Clinical Characteristics of Central Serous Chorioretinopathy in Women

Ophthalmology. Feb, 2002  |  Pubmed ID: 11825806

To describe the clinical features of central serous chorioretinopathy (CSC) in women and identify factors predictive of complete recovery, moderate visual loss, and prolonged duration.

Time-resolved Resonance Raman Analysis of Chromophore Structural Changes in the Formation and Decay of Rhodopsin's BSI Intermediate

Journal of the American Chemical Society. May, 2002  |  Pubmed ID: 11971736

Time-resolved resonance Raman microchip flow experiments are performed to obtain the vibrational spectrum of the chromophore in rhodopsin's BSI intermediate and to probe structural changes in the bathorhodopsin-to-BSI and BSI-to-lumirhodopsin transitions. Kinetic Raman spectra from 250 ns to 3 micros identify the key vibrational features of BSI. BSI exhibits relatively intense HOOP modes at 886 and 945 cm(-1) that are assigned to C(14)H and C(11)H=C(12)H A(u) wags, respectively. This result suggests that in the bathorhodopsin-to-BSI transition the highly strained all-trans chromophore has relaxed in the C(10)-C(11)=C(12)-C(13) region, but is still distorted near C(14). The low frequency of the 11,12 A(u) HOOP mode in BSI compared with that of lumirhodopsin and metarhodopsin I indicates weaker coupling between the 11H and 12H wags due to residual distortion of the BSI chromophore near C(11)=C(12). The C=NH(+) stretching mode in BSI at 1653 cm(-1) exhibits a normal deuteriation induced downshift of 23 cm(-1), implying that there is no significant structural rearrangement of the Schiff base counterion region in the transition of bathorhodopsin to BSI. However, a dramatic Schiff base environment change occurs in the BSI-to-lumirhodopsin transition, because the 1638 cm(-1) C=NH(+) stretching mode in lumirhodopsin is unusually low and shifts only 7 cm(-1) in D(2)O, suggesting that it has essentially no H-bonding acceptor. With these data we can for the first time compare and discuss the room temperature resonance Raman vibrational structure of all the key intermediates in visual excitation.

Fluorescein Angiographic Findings in an Infected Scleral Buckle

Ophthalmic Surgery and Lasers. Sep-Oct, 2002  |  Pubmed ID: 12358300

This report presents fluorescein angiographic (FA) findings in a patient with scleral buckle infection. Ten days following scleral buckling surgery, FA demonstrated dilated choroidal vessels over the buckle with leakage of fluorescein into the subretinal space. Irregular diffuse scleral thickening was noted on the computed tomography (CT). The findings of focal choroiditis with dilated leaky choroidal vessels seen on FA, or diffuse scleral thickening demonstrated by a CT may aid in establishing the diagnosis of scleral buckle infection.

Vibrational Relaxation in Beta-Carotene Probed by Picosecond Stokes and Anti-Stokes Resonance Raman Spectroscopy

The Journal of Physical Chemistry. A. Jun, 2002  |  Pubmed ID: 17235377

Picosecond time-resolved Stokes and anti-Stokes resonance Raman spectra of all-trans-beta-carotene are obtained and analyzed to reveal the dynamics of excited-state (S(1)) population and decay, as well as ground-state vibrational relaxation. Time-resolved Stokes spectra show that the ground state recovers with a 12.6 ps time constant, in agreement with the observed decay of the unique S(1) Stokes bands. The anti-Stokes spectra exhibit no peaks attributable to the S(1) (2A(g) (-)) state, indicating that vibrational relaxation in S(1) must be nearly complete within 2 ps. After photoexcitation there is a large increase in anti-Stokes scattering from ground-state modes that are vibrationally excited through internal conversion. The anti-Stokes data are fit to a kinetic scheme in which the C=C mode relaxes in 0.7 ps, the C-C mode relaxes in 5.4 ps and the C-CH(3) mode relaxes in 12.1 ps. These results are consistent with a model for S(1)-S(0) internal conversion in which the C=C mode is the primary acceptor, the C-C mode is a minor acceptor, and the C-CH(3) mode is excited via intramolecular vibrational energy redistribution.

Anti-stokes Raman Study of Vibrational Cooling Dynamics in the Primary Photochemistry of Rhodopsin

The Journal of Physical Chemistry. A. Sep, 2002  |  Pubmed ID: 16552447

Picosecond Stokes and anti-Stokes Raman spectra are used to probe the structural dynamics and reactive energy flow in the primary cis-to-trans isomerization reaction of rhodopsin. The appearance of characteristic ethylenic, hydrogen out-of-plane (HOOP), and low-wavenumber photoproduct bands in the Raman spectra is instrument-response-limited, consistent with a subpicosecond product appearance time. Intense high and low-frequency anti-Stokes peaks demonstrate that the all-trans photoproduct is produced vibrationally hot on the ground-state surface. Specifically, the low-frequency modes at 282, 350, and 477 cm(-1) are highly vibrationally excited (T > 2000 K) immediately following isomerization, revealing that these low-frequency motions directly participate in the reactive curve-crossing process. The anti-Stokes modes are characterized by a approximately 2.5 ps temporal decay that coincides with the conversion of photorhodopsin to bathorhodopsin. This correspondence shows that the photo-to-batho transition is a ground-state cooling process and that energy storage in the primary visual photoproduct is complete on the picosecond time scale. Finally, unique Stokes vibrations at 290, 992, 1254, 1290, and 1569 cm(-1) arising from the excited state of rhodopsin are observed only at 0 ps delay.

