Two-Photon Excited Fluorescence Lifetime Imaging of Tetracycline-Labeled Retinal Calcification.

Deposition of calcium-containing minerals such as hydroxyapatite and whitlockite in the subretinal pigment epithelial (sub-RPE) space of the retina is linked to the development of and progression to the end-stage of age-related macular degeneration (AMD). AMD is the most common eye disease causing blindness amongst the elderly in developed countries; early diagnosis is desirable, particularly to begin treatment where available. Calcification in the sub-RPE space is also directly linked to other diseases such as Pseudoxanthoma elasticum (PXE).

Fluorescence Lifetime Imaging of Human Sub-RPE Calcification In Vitro Following Chlortetracycline Infusion.

We have shown that all sub-retinal pigment epithelial (sub-RPE) deposits examined contain calcium phosphate minerals: hydroxyapatite (HAP), whitlockite (Wht), or both. These typically take the form of ca. 1 μm diameter spherules or >10 μm nodules and appear to be involved in the development and progression of age-related macular degeneration (AMD).

TLR5-Derived, TIR-Interacting Decoy Peptides to Inhibit TLR Signaling.

TLR5, which is activated by flagellin, plays an important role in initiating immune response to a broad spectrum of motile bacterial pathogens. TLRs induce intracellular signaling via dimerization of their TIR domains followed by adapter recruitment through multiple interactions of receptor and adapter TIRs. Here, a library of cell-permeable decoy peptides derived from the TLR5 TIR was screened for TLR5 signaling inhibition in the HEK-Blue-mTLR5 reporter cell line.

Sulforaphane covalently interacts with the transglutaminase 2 cancer maintenance protein to alter its structure and suppress its activity.

Type 2 transglutaminase (TG2) functions as an important cancer cell survival protein in a range of cancers including epidermal squamous cell carcinoma. TG2 exists in open and closed conformations each of which has a distinct and mutually exclusive activity. The closed conformation has GTP-binding/GTPase activity while the open conformation functions as a transamidase to catalyze protein-protein crosslinking.

Sodium-Sensitive Contact Lens for Diagnostics of Ocular Pathologies.

The ability to measure all the electrolyte concentrations in tears would be valuable in ophthalmology for research and diagnosis of dry eye disease (DED) and other ocular pathologies. However, tear samples are difficult to collect and analyze because the total volume is small and the chemical composition changes rapidly. Measurements of electrolytes in tears is challenging because typical clinical assays for proteins and other biomarkers cannot be used to detect ion concentrations tears.

Fluorescent contact lens for continuous non-invasive measurements of sodium and chloride ion concentrations in tears.

Rapid and non-invasive measurement of hydration status is medically important because even mild levels of dehydration can have a significant impact on physical and cognitive performance. Despite the potential value of determining whole-body hydration based on the electrolytes found in tears, very few tests are available. An area of intense interest is the development of a contact lens which could measure ion concentrations in tears, specifically that of sodium (Na) and chloride (Cl) ions, the dominant electrolytes in blood plasma and tears.

Imaging hydroxyapatite in sub-retinal pigment epithelial deposits by fluorescence lifetime imaging microscopy with tetracycline staining.

Significance: Recent evidence suggests that hydroxyapatite (HAP) in sub-retinal pigment epithelial (sub-RPE) deposits in aged human eyes may act to nucleate and contribute to their growth to clinically detectable size. Sub-RPE deposits such as drusen are clinical hallmarks of age-related macular degeneration (AMD), therefore enhanced and earlier detection is a clinical need. We found that tetracycline-family antibiotics, long known to stain HAP in teeth and bones, can also label the HAP in sub-RPE deposits.

Blocking TIR Domain Interactions in TLR9 Signaling.

Interaction of TLR9 with ligands activates NF-κB, leading to proinflammatory cytokine production. Excessive TLR activation is a pathogenic factor for inflammatory diseases. This study has examined cell-permeating decoy peptides (CPDPs) derived from the TLR9 Toll/IL-1R resistance (TIR) domain.

Supramolecular polymer formation by cyclic dinucleotides and intercalators affects dinucleotide enzymatic processing.

Background: Cyclic dinucleotides form supramolecular aggregates with intercalators, and this property could be utilized in nanotechnology and medicine.

Methods & Results: Atomic force microscopy and electrophoretic mobility shift assays were used to show that cyclic diguanylic acid (c-di-GMP) forms G-wires in the presence of intercalators. The average fluorescence lifetime of thiazole orange, when bound to c-di-GMP was greater than when bound to DNA G-quadruplexes or dsDNA.

Transamidase site-targeted agents alter the conformation of the transglutaminase cancer stem cell survival protein to reduce GTP binding activity and cancer stem cell survival.

Type 2 transglutaminase (TG2) is an important cancer stem cell survival protein that exists in open and closed conformations. The major intracellular form is the closed conformation that functions as a GTP-binding GTPase and is required for cancer stem cell survival. However, at a finite rate, TG2 transitions to an open conformation that exposes the transamidase catalytic site involved in protein-protein crosslinking.

Metal-Dielectric Waveguides for High Efficiency Coupled Emission.

We report a metal-dielectric planar structure which provides high efficiency coupling of fluorescence at distances over 100 nm away from the metal surface. This hybrid metal-dielectric waveguide (MDW) consists of a continuous metal film coated with a dielectric layer. We observed efficient long-range coupling of Rhodamine B on top of a 130 nm layer of silica resulting in a narrow angular distribution of the emission.

Fluorescence Spectroscopy with Metal-Dielectric Waveguides.

