Reducing Zinc Oxide Toxicity via Surface Modification: Emerging Strategies for Cosmetic Applications

Photoinitiators and UV filters as triggers of oxidative stress damage: evidence from national monitoring to in vitro and in silico studies.

Chlorella vulgaris is a unicellular green alga resistant to heavy metals. Chlorella is a rich nutritional ingredient because it contains high levels of antioxidants. The objective of this research was to study if C. vulgaris protect renal cells against mercury-chloride-caused oxidative stress and cellular damage in the kidney. Our results demonstrated that HgCl2 causes oxidative stress and cellular damage, and that C. vulgaris administration prevents oxidative stress and cellular damage in kidney

Our objective was to study if the phycobiliproteins of the cyanobacterium Pseudanabanea tenuis rich in phycoerythrin protect renal cells against mercury-caused oxidative stress and cellular damage in the kidney. We used 40 male mice that were assigned into five groups: a control group that received phosphate buffer (PB) and saline and four treatment groups which received either PB+HgCl2, PB+phycobiliproteins, or HgCl2+phycobiliproteins. The kidneys of the mice were used to determine lipid peroxidation and quantification of reactive oxygen species, oxidized glutathione, and peroxidase activities (catalase and glutathione peroxidase) and were also examined histologically. Our results demonstrated that HgCl2 causes oxidative stress and cellular damage and that all doses of phycobiliproteins prevented the increase of oxidative markers and partially protected against HgCl2-caused cell damage. This is the first report which applied phycobiliproteins of P. tenuis rich in c-phycoerythrin, like antioxidants against mercury chloride-caused oxidative stress and renal damage.

Mechanistic Skin Modeling of Plasma Concentrations of Sunscreen Active Ingredients Following Facial Application

Comparison of toxicological effects of oxybenzone, avobenzone, octocrylene, and octinoxate sunscreen ingredients on cucumber plants (Cucumis sativus L.)

Ultraviolet (UV) filters are active compounds which absorb, block, or scatter UV radiation, and are added to personal-care products (PCPs), such as sunscreens and cosmetics, to protect against photoaging and photocarcinogenesis. UV filters can be mineral-based (inorganic) or chemical-based (organic) and are considered contaminants of emerging concern (CEC) due to their widespread use and ubiquitous presence in the environment. UV filters can enter the marine environment through various pathways, including direct skin wash-off, wastewater effluent and industrial spills. UV filters have been detected globally in wastewater, freshwater and marine environments; and have been recorded in multiple matrices including accumulation within aquatic biota, sediments and water columns. Media attention has particularly focused on the effects of sunscreens on corals in recent years; resulting in regulatory implementation on such compounds in areas such as Hawaii and Palau; however, very little research has been conducted on the effects of these compounds on temperate marine organisms. Adopting a multidisciplinary approach, this research highlights the ecotoxicological effects of two contrasting sunscreen derived UV filters (benzophenone-3 and nano-titanium dioxide) on temperate marine organisms; including Isochrysis galbana and Mytilus galloprovincinalis, and analyses the potential of these compounds to bioaccumulate and undergo trophic transfer. The ecotoxicological effects are elucidated at a molecular, cellular and individual level. Here, we subjected Isochrysis galbana to varying environmentally relevant concentrations of benzophenone-3 and nano-titanium dioxide in a series of experiments under ultraviolet lighting. Ecotoxicological effects including cell viability, growth, chlorophyll-a content, dimethylsulfoniopropionate (DMSP) concentration, oxidative stress markers, enzymatic activity and bioaccumulation was assessed using a range of techniques including flow cytometry, fluorescence, electron microscopy, gas chromatography, LC-MS and ICP-MS. Results so far have indicated that both benzophenone-3 and nano-titanium dioxide can result in significant effects to cell density and chlorophyll-a content, whilst nano-titanium dioxide can cause oxidative stress in the form of lipid peroxidation. This research aligns with the UN Ocean Decade Vision of ‘A Clean Ocean – Where sources of pollution are identified, reduced, or removed.’ Whilst sunscreens and UV filters are important for human health, their environmental fate and effects on marine life must be elucidated to accurately target which compounds are safer for use and which should be highlighted for regulatory approaches. Please note – this work is still ongoing, and results are still being collated. By the time of the congress in June 2025 – more results will be made available to share, particularly relating to the trophic-transfer potential to Mytilus galloprovincinalis.

