Enhancing single-molecule photostability by optical feedback from quantum-jump detection

We report an optical technique that yields an enhancement of single-molecule photostability, by greatly suppressing photobleaching pathways which involve photoexcitation from the triplet state. This is accomplished by dynamically switching off the excitation laser when a quantum-jump of the molecule to the triplet state is optically detected. This procedure leads to a lengthened single-molecule observation time and an increased total number of detected photons. The resulting improvement in photostability unambiguously confirms the importance of photoexcitation from the triplet state in photobleaching dynamics, and may allow the investigation of new phenomena at the single-molecule level

Automatic covariate selection in logistic models for chest pain diagnosis: A new approach

A newly established method for optimizing logistic models via a minorization-majorization procedure is applied to the problem of diagnosing acute coronary syndromes (ACS). The method provides a principled approach to the selection of covariates which would otherwise require the use of a suboptimal method owing to the size of the covariate set. A strategy for building models is proposed and two models optimized for performance and for simplicity are derived via ten-fold cross-validation. These models confirm that a relatively small set of covariates including clinical and electrocardiographic features can be used successfully in this task. The performance of the models is comparable with previously published models using less principled selection methods. The models prove to be portable when tested on data gathered from three other sites. Whilst diagnostic accuracy and calibration diminishes slightly for these new settings, it remains satisfactory overall. The prospect of building predictive models that are as simple as possible for a required level of performance is valuable if data-driven decision aids are to gain wide acceptance in the clinical situation owing to the need to minimize the time taken to gather and enter data at the bedside

Tissue-specific expression of high-voltage-activated dihydropyridine-sensitive L-type calcium channels

The cloning of the cDNA for the α1 subunit of L-type calcium channels revealed that at least two genes (CaCh1 and CaCh2) exist which give rise to several splice variants. The expression of mRNA for these α1 subunits and the skeletal muscle α2/δ, β and γ subunits was studied in rabbit tissues and BC3H1 cells. Nucleic-acid-hybridization studies showed that the mRNA of all subunits are expressed in skeletal muscle, brain, heart and aorta. However, the α1-, β- and γ-specific transcripts had different sizes in these tissues. Smooth muscle and heart contain different splice variants of the CaCh2 gene. The α1, β and γ mRNA are expressed together in differentiated but not in proliferating BC3H1 cells. A probe specific for the skeletal muscle α2/δ subunit did not hybridize to poly(A)-rich RNA from BC3H1 cells. These results suggest that different splice variants of the genes for the α1, β and γ subunits exist in tissues containing L-type calcium channels, and that their expression is regulated in a coordinate manner

Conditional Knockout of NMDA Receptors in Dopamine Neurons Prevents Nicotine-Conditioned Place Preference

Nicotine from smoking tobacco produces one of the most common forms of addictive behavior and has major societal and health consequences. It is known that nicotine triggers tobacco addiction by activating nicotine acetylcholine receptors (nAChRs) in the midbrain dopaminergic reward system, primarily via the ventral tegmental area. Heterogeneity of cell populations in the region has made it difficult for pharmacology-based analyses to precisely assess the functional significance of glutamatergic inputs to dopamine neurons in nicotine addiction. By generating dopamine neuron-specific NR1 knockout mice using cre/loxP-mediated method, we demonstrate that genetic inactivation of the NMDA receptors in ventral tegmental area dopamine neurons selectively prevents nicotine-conditioned place preference. Interestingly, the mutant mice exhibit normal performances in the conditioned place aversion induced by aversive air puffs. Therefore, this selective effect on addictive drug-induced reinforcement behavior suggests that NMDA receptors in the dopamine neurons are critical for the development of nicotine addiction

Optogenetics and deep brain stimulation neurotechnologies

Brain neural network is composed of densely packed, intricately wired neurons whose activity patterns ultimately give rise to every behavior, thought, or emotion that we experience. Over the past decade, a novel neurotechnique, optogenetics that combines light and genetic methods to control or monitor neural activity patterns, has proven to be revolutionary in understanding the functional role of specific neural circuits. We here briefly describe recent advance in optogenetics and compare optogenetics with deep brain stimulation technology that holds the promise for treating many neurological and psychiatric disorders

Virtual finger boosts three-dimensional imaging and microsurgery as well as terabyte volume image visualization and analysis

Three-dimensional (3D) bioimaging, visualization and data analysis are in strong need of powerful 3D exploration techniques. We develop virtual finger (VF) to generate 3D curves, points and regions-of-interest in the 3D space of a volumetric image with a single finger operation, such as a computer mouse stroke, or click or zoom from the 2D-projection plane of an image as visualized with a computer. VF provides efficient methods for acquisition, visualization and analysis of 3D images for roundworm, fruitfly, dragonfly, mouse, rat and human. Specifically, VF enables instant 3D optical zoom-in imaging, 3D free-form optical microsurgery, and 3D visualization and annotation of terabytes of whole-brain image volumes. VF also leads to orders of magnitude better efficiency of automated 3D reconstruction of neurons and similar biostructures over our previous systems. We use VF to generate from images of 1,107 Drosophila GAL4 lines a projectome of a Drosophila brain. © 2014 Macmillan Publishers Limited. All rights reserved

“cAMP Sponge”: A Buffer for Cyclic Adenosine 3′, 5′-Monophosphate

Background: While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP). Methods/Principal Findings: Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit $RI\beta$ of protein kinase A (PKA). Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named “cAMP sponge”) was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets. Conclusions: This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events