Photophysics of EGFP (E222H) Mutant, with Comparisons to Model Chromophores: Excited State pK's, Progressions, Quenching and Exciton Interaction.

A novel version of the well-known and commercially successful Green Fluorescent Protein (GFP) variant known as EGFP, with an introduced E222H mutation, was produced in this laboratory. Given the current state of hypotheses about the role of glutamate 222, and the observed dominance of the phenolate absorption with an E222H variant observed from earlier study, the new mutant was considered a natural choice to investigate more fully the acid-base behavior of the chromophore in absorption and fluorescence. The bulk of this investigation concerns fitting the excitation, emission and absorption spectra to vibrational progressions of a novel 'q-deformed' type at various values of pH, and protein concentration. From these data, and from temperature-dependent fluorescence lifetime data and other experiments (with lanthanide doped gels into which H/EGFP is embedded), we construct a picture of excited inter- state conversion mechanisms, and quenching mechanisms, that attempts to explain many features of the GFP system. Graphical Abstract Hypothetical proton current loop (orange) upon excitation; electron motion in purple H/EGFP. Solid boxes about waters project toward viewer, dashed boxes project away.

Prevention of biofilm-induced capsular contracture with antibiotic-impregnated mesh in a porcine model.

BACKGROUND: A growing body of evidence implicates subclinical (biofilm) infection around breast implants as an important cause of capsular contracture (CC). OBJECTIVES: The authors use an in vivo porcine model to investigate the potential of antibiotic-impregnated mesh as a prophylactic measure against biofilm formation and CC. METHODS: A total of 28 implants (14 untreated controls, 14 treated with antibiotic mesh) were inserted into 5 adult female pigs. All implants and pockets were inoculated with a human clinical strain of Staphylococcus epidermidis. The implants were left in situ for 16 weeks and then analyzed for contracture using both Baker grading and applanation tonometry. The presence of biofilm infection was assessed by subsequent microbiological analysis of implants and capsules. RESULTS: One untreated implant had extruded and was excluded from analysis. The tissue surrounding the 13 untreated control implants had Baker Grade III/IV CC, whereas no CC was identified around the 14 antibiotic mesh-treated implants. This difference was highly significant (P < .001). Tonometry findings were consistent with the Baker assessments. Although bacterial biofilm was detected on all implants and capsules, the biofilms on the antibiotic-treated implants and surrounding capsules were generally single-layered or isolated in contrast to the multilayer biofilms found on untreated implants and capsules. CONCLUSIONS: Based on the findings from this study of a porcine model, the use of antibiotic-impregnated mesh reduces bacterial access to breast implants at the time of surgical insertion and may subsequently protect against subclinical infection and CC.

Human GLTP: Three distinct functions for the three tryptophans in a novel peripheral amphitropic fold.

Human glycolipid transfer protein (GLTP) serves as the GLTP-fold prototype, a novel, to our knowledge, peripheral amphitropic fold and structurally unique lipid binding motif that defines the GLTP superfamily. Despite conservation of all three intrinsic Trps in vertebrate GLTPs, the Trp functional role(s) remains unclear. Herein, the issue is addressed using circular dichroism and fluorescence spectroscopy along with an atypical Trp point mutation strategy. Far-ultraviolet and near-ultraviolet circular dichroism spectroscopic analyses showed that W96F-W142Y-GLTP and W96Y-GLTP retain their native conformation and stability, whereas W85Y-W96F-GLTP is slightly altered, in agreement with relative glycolipid transfer activities of >90%, ∼85%, and ∼45%, respectively. In silico three-dimensional modeling and acrylamide quenching of Trp fluorescence supported a nativelike folding conformation. With the Trp96-less mutants, changes in emission intensity, wavelength maximum, lifetime, and time-resolved anisotropy decay induced by phosphoglyceride membranes lacking or containing glycolipid and by excitation at different wavelengths along the absorption-spectrum red edge indicated differing functions for W142 and W85. The data suggest that W142 acts as a shallow-penetration anchor during docking with membrane interfaces, whereas the buried W85 indole helps maintain proper folding and possibly regulates membrane-induced transitioning to a glycolipid-acquiring conformation. The findings illustrate remarkable versatility for Trp, providing three distinct intramolecular functions in the novel amphitropic GLTP fold.

Reconstruction of the lower eye lid with a rotation-advancement tarsoconjunctival cheek flap.

