CD63 as novel target for nanoemulsion-based F MRI imaging and drug delivery to activated cardiac fibroblasts.

Cardiac fibroblasts are activated following myocardial infarction (MI) and cardiac fibrosis is a major driver of the growing burden of heart failure. A non-invasive targeting method for activated cardiac fibroblasts would be advantageous because of their importance for imaging and therapy. Targeting was achieved by linking a 7-amino acid peptide (EP9) to a perfluorocarbon-containing nanoemulsion (PFC-NE) for visualization by F-combined with H-MRI.

Inhibition of myeloperoxidase to treat left ventricular dysfunction in non-ischaemic cardiomyopathy.

Aims: Non-ischaemic cardiomyopathy (NICMP), an incurable disease terminating in systolic heart failure (heart failure with reduced ejection fraction [HFrEF]), causes immune activation, however anti-inflammatory treatment strategies so far have failed to alter the course of this disease. Myeloperoxidase (MPO), the principal enzyme in neutrophils, has cytotoxic, pro-fibrotic and nitric oxide oxidizing effects. Whether MPO inhibition ameliorates the phenotype in NICMP remains elusive.

RhoGDI1 regulates cell-cell junctions in polarized epithelial cells.

Cell-cell contact formation of polarized epithelial cells is a multi-step process that involves the co-ordinated activities of Rho family small GTPases. Consistent with the central role of Rho GTPases, a number of Rho guanine nucleotide exchange factors (GEFs) and Rho GTPase-activating proteins (GAPs) have been identified at cell-cell junctions at various stages of junction maturation. As opposed to RhoGEFs and RhoGAPs, the role of Rho GDP dissociation inhibitors (GDIs) during cell-cell contact formation is poorly understood.

Power Density Titration of Reversible Photoisomerization of a Fluorescent Protein Chromophore in the Presence of Thermally Driven Barrier Crossing Shown by Quantitative Millisecond Serial Synchrotron X-ray Crystallography.

We present millisecond quantitative serial X-ray crystallography at 1.7 Å resolution demonstrating precise optical control of reversible population transfer from Trans-Cis and Cis-Trans photoisomerization of a reversibly switchable fluorescent protein, rsKiiro. Quantitative results from the analysis of electron density differences, extrapolated structure factors, and occupancy refinements are shown to correspond to optical measurements of photoinduced population transfer and have sensitivity to a few percent in concentration differences.

An electropneumatic cleaning device for piezo-actuator-driven picolitre-droplet dispensers.

Recently, we introduced the liquid application method for time-resolved analyses (LAMA). The time-consuming cleaning cycles required for the substrate solution exchange and storage of the sensitive droplet-dispenser nozzles present practical challenges. In this work, a dispenser cleaning system for the semi-automated cleaning of the piezo-actuator-driven picolitre-droplet dispensers required for LAMA is introduced to streamline typical workflows.

Protective effects of Gα deficiency in a murine heart-failure model of β-adrenoceptor overexpression.

We have shown that in murine cardiomyopathy caused by overexpression of the β-adrenoceptor, Gα-deficiency is detrimental. Given the growing evidence for isoform-specific Gα-functions, we now examined the consequences of Gα deficiency in the same heart-failure model. Mice overexpressing cardiac β-adrenoceptors with (β-tg) or without Gα-expression (β-tg/Gα) were compared to C57BL/6 wildtypes and global Gα-knockouts (Gα).

Millisecond cryo-trapping by the spitrobot crystal plunger simplifies time-resolved crystallography.

We introduce the spitrobot, a protein crystal plunger, enabling reaction quenching via cryo-trapping with a time-resolution in the millisecond range. Protein crystals are mounted on canonical micromeshes on an electropneumatic piston, where the crystals are kept in a humidity and temperature-controlled environment, then reactions are initiated via the liquid application method (LAMA) and plunging into liquid nitrogen is initiated after an electronically set delay time to cryo-trap intermediate states. High-magnification images are automatically recorded before and after droplet deposition, prior to plunging.

Polarity signaling balances epithelial contractility and mechanical resistance.

Epithelia maintain a functional barrier during tissue turnover while facing varying mechanical stress. This maintenance requires both dynamic cell rearrangements driven by actomyosin-linked intercellular adherens junctions and ability to adapt to and resist extrinsic mechanical forces enabled by keratin filament-linked desmosomes. How these two systems crosstalk to coordinate cellular movement and mechanical resilience is not known.