Analysis of the Mode-specific Excited-state Energy Distribution and Wavelength-dependent Photoreaction Quantum Yield in Rhodopsin

Biophysical Journal. Apr, 2003  |  Pubmed ID: 12668457

The photoreaction quantum yield of rhodopsin is wavelength dependent: phi(lambda) is reduced by up to 5% at wavelengths to the red of 500 nm but is invariant (phi = 0.65 +/- 0.01) between 450 and 500 nm (Kim et al., 2001). To understand this nonstatistical internal conversion process, these results are compared with predictions of a Landau-Zener model for dynamic curve crossing. The initial distribution of excess photon energy in the 28 Franck-Condon active vibrational modes of rhodopsin is defined by a fully thermalized sum-over-states vibronic calculation. This calculation reveals that absorption by high-frequency unreactive modes (e.g., C[double bond]C stretches) increases as the excitation wavelength is shifted from 570 to 450 nm whereas relatively less energy is deposited into reactive low-frequency modes. This result qualitatively explains the experimentally observed wavelength dependence of phi(lambda) for rhodopsin and reveals the importance of delocalized, torsional modes in the reactive pathway.

Retinal Detachment in Eyes Undergoing Pars Plana Vitrectomy for Removal of Retained Lens Fragments

Ophthalmology. Apr, 2003  |  Pubmed ID: 12689890

To investigate the incidence and outcomes of retinal detachment (RD) associated with retained lens fragments removed by pars plana vitrectomy (PPV).

Choroidal Metastases from a Primary Ovarian Mucinous Cystadenocarcinoma

Retina (Philadelphia, Pa.). Apr, 2003  |  Pubmed ID: 12707607

Picosecond Dynamics of G-protein Coupled Receptor Activation in Rhodopsin from Time-resolved UV Resonance Raman Spectroscopy

Biochemistry. May, 2003  |  Pubmed ID: 12731857

The protein response to retinal chromophore isomerization in the visual pigment rhodopsin is studied using picosecond time-resolved UV resonance Raman spectroscopy. High signal-to-noise Raman spectra are obtained using a 1 kHz Ti:Sapphire laser apparatus that provides <3 ps visible (466 nm) pump and UV (233 nm) probe pulses. When there is no time delay between the pump and probe events, tryptophan modes W18, W16, and W3 exhibit decreased Raman scattering intensity. At longer pump-probe time delays of +5 and +20 ps, both tryptophan (W18, W16, W3, and W1) and tyrosine (Y1 + 2xY16a, Y7a, Y8a) peak intensities drop by up to 3%. These intensity changes are attributed to decreased hydrophobicity in the microenvironment near at least one tryptophan and one tyrosine residue that likely arise from weakened interaction with the beta-ionone ring of the chromophore following cis-to-trans isomerization. Examination of the crystal structure suggests that W265 and Y268 are responsible for these signals. These UV Raman spectral changes are nearly identical to those observed for the rhodopsin-to-Meta I transition, implying that impulsively driven protein motion by the isomerizing chromophore during the 200 fs primary transition drives key structural changes that lead to protein activation.

Voriconazole Treatment of Fungal Scleritis and Epibulbar Abscess Resulting from Scleral Buckle Infection

Archives of Ophthalmology. May, 2003  |  Pubmed ID: 12742860

Determination of Vitreous, Aqueous, and Plasma Concentration of Orally Administered Voriconazole in Humans

Archives of Ophthalmology. Jan, 2004  |  Pubmed ID: 14718293

To investigate the penetration of voriconazole, a new-generation triazole antifungal agent, into the vitreous and aqueous humor after oral administration.

Endogenous Ocular Nocardiosis

Journal of AAPOS : the Official Publication of the American Association for Pediatric Ophthalmology and Strabismus / American Association for Pediatric Ophthalmology and Strabismus. Apr, 2004  |  Pubmed ID: 15088059

Subconjunctival Antibiotics for Acute Postcataract Extraction Endophthalmitis--is It Necessary?

American Journal of Ophthalmology. Jun, 2004  |  Pubmed ID: 15183800

To evaluate the efficacy of adjunctive subconjunctival antibiotic injection in the treatment of acute postcataract extraction endophthalmitis.

Zinc-porphyrin Solvation in Folded and Unfolded States of Zn-cytochrome C

Inorganic Chemistry. Dec, 2004  |  Pubmed ID: 15578829

After a brief review of the use of photochemical triggers and heme metal substitution to probe the folding dynamics of cytochrome c, we present new results on the photophysics and photochemistry of folded and unfolded states of the zinc-substituted protein (Zn-cyt c). Our measurements of Zn-cyt c triplet state decay kinetics reveal a systematic isotope effect on lifetimes: the decay in the folded protein (tau(H)2(O) approximately 10 ms) is only modestly affected by isotopically substituted buffers (k(H)2(O)/k(D)2(O) = 1.2), whereas a reduced triplet lifetime (approximately 1.3 ms) and greater isotope effect (1.4) were found for the chemically denatured, fully unfolded protein. The shortest lifetime (0.1-0.4 ms) and greatest isotope effect (1.5) were found for a fully exposed model compound, zinc-substituted N-acetyl-microperoxidase-8 (ZnAcMP8), implying that the unfolded protein provides some protection to the Zn-porphyrin group even under fully denaturing conditions. Further evidence for partial structure in unfolded Zn-cyt c comes from bimolecular quenching experiments using Ru(NH(3))(6)(3+) as an external Zn-porphyrin triplet state quencher. In the presence of quencher, partially unfolded protein at midpoint guanidinium chloride (GdmCl) and urea concentrations exhibits biphasic triplet decay kinetics, a fast component corresponding to an extended, solvent-exposed state (6.6 x 10(8) M(-1) s(-1) in GdmCl, 6.3 x 10(8) M(-1) s(-1) in urea) and a slow component attributable to a compact, relatively solvent-inaccessible, state (5.9 x 10(7) M(-1) s(-1) in GdmCl, 8.6 x 10(6) M(-1) s(-1) in urea). The variation in Zn-porphyrin solvation for the compact states in the two denaturants reveals that the cofactor in the partially unfolded protein is better protected in urea solutions.

