Transient radical species and oxygen colorimetric indicators grounded on phenothiazinium dyes.

This study investigates four phenothiazinium dyes including methylene blue (MB) and three analogues containing auxochrome variation 2-4 as oxygen colorimetric indicators prepared by their incorporation into two types of oxygen permeable materials containing: a) polyvinyl alcohol substrate with additional TiO2 photocatalysts (PhOxIn), and b) carboxymethylcellulose polymer matrix containing glucose and KOH (ChOxIn). In vacuum packages where volumetric concentrations of oxygen were below 0.01 %, the leuco forms of the tested phenothiazinum dyes were readily oxidised, initiating the colour turn from white to blue. The redox processes involved were explored by experimental electron paramagnetic resonance EPR and UV-vis spectroscopic methods and further supported by theoretical quantum chemistry. The EPR experiments showed that the chemical oxidation of the leuco-form of the phenothiazinium dyes 1-4 produced transient aminyl radical species with the single electron mainly located at the nitrogen atom of the heterocyclic core. The formation of these transient aminyl species was also perceived by UV-vis spectroscopy, their absorption maxima situated in the 552-592 nm range being also supported by TD-DFT theoretical calculations.

A biomimetic assay for antioxidant reactivity, based on liposomes and myoglobin.

Antioxidant assays are typically based on non-physiologically relevant reagents. We describe here a quantitative assay based on the inhibition of the liposome autooxidation in the presence of myoglobin (ILA-Mb), an oxidative process with direct biomedical relevance. Additional advantages of the assay include the use of standard and readily available reagents (lecithin and myoglobin) and the applicability to lipophilic antioxidants. The ILA-Mb assay is based on previously reported qualitative or semi-quantitative ones that employed cytochrome c instead of myoglobin. A number of antioxidants are tested, and their IC50 parameters are discussed and interpreted to involve direct interaction with both myoglobin and the liposomes.

Decarbonylation Products of Binuclear Methylphosphinidene Complexes of Cyclopentadienyliron Carbonyls: Triplet and Quintet Structures Are Favored Energetically over Singlet Structures with Iron-Iron Multiple Bonding.

The structures, energetics, and energetically preferred spin states of methylphosphinidene-bridged binuclear cyclopentadienyliron carbonyl complexes MePFe2(CO)nCp2 (n = 4, 3, 2, and 1) related to the experimentally known (µ-RP)Fe2(µ-CO)(CO)2Cp2 (R = cyclohexyl, phenyl, mesityl, and 2,4,6-tBu3C6H2) complexes have been investigated by density functional theory. Singlet structures having a pyramidal pseudotetrahedral phosphorus environment with 18-electron iron configurations are energetically preferred in the tricarbonyl and tetracarbonyl systems MePFe2(CO)nCp2 (n = 4 and 3) with the lowest energy structures of the tricarbonyl very closely resembling the experimentally determined structures. For the more unsaturated dicarbonyl and monocarbonyl systems MePFe2(CO)nCp2 (n = 2 and 1), higher spin state triplet and quintet structures are energetically preferred over singlet structures. These more highly unsaturated structures can be derived from the lowest energy singlet MePFe2(CO)nCp2 (n = 4, 3) by the removal of carbonyl groups. The iron atoms giving up carbonyl groups in their 16- and 14-electron configurations bear the spin density of the unpaired electrons in the higher spin states. The lowest energy singlet structure of the monocarbonyl MePFe2(CO)Cp2, although a relatively high energy isomer, is unusual among the collection of MePFe2(CO)nCp2 (n = 4, 3, 2, and 1) structures by having both the formal Fe=Fe double bond and the four-electron donor MeP unit with the planar phosphorus coordination required to allow each of its iron atoms to attain the favored 18-electron configuration.

Redox Reactivity of Nonsymbiotic Phytoglobins towards Nitrite.

