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The discovery that plant smoke contains germination stimuli has led to the identification of a new class of signaling molecules named karrikins. Here we report a potential second role for these molecules: in various bacterial species – A. tumefaciens, P. aeruginosa and V. harveyi – they modulate bacterial quorum-sensing (QS), with very different outcomes.

Sulforaphane and erucin, two natural isothiocyanates that are highly abundant in broccoli and other cruciferous vegetables, were found to strongly inhibit quorum sensing and virulence in Pseudomonas aeruginosa. Mechanistic evaluations of these effects suggest that these isothiocyanates are antagonists of the transcriptional activator LasR.

The 2:1 adducts of dimedone and phenalen-1,3-dione with 2- and 4-pyridine carboxaldehyde, in spite of similar chemical behavior of their diketone precursors, have quite different tautomeric structure both in solid state and in solution. 2,2′-(Pyridin-2-ylmethanediyl)-bis(5,5-dimethyl-cyclohexane-1,3-dione) 5 exists as an equilibrium mixture of its dienol tautomer 5а′ with two intramolecular H-bonds ОН⋯О С and OH⋯N and the epimeric products of its reversible cyclization, that is, 4a-hydroxy-9-(pyridin-2-yl)-2,3,4,4a,6,7,9,9a-octahydro-5-H-xanthene-1,8-diones 5b (major) and 5c (minor), the latter appears only in polar media like DMSO. 2,2′-(Pyridin-4-ylmethanediyl)bis(5,5-dimethylcyclohexane-1,3-dione) 4, like other 2:1 dimedone–aldehyde adducts, both in solution and in solid state exists as dienol with two intramolecular H-bonds ОН⋯О С. 4-[Bis(1H-phenalen-1,3(2H)-dione)methyl]pyridine 6 in nonpolar media like chloroform exists as dienol, but crystallizes from this solvent as zwitter-ion 6b with one very strong ionic hydrogen bond O H⋯O and protonated pyridine nitrogen. The same zwitterion is formed in polar media (DMSO). For 2-[bis(1H-phenalen-1,3(2H)-dione)-methyl]-pyridine 7, fast exchange between its dienol tautomer 7a and zwitter-ion 7b occurs even in CD2Cl2, whereas in DMSO the equilibrium shifts towards zwitter-ion 7b.

Live cell fluorescent labeling of proteins has become a seminal tool in biology and has led to hallmark discoveries in diverse research areas such as protein trafficking, cell-to-cell interactions, and intracellular network dynamics. One of the main challenges, however, remains the ability to label intracellular proteins using fluorescent ligands with high specificity, all the while retaining viability of the targeted cells. Here, we present the first example of live cell labeling and imaging of an intracellular bacterial receptor involved in cell-to-cell communication (i.e., quorum sensing), using a novel two-step approach involving covalent attachment of a reactive mimic of the primary endogenous Pseudomonas aeruginosa quorum-sensing signal to its receptor, LasR, followed by aniline-catalyzed oxime formation between the modified receptor and a fluorescent BODIPY derivative. Our results indicate that LasR is not distributed evenly throughout the cytoplasmic membrane but is instead concentrated at the poles of the P. aeruginosa cell.

Chemical coordination of gene expression among bacteria as a function of population density is regulated by a mechanism known as 'quorum sensing' (QS). QS in Pseudomonas aeruginosa, an opportunistic pathogen that causes disease in immunocompromised patients, is mediated by binding of the transcriptional activator, LasR, to its ligand, 3-oxo-C(12)-HSL, leading to population-wide secretion of virulence factors and biofilm formation. We have targeted QS in P. aeruginosa with a set of electrophilic probes designed to covalently bind Cys79 in the LasR binding pocket, leading to specific inhibition of QS-regulated gene expression and concomitant reduction of virulence factor secretion and biofilm formation. This first example of covalent modification of a QS receptor provides a new tool to study molecular mechanisms of bacterial group behavior and could lead to new strategies for targeting bacterial virulence.