Background Phototherapy induces DNA damage by direct and indirect (oxidative) effects, which are prevented by ultraviolet (UV) filters and antioxidants. Aim Our goal was to assess DNA damage and oxidative stress associated with phototherapy and to prove the efficacy of UV filters and antioxidants for the prevention of DNA damage. Patients and methods The study included 160 jaundiced neonates who had been exposed to phototherapy for at least 48 h. The neonates were divided into four groups, 40 neonates in each group. The first (control) group received phototherapy only; the second group received antioxidants before and during phototherapy; the third group received phototherapy under an umbrella of UV filters; and the fourth group received phototherapy under both UV filters and antioxidants. DNA damage was assayed by the comet assay. Plasma total antioxidant capacity and total oxidant status levels were also measured and then oxidative stress index was calculated for all the four groups before and 48 h after phototherapy. Results The first group showed significant DNA damage accompanied with severe deterioration in all oxidative stress parameters by about 19%; the second group showed a decreasing trend as regards DNA damage and oxidative stress parameter deterioration to about 9%; the third group showed a more decreasing trend than group II to about 5%; but on the other hand, the fourth group showed complete DNA protection from damage with no changes in oxidative stress parameters. Conclusion Phototherapy causes DNA damage that can be completely prevented by combined concurrent use of UV filters and antioxidants.

Combined exposure to nano-silica and lead induced potentiation of oxidative stress and DNA damage in human lung epithelial cells

Cytoprotective effect against UV-induced DNA damage and oxidative stress: Role of new biological UV filter

A global tendency for products considered environmentally sustainable, and ecologically obtained led the industry related to personal care formulations to fund the research and the development of personal care/cosmetics containing ingredients from natural resources. Furthermore, consumers are aware of environmental and sustainability issueans, thus not harming the environment represents a key consideration when developing a new cosmetic ingredient. In this study we review some examples of active ingredients or raw materials used in cosmetics/personal care/biomedical products that are coming from either through biotechnological systems, or as byproducts of several industries. A skin formulation containing biosynthetic actives, prepared by us and the study regarding its dermocosmetic properties are also described. The need for the standardization processes, the safety assessment tools, the improvement of the exploitation methods of these renewable sources in order the production to be ecologically and economically better are also discussed.

Septic lung injury is a severe clinical problem with high mortality, and oxidative stress plays a critical role in its pathogenesis. This research focused on investigating how IGF2BP3 stabilizes CCL25 mRNA and its subsequent effects on cellular oxidative stress and septic lung injury. An in vitro cell injury model was established using LPS. The CCK-8 assay, flow cytometry and ELISA were employed to evaluate cell viability, death and inflammatory cytokine levels respectively. ROS accumulation, MDA content and antioxidant enzyme activities (SOD and GSH-Px) were quantified. Analysis of IGF2BP3 and CCL25 expression was conducted through qPCR and Western Blot. To confirm the interaction between IGF2BP3 and CCL25 mRNA, RIP and RNA pull-down were conducted. CCL25 mRNA stability was assessed following actinomycin D administration. Sepsis-caused lung injury model was created using CLP in mice. Further validation of CCL25's involvement in septic lung injury was conducted through qPCR, Western Blot, HE, ELISA and measurement of oxidative stress indicators. After LPS treatment, the cell viability was significantly decreased and the cell death, inflammation as well as oxidative stress were induced, the expressions of IGF2BP3 as well as CCL25 were markedly increased. Knockdown of CCL25 alleviated cellular oxidative stress and cellular damage caused by LPS. In addition, IGF2BP3 bound CCL25 and stabilized CCL25 mRNA, thus regulating LPS-induced cellular oxidative stress and cellular damage. In vivo, knockdown of CCL25 alleviated oxidative stress and inflammatory injury of lung tissue. CCL25 stabilized by IGF2BP3 could promote septic lung injury by inducing cellular oxidative stress.

