Emature: Intergenerational Networks in Digital Media Spaces

While the literature on adolescent usage of the Internet and mobile communication technology is burgeoning, the technological affordance of increased intergenerational contacts and intergenerational friendships received less attention. This project documents how intergenerational virtual networks operate in one particular massively multiplayer online game (World of Warcraft) from the standpoint of adolescent players. Online social worlds are similar to offline settings in a lot of ways. Users must obey certain behavioral standards and follow established rules and moral codes to participate. Despite accounts of online democracy and networked individualism, control and authority is central to the functioning of these environments. Power-relationships are structured by technological protocols and affordances. However, within the social space created by technology, participants actively create and re-create cultural customs shaping experience. Using the ethnographic methods of participant observation and semi-structured, open-ended interviews, this project documents the ways a different form of mature behavior, ematurity, is emerging in online spaces. Ematurity means that skilled adolescents with the right set of cultural toolkits and impression management techniques are able to participate as social equals in adult governed environments. The main foundation of emature habitus is social class. Ematurity allows young people to maintain desirable and acceptable social selves in adultist environments. While ematurity is redefining the currently narrow means of adult-youth interaction and friendship, it does not signal the end of childhood

Infrared spectroscopy reveals multi-step multi-timescale photoactivation in the photoconvertible protein archetype dronpa

Photochromic fluorescent proteins play key roles in super-resolution microscopy and optogenetics. The light-driven structural changes that modulate the fluorescence involve both trans-to-cis isomerization and proton transfer. The mechanism, timescale and relative contribution of chromophore and protein dynamics are currently not well understood. Here, the mechanism of off-to-on-state switching in dronpa is studied using femtosecond-to-millisecond time-resolved infrared spectroscopy and isotope labelling. Chromophore and protein dynamics are shown to occur on multiple timescales, from picoseconds to hundreds of microseconds. Following excitation of the trans chromophore, a ground-state primary product is formed within picoseconds. Surprisingly, the characteristic vibrational spectrum of the neutral cis isomer appears only after several tens of nanoseconds. Further fluctuations in protein structure around the neutral cis chromophore are required to form a new intermediate, which promotes the final proton-transfer reaction. These data illustrate the interplay between chromophore dynamics and the protein environment underlying fluorescent protein photochromism

Zero fluoroscopy catheter ablation for atrial fibrillation: a systematic review and meta-analysis

IntroductionCatheter ablation for atrial fibrillation (AF) is the most frequently performed cardiac ablation procedure worldwide. The majority of ablations can now be performed safely with minimal radiation exposure or even without the use of fluoroscopy, thanks to advances in 3-dimensional electroanatomical mapping systems and/or intracardiac echocardiography. The aim of this study was to conduct a meta-analysis to compare the effectiveness of zero fluoroscopy (ZF) versus non-zero fluoroscopy (NZF) strategies for AF ablation procedures.MethodsElectronic databases were searched and systematically reviewed for studies comparing procedural parameters and outcomes of ZF vs. NZF approaches in patients undergoing catheter ablation for AF. We used a random-effects model to derive the mean difference (MD) and risk ratios (RR) with a 95% confidence interval (CI).ResultsOur meta-analysis included seven studies comprising 1,593 patients. The ZF approach was found to be feasible in 95.1% of patients. Compared to the NZF approach, the ZF approach significantly reduced procedure time [mean difference (MD): −9.11 min (95% CI: −12.93 to −5.30 min; p < 0.01)], fluoroscopy time [MD: −5.21 min (95% CI: −5.51 to −4.91 min; p < 0.01)], and fluoroscopy dose [MD: −3.96 mGy (95% CI: −4.27 to −3.64; p < 0.01)]. However, there was no significant difference between the two groups in terms of total ablation time [MD: −104.26 s (95% CI: −183.37 to −25.14; p = 0.12)]. Furthermore, there was no significant difference in the acute [risk ratio (RR): 1.01, 95% CI: 1.00–1.02; p = 0.72] and long-term success rates (RR: 0.96, 95% CI: 0.90–1.03; p = 0.56) between the ZF and NZF methods. The complication rate was 2.76% in the entire study population and did not differ between the groups (RR: 0.94, 95% CI: 0.41–2.15; p = 0.89).ConclusionThe ZF approach is a feasible method for AF ablation procedures. It significantly reduces procedure time and radiation exposure without compromising the acute and long-term success rates or complication rates

Femtosecond To Millisecond Dynamics Of Light Induced Allostery In The Avena Sativa LOV Domain

The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatio-temporal control of cell signalling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds timescale, which then modulate the activity of output domains responsible for biological signalling. Using time resolved vibrational spectroscopy coupled with isotope labelling we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 femtoseconds and one millisecond after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labelled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a sub-picosecond perturbation of the protein matrix occurs. In this perturbed environment the previously characterised reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the -sheet then -helix regions of the AsLOV2 domain, which ultimately gives rise to J-helix unfolding, yielding the signalling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513

