Excitation (panel <i>A</i>) and emission (panel <i>B</i>) spectra of RLC–C5–MDCC exchanged into fibers.

<p>Fibers were mounted in a fluorescence cuvette as described in Methods and observed first in a rigor solution followed by a relaxing solution. Intensities are shown in arbitrary units. For excitation spectra the emission was observed at 525nm, and for emission spectra the fibers were excited at 387nm. As can be seen there was a shift in the emission spectrum to shorter wavelengths in the relaxing solution. There was also an increase in absolute intensity, whose magnitude was determined by quantitative epi-fluorescence microscopy as described in Methods. There was no change in the shape of the excitation spectrum.</p

Fraction of the Fluorescent Nucleotides Released Slowly during the Chase Phase.

<p>Fraction of the Fluorescent Nucleotides Released Slowly during the Chase Phase.</p

Gel electrophoresis of fibers and proteins used to quantitate exchange of mutant proteins into the fibers.

<p>Lane 1, 2 myosin; Lane 3,4 RLC-C5 uncut; Lanes 5,6, control unexchanged fibers; lanes 7,8 fibers exchanged with RLC-C44-MSL; lanes 9, 10, fibers exchanged with RLC-C44-MMTS. As can be seen in lanes 7–10 the intensity of the endogenous RLC (LC-2) is diminished with a new band showing the presence of the uncut mutant. Quantitation of the bands showed that the intensity of LC-2 is diminished by 68%, 71%, 73% and 82% in lanes 7–10 respectively. The intensity of the RLC bound to the fibers relative to LC-2 in the controls is 106%, 103%, 96% and 90% in lanes 7–10 respectively. Thus about 70% of the LC-2 in the preparations has been replaced by mutant RLC, with an additional 30% mutant bound non-specifically.</p

The intensity of the fiber fluorescence, relative to the pre-chase value, is plotted as a function of time during the chase phase of the single nucleotide turnover measurements.

<p>The fibers were previously relaxed in 250μM mantATP, and then chased with 4mM ATP. Fiber fluorescence decreases in two phases, a fast phase that is largely over in about 20–30 seconds, followed by a slow phase with a lifetime of minutes. The slow phase arises from the slow release of nucleotides by myosin in the SRX. The top trace (solid squares) shows a control fiber. The middle trace (solid triangles) shows a fiber exchanged with RLC-C6 labeled with MDCC. As shown the slower phase has a decreased population and a faster lifetime, indicating that the SRX has been partially destabilized. The lowest trace (solid circles) shows a fiber exchanged with RLC-C44 labeled with BIMANE. Here the slow phase is completely absent, indicating complete destabilization of the SRX.</p

Changes in the Intensity of the Fluorescence.

<p>Changes in the Intensity of the Fluorescence.</p

The intensity of the fluorescence of fibers exchanged with RLC-C31-MDCC is shown as a function of time.

<p>The fiber starts in rigor. At 120 seconds ATP is added to relax the fiber and populate the SRX. At 450 seconds the ATP is replaced by GTP, which keeps the fiber mechanically relaxed but de-populates the SRX.</p

The structure of the tarantula IHM (3DTP) [9].

<p>(A) The myosin heavy chain of the blocked head is light blue, and that of the free head is dark blue. The essential light chains are in light green and green, and the RLCs are in white and grey in the lower part of the structure. (B) The structure of the RLC domain as seen from the back of panel a, enlarged. Mutants are in yellow and red, C128 residues are highlighted. The first 50 residues in the N-terminus of the RLCs are not shown since they are very mobile and not present in the mouse RLC. (C) A closer view of the two RLC structures, as seen in panel b, with the mutants highlighted.</p

Live Cell Imaging in Microfluidic Device Proves Resistance to Oxygen/Glucose Deprivation in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Analyses of cellular responses to fast oxygen dynamics are challenging and require ad hoc technological solutions, especially when decoupling from liquid media composition is required. In this work, we present a microfluidic device specifically designed for culture analyses with high resolution and magnification objectives, providing full optical access to the cell culture chamber. This feature allows fluorescence-based assays, photoactivated surface chemistry, and live cell imaging under tightly controlled pO₂ environments. The device has a simple design, accommodates three independent cell cultures, and can be employed by users with basic cell culture training in studies requiring fast oxygen dynamics, defined media composition, and in-line data acquisition with optical molecular probes. We apply this technology to produce an oxygen/glucose deprived (OGD) environment and analyze cell mortality in murine and human cardiac cultures. Neonatal rat ventricular cardiomyocytes show an OGD time-dependent sensitivity, resulting in a robust and reproducible 66 ± 5% death rate after 3 h of stress. Applying an equivalent stress to human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) provides direct experimental evidence for fetal-like OGD-resistant phenotype. Investigation on the nature of such phenotype exposed large glycogen deposits. We propose a culture strategy aimed at depleting these intracellular energy stores and concurrently activate positive regulation of aerobic metabolic molecular markers. The observed process, however, is not sufficient to induce an OGD-sensitive phenotype in hiPS-CMs, highlighting defective development of mature aerobic metabolism <i>in vitro</i>

Measuring the SRX in a plate reader.

<p>Fibers labeled with C5-MDCC were minced and pipetted into wells of a 384 well plate. Images were obtained using quantitative epifluorescence at both short and long emission wavelengths. Images were analyzed to determine fiber fluorescence and the ratio of intensities is shown. The first 32 wells show data obtained in the presence of ATP. The average ratio is 1.195 ± 0.014 (st dev), characteristic of the SRX. The next 32 wells show data obtained in rigor, where the ratio is normalized to 1.0 ± 0.013 (st dev). The Z’ factor calculated from the data was 0.57 indicating that a more extensive screen using these methods is realistic.</p

Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability

[[abstract]]Hepatitis B virus (HBV) is a driver of hepatocellular carcinoma, and two viral products, X and large surface antigen (LHBS), are viral oncoproteins. During chronic viral infection, immune-escape mutants on the preS2 region of LHBS (preS2-LHBS) are gain-of-function mutations that are linked to preneoplastic ground glass hepatocytes (GGHs) and early disease onset of hepatocellular carcinoma. Here, we show that preS2-LHBS provoked calcium release from the endoplasmic reticulum (ER) and triggered stored-operated calcium entry (SOCE). The activation of SOCE increased ER and plasma membrane (PM) connections, which was linked by ER- resident stromal interaction molecule-1 (STIM1) protein and PM-resident calcium release- activated calcium modulator 1 (Orai1). Persistent activation of SOCE induced centrosome overduplication, aberrant multipolar division, chromosome aneuploidy, anchorage-independent growth, and xenograft tumorigenesis in hepatocytes expressing preS2- LHBS. Chemical inhibitions of SOCE machinery and silencing of STIM1 significantly reduced centrosome numbers, multipolar division, and xenograft tumorigenesis induced by preS2-LHBS. These results provide the first mechanistic link between calcium homeostasis and chromosome instability in hepatocytes carrying preS2-LHBS. Therefore, persistent activation of SOCE represents a novel pathological mechanism in HBV-mediated hepatocarcinogenesis