Repair of full-thickness defects of the lower eyelids poses a challenge because a graft in combination with a flap is typically used to replace either the posterior or the anterior lamella. This often results in aesthetically and functional unsatisfactory outcomes. A rotation-advancement tarsoconjunctival cheek flap, which reconstructs both posterior and anterior lamellae with vascularized tissue similar to the native eyelid, is described. Nine patients underwent reconstruction with a rotation-advancement tarsoconjunctival cheek flap. Indications, complications, and outcomes were evaluated. The follow-up time ranged from 6 to 60 months, with a mean of 23 months. The main indication for use of this flap is full-thickness defect of the lower eyelid between 25% and 75%, typically after tumor ablation. All patients had a functional and aesthetically satisfactory outcome. One patient underwent a revision canthoplasty. The rotation-advancement tarsoconjunctival cheek flap adheres to basic plastic surgery principles resulting in a satisfactory outcome: vascularized tissue is used to reconstruct the defect, the flap composition is similar to the native eyelid, that is, replace like with like, and the flap makes use of tissue that is in excess and therefore limits donor morbidity.

Photophysics of ANS. IV. Electron transfer quenching of ANS in alcoholic solvents and mixtures.

ANS is observed to decay from the fluorescent state with distributed kinetics in nearly pure ethanol solvent, notwithstanding that in mixed ethanol/water solvents the decay is discrete and biexponential. The origin of this behavior is investigated. In particular, a theory of electron transfer theory in the adiabatic regime is adduced, with specific involvement of solvent cage structure in the form of the solvent-electron polaron wave function. Properties of various polarons for various solvent systems are predicted and, for the case of ethanol and cyclohexanol, employed to generate the observed Arrhenius-type decay parameters in a quantitative fashion.

Photophysics of ANS. V. Decay modes of ANS in proteins: the IFABP-ANS complex.

The fluorescence properties of ANS as bound to proteins are treated. Several points of view concerning the origin of these properties are reviewed and synthesized into one framework. On proteins where the quantum yield (QY) is appreciable, as in organic solvents, the preferred conformation of ANS is often with the phenyl ring nearly (65 degrees -85 degrees ) orthogonal to the naphthalene. The major consequence of this geometry is water exclusion from the critical zone of ANS at which the largest amount of solvent dipolar relaxation originates. This, in turn, leads to a depression of the rate of electron transfer to the surroundings, together with other effects, as noted in the literature and in our lab. Alternative quenching pathways for ANS on the protein vs in water are also elucidated.

Photophysics of ANS. I. Protein-ANS complexes: Intestinal fatty acid binding protein and single-trp mutants.

We continue investigations into the physical chemistry of intestinal fatty acid binding protein, I-FABP, and its interaction with ANS and other ligands [cf references [Kirk, W., E. Kurian, and F. Prendergast. 1996. Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-anilinonaphthalene binding to intestinal fatty acid binding protein. Biophys. J. 70: 69-83., Kurian, E., W. Kirk, and F. Prendergast. 1996. Affinity of fatty acid for rRat intestinal fatty acid binding protein: Further examination. Biochemistry. 35:3865-74]. The photophysics of the wt protein is compared with that in two mutants which lack respectively one or the other of two trp moieties, one of which, trp 82, is located near the binding region for the polar head group of ligands. These studies afford a look into how the fluorescence of the wt protein is established, that is, as an almost direct sum of the fluorescence of the two individual trp residues, and how this fluorescence is quenched upon binding to ANS. Though we have access to all the relevant spectroscopic and geometric information necessary to specify in detail the Foerster-Dexter energy transfer model, the quenching process is not explicable in terms of very-weak coupling, as is usually assumed in fluorescence studies in protein systems, but in terms of a stronger effect which goes beyond the simple very-weak dipole:dipole formalism. The quenching of trp emission by bound ANS is not as great as that anticipated by ordinary resonance energy transfer, neither is the quenching observed in the reduced lifetimes of the trp emission upon ANS binding as great as that observed in steady-state intensity. However the observed steady-state quenching is explicable in terms derived from the lifetime measurements, together with observed spectral band shifts, by the exciton coupling model we invoke here.

Breast Implant-Associated Anaplastic Large Cell Lymphoma in Australia and New Zealand: High-Surface-Area Textured Implants Are Associated with Increased Risk.