Photoactivation of the BLUF protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues

The flavin chromophore in blue light using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppA by the introduction of fluorotyrosine (F-Tyr) analogs that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark to light adapted form) photoreaction was observed, the change in Y21 pKa led to a 4,000-fold increase in the rate of dark state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppA, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppA, despite their sharing highly conserved FAD binding architectures

Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii

Acinetobacter baumannii is an important human pathogen that can form biofilms and persist under harsh environmental conditions. Biofilm formation and virulence are modulated by blue light, which is thought to be regulated by a BLUF protein, BlsA. To understand the molecular mechanism of light sensing, we have used steady-state and ultrafast vibrational spectroscopy to compare the photoactivation mechanism of BlsA to the BLUF photosensor AppA from Rhodobacter sphaeroides. Although similar photocycles are observed, vibrational data together with homology modeling identify significant differences in the β5 strand in BlsA caused by photoactivation, which are proposed to be directly linked to downstream signaling

BLUF Domain Function Does Not Require a Metastable Radical Intermediate State

BLUF (blue light using flavin) domain proteins are an important family of blue light-sensing proteins which control a wide variety of functions in cells. The primary light-activated step in the BLUF domain is not yet established. A number of experimental and theoretical studies points to a role for photoinduced electron transfer (PET) between a highly conserved tyrosine and the flavin chromophore to form a radical intermediate state. Here we investigate the role of PET in three different BLUF proteins, using ultrafast broadband transient infrared spectroscopy. We characterize and identify infrared active marker modes for excited and ground state species and use them to record photochemical dynamics in the proteins. We also generate mutants which unambiguously show PET and, through isotope labeling of the protein and the chromophore, are able to assign modes characteristic of both flavin and protein radical states. We find that these radical intermediates are not observed in two of the three BLUF domains studied, casting doubt on the importance of the formation of a population of radical intermediates in the BLUF photocycle. Further, unnatural amino acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines, thus modifying the driving force for the proposed electron transfer reaction; the rate changes observed are also not consistent with a PET mechanism. Thus, while intermediates of PET reactions can be observed in BLUF proteins they are not correlated with photoactivity, suggesting that radical intermediates are not central to their operation. Alternative nonradical pathways including a keto–enol tautomerization induced by electronic excitation of the flavin ring are considered

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule

Site Specific Protein Dynamics Probed by Ultrafast Infrared Spectroscopy of a Noncanonical Amino Acid

Real-time observation of structure change associated with protein function remains a major challenge. Ultrafast pump-probe methods record dynamics in light activated proteins, but the assignment of spectroscopic observables to specific structure changes can be difficult. The BLUF (blue light using flavin) domain proteins are an important class of light sensing flavoprotein. Here we incorporate the unnatural amino acid (UAA) azidophenylalanine (AzPhe) at key positions in the H-bonding environment of the isoalloxazine chromophore of two BLUF domains, PixD and AppABLUF; both proteins retain the red shift on irradiation characteristic of photoactivity. Steady state and ultrafast time resolved infrared (TRIR) difference measurements of the azido mode reveal site-specific information on the nature and dynamics of light driven structure change. AzPhe dynamics are thus shown to be an effective probe of BLUF domain photoactivation, revealing significant differences between the two proteins, and a differential response of the two sites to chromophore excitation

Variation in LOV Photoreceptor Activation Dynamics Probed by Time-Resolved Infrared Spectroscopy

The light, oxygen, voltage (LOV) domain proteins are blue light photoreceptors that utilize a non-covalently bound flavin mononucleotide (FMN) cofactor as the chromophore. The modular nature of these proteins has led to their wide adoption in the emerging fields of optogenetics and optobiology, where the LOV domain has been fused to a variety of output domains leading to novel light-controlled applications. In the present work, we extend our studies of the sub-picosecond to sub-millisecond transient infrared spectroscopy of the isolated LOV domain AsLOV2 to three full-length photoreceptors in which the LOV domain is fused to an output domain: the LOV-STAS protein, YtvA, the LOV-HTH transcription factor, EL222, and the LOV-histidine kinase, LovK. Despite differences in tertiary structure, the overall pathway leading to cysteine adduct formation from the FMN triplet state is highly conserved, although there are slight variations in rate. However significant differences are observed in the vibrational spectra and kinetics after adduct formation, which are directly linked to the specific output function of the LOV domain. While the rate of adduct formation varies by only 3.6-fold amongst the proteins, the subsequent large-scale structural changes in the full-length LOV photoreceptors occur over the micro- to sub-millisecond timescales and vary by orders of magnitude depending on the different output function of each LOV domain