Binding of muscimol-conjugated quantum dots to GABAC receptors

Functionalization of highly fluorescent CdSe/ZnS core-shell nanocrystals (quantum dots, qdots) is an emerging technology for labeling cell surface proteins. We have synthesized a conjugate consisting of approximately 150-200 muscimols (a GABA receptor agonist) covalently joined to the qdot via a poly(ethylene glycol) (PEG) linker (approximately 78 ethylene glycol units) and investigated the binding of this muscimol-PEG-qdot conjugate to homomeric rho1 GABAC receptors expressed in Xenopus oocytes. GABAC receptors mediate inhibitory synaptic signaling at multiple locations in the central nervous system (CNS). Binding of the conjugate was analyzed quantitatively by determining the fluorescence intensity of the oocyte surface membrane in relation to that of the surrounding incubation medium. Upon 5- to 10-min incubation with muscimol-PEG-qdots (34 nM in qdot concentration), GABAC-expressing oocytes exhibited a fluorescent halo at the surface membrane that significantly exceeded the fluorescence of the incubation medium. This halo was absent following muscimol-PEG-qdot treatment of oocytes lacking GABAC receptors. Incubation of the oocyte with free muscimol (100 microM-5 mM), PEG-muscimol (500 microM), or GABA (100 microM - 5 mM) substantially reduced or eliminated the fluorescence halo produced by muscimol-PEG-qdots, and the removal of GABA or free muscimol led to a recovery of muscimol-PEG-qdot binding. Unconjugated qdots and PEG-qdots that lacked conjugated muscimol neither exhibited significant binding activity nor diminished the subsequent binding of muscimol-PEG-qdots. The results indicate that muscimol joined to qdots via a long-chain PEG linker exhibits specific binding activity at the ligand-binding pocket of expressed GABAC receptors, despite the presence of both the long PEG linker and the sterically bulky qdot.     

Coupling versus surface-etching reactions of alkyl halides on GaAs(100): I. CF3CH2I reactions

We report the rich surface chemistry exhibited by the reactions of 1,1,1-trifluoroethyl iodide (CF3CH2I) adsorbed onto gallium-rich GaAs(100)-(4 x 1), studied by temperature-programmed desorption (TPD) and low-energy electron diffraction (LEED) studies and X-ray photoelectron spectroscopy (XPS). CF3CH2I adsorbs molecularly at 150 K but dissociates, below room temperature, to form a chemisorbed monolayer of CF3CH2 and I species. Recombinative desorption of molecular CF3CH2I competes with the further reactions of the CF3CH2 and I chemisorbed species. The CF3CH2 species can either undergo beta-fluoride elimination to yield gaseous CF2=CH2 or it can undergo self-coupling to form the corresponding higher alkane, CF3CH2CH2CF3. A second coupling product, CF3CH2CH=CF2, is also evolved, and it is postulated that migratory insertion of the liberated CF2=CH2 into the surface-carbon bond of the chemisorbed CF3CH2 is responsible for its formation. The iodines, formed by C-I scission in the chemisorbed CF3CH2I, and the fluorines, derived from beta-fluoride elimination in CF3CH2, react with the surface gallium dimers, and Ga-As back-bonds to generate five etch products (GaF, AsF, GaI, AsI, and As2) that desorb in the temperature range of 420 to >600 K. XPS data reveal that the surface stoichiometry remains constant throughout the entire annealing temperature range because of the desorption of both gallium- and arsenic-containing etch products, which occur sequentially. In this article, plausible mechanisms by which all products form and the binding sites of these reactions in the (4 x 1) reconstruction are discussed. Factors that control the rate constants of etch product versus hydrocarbon product formation and in particular how they impact on the respective desorption temperatures will be discussed.     

PNIPAM chain collapse depends on the molecular weight and grafting density

This study demonstrates that the thermally induced collapse of end-grafted poly(N-isopropylacrylamide) (PNIPAM) above the lower critical solution temperature (LCST) of 32 degrees C depends on the chain grafting density and molecular weight. The polymer was grafted from the surface of a self-assembled monolayer containing the initiator (BrC(CH3)2COO(CH2)11S)2, using surface-initiated atom transfer radical polymerization. Varying the reaction time and monomer concentration controlled the molecular weight, and diluting the initiator in the monolayer altered the grafting density. Surface force measurements of the polymer films showed that the chain collapse above the LCST decreases with decreasing grafting density and molecular weight. At T > LCST, the advancing water contact angle increases sharply on PNIPAM films of high molecular weight and grafting density, but the change is less pronounced with films of low-molecular-weight chains at lower densities. Below the LCST, the force-distance profiles exhibit nonideal polymer behavior and suggest that the brush architecture comprises dilute outer chains and much denser chains adjacent to the surface.     

