Hue-shifted monomeric variants of Clavularia cyan fluorescent protein: identification of the molecular determinants of color and applications in fluorescence imaging

<p>Abstract</p> <p>Background</p> <p>In the 15 years that have passed since the cloning of <it>Aequorea victoria </it>green fluorescent protein (avGFP), the expanding set of fluorescent protein (FP) variants has become entrenched as an indispensable toolkit for cell biology research. One of the latest additions to the toolkit is monomeric teal FP (mTFP1), a bright and photostable FP derived from <it>Clavularia </it>cyan FP. To gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. We also employed site-directed and random mutagenesis in combination with library screening to create new hues of mTFP1-derived variants with wavelength-shifted excitation and emission spectra.</p> <p>Results</p> <p>Our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mTFP1 operate independently of the chromophore structure. This conclusion is supported by the observation that the Tyr67Trp and Tyr67His mutants of mTFP1 retain a blue-shifted fluorescence emission relative to their avGFP counterparts (that is, Tyr66Trp and Tyr66His). Based on previous work with close homologs, His197 and His163 are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. Indeed we have identified the substitutions His163Met and Thr73Ala that abolish or disrupt the interactions of these residues with the chromophore. The mTFP1-Thr73Ala/His163Met double mutant has an emission peak that is 23 nm red-shifted from that of mTFP1 itself. Directed evolution of this double mutant resulted in the development of mWasabi, a new green fluorescing protein that offers certain advantages over enhanced avGFP (EGFP). To assess the usefulness of mTFP1 and mWasabi in live cell imaging applications, we constructed and imaged more than 20 different fusion proteins.</p> <p>Conclusion</p> <p>Based on the results of our mutagenesis study, we conclude that the two histidine residues in close proximity to the chromophore are approximately equal determinants of the blue-shifted fluorescence emission of mTFP1. With respect to live cell imaging applications, the mTFP1-derived mWasabi should be particularly useful in two-color imaging in conjunction with a Sapphire-type variant or as a fluorescence resonance energy transfer acceptor with a blue FP donor. In all fusions attempted, both mTFP1 and mWasabi give patterns of fluorescent localization indistinguishable from that of well-established avGFP variants.</p

Probing the momentum relaxation time of charge carriers in ultrathin semiconductor layers

We report on a terahertz time-domain technique for measuring the momentum relaxation time of charge carriers in ultrathin semiconductor layers. The phase sensitive modulation technique directly provides the relaxation time. Time-resolved THz experiments were performed on n-doped GaAs and show precise agreement with data obtained by electrical characterization. The technique is well suited for studying novel materials where parameters such as the charge carriers' effective mass or the carrier density are not known a priori

Economics, Agency, and Causal Explanation

The paper considers three questions. First, what is the connection between economics and agency? It is argued that causation and explanation in economics fundamentally depend on agency. So a philosophical understanding of economic explanation must be sensitive to an understanding of agency. Second, what is the connection between agency and causation? A causal view of agency-involving explanation is defended against a number of arguments from the resurgent noncausalist tradition in the literature on agency and action-explanation. If agency is fundamental to economic explanation, it is argued, then so is causation. Third, what is the connection between causal explanation and the natural sciences? It is argued that, though the explanations given in economics and other social sciences are causal explanations, they are different in kind from the causal explanations of the natural sciences. On the one hand, then, the causal explanations of the social sciences are irreducible to those found in the natural sciences. On the other hand, the causal relations described by the social sciences are not completely autonomous; they do not float free of, or operate independently from, the causal relations charted by the natural sciences

Practical applications of multi-agent systems in electric power systems

The transformation of energy networks from passive to active systems requires the embedding of intelligence within the network. One suitable approach to integrating distributed intelligent systems is multi-agent systems technology, where components of functionality run as autonomous agents capable of interaction through messaging. This provides loose coupling between components that can benefit the complex systems envisioned for the smart grid. This paper reviews the key milestones of demonstrated agent systems in the power industry and considers which aspects of agent design must still be addressed for widespread application of agent technology to occur

New physics searches at near detectors of neutrino oscillation experiments

We systematically investigate the prospects of testing new physics with tau sensitive near detectors at neutrino oscillation facilities. For neutrino beams from pion decay, from the decay of radiative ions, as well as from the decays of muons in a storage ring at a neutrino factory, we discuss which effective operators can lead to new physics effects. Furthermore, we discuss the present bounds on such operators set by other experimental data currently available. For operators with two leptons and two quarks we present the first complete analysis including all relevant operators simultaneously and performing a Markov Chain Monte Carlo fit to the data. We find that these effects can induce tau neutrino appearance probabilities as large as O(10^{-4}), which are within reach of forthcoming experiments. We highlight to which kind of new physics a tau sensitive near detector would be most sensitive.Comment: 20 pages, 2 figures, REVTeX

