Cis- and trans-membrane interactions of synaptotagmin-1.

In neurotransmission synaptotagmin-1 tethers synaptic vesicles to the presynaptic plasma membrane by binding to acidic membrane lipids and SNAREs and promotes rapid SNARE-mediated fusion upon Ca(2+) triggering. However, recent studies suggested that upon membrane contact synaptotagmin may not only bind in trans to the target membrane but also in cis to its own membrane. Using a sensitive membrane tethering assay we have now dissected the structural requirements and concentration ranges for Ca(2+)-dependent and -independent cis-binding and trans-tethering in the presence and absence of acidic phospholipids and SNAREs. Using variants of membrane-anchored synaptotagmin in which the Ca(2+)-binding sites in the C2 domains and a basic cluster involved in membrane binding were disrupted we show that Ca(2+)-dependent cis-binding prevents trans-interactions if the cis-membrane contains 12-20% anionic phospholipids. Similarly, no trans-interactions were observable using soluble C2AB-domain fragments at comparable concentrations. At saturating concentrations, however, tethering was observed with soluble C2AB domains, probably due to crowding on the vesicle surface and competition for binding sites. We conclude that trans-interactions of synaptotagmin considered to be essential for its function are controlled by a delicate balance between cis- and trans-binding, which may play an important modulatory role in synaptic transmission.

Harvesting highly electronically excited energy to triplet manifolds: state-dependent intersystem crossing rate in Os(II) and Ag(I) complexes.

A series of newly synthesized Os(II) and Ag(I) complexes exhibit remarkable ratiometric changes of intensity for phosphorescence versus fluorescence that are excitation wavelength dependent. This phenomenon is in stark contrast to what is commonly observed in condensed phase photophysics. While the singlet to triplet intersystem crossing (ISC) for the titled complexes is anomalously slow, approaching several hundred picoseconds in the lowest electronic excited state (S(1) → T(1)), higher electronic excitation leads to a much accelerated rate of ISC (10(11)-10(12) s(-1)), which is competitive with internal conversion and/or vibrational relaxation, as commonly observed in heavy transition metal complexes. The mechanism is rationalized by negligible metal d orbital contribution in the S(1) state for the titled complexes. Conversely, significant ligand-to-metal charge transfer character in higher-lying excited states greatly enhances spin-orbit coupling and hence the ISC rate. The net result is to harvest high electronically excited energy toward triplet states, enhancing the phosphorescence.

Systematic investigation of the metal-structure-photophysics relationship of emissive d10-complexes of group 11 elements: the prospect of application in organic light emitting devices.

A series of new emissive group 11 transition metal d(10)-complexes 1-8 bearing functionalized 2-pyridyl pyrrolide together with phosphine ancillary such as bis[2-(diphenylphosphino)phenyl] ether (POP) or PPh(3) are reported. The titled complexes are categorized into three classes, i.e. Cu(I) complexes (1-3), Ag(I) complexes (4 and 5), and Au(I) metal complexes (6-8). Via combination of experimental and theoretical approaches, the group 11 d(10)-metal ions versus their structural variation, stability, and corresponding photophysical properties have been investigated in a systematic and comprehensive manner. The results conclude that, along the same family, how much a metal d-orbital is involved in the electronic transition plays a more important role than how heavy the metal atom is, i.e. the atomic number, in enhancing the spin-orbit coupling. The metal ions with and without involvement of a d orbital in the lowest lying electronic transition are thus classified into internal and external heavy atoms, respectively. Cu(I) complexes 1-3 show an appreciable metal d contribution (i.e., MLCT) in the lowest lying transition, so that Cu(I) acts as an internal heavy atom. Despite its smallest atomic number among group 11 elements, Cu(I) complexes 1-3 exhibit a substantially larger rate of intersystem crossing (ISC) and phosphorescence radiative decay rate constant (k(r)(p)) than those of Ag(I) (4 and 5) and Au(I) (6-8) complexes possessing pure π → π* character in the lowest transition. Since Ag(I) and Au(I) act only as external heavy atoms in the titled complexes, the spin-orbit coupling is mainly governed by the atomic number, such that complexes associated with the heavier Au(I) (6-8) show faster ISC and larger k(r)(p) than the Ag(I) complexes (4 and 5). This trend of correlation should be universal and has been firmly supported by experimental data in combination with empirical derivation. Along this line, Cu(I) complex 1 exhibits intensive phosphorescence (Φ(p) = 0.35 in solid state) and has been successfully utilized for fabrication of OLEDs, attaining peak EL efficiencies of 6.6%, 20.0 cd/A, and 14.9 lm/W for the forward directions.