Sex differences in the relationship between white matter microstructure and impulsivity in adolescents

Rapid maturational brain changes occur during adolescence--a time associated with risk-taking behaviors and improvements in cognition. The present study examined the relationship between white matter (WM) microstructure, impulsive behavior and response inhibition in female and male adolescents. Twenty-one healthy adolescents underwent diffusion tensor imaging using a 3.0-T magnetic resonance imaging system. Impulse control was assessed using the Bar-On Emotional Quotient Inventory, Youth Version. Response inhibition was assessed using the Stroop Color-Word Interference Test. Fractional anisotropy (FA), a measure of WM coherence, and trace, a measure of overall diffusivity, were determined from voxels manually placed in the midline and in the left and right forward-projecting arms of the genu and the splenium of the corpus callosum. Sex-specific differences were observed for the relationship between FA and impulsive behavior in the right anterior callosum for males and in the splenium for females. Males, compared to females, displayed significantly higher FA in the left WM region. Although trace was not associated with impulse control, trace in the genu (for females) and splenium (males and females) was associated with Stroop performance. Regional differences in trace also were evident, with lower values in the splenium observed than in all other regions. Although the latter significantly improved with age, no sex differences in impulse control or in Stroop performance were detected. The present findings provide supporting evidence for sex-related differences in the development of WM microstructure during adolescence. These data further suggest a neurobiological mechanism underlying some of the emotional and cognitive changes commonly observed in males versus females during the adolescent period.     

Furthering Precision in Sentinel Node Navigational Surgery for Oral Cancer: a Novel Triple Targeting System

To describe an innovative sentinel lymph node (SLN) guidance approach using a radionuclide tracer, 3D augmented reality-guided imaging, and near infrared (NIR) fluorescence over-lay imaging with hand-held probes to optimize accuracy, efficiency, and precise navigation for sentinel node (SN) localization in head and neck cancer. In a cT1N0M0 squamous cell carcinoma of the tongue, pre-operative radionuclide lymphoscintigraphy was performed with a sentinel node-specific radiolabeled tracer. Intraoperatively, a 3D hand-held augmented reality (AR) scanning SPECT probe assessed concordance of the SN with pre-operative SPECT-CT images. The real-time optical video was linked to the SPECT-CT images for added precision. Final guidance to the SN was performed using ICG fluorescence imaging. Dynamic and SPECT-CT showed bilateral lymphatic drainage from the tumor. The 3D hand-held AR SPECT probe SN localization was concordant with pre-operative imaging. The optical video successfully demonstrated the lymphatic drainage in real-time through a unique overlay fluorescence image. The ICG localized to the same nodes identified by both the SPECT-CT and hand-held SPECT images. The use of dual radiation and fluorescence tracers improved SN detection, especially for SN close to the injection site. The hand-held probes allowed the surgeon to dissect continuously, without needing to change tools. The combination of augmented reality, nuclear medicine, and over-lay fluorescence imaging allowed greater accuracy for matching the preoperative imaging with intraoperative identification and precisely guiding the dissection. This method uniquely permitted the surgeon to efficiently dissect the SN with accurate visualization and optimal precision.     

Continuous Fluorescence Depletion Anisotropy Measurement of Protein Rotation

Protein rotation in viscous environments can be measured by fluorescence depletion anisotropy (FDA) which combines long lifetimes of chromophore triplet states with the sensitivity of fluorescence excitation and detection. FDA achieves sensitivity well beyond that attainable by the more common technique of time-resolved phosphorescence anisotropy (TPA). We have now combined benefits of both time-domain and frequency-domain FDA into a single continuous technique (CFDA). Intensity and polarization of a single laser beam are modulated continuously according to a complex, repeating waveform. Fluorescence signals excited from triplet-forming fluorescent probes are digitized over recurring waveform periods by a high-speed signal averager. CFDA experiments typically involve substantial ground state depletion. Thus signals, unlike those of TPA, are not linear in the exciting light intensity and simple data analysis based on such linearity is not appropriate. An exact solution of the coupled diffusion and triplet production/decay equation describing CFDA within individual data points has been combined with simulated annealing optimization to extract triplet and anisotropy decay kinetics from experimental data. Related calculations compare possible excitation waveforms with respect to rotational information provided per fluorescence photon. We present CFDA results for the model system of eosin conjugates of carbonic anhydrase, BSA and immunoglobulin G in 90% glycerol at various temperatures and initial cellular results on eosin-IgE bound to 2H3 cell Type I Fcε receptors. We explore how CFDA reflects rotational parameters of heterogeneous systems and discuss challenges of extending this method to single cell microscopic measurements.     