Lightning-induced Maculopathy

Retina (Philadelphia, Pa.). Apr-May, 2005  |  Pubmed ID: 15805923

Neurofibromatosis Type 2 in an Infant Presenting with Visual Impairment Confirmed by Genetic Mutation Analysis

Retina (Philadelphia, Pa.). Oct-Nov, 2005  |  Pubmed ID: 16205576

Optic Pit Maculopathy Without Inner Retinal Schisis Cavity

Retina (Philadelphia, Pa.). Jan, 2006  |  Pubmed ID: 16395153

Transpupillary Thermotherapy with Indocyanine Green Dye Enhancement for the Treatment of Occult Subfoveal Choroidal Neovascularization in Age-related Macular Degeneration

Ophthalmic Surgery, Lasers & Imaging : the Official Journal of the International Society for Imaging in the Eye. Jul-Aug, 2006  |  Pubmed ID: 16898386

Transpupillary thermotherapy (TTT) with indocyanine green (ICG) dye enhancement (TTT+) and TTT alone were compared for safety and effectiveness as a treatment of occult subfoveal choroidal neovascularization in age-related macular degeneration.

Probing Folded and Unfolded States of Outer Membrane Protein a with Steady-state and Time-resolved Tryptophan Fluorescence

The Journal of Physical Chemistry. B. Sep, 2006  |  Pubmed ID: 16942111

Steady-state and time-resolved fluorescence measurements on each of five native tryptophan residues in full-length and truncated variants of E. coli outer-membrane protein A (OmpA) have been made in folded and denatured states. Tryptophan singlet excited-state lifetimes are multiexponential and vary among the residues. In addition, substantial increases in excited-state lifetimes accompany OmpA folding, with longer lifetimes in micelles than in phospholipid bilayers. This finding suggests that the Trp environments of OmpA folded in micelles and phospholipid bilayers are different. Measurements of Trp fluorescence decay kinetics with full-length OmpA folded in brominated lipid vesicles reveal that W102 is the most distant fluorophore from the hydrocarbon core, while W7 is the closest. Steady-state and time-resolved polarized fluorescence measurements indicate reduced Trp mobility when OmpA is folded in a micelle, and even lower mobility when the protein is folded in a bilayer. The fluorescence properties of truncated OmpA, in which the soluble periplasmic domain is removed, only modestly differ from those of the full-length form, suggesting similar folded structures for the two forms under these conditions.

Intravitreal Triamcinolone Acetonide for Treatment of Cystoid Macular Edema Associated with Retinitis Pigmentosa

Retina (Philadelphia, Pa.). Nov-Dec, 2006  |  Pubmed ID: 17151506

Peptide Insertion, Positioning, and Stabilization in a Membrane: Insight from an All-atom Molecular Dynamics Simulation

Biopolymers. Apr 5-15, 2007  |  Pubmed ID: 17274025

Peptide insertion, positioning, and stabilization in a model membrane are probed via an all-atom molecular dynamics (MD) simulation. One peptide (WL5) is simulated in each leaflet of a solvated dimyristoylglycero-3-phosphate (DMPC) membrane. Within the first 5 ns, the peptides spontaneously insert into the membrane and then stabilize during the remaining 70 ns of simulation time. In both leaflets, the peptides localize to the membrane interface, and this localization is attributed to the formation of peptide-lipid hydrogen bonds. We show that the single tryptophan residue in each peptide contributes significantly to these hydrogen bonds; specifically, the nitrogen heteroatom of the indole ring plays a critical role. The tilt angles of the indole rings relative to the membrane normal in the upper and lower leaflets are approximately 26 degrees and 54 degrees , respectively. The tilt angles of the entire peptide chain are 62 degrees and 74 degrees . The membrane induces conformations of the peptide that are characteristic of beta-sheets, and the peptide enhances the lipid ordering in the membrane. Finally, the diffusion rate of the peptides in the membrane plane is calculated (based on experimental peptide concentrations) to be approximately 6 A(2)/ns, thus suggesting a 500 ns time scale for intermolecular interactions.

Reproducibility of Macular Thickness and Volume Using Zeiss Optical Coherence Tomography in Patients with Diabetic Macular Edema

Ophthalmology. Aug, 2007  |  Pubmed ID: 17353052

To evaluate optical coherence tomography (OCT) reproducibility in patients with diabetic macular edema (DME).

Subretinal Perfluorocarbon Removal: Perfluorocarbon Volume Estimation and Cannula Choice

Retina (Philadelphia, Pa.). Jan, 2008  |  Pubmed ID: 18185156

ISIS-DME: a Prospective, Randomized, Dose-escalation Intravitreal Steroid Injection Study for Refractory Diabetic Macular Edema

Retina (Philadelphia, Pa.). May, 2008  |  Pubmed ID: 18463518

: To determine safety and efficacy of intravitreal triamcinolone acetonide (IVTA) for refractory clinically significant diabetic macular edema (DME).

