Utilizing pHluorin-Tagged Receptors to Monitor Subcellular Localization and Trafficking

Understanding membrane protein trafficking, assembly, and expression requires an approach that differentiates between those residing in intracellular organelles and those localized on the plasma membrane. Traditional fluorescence-based measurements lack the capability to distinguish membrane proteins residing in different organelles. Cutting edge methodologies transcend traditional methods by coupling pH-sensitive fluorophores with total internal reflection fluorescence microscopy (TIRFM). TIRF illumination excites the sample up to approximately 150 nm from the glass-sample interface, thus decreasing background, increasing the signal to noise ratio, and enhancing resolution. The excitation volume in TIRFM encompasses the plasma membrane and nearby organelles such as the peripheral ER. Superecliptic pHluorin (SEP) is a pH sensitive version of GFP. Genetically encoding SEP into the extracellular domain of a membrane protein of interest positions the fluorophore on the luminal side of the ER and in the extracellular region of the cell. SEP is fluorescent when the pH is greater than 6, but remains in an off state at lower pH values. Therefore, receptors tagged with SEP fluoresce when residing in the endoplasmic reticulum (ER) or upon insertion in the plasma membrane (PM) but not when confined to a trafficking vesicle or other organelles such as the Golgi. The extracellular pH can be adjusted to dictate the fluorescence of receptors on the plasma membrane. The difference in fluorescence between TIRF images at neutral and acidic extracellular pH for the same cell corresponds to a relative number of receptors on the plasma membrane. This allows a simultaneous measurement of intracellular and plasma membrane resident receptors. Single vesicle insertion events can also be measured when the extracellular pH is neutral, corresponding to a low pH trafficking vesicle fusing with the plasma membrane and transitioning into a fluorescent state. This versatile technique can be exploited to study localization, expression, and trafficking of membrane proteins

High-precision measurement of the half-life of 62^{62}Ga

The beta-decay half-life of 62Ga has been studied with high precision using on-line mass separated samples. The decay of 62Ga which is dominated by a 0+ to 0+ transition to the ground state of 62Zn yields a half-life of T_{1/2} = 116.19(4) ms. This result is more precise than any previous measurement by about a factor of four or more. The present value is in agreement with older literature values, but slightly disagrees with a recent measurement. We determine an error weighted average value of all experimental half-lives of 116.18(4) ms.Comment: 9 pages, 5 figures, accepted for publication in PR

Women's views of continuity of information provided during and after pregnancy: A qualitative interview study

Straightforward transfer of care from pregnancy to the postpartum period is associated with health benefits and is desired by women worldwide. Underpinning this transfer of care is the sharing of information between healthcare professionals and the provision of consistent information to women. In this qualitative study, two aspects of continuity of information were examined; first the information passed on from midwife to health visitor regarding a woman and her baby before the health visitor meets the woman postnatally and second, the consistency of information received by women from these two healthcare professionals (the main healthcare providers during and after pregnancy in England). To be eligible for the study, women had to have had a baby in England within 12 months prior to the interview. Participants also needed to be able to read and speak English and be over 18 years old. Recruitment of participants was via word of mouth and social media. Twenty-nine mothers were interviewed of whom 19 were first time mothers. The interviews took place in the summer and autumn of 2016 and were transcribed verbatim and analysed using Framework Analysis. Two overarching themes were identified: not feeling listened to and information inconsistencies. Women reported little experience of midwives and health visitors sharing information about their care, forcing women to repeat information. This made women feel not listened to and participants recommended that healthcare professionals share information; prioritising information about labour, mental health, and chronic conditions. Women had mixed experiences regarding receiving information from midwives and health visitors, with examples of both consistent and inconsistent information received. To avoid inconsistent information, joint appointments were recommended. Findings from this study clearly suggest that better communication pathways need to be developed and effectively implemented for midwives and health visitors to improve the care that they provide to women

Order in driven vortex lattices in superconducting Nb films with nanostructured pinning potentials

Driven vortex lattices have been studied in a material with strong pinning, such as Nb films. Samples in which natural random pinning coexists with artificial ordered arrays of defects (submicrometric Ni dots) have been fabricated with different geometries (square, triangular and rectangular). Three different dynamic regimes are found: for low vortex velocities, there is a plastic regime in which random defects frustrate the effect of the ordered array; then, for vortex velocities in the range 1-100 m/s, there is a sudden increase in the interaction between the vortex lattice and the ordered dot array, independent on the geometry. This effect is associated to the onset of quasi long range order in the vortex lattice leading to an increase in the overlap between the vortex lattice and the magnetic dots array. Finally, at larger velocities the ordered array-vortex lattice interaction is suppresed again, in agreement with the behavior found in numerical simulations.Comment: 8 text pages + 4 figure