Diabetes Mellitus is a metabolic disease that leads to microvascular complications like Diabetic retinopathy (DR), a major cause of blindness worldwide. Current medications for DR are expensive and report multiple side effects; therefore, an alternative medication that alleviates the disease condition is required. An interventional approach targeting the vascular endothelial growth factor (VEGF) remains a treatment strategy for DR. Anti-VEGF medicines are being investigated as the main therapy for managing vision-threatening complications of DR, such as diabetic macular oedema. Therefore, this study investigated the effect of flavonoid naringenin (NG) from citrus fruits on inhibiting early DR in zebrafish. When exposed to 130 mM glucose, the zebrafish larvae developed a hyperglycaemic condition accompanied by oxidative stress, cellular damage, and lipid peroxidation. Similarly, when adult zebrafish were exposed to 4% Glucose, high glucose levels were observed in the ocular region and massive destruction in the retinal membrane. High glucose upregulated the expression of VEGF. In comparison, the co-exposure to NG inhibited oxidative stress and cellular damage and restored the glutathione levels in the ocular region of the zebrafish larvae. NG regressed the glucose levels and cellular damage along with an inhibition of macular degeneration in the retina of adult zebrafish and normalized the overexpression of VEGF as a promising strategy for treating DR. Therefore, intervention of NG could alleviate the domestication of alternative medicine in ophthalmic research.

Transcriptomic analysis reveals Brassica rapa ssp. chinensis (pakchoi) responses to different UV filters