Ultraviolet Resonance Raman Spectroscopy of Folded and Unfolded States of an Integral Membrane Protein

The Journal of Physical Chemistry. B. Aug, 2008  |  Pubmed ID: 18588328

The vibrational structure of native anchoring tryptophan (Trp) and tyrosine residues in an integral membrane protein, bacterial outer membrane protein A (OmpA), have been investigated using UV resonance Raman (UVRR) spectroscopy for the first time. Spectra of native OmpA, a single-Trp mutant, and a Trp-less mutant were recorded in folded and unfolded states, and reveal significant changes in tryptophan structure and local environment. Salient alterations upon folding include loss of hydrogen-bonding character of indole N1H, evidenced by a shift in W17 frequency from 874 and 878 cm(-1), and growth in hydrophobicity of the local tryptophan environment, supported by increase in the ratio I1361/I1340. In addition to these site-specific changes in a single tryptophan residue, modification of the vibrational structure of the remaining native tryptophan and tyrosine amino acids is also evident. Finally, the UVRR data presented here indicate that the structures of OmpA folded in vesicle and folded in detergent may differ, and provide important foundations for ongoing studies of membrane protein folding.

Marking Embryonic Stem Cells with a 2A Self-cleaving Peptide: a NKX2-5 Emerald GFP BAC Reporter

PloS One. 2008  |  Pubmed ID: 18596956

Fluorescent reporters are useful for assaying gene expression in living cells and for identifying and isolating pure cell populations from heterogeneous cultures, including embryonic stem (ES) cells. Multiple fluorophores and genetic selection markers exist; however, a system for creating reporter constructs that preserve the regulatory sequences near a gene's native ATG start site has not been widely available.

Risk Factors for Scleral Buckle Removal: a Matched, Case-control Study

American Journal of Ophthalmology. Sep, 2008  |  Pubmed ID: 18614132

To identify preoperative, perioperative, and postoperative risk factors for scleral buckle (SB) removal.

Thermodynamics of Peptide Insertion and Aggregation in a Lipid Bilayer

The Journal of Physical Chemistry. B. Aug, 2008  |  Pubmed ID: 18681475

A variety of biomolecules mediate physiological processes by inserting and reorganizing in cell membranes, and the thermodynamic forces responsible for their partitioning are of great interest. Recent experiments provided valuable data on the free energy changes associated with the transfer of individual amino acids from water to membrane. However, a complete picture of the pathways and the associated changes in energy of peptide insertion into a membrane remains elusive. To this end, computational techniques supplement the experimental data with atomic-level details and shed light on the energetics of insertion. Here, we employed the technique of umbrella sampling in a total 850 ns of all-atom molecular dynamics simulations to study the free energy profile and the pathway of insertion of a model hexapeptide consisting of a tryptophan and five leucines (WL5). The computed free energy profile of the peptide as it travels from bulk solvent through the membrane core exhibits two minima: a local minimum at the water-membrane interface or the headgroup region and a global minimum at the hydrophobic-hydrophilic interface close to the lipid glycerol region. A rather small barrier of roughly 1 kcal mol (-1) exists at the membrane core, which is explained by the enhanced flexibility of the peptide when deeply inserted. Combining our results with those in the literature, we present a thermodynamic model for peptide insertion and aggregation which involves peptide aggregation upon contact with the membrane at the solvent-lipid headgroup interface.

A Randomized Trial Comparing Intravitreal Triamcinolone and Focal/grid Photocoagulation for Diabetic Macular Edema: Baseline Features

Retina (Philadelphia, Pa.). Jul-Aug, 2008  |  Pubmed ID: 18698292

To compare baseline demographic, systemic, and ocular characteristics within age and racial subgroups among participants in this Diabetic Retinopathy Clinical Research Network clinical trial and to compare this cohort with other cohorts enrolled in phase 3 clinical trials for diabetic retinopathy.

Short-term Intraocular Pressure Changes Immediately After Intravitreal Injections of Anti-vascular Endothelial Growth Factor Agents

American Journal of Ophthalmology. Dec, 2008  |  Pubmed ID: 18775528

To assess short-term trends and the need to monitor intraocular pressure (IOP) changes immediately after intravitreal injections of ranibizumab, bevacizumab, pegaptanib, and triamcinolone acetonide.

Effects of Tryptophan Microenvironment, Soluble Domain, and Vesicle Size on the Thermodynamics of Membrane Protein Folding: Lessons from the Transmembrane Protein OmpA

Biochemistry. Dec, 2008  |  Pubmed ID: 18991402

Refolding curves of the integral membrane protein outer membrane protein A (OmpA) were measured to determine the conformational stabilities of this model system for membrane protein folding. Wild-type OmpA exhibits a free energy of unfolding (DeltaG degrees H2O) of 10.5 kcal/mol. Mutants, containing a single tryptophan residue at the native positions 7, 15, 57, 102, or 143, are less stable than wild-type OmpA, with DeltaG degrees H2O values of 6.7, 4.8, 2.4, 4.7, and 2.8 kcal/mol, respectively. The trend observed here is discussed in terms of noncovalent interactions, including aromatic interactions and hydrogen bonding. The effect of the soluble tail on the conformational stability of the transmembrane domain of OmpA was also investigated via truncated single-Trp mutants; DeltaG degrees H2O values for four of the five truncated mutants are greater by >2.7 kcal/mol relative to the full-length versions, suggesting that the absence of the soluble domain may destabilize the unfolded transmembrane domain. Finally, dynamic light scattering experiments were performed to measure the effects of urea and protein on vesicle size and stability. Urea concentrations greater than 1 M cause an increase in vesicle size, and these diameters are unaltered in the presence of protein. These dynamic light scattering results complement the fluorescence studies and illustrate the important effects of vesicle size on protein conformational stability.

Risk Factors for Scleral Buckle Removal: a Matched, Case-control Study

Transactions of the American Ophthalmological Society. 2008  |  Pubmed ID: 19277232

To identify preoperative, perioperative, and postoperative risk factors for scleral buckle (SB) removal.

