The RNA Capping Enzyme Domain in Protein A is Essential for Flock House Virus Replication

The nodavirus flock house virus (FHV) and the alphavirus Semliki Forest virus (SFV) show evolutionarily intriguing similarities in their replication complexes and RNA capping enzymes. In this study, we first established an efficient FHV trans-replication system in mammalian cells, which disjoins protein expression from viral RNA synthesis. Following transfection, FHV replicase protein A was associated with mitochondria, whose outer surface displayed pouch-like invaginations with a ‘neck’ structure opening towards the cytoplasm. In mitochondrial pellets from transfected cells, high-level synthesis of both genomic and subgenomic RNA was detected in vitro and the newly synthesized RNA was of positive polarity. Secondly, we initiated the study of the putative RNA capping enzyme domain in protein A by mutating the conserved amino acids H93, R100, D141, and W215. RNA replication was abolished for all mutants inside cells and in vitro except for W215A, which showed reduced replication. Transfection of capped RNA template did not rescue the replication activity of the mutants. Comparing the efficiency of SFV and FHV trans-replication systems, the FHV system appeared to produce more RNA. Using fluorescent marker proteins, we demonstrated that both systems could replicate in the same cell. This work may facilitate the comparative analysis of FHV and SFV replication.The nodavirus flock house virus (FHV) and the alphavirus Semliki Forest virus (SFV) show evolutionarily intriguing similarities in their replication complexes and RNA capping enzymes. In this study, we first established an efficient FHV trans-replication system in mammalian cells, which disjoins protein expression from viral RNA synthesis. Following transfection, FHV replicase protein A was associated with mitochondria, whose outer surface displayed pouch-like invaginations with a 'neck' structure opening towards the cytoplasm. In mitochondrial pellets from transfected cells, high-level synthesis of both genomic and subgenomic RNA was detected in vitro and the newly synthesized RNA was of positive polarity. Secondly, we initiated the study of the putative RNA capping enzyme domain in protein A by mutating the conserved amino acids H93, R100, D141, and W215. RNA replication was abolished for all mutants inside cells and in vitro except for W215A, which showed reduced replication. Transfection of capped RNA template did not rescue the replication activity of the mutants. Comparing the efficiency of SFV and FHV trans-replication systems, the FHV system appeared to produce more RNA. Using fluorescent marker proteins, we demonstrated that both systems could replicate in the same cell. This work may facilitate the comparative analysis of FHV and SFV replication.Peer reviewe

Intramolecular monomer-on-monomer (MoM) mitsunobu cyclization for the synthesis of benzofused thiadiazepine-dioxides

The utilization of a monomer-on-monomer (MoM) intramolecular Mitsunobu cyclization reaction employing norbornenyl-tagged (Nb-tagged) reagents is reported for the synthesis of benzofused thiadiazepine-dioxides. Facile purification was achieved via ring-opening metathesis (ROM) polymerization initiated by one of three metathesis catalyst methods: (i) free metathesis catalyst, (ii) surface-initiated catalyst-armed silica, or (iii) surface-initiated catalyst-armed Co/C magnetic nanoparticles

Individual differences in anxiety and automatic amygdala response to fearful faces: A replication and extension of Etkin et al. (2004)

Trait anxiety refers to the stable tendency to attend to threats and experience fears and worries across many situations. According to the widely noticed, pioneering investigation by Etkin et al. (2004) trait anxiety is strongly associated with reactivity in the right basolateral amygdala to non-conscious threat. Although this observation was based on a sample of only 17 individuals, no replication effort has been reported yet. We reexamined automatic amygdala responsiveness as a function of anxiety in a large sample of 107 participants. Besides self-report instruments, we administered an indirect test to assess implicit anxiety. To assess early, automatic stages of emotion processing, we used a color-decision paradigm presenting brief (33 ms) and backward-masked fearful facial expressions. N = 56 participants were unaware of the presence of masked faces. In this subset of unaware participants, the relationship between trait anxiety and basolateral amygdala activation by fearful faces was successfully replicated in region of interest analyses. Additionally, a relation of implicit anxiety with masked fear processing in the amygdala and temporal gyrus was observed. We provide evidence that implicit measures of affect can be valuable predictors of automatic brain responsiveness and may represent useful additions to explicit measures. Our findings support a central role of amygdala reactivity to non-consciously perceived threat in understanding and predicting dispositional anxiety, i.e. the frequency of spontaneously occurring anxiety in everyday life

Developing mHealth Apps with researchers: multi-stakeholder design considerations