Autophagy regulates neuronal excitability by controlling cAMP/protein kinase A signaling at the synapse.

Autophagy provides nutrients during starvation and eliminates detrimental cellular components. However, accumulating evidence indicates that autophagy is not merely a housekeeping process. Here, by combining mouse models of neuron-specific ATG5 deficiency in either excitatory or inhibitory neurons with quantitative proteomics, high-content microscopy, and live-imaging approaches, we show that autophagy protein ATG5 functions in neurons to regulate cAMP-dependent protein kinase A (PKA)-mediated phosphorylation of a synapse-confined proteome.

Peptide Mass Spectra from Micrometer-Thick Ice Films Produced with Femtosecond Pulses.

We present a cryogenic mass spectrometry protocol with the capability to detect peptides in the attomole dilution range from ice films. Our approach employs femtosecond laser pulses and implements neither substrate modification nor proton donor agents in the aqueous solution, known to facilitate analyte detection in mass spectrometry. In a systematic study, we investigated the impact of temperature, substrate composition, and irradiation wavelength (513 and 1026 nm) on the bradykinin signal onset.

Torsionally broken symmetry assists infrared excitation of biomimetic charge-coupled nuclear motions in the electronic ground state.

The concerted interplay between reactive nuclear and electronic motions in molecules actuates chemistry. Here, we demonstrate that out-of-plane torsional deformation and vibrational excitation of stretching motions in the electronic ground state modulate the charge-density distribution in a donor-bridge-acceptor molecule in solution. The vibrationally-induced change, visualised by transient absorption spectroscopy with a mid-infrared pump and a visible probe, is mechanistically resolved by molecular dynamics simulations.

Two-dimensional confinement for generating thin single crystals for applications in time-resolved electron diffraction and spectroscopy: an intramolecular proton transfer study.

Thin single organic crystals (≤1 μm) with large area (≥100 × 100 μm) are desirable to explore photoinduced processes using ultrafast spectroscopy and electron-diffraction. Here, we present a general method based on spatial confinement to grow such crystals using the prototypical proton transfer system, 1,5-dihydroxyanthraquinone, as an example, and provide the protocol for optically characterizing structural dynamics to enable proper assignments using diffraction methods.

The effect of secondary electrons on radiolysis as observed by in liquid TEM: The role of window material and electrical bias.

The effect of window material on electron beam induced phenomena in liquid phase electron microscopy (LPEM) is an interesting yet under-explored subject. We have studied the differences of electron beam induced gold nanoparticle (AuNP) growth subject to three encapsulation materials: Silicon Nitride (SiN), carbon and formvar. We find SiN liquid cells (LCs) to result in significantly higher AuNP growth yield as compared to LCs employing the other two materials.

Fundamental Flaw in the Current Construction of the TiO Electron Transport Layer of Perovskite Solar Cells and Its Elimination.

The top-performing perovskite solar cells (efficiency > 20%) generally rely on the use of a nanocrystal TiO electron transport layer (ETL). However, the efficacies and stability of the current stereotypically prepared TiO ETLs employing commercially available TiO nanocrystal paste are far from their maximum values. As revealed herein, the long-hidden reason for this discrepancy is that acidic protons (∼0.

Serial femtosecond and serial synchrotron crystallography can yield data of equivalent quality: A systematic comparison.

For the two proteins myoglobin and fluoroacetate dehalogenase, we present a systematic comparison of crystallographic diffraction data collected by serial femtosecond (SFX) and serial synchrotron crystallography (SSX). To maximize comparability, we used the same batch of micron-sized crystals, the same sample delivery device, and the same data analysis software. Overall figures of merit indicate that the data of both radiation sources are of equivalent quality.

Environmental Liquid Cell Technique for Improved Electron Microscopic Imaging of Soft Matter in Solution.

Liquid-phase transmission electron microscopy is a technique for simultaneous imaging of the structure and dynamics of specimens in a liquid environment. The conventional sample geometry consists of a liquid layer tightly sandwiched between two Si3N4 windows with a nominal spacing on the order of 0.5 μm.

Proteomics of Galápagos Marine Iguanas Links Function of Femoral Gland Proteins to the Immune System.

Communication between individuals via molecules, termed chemosignaling, is widespread among animal and plant species. However, we lack knowledge on the specific functions of the substances involved for most systems. The femoral gland is an organ that secretes a waxy substance involved in chemical communication in lizards.

Compact Ho:YLF-pumped ZnGeP-based optical parametric amplifiers tunable in the molecular fingerprint regime.

