Fold Increase In Fluorescence Research Articles

A fluorogenic peptide-based smartprobe for the detection of neutrophil extracellular traps and inflammation.

A highly sensitive optical probe for the detection of activated neutrophils and Neutrophil Extracellular Traps (NETs) is reported. It is based on a triple-quenched, super-silent tri-branched probe that generates >20 fold increase in fluorescence upon cleavage. The probe was highly specific for human neutrophil elastase, a protease that mediates a variety of inflammatory diseases, and detected NETosis and neutrophil activation in in vitro differentiated neutrophils and isolated human neutrophils.

A Combined Single Molecule FRET/Magnetic Tweezers Instrument to Calibrate Molecular Tension - Based Fluorescence Probes

Despite the importance of mechanical forces in biology, few methods are available to measure molecular forces in living systems. To address this issue, we developed molecular tension - based fluorescence microscopy (MTFM), a technique that allows one to visualize piconewton forces using a conventional fluorescence microscope. In MTFM, a flexible linker (polymer) is flanked by a fluorophore and a quencher, such that tension leads to extension of the linker and a 10-30 fold increase in fluorescence. Thus far, MTFM provides the only approach to map piconewton forces at the cell membrane.To accurately calibrate the conversion from fluorescence to tension, we integrate magnetic tweezers to a total internal reflection fluorescence (TIRF) microscope for single molecule Forster resonance energy transfer (smFRET) - force measurement. To minimize autofluorescence from paramagnetic beads, we use 4.4 micron lambda-DNA fragments to link a MTFM probe to each bead. The setup can apply forces ranging from 1-50 pN to calibrate the probes. Comparing its force-extension data to that of theoretical models, this direct measurement of force-induced extension change provides a good tool for determining cellular force with higher accuracy.

Abstract 364: In vivo targeting of CD44+ ovarian cancer stem cells (CSC) by Clostridium Perfringens Enterotoxin binding domain (CPE-peptide) conjugated to poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP)

Abstract Recent evidence strongly supports the hypothesis that the capability to sustain tumor formation, growth and resistance to chemotherapy in ovarian cancer exclusively resides in a small proportion of tumor cells termed cancer stem cells (CSC) overexpressing the CD44 marker. If this hypothesis is correct, development of novel, more specific approaches to target and eradicate the CD44+ ovarian CSC population remains a high priority. Our group has recently reported high expression of claudin-4 tight junction protein in CD44+ ovarian CSC. Because this protein is the natural high affinity receptor for Clostridium Perfringens Enterotoxin (CPE), we have evaluated the in vitro targeting potential of fluorescent (i.e. Coumarin 6 loaded) poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) conjugated with the C-terminus binding domain of CPE (CPE-peptide) against ovarian CSC and the in vivo biodistribution of these NP after intravenous (IV) injection in SCID mice harboring intraperitoneal CD44+ ovarian CSC tumor. Using flow cytometry, we report a higher time and dose-dependent in vitro uptake of fluorescent PLGA NP conjugated to CPE peptide (CPE-peptide NP) in CD44+ ovarian CSC when compared to unconjugated NP (i.e., control NP, Mean-Fluorescence-Intensity = MFI of 37.1 and 15.0 respectively; p=0.037). Of interest, confocal microcopy images clearly show internalization of CPE-peptide NP within tumor cells after NP exposure. More importantly, when SCID mice harboring claudin-4 overexpressing CD44+ ovarian CSC tumors were injected via the tail vein (IV) with 25 μg/g of CPE-peptide NP or the same concentration of unconjugated NP, a 4 fold increase in fluorescence was detected in tumors excised from mice injected with CPE-peptide NP compared to controls 48 hours after NP administration (mean ± STDV 33159.6 2707 and 6655.8 ±1391 respectively; p<0.01). Finally, non specific NP uptake in healthy organs calculated as the ratio between organs and tumor fluorescence was found to be significantly lower after injection of CPE-peptide NP than the unconjugated control NP. Taken together these results suggest that the CPE-peptide, when conjugated to NP, may confer high specificity in targeting claudin-4 overexpressing CD44+ ovarian CSC tumors in vivo. CPE-peptide NP may represent an unconventional, potentially highly effective strategy to specifically target chemotherapy-resistant CD44+ ovarian CSC for diagnostic and/or therapeutic purposes. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 364. doi:1538-7445.AM2012-364