Beyond power:unbridging Foucault and Weber

Today, very few would doubt that there are plenty of reasons to liken Weber’s and Foucault’s theories of power. Nevertheless, their respective works have divergent ethical and ontological preoccupations which should be reconsidered. This paper explores Foucault’s account of a historical episode in Discipline and Punish and Weber’s theory of life spheres, uncovering evidence that there is a need to reassess the conceptual bridges which have been built so far. The exploration reveals a radical difference between a monological theory of power (Foucault) and a multidimensional approach to power (Weber). Yet by unbridging the two thinkers and focusing on other aspects of their theories along with their ideas about power, we also find that alternative links between the two frameworks may offer a more promising critical theory

A new twist on the geometry of gravitational plane waves

The geometry of twisted null geodesic congruences in gravitational plane wave spacetimes is explored, with special focus on homogeneous plane waves. The role of twist in the relation of the Rosen coordinates adapted to a null congruence with the fundamental Brinkmann coordinates is explained and a generalised form of the Rosen metric describing a gravitational plane wave is derived. The Killing vectors and isometry algebra of homogeneous plane waves (HPWs) are described in both Brinkmann and twisted Rosen form and used to demonstrate the coset space structure of HPWs. The van Vleck-Morette determinant for twisted congruences is evaluated in both Brinkmann and Rosen descriptions. The twisted null congruences of the Ozsvath-Schucking,`anti-Mach' plane wave are investigated in detail. These developments provide the necessary geometric toolkit for future investigations of the role of twist in loop effects in quantum field theory in curved spacetime, where gravitational plane waves arise generically as Penrose limits; in string theory, where they are important as string backgrounds; and potentially in the detection of gravitational waves in astronomy.Comment: 60 pages, 2 figures. Extended version with new material on Rosen geodesics and isometries. Title change

An AI-powered patient triage platform for future viral outbreaks using COVID-19 as a disease model

Over the last century, outbreaks and pandemics have occurred with disturbing regularity, necessitating advance preparation and large-scale, coordinated response. Here, we developed a machine learning predictive model of disease severity and length of hospitalization for COVID-19, which can be utilized as a platform for future unknown viral outbreaks. We combined untargeted metabolomics on plasma data obtained from COVID-19 patients (n = 111) during hospitalization and healthy controls (n = 342), clinical and comorbidity data (n = 508) to build this patient triage platform, which consists of three parts: (i) the clinical decision tree, which amongst other biomarkers showed that patients with increased eosinophils have worse disease prognosis and can serve as a new potential biomarker with high accuracy (AUC = 0.974), (ii) the estimation of patient hospitalization length with ± 5 days error (R2 = 0.9765) and (iii) the prediction of the disease severity and the need of patient transfer to the intensive care unit. We report a significant decrease in serotonin levels in patients who needed positive airway pressure oxygen and/or were intubated. Furthermore, 5-hydroxy tryptophan, allantoin, and glucuronic acid metabolites were increased in COVID-19 patients and collectively they can serve as biomarkers to predict disease progression. The ability to quickly identify which patients will develop life-threatening illness would allow the efficient allocation of medical resources and implementation of the most effective medical interventions. We would advocate that the same approach could be utilized in future viral outbreaks to help hospitals triage patients more effectively and improve patient outcomes while optimizing healthcare resources

Quantum dynamics in strong fluctuating fields

A large number of multifaceted quantum transport processes in molecular systems and physical nanosystems can be treated in terms of quantum relaxation processes which couple to one or several fluctuating environments. A thermal equilibrium environment can conveniently be modelled by a thermal bath of harmonic oscillators. An archetype situation provides a two-state dissipative quantum dynamics, commonly known under the label of a spin-boson dynamics. An interesting and nontrivial physical situation emerges, however, when the quantum dynamics evolves far away from thermal equilibrium. This occurs, for example, when a charge transferring medium possesses nonequilibrium degrees of freedom, or when a strong time-dependent control field is applied externally. Accordingly, certain parameters of underlying quantum subsystem acquire stochastic character. Herein, we review the general theoretical framework which is based on the method of projector operators, yielding the quantum master equations for systems that are exposed to strong external fields. This allows one to investigate on a common basis the influence of nonequilibrium fluctuations and periodic electrical fields on quantum transport processes. Most importantly, such strong fluctuating fields induce a whole variety of nonlinear and nonequilibrium phenomena. A characteristic feature of such dynamics is the absence of thermal (quantum) detailed balance.Comment: review article, Advances in Physics (2005), in pres