Hypocrates is a genetically encoded fluorescent biosensor for (pseudo)hypohalous acids and their derivatives

The lack of tools to monitor the dynamics of (pseudo)hypohalous acids in live cells and tissues hinders a better understanding of inflammatory processes. Here we present a fluorescent genetically encoded biosensor, Hypocrates, for the visualization of (pseudo)hypohalous acids and their derivatives. Hypocrates consists of a circularly permuted yellow fluorescent protein integrated into the structure of the transcription repressor NemR from Escherichia coli. We show that Hypocrates is ratiometric, reversible, and responds to its analytes in the 106 M−1s−1 range. Solving the Hypocrates X-ray structure provided insights into its sensing mechanism, allowing determination of the spatial organization in this circularly permuted fluorescent protein-based redox probe. We exemplify its applicability by imaging hypohalous stress in bacteria phagocytosed by primary neutrophils. Finally, we demonstrate that Hypocrates can be utilized in combination with HyPerRed for the simultaneous visualization of (pseudo)hypohalous acids and hydrogen peroxide dynamics in a zebrafish tail fin injury model.

Heme Peroxidases at Unperturbed and Inflamed Mucous Surfaces

In our organism, mucous surfaces are important boundaries against the environmental milieu with defined fluxes of metabolites through these surfaces and specific rules for defense reactions. Major mucous surfaces are formed by epithelia of the respiratory system and the digestive tract. The heme peroxidases lactoperoxidase (LPO), myeloperoxidase (MPO), and eosinophil peroxidase (EPO) contribute to immune protection at epithelial surfaces and in secretions. Whereas LPO is secreted from epithelial cells and maintains microbes in surface linings on low level, MPO and EPO are released from recruited neutrophils and eosinophils, respectively, at inflamed mucous surfaces. Activated heme peroxidases are able to oxidize (pseudo)halides to hypohalous acids and hypothiocyanite. These products are involved in the defense against pathogens, but can also contribute to cell and tissue damage under pathological conditions. This review highlights the beneficial and harmful functions of LPO, MPO, and EPO at unperturbed and inflamed mucous surfaces. Among the disorders, special attention is directed to cystic fibrosis and allergic reactions.

Bleaching the Record: After 200 Years, Single Crystal X-Ray Crystallography Reveals the Structure and Hydrogen-Bonding Properties of Hypochlorite and Hypobromite Ions in the Solid State

<div>We report the first single crystal structures of hypochlorite and hypobromite salts, including hydrated sodium hypochlorite - a ubiquitous bleaching and disinfection agent in use for almost 200 years. The structures represent the first characterization of fundamentally important hypochlorite and hypobromite anions in the solid state, by X-ray crystallography and are supported by Raman spectroscopy on individual crystals. The structural analysis provides insight into supramolecular chemistry of the hypohalite ions in the hydrated environment of the NaOCl^.5H₂O and NaOBr^.5H₂O solid salts, and reveals measured Cl-O and Br-O bond lengths of 1.69 A and 1.82 A, respectively, which are significantly longer than those for corresponding higher-valence oxoanions, and in agreement with the values spectroscopically determined for hypohalous acids and corresponding oxides in the gas phase.<br></div>

Bleaching the Record: After 200 Years, Single Crystal X-Ray Crystallography Reveals the Structure and Hydrogen-Bonding Properties of Hypochlorite and Hypobromite Ions in the Solid State

<div>We report the first single crystal structures of hypochlorite and hypobromite salts, including hydrated sodium hypochlorite - a ubiquitous bleaching and disinfection agent in use for almost 200 years. The structures represent the first characterization of fundamentally important hypochlorite and hypobromite anions in the solid state, by X-ray crystallography and are supported by Raman spectroscopy on individual crystals. The structural analysis provides insight into supramolecular chemistry of the hypohalite ions in the hydrated environment of the NaOCl^.5H₂O and NaOBr^.5H₂O solid salts, and reveals measured Cl-O and Br-O bond lengths of 1.69 A and 1.82 A, respectively, which are significantly longer than those for corresponding higher-valence oxoanions, and in agreement with the values spectroscopically determined for hypohalous acids and corresponding oxides in the gas phase.<br></div>

The Role of Inflammation and Myeloperoxidase-Related Oxidative Stress in the Pathogenesis of Genetically Triggered Thoracic Aortic Aneurysms

Genetically triggered thoracic aortic aneurysms (TAAs) are usually considered to exhibit minimal levels of inflammation. However, emerging data demonstrate that specific features of an inflammatory response can be observed in TAA, and that the extent of the inflammatory response can be correlated with the severity, in both mouse models and in human studies. Myeloperoxidase (MPO) is a key mediator of the inflammatory response, via production of specific oxidative species, e.g., the hypohalous acids. Specific tissue modifications, mediated by hypohalous acids, have been documented in multiple cardiovascular pathologies, including atherosclerosis associated with coronary artery disease, abdominal aortic, and cerebral aneurysms. Similarly, data are now emerging that show the capacity of MPO-derived oxidative species to regulate mechanisms important in TAA pathogenesis, including alterations in extracellular matrix homeostasis, activation of matrix metalloproteinases, induction of endothelial dysfunction and vascular smooth muscle cell phenotypic switching, and activation of ERK1/2 signaling. The weight of evidence supports a role for inflammation in exacerbating the severity of TAA progression, expanding our understanding of the pathogenesis of TAA, identifying potential biomarkers for early detection of TAA, monitoring severity and progression, and for defining potential novel therapeutic targets.

The Role of Thiocyanate in Modulating Myeloperoxidase Activity during Disease

Thiocyanate (SCN−) is a pseudohalide anion omnipresent across mammals and is particularly concentrated in secretions within the oral cavity, digestive tract and airway. Thiocyanate can outcompete chlorine anions and other halides (F−, Br−, I−) as substrates for myeloperoxidase by undergoing two-electron oxidation with hydrogen peroxide. This forms their respective hypohalous acids (HOX where X− = halides) and in the case of thiocyanate, hypothiocyanous acid (HOSCN), which is also a bactericidal oxidative species involved in the regulation of commensal and pathogenic microflora. Disease may dysregulate redox processes and cause imbalances in the oxidative profile, where typically favoured oxidative species, such as hypochlorous acid (HOCl), result in an overabundance of chlorinated protein residues. As such, the pharmacological capacity of thiocyanate has been recently investigated for its ability to modulate myeloperoxidase activity for HOSCN, a less potent species relative to HOCl, although outcomes vary significantly across different disease models. To date, most studies have focused on therapeutic effects in respiratory and cardiovascular animal models. However, we note other conditions such as rheumatic arthritis where SCN− administration may worsen patient outcomes. Here, we discuss the pathophysiological role of SCN− in diseases where MPO is implicated.