Human serum albumin (HSA) is the frontline antioxidant protein in blood with established anti-inflammatory and anticoagulation functions. Here, we report that COVID-19-induced oxidative stress inflicts structural damages to HSA and is linked with mortality outcome in critically ill patients. We recruited 39 patients who were followed up for a median of 12.5 days (1–35 days), among them 23 had died. Analyzing blood samples from patients and healthy individuals (n=11), we provide evidence that neutrophils are major sources of oxidative stress in blood and that hydrogen peroxide is highly accumulated in plasmas of non-survivors. We then analyzed electron paramagnetic resonance spectra of spin-labeled fatty acids (SLFAs) bound with HSA in whole blood of control, survivor, and non-survivor subjects (n=10–11). Non-survivors’ HSA showed dramatically reduced protein packing order parameter, faster SLFA correlational rotational time, and smaller S/W ratio (strong-binding/weak-binding sites within HSA), all reflecting remarkably fluid protein microenvironments. Following loading/unloading of 16-DSA, we show that the transport function of HSA may be impaired in severe patients. Stratified at the means, Kaplan–Meier survival analysis indicated that lower values of S/W ratio and accumulated H2O2 in plasma significantly predicted in-hospital mortality (S/W≤0.15, 81.8% (18/22) vs. S/W>0.15, 18.2% (4/22), p=0.023; plasma [H2O2]>8.6 μM, 65.2% (15/23) vs. 34.8% (8/23), p=0.043). When we combined these two parameters as the ratio ((S/W)/[H2O2]) to derive a risk score, the resultant risk score lower than the mean (<0.019) predicted mortality with high fidelity (95.5% (21/22) vs. 4.5% (1/22), log-rank χ2=12.1, p=4.9×10−4). The derived parameters may provide a surrogate marker to assess new candidates for COVID-19 treatments targeting HSA replacements and/or oxidative stress.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Human serum albumin (HSA) is the frontline antioxidant protein in blood with established anti-inflammatory and anticoagulation functions. Here, we report that COVID-19-induced oxidative stress inflicts structural damages to HSA and is linked with mortality outcome in critically ill patients. We recruited 39 patients who were followed up for a median of 12.5 days (1–35 days), among them 23 had died. Analyzing blood samples from patients and healthy individuals (n=11), we provide evidence that neutrophils are major sources of oxidative stress in blood and that hydrogen peroxide is highly accumulated in plasmas of non-survivors. We then analyzed electron paramagnetic resonance spectra of spin-labeled fatty acids (SLFAs) bound with HSA in whole blood of control, survivor, and non-survivor subjects (n=10–11). Non-survivors' HSA showed dramatically reduced protein packing order parameter, faster SLFA correlational rotational time, and smaller S/W ratio (strong-binding/weak-binding sites within HSA), all reflecting remarkably fluid protein microenvironments. Following loading/unloading of 16-DSA, we show that the transport function of HSA may be impaired in severe patients. Stratified at the means, Kaplan–Meier survival analysis indicated that lower values of S/W ratio and accumulated H2O2 in plasma significantly predicted in-hospital mortality (S/W≤0.15, 81.8% (18/22) vs. S/W>0.15, 18.2% (4/22), p=0.023; plasma [H2O2]>8.6 μM, 65.2% (15/23) vs. 34.8% (8/23), p=0.043). When we combined these two parameters as the ratio ((S/W)/[H2O2]) to derive a risk score, the resultant risk score lower than the mean (<0.019) predicted mortality with high fidelity (95.5% (21/22) vs. 4.5% (1/22), log-rank χ2=12.1, p=4.9×10−4). The derived parameters may provide a surrogate marker to assess new candidates for COVID-19 treatments targeting HSA replacements and/or oxidative stress. Editor's evaluation This submission is novel since it provides information on the structure changes of albumin in COVID-19. https://doi.org/10.7554/eLife.69417.sa0 Decision letter Reviews on Sciety eLife's review process Introduction COVID-19 pandemic continues as a global health crisis while the underlying SARS-CoV-2 virus defies all attempted treatment strategies. While writing this report, there have been more than 135 million confirmed cases including around 3 million deaths worldwide according to the World Health Organization Coronavirus Disease Dashboard (https://covid19.who.int/). Although 50% of cases are reported to be in the 25–64 age group, the percentage of deaths increases dramatically with age, and approximately 75% of deaths are in those aged 65 years and above (COVID-19 Hospitalization and Death by Age | CDC). People in the age groups 30–39 years, 40–49 years, and 50–64 years are 4, 10, and 30 times more likely to die from COVID-19 complications compared to the 18–29 years age group. Nevertheless, molecular and cellular factors contributing to mortality outcome in a homogeneous cohort of patients are not yet clear. Lack of diagnostic markers that predict mortality in COVID-19 patients impedes current efforts to siege the pandemic. It is thus critical to identify prognostic tests that can assess the risk of death in critically ill patients to guide clinical protocols and prioritize interventions. Furthermore, mechanistic clues for determining the underlying molecular factors contributing to the hypercoagulability, inflammation, and cytokine storm have been so far illusive. It is therefore imperative