Fluorescence Polarimetry Research Articles

Characterization of novel compounds that directly bind to and inactivate the HPV E6 protein for treatment of HPV-associated cervical and oropharyngeal cancers.

e15122 Background: The human papillomavirus (HPV) E6 protein is necessary for viral genome replication and is always expressed in HPV-induced malignancies. We performed in silico screening of chemical libraries for molecules that bind to a pocket on the cancer associated HPV-16 E6 protein. We focused on the protein-protein interaction of E6 with one of its cellular binding partners, the ubiquitin ligase E6AP. In a trimolecular complex with E6, E6AP ubiquitinates p53, leading to its destruction by the proteasome. We designed, synthesized, and characterized >300 compounds that 1) bind to the E6AP interaction surface of the HPV-16 E6 protein; 2) are armed with a ‘warhead’ to make an irreversible bond with a specific cysteine in this HPV-16 E6 binding pocket; 3) and restore levels of p53 and selectively decrease viability of HPV-16 expressing tumor derived cells. Methods: E6 inhibitor optimization was guided by in silico docking strategies, co-crystallization, and medicinal chemistry, followed by biochemical and cellular assays to iteratively design and synthesize molecules with increased E6 inhibitory activities. Results: We used whole protein mass spectrometry to interrogate covalent bonding to E6, showing irreversible binding to the cysteine in the targeted E6 binding pocket without the other 13 cysteines in E6, with Ki in the range of seconds. Biolayer interferometry and fluorescence polarimetry measured blockade of E6 with E6AP, with IC50 in the range of 400-600 nM. To establish in cell inactivation of E6, human cervical cancer derived SiHa cells that express HPV-16 E6 were incubated with compounds, showing dose dependent increase of p53 levels up to 8-fold after 24 hrs, an effect that was not observed in the HPV-16- cell line RPE-1 expressing wild-type p53. Viability of two cervical (SiHa, CaSki) and two oropharyngeal HPV-16+ cancer cell lines (UM-SC-47, UM-SCC-104) was reduced with an IC50 of ~2-3 µM, with an IC50>30µM in RPE-1 cells and telomerase immortalized human keratinocytes. These in vitro and cell-based experiments revealed dependence on the cysteine residue in the E6AP binding pocket of E6. For in vivo proof-of-concept studies, we developed mouse xenograft models, performed by engrafting SiHa, UM-SC-47 and UM-SCC-104 cancer derived cells subcutaneously into the flank of immunodeficient mice. Lead compounds were administered by intraperitoneal injection and demonstrated nearly complete inhibition (> 70% tumor reduction) in HPV-16 tumor growth compared to vehicle. Anti-tumor activity was not observed with the HPV-negative tumor cell line C33A. Conclusions: HPV-16 is identified in about 50% of all cervical cancers and the majority of head and neck malignancies. Inactivation of the HPV E6 protein represents the opportunity for a novel molecular targeted approach for medical treatment of HPV-16 expressing cancers of the cervix and oropharynx.

Soleillet's formalism of coherence and partial polarization in 2D and 3D: application to fluorescence polarimetry.

We review here the pioneering research conducted by Paul Soleillet on the statistical properties of light in his doctoral thesis from 1929 [Ann. Phys.10, 23 (1929)]. Soleillet's wide-reaching work on polarization, coherence, fluorescence scattering, and three-dimensional fields has remained largely unrecognized; yet, his original contributions rival the modern rediscoveries in both generality and form. Only now, 89 years after Soleillet's original publication and stimulated by our current research on fluorescence polarimetry, have we been able to fully understand and recognize the significance of his results.

Thio-Linked UDP-Peptide Conjugates as O-GlcNAc Transferase Inhibitors.

O-GlcNAc transferase (OGT) is an essential glycosyltransferase that installs the O-GlcNAc post-translational modification on the nucleocytoplasmic proteome. We report the development of S-linked UDP–peptide conjugates as potent bisubstrate OGT inhibitors. These compounds were assembled in a modular fashion by photoinitiated thiol–ene conjugation of allyl-UDP and optimal acceptor peptides in which the acceptor serine was replaced with cysteine. The conjugate VTPVC(S-propyl-UDP)TA (Ki = 1.3 μM) inhibits the OGT activity in HeLa cell lysates. Linear fusions of this conjugate with cell penetrating peptides were explored as prototypes of cell-penetrant OGT inhibitors. A crystal structure of human OGT with the inhibitor revealed mimicry of the interactions seen in the pseudo-Michaelis complex. Furthermore, a fluorophore-tagged derivative of the inhibitor works as a high affinity probe in a fluorescence polarimetry hOGT assay.