Nonsymbiotic phytoglobins (nsHbs) are a diverse superfamily of hemoproteins grouped into three different classes (1, 2, and 3) based on their sequences. Class 1 Hb are expressed under hypoxia, osmotic stress, and/or nitric oxide exposure, while class 2 Hb are induced by cold stress and cytokinins. Both are mainly six-coordinated. The deoxygenated forms of the class 1 and 2 nsHbs from A. thaliana (AtHb1 and AtHb2) are able to reduce nitrite to nitric oxide via a mechanism analogous to other known globins. NsHbs provide a viable pH-dependent pathway for NO generation during severe hypoxia via nitrite reductase-like activity with higher rate constants compared to mammalian globins. These high kinetic parameters, along with the relatively high concentrations of nitrite present during hypoxia, suggest that plant hemoglobins could indeed serve as anaerobic nitrite reductases in vivo. The third class of nsHb, also known as truncated hemoglobins, have a compact 2/2 structure and are pentacoordinated, and their exact physiological role remains mostly unknown. To date, no reports are available on the nitrite reductase activity of the truncated AtHb3. In the present work, three representative nsHbs of the plant model Arabidopsis thaliana are presented, and their nitrite reductase-like activity and involvement in nitrosative stress is discussed. The reaction kinetics and mechanism of nitrite reduction by nsHbs (deoxy and oxy form) at different pHs were studied by means of UV-Vis spectrophotometry, along with EPR spectroscopy. The reduction of nitrite requires an electron supply, and it is favored in acidic conditions. This reaction is critically affected by molecular oxygen, since oxyAtHb will catalyze nitric oxide deoxygenation. The process displays unique autocatalytic kinetics with metAtHb and nitrate as end-products for AtHb1 and AtHb2 but not for the truncated one, in contrast with mammalian globins.

The Iodine/Iodide/Starch Supramolecular Complex.

The nature of the blue color in the iodine-starch reaction (or, in most cases, iodine-iodide-starch reaction, i.e., I2 as well as I- are typically present) has for decades elicited debate. The intensity of the color suggests a clear charge-transfer nature of the band at ~600 nm, and there is consensus regarding the fact that the hydrophobic interior of the amylose helix is the location where iodine binds. Three types of possible sources of charge transfer have been proposed: (1) chains of neutral I2 molecules, (2) chains of poly-iodine anions (complicated by the complex speciation of the I2-I- mixture), or (3) mixtures of I2 molecules and iodide or polyiodide anions. An extended literature review of the topic is provided here. According to the most recent data, the best candidate for the "blue complex" is an I2-I5--I2 unit, which is expected to occur in a repetitive manner inside the amylose helix.

Stachys Species: Comparative Evaluation of Phenolic Profile and Antimicrobial and Antioxidant Potential.

This study aimed to investigate the polyphenolic composition and antioxidant and antimicrobial potential of six Romanian Stachys species: S. officinalis, S. germanica, S. byzantina, S. sylvatica, S. palustris, and S. recta. The LC-MS/MS method was used to analyze the polyphenolic profile, while the phenolic contents were spectrophotometrically determined. The antioxidant activity was evaluated using the following methods: DPPH, FRAP, nitrite-induced autooxidation of hemoglobin, inhibition of cytochrome c-catalyzed lipid peroxidation, and electron paramagnetic resonance spectroscopy. The in vitro antimicrobial properties were assessed using agar-well diffusion, broth microdilution, and antibiofilm assays. Fifteen polyphenols were identified using LC-MS and chlorogenic acid was the major component in all the samples (1131.8-6761.4 µg/g). S. germanica, S. palustris, and S. byzantina extracts each displayed an intense antiradical action in relation to high contents of TPC (6.40 mg GAE/mL), flavonoids (3.90 mg RE/mL), and caffeic acid derivatives (0.89 mg CAE/mL). In vitro antimicrobial and antibiofilm properties were exhibited towards Candida albicans, Gram-positive and Gram-negative strains, with the most intense efficacy recorded for S. germanica and S. byzantina when tested against S. aureus. These results highlighted Stachys extracts as rich sources of bioactive compounds with promising antioxidant and antimicrobial efficacies and important perspectives for developing phytopharmaceuticals.