Effects of multibranching on 3-hydroxyflavone-based chromophores and the excited-state intramolecular proton transfer dynamics.

A series of one-, two-, and three-branched chromophores based on 3-hydroxyflavones (1-3) have been synthesized as the first example of multibranched chromophores demonstrating excited-state intramolecular proton transfer (ESIPT). Coupling between the 3-hydroxyflavone branches connected by an electron-donating triphenylamine core is manifested in the red-shifted and asymmetric absorption band of 2, whereas the absorption of 3 is governed by the divided donor strength. Their excited-state charge-transfer (ESCT)-coupled ESIPT dynamics is investigated via femtosecond fluorescence upconversion and is proved to be well correlated with the ratio of normal/tautomer emission in the fluorescence spectra. For 1 and 2, with increased donor strength compared with the 4'-N,N-dialkylamino-3-hydroxyflavone analogue, ESIPT appears to cease in the more polar solvent of acetonitrile. Nevertheless, similar dependence of 1-3 on solvent polarity signifies resembling charge-transfer character at the normal excited states (N*), despite their varying structures. As evidenced by the theoretical approach, the frontier orbitals of vibrationally relaxed (geometry-optimized) N*, from which fluorescence and ESIPT should take place, are localized on one specific branch, leading to similar emission patterns and dynamics, whereas the orbitals contributing to Franck-Condon excitation (absorption) spread over the entire molecule. The localization is found to be facilitated by rotation of a specific branch pivoting on the central nitrogen atom, while planarity is maintained within each 3-hydroxyflavone chromophore.

Effects of using relaxation breathing training to reduce music performance anxiety in 3rd to 6th graders.

The current study examined the effects of applying relaxation breathing training (RBT) as a means to reduce music performance anxiety (MPA) in young, talented musicians. A group of 59 young musicians from 3rd to 6th grade participated in this study, and all of them started RBT twice a week for 2 months prior to the examination. Four tests--2 mos, 1 mos, half an hour and 5 min before the examination--were conducted to examine the level of MPA after the application of RBT. Results show that the degree of MPA 5 min before the trial was lower than the degree of performance anxiety half an hour before the jury (t = -3.683, p < 0.01), which indicated that the RBT was associated with a decrease in MPA. Although a series of RBT exercises was applied, results indicated that when approaching the date of examination, the degree of performance anxiety still increased and reached its maximum half an hour before the jury. The recommendation for future studies is to combine the application of RBT with other methods to expand its effect in reducing MPA.

Dual fluorescent photochromic colorants bearing pyrano[3,2-c]chromen-5-one moiety.

In this study, the photochromic processes of 8-N,N-dimethylamino-2,2-dimethyl-2H-pyrano[3,2-c]chromen-5-one (1) and its derivatives (2, 3) are investigated with steady-state, temperature-dependent and time-resolved absorption and emission spectroscopy. The differences among compounds 1-3 lie in their various substituents anchored at the pyran moiety that is subject to the photoinduced ring-opening reaction. Compounds 1 and 2 exhibit salient photochromism with a very unique phenomenon, in which fluorescence is observed in 1 for both the ring-closed form (1-CF, lambda(max) approximately 445 nm) and the ring-open form (1-OF, lambda(max) approximately 650 nm in CH2Cl2). The yields of forward and reverse photochromism processes were determined to be 0.40 and 1.0% for 1. Along with fluorescence quantum yields of 9.5 x 10(-2) and 5.8 x 10(-3) for 1-CF and 1-OF, respectively, 1 enables fluorescence detection while it exhibits photochromism in both directions, that is, a photoinduced on/off fluorescence switch. An increase in on/off ratiometric fluorescence between 1-OF and 1-CF can reach a factor of 4.0 upon excitation at the absorption isosbestic point. The activation energies for the ground-state OFtrans --> CF thermal bleaching processes were determined to be 58.2 and 54.8 kJ/mol, with frequency factors of 1.7 x 10(5) and 3.6 x 10(5) s(-1) for 1 and 2, respectively. Conversely, bromo-substituted 3 did not undergo photochromic reaction, as evidenced by the lack of changes in the absorption spectrum after a prolonged (2 h) 354 nm (0.2 W/cm2) photolysis, manifesting the fact that other relaxation processes, such as enhanced intersystem crossing, may govern the deactivation of 3 (3-CF) upon excitation.

Photogeneration and thermal generation of pentacene from soluble precursors for OTFT applications.