Probing intrinsic anisotropies of fluorescence: Mueller matrix approach.

We demonstrate that information on “intrinsic” anisotropies of fluorescence originating from preferential orientation/organization of fluorophore molecules can be probed using a Mueller matrix of fluorescence. For this purpose, we have developed a simplified model to decouple and separately quantify the depolarization property and the intrinsic anisotropy properties of fluorescence from the experimentally measured fluorescence Mueller matrix. Unlike the traditionally defined fluorescence anisotropy parameter, the Mueller matrix-derived fluorescence polarization metrics, namely, fluorescence diattenuation and polarizance parameters, exclusively deal with the intrinsic anisotropies of fluorescence. The utility of these newly derived fluorescence polarimetry parameters is demonstrated on model systems exhibiting multiple polarimetry effects, and an interesting example is illustrated on biomedically important fluorophores, collagen.

Growth of α-sexithiophene nanostructures on C60 thin film layers

Organic molecular beam grown α-sexithiophene (α-6T) forms nanostructured thin films on buckminsterfullerene (C60) thin film layers. At substrate temperatures of 300K during growth a rough continuous film is observed, which develops to larger elongated islands and dendritic- as well as needle-like structures at temperatures around 390K. X-ray diffractrometry reveals (100) crystalline orientation in the films grown at higher surface temperatures, which corresponds to upright oriented molecules. Rocking curves reveal the lowest mosaicity for samples grown at the highest substrate temperatures. From fluorescence polarimetry measurements the in-plane orientation within the needle like structures is determined. The polarimetry investigations together with the intensity of the fluorescence strongly indicate that the needle like structures consist of lying molecules.

Membrane Orientation and Lateral Diffusion of BODIPY-Cholesterol as a Function of Probe Structure

Cholesterol tagged with the BODIPY fluorophore via the central difluoroboron moiety of the dye (B-Chol) is a promising probe for studying intracellular cholesterol dynamics. We synthesized a new BODIPY-cholesterol probe (B-P-Chol) with the fluorophore attached via one of its pyrrole rings to carbon-24 of cholesterol (B-P-Chol). Using two-photon fluorescence polarimetry in giant unilamellar vesicles and in the plasma membrane (PM) of living intact and actin-disrupted cells, we show that the BODIPY-groups in B-Chol and B-P-Chol are oriented perpendicular and almost parallel to the bilayer normal, respectively. B-Chol is in all three membrane systems much stronger oriented than B-P-Chol. Interestingly, we found that the lateral diffusion in the PM was two times slower for B-Chol than for B-P-Chol, although we found no difference in lateral diffusion in model membranes. Stimulated emission depletion microscopy, performed for the first time, to our knowledge, with fluorescent sterols, revealed that the difference in lateral diffusion of the BODIPY-cholesterol probes was not caused by anomalous subdiffusion, because diffusion of both analogs in the PM was free but not hindered. Our combined measurements show that the position and orientation of the BODIPY moiety in cholesterol analogs have a severe influence on lateral diffusion specifically in the PM of living cells.

Probing Orientational Order of MHC Class I Protein and Lipids in Cell Membranes by Fluorescence Polarization-Resolved Microscopy Imaging

Biomolecular orientational organization of lipids and proteins in plasma membrane is a crucial factor in biological processes where functions can be closely related to orientation and ordering mechanisms. The concept of transient nanosized phase separations in ordered and disordered domains, called “lipid rafts” is now widely accepted. Furthermore, the ordered domains are enriched in signalling proteins, which highlights the crucial impact of phase separation during the signalling processes. While this field has been so far largely addressed by studying the translational diffusion behaviour of membrane proteins by Single Molecule Tracking or Fluorescence Correlation Spectroscopy, only little is known about the orientational behaviour of signalling proteins in plasma membranes, mainly due to the lack of appropriate rigid fluorescent label which would be able to act as a proper orientation reporter. In this work we develop a fully polarization-resolved fluorescence imaging technique using a tuneable incident polarization state (“fluorescence polarimetry”), in combination with fluorescence anisotropy imaging, in order to provide orientational order information in very general cell membranes shapes.We apply this technique to the measurement of quantitative orientational distribution of MHC Class I proteins in the plasma and nuclear membranes, benefiting from a rigidly attached GFP probe. The surrounding lipid orientational order in the plasma membrane is additionally probed using the fluorescent reporter di-8-ANEPPQ.The MHC Class I protein is found to be more ordered in the plasma membrane as compared to the nuclear membrane. Both MHC I and di-8-ANEPPQ orientational orders in the plasma membrane are furthermore seen to be highly affected by actin depolymerisation upon Latrunculin A treatment, with variations that indicate both a structural change in the membrane morphology and a disruption of MHC I - actin interactions.

