Light-Induced Charge Separation in Photosystem I from Different Biological Species Characterized by Multifrequency Electron Paramagnetic Resonance Spectroscopy.

Photosystem I (PSI) serves as a model system for studying fundamental processes such as electron transfer (ET) and energy conversion, which are not only central to photosynthesis but also have broader implications for bioenergy production and biomimetic device design. In this study, we employed electron paramagnetic resonance (EPR) spectroscopy to investigate key light-induced charge separation steps in PSI isolated from several green algal and cyanobacterial species. Following photoexcitation, rapid sequential ET occurs through either of two quasi-symmetric branches of donor/acceptor cofactors embedded within the protein core, termed the A and B branches. Using high-frequency (130 GHz) time-resolved EPR (TR-EPR) and deuteration techniques to enhance spectral resolution, we observed that at low temperatures prokaryotic PSI exhibits reversible ET in the A branch and irreversible ET in the B branch, while PSI from eukaryotic counterparts displays either reversible ET in both branches or exclusively in the B branch. Furthermore, we observed a notable correlation between low-temperature charge separation to the terminal [4Fe-4S] clusters of PSI, termed FA and FB, as reflected in the measured FA/FB ratio. These findings enhance our understanding of the mechanistic diversity of PSI's ET across different species and underscore the importance of experimental design in resolving these differences. Though further research is necessary to elucidate the underlying mechanisms and the evolutionary significance of these variations in PSI charge separation, this study sets the stage for future investigations into the complex interplay between protein structure, ET pathways, and the environmental adaptations of photosynthetic organisms.

Visible light triggers the formation of reactive oxygen species in monoclonal antibody formulations.

Most monoclonal antibody formulations require the presence of a surfactant, such as polysorbate, to ensure protein stability. The presence of high concentrations of polysorbate have been shown to enhance photooxidation of certain protein drug products when exposed to visible light. The current literature, however, suggest that photooxidation of polysorbate only occurs when exposed to visible light in combination with UVA light. This is probable as peroxides present in polysorbate solutions can be cleaved homolytically in the UVA region. In the visible region, photooxidation is not expected to occur as cleavage of peroxides is not expected at these wavelengths. This report presents findings suggesting that the presence of one or more photosensitiser(s) in polysorbate must be a cause and is required to catalyse the aerobic oxidation of polysorbate solutions upon exposure to visible light. Our investigation aimed to clarify the mechanism(s) of polysorbate photooxidation and explore the kinetics and the identity of the generated radicals and their impact on monoclonal antibody (mAb) degradation. Our study reveals that when polysorbate solutions are exposed to visible light between 400 - 800 nm in the absence of proteins, discolouration, radical formation, and oxygen depletion occur. We discuss the initial formation of reactive species, most likely occurring directly after reaction of molecular oxygen, with the presence of a triplet state photosensitiser, which is generated by intersystem crossing of the excited singlet state. When comparing the photooxidation of PS20 and PS80 in varying quality grades, we propose that singlet oxygen possesses potential for reacting with unsaturated fatty acids in PS80HP, however, PS20HP itself exhibited no measurable oxidation under the tested conditions. The study's final part delves into the photooxidation behaviour of different PS grades, examining its influence on the integrity of a mAb in the formulation. Finally, we examined the effect of photooxidation on the integrity of monoclonal antibodies. Our findings show that the exposure to visible light in polysorbate-containing mAb solutions at high PS concentrations of 4 mg·ml-1 results in increased monoclonal antibody degradation, highlighting the need for cautious evaluation of the correct PS concentration to stabilise protein therapeutics.

Spin-labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells.

As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction. Continuous wave X-band electron paramagnetic resonance spectroscopy is employed to characterize how the chronological sequence of chemical fixation and spin labeling impacts the local mobility and accessibility of the nitroxide-labeled glycans in the glycocalyx of HeLa cells. Results demonstrate that chemical fixation with paraformaldehyde can alter local glycan mobility and care should be taken in the analysis of data in any study where chemical fixation and cellular labeling occur.