A CO adduct of pentacene with an unsymmetrical structure is synthesized; it is soluble and can be spin-coated into thin films. Pentacene is regenerated in near quantitative yield by either thermal or photoinduced elimination of CO. OTFT devices fabricated by this compound exhibit typical FET characteristics.

Tunable Membrane Potential Reconstituted in Giant Vesicles Promotes Permeation of Cationic Peptides at Nanomolar Concentrations.

We investigate the influence of membrane potential on the permeation of cationic peptides. Therefore, we employ a microfluidic chip capable of capturing giant unilamellar vesicles (GUVs) in physical traps and fast exchange of chemical compounds. Control experiments with calcein proved that the vesicle membranes' integrity is not affected by the physical traps and applied shear forces. Combined with fluorescence correlation spectroscopy, permeation of fluorescently labeled peptides across vesicle membranes can be measured down to the nanomolar level. With the addition of a lipophilic ruthenium(II) complex Ru(C17)22+, GUVs consisting of mixed acyl phospholipids are prepared with a negative membrane potential, resembling the membrane asymmetry in cells. The membrane potential serves as a driving force for the permeation of cationic cell-penetrating peptides (CPPs) nonaarginine (Arg9) and the human immunodeficiency virus trans-activator of transcription (TAT) peptide already at nanomolar doses. Hyperpolarization of the membrane by photo-oxidation of Ru(C17)22+ enhances permeation significantly from 55 to 78% for Arg9. This specific enhancement for Arg9 (cf. TAT) is ascribed to the higher affinity of the arginines to the phosphoserine head groups. On the other hand, permeation is decreased by introducing an additional negative charge in close proximity to the N-terminal arginine residue when changing the fluorophore. In short, with the capability to reconstitute membrane potential as well as shear stress, our system is a suitable platform for modeling the membrane permeability of pharmaceutics candidates. The results also highlight the membrane potential as a major cause of discrepancies between vesicular and cellular studies on CPP permeation.

A One Donor-Two Acceptor Lipid Bilayer FRET Assay Based on Asymmetrically Labeled Liposomes.

The fusion of two opposing membranes is essential in biological functions such as fertilization, viral entry, membrane trafficking and synaptic transmission. Before the membrane bilayers are fully connected, at some stage a hemifusion intermediate-when the outer leaflets are merged but not the inner leaflets-is formed. However, the position of hemifusion in the energy landscape and the duration of it vary and have not been fully mapped out. To date, there has not been a way to differentiate lipid mixing of the two leaflets directly in a single experiment. Herein we demonstrate labeling of the outer and inner leaflets with different fluorophores, which can be distinguished by their fluorescence lifetimes. As a proof of concept, the asymmetrically labeled liposomes were used as acceptor liposomes in a novel one donor-two acceptor Förster resonance energy transfer (FRET) assay to monitor membrane fusion reactions mediated by the synaptic proteins soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) in microfluidic devices. Initial hemifusion was clearly indicated by the acceptor fluorescence lifetime originating solely from FRET acceptors on the outer leaflet (Oregon Green 488; τFl ∼ 4.8 ns). Progression to full fusion was then indicated by the significantly increasing lifetime contribution from acceptors on the inner leaflet (nitrobenzoxadiazole; τFl ∼ 6.7 ns). The new labeling strategy creates many possibilities in the design of bulk and single-molecule experiments.

Control of membrane gaps by synaptotagmin-Ca2+ measured with a novel membrane distance ruler.

Fast synchronous neurotransmitter release is triggered by calcium that activates synaptotagmin-1 (syt-1), resulting in fusion of synaptic vesicles with the presynaptic membrane. Syt-1 possesses two Ca(2+)-binding C2 domains that tether membranes via interactions with anionic phospholipids. It is capable of crosslinking membranes and has recently been speculated to trigger fusion by decreasing the gap between them. As quantitative information on membrane gaps is key to understanding general cellular mechanisms, including the role of syt-1, we developed a fluorescence-lifetime based inter-membrane distance ruler using membrane-anchored DNAs of various lengths as calibration standards. Wild-type and mutant data provide evidence that full-length syt-1 indeed regulates membrane gaps: without Ca(2+), syt-1 maintains membranes at distances of ~7-8 nm. Activation with 100 µM Ca(2+) decreases the distance to ~5 nm by binding the C2 domains to opposing membranes, respectively. These values reveal that activated syt-1 adjusts membrane distances to the level that promotes SNARE complex assembly.

Identification of Rickettsia felis in fleas but not ticks on stray cats and dogs and the evidence of Rickettsia rhipicephali only in adult stage of Rhipicephalus sanguineus and Rhipicephalus haemaphysaloides.