Förster Resonance Energy Transfer and Conformational Stability of Proteins: An Advanced Biophysical Module for Physical Chemistry Students

Journal of Chemical Education. Sep, 2008  |  Pubmed ID: 19756254

Protein folding is an exploding area of research in biophysics and physical chemistry. Here, we describe the integration of several techniques, including absorption spectroscopy, fluorescence spectroscopy, and Förster resonance energy transfer (FRET) measurements, to probe important topics in protein folding. Cytochrome c is used as a model protein; comparison of conformational stabilities ( ΔGH2O∘) measured via two chemical denaturants, urea and guanidinium hydrochloride, illustrate important concepts in protein folding and intermolecular interactions. In addition, the determination of intraprotein distances based upon the FRET pair Trp-59 and the heme group for unfolded states of cytochrome c highlights the evolution of the protein structure under unfolding conditions. Analysis and discussion of these results provide opportunities to gain in-depth understanding of models for protein folding while enhancing students' skills with optical techniques. Collectively, the combination of optical spectroscopy, rigorous quantitative analysis, and a focus on biophysics illustrates the significance of fundamental research at the growing intersection of chemistry, biology, and physics.

Phenotypic Heterogeneity and Lesion Size Measurements in Stargardt Macular Dystrophy

Ophthalmic Surgery, Lasers & Imaging : the Official Journal of the International Society for Imaging in the Eye. Sep-Oct, 2009  |  Pubmed ID: 19772279

Fundus autofluorescence has been used in Stargardt macular dystrophy and fundus flavimaculatus to better understand disease status and progression. Fundus autofluorescence images delineate atrophic regions with great clarity and high contrast. The authors investigated whether fundus autofluorescence images revealed more atrophy than color fundus photographs by studying a family of three siblings with long-standing disease that demonstrated a heterogeneous fundus appearance. Findings on fundus autofluorescence were compared with color fundus photographs and fluorescein images. Optical coherence tomography images were also analyzed. Areas of atrophic lesions were measured and compared on fundus autofluorescence and color fundus photographs. Autofluorescence images delineated atrophic regions more clearly than color fundus photographs. However, the measured areas of atrophy were only modestly larger in fundus autofluorescence images than in color fundus photographs. The optical coherence tomography images showed severe outer retinal atrophy and loss of foveal contour.

Near-peer Videos for Physical Examination Instruction

Medical Education. Nov, 2009  |  Pubmed ID: 19788641

Hydrogen Bonding and Solvent Polarity Markers in the Uv Resonance Raman Spectrum of Tryptophan: Application to Membrane Proteins

The Journal of Physical Chemistry. B. Nov, 2009  |  Pubmed ID: 19817473

Ultraviolet resonance Raman (UVRR) spectra of tryptophan compounds in various solvents and a model peptide are presented and reveal systematic changes that reflect solvent polarity, hydrogen bond strength, and cation-pi interaction. The commonly utilized UVRR spectral marker for environment polarity that has been based on off-resonance Raman data, the tryptophan Fermi doublet ratio I1360/I1340, exhibits different values in on- and off-resonance Raman spectra as well as for different tryptophan derivatives. Specifically, the UVRR Fermi doublet ratio for indole ranges from 0.3 in polar solvents to 0.8 in nonpolar solvents, whereas the respective values reported here and previously for off-resonance Raman spectra are 0.5-1.3. UVRR Fermi doublet ratios for the more biologically relevant molecule, N-acetyl tryptophan ethyl ester (NATEE), are in a smaller range of 1.1 (polar solvent) to 1.7 (nonpolar solvent) and correlate to the solvent polarity/polarization parameters pi* and ETN. As has been reported previously, several UVRR modes are also sensitive to the hydrogen bond strength of the indole N-H moiety. Here, we report a new unambiguous marker for H-bonding: the ratio of the W10 (approximately 1237 cm-1) intensity to that of the W9 (approximately 1254 cm-1) mode (RW10). This ratio is 0.7 for NATEE in the absence of hydrogen bond acceptors and increases to 3.1 in the presence of strong hydrogen bond acceptors, with a value of 2.3 in water. The W8 and W17 modes shift more than +10 and approximately -5 cm-1 upon increase in hydrogen bond strength; this range for W17 is smaller than that reported previously and reflects a more realistic range for proteins and peptides in solution. Finally, our data provide evidence for change in the W18 and W16 relative intensity in the presence of cation-pi interactions. These UVRR markers are utilized to interpret spectra of model membrane-bound systems tryptophan octyl ester and the peptide toxin melittin. These spectra reveal the importance of intra- and intermolecular hydrogen bonding and cation-pi interactions that likely influence the partitioning of membrane-associated biomolecules to lipid bilayers or self-associated soluble oligomers. The UVRR analysis presented here modifies and augments prior reports and provides an unambiguous set of spectral makers that can be applied to elucidate the molecular microenvironment and structure of a wide range of complex systems, including anchoring tryptophan residues in membrane proteins and peptides.

Fluorescence and UV Resonance Raman Study of Peptide-vesicle Interactions of Human Cathelicidin LL-37 and Its F6W and F17W Mutants

Biochemistry. Dec, 2009  |  Pubmed ID: 19894716

LL-37 is a broad-spectrum human antimicrobial peptide in the cathelicidin family. Potency assays in the form of minimal inhibitory concentration and vesicle leakage indicate that the single-tryptophan mutants, F6W and F17W, are as effective at killing bacteria and disrupting membranes as the native, tryptophan-free LL-37 peptide. Steady-state fluorescence and UV resonance Raman spectroscopy of F6W and F17W reveal molecular details of these tryptophan residues. The local environment polarity, hydrogen bond strength of the indole N-H moiety, and rotational freedom decrease for both F6W and F17W in the presence of carbonate ions relative to in pure distilled water; these results are consistent with burial of the hydrophobic region of alpha-helical LL-37 in oligomeric cores induced in the presence of carbonate ions. Differences in the spectroscopic properties of the carbonate-induced alpha-helical forms of F6W and F17W reflect the presence of a local lysine residue near F6W that makes the microenvironment of F6W more polar than that of F17W. In the presence of lipid vesicles, the mutants undergo additional loss of environment polarity, hydrogen bond strength, and rotational freedom. Quenching experiments utilizing brominated lipids reveal that the tryptophan residues in both mutants are essentially equidistant from the bilayer center and that bromines closer to the bilayer center, in the 9,10 positions, quench fluorescence more efficiently than those closer to the headgroups (6,7 positions). These results support carpeting or toroidal pore mechanisms of membrane disruption by LL-37 and demonstrate that the combination of tryptophan mutants and sensitive spectroscopic tools may provide important molecular clues about antimicrobial action.