Removal and degradation of sodium diclofenac via radical-based mechanisms using S. sclerotiorum laccase.

The recently isolated Sclerotinia sclerotiorum laccase was used for the degradation of sodium diclofenac, a nonsteroidal anti-inflammatory drug widely found in the aquatic environment. The Michaelis-Menten parameters, half-life of diclofenac at different pH values in presence of this enzyme and potential inhibitors were evaluated. Diclofenac-based radicals formed in presence of laccase were spin-trapped and detected using EPR spectroscopy. Almost complete diclofenac degradation (> 96%) occurred after a 30-h treatment via radical-based generated oligomers and their rapid precipitation, thus ensuring an unprecedented green formula suitable not only for degradation but also for straightforward removal of the degradation products. High performance liquid chromatography coupled with atmospheric pressure chemical ionization-ion trap mass spectrometry (HPLC-APCI-MS) analyses of the degradation products of diclofenac in aqueous dosage revealed the presence of at least seven products while HR Orbitrap MS analysis showed that the enzymatic treatment produced high molecular weight metabolites through a radical oligomerization mechanism of diclofenac. The enzymatically formed products precipitated and its constituting components were also characterized using UV-vis spectroscopy, infrared spectroscopy (FTIR) and thermogravimetric analysis (TGA).

Trends in the texts of national anthems: A comparative study.

In a recent previous investigation of national anthems, preferred topics and their bias (e.g., towards identity, fighting, or well-being) were identified subjectively (Silaghi-Dumitrescu, 2020). The present report aims to verify whether a more objective, automated, comparison of the texts of national anthems across the world can also reveal systematic trends - and to what extent. To this end, the Tropes and Semantria software packages are employed, revealing preferred topics (e.g., state, feeling, body, time, land, religion, family, fight), how their relative weights differ across continents and cultures, and how the conveyed sentiments vary. For instance, "liberty" is more common in Latin-country anthems while almost absent in Asia, "feelings" are less mentioned in Germanic-language anthems, and the first-person singular "I" is essentially absent African anthems. The sentiment scores of the anthems vary from neutral in Latin and Mediterranean anthems to much more positive in Central and Western Asian, Germanic and Slavic countries.

Comorbidities of COVID-19 Patients.

The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) responsible for the coronavirus disease outbreak initiated in 2019 (COVID-19) has been shown to affect the health of infected patients in a manner at times dependent on pre-existing comorbidities. Reported here is an overview of the correlation between comorbidities and the exacerbation of the disease in patients with COVID-19, which may lead to poor clinical outcomes or mortality. General medical issues are also reviewed, such as the types of symptoms present in people infected with SARS-CoV-2, the long-term effects of COVID-19 disease, and the types of treatment that are currently used.

The adducts of cyano- and aquacobalamin with hypochlorite.

Hypochlorite is known to oxidatively degrade the corrin ring of cobalamin. Here, transient reaction intermediates are described in the reaction of aqua as well as of cyano-cobalamin with hypochlorite, using stopped-flow UV-vis kinetics. For aqua-cobalamin, the intermediate is assigned as arising from substitution of the aqua ligand with hypochlorite. For cyano-cobalamin, the intermediate is proposed to arise from substitution of the benzimidazole ligand trans to the cyanide. In both cases, the intermediates would feature a new Co(III)-OCl-bond-which is also supported by density functional theory (DFT) calculations.

Selective Attachment of Polyethylene Glycol to Hemerythrin for Potential Use in Blood Substitutes.