Flow Induced Organization and Memory of a Vortex Lattice

We report on experiments probing the evolution of a vortex state in response to a driving current in 2H-NbSe$_2$ crystals. By following the vortex motion with fast transport measurements we find that the current enables the system to reorganize and access new configurations. During this process the system exhibits a long-term memory: if the current is turned off the vortices freeze in place remembering their prior motion. When the current is restored the motion resumes where it stopped. The experiments provide evidence for a dynamically driven structural change of the vortex lattice and a corresponding dynamic phase diagram that contains a previously unknown regime where the critical current can be either $increased$ or $decreased$ by applying an appropriate driving current.Comment: 5 pages, 4figure

Quantum dynamics of local phase differences between reservoirs of driven interacting bosons separated by simple aperture arrays

We present a derivation of the effective action for the relative phase of driven, aperture-coupled reservoirs of weakly-interacting condensed bosons from a (3+1)-D microscopic model with local U(1) gauge symmetry. We show that inclusion of local chemical potential and driving velocity fields as a gauge field allows derivation of the hydrodynamic equations of motion for the driven macroscopic phase differences across simple aperture arrays. For a single aperture, the current-phase equation for driven flow contains sinusoidal, linear, and current-bias contributions. We compute the renormalization group (RG) beta function of the periodic potential in the effective action for small tunneling amplitudes and use this to analyze the temperature dependence of the low-energy current-phase relation, with application to the transition from linear to sinusoidal current-phase behavior observed in experiments by Hoskinson et al. \cite{packard} for liquid $^{4}$He driven through nanoaperture arrays. Extension of the microscopic theory to a two-aperture array shows that interference between the microscopic tunneling contributions for individual apertures leads to an effective coupling between apertures which amplifies the Josephson oscillations in the array. The resulting multi-aperture current-phase equations are found to be equivalent to a set of equations for coupled pendula, with microscopically derived couplings.Comment: 16 pages, 5 figures v2: typos corrected, RG phase diagram correcte

Classical information deficit and monotonicity on local operations

We investigate classical information deficit: a candidate for measure of classical correlations emerging from thermodynamical approach initiated in [Phys. Rev. Lett 89, 180402]. It is defined as a difference between amount of information that can be concentrated by use of LOCC and the information contained in subsystems. We show nonintuitive fact, that one way version of this quantity can increase under local operation, hence it does not possess property required for a good measure of classical correlations. Recently it was shown by Igor Devetak, that regularised version of this quantity is monotonic under LO. In this context, our result implies that regularization plays a role of "monotoniser".Comment: 6 pages, revte

Multi-confocal fluorescence correlation spectroscopy in living cells using a complementary metal oxide semiconductor-single photon avalanche diode array

International audienceLiving cells are heterogeneous and rapidly changing biological samples. It is thus desirable to measure molecular concentration and dynamics in many locations at the same time. In this note, we present a multi-confocal setup capable of performing simultaneous fluorescence correlation spectroscopy measurements, by focusing the spots with a spatial light modulator and acquiring data with a monolithic 32 × 32 single-photon avalanche photodiode array. A post-processing method is proposed to correct cross-talk effects between neighboring spots. We demonstrate the applicability of our system by simultaneously measuring the diffusion of free enhanced Green Fluorescent Protein (eGFP) molecules at nine different points in living cells

Nonlinear analysis of spacecraft thermal models

We study the differential equations of lumped-parameter models of spacecraft thermal control. Firstly, we consider a satellite model consisting of two isothermal parts (nodes): an outer part that absorbs heat from the environment as radiation of various types and radiates heat as a black-body, and an inner part that just dissipates heat at a constant rate. The resulting system of two nonlinear ordinary differential equations for the satellite's temperatures is analyzed with various methods, which prove that the temperatures approach a steady state if the heat input is constant, whereas they approach a limit cycle if it varies periodically. Secondly, we generalize those methods to study a many-node thermal model of a spacecraft: this model also has a stable steady state under constant heat inputs that becomes a limit cycle if the inputs vary periodically. Finally, we propose new numerical analyses of spacecraft thermal models based on our results, to complement the analyses normally carried out with commercial software packages.Comment: 29 pages, 4 figure