CardiLect: A combined cross-species lectin histochemistry protocol for the automated analysis of cardiac remodelling.

Background: Cardiac remodelling, a crucial aspect of heart failure, is commonly investigated in preclinical models by quantifying cardiomyocyte cross-sectional area (CSA) and microvascular density (MVD) via histological methods, such as immunohistochemistry. To achieve this, optimized protocols are needed, and the species specificity is dependent on the antibody used. Lectin histochemistry offers several advantages compared to antibody-based immunohistochemistry, including as cost-effectiveness and cross-species applicability.

Addressing burning questions on axon regeneration.

Regeneration of sensory axons after a burn injury depends on early keratinocyte responses regulated by the wound microenvironment.

A genetically encoded sensor for visualizing leukotriene B4 gradients in vivo.

Leukotriene B (LTB) is a potent lipid chemoattractant driving inflammatory responses during host defense, allergy, autoimmune and metabolic diseases. Gradients of LTB orchestrate leukocyte recruitment and swarming to sites of tissue damage and infection. How LTB gradients form and spread in live tissues to regulate these processes remains largely elusive due to the lack of suitable tools for monitoring LTB levels in vivo.

Hydrogen peroxide production by epidermal dual oxidase 1 regulates nociceptive sensory signals.

Keratinocytes of the mammalian skin provide not only mechanical protection for the tissues, but also transmit mechanical, chemical, and thermal stimuli from the external environment to the sensory nerve terminals. Sensory nerve fibers penetrate the epidermal basement membrane and function in the tight intercellular space among keratinocytes. Here we show that epidermal keratinocytes produce hydrogen peroxide upon the activation of the NADPH oxidase dual oxidase 1 (DUOX1).

Optimization of the Heterologous Expression of the Cannabinoid Type-1 (CB) Receptor.

The G protein-coupled type 1 cannabinoid receptor (CBR) mediates virtually all classic cannabinoid effects, and both its agonists and antagonists hold major therapeutic potential. Heterologous expression of receptors is vital for pharmacological research, however, overexpression of these proteins may fundamentally alter their localization pattern, change the signalling partner preference and may also spark artificial clustering. Additionally, recombinant CBRs are prone to intense proteasomal degradation, which may necessitate substantial modifications, such as N-terminal truncation or signal sequence insertion, for acceptable cell surface expression.

A Photo-clickable ATP-Mimetic Reveals Nucleotide Interactors in the Membrane Proteome.

ATP is an important energy metabolite and allosteric signal in health and disease. ATP-interacting proteins, such as P2 receptors, control inflammation, cell death, migration, and wound healing. However, identification of allosteric ATP sites remains challenging, and our current inventory of ATP-controlled pathways is likely incomplete.

Quantitative Imaging of Endogenous and Exogenous HO Gradients in Live Zebrafish Larvae.

Quantitative aspects of extracellular HO signaling in animals, such as its spatiotemporal dynamics within tissues, remain little understood. Here we detail an optimized, experimental setup for measuring the dynamics and physiological consequences of extracellular HO application to live tissues by intravital biosensor imaging in zebrafish larvae.

Imaging Intracellular HO with the Genetically Encoded PerFRET and OxyFRET Probes.

Hydrogen peroxide (HO) is an important signaling intermediate with various regulatory and effector functions. Despite its significance, the subcellular organization of HO signals is poorly understood. Introducing novel techniques for the intracellular detection of HO would be essential for a more complete understanding of its role in cellular signaling.

Live imaging of leukocyte recruitment in a zebrafish model of chemical liver injury.

Studying early immune responses to organ damage in situ requires animal models amenable to intravital imaging. Here, we used transparent zebrafish larvae, a powerful animal model for innate immunity, to measure leukocyte recruitment to damaged livers. Bath application of metronidazole (Mtz) to fish expressing nitroreductase (NTR) under a liver-specific promoter damaged the organ within 24 hours causing oxidative stress, distorted liver morphology, accumulation of TUNEL-positive cells, and transcriptional upregulation of apoptotic and antioxidant genes.

Peroxidasin-mediated crosslinking of collagen IV is independent of NADPH oxidases.

Collagen IV is a major component of the basement membrane in epithelial tissues. The NC1 domains of collagen IV protomers are covalently linked together through sulfilimine bonds, the formation of which is catalyzed by peroxidasin. Although hydrogen peroxide is essential for this reaction, the exact source of the oxidant remains elusive.

Interaction between p22 and Nox4 in the endoplasmic reticulum suggests a unique mechanism of NADPH oxidase complex formation.

The p22 protein is an essential component of the phagocytic- and inner ear NADPH oxidases but its relationship to other Nox proteins is less clear. We have studied the role of p22 in the TGF-β1-stimulated HO production of primary human and murine fibroblasts. TGF-β1 induced HO release of the examined cells, and the response was dependent on the expression of both Nox4 and p22.

Image-Based Measurement of HO Reaction-Diffusion in Wounded Zebrafish Larvae.

Epithelial injury induces rapid recruitment of antimicrobial leukocytes to the wound site. In zebrafish larvae, activation of the epithelial NADPH oxidase Duox at the wound margin is required early during this response. Before injury, leukocytes are near the vascular region, that is, ∼100-300 μm away from the injury site.

Nuclear membrane stretch and its role in mechanotransduction.

Most research in nuclear mechanotransduction has focused on the nuclear lamina and lamin binding proteins. These structures provide mechanical stability to the nucleus, establish a link between the cytoskeleton and chromatin, and can transmit mechanical signals. At the same time, mechanical perturbations to the nucleus also affect its phospholipid membranes.