The Prospects of Carrying and Releasing Drugs Via Biodegradable Magnesium Foam

AbstractPowder metallurgy technology was used to produce magnesium foams in order to evaluate their ability to perform as a solid biodegradable platform for drug delivery. The amount and delivery time of the released drug (gentamicin) was controlled by the level of space‐holding particles (spacer) that was mixed with the magnesium powder prior to the sintering process. Metallurgical examination of the magnesium foams was carried out using optical and scanning electron microscopy (SEM) and X‐ray diffraction analysis. Microtomography CT analysis was used to evaluate the structural characteristics of the magnesium foams and their internal interconnected porosity configuration. The corrosion behavior of the magnesium foams was evaluated by immersion test in a simulated physiological environment (PBS solution). The absorption of gentamicin was obtained by immersing magnesium foams in concentrated gentamicin solutions within a vacuum chamber, followed by water evaporation. The detection of gentamicin in PBS solution was carried out using a Fluorescence Polarimetry analyzer. The results show that the release profile of gentamicin from magnesium foam with 10 and 25% spacer in PBS solution was in accord with common dissolution kinetics of an active ingredient from polymeric drug delivery systems.

Quantitative Imaging of Molecular Order in Lipid Membranes Using Two-Photon Fluorescence Polarimetry

We present a polarimetric two-photon microscopy technique to quantitatively image the local static molecular orientational behavior in lipid and cell membranes. This approach, based on a tunable excitation polarization state complemented by a polarized readout, is easily implementable and does not require hypotheses on the molecular angular distribution such as its mean orientation, which is a main limitation in traditional fluorescence anisotropy measurements. The method is applied to the investigation of the molecular angular distribution in giant unilamellar vesicles formed by liquid-ordered and liquid-disordered micro-domains, and in COS-7 cell membranes. The highest order contrast between ordered and disordered domains is obtained for dyes locating within the membrane acyl chains.

Studies on CdSe/l-cysteine Quantum Dots Synthesized in Aqueous Solution for Biological Labeling

Water-soluble, biologically compatible CdSe quantum dots (QDs) with l-cysteine as capping agent were synthesized in aqueous medium. Fluorescence (FL) spectra, absorption spectra, and transmission electron microscopy studies showed that both the molar ratio of Se/Cd and the reaction time are the determining factors for the size distribution of CdSe/l-cysteine QDs. The interaction of QDs bioconjugated to bovine serum albumin (BSA) was also studied by absorption and FL titration experiments. With addition of QDs, the FL intensity of BSA was largely quenched, which can be explained by static mechanism. However, when BSA was added to the solution of QDs, FL intensity of QDs was faintly enhanced at first and then quenched. Lysozyme could change the Escherchia coli (E. coli) membrane fluidity and permeability by FL polarimetry. Fluorescent imaging suggested that QDs can be designed as a probe to label the E. coli cell. These results suggested CdSe/l-cysteine QDs can be used as a probe for labeling biological mol...

Polarization distortion effects in polarimetric two-photon microscopy

We present a global analysis of experimental factors affecting polarization responses in two-photon inverted microscopy. The role of reflection optics and high numerical aperture focusing is investigated in two-photon fluorescence, which can be extended to other nonlinear processes. We show that both effects strongly distort polarization responses and can lead to misleading extraction of molecular order information from polarimetric measurements. We describe a model accounting for these effects and develop a calibration technique for the determination of polarization parameters in the sample plane using two-photon fluorescence polarimetry in liquids.

Partial wave analysis of interfering Kr 3d95p resonant Raman Auger transitions based on measurements of alignment and orientation parameters within the natural line width

The alignment parameter A20 and the orientation parameter O10 for the 4p4(1D)5p2F7/2 and the 4p4(1D)5p2D5/2 states of Kr+ populated in resonant Auger transitions from 3d95p/6p photo-excited states of Kr were measured at BESSY II for photon energies between 91 eV and 92.8 eV. The method of fluorescence polarimetry was applied. A bandwidth of the exciting photons of 10 meV allowed to study A20 and O10 within the natural line width. Interference effects for resonances overlapping due to lifetime broadening were predicted and observed even when separated by 15 times the natural line width. The relative contribution of the three allowed electron partial waves was derived from A20 and O10 as a function of the exciting-photon energy and compared with the calculated contributions. The resulting good overall agreement between experiment and prediction is considered as a noticeable improvement of the understanding of excitation and decay of the prominent Kr 3d95p resonances.