Determining the Oxidation Mechanism through Radical Intermediates in Polysorbates 80 and 20 by Electron Paramagnetic Resonance Spectroscopy.

Polysorbates 20 and 80 (PS20 and PS80) are added to many commercial biologic and vaccine pharmaceuticals. It is commonly known that these polysorbates undergo a radical oxidation mechanism; however, the identity of these radical intermediates has not been clearly determined. Furthermore, PS20 and PS80 differ by the presence of a lauric acid instead of an oleic acid, respectively. The oxidation of PS80 is thought to be centered around the double bond of the oleic acid even though PS20 also undergoes oxidation, making the mechanism of oxidation unclear for PS20. Using commercial stocks of PS20 and PS80 alkyl (R•), alkoxyl (C-O•) and peroxyl (C-OO•) radicals were detected by electron paramagnetic resonance spectroscopy likely originating from radical-initiating species already present in the material. When dissolved in water, the peroxyl radicals (C-OO•) originally in the stocks were not detected but poly(ethylene oxide) radicals were. An oxidative pathway for polysorbates was suggested based on the radical species identified in the polysorbate stock material and solutions.

Evidence of the protective effect of anti-pollution products against oxidative stress in skin ex vivo using EPR spectroscopy and autofluorescence measurements.

The concentration of air pollution is gradually increasing every year so that daily skin exposure is unavoidable. Dietary supplements and topical formulations currently represent the protective strategies to guard against the effects of air pollution on the body and the skin. Unfortunately, there are not yet enough methods available to measure the effectiveness of anti-pollution products on skin. Here, we present two ex vivo methods for measuring the protective effect against air pollution of different cream formulations on the skin: Electron paramagnetic resonance (EPR) spectroscopy and autofluorescence excited by 785 nm using a confocal Raman microspectrometer (CRM). Smoke from one cigarette was used as a model pollutant. EPR spectroscopy enables the direct measurement of free radicals in excised porcine skin after smoke exposure. The autofluorescence in the skin was measured ex vivo, which is an indicator of oxidative stress. Two antioxidants and a chelating agent in a base formulation and a commercial product containing an antioxidant mixture were investigated. The ex vivo studies show that the antioxidant epigallocatechin-3-gallate (EGCG) in the base cream formulation provided the best protection against oxidative stress from smoke exposure for both methods.

Quantitative MRI Evaluation of Ferritin Overexpression in Non-Small-Cell Lung Cancer.

Cancer cells frequently present elevated intracellular iron levels, which are thought to facilitate an enhanced proliferative capacity. Targeting iron metabolism within cancer cells presents an avenue to enhance therapeutic responses, necessitating the use of non-invasive models to modulate iron manipulation to predict responses. Moreover, the ubiquitous nature of iron necessitates the development of unique, non-invasive markers of metabolic disruptions to develop more personalized approaches and enhance the clinical utility of these approaches. Ferritin, an iron storage enzyme that is often upregulated as a response to iron accumulation, plays a central role in iron metabolism and has been frequently associated with unfavorable clinical outcomes in cancer. Herein, we demonstrate the successful utility, validation, and functionality of a doxycycline-inducible ferritin heavy chain (FtH) overexpression model in H1299T non-small-cell lung cancer (NSCLC) cells. Treatment with doxycycline increased the protein expression of FtH with a corresponding decrease in labile iron in vitro and in vivo, as determined by calcein-AM staining and EPR, respectively. Moreover, a subsequent increase in TfR expression was observed. Furthermore, T2* MR mapping effectively detected FtH expression in our in vivo model. These results demonstrate that T2* relaxation times can be used to monitor changes in FtH expression in tumors with bidirectional correlations depending on the model system. Overall, this study describes the development of an FtH overexpression NSCLC model and its correlation with T2* mapping for potential use in patients to interrogate iron metabolic alterations and predict clinical outcomes.

Insights into the LiI Redox Mediation in Aprotic Li-O2 Batteries: Solvation Effects and Singlet Oxygen Evolution.

