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Bacterial persistence is a phenomenon in which a small fraction of a bacterial population enters dormancy in otherwise growth-promoting conditions to survive future stress. These survivors are responsible for the relapse of biofilm infections, and thus a greater understanding of their formation will lead to more effective therapies against biofilm-utilizing pathogens, such as Pseudomonas aeruginosa, Escherichia coli, Acinetobacter baumannii, and Staphylococcus aureus. We have discovered that diauxic carbon shifts stimulate the generation of persisters in planktonic cultures, and hypothesized that metabolic transitions generate persisters in biofilms. In this project, we are identifying metabolic transitions in biofilm communities of E. coli, P. aeruginosa, and S. aureus that generate persisters, and using genetic and biochemical techniques to reconstruct the underlying signaling pathways. During this reporting period we discovered that specific carbon source transitions in E. coli biofilms stimulate persister formation through a ppGpp and nucleoid-associated protein dependent pathway. In addition, preliminary data suggests that nitrogen source transitions in P. aeruginosa biofilms and carbon source transitions in S. aureus biofilms stimulate persister formation. Further, we have developed a rapid method to assay persister metabolic activity. Support from this grant has led to three publications and one submitted manuscript thus far., The original document contains color images.

Work supported by this grant led to major theoretical and experimental advances in the physics of sodium (Na) guide stars for use in adaptive optics. We developed a powerful and realistic new theory of the effect of velocity-changing and spin changing collisions on optically pumped atoms at low pressures, similar to those of Na guide star atoms at altitude of about 100 km in the earth's atmosphere. Particularly useful was the development of new cusp kernels to describe the combined effects of strong velocity changing collisions acting simultaneously with spin precession in geomagnetic fields and spin-exchange collisions with oxygen molecules and dissociated oxygen atoms in the upper atmosphere. We implemented laboratory experiments that mimicked as closely as possible the collisional conditions of the upper atmosphere, given the unavoidable need for constraining cell walls. We developed a new, temperature-insensitive way to use Faraday rotation to lock a tunable diode laser to an arbitrary frequency in the absorption spectrum of an atomic resonance line., The original document contains color images.

This report summarizes advanced quantum cascade laser (QCL) development during the Phase Ib performance period of DARPA's EMIL program. Examining tunneling in QCLs, we found that strong dephasing from interface roughness scattering poses a likely bottleneck for electron transport. We therefore proposed an ultra-strong coupling design strategy and calculated the optimal coupling strength with a density matrix based model. By implementing the ultra-strong coupling design strategy in real QCLs, we demonstrated a major step forward in the overall device performance and achieved a record breaking wall plug efficiency (WPE) of 50% (pulsed at cryogenic temperatures). Ultra-strong coupling also produced QCLs with exceedingly broad optical gain (e.g. 400 cm-1 at peak emission wavelengths around 4.x m), as well as low threshold current density, large slope efficiency (5 W/A), and high WPE (23 %) in pulsed mode at 295 K. To further study transport in QCLs, electron transit times of different QCLs were measured and compared to calculated upper laser level lifetimes with and without interface roughness scattering. A better correlation is found when including interface roughness, suggesting that it plays a crucial role in determining intersubband lifetimes in mid-infrared QCLs and should routinely be included in design. Quantum Cascade (QC) lasers have seen and continue to see rapid performance improvements driven by applications such as chemical trace gas sensing or infrared countermeasures. The DARPA EMIL program has contributed immeasurable value to the development of high-performance QC lasers, especially in the short 4 5 m wavelength range. High wall-plug efficiency (WPE) was one of this program s main focus. This report summarizes our team s main activities and results during the Phase Ib performance period.

We are creating an anti-microbial sense-and-destroy system by engineering sentinel/killer cells that detect the presence of pathogenic bacteria, report the identity of the pathogen with a coded output signal, and secrete specific bacteriocins/lysins that destroy the pathogen. Engineering cells for detection and targeted destruction of pathogens offers a single integrated solution to eradicate multiple threats with an approach that is a rapid, selective, and highly sensitive. We are engineering a single cell type that detects chemical signals naturally produced by different classes of pathogens and initiates multiple specific responses, each tailored to particular members of the classes. The system will thus be able to analyze environmental conditions and execute an "intelligent" response by utilizing multiple, customized treatments., The original document contains color images.

The electronic properties of the two-dimensional (2D) electron systems in modulation-doped AlAs quantum wells are investigated, with the goal of developing this material as a sensor with an unprecedented, large sensitivity to stress and/or strain. Such a sensor may find use in various scanning probe microscopes, and in other applications where minute amounts of variables such as force, displacement, or pressure need to be measured., The original document contains color images.

We combined microfluidic technology with recently developed algorithms of nucleic acid-based computing to demonstrate a compact, automated nucleotide-based computational device capable of rapid detection of computational output. We explored both experimental and theoretical aspects of the construction of microfluidic nucleic acid computing. Microreactors implementing Boolean operators lent themselves to a relatively simple implementation of DNA computing. On the theoretical side, we simulated the performance of individual reactors and designed small reactor systems (sorters) mimicking AND and OR gates and systems capable of solving complex Boolean expressions. We compared positive vs. negative selection and parallel vs. series arrangements of reactors, considering both the accuracy of and disproportionate representation ( bias ) within the final pool of solutions. We also developed O(P2) software that designs operating instructions for an O(NL) system capable of solving any Boolean expression. We developed software for automated data collection and signal analysis of electronic detection of labeled DNA and explored novel nanocrystal probes that enable detection of DNA for readout in on-chip electrical assays. We focused in the areas of methods to produce nanocrystals bearing precise numbers of oligonucleotides, and development of nanocrystal precise groupings that bear a collective signature., The original document contains color images. Funded in part by DARPA.

The long-term goal of this research is to explore the molecular mechanisms that marine bacteria use for intra-species and inter-species cell-cell communication. To do this we will investigate the functions controlled by the two quorum sensing signalling pathways of the model luminous bacterium Vibrio harveyi. We have shown that V. harveyi uses Signalling System 1 for intra-species communication and Signalling System 2 for inter-species cell-cell communication. We will identify target genes that are regulated exclusively by System I and System 2, and those target genes that are jointly controlled by both Systems. Our analysis of this regulatory network will lead to a molecular understanding of how bacteria integrate, process and transduce sensory information to control gene expression in response to inter-cellular communication.