The interaction between an argon plasma jet excited using microsecond duration voltage pulses and a liquid target was examined using Thomson scattering to quantify the temporal evolution of the electron density and temperature. The electrical resistance between a liquid target and the electrical ground was varied from 1 to [Formula: see text] to mimic different conductivity liquids while the influence of the varying electrical properties on the electron dynamics within the plasma were examined. It was demonstrated that the interaction between the plasma jet and a liquid target grounded via a high resistance resulted in typical dielectric barrier discharge behaviour, with two discharge events per applied voltage pulse. Under such conditions, the electron density and temperature reached a peak of [Formula: see text] and 3.4 eV, respectively; with both rapidly decaying over several hundreds of nanoseconds. For liquid targets grounded via a low resistance, the jet behaviour transitioned to a DC-like discharge, with a single breakdown event being observed and sustained throughout the duration of each applied voltage pulse. Under such conditions, electron densities of [Formula: see text] were detected for several microseconds. The results demonstrate that the electron dynamics in a pulsed argon plasma jet are extremely sensitive to the electrical characteristics of the target, which in the case of water, can evolve during exposure to the plasma.

To examine the ability of retinal pigment epithelial (RPE) cells derived from human embryonic stem cells (HESC) to phagocytose photoreceptor outer segments, and to determine whether exposure to human retina induces any morphological changes in these cells.

Uncoating of the metastable HIV-1 capsid is a tightly regulated disassembly process required for release of the viral cDNA prior to nuclear import. To understand the intrinsic capsid disassembly pathway and how it can be modulated, we have developed a single-particle fluorescence microscopy method to follow the real-time uncoating kinetics of authentic HIV capsids in vitro immediately after permeabilizing the viral membrane. Opening of the first defect in the lattice is the rate-limiting step of uncoating, which is followed by rapid, catastrophic collapse. The capsid-binding inhibitor PF74 accelerates capsid opening but stabilizes the remaining lattice. In contrast, binding of a polyanion to a conserved arginine cluster in the lattice strongly delays initiation of uncoating but does not prevent subsequent lattice disassembly. Our observations suggest that different stages of uncoating can be controlled independently with the interplay between different capsid-binding regulators likely to determine the overall uncoating kinetics.

Two-photon direct laser writing is an additive fabrication process that utilizes two-photon absorption of tightly focused femtosecond laser pulses to implement spatially controlled polymerization of a liquid-phase photoresist. Two-photon direct laser writing is capable of nanofabricating arbitrary three-dimensional structures with nanometer accuracy. Here, we explore direct laser writing for high-resolution optical microscopy by fabricating unique 3D optical fiducials for single-molecule tracking and 3D single-molecule localization microscopy. By having control over the position and three-dimensional architecture of the fiducials, we improve axial discrimination and demonstrate isotropic subnanometer 3D focusing (<0.8 nm) over tens of micrometers using a standard inverted microscope. We perform 3D single-molecule acquisitions over cellular volumes, unsupervised data acquisition and live-cell single-particle tracking with nanometer accuracy.

Efficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme's active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular dynamics simulations identified a secondary CA binding site on CypA that allows a bridging interaction with two hexameric subunits of the assembled CA lattice, leading to capsid stabilization (Liu et al. in Nat Commun 7:10714, 2016).

Exertional dyspnoea is the primary diagnostic symptom for chronic cardiopulmonary disease populations. Whilst a number of exercise tests are used, there remains no gold standard clinical measure of exertional dyspnoea. The aim of this review was to comprehensively describe and evaluate all types of fixed-intensity exercise tests used to assess exertional dyspnoea in chronic cardiopulmonary populations and, where possible, report the reliability and responsiveness of the tests.