Lithium-oxygen aprotic batteries (aLOBs) are highly promising next-generation secondary batteries due to their high theoretical energy density. However, the practical implementation of these batteries is hindered by parasitic reactions that negatively impact their reversibility and cycle life. One of the challenges lies in the oxidation of Li2O2, which requires large overpotentials if not catalyzed. To address this issue, redox mediators (RMs) have been proposed to reduce the oxygen evolution reaction (OER) overpotentials. In this study, we focus on a lithium iodide RM and investigate its role on the degradation chemistry and the release of singlet oxygen in aLOBs, in different solvent environments. Specifically, we compare the impact of a polar solvent, dimethyl sulfoxide (DMSO), and a low polarity solvent, tetraglyme (G4). We demonstrate a strong interplay between solvation, degradation, and redox mediation in OER by LiI in aLOBs. The results show that LiI in DMSO-based electrolytes leads to extensive degradation and to 1O2 release, affecting the cell performance, while in G4-based electrolytes, the release of 1O2 appears to be suppressed, resulting in better cyclability.

Differentiating between Label and Protein Conformers in Pulsed Dipolar EPR Spectroscopy with the dHis-Cu2+ (NTA) Motif.

Pulsed dipolar EPR spectroscopy (PDS) in combination with site-directed spin labeling is a powerful tool in structural biology. However, the commonly used spin labels are conjugated to biomolecules via rather long and flexible linkers, which hampers the translation of distance distributions into biomolecular conformations. In contrast, the spin label copper(II)-nitrilotriacetic acid [Cu2+ (NTA)] bound to two histidines (dHis) is rigid and yields narrow distance distributions, which can be more easily translated into biomolecular conformations. Here, we use this label on the 71 kDa Yersinia outer protein O (YopO) to decipher whether a previously experimentally observed bimodal distance distribution is due to two conformations of the biomolecule or of the flexible spin labels. Two different PDS experiments, that is, pulsed electron-electron double resonance (PELDOR aka DEER) and relaxation-induced dipolar modulation enhancement (RIDME), yield unimodal distance distribution with the dHis-Cu2+ (NTA) motif; this result suggests that the α-helical backbone of YopO adopts a single conformation in frozen solution. In addition, we show that the Cu2+ (NTA) label preferentially binds to the target double histidine (dHis) sites even in the presence of 22 competing native histidine residues. Our results therefore suggest that the generation of a His-null background is not required for this spin labeling methodology. Together these results highlight the value of the dHis-Cu2+ (NTA) motif in PDS experiments.

DODAB vesicles containing lysophosphatidylcholines: The relevance of acyl chain saturation on the membrane structure and thermal properties.

The saturated LPC18:0 and unsaturated LPC18:1 lysophosphatidylcholines have important roles in inflammation and immunity and are interesting targets for immunotherapy. The synthetic cationic lipid DODAB has been successfully employed in delivery systems, and would be a suitable carrier for those lysophosphatidylcholines. Here, assemblies of DODAB and LPC18:0 or LPC18:1 were characterized by Differential Scanning Calorimetry (DSC) and Electron Paramagnetic Resonance (EPR) spectroscopy. LPC18:0 increased the DODAB gel-fluid transition enthalpy and rigidified both phases. In contrast, LPC18:1 caused a decrease in the DODAB gel-fluid transition temperature and cooperativity, associated with two populations with distinct rigidities in the gel phase. In the fluid phase, LPC18:1 increased the surface order but, differently from LPC18:0, did not affect viscosity at the membrane core. The impact of the different acyl chains of LPC18:0 and 18:1 on structure and thermotropic behavior should be considered when developing applications using mixed DODAB membranes.

Deep Levels and Electron Paramagnetic Resonance Parameters of Substitutional Nitrogen in Silicon from First Principles.