We describe a hybrid metal-dielectric waveguide structures (MDWs) with numerous potential applications in the biosciences. These structures consist of a thin metal film coated with a dielectric layer. Depending on the thickness of the dielectric layer, the modes can be localized near the metal, within the dielectric, or at the top surface of the dielectric.

Several hundred-fold enhanced fluorescence from single fluorophores assembled on silver nanoparticle-dielectric-metal substrate.

We observed over 400-fold enhanced fluorescence from single Cy5 molecules assembled on multilayer silver nanoparticle-dielectric-metal (PDM) substrate. This substantial enhancement is due to the near-field enhanced excitation, emission, and interaction of Cy5 with plasmonic nanostructures. Experimental observation is supported by finite-element method calculations.

A Decoy Peptide that Disrupts TIRAP Recruitment to TLRs Is Protective in a Murine Model of Influenza.

Toll-like receptors (TLRs) activate distinct, yet overlapping sets of signaling molecules, leading to inflammatory responses to pathogens. Toll/interleukin-1 receptor (TIR) domains, present in all TLRs and TLR adapters, mediate protein interactions downstream of activated TLRs. A peptide library derived from TLR2 TIR was screened for inhibition of TLR2 signaling.

Binding of fusion protein FLSC IgG1 to CCR5 is enhanced by CCR5 antagonist Maraviroc.

The CCR5 chemokine receptor is crucial for human immunodeficiency virus type 1 (HIV-1) infection, acting as the principal coreceptor for HIV-1 entry and transmission and is thus an attractive target for antiviral therapy. Studies have suggested that CCR5 surface density and its conformational changes subsequent to virion engagement are rate limiting for entry, and consequently, infection. Not all CCR5 antibodies inhibit HIV-1 infection, suggesting a need for more potent reagents.

Application of phasor plot and autofluorescence correction for study of heterogeneous cell population.

Protein-protein interactions in cells are often studied using fluorescence resonance energy transfer (FRET) phenomenon by fluorescence lifetime imaging microscopy (FLIM). Here, we demonstrate approaches to the quantitative analysis of FRET in cell population in a case complicated by a highly heterogeneous donor expression, multiexponential donor lifetime, large contribution of cell autofluorescence, and significant presence of unquenched donor molecules that do not interact with the acceptor due to low affinity of donor-acceptor binding. We applied a multifrequency phasor plot to visualize FRET FLIM data, developed a method for lifetime background correction, and performed a detailed time-resolved analysis using a biexponential model.

Fluorescence lifetime imaging of physiological free Cu(II) levels in live cells with a Cu(II)-selective carbonic anhydrase-based biosensor.

Copper is a required trace element that plays key roles in a number of human enzymes, such that copper deficiency or genetic defects in copper transport lead to serious or fatal disease. Rae, et al., had famously predicted that free copper ion levels in the cell cytoplasm were extremely low, typically too low to be observable.

Unexpected complex formation between coralyne and cyclic diadenosine monophosphate providing a simple fluorescent turn-on assay to detect this bacterial second messenger.

Cyclic diadenosine monophosphate (c-di-AMP) has emerged as an important dinucleotide that is involved in several processes in bacteria, including cell wall remodeling (and therefore resistance to antibiotics that target bacterial cell wall). Small molecules that target c-di-AMP metabolism enzymes have the potential to be used as antibiotics. Coralyne is known to form strong complexes with polyadenine containing eight or more adenine stretches but not with short polyadenine oligonucleotides.

Large Fluorescence Enhancements of Fluorophore Ensembles with Multilayer Plasmonic Substrates: Comparison of Theory and Experimental Results.

Multilayer substrates consisting of a glass slide, silver mirror, silica layer, and silver nanoparticles were fabricated using magnetron sputtering. This new geometry of substrates with backplane mirror and dielectric photonic cavity produced large average fluorescence enhancements up to 190-fold. Fluorescence enhancements of five fluorescent probes were measured over the broad spectral range from 470 to 800 nm.

Imaging of Protein Secretion from a Single Cell Using Plasmonic Substrates.

Detecting, imaging, and monitoring cell function on a single cell basis is very important in the field of immunology research where many molecules are secreted from cells in response to external stimuli including immunization. Here we introduce substrates with plasmonic nanoparticles and fluorescence microscopy as promising imaging methods for studies on molecular processes controlling cell behavior, particularly secretion of cytokines. We developed unique composition of silver and silica layers of plasmonic nanostructures which resulted in fluorescence enhancement of more than 200-fold for ensemble of molecules in the immunoassay.

BfpL is essential for type IV bundle-forming pilus biogenesis and interacts with the periplasmic face of BfpC.

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea among infants in developing countries. The bundle-forming pilus (BFP), a type IV pilus found on the surface of EPEC, is essential for full virulence of typical EPEC strains. The machinery for BFP assembly and function is encoded by an operon of 14 genes.

Sometimes it takes two to tango: contributions of dimerization to functions of human α-defensin HNP1 peptide.

Human myeloid α-defensins called HNPs play multiple roles in innate host defense. The Trp-26 residue of HNP1 was previously shown to contribute importantly to its ability to kill S. aureus, inhibit anthrax lethal factor (LF), bind gp120 of HIV-1, dimerize, and undergo further self-association.

Fabrication and Characterization of Planar Plasmonic Substrates with High Fluorescence Enhancement.

The use of plasmonic nanostructures for fluorescence signal amplification is currently a very active research field. The detection of submonolayers of proteins labeled with organic dyes is a widely used technique in surface-based immunoassays and DNA hybridization. There is a strong interest in the development of new optical and chemical methods to increase the signal from ultralow concentrations of dyes on the surface of sensor substrates.