BACKGROUND: The association between breast implants and breast implant-associated anaplastic large cell lymphoma (ALCL) has been confirmed. Implant-related risk has been difficult to estimate to date due to incomplete datasets. METHODS: All cases in Australia and New Zealand were identified and analyzed. Textured implants reported in this group were subjected to surface area analysis. Sales data from three leading breast implant manufacturers (i.e., Mentor, Allergan, and Silimed) dating back to 1999 were secured to estimate implant-specific risk. RESULTS: Fifty-five cases of breast implant-associated ALCL were diagnosed in Australia and New Zealand between 2007 and 2016. The mean age of patients was 47.1 years and the mean time of implant exposure was 7.46 years. There were four deaths in the series related to mass and/or metastatic presentation. All patients were exposed to textured implants. Surface area analysis confirmed that higher surface area was associated with 64 of the 75 implants used (85.3 percent). Biocell salt loss textured (Allergan, Inamed, and McGhan) implants accounted for 58.7 percent of the implants used in this series. Comparative analysis showed the risk of developing breast implant-associated ALCL to be 14.11 times higher with Biocell textured implants and 10.84 higher with polyurethane (Silimed) textured implants compared with Siltex textured implants. CONCLUSIONS: This study has calculated implant-specific risk of breast implant-associated ALCL. Higher-surface-area textured implants have been shown to significantly increase the risk of breast implant-associated ALCL in Australia and New Zealand. The authors present a unifying hypothesis to explain these observations.

Electrophoretic mobility of weakly-charged (dipolar) hydrogels in water: contribution of hydrogen-bonding in the solvent dipole layer.

Counterintuitive observations by dynamic light-scattering experiments of negative electrophoretic mobility in uncharged, lightly charged, and later, densely-charged hydrogel nanoparticles are presented. A tentative theory, emphasizing the roles of electric field energy density and induced dipole moments in the dipolar and hydrogen-bonding solvent layer surrounding the particle, is introduced to explain and rationalize these observations. Addition of co-solvent glycine seems to produce a Kohlrausch boundary regulating effect which again illustrates the importance of the dipole layer and hydrogen bonds within it. Further alternative theories involving electric field gradients are discussed which may be relevant to other uncharged systems (such as gold nanoparticles). A contribution to the dipolar solvent-induced mobility is derived in Appendix A. A proposal for a new treatment of traditional (i.e. charged colloid particle) electrokinetic phenomena is given in a second Appendix (Appendix B).

In vitro and in vivo investigation of the influence of implant surface on the formation of bacterial biofilm in mammary implants.

BACKGROUND: Capsular contracture remains the most common complication following breast augmentation surgery, and evidence suggests that bacterial biofilm on the implant surface is responsible. The authors investigated whether the interaction of bacterial biofilm with implants independently determines progression to capsule formation. They also studied the rate of bacterial growth and adhesion to implants. METHODS: Sixteen adult female pigs had 121 breast implants inserted. Sixty-six implants-23 smooth and 43 textured-were inoculated with a human strain of Staphylococcus epidermidis and received no other treatment. After an average period of 19 weeks, Baker grading was performed and implants were retrieved. For the in vitro study, samples underwent both quantitative bacterial analysis and imaging using confocal laser scanning and scanning electron microscopy. RESULTS: At explantation, there was no significant difference (p = 1.0) in the presence of capsular contracture (Baker grade III and IV) between smooth (83 percent) and textured implants (84 percent). Biofilm was confirmed on 60 of the 66 capsules. Capsules from smooth and textured implants had the same number of infecting bacteria (textured: 3.01 × 10 bacteria/g; smooth: 3.00 × 10 bacteria/g). In vitro, the surface of textured implants showed 11-, 43-, and 72-fold more bacteria at 2, 6, and 24 hours, respectively, compared with smooth implants (p < 0.001). These findings were confirmed by imaging analysis. CONCLUSIONS: These results show that textured implants develop a significantly higher load of bacterial biofilm in comparison with smooth implants. Furthermore, in vivo, once a threshold of biofilm forms on either smooth or textured implant surfaces, there seems to be an equal propensity to progress to capsular contracture.

Transverse ionic mobility measured in a dynamic light scattering device.