Nitrogen is commonly implanted in silicon to suppress the diffusion of self-interstitials and the formation of voids through the creation of nitrogen-vacancy complexes and nitrogen-nitrogen pairs. Yet, identifying a specific N-related defect via spectroscopic means has proven to be non-trivial. Activation energies obtained from deep-level transient spectroscopy are often assigned to a subset of possible defects that include non-equivalent atomic structures, such as the substitutional nitrogen and the nitrogen-vacancy complex. Paramagnetic N-related defects were the object of several electron paramagnetic spectroscopy investigations which assigned the so-called SL5 signal to the presence of substitutional nitrogen (NSi). Nevertheless, its behaviour at finite temperatures has been imprecisely linked to the metastability of the NSi center. In this work, we build upon the robust identification of the SL5 signature and we establish a theoretical picture of the substitutional nitrogen. Through an understanding of its symmetry-breaking mechanism, we provide a model of its fundamental physical properties (e.g., its energy landscape) based on ab initio calculations. Moreover by including more refined density functional theory-based approaches, we calculate EPR parameters (↔g and ↔A tensors), elucidating the debate on the metastability of NSi. Finally, by computing thermodynamic charge transition levels within the GW method, we present reference values for the donor and acceptor levels of NSi.

Spin-labeling Insights into How Chemical Fixation Impacts Glycan Organization on Cells.

As new methods to interrogate glycan organization on cells develop, it is important to have a molecular level understanding of how chemical fixation can impact results and interpretations. Site-directed spin labeling technologies are well suited to study how the spin label mobility is impacted by local environmental conditions, such as those imposed by cross-linking effects of paraformaldehyde cell fixation methods. Here, we utilize three different azide-containing sugars for metabolic glycan engineering with HeLa cells to incorporate azido glycans that are modified with a DBCO-based nitroxide moiety via click reaction. Continuous wave X-band electron paramagnetic resonance spectroscopy is employed to characterize how the chronological sequence of chemical fixation and spin labeling impacts the local mobility and accessibility of the nitroxide-labeled glycans in the glycocalyx of HeLa cells. Results demonstrate that chemical fixation with paraformaldehyde can alter local glycan mobility and care should be taken in the analysis of data in any study where chemical fixation and cellular labeling occur.

Influence of Cupric (Cu2+) Ions on the Iron Oxidation Mechanism by DNA-Binding Protein from Starved Cells (Dps) from Marinobacter nauticus.

Dps proteins (DNA-binding proteins from starved cells) are multifunctional stress defense proteins from the Ferritin family expressed in Prokarya during starvation and/or acute oxidative stress. Besides shielding bacterial DNA through binding and condensation, Dps proteins protect the cell from reactive oxygen species by oxidizing and storing ferrous ions within their cavity, using either hydrogen peroxide or molecular oxygen as the co-substrate, thus reducing the toxic effects of Fenton reactions. Interestingly, the interaction between Dps and transition metals (other than iron) is a known but relatively uncharacterized phenomenon. The impact of non-iron metals on the structure and function of Dps proteins is a current topic of research. This work focuses on the interaction between the Dps from Marinobacter nauticus (a marine facultative anaerobe bacterium capable of degrading petroleum hydrocarbons) and the cupric ion (Cu2+), one of the transition metals of greater biological relevance. Results obtained using electron paramagnetic resonance (EPR), Mössbauer and UV/Visible spectroscopies revealed that Cu2+ ions bind to specific binding sites in Dps, exerting a rate-enhancing effect on the ferroxidation reaction in the presence of molecular oxygen and directly oxidizing ferrous ions when no other co-substrate is present, in a yet uncharacterized redox reaction. This prompts additional research on the catalytic properties of Dps proteins.

Recent Advances in Characterization of Melanin Pigments in Biological Samples.

The melanin pigments eumelanin (EM) and pheomelanin (PM), which are dark brown to black and yellow to reddish-brown, respectively, are widely found among vertebrates. They are produced in melanocytes in the epidermis, hair follicles, the choroid, the iris, the inner ear, and other tissues. The diversity of colors in animals is mainly caused by the quantity and quality of their melanin, such as by the ratios of EM versus PM. We have developed micro-analytical methods to simultaneously measure EM and PM and used these to study the biochemical and genetic fundamentals of pigmentation. The photoreactivity of melanin has become a major focus of research because of the postulated relevance of EM and PM for the risk of UVA-induced melanoma. Our biochemical methods have found application in many clinical studies on genetic conditions associated with alterations in pigmentation. Recently, besides chemical degradative methods, other methods have been developed for the characterization of melanin, and these are also discussed here.