Calcium ionophore A23187 reveals calcium related cellular stress as “I-Bodies”: An old actor in a new role

Calcimycin (A23187) is an ionophore widely used in studies related to calcium dynamics in cells, but its fluorometric potential to reveal intracellular physiology has not been explored. Exploiting the microenvironment-induced changes in its fluorescence, we show that a brief exposure of cells to non-toxic concentrations (≤3 μM) of the ionophore results in the characteristic organization of the ionophore forming brightly fluorescent cytoplasmic bodies termed “I-Bodies”, which are closely related to stress linked disturbances/changes in calcium homeostasis. “I-Bodies” appear to be Ca 2+ rich intracellular sites formed during stress-induced release of intracellular Ca 2+, causing dysfunction and aggregation of mitochondria, providing scaffold for high density packing of A23187. Formation of “I-Bodies” in cells exposed to ionizing radiation and certain anticancer drugs suggest their potential in revealing alterations in calcium signaling and mitochondrial function during (related to) macromolecular damage-induced cell death. The absence of “I-Bodies” in non-malignant cells and their varying numbers in malignant cells with 5 fold increase in fluorescence imply that they can be potential biomarkers of cancer. Thus, “I-Bodies” are novel indicators of endogenous and induced stress linked to disturbances in calcium homeostasis in cells, with a potential to serve as biomarker of cancer.

Preclinical Strategies to Optimize Optical Imaging in Head and Neck Cancer

Objective: There is a need for cancer imaging in the clinic and operating room. We have shown that fluorescently labeled antibodies can detect microscopic islands of head and neck cancer cells using modified intraoperative microscopes in preclinical animal studies. Here we evaluated the optimal targeting molecule for clinical translation. Method: We assessed in vitro binding of fluorescently labeled antibodies targeting EGFR (cetuximab/Erbitux and panitumumab/Vectibix) and an antibody mimetic (anti-EGFR affibody) in multiple cell lines (SCC5, OSC19, FADU). To compare in vivo imaging characteristics, HNSCC xenografts (SCC5, SCC1, FADU) were imaged after systemic injection of fluorescently labeled antibodies over 5 days. Results: The binding affinity was lower for the control antibody IgG (KD = 9.55 nM) compared with either panitumumab (KD = 6.82 nM) or cetuximab (KD = 2.86 nM). In vitro, panitumumab had a two fold increase in relative fluorescence compared with cetuximab and a four fold increase in fluorescence compared with control antibody (nonspecific IgG). Xenograft imaging in vivo demonstrated a variation in fluorescence intensity that correlated with circulating half-lives: peaks in fluorescence intensity were seen at 1.5 h (affibody) and 48 to 72 h (cetuximab, panitunumab). The pantitumumab and affibody images demonstrated greater tumor fluorescence and lower background signal compared to cetuximab. Conclusion: Panitumumab/Vectibix demonstrated optimal imaging characteristics which appeared to be related to its binding affinity and increased circulation time. These characteristics may allow for increased localization to the tumor site. Panitumumab may be the optimal antibody for clinical translation since it will decrease false positive rates associated with non-specific uptake.

A nonfluorescent, broad-range quencher dye for Förster resonance energy transfer assays

We report here a novel, water-soluble, nonfluorescent dye that efficiently quenches fluorescence from a broad range of visible and near-infrared (NIR) fluorophores in Förster resonance energy transfer (FRET) systems. A model FRET-based caspase-3 assay system was used to test the performance of the quencher dye. Fluorogenic caspase-3 substrates were prepared by conjugating the quencher, IRDye ® QC-1, to a GDEVDGAK peptide in combination with fluorescein (emission maximum ∼540 nm), Cy3 (∼570 nm), Cy5 (∼670 nm), IRDye 680 (∼700 nm), IRDye 700DX (∼690 nm), or IRDye 800CW (∼790 nm). The Förster distance R 0 values are calculated as 41 to 65 Å for these dye/quencher pairs. The fluorescence quenching efficiencies of these peptides were determined by measuring the fluorescence change on complete cleavage by recombinant caspase-3 and ranged from 97.5% to 98.8%. The fold increase in fluorescence on caspase cleavage of the fluorogenic substrates ranged from 40 to 83 depending on the dye/quencher pair. Because IRDye QC-1 effectively quenches both the NIR fluorophores (e.g., IRDye 700DX, IRDye 680, IRDye 800CW) and the visible fluorophores (e.g., fluorescein, Cy3, Cy5), it should find broad applicability in FRET assays using a wide variety of fluorescent dyes.