We report on a compact mid-infrared laser architecture, comprising a chain of $ {\rm ZnGeP}_2 $ZnGeP-based optical parametric amplifiers (OPAs), which afford a higher energy yield ($ \mathbin{\lower.3ex\hbox{$\buildrel \lt \over{\smash{\scriptstyle\sim}\vphantom{_x}}$}} 60\;\unicode{x00B5} {\rm J} $∼<60µJ at 1 kHz) compared to most conventional OPA gain media transparent in the 2-8-µm wavelength range. Specifically, our OPA scheme allows ready tunability in the molecular fingerprint regime and is tailored for strong-field excitation and coherent control of both stretch and bend (or torsional) vibrational modes in molecules.

The HARE chip for efficient time-resolved serial synchrotron crystallography.

Serial synchrotron crystallography (SSX) is an emerging technique for static and time-resolved protein structure determination. Using specifically patterned silicon chips for sample delivery, the `hit-and-return' (HARE) protocol allows for efficient time-resolved data collection. The specific pattern of the crystal wells in the HARE chip provides direct access to many discrete time points.

Murine Epidermal Ceramide Synthase 4 Is a Key Regulator of Skin Barrier Homeostasis.

Epidermal barrier dysfunction is associated with a wide range of highly prevalent inflammatory skin diseases. However, the molecular processes that drive epidermal barrier maintenance are still largely unknown. Here, using quantitative proteomics, lipidomics, and mouse genetics, we characterize epidermal barrier maintenance versus a newly established barrier and functionally identify differential ceramide synthase 4 protein expression as one key difference.

Liquid application method for time-resolved analyses by serial synchrotron crystallography.

We introduce a liquid application method for time-resolved analyses (LAMA), an in situ mixing approach for serial crystallography. Picoliter-sized droplets are shot onto chip-mounted protein crystals, achieving near-full ligand occupancy within theoretical diffusion times. We demonstrate proof-of-principle binding of GlcNac to lysozyme, and resolve glucose binding and subsequent ring opening in a time-resolved study of xylose isomerase.

Time-resolved crystallography reveals allosteric communication aligned with molecular breathing.

A comprehensive understanding of protein function demands correlating structure and dynamic changes. Using time-resolved serial synchrotron crystallography, we visualized half-of-the-sites reactivity and correlated molecular-breathing motions in the enzyme fluoroacetate dehalogenase. Eighteen time points from 30 milliseconds to 30 seconds cover four turnover cycles of the irreversible reaction.

Rapid mixing of colliding picoliter liquid droplets delivered through-space from piezoelectric-actuated pipettes characterized by time-resolved fluorescence monitoring.

Rapid mixing of aqueous solutions is a crucial first step to study the kinetics of fast biochemical reactions with high temporal resolution. Remarkable progress toward this goal has been made through the development of advanced stopped-flow mixing techniques resulting in reduced dead times, and thereby extending reaction monitoring capabilities to numerous biochemical systems. Concurrently, piezoelectric actuators for through-space liquid droplet sample delivery have also been applied in several experimental systems, providing discrete picoliter sample volume delivery and precision sample deposition onto a surface, free of confinement within microfluidic devices, tubing, or other physical constraints.

The hit-and-return system enables efficient time-resolved serial synchrotron crystallography.

We present a 'hit-and-return' (HARE) method for time-resolved serial synchrotron crystallography with time resolution from milliseconds to seconds or longer. Timing delays are set mechanically, using the regular pattern in fixed-target crystallography chips and a translation stage system. Optical pump-probe experiments to capture intermediate structures of fluoroacetate dehalogenase binding to its ligand demonstrated that data can be collected at short (30 ms), medium (752 ms) and long (2,052 ms) intervals.

Multi-octave supercontinuum generation in YAG pumped by mid-infrared, multi-picosecond pulses.

High-energy, multi-octave supercontinuum (SC) generation in bulk media pumped with picosecond pulses in the mid-infrared, though pivotal in a myriad of applications, poses severe constraints due to wavelength scaling of the critical power criterion and the propensity to induce avalanche-ionization-seeded breakdown mechanisms. Here, we demonstrate a simple experimental geometry, relying on a very low numerical aperture for the pump pulse, and a crystal length commensurate with the Rayleigh length of the focusing geometry, generating a multi-octave, stable SC in yttrium aluminum garnet (YAG). The SC ranges from 500 nm to 3.