Physician Assessment of Changing Lesion Size of Predominantly Classic Choroidal Neovascular Membranes in Age-related Macular Degeneration

Ophthalmic Surgery, Lasers & Imaging : the Official Journal of the International Society for Imaging in the Eye. Nov-Dec, 2009  |  Pubmed ID: 19928720

To quantify the interpretation of fluorescein angiograms of evolving predominantly classic choroidal neovascularization in age-related macular degeneration.

Microbiological Spectrum of Organisms Isolated from Explanted Scleral Buckles

Ophthalmic Surgery, Lasers & Imaging : the Official Journal of the International Society for Imaging in the Eye. Mar-Apr, 2009  |  Pubmed ID: 19320315

Scleral buckle removal is an uncommon procedure performed for various reasons. Microbiological information on explanted scleral buckles remains limited. The authors identified 37 cases of scleral buckle removal during an 18-year period. Bacterial cultures isolated an organism in 4 of 9 patients (44%) with clinical infection. Organisms identified included methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, Mycobacterium abscessus, and coagulase-negative Staphylococcus species. Bacterial cultures isolated an organism in 3 of 11 patients (27%) without clinical infection. Organisms identified included Nocardia species, Alcaligenes xylosoxidans, and Mycobacterium chelonae. Scleral buckles appearing clinically infected may be associated with more virulent organisms and a greater chance of identifying an organism. Bacterial cultures may be of value for scleral buckles upon removal.

Resonance Raman Studies of the (His)(Cys)3 2Fe-2S Cluster of MitoNEET: Comparison to the (Cys)4 Mutant and Implications of the Effects of PH on the Labile Metal Center

Biochemistry. Jun, 2009  |  Pubmed ID: 19388667

MitoNEET is a 2Fe-2S outer mitochondrial membrane protein that was initially identified as a target for anti-diabetic drugs. It exhibits a novel protein fold, and in contrast to other 2Fe-2S proteins such as Rieske proteins and ferredoxins, the metal clusters in the mitoNEET homodimer are each coordinated by one histidine residue and three cysteine residues. The interaction of the ligating His87 residue with the 2Fe-2S moiety is especially significant because previous studies have shown that replacement with Cys in the H87C mutant stabilizes the cluster against release. Here, we report the resonance Raman spectra of this naturally occurring Fe(2)S(2)(His)(Cys)(3) protein to assess local structural changes associated with cluster lability. Comparison of mitoNEET to its ferredoxin-like H87C mutant indicates that Raman peaks in the approximately 250-300 cm(-1) region of mitoNEET are influenced by the Fe-His87 moiety. Systematic pH-dependent resonance Raman spectral changes were observed in this spectral region for native mitoNEET but not the H87C mutant. The approximately 250-300 cm(-1) region of native mitoNEET is also sensitive to phosphate buffer. Thus, conditions that influence cluster release are shown here to concomitantly affect the resonance Raman spectrum in the region with Fe-His contribution. These results support the hypothesis that the Fe-N(His87) interaction is modulated within the physiological pH range, and this modulation may be critical to the function of mitoNEET.

Genetic and Clinical Evaluation of Juvenile Retinoschisis

Journal of AAPOS : the Official Publication of the American Association for Pediatric Ophthalmology and Strabismus / American Association for Pediatric Ophthalmology and Strabismus. Apr, 2009  |  Pubmed ID: 19393523

Juvenile retinoschisis is a rare retinal dystrophy caused by RS1 gene mutations.(1) Clinical examinations and molecular testing definitively diagnosed juvenile retinoschisis in 2 male infants, one of whom had a novel mutation not previously reported in the United States. Genetic testing may be the simplest way to confirm this diagnosis in infants.

Transconjunctival 20-gauge Pars Plana Vitrectomy Using a Single Entry Cannulated Sutureless System

Retina (Philadelphia, Pa.). Oct, 2009  |  Pubmed ID: 19696700

Pars plana vitrectomy with a 20-gauge transconjunctival cannulated sutureless (TCS) system has the potential of combining the advantages of smaller-gauge vitrectomy systems with the economical advantage of not needing to purchase any additional handheld instruments. However, the sclerotomy size is much larger, and self-sealing sclerotomies may be more difficult to construct. Therefore, we evaluated the need for sclerotomy suturing after performing 20-gauge TCS vitrectomy.