Heme degradation and oxidative stress in murine models for hemoglobinopathies: Thalassemia, sickle cell disease and hemoglobin C disease

Red blood cells with abnormal hemoglobins (Hb) are frequently associated with increased hemoglobin autoxidation, accumulation of iron in membranes, increased membrane damage and a shorter red cell life span. The mechanisms for many of these changes have not been elucidated. We have shown in our previous studies that hydrogen peroxide formed in association with hemoglobin autoxidation reacts with hemoglobin and initiates a cascade of reactions that results in heme degradation with the formation of two fluorescent emission bands and the release of iron. Heme degradation was assessed by measuring the fluorescent band at ex 321 nm. A 5.6 fold increase in fluorescence was found in red cells from sickle transgenic mice that expressed exclusively human globins when compared to red cells from control mice. When sickle transgenic mice co-express the γM transgene, that expresses HbF and inhibits polymerization, heme degradation is decreased. Mice expressing exclusively hemoglobin C had a 6.9 fold increase in fluorescence compared to control. Heme degradation was also increased 3.5 fold in β-thalassemic mice generated by deletion of murine β major. Membrane bound IgG and red cell metHb were highly correlated with the intensity of the fluorescent heme degradation band. These results suggest that degradation of the heme moiety in intact hemoglobin and/or degradation of free heme by peroxides are higher in pathological RBCs. Concomitant release of iron appears to be responsible for the membrane damage that leads to IgG binding and the removal of red cells from circulation.

Restricted Active Site Docking by Enzyme-bound Substrate Enforces the Ordered Cleavage of Prothrombin by Prothrombinase

The preferred pathway for prothrombin activation by prothrombinase involves initial cleavage at Arg(320) to produce meizothrombin, which is then cleaved at Arg(271) to liberate thrombin. Exosite binding drives substrate affinity and is independent of the bond being cleaved. The pathway for cleavage is determined by large differences in V(max) for cleavage at the two sites within intact prothrombin. By fluorescence binding studies in the absence of catalysis, we have assessed the ability of the individual cleavage sites to engage the active site of Xa within prothrombinase at equilibrium. Using a panel of recombinant cleavage site mutants, we show that in intact prothrombin, the Arg(320) site effectively engages the active site in a 1:1 interaction between substrate and enzyme. In contrast, the Arg(271) site binds to the active site poorly in an interaction that is approximately 600-fold weaker. Perceived substrate affinity is independent of active site engagement by either cleavage site. We further show that prior cleavage at the 320 site or the stabilization of the uncleaved zymogen in a proteinase-like state facilitates efficient docking of Arg(271) at the active site of prothrombinase. Therefore, we establish direct relationships between docking of either cleavage site at the active site of the catalyst, the V(max) for cleavage at that site, substrate conformation, and the resulting pathway for prothrombin cleavage. Exosite tethering of the substrate in either the zymogen or proteinase conformation dictates which cleavage site can engage the active site of the catalyst and enforces the sequential cleavage of prothrombin by prothrombinase.

Thiol reactive dyes as probes for kinase assays

Protein kinases are involved in signaling, making them favorable drug targets; hence the need to develop methods adaptable to high throughput assays and screening. We recently reported a UV Vis based kinase assay (Chiku et al, J Biomol Screen 2006 11: –853) involving UV Vis absorbing species. The assay is based on the differential reactivity of ADP(S and ATP(S toward dithiols. We now report an efficient fluorescence version of this assay, where dithionitrobenzoic acid (DTNB) is replaced by a disulfide bridged fluorophore and the assay follows the removal of fluorescence quenching. The fluorescent probe has a cystamine linker joining a blackhole quencher and fluorescein, and has negligible background fluorescence. Removal of quenching gives greater than ten fold increase in fluorescence, and the current assay has advantages over the traditional kinase assays. A similar strategy is applied using fluorescence resonance energy transfer (FRET), and in this case kinase activity is quantitated by decrease and increase in acceptor (fluorescein) and donor (coumarin) fluorescence respectively. The FRET probes offer the possibility of in cell quantitation of kinase activity and we also pursuing a novel synthesis of ADPbS. Research supported in part by BTA grant (#LEGFY06-12368 01KMS), AHA grant (05303072) and NIH_NSF instrumentation grants (S10RR019012 and CHE-0521323).