Derivatives 2–6 were prepared as models for studying intra- and intermolecular C(sp2)–H···O hydrogen bonding. Their X-ray structures confirm the presence of intramolecular hydrogen bonds in derivatives of the “a” series: the corresponding C···O distances vary between 2.91 and 2.97 A. The corresponding 13C–1H coupling constants are increased by about 7.5 Hz, and the 1H chemical shifts in CDCl3 are 9.1–10.7 ppm. No intramolecular hydrogen bonds can form in derivatives of the isomeric “b” series. In this series, the chemical shifts of the corresponding aromatic protons exhibit strong solvent dependency; in particular, they are as sensitive as the proton in chloroform to the presence of DMSO. The vinylic protons activated by the electron-accepting COOR groups behave similarly. Quantum mechanical calculations in the gas phase and in DMSO reproduce the experimental observations. Energies of the intramolecular hydrogen bonds evaluated by two independent approaches vary between 3.7 and 4.4 kcal mol–1 in the gas phase and still amount to at least 2.5 kcal mol–1 in 2a in DMSO. These estimates are practically independent of the computational method (HF, MP2, and DFT B3LYP were employed for derivatives 2). We conclude that the behavior of both activated aromatic and vinylic C(sp2)–H atoms in the studied derivatives is qualitatively and quantitatively similar to the behavior of the C(sp3)–H atom in chloroform. The existence of hydrogen bonds involving these atoms can easily be detected by NMR spectroscopy.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Derivatives of a series of 1,3-indandione (1) self-condensation products (2–4) and their anions (2a–4a) were investigated by NMR and UV/Vis spectroscopy and quantum mechanical calculations. These compounds exhibit a number of unusual features in their NMR spectra. Parent compound 1 is nearly not ionized in DMSO, whereas 3 is ionized completely. In contrast, derivatives 2 and 4 undergo partial ionization in DMSO and interaction between the neutral and anionic species affords the anionic intermolecular complexes, which exhibit both temperature- and concentration-dependent NMR spectra. The NMR spectra of pure salts 2a and 3a are concentration independent, but temperature dependent. Charge delocalization in anions 2a and 3a gives rise to restricted rotation over the partial double bonds connecting the indane fragments. Energies of barriers to rotation were estimated from the temperature dependence of the 13C NMR spectra. The intramolecular aromatic C–H···O hydrogen bonding found previously in 2–4 is also observed in corresponding anions 2a–4a.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Efficient syntheses, X-ray structures and spectroscopic properties of s-indacene-1,3,5,7(2H,6H)-tetraone (1) and 1,3-dioxo-5,6-indanedicarboxylic acid (2), their precursors and derivatives, are described for the first time.

The synthesis and evaluation of a 'tag-free' probe to isolate and identify receptors for N-acyl homoserine lactones is described.

The primary quorum sensing system in the opportunistic pathogen Pseudomonas aeruginosa is regulated through the synthesis and secretion of N-3-oxo-dodecanoyl-L-homoserine lactone (C12) which binds the transcriptional activator LasR. In this study we report the design, synthesis and biological evaluation of new analogs of C12. Analysis of the autoinducer binding site cavity of LasR revealed a positively charged cavity near the center of bound C12. Accordingly, we synthesized two piperidine-C12 diastereoisomers and tested their biological activity. Both analogs proved to be strong LasR agonists that showed a synergistic effect when presented together with the natural ligand. Moreover, binding of the analogs resulted in phenotypic changes characteristic of QS controlled receptor activation.