Due to its ability to reversibly bind O2, alongside a relatively low redox reactivity and a limited cytotoxicity, the oxygen-carrying protein hemerythrin has been considered as an alternative to hemoglobin in preparing blood substitutes. In order to increase the hydrodynamic volume and lower antigenicity, two site-directed variants, H82C and K92C, were engineered that contained a single cysteine residue on the surface of each hemerythrin octamer for the specific attachment of polyethylene glycol (PEG). A sulfhydryl-reactive PEGylation reagent with a 51.9 Å spacer arm was used for selective cysteine derivatization. The mutants were characterized by UV-vis spectroscopy, size-exclusion chromatography, oxygen affinity, and autooxidation rate measurements. The H82C variant showed altered oligomeric behavior compared to the wild-type and was unstable in the met form. The PEGylated K92C variant is reasonably stable, displays an oxygen affinity similar to that of the wild-type, and shows an increased rate of autoxidation; the latter disadvantage may be counteracted by further chemical modifications.

On the Origin of the Blue Color in The Iodine/Iodide/Starch Supramolecular Complex.

The nature of the blue color in the iodine-starch reaction is still a matter of debate. Some textbooks still invoke charge-transfer bands within a chain of neutral I2 molecules inside the hydrophobic channel defined by the interior of the amylose helical structure. However, the consensus is that the interior of the helix is not altogether hydrophobic-and that a mixture of I2 molecules and iodide anions reside there and are responsible for the intense charge-transfer bands that yield the blue color of the "iodine-starch complex". Indeed, iodide is a prerequisite of the reaction. However, some debate still exists regarding the nature of the iodine-iodine units inside the amylose helix. Species such as I3-, I5-, I7- etc. have been invoked. Here, we report UV-vis titration data and computational simulations using density functional theory (DFT) for the iodine/iodide chains as well as semiempirical (AM1, PM3) calculations of the amylose-iodine/iodide complexes, that (1) confirm that iodide is a pre-requisite for blue color formation in the iodine-starch system, (2) propose the nature of the complex to involve alternating sets of I2 and Ix- units, and (3) identify the nature of the charge-transfer bands as involving transfer from the Ix- σ* orbitals (HOMO) to I2 σ* LUMO orbitals. The best candidate for the "blue complex", based on DFT geometry optimizations and TD-DFT spectral simulations, is an I2-I5-I2 unit, which is expected to occur in a repetitive manner inside the amylose helix.

Glutaraldehyde-Polymerized Hemerythrin: Evaluation of Performance as an Oxygen Carrier in Hemorrhage Models.

Hemoglobin-based oxygen carriers (HBOCs) have been proposed and tested for several decades for the treatment of hemorrhage. We have previously proposed replacing hemoglobin (Hb) in HBOC with the oxygen-carrying protein hemerythrin (Hr), from marine worms, showing that Hr-based derivatives can perform at least as well or even better than Hb-based HBOC in a range of in vitro assays involving oxidative and nitrosative stress as well as in top-up animal models, where small amounts of Hr- or Hb-HBOC were injected into rats. Here, these experiments are extended to a hemorrhage experiment, in which Hr polymerized with glutaraldehyde, alone or conjugated with human serum albumin, is administered after a loss of 20-30% blood volume. The performance of these preparations is compared with that of Hb-based HBOC measured under the same conditions. Polymerized Hr is found to decrease the survival rate and can hence cannot be used as an oxygen carrier in transfusions. On the other hand, an Hr-albumin copolymer restores survival rates to 100% and generally yields biochemical and histological parameters similar to those of glutaraldehyde-polymerized bovine hemoglobin, with the exception of an acid-base imbalance. The latter may be solved by employing an allogeneic albumin as opposed to the human albumin employed in the present study.

Adduct of Aquacobalamin with Hydrogen Peroxide.

Aquacobalamin binds hydrogen peroxide reversibly to form a cobalt(III) hydroperoxo adduct with a 0.25 mM dissociation constant, as evidenced by UV-vis absorption spectroscopy and corroborated by NMR, Raman spectroscopy, stopped-flow UV-vis measurements, and density functional theory calculations.