Resonance Raman Characterization of a Stable Tryptophan Radical in an Azurin Mutant

The Journal of Physical Chemistry. B. Jan, 2009  |  Pubmed ID: 19072535

Tryptophan radicals play a significant role in mediating biological electron transfer and catalytic processes. Here, we employ visible and UV resonance Raman, EPR, and absorption spectroscopy along with pH/isotope studies and calculations to probe a neutral closed-shell tryptophan and its oxidized radical counterpart in a modified azurin protein. Comparison of the resonance Raman spectra of the radical and closed-shell species combined with vibrational analysis reveals important structural differences between these two tryptophan species. We experimentally observe a significant reduction in bond order of the pyrrole ring of the radical, as evidenced by a 208 cm(-1) downshift of the W3 mode (predominantly C(2)-C(3) stretch). Analysis of the spectra acquired at acidic pH and in deuterated buffer highlights those vibrational modes of the radical that are sensitive to the hydrogen-bonding environment. The most significant change caused by the deuterated buffer is a 45 cm(-1) downshift of an indole nitrogen displacement mode (W17). Our spectra provide evidence that the radical species is a strong hydrogen bond acceptor, particularly in an acidic environment. Furthermore, the pK(a) for this tryptophan radical must be less than 4.0, which falls below previously reported values for l-tryptophan in aqueous solution. The normal mode assignments of the tryptophan radical help characterize its local environment, conformation, hydrogen bonding, and protonation state within a protein.

Three-year Follow-up of a Randomized Trial Comparing Focal/grid Photocoagulation and Intravitreal Triamcinolone for Diabetic Macular Edema

Archives of Ophthalmology. Mar, 2009  |  Pubmed ID: 19273785

To report 3-year outcomes of patients who participated in a randomized trial evaluating 1-mg and 4-mg doses of preservative-free intravitreal triamcinolone compared with focal/grid photocoagulation for treatment of diabetic macular edema.

Selective Antimicrobial Action is Provided by Phenol-soluble Modulins Derived from Staphylococcus Epidermidis, a Normal Resident of the Skin

The Journal of Investigative Dermatology. Jan, 2010  |  Pubmed ID: 19710683

Antimicrobial peptides serve as a first line of innate immune defense against invading organisms such as bacteria and viruses. In this study, we hypothesized that peptides produced by a normal microbial resident of human skin, Staphylococcus epidermidis, might also act as an antimicrobial shield and contribute to normal defense at the epidermal interface. We show by circular dichroism and tryptophan spectroscopy that phenol-soluble modulins (PSMs) gamma and delta produced by S. epidermidis have an alpha-helical character and a strong lipid membrane interaction similar to mammalian AMPs such as LL-37. Both PSMs directly induced lipid vesicle leakage and exerted selective antimicrobial action against skin pathogens such as Staphylococcus aureus. PSMs functionally cooperated with each other and LL-37 to enhance antimicrobial action. Moreover, PSMs reduced Group A Streptococcus (GAS) but not the survival of S. epidermidis on mouse skin. Thus, these data suggest that the production of PSMgamma and PSMdelta by S. epidermidis can benefit cutaneous immune defense by selectively inhibiting the survival of skin pathogens while maintaining the normal skin microbiome.

Staphylococcus Epidermidis Antimicrobial Delta-toxin (phenol-soluble Modulin-gamma) Cooperates with Host Antimicrobial Peptides to Kill Group A Streptococcus

PloS One. 2010  |  Pubmed ID: 20052280

Antimicrobial peptides play an important role in host defense against pathogens. Recently, phenol-soluble modulins (PSMs) from Staphylococcus epidermidis (S. epidermidis) were shown to interact with lipid membranes, form complexes, and exert antimicrobial activity. Based on the abundance and innocuity of the cutaneous resident S. epidermidis, we hypothesized that their PSMs contribute to host defense. Here we show that S. epidermidis delta-toxin (PSMgamma) is normally present in the epidermis and sparsely in the dermis of human skin using immunohistochemistry. Synthetic delta-toxin interacted with neutrophil extracellular traps (NETs) and colocalized with cathelicidin while also inducing NET formation in human neutrophils. In antimicrobial assays against Group A Streptococcus (GAS), delta-toxin cooperated with CRAMP, hBD2, and hBD3. In whole blood, addition of delta-toxin exerted a bacteriostatic effect on GAS, and in NETs, delta-toxin increased their killing capacity against this pathogen. Coimmunoprecipitation and tryptophan spectroscopy demonstrated direct binding of delta-toxin to host antimicrobial peptides LL-37, CRAMP, hBD2, and hBD3. Finally, in a mouse wound model, GAS survival was reduced (along with Mip-2 cytokine levels) when the wounds were pretreated with delta-toxin. Thus, these data suggest that S. epidermidis-derived delta-toxin cooperates with the host-derived antimicrobial peptides in the innate immune system to reduce survival of an important human bacterial pathogen.

Near-peer Workshops for Pre-clerkship Physical Examination Skills

Medical Education. May, 2010  |  Pubmed ID: 20374469

Spectroscopic Comparison of Photogenerated Tryptophan Radicals in Azurin: Effects of Local Environment and Structure

Journal of the American Chemical Society. Jul, 2010  |  Pubmed ID: 20536238

Tryptophan radicals play a significant role in mediating biological electron transfer. We report the photogeneration of a long-lived, neutral tryptophan radical (Az48W*) from the native residue tryptophan-48 in the hydrophobic core of azurin. The optical absorption, electron paramagnetic resonance, and resonance Raman spectra strongly support the formation of a neutral radical, and the data are consistent with direct electron transfer between tryptophan and the copper(II) center. Spectra of the long-lived Az48W* species are compared to those of a previously studied, solvent-exposed radical at position 108 to identify signatures of tryptophan radicals that are sensitive to the local environment. The absorption maxima of Az48W* display an approximately 23 nm hypsochromic shift in the nonpolar environment. The majority of the resonance Raman frequencies are downshifted by approximately 7 cm(-1) relative to the solvent-exposed radical, and large changes in intensity are observed for some modes. The resonance Raman excitation profiles for Az48W* exhibit distinct maxima within the absorption envelope. Electron paramagnetic resonance spectroscopy yields spectra with partially resolved lines caused by hyperfine couplings; the differences between the coupling constants for the buried and solvent-exposed radical are primarily caused by variations in structure. The insights gained by electronic, vibrational, and magnetic resonance spectroscopy enhance our fundamental understanding of the effects of protein environment on radical properties. Hypotheses for the proton transfer pathway within azurin and a deprotonation rate of approximately 5 x 10(6) s(-1) are proposed.