Interaction of 2-dodecylcyclobutanone (2-DCB) and internal standard in irradiated chicken jerkies and pig ears in solid-phase microextraction (SPME)-coupled gas chromatography-mass spectrometry (GC-MS) analysis.

The radiolysis of palmitic acid in chicken jerky (CJ) and pig ears (PE) can form 2-dodecylcyclobutanone (2-DCB). A solid-phase micro extraction-coupled GC-MS technique can be used for quantitative analysis of 2-DCB with the adequate use of an internal standard (IS). The objectives of this study are to: 1) investigate the IS and 2-DCB interactions as a function of IS concentration and irradiation dose; 2) elucidate the effects of bound 2-DCB; 3) use electron paramagnetic resonance spectroscopy to complement 2-DCB measurements. The measurement of 2-DCB formed by irradiation was significantly (P < 0.05) affected by the palmitic acid content and IS concentration. The amount of 2-DCB measured in irradiated (10 kGy) CJ and PE increased 70% and 300%, respectively, when the IS concentration increased from 8 to 800 ppb. Our findings serve as a guide for the adequate use of IS for quantitative analysis of 2-DCB formed in irradiated meat matrixes.

Xanthine Oxidase-A Personal History.

A personal perspective is provided regarding the work in several laboratories, including the author's, that has established the reaction mechanism of xanthine oxidase and related enzymes.

Conditioned place avoidance is associated with a distinct hippocampal phenotype, partly preserved pattern separation, and reduced reactive oxygen species production after stress.

Stress is associated with contextual memory deficits, which may mediate avoidance of trauma-associated contexts in posttraumatic stress disorder. These deficits may emerge from impaired pattern separation, the independent representation of similar experiences by the dentate gyrus-Cornu Ammonis 3 (DG-CA3) circuit of the dorsal hippocampus, which allows for appropriate behavioral responses to specific environmental stimuli. Neurogenesis in the DG is controlled by mitochondrial reactive oxygen species (ROS) production, and may contribute to pattern separation. In Experiment 1, we performed RNA sequencing of the dorsal hippocampus 16 days after stress in rats that either develop conditioned place avoidance to a predator urine-associated context (Avoiders), or do not (Non-Avoiders). Weighted genome correlational network analysis showed that increased expression of oxidative phosphorylation-associated gene transcripts and decreased expression of gene transcripts for axon guidance and insulin signaling were associated with avoidance behavior. Based on these data, in Experiment 2, we hypothesized that Avoiders would exhibit elevated hippocampal (HPC) ROS production and degraded object pattern separation (OPS) compared with Nonavoiders. Stress impaired pattern separation performance in Non-Avoider and Avoider rats compared with nonstressed Controls, but surprisingly, Avoiders exhibited partly preserved pattern separation performance and significantly lower ROS production compared with Non-Avoiders. Lower ROS production was associated with better OPS performance in Stressed rats, but ROS production was not associated with OPS performance in Controls. These results suggest a strong negative association between HPC ROS production and pattern separation after stress, and that stress effects on these outcome variables may be associated with avoidance of a stress-paired context.

Sustainable Extraction Methods Affect Metabolomics and Oxidative Stability of Myrtle Seed Oils Obtained from Myrtle Liqueur By-Products: An Electron Paramagnetic Resonance and Mass Spectrometry Approach.