Glutaraldehyde-Polymerized Hemoglobin: In Search of Improved Performance as Oxygen Carrier in Hemorrhage Models.

Hemoglobin- (Hb-) based oxygen carriers (HBOC) have for several decades been explored for treatment of hemorrhage. In our previous top-up tests, HBOC with lower in vitro prooxidant reactivity (incorporating a peroxidase or serum albumin to this end) showed a measurable but small improvement of oxidative stress-related parameters. Here, such HBOCs are tested in a hemorrhage set-up; ovine hemoglobin is also tested for the first time in such a setting, based on in vitro data showing its improved performance versus bovine Hb against oxidative and nitrosative stress agents. Indeed, ovine Hb performs better than bovine Hb in terms of survival rates, arterial tension, immunology, and histology. On the other hand, unlike in the top-up models, where the nonheme peroxidase rubrerythrin as well as bovine serum albumin copolymerized with Hb were shown to improve the performance of HBOC, in the present hemorrhage models rubrerythrin fails dramatically as HBOC ingredient (with a distinct immunological reaction), whereas serum albumin appears not feasible if its source is a different species (i.e., bovine serum albumin fares distinctly worse than rat serum albumin, in HBOC transfusions in rats). An effect of the matrix in which the HBOCs are dissolved (PBS versus gelofusine versus plasma) is noted.

On the Stability of Glutaraldehyde in Biocide Compositions.

Glutaraldehyde (GA) is used as biocide in hospitals. Recent public investigations on the chemical composition of biocides used in Romania have in some cases found GA, as a key ingredient, to be apparently diluted. However, these data did not explicitly consider the complex chemical equilibria inherent to GA. An investigation of experimental and theoretical data is reported here, assessing the stability of GA solutions relevant for biocide compositions. GA solutions of various chemical composition and under varying circumstances were analyzed using spectroscopy (UV-VIS, Raman, NMR) coupled with density functional theory (DFT) calculations, as well as chemically, such as via the formation of imines in reaction/titration with glycine monitored at 270 nm; using LC-MS; or using SDS-PAGE analysis with GA as reagent in the polymerization of two test proteins- hemoglobin and myoglobin. The spectral properties of GA changed significantly over time, in a temperature-dependent manner; titration with glycine confirmed the spectral data. SDS-PAGE experiments demonstrated a non-linear and apparently unpredictable change in the reactivity of GA over time. The results may be relevant for the determination of GA concentration in various settings such as biocide analysis, hospital wastewaters, and others.

Excess Ascorbate is a Chemical Stress Agent against Proteins and Cells.

Excess ascorbate (as expected in intravenous treatment proposed for COVID-19 management, for example) oxidizes and/or degrades hemoglobin and albumin, as evidenced by UV-vis spectroscopy, gel electrophoresis, and mass spectrometry. It also degrades hemoglobin in intact blood or in isolated erythrocytes. The survival rates and metabolic activities of several leukocyte subsets implicated in the antiviral cellular immune response are also affected. Excess ascorbate is thus an unselective biological stress agent.

On the Apparent Redox Reactivity of "Oxygen-Enriched Water".

Molecular oxygen-enriched water (OxEW) is advocated in popular media as useful for various health issues, presumably due to involvement of a purported antioxidant activity and to such notions as "active oxygen." To our knowledge, there are no explicit reports in the scientific literature where such redox reactivity would be described and explained. Reported here are data showing that a commercial preparation of OxEW does display a measurable, albeit very small, antioxidant activity as monitored by reaction with a standard reagent, DPPH. Moreover, OxEW also displays an apparent pro-oxidant reactivity, against caffeic acid. This does not correlate with any UV-vis-detectable contents of chemical substances in the water, nor can it be explained by typical chemical impurities (e.g., hydrogen peroxide or molecular hydrogen) that would arise upon enrichment with molecular oxygen of pure water by the two most common procedures: purging with gaseous O2 or electrolysis. Instead, this apparent redox reactivity is revealed to be due to differences in pH and in chemical content - and the differences in turn are most likely due to the trace amounts of inorganic ions/elements in the OxEW; importantly, electrolysis, which is often employed as a means to generate O2 in OxEW preparation, is also found to enhance the redox effect of OxEW-like preparations. Thus, in line with expectations, the herein-reported data show that there are no long-lived reactive oxygen species, no activated oxygen, and no extra reducing agents in OxEW - but that an apparent weak redox reactivity can still be measured and assigned to simple side effects of the electrolysis procedure presumably performed in order to enrich the sample in oxygen.

