The transition of an α-helix to a β-sheet in proteins is among the most complex conformational changes seen in biomolecular systems. Due to long time scales involved in the transition...

Biological processes performed by proteins interacting with and processing DNA and RNA are key to cell metabolism and life. Detailed insights into these processes provide essential information for understanding the molecular basis of life and the pathological conditions that develop when such processes go awry. The next scientific breakthrough consists in the actual, direct, real-time observations and measurements of the individual mechanisms involved, in order to validate and complete the current biological models. Single-molecule technologies offer an exciting opportunity to meet these challenges and to study protein function and activity in real-time and at the single-molecule level. Here, we present our efforts for further enabling discoveries in the field of biology and biophysics using both the combination of optical tweezers with singlemolecule fluorescence microscopy (C-Trap TM ). We show the latest applications of these technologies that can enhance our understanding not only in the field of DNA/RNA-protein interactions but also in the fields of molecular motors, protein folding/unfolding, cell membranes and genome structure and organization. These experiments show that the technological advances in hybrid single-molecule methods can be turned into an easy-to-use and stable instrument that has the ability to open up new venues in many research areas.

Bacterial infections are a major health problem worldwide. Identification of disease-causing organisms by culture-based approaches is time-consuming and often lacks sensitivity. Molecular approaches such as PCR and microwaveaccelerated metal-enhanced fluorescence (MAMEF) assays1, are more sensitive and faster than traditional culture-based approaches, but require isolation of the target DNA. In order to determine the effect of both boiling and microwave irradiation on microbial lysing and DNA fragmentation, cultures of Neisseria gonorrhoeae and Listeria monocytogenes (108 CFU /mL) were either boiled (range 40 -70 C) or lysed in a 900-watt microwave on isolator-mounted microscope slides, both with and without the assistance of disjointed antenna gold bow-tie structures. The temperatures of cultures were obtained prior to and after lysing and the resulting lysate cultured on selective agar plates. DNA isolation and fragmentation efficiency were determined by gel electrophoresis and PCR. N. gonorrhoeae lysed at a lower temperature ( C) than L. monocytogenes. Microbial lysing and DNA fragmentation was more effectively carried out in the presence disjointed gold triangle structures, but only when small sample volume were used. Standard boiling was successful for bacterial lysing and DNA fragmentation, but required higher temperatures and longer times than microwave focusing. PCR results suggest that low power microwave irradiation is ideal for PCR methods while higher microwave powers are required to generate DNA fragments 1586-Pos Board B537 Molecular Modeling of Self-Assembly of Anticancer Drug Amphiphiles

Uropathogenic strains of E. coli deliver the toxin alpha-hemolysin (HlyA) to optimize the host environment for the spread of infection. It was reported that at high concentrations, the toxin forms pores in eukaryotic membranes, leading to cell lysis, while lower concentrations might interfere with host-cell-signaling pathways, causing apoptosis. In the present investigation we demonstrate that a relatively low concentration of HlyA induces morphological changes and phosphatidylserine (PS) externalization of human erythrocytes. On the other hand, the unacylated nonhemolytic form of HlyA, ProHlyA induces similar morphological changes but no PS externalization. We performed osmoscan experiments to test the effect of both proteins on erythrocytes structure. HlyA treated erythrocytes show increased membrane fragility and cell volume as well as diminished cytoplasmic viscosity and S/V ration. ProHlyA-treated erythrocyte are not different from control ones. Since PS exposure of erythrocytes is known to induce cell adhesion, we used a dynamic cell adhesion platform to study the consequences of HlyA vs ProHlya exposure of erythrocytes on their adhesion to human endothelial cells (HMEC). Results indicate that HlyA-treated erythrocytes adhere more to endothelial cells than Pro-treated erythrocytes at low flux (0.5 din). At higher fluxes (1 and 2 din), however, HlyA-treated erythrocytes detached easily than control ones, indicating that the adherence is weak. We also study the efflux of ATP from erythrocytes treated with both toxins by luciferinluciferase luminescence. Results demonstrate that HlyA induces the efflux of ATP while ProHlyA does not. Since PS exposure was suggested to simultaneously increase extracellular ATP and adhesion to the vascular endothelium, and erythrocyte derived ATPe can alter the caliber of the vascular lumen, future experiments will be designed to relate HlyA induced efflux of ATP of erythrocytes with their adhesion and interaction with endothelial cells.

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