Demographic Changes and Response to Laser Treatment in Threshold Retinopathy of Prematurity: 10-year Experience

Journal of Pediatric Ophthalmology and Strabismus. Jul-Aug, 2010  |  Pubmed ID: 19791711

To evaluate the outcome of indirect diode laser treatment for threshold retinopathy of prematurity (ROP) and to report demographic changes observed over 10 years.

Standard Care Vs Corticosteroid for Retinal Vein Occlusion (SCORE) Study System for Evaluation of Stereoscopic Color Fundus Photographs and Fluorescein Angiograms: SCORE Study Report 9

Archives of Ophthalmology. Sep, 2010  |  Pubmed ID: 20837797

To describe the procedures and reproducibility for grading stereoscopic color fundus photographs and fluorescein angiograms of participants in the SCORE Study.

Tryptophan-lipid Interactions in Membrane Protein Folding Probed by Ultraviolet Resonance Raman and Fluorescence Spectroscopy

Biophysical Journal. May, 2011  |  Pubmed ID: 21539779

Aromatic amino acids of membrane proteins are enriched at the lipid-water interface. The role of tryptophan on the folding and stability of an integral membrane protein is investigated with ultraviolet resonance Raman and fluorescence spectroscopy. We investigate a model system, the β-barrel outer membrane protein A (OmpA), and focus on interfacial tryptophan residues oriented toward the lipid bilayer (trp-7, trp-170, or trp-15) or the interior of the β-barrel pore (trp-102). OmpA mutants with a single tryptophan residue at a nonnative position 170 (Trp-170) or a native position 7 (Trp-7) exhibit the greatest stability, with Gibbs free energies of unfolding in the absence of denaturant of 9.4 and 6.7 kcal/mol, respectively. These mutants are more stable than the tryptophan-free OmpA mutant, which exhibits a free energy of unfolding of 2.6 kcal/mol. Ultraviolet resonance Raman spectra of Trp-170 and Trp-7 reveal evolution of a hydrogen bond in a nonpolar environment during the folding reaction, evidenced by systematic shifts in hydrophobicity and hydrogen bond markers. These observations suggest that the hydrogen bond acceptor is the lipid acyl carbonyl group, and this interaction contributes significantly to membrane protein stabilization. Other spectral changes are observed for a tryptophan residue at position 15, and these modifications are attributed to development of a tryptophan-lipid cation-π interaction that is more stabilizing than an intraprotein hydrogen bond by ∼2 kcal/mol. As expected, there is no evidence for lipid-protein interactions for the tryptophan residue oriented toward the interior of the β-barrel pore. These results highlight the significance of lipid-protein interactions, and indicate that the bilayer provides more than a hydrophobic environment for membrane protein folding. Instead, a paradigm of lipid-assisted membrane protein folding and stabilization must be adopted.

Using Charge to Control the Functional Properties of Self-assembled Nanopores in Membranes

Small (Weinheim an Der Bergstrasse, Germany). Jul, 2011  |  Pubmed ID: 21626687

Hydrogen Bonding of Tryptophan Radicals Revealed by EPR at 700 GHz

Journal of the American Chemical Society. Nov, 2011  |  Pubmed ID: 22007694

Redox-active tryptophans are important in biological electron transfer and redox biochemistry. Proteins can tune the electron transfer kinetics and redox potentials of tryptophan via control of the protonation state and the hydrogen-bond strength. We examine the local environment of two neutral tryptophan radicals (Trp108 on the solvent-exposed surface and Trp48 buried in the hydrophobic core) in two azurin variants. Ultrahigh-field EPR spectroscopy at 700 GHz and 25 T allowed complete resolution of all of the principal components of the g tensors of the two radicals and revealed significant differences in the g tensor anisotropies. The spectra together with (2)H ENDOR spectra and supporting DFT calculations show that the g tensor anisotropy is directly diagnostic of the presence or absence as well as the strength of a hydrogen bond to the indole nitrogen. The approach is a powerful one for identifying and characterizing hydrogen bonds that are critical in the regulation of tryptophan-assisted electron transfer and tryptophan-mediated redox chemistry in proteins.

Photodynamic Therapy for Steroid-associated Central Serous Chorioretinopathy

The British Journal of Ophthalmology. Apr, 2011  |  Pubmed ID: 20679080

To evaluate the short-term efficacy of photodynamic therapy (PDT) for steroid-associated chronic or recurrent central serous chorioretinopathy (CSC).

Förster Resonance Energy Transfer As a Probe of Membrane Protein Folding

Biochimica Et Biophysica Acta. Feb, 2012  |  Pubmed ID: 21925139

The folding reaction of a β-barrel membrane protein, outer membrane protein A (OmpA), is probed with Förster resonance energy transfer (FRET) experiments. Four mutants of OmpA were generated in which the donor fluorophore, tryptophan, and acceptor molecule, a naphthalene derivative, are placed in various locations on the protein to report the evolution of distances across the bilayer and across the protein pore during a folding event. Analysis of the FRET efficiencies reveals three timescales for tertiary structure changes associated with insertion and folding into a synthetic bilayer. A narrow pore forms during the initial stage of insertion, followed by bilayer traversal. Finally, a long-time component is attributed to equilibration and relaxation, and may involve global changes such as pore expansion and strand extension. These results augment the existing models that describe concerted insertion and folding events, and highlight the ability of FRET to provide insight into the complex mechanisms of membrane protein folding. This article is part of a Special Issue entitled: Membrane protein structure and function.