We describe here some novel experiments with a commercial dynamic light scattering device. By inserting a quarter-wave plate in the light beam of the HeNe laser used in the Malvern DLS 'Zetasizer', one can obtain right handed (RH) or left-handed (LH) circularly polarized light from the incoming horizontally polarized laser light. This RH vs LH light is used in the ionic mobility (ζ-potential) measuring mode to detect what we believe are phenomena related to transverse ionic mobility, i.e. speed of a particle (or portions of the particle) as a function of applied static electric field, in directions transverse to those fields, and which, we suggest, arise from surface impedence phenomena related to the (1) parity-biased mechanical flexing of charged molecular moieties at the surface of a chiral particle or of an achiral particle+chiral co-solvents, possibly driven by the electrophoresis field and (2) electro-optic effects (induced currents) arising from the interaction of chiral co-solvents upon the surface of charged colloid particles in the presence of a (high frequency) electric field. Fluctuations of structure induce currents which are chirally biased either in themselves (in a chiral particle) or which 'borrow' chirality from chiral co-solvents conditioning the local high frequency E-field, and advance or retard the scattered phase of RH or LH polarized light. In either case the 'differential mobility' observed is related to the relative extent of motion in internal portions of the colloid particle - i.e. 'floppiness' in the particle.

Environment and mobility of a series of fluorescent reporters at the amino terminus of structurally related peptide agonists and antagonists bound to the cholecystokinin receptor.

Fluorescence is a powerful biophysical tool for the analysis of the structure and dynamics of proteins. Here, we have developed two series of new fluorescent probes of the cholecystokinin (CCK) receptor, representing structurally related peptide agonists and antagonists. Each ligand had one of three distinct fluorophores (Alexa(488), nitrobenzoxadiazolyl, or acrylodan) incorporated in analogous positions at the amino terminus just outside the hormone's pharmacophore. All of the probes bound to the CCK receptor specifically and with high affinity, and intracellular calcium signaling studies showed the chemically modified peptides to be fully biologically active. Quenching by iodide and measurement of fluorescence spectra, anisotropy, and lifetimes were used to characterize the response of the fluorescence of the probe in the peptide-receptor complex for agonists and antagonists. All three fluorescence indicators provided the same insights into differences in the environment of the same indicator in the analogous position for agonist and antagonist peptides bound to the CCK receptor. Each agonist had its fluorescence quenched more easily and showed lower anisotropy (higher mobility of the probe) and shorter lifetime than the analogous antagonist. Treatment of agonist-occupied receptors with a non-hydrolyzable GTP analogue shifted the receptor into its inactive low affinity state and increased probe fluorescence lifetimes toward values observed with antagonist probes. These data are consistent with a molecular conformational change associated with receptor activation that causes the amino terminus of the ligand (situated above transmembrane segment six) to move away from its somewhat protected environment and toward the aqueous milieu.

Measurement of intermolecular distances for the natural agonist Peptide docked at the cholecystokinin receptor expressed in situ using fluorescence resonance energy transfer.

Fluorescence resonance energy transfer is a powerful biophysical technique used to analyze the structure of membrane proteins. Here, we used this tool to determine the distances between a distinct position within a docked agonist and a series of distinct sites within the intramembranous confluence of helices and extracellular loops of the cholecystokinin (CCK) receptor. Pseudo-wild-type CCK receptor constructs having single reactive cysteine residues inserted into each of these sites were developed. The experimental strategy included the use of the full agonist, Alexa488-CCK, bound to these receptors as donor, with Alexa568 covalently bound to the specific sites within the CCK receptor as acceptor. Site-labeling was achieved by derivatization of intact cells with a novel fluorescent methanethiosulfonate reagent. A high degree of spectral overlap was observed between receptor-bound donor and receptor-derivatized acceptors, with no transfer observed for a series of controls representing saturation of the receptor binding site with nonfluorescent ligand and use of a null-reactive CCK receptor construct. The measured distances between the fluorophore within the docked agonist and the sites within the first (residue 102) and third (residue 341) extracellular loops of the receptor were shorter than those directed to the second loop (residue 204) or to intramembranous helix two (residue 94). These distances were accommodated well within a refined molecular model of the CCK-occupied receptor that is fully consistent with all existing structure-activity and photoaffinity-labeling studies. This approach provides the initial insights into the conformation of extracellular loop regions of this receptor and establishes clear differences from analogous loops in the rhodopsin crystal structure.