Universal scaling of resonances in vector correlation photoionization parameters

In the present work it is shown that in atomic photoionization the Fano-like behaviour of vector correlation parameters in the region of an isolated autoionizing resonance possesses two universal values: the width and the energy shift with respect to the energy of the autoionizing state. This property is confirmed by fluorescence polarimetry studies, which provide, in the particular case of Xe 4d−15/26p(J = 1) photoexcitation, the experimental determination of the alignment and the orientation of the final photoion across the resonance.

Resonant Auger decay ofXe*4d5∕2−16p: A contribution to the complete experiment from fluorescence polarization studies

Fluorescence polarimetry has been used to determine the relative partial-wave Auger decay widths for transitions to states of the $\mathrm{Xe}\phantom{\rule{0.2em}{0ex}}II$ $5{p}^{4}6p$ multiplet after photoexcitation of the ${\mathrm{Xe}}^{*}\phantom{\rule{0.3em}{0ex}}4{d}_{5∕2}^{\ensuremath{-}1}6p({J}^{*}=1)$ resonance by linearly and circularly polarized synchrotron radiation. Combination with data on the angular distribution and spin polarization of the Auger electrons, providing information on the relative phases of the amplitudes, constitutes the complete experiment on the Auger decay. Multiconfiguration relativistic calculations of the amplitudes have been performed and compared to the measurements.

Circular Polarization of Ion Fluorescence Completing the Analysis of Resonant Xe*4d5/2−16pAuger Decay

The relative contributions of the partial electron waves emitted in the Auger decay of the Xe* 4d(-1)(5/2)6p(J(*)=1) resonance have been determined by fluorescence polarimetry after excitation with circularly polarized synchrotron radiation. The analysis of circularly polarized fluorescence of the photoion leads to an independent determination of the orientation parameters for all states of the Xe II 5p(4)6p multiplet. The present study provides, in combination with data on the angular distribution and spin polarization of the Auger electrons, complete quantum mechanical information on the resonant Auger decay, i.e., branching ratios and relative phases of the Auger decay amplitudes.

Does preeclampsia influence fetal lung maturity?

This retrospective study compared the fetal lung maturity biochemical profile of patients having preeclampsia with that of patients having preterm labor. Amniotic fluid was obtained by transabdominal amniocentesis in 90 patients, 59 patients with preterm labor (PTL) and 31 patients with preeclampsia (PRE). Pregnancies with fetal growth restriction were excluded. Fetal lung maturity was assessed by lecithin/sphingomyelin ratio (L/S) and by a fluorescence polarimetry assay (FLM). Mean values of L/S ratios and FLM were compared between the PTL and the PRE groups, each within two gestational age subgroups (27-32.9 weeks gestation and 33-36 weeks gestation). Student t-test, Chi-square test Fisher's exact test were used for statistical analysis. A p value < 0.05 was considered significant. During the gestational age interval of 33-36 weeks, the mean L/S ratios were significantly lower in pregnancies complicated by PRE than in those complicated by PTL (1.99 +/- 0.26 and 2.4 +/- 0.57, respectively; p = 0.01). Similarly, during this gestational age interval, the FLM values were also lower in PRE than in PTL, although the difference did not reach statistical significance. During the gestational age between 33 and 36 weeks of gestation, the biochemical profile of preeclamptic patients without IUGR has a significant lower L/S ratio compared to that of preterm patients.

Fetal Maturity Cascade

One hundred ninety-three amniotic fluid samples were tested for fetal lung maturity using a maturity cascade scheme involving the sequential use of, in order, the shake test, fluorescence polarimetry, and lecithin: sphingomyelin (L:S) ratio. If any of these tests indicated maturity, the sequence was terminated and no further test was performed, and the fetus was considered mature. Seventy percent of the tests yielded mature values and of these, 85 (63%) required a shake test only, 37 (27%) had a shake test and a fluorescence polarimetry, and only 14 (10%) required all three tests. From these 193 amniocenteses, 111 patients delivered within 72 hours of the procedure. One of 94 infants had respiratory distress syndrome after a mature test (1% false maturity) and ten of 17 had respiratory distress syndrome after an immature cascade (41% falsely immature). This approach saves time and cost and by confirming immaturity with multiple tests only when necessary and may improve predictability of neonatal respiratory distress syndrome.