PACAP is a critical regulator of long-term catecholamine secretion from the adrenal medulla in vivo, however the receptor or pathways for Ca(2+) entry triggering acute and sustained secretion have not been adequately characterized. We have previously cloned the bovine adrenal chromaffin cell PAC1 receptor that contains the molecular determinants required for PACAP-induced Ca(2+) elevation and is responsible for imparting extracellular Ca(2+) influx-dependent secretory competence in PC12 cells. Here, we use this cell model to gain mechanistic insights into PAC1hop-dependent Ca(2+) pathways responsible for catecholamine secretion. PACAP-modulated extracellular Ca(2+) entry in PC12 cells could be partially blocked with nimodipine, an inhibitor of L-type VGCCs and partially blocked by 2-APB, an inhibitor and modulator of various transient receptor potential (TRP) channels. Despite the co-existence of these two modes of Ca(2+) entry, sustained catecholamine secretion in PC12 cells was exclusively modulated by 2-APB-sensitive Ca(2+) channels. While IP3 generation occurred after PACAP exposure, most PACAP-induced Ca(2+) mobilization involved release from ryanodine-gated cytosolic stores. 2-APB-sensitive Ca(2+) influx, and subsequent catecholamine secretion was however not functionally related to intracellular Ca(2+) mobilization and store depletion. The reconstituted PAC1hop-expessing PC12 cell model therefore recapitulates both PACAP-induced Ca(2+) release from ER stores and extracellular Ca(2+) entry that restores PACAP-induced secretory competence in neuroendocrine cells. We demonstrate here that although bPAC1hop receptor occupancy induces Ca(2+) entry through two independent sources, VGCCs and 2-APB-sensitive channels, only the latter contributes importantly to sustained vesicular catecholamine release that is a fundamental characteristic of this neuropeptide system. These results emphasize the importance of establishing functional linkages between Ca(2+) signaling pathways initiated by pleotrophic signaling molecules such as PACAP, and physiologically important downstream events, such as secretion, triggered by them.

Tropomyosins regulate the dynamics and functions of the actin cytoskeleton by forming long chains along the two strands of actin filaments that act as gatekeepers for the binding of other actin-binding proteins. The fundamental molecular interactions underlying the binding of tropomyosin to actin are still poorly understood. Using microfluidics and fluorescence microscopy, we observed the binding of the fluorescently labeled tropomyosin isoform Tpm1.8 to unlabeled actin filaments in real time. This approach, in conjunction with mathematical modeling, enabled us to quantify the nucleation, assembly, and disassembly kinetics of Tpm1.8 on single filaments and at the single-molecule level. Our analysis suggests that Tpm1.8 decorates the two strands of the actin filament independently. Nucleation of a growing tropomyosin domain proceeds with high probability as soon as the first Tpm1.8 molecule is stabilized by the addition of a second molecule, ultimately leading to full decoration of the actin filament. In addition, Tpm1.8 domains are asymmetrical, with enhanced dynamics at the edge oriented toward the barbed end of the actin filament. The complete description of Tpm1.8 kinetics on actin filaments presented here provides molecular insight into actin-tropomyosin filament formation and the role of tropomyosins in regulating actin filament dynamics.

A key part of any super-resolution technique involves accurately correcting for mechanical motion of the sample and setup during acquisition. If left uncorrected, drift degrades the resolution of the final reconstructed image and can introduce unwanted artifacts. Here, we describe how to implement active stabilization, thereby reducing drift to ~1 nm across all three dimensions. In this protocol, we show how to implement our method on custom and standard microscopy hardware. We detail the construction of a separate illumination and detection path, dedicated exclusively to acquiring the diffraction pattern of fiducials deposited on the imaging slide. We also show how to focus lock and adjust the focus in arbitrary nanometer step size increments. Our real-time focus locking is based on kHz calculations performed using the graphics processing unit. The fast calculations allow for rapid repositioning of the sample, which reduces drift below the photon-limited localization precision. Our approach allows for a single-molecule and/or super-resolution image acquisition free from movement artifacts and eliminates the need for complex algorithms or hardware installations. The method is also useful for long acquisitions which span over hours or days, such as multicolor super resolution. Installation does not require specialist knowledge and can be implemented in standard biological laboratories. The full protocol can be implemented within ~2 weeks.