The frequency of hard glottal attacks in patients with muscle tension dysphonia, unilateral benign masses and bilateral benign masses

Hard or abrupt glottal attack (HGA) is one of the vocal behaviors often associated with benign lesion of the vocal folds. This study was designed to determine whether the frequency of HGA was different in hyperfunctional voice patients with and without vocal fold masses. One hundred and forty-seven subjects were studied. All subjects received a complete otolaryngological evaluation including strobovideolaryngoscopy, objective voice measures, and evaluation by a speech-language pathologist. Thirty-two patients were diagnosed with muscle tension dysphonia (19 male, 13 female) without vocal fold masses. Fifty-seven patients were diagnosed with unilateral vocal fold masses (29 male, 28 female), most of which were cysts. Fifty-eight patients were diagnosed with bilateral vocal fold masses (13 male, 45 female). Of the 45 females with bilateral vocal fold masses. 26 had a vocal cyst and reactive nodule and 19 had bilateral vocal fold nodules. The control group was balanced and matched based on sex and on percentage of singers and nonsingers. It consisted of 49 subjects with no vocal fold pathology (20 male, 29 female). The group was composed of professional speakers, singers, and nonprofessional speakers. All voice disordered groups demonstrated higher frequencies of HGA than the control group. Differences were found between the male and female subjects in this study. No differences were found between the various disorders. Differences were also found between the subgroups of bilateral masses, where the bilateral nodules group presented a higher frequency of HGA than the cyst and contralateral reactive nodule.     

Modeling the interface between islet amyloid polypeptide and insulin-based aggregation inhibitors: correlation to aggregation kinetics and membrane damage

Human islet amyloid polypeptide (hIAPP) forms cytotoxic fibrils in type-2 diabetes and insulin is known to inhibit formation of these aggregates. In this study, a series of insulin-based inhibitors were synthesized and assessed for their ability to slow aggregation and impact hIAPP-induced membrane damage. Computational studies were employed to examine the underlying mechanism of inhibition. Overall, all compounds were able to slow aggregation at sufficiently high concentrations (10× molar excess); however, only two peptides showed any inhibitory capability at the 1:1 molar ratio (EALYLV and VEALYLV). The results of density functional calculations suggest this is due to the strength of a salt bridge formed with the Arg11 side chain of hIAPP and the inhibitors' ability to span from the Arg11 to past the Phe15 residue of hIAPP, blocking one of the principal amyloidogenic regions of the molecule. Unexpectedly, slowing fibrillogenesis actually increased damage to lipid membranes, suggesting that the aggregation process itself, rather than the fibrilized peptide, may be the cause of cytotoxicity in vivo.     

Investigating the effect of antibiotics on quorum sensing with whole-cell biosensing systems

Quorum sensing (QS) allows bacteria to communicate with one another by means of QS signaling molecules and control certain behaviors in a group-based manner, including pathogenicity and biofilm formation. Bacterial gut microflora may play a role in inflammatory bowel disease pathogenesis, and antibiotics are one of the available therapeutic options for Crohn’s disease. In the present study, we employed genetically engineered bioluminescent bacterial whole-cell sensing systems as a tool to evaluate the ability of antibiotics commonly employed in the treatment of chronic inflammatory conditions to interfere with QS. We investigated the effect of ciprofloxacin, metronidazole, and tinidazole on quorum sensing. Several concentrations of individual antibiotics were allowed to interact with two different types of bacterial sensing cells, in both the presence and absence of a fixed concentration of N-acylhomoserine lactone (AHL) QS molecules. The antibiotic effect was then determined by monitoring the biosensor’s bioluminescence response. Ciprofloxacin, metronidazole, and tinidazole exhibited a dose-dependent augmentation in the response of both bacterial sensing systems, thus showing an AHL-like effect. Additionally, such an augmentation was observed, in both the presence and absence of AHL. The data obtained indicate that ciprofloxacin, metronidazole, and tinidazole may interfere with bacterial communication systems. The results suggest that these antibiotics, at the concentrations tested, may themselves act as bacterial signaling molecules. The beneficial effect of these antibiotics in the treatment of intestinal inflammation may be due, at least in part, to their effect on QS-related bacterial behavior in the gut.     

Average orientation of a molecular rotor embedded in a Langmuir-Blodgett monolayer

A molecular rotor in which a naphthalene rotator is attached through a silicon atom to three fatty acid chains has been synthesized, and Langmuir-Blodgett techniques were used to deposit on silica surfaces monolayers of its calcium salt, both neat and diluted with stearic acid salts. The monolayer films have been characterized by ellipsometry and Fourier transform infrared (FT-IR) grazing-incidence attenuated total internal reflection (GATR) spectroscopy on Si-SiO(2) and by UV-vis absorption spectroscopy on SiO(2). The measurements were combined with calculations of the electronic (INDO/S) and vibrational (DFT) transition moment directions to deduce the average orientation of the rotor molecules, including the naphthalene ring, relative to the surface. In both neat and mixed films, the naphthalene ring is found to preferentially tilt toward the surface, enough that its rotation is most likely hindered. A comparable picture was obtained from molecular mechanics calculations on a mixed film of the naphthalene rotor and stearic acid.