The bacterial N-3-oxo-dodecanoyl-L-homoserine lactone (C12) has critical roles in both inter-bacterial communication and inter-kingdom signaling. The ability of C12 to down-regulate production of the key pro-inflammatory cytokine tumor necrosis factor α (TNFα) in stimulated macrophages was suggested to contribute to the establishment of chronic infections by opportunistic Gram-negative bacteria, such as Pseudomonas aeruginosa. We show that in contrast to TNFα suppression, C12 amplifies production of the major anti-inflammatory cytokine interleukin-10 (IL-10) in lipopolysaccharide (LPS)-stimulated murine RAW264.7 macrophages as well as peritoneal macrophages. Furthermore, C12 increases IL-10 mRNA level and IL-10 promoter reporter activity in LPS-stimulated RAW264.7 macrophages, indicating that C12 modulates IL-10 expression at the transcriptional level. Finally, C12 substantially potentiated LPS-stimulated NFκB DNA-binding level, and prolonged p38 MAP kinase phosphorylation in the RAW264.7 macrophages, suggesting that increased transcriptional activity of NFκB and/or p38-activated transcription factors serves to up-regulate IL-10 production in macrophages exposed to both LPS and C12. These findings reveal another part of the complex array of host transitions through which opportunistic bacteria down-regulate immune responses in order to flourish and establish a chronic infection.

The synthesis and comparison of activities of ‘tag-free’ probes with diazirines at various positions are described. Remarkable differences in their effects on P. aeruginosa and on human bronchial epithelial cells were observed, supporting the efforts to isolate and identify receptors for N-acyl homoserine lactones.

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To date, a full-scale solar sail has never flown in space. Furthermore, solar sail technology development represents a field that only recently has enjoyed significant support. The goal of this work is to contribute to the development of a low-mass ODS for solar sails that would include research and development in the areas of photogrammetry and thermography. The focus of this work was on the development of the thermography system. A measurement protocol was designed for obtaining accurate temperature measurements using thermal imaging when heat was applied to the membrane surface. Two main limitations were considered during the experimental process. The first is that conventional infrared detector arrays must be kept cool. To minimize the effect that an imager's operating temperature would have on the ODS, a miniature, un-cooled microbolometer was used to acquire temperature measurements from the membrane surface. A second limitation is that a detector array cannot distinguish between emitted and reflected photons, thus presenting a significant problem if one cannot predict the reflected component or if the reflected component is significantly greater than the emitted. To address this limitation, spectral properties of the membrane, including reflectance and transmission, were analyzed using a Hemispherical Directional Reflectometer (HDR) to predict the effects that optical properties would have on sail membrane temperatures. A thermal modeling strategy was also developed. The results of this investigation are presented.

Efficient syntheses, X-ray structures and spectroscopic properties of s-indacene-1,3,5,7(2H,6H)-tetraone (1) and 1,3-dioxo-5,6-indanedicarboxylic acid (2), their precursors and derivatives, are described for the first time.

Multi-Photon Laser Scanning Microscopy (MPLSM) requires efficient two-photon absorbing fluorescent (TPF) probes. In particular, probes exhibiting bio-functionality are very attractive for MPLSM studies of biological samples. We have synthesized and studied a new class of TPF probes capable of caging metal ions, such as Ca +2 and Na + , which play an important role in neuronal mechanisms. The TPF probes are based on a tetraketo derivative with a symmetric Donor-Acceptor-Donor (D-A-D) structure. The donor is an azacrown moiety, which also serves as a metal ion-caging unit. We studied the linear and the non-linear spectroscopic properties of these TPF probes as a function of conjugation length and the size of the crown ring. We find that this new class of TPF probes possesses very large two-photon excitation cross-section coefficients (∼1000GM) at near IR wavelengths as well as affinity to metal ions. In the presence of changing sodium ion concentration the dye spectra reveals four distinguishable forms and the TPF efficiency changes strongly. We therefore conclude that the dye can perform as a sensitive metal ion TPF probe.