Blocking of Interleukin-17 during Reactivation of Experimental Arthritis Prevents Joint Inflammation and Bone Erosion by Decreasing RANKL and Interleukin-1

Rheumatoid arthritis is characterized by an intermittent course of disease with alternate periods of remission and relapse. T cells, and in particular the T-cell cytokine interleukin-17 (IL-17), are expected to be involved in arthritic flares. Here, we report that neutralizing endogenous IL-17 during reactivation of antigen-induced arthritis prevents joint inflammation and bone erosion. Synovial IL-17 mRNA expression was clearly up-regulated during primary arthritis and was further enhanced after antigen rechallenge. Neutralization of IL-17 significantly prevented joint swelling at day 1 of flare and significantly suppressed joint inflammation and cartilage proteoglycan depletion at day 4, as assessed by histology. Blocking IL-17 also clearly reduced bone erosions. Cathepsin K, a marker of osteoclast-like activity, and synovial RANKL mRNA expression were both suppressed. The degree of bone erosions strongly correlated with the severity of joint inflammation, suggesting that anti-IL-17 treatment reduced bone erosion by suppressing joint inflammation. Interestingly, blocking IL-17 suppressed synovial expression of both IL-1beta and tumor necrosis factor-alpha, whereas blocking IL-1 did not affect tumor necrosis factor-alpha levels. These data indicate that IL-17 is an important upstream mediator in joint pathology during flare-up of experimental arthritis.

Determination of P-gp and MRP1 expression and function in peripheral blood mononuclear cells in vivo

P-glycoprotein (P-gp) and multidrug resistance-associated protein (MRP) mediate the efflux of many therapeutic agents and have been implicated in the treatment failure of many infectious diseases and cancers. The ability to characterise the expression and function of these transporters in vivo is important when assessing the pharmacological activity of drugs. We investigated some of the problems involved in screening the multidrug resistance status of individuals using flow cytometry. Expression of P-gp and MRP1 on the surface of lymphocytes isolated from blood samples (30 ml) was determined by indirect immunofluorescence. Functional ability was assessed by measuring the efflux of specific fluorescent dyes. Results were expressed as a mean fold increase in fluorescence from the isotype control (expression) and a change in fluorescence compared to the load (function). Using these assays, we determined the expression of P-gp to be 2.01±0.40, n=30 and MRP1 to be 1.46±0.23, n=25. Functional ability was 6.98±4.97, n=25 for P-gp and 1.55±0.25, n=25 for MRP1. The dye efflux studies were associated with a lack of specificity and a number of methodological difficulties. There was no correlation between the expression and function of P-gp ( r=0.338; p=0.10) or MRP1 ( r=0.283; p=0.17). Therefore, we considered determination of P-gp and MRP1 expression to be a more reproducible and accurate approach to clinical investigation into the role of multidrug resistance.

High-performance liquid chromatography of the fluorescent dyes Fura-2 and Mag-Fura stability in organic solvents

The fluorescent dye indicators Fura-2/AM and Mag-fura are used to estimate changes in intracellular concentrations of calcium and magnesium, respectively. HPLC, coupled to fluorescence and ultraviolet detectors, indicated that these dyes are unstable when dissolved in methanol and are especially unstable in glass containers compared to plastic. Both dyes were very stable in acetonitrile and dimethyl sulfoxide. The glass vials used in these studies appear to contain an unidentified substance which causes changes in the spectral intensities of these fluorescent dyes in the presence of methanol but not other solvents. With Fura-2/AM, there was an initial two fold increase in fluorescence during the first four hours in methanol and glass with no change in retention time, peak shape or ultraviolet absorption. With Mag-fura, no increase in fluorescence was observed but instead there was a reduction of more than 90% in both fluorescence and ultraviolet absorption within 30 min, indicating that significant decomposition occurs under these conditions. These results confirm previous studies which suggest that acetonitrile is preferable to methanol for sample preparation and chromatographic analysis of Fura-2 and Mag-Fura. These data also indicate that glass vials contain an extractable substance which markedly enhances the spectral intensities of Fura-2/AM.