The authors have been involved with developing a number of mHealth smartphone Apps for use in health or wellness research in collaboration with researchers, clinicians and patient groups for clinical areas including Sickle Cell Disease, Attention Deficit Hyperactivity Disorder, asthma and infertility treatment. In these types of applications, end-users self-report their symptoms and quality of life or conduct psychometric tests. Physiological data may also be captured using sensors that are internal or external to the device. Following a discussion of the multiple stakeholders that are typically involved in small scale research projects involving end-user data collection, four Apps are used as case studies to explore the issue of non-functional requirements

Development of a GEM-TPC prototype

The use of GEM foils for the amplification stage of a TPC instead of a con- ventional MWPC allows one to bypass the necessity of gating, as the backdrift is suppressed thanks to the asymmetric field configuration. This way, a novel continuously running TPC, which represents one option for the PANDA central tracker, can be realized. A medium sized prototype with a diameter of 300 mm and a length of 600 mm will be tested inside the FOPI spectrometer at GSI using a carbon or lithium beam at intermediate energies (E = 1-3AGeV). This detector test under realistic experimental conditions should allow us to verify the spatial resolution for single tracks and the reconstruction capability for displaced vertexes. A series of physics measurement implying pion beams is scheduled with the FOPI spectrometer together with the GEM-TPC as well.Comment: 5 pages, 4 figures, Proceedings for 11th ICATTP conference in como (italy

Dendritic calcium signals in rhesus macaque motor cortex drive an optical brain-computer interface

Calcium imaging is a powerful tool for recording from large populations of neurons in vivo. Imaging in rhesus macaque motor cortex can enable the discovery of fundamental principles of motor cortical function and can inform the design of next generation brain-computer interfaces (BCIs). Surface two-photon imaging, however, cannot presently access somatic calcium signals of neurons from all layers of macaque motor cortex due to photon scattering. Here, we demonstrate an implant and imaging system capable of chronic, motion-stabilized two-photon imaging of neuronal calcium signals from macaques engaged in a motor task. By imaging apical dendrites, we achieved optical access to large populations of deep and superficial cortical neurons across dorsal premotor (PMd) and gyral primary motor (M1) cortices. Dendritic signals from individual neurons displayed tuning for different directions of arm movement. Combining several technical advances, we developed an optical BCI (oBCI) driven by these dendritic signalswhich successfully decoded movement direction online. By fusing two-photon functional imaging with CLARITY volumetric imaging, we verified that many imaged dendrites which contributed to oBCI decoding originated from layer 5 output neurons, including a putative Betz cell. This approach establishes new opportunities for studying motor control and designing BCIs via two photon imaging

Acousto-optical Scanning-Based High-Speed 3D Two-Photon Imaging In Vivo.

Recording of the concerted activity of neuronal assemblies and the dendritic and axonal signal integration of downstream neurons pose different challenges, preferably a single recording system should perform both operations. We present a three-dimensional (3D), high-resolution, fast, acousto-optic two-photon microscope with random-access and continuous trajectory scanning modes reaching a cubic millimeter scan range (now over 950 × 950 × 3000 μm3) which can be adapted to imaging different spatial scales. The resolution of the system allows simultaneous functional measurements in many fine neuronal processes, even in dendritic spines within a central core (>290 × 290 × 200 μm3) of the total scanned volume. Furthermore, the PSF size remained sufficiently low (PSFx < 1.9 μm, PSFz < 7.9 μm) to target individual neuronal somata in the whole scanning volume for simultaneous measurement of activity from hundreds of cells. The system contains new design concepts: it allows the acoustic frequency chirps in the deflectors to be adjusted dynamically to compensate for astigmatism and optical errors; it physically separates the z-dimension focusing and lateral scanning functions to optimize the lateral AO scanning range; it involves a custom angular compensation unit to diminish off-axis angular dispersion introduced by the AO deflectors, and it uses a high-NA, wide-field objective and high-bandwidth custom AO deflectors with large apertures. We demonstrate the use of the microscope at different spatial scales by first showing 3D optical recordings of action potential back propagation and dendritic Ca2+ spike forward propagation in long dendritic segments in vitro, at near-microsecond temporal resolution. Second, using the same microscope we show volumetric random-access Ca2+ imaging of spontaneous and visual stimulation-evoked activity from hundreds of cortical neurons in the visual cortex in vivo. The selection of active neurons in a volume that respond to a given stimulus was aided by the real-time data analysis and the 3D interactive visualization accelerated selection of regions of interest