"Yellow" laccase from Sclerotinia sclerotiorum is a blue laccase that enhances its substrate affinity by forming a reversible tyrosyl-product adduct.

Yellow laccases lack the typical blue type 1 Cu absorption band around 600 nm; however, multi-copper oxidases with laccase properties have been reported. We provide the first evidence that the yellow laccase isolated from Sclerotinia sclerotiorum is obtained from a blue form by covalent, but nevertheless reversible modification with a phenolic product. After separating the phenolics from the extracellular medium, a typical blue laccase is obtained. With ABTS as model substrate for this blue enzyme, a non-natural purple adduct is formed with a spectrum nearly identical to that of the 1:1 adduct of an ABTS radical and Tyr. This modification significantly increases the stability and substrate affinity of the enzyme, not by acting primarily as bound mediator, but by structural changes that also alters the type 1 Cu site. The HPLC-MS analyses of the ABTS adduct trypsin digests revealed a distinct tyrosine within a unique loop as site involved in the modification of the blue laccase form. Thus, S. sclerotiorum yellow laccase seems to be an intrinsically blue multi-copper oxidase that boosts its activity and stability with a radical-forming aromatic substrate. This particular case could, at least in part, explain the enigma of the yellow laccases.

Importance of the iron-sulfur component and of the siroheme modification in the resting state of sulfite reductase.

The active site of sulfite reductase (SiR) consists of an unusual siroheme-Fe4S4 assembly coupled via a cysteinate sulfur, and serves for multi-electron reduction reactions. Clear explanations have not been demonstrated for the reasons behind the choice of siroheme (vs. other types of heme) or for the single-atom coupling to an Fe4S4 center (as opposed to simple adjacency or to coupling via chains consisting of more than one atom). Possible explanations for these choices have previously been invoked, relating to the control of the spin state of the substrate-binding (siro)heme iron, modulation of the trans effect of the (Fe4S4-bound) cysteinate, or modulation of the redox potential. Reported here is a density functional theory (DFT) investigation of the structural interplay (in terms of geometry, molecular orbitals and magnetic interactions) between the siroheme and the Fe4S4 center as well as the importance of the covalent modifications within siroheme compared to the more common heme b, aiming to verify the role of the siroheme modification and of the Fe4S4 cluster at the SiR active site, with focus on previously-formulated hypotheses (geometrical/sterics, spin state, redox and electron-transfer control). A calibration of various DFT methods/variants for the correct description of ground state spin multiplicity is performed using a set of problematic cases of bioinorganic Fe centers; out of 11 functionals tested, M06-L and B3LYP offer the best results - though none of them correctly predict the spin state for all test cases. Upon examination of the relative energies of spin states, reduction potentials, energy decomposition (electrostatic, exchange-repulsion, orbital relaxation, correlation and dispersion interactions) and Mayer bond indices in SiR models, the following main roles of the siroheme and cubane are identified: (1) the cubane cofactor decreases the reduction potential of the siroheme and stabilizes the siroheme-cysteine bond interaction, and (2) the siroheme removes the quasi-degeneracy between the intermediate and high-spin states found in ferrous systems by preserving the latter as ground state; the higher-spin preference and the increased accessibility of multiple spin states are likely to be important in selective binding of the substrate and of the subsequent reaction intermediates, and in efficient changes in redox states throughout the catalytic cycle.