A Case for the Nuclear Membrane as a Mechanotransducer.

The cell nucleus is becoming increasingly recognized as a mechanosensitive organelle. Most research on nuclear mechanosignaling focuses on the nuclear lamina and coupled actin structures. In this commentary, we discuss the possibility that the nuclear membrane senses and transduces mechanical signals similar to the plasma membrane.

Redox Nanodomains Are Induced by and Control Calcium Signaling at the ER-Mitochondrial Interface.

The ER-mitochondrial interface is central to calcium signaling, organellar dynamics, and lipid biosynthesis. The ER and mitochondrial membranes also host sources and targets of reactive oxygen species (ROS), but their local dynamics and relevance remained elusive since measurement and perturbation of ROS at the organellar interface has proven difficult. Employing drug-inducible synthetic ER-mitochondrial linkers, we overcame this problem and demonstrate that the ER-mitochondrial interface hosts a nanodomain of H2O2, which is induced by cytoplasmic [Ca(2+)] spikes and exerts a positive feedback on calcium oscillations.

The Cell Nucleus Serves as a Mechanotransducer of Tissue Damage-Induced Inflammation.

Tissue damage activates cytosolic phospholipase A2 (cPLA2), releasing arachidonic acid (AA), which is oxidized to proinflammatory eicosanoids by 5-lipoxygenase (5-LOX) on the nuclear envelope. How tissue damage is sensed to activate cPLA2 is unknown. We investigated this by live imaging in wounded zebrafish larvae, where damage of the fin tissue causes osmotic cell swelling at the wound margin and the generation of a chemotactic eicosanoid signal.

Hyperspectral Microscopy of Near-Infrared Fluorescence Enables 17-Chirality Carbon Nanotube Imaging.

The intrinsic near-infrared photoluminescence (fluorescence) of single-walled carbon nanotubes exhibits unique photostability, narrow bandwidth, penetration through biological media, environmental sensitivity, and both chromatic variety and range. Biomedical applications exploiting this large family of fluorophores will require the spectral and spatial resolution of individual (n,m) nanotube species' fluorescence and its modulation within live cells and tissues, which is not possible with current microscopy methods. We present a wide-field hyperspectral approach to spatially delineate and spectroscopically measure single nanotube fluorescence in living systems.

Mechanisms of epithelial wound detection.

Efficient wound healing requires the coordinated responses of various cell types within an injured tissue. To react to the presence of a wound, cells have to first detect it. Judging from their initial biochemical and morphological responses, many cells including leukocytes, epithelial cells, and endothelial cells detect wounds from over hundreds of micrometers within seconds-to-minutes.

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide.

The endoplasmic reticulum (ER) is a metabolically active organelle, which has a central role in proteostasis by translating, modifying, folding, and occasionally degrading secretory and membrane proteins. The lumen of the ER represents a separate compartment of the eukaryotic cell, with a characteristic proteome and metabolome. Although the redox metabolome and proteome of the compartment have not been holistically explored, it is evident that proper redox conditions are necessary for the functioning of many luminal pathways.

Osmotic surveillance mediates rapid wound closure through nucleotide release.

Osmotic cues from the environment mediate rapid detection of epithelial breaches by leukocytes in larval zebrafish tail fins. Using intravital luminescence and fluorescence microscopy, we now show that osmolarity differences between the interstitial fluid and the external environment trigger ATP release at tail fin wounds to initiate rapid wound closure through long-range activation of basal epithelial cell motility. Extracellular nucleotide breakdown, at least in part mediated by ecto-nucleoside triphosphate diphosphohydrolase 3 (Entpd3), restricts the range and duration of osmotically induced cell migration after injury.

Tissue damage detection by osmotic surveillance.

How tissue damage is detected to induce inflammatory responses is unclear. Most studies have focused on damage signals released by cell breakage and necrosis. Whether tissues use other cues in addition to cell lysis to detect that they are damaged is unknown.

H2O2: a chemoattractant?

H2O2 is a relatively stable, rapidly diffusing reactive oxygen species that has been recently implicated as a mediator of leukocyte recruitment to epithelial wounds and transformed cells in zebrafish. Whether H2O2 activates the innate immune response by acting as a bona fide chemoattractant, enhancing chemoattractant sensing, or triggering production of other chemoattractive ligands remains largely unclear. Here, we describe the basic experimental procedures required to study these questions.

Spatial and temporal analysis of NADPH oxidase-generated hydrogen peroxide signals by novel fluorescent reporter proteins.

Aims: Hydrogen peroxide (H2O2) is an emerging signaling molecule with diverse regulatory functions. Despite its significance, the spatial and temporal organization of H2O2 signals within cells is basically unknown. Our limited knowledge about H2O2 signals is largely due to the lack of appropriate techniques for measuring intracellular H2O2.

The effect of OPA1 on mitochondrial Ca²⁺ signaling.

The dynamin-related GTPase protein OPA1, localized in the intermembrane space and tethered to the inner membrane of mitochondria, participates in the fusion of these organelles. Its mutation is the most prevalent cause of Autosomal Dominant Optic Atrophy. OPA1 controls the diameter of the junctions between the boundary part of the inner membrane and the membrane of cristae and reduces the diffusibility of cytochrome c through these junctions.

Urothelial cells produce hydrogen peroxide through the activation of Duox1.

Hydrogen peroxide (H(2)O(2)) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H(2)O(2) to kill pathogens, and epithelial cells of large airways have also been reported to produce H(2)O(2) for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H(2)O(2) in response to a calcium signal.