Rickettsia spp. are zoonotic pathogens and mainly transmitted by various arthropod vectors, such as fleas, ticks, and lice. Previous epidemiological studies indicated that ectoparasites infested on dogs or cats may be infected by Rickettsia spp., and transmit them to human beings accidentally. In this study, the prevalence of Rickettsia infection was evaluated using fleas and ticks from stray dogs and cats in Taiwan. A total of 158 pools made by 451 cat fleas (Ctenocephalides felis) from 37 dogs and 4 cats were used for analysis. Besides, 386 Rhipicephalus ticks collected from the other 62 stray dogs were included in this study. Nymphal and adult ticks were individually analyzed but larvae were separated into 21 pools for molecular detection. Partial sequencing analysis of the gltA gene was applied for Rickettsia identification. The results showed that 44.3% (70/158) of the cat flea pools were harboring Rickettsia DNA. Although 6.9% (13/187) of adult ticks were infected with Rickettsia, neither larval pools nor nymphal ticks were found to contain Rickettsia DNA. According to the results of sequencing analyses, all Rickettsia PCR-positive cat flea pools were infected with R. felis, and all Rickettsia PCR-positive adult ticks were infected with R. rhipicephali. The results of this study demonstrated that C. felis but not Rhipicephlus sanguineus (the brown dog tick) and Rh. haemaphysaloides collected from stray animals in Taiwan could be infected the zoonotic pathogen R. felis. Moreover, R. rhipicephali was only identified in adult stage of Rhipicephalus sanguineus and Rh. haemaphysaloides.

Bartonella infection in shelter cats and dogs and their ectoparasites.

Mainly through vector transmission, domestic cats and dogs are infected by several Bartonella spp. and represent a large reservoir for human infections. This study investigated the relationship of prevalences of Bartonella infection in shelter dogs and cats and various ectoparasite species infesting them (fleas, ticks, and lice). Moreover, relationships between Bartonella infection and animal gender and age and presence of ectoparasites were analyzed. Blood samples were collected from 120 dogs and 103 cats. There were 386 ticks and 36 fleas harvested on these dogs, and 141 fleas, 4 ticks, and 2 lice harvested on these cats. Isolation/detection of Bartonella sp. was performed by culture, polymerase chain reaction (PCR), and partial sequencing. Bartonella was isolated from 21 (20.4%) cats and detected by PCR from 20 (19.4%) cats, 2 (1.7%) dogs, 55 (39%) fleas collected from cats, 28 (10%) ticks DNA samples, and 1 (2.8%) flea collected from dogs. When combining culture and PCR data, 27 cats and 55 fleas collected on cats were positive for Bartonella henselae or Bartonella clarridgeiae, but none were coinfected. Approximately half of the B. henselae isolates from 21 cats were B. henselae type I. Moreover, B. henselae, Bartonella phoceensis, Bartonella queenslandensis, Bartonella rattimassiliensis, Bartonella elizabethae DNA was detected in ticks collected from dogs and one flea was B. clarridgeiae PCR positive. This is the first report of such a wide variety of Bartonella spp. detected in Rhipicephalus sanguineus. Further studies are required to understand the relative importance of these ectoparasites to transmit Bartonella spp. in dogs and cats.

Synthesis, photophysical and theoretical studies of luminescent silver(I)-copper(I) alkynyl-diphosphine complexes.

The novel heterometallic aggregates [Ag(8)Cu(2)(C(2)C(6)H(4)X)(6)(PPh(2)C(6)H(4)PPh(2))(6)][PF(6)](4) (X = H, 1; OMe, 2; NMe(2), 3) were prepared by the self-assembly reactions starting from simple precursors. These compounds consist of the central [Ag(6)Cu(2)(C(2)C(6)H(4)X)(6)](2+) clusters capped by two tripodal fragments [Ag(diphosphine)(3)](+). The solid state structures of complexes 1 and 3 have been determined by X-ray crystallographic studies, and NMR spectroscopy confirmed that the compounds remain intact in solution. In solution such as CH(3)CN, complexes 1-3 show a weak to moderate room-temperature phosphorescence in the blue region with a maximum quantum yield of 0.038 (2), while strong phosphorescence was recorded in the solid film of with a quantum yield of 0.26. Photophysical experiments revealed a relatively small effect of luminescence quenching by O(2) for 1, but more prominent quenching for 2 and 3 in solution. Computational approaches are in good agreement with the experimental data, supporting the proposed structural motif, and provide an additional insight into the electronic properties of these compounds to rationalize the different behavior of 1 compared to 2 and 3.