Myrtle liqueur production generates high amounts of by-products that can be employed for the extraction of bioactive compounds. Bio-based, non-toxic and biodegradable solvents (ethyl acetate and 2-methyltetrahydrofuran), and a mechanical extraction were applied to myrtle seeds, by-products of the liqueur production, to extract oils rich in phenolic compounds. The oils obtained were characterized for yield, peroxide value (PV), lipid composition, and total phenolic concentration (TPC). The phenolic profile of the oils, determined by LC-MS, the antioxidant activity, and the oxidative stability were also analyzed. A validated UHPLC-ESI-QTRAP-MS/MS analytical method in multiple reaction monitoring (MRM) mode was applied to quantify myricetin and its main derivatives in myrtle oils. The results pointed out clear differences among extraction methods on myricetin concentration. The oxidative stability of myrtle oils was studied with electron paramagnetic resonance (EPR) spectroscopy highlighting the effect of the extraction method on the oxidation status of the oils and the role of phenolic compounds in the evolution of radical species over time. A principal component analysis applied to LC-MS data highlighted strong differences among phenolic profiles of the oils and highlighted the role of myricetin in the oxidative stability of myrtle oils. Myrtle oil, obtained from the by-products of myrtle liqueur processing industry, extracted with sustainable and green methods might have potential application in food or cosmetic industries.

Biological Assessment of the NO-Dependent Endothelial Function.

Nitric oxide (NO) is implicated in numerous physiological processes, including vascular homeostasis. Reduced NO bioavailability is a hallmark of endothelial dysfunction, a prequel to many cardiovascular diseases. Biomarkers of an early NO-dependent endothelial dysfunction obtained from routine venous blood sampling would be of great interest but are currently lacking. The direct measurement of circulating NO remains a challenge due by its high reactivity and short half-life. The current techniques measure stable products from the NO signaling pathway or metabolic end products of NO that do not accurately represent its bioavailability and, therefore, endothelial function per se. In this review, we will concentrate on an original technique of low temperature electron paramagnetic resonance spectroscopy capable to directly measure the 5-α-coordinated heme nitrosyl-hemoglobin in the T (tense) state (5-α-nitrosyl-hemoglobin or HbNO) obtained from fresh venous human erythrocytes. In humans, HbNO reflects the bioavailability of NO formed in the vasculature from vascular endothelial NOS or exogenous NO donors with minor contribution from erythrocyte NOS. The HbNO signal is directly correlated with the vascular endothelial function and inversely correlated with vascular oxidative stress. Pilot studies support the validity of HbNO measurements both for the detection of endothelial dysfunction in asymptomatic subjects and for the monitoring of such dysfunction in patients with known cardiovascular disease. The impact of therapies or the severity of diseases such as COVID-19 infection involving the endothelium could also be monitored and their incumbent risk of complications better predicted through serial measurements of HbNO.

Establishment of a method to expose and measure pollution in excised porcine skin with electron paramagnetic resonance spectroscopy.

Health problems associated with the amount of air pollutants are increasing worldwide. Pollution damages not only the lungs; it also has an impact on skin health and is co-responsible for the development of skin diseases. Anti-pollution products are on the rise in the cosmetic market but so far, there is no established method to directly assess the impact of pollution on the skin and to test the efficacy of anti-pollution products. To address this problem, two different chambers were developed for the reproducible exposure to realistic air pollutant concentrations. One chamber for the exclusive use of excised skin and hair samples, the second chamber for ex vivo and in vivo measurements. Measurements of nicotine next to the investigated skin area allow conclusions to be drawn on the particle concentration to which the skin is exposed. Electron paramagnetic resonance spectroscopy, which enables the detection of free radicals in different systems, was applied to assess the hazard potential of pollution in the skin. A direct proof of the formation of free radicals in the skin by the model pollutant cigarette smoke could be demonstrated. An additional application of UV irradiation even increased the formation of free radicals in the skin seven-fold (sum parameter). Depending on the question of interest, the use of different spin probes allows various assessments of the radical formation in skin: the amount of radicals but also the antioxidant status of the microenvironment can be estimated. Using two exposure chambers, the direct formation of oxidative stress by cigarette smoke on ex vivo skin, with and without additional UV exposure, could be reproducibly examined. This measurement method is promising for the assessment of anti-pollution products and could allow a direct causal connection between pollutant, effect on the skin and the protective function of skin care products.