta, was more effective than other tested traps. Orange delta traps caught more moths than other tested colors. ETC males are caught at all tested heights within the tree canopy. For monitoring flights, setting traps at 1.5 m would allow easy counting of moths. A 9:1 blend of (E,Z)-5,7-dodecadienal (ETC-Ald) and (E,Z)-5,7-dodecadienol (ETC-OH) was most effective in capturing males. Increasing loading doses of a 3:1 blend (Ald:OH) resulted in the capture of increasing numbers of moths, but a 9:1 blend was more effective than 3:1 blend even at a nine-fold lower loading rate. Pheromone-impregnated white septa caught more moths than gray septa at the same loading dose. The advantages and limitations of using pheromone traps for monitoring M. americanum are discussed.     

Abnormal development and increased 3-nitrotyrosine in copper-deficient mouse embryos

Copper-deficient rat embryos are characterized by brain and heart anomalies, low superoxide dismutase activity, and high superoxide anion concentrations. One consequence of increased superoxide anions can be the formation of peroxynitrite, a strong biological oxidant. To investigate developmentally important features of copper deficiency, GD 8.5 mouse embryos from copper-adequate and copper-deficient dams were cultured in media that were adequate or deficient in copper. After 48 h, copper-deficient embryos exhibited brain and heart anomalies, and a high incidence of yolk sac vasculature abnormalities compared to controls. Immunohistochemistry of 4-hydroxynonenal and 8-hydroxy-2'-deoxyguanosine for lipid and DNA damage, respectively, was similar between groups. In contrast, 3-nitrotyrosine, taken as a measure of protein nitration, was markedly higher in the neuroepithelium of the anterior neural tube of copper-deficient embryos than in controls. Repletion of copper-deficient media with copper, or supplementation with copper-zinc superoxide dismutase, Tiron, or glutathione peroxidase did not ameliorate the abnormal development, but did decrease 3-nitrotyrosine in neuroepithelium of copper-deficient embryos. These data support the concept that while copper deficiency compromises oxidant defense and increases protein nitration, additional mechanisms, e.g., altered nitric oxide metabolism may contribute to copper-deficiency-induced teratogenesis.