Pnina Krief

Live Cell Labeling of Native Intracellular Bacterial Receptors Using Aniline-Catalyzed Oxime Ligation

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.

Do π-dimers of tetrathiafulvalene cation radicals really exist at room temperature?

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.

Sulforaphane and erucin, natural isothiocyanates from broccoli, inhibit bacterial quorum sensing

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 Bacterial Quorum-Sensing Signal Molecule N-3-Oxo-Dodecanoyl-l-Homoserine Lactone Reciprocally Modulates Pro- and Anti-Inflammatory Cytokines in Activated Macrophages

The bacterial molecule N-3-oxo-dodecanoyl-l-homoserine lactone (C12) has critical roles in both interbacterial communication and interkingdom signaling. The ability of C12 to downregulate production of the key proinflammatory cytokine TNF-α 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 TNF-α suppression, C12 amplifies production of the major anti-inflammatory cytokine IL-10 in LPS-stimulated murine RAW264.7 macrophages, as well as peritoneal macrophages. Furthermore, C12 increased IL-10 mRNA levels 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 NF-κB DNA-binding levels and prolonged p38 MAPK phosphorylation in RAW264.7 macrophages, suggesting that increased transcriptional activity of NF-κB and/or p38-activated transcription factors serves to upregulate 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 downregulate immune responses to flourish and establish a chronic infection.

Resonance energy transfer in a novel two-component system: two-photon fluorophore and a photo-chromic acceptor molecule

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 imagers 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.

Studies of Langmuir and Langmuir–Blodgett films of NLO-active amphiphilic 1,3-indanedione derivatives

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.

New two-photon fluorescent probe for multiphoton microscopy in biological media

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 λmax = 980 nm.

New long-wave and highly efficient two-photon fluorophores for multiphoton microscopy

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.

Studies of new two-photon fluorescent probes suitable for multiphoton microscopy in biological settings

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.

Optimization of π-A isotherms to give highly efficient SHG from Langmuir-Blodgett films

Langmuir-Blodgett films have been prepared from amphiphilc molecules containing an indandione-based nonlinear chromophore. Study of the pressure-area (π-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.