An important ingredient in improving Multi Photon Laser Scanning Microscopy, MPLSM, is the development of efficient two-photon fluorescent (TPF) probes. We previously reported on a new class of TPF probes, specifically designed in order to maximize their efficiency in potential MPLSM applications. The fluorophores are based on a tetraketo derivative (TK) with a symmetric structure Donor-Acceptor-Donor (D-A-D). Those fluorophores have the following properties: a) Very large two-photon absorption coefficients (δ ~ 1000GM); b) Two-photon excitation (TPE) peak wavelength strongly shifted to the red (λ ~ 1µm); c) High fluorescence quantum efficiency; d) Large Stokes shifts of the fluorescence bands. We extended our work to a new fluorophore from this class that is more suitable for biological settings. This new fluorophore has a structure of crown-TK-crown that incorporates the ability to trap metal ions such as calcium. The TPE wavelength dependence of the TK-crown derivative is very similar to its analogous linear derivative with enhancement in the value of the cross-section, due to the stronger donor moieties. The TPE cross-section for the TK-crown derivative was about δ = 950 GM at λ max = 980 nm.

Langmuir-Blodgett films have been prepared from amphiphilc molecules containing an indandione-based nonlinear chromophore. Study of the pressure-area ( -A) isotherm enabled us to find optimal conditions for monolayer transfer to a glass substrate. The multilayer films thus formed exhibited strong optical second harmonic generation with a bulk nonlinear co-efficient equal to the ideal value predicted by the product of the chromophore density and its known molecular hyperpolarizability. Keywords: Langmuir-Blodgett film, second harmonic generation, organic molecules 1. INTRODUCTION The search for efficient second order organic nonlinear optical (NLO) materials and devices has generally been conducted by first finding molecules (or chromophores) with large NLO polarizability ( ) and then incorporating the optimized molecular species into an appropriate non-centrosymmetric bulk system. Since the overwhelming majority of high organic molecules do not spontaneously crystallize in non-centrosymmetric crystals, alternative techniques must be used to create non-centric structures. Two main methods have been used: a) Polymer poling

An important ingredient in improving Multi Photon Laser Scanning Microscopy, MPLSM, is the development of efficient exogenous two-photon fluorescent (TPF) probes. Here we report on a new class of two-photon fluorophores, specifically designed in order to maximize their efficiency in potential MPLSM applications. The fluorophores possess a symmetric Donor-Acceptor-Donor (D-n-A-n-D) structure with varying conjugation length and have strong donors and acceptors. We have studied the two-photon excitation (TPE) properties of these fluorophores and found the following properties: (1) Very large two-photon absorption coefficients (6 > 1000 GM); (2) Peak TP excitation wavelength which are strongly shifted to the red ((lambda) 1 micrometer); (3) Large fluorescence quantum efficiency; (4) Large Stokes shifts of the fluorescence bands. These properties make them particularly suitable for imaging thicker samples, relying on the large improvement in TPE cross-sections and the reduced attenuation at both the excitation and emission wavelengths. We also describe TPE fluorescence anisotropy experiments revealing the tensorial shape of the fluorophores.

The longest wave absorption band of the tetramethylthio- tetrathiafulvalene cation radical, which is usually interpreted as a π-dimer band, is shown to be the intrinsic cation radical absorption, all studied cation radicals in solution at room temperature exist as paramagnetic monomers and only tetrathiafulvelene and tetramethyltetrathiafulvalene cation radicals undergo π-dimerization at low temperatures.

A one-step label-free electrochemical assay for quorum sensing inhibition in Pseudomonas aeruginosa is presented.

We studied Langmuir and Langmuir–Blodgett (LB) films of several strong nonlinear optical (NLO) active amphiphilic derivatives of 1,3-indanedione-5,6-dicarboxylic acid. The surface pressure–area (π–A) isotherms at the air–water interface were investigated at different temperatures and pH values. Non-centrosymmetric Z-type LB films were deposited. UV–visible spectra indicated a uniform film transfer. The orientation of the molecular transition moments was calculated from the polarized UV–visible spectra. Packing within the LB films was observed by UV–visible absorption and fluorescence spectroscopy. The morphology of the LB films was examined by AFM and compared to ellipsometric measurements, and the nature of the films was studied by contact angle measurements. Significant second harmonic generation (SHG) by the LB films was observed. Alternate layer Y-type LB films of two different dyes were fabricated to achieve enhanced stability and SHG.