to intensify efforts focusing on understanding the molecular pathophysiology of COVID-19 infection and to identify prognostic markers to guide and prioritize clinical decisions. Human serum albumin (HSA) is the most abundant constituent of soluble proteins in the circulatory system. HSA has been suggested and used as a diagnostic and prognostic marker of numerous diseases and conditions including ischemia, rheumatoid arthritis, cancer, septic shock, among many others. In addition to its numerous physiological and pharmacological functions including the maintenance of blood/tissue osmotic balance (Singh-Zocchi et al., 1999), blood pH, metal cation transport and homeostasis (Bal et al., 2013; Stewart et al., 2003), nutrients and drug shuttling (Fujiwara and Amisaki, 2013; Wishart et al., 2018), and toxin neutralization (Ascenzi et al., 2006; Vorum and Honoré, 1996), HSA is suggested to be a major circulating antioxidant (Cha and Kim, 1996; Loban et al., 1997). HSA can remarkably bind with a diverse array of drugs and toxins thus controlling their bioavailability and pharmacologic effects (Fasano et al., 2005). It has been previously shown that more than 70% of the free radical-trapping capacity of serum was due to HSA (reviewed in Roche et al., 2008). Importantly, several reports indicated that inflammation enhances vascular permeability of various tissues to HSA apparently to confer antioxidant beneficial effects against reactive species released by activated neutrophils (Cross et al., 1994; Halliwell, 1988; Sitar et al., 2013). Although currently without direct experimental evidence, neutrophilia-mediated oxidative stress was implicated in the COVID-19 pathology and speculated to exacerbate the inflammatory immune response eventually causing multi-organ failure and death (Laforge et al., 2020). We hypothesized that COVID-19-mediated oxidative stress may be differentially reflected in HSA's structure and functions and employed electron paramagnetic resonance (EPR) spin labeling spectroscopy to explore HSA's structural changes in correlation with severity and mortality of critically ill COVID-19 patients. Spin-labeled fatty acids (SLFAs) are established probes to explore structural and functional changes in albumin by EPR spectroscopy (Ge et al., 1990; Haeri et al., 2019). This approach relies on the well-studied ability of albumin to strongly and exclusively bind with fatty acids in blood. Albumin has at least seven different specific binding sites for long-chain fatty acids located in different domains within the protein (Bhattacharya et al., 2000; Curry et al., 1999; Simard et al., 2006). Effectively, structural and functional changes in HSA may be assessed through the detection of parallel changes in mobility and binding affinity of SLFAs, in addition to the distribution of the spin labels on the albumin molecule (Haeri et al., 2019). EPR spectra of spin labels bound to different domains of the protein provide information on the local fatty acids/protein interactions, which may probe changes in the overall structure of the protein under unfolding or damaging conditions (Figure 1A; Bhattacharya et al., 2000). Here, we compare changes that occur to the mobility, binding affinity, and distribution of the HSA-bound SLFA in whole blood and plasma from COVID-19 patients in critical care unit relative to those observed in normal healthy individuals. Figure 1 Download asset Open asset Probing structual changes of serum albumin through spin labeling EPR spectroscopy. (A) HSA crystal structure containing seven copies of stearic acid. (B) Representative EPR spectra of free and HSA-bound 5-DSA (B) and 16-DSA (C) in whole blood from the same COVID-19 recovered patient. Chemical structures of the two spin-labeled fatty acids are given on the right side of the figure. EPR, electron paramagnetic resonance; HSA, human serum albumin. Results Demographic, clinical, and laboratory hematologic characteristics of COVID-19 patients Table 1 lists demographic data, comorbidities, ongoing medications, and administered anti-COVID-19 medications applied to treat current study participants that were divided into survivors (Sev-R) and deceased (Sev-D). No clinical or demographic characteristic showed statistically significant difference between Sev-R and Sev-D groups when analyzed by Pearson's Chi-square test. In Table 2, we show and statistically compare laboratory results of survivors versus non-survivor COVID-19 groups. Although when comparing all parameters in the two COVID-19 groups, we observed changes following the same reported trends in the literature, means' comparisons by Tukey test reported non-significant changes in all parameters except for a significant decrease in albumin level (p<0.05) and a strong trend observed for C-reactive protein (CRP) (greater levels in Sev-D group, p=0.06). Nevertheless, non-survivors' blood carried the frequently observed hallmarks of increased CRP, D-dimer, IL-6, ferritin, and the liver enzymes ALT and AST (reviewed in: Singh et al., 2021; Velavan and Meyer, 2020). However, it is conceivable that the clinical severe category and the same ICU status of patients in the two groups in addition to relatively small sample sizes underlie the observed lack of robust statistical differences between these parameters. Table 1 Demographic and clinical characteristics of the studied subjects. Sev-RSev-DTukey 95% CIpn1623Age (mean ± SD)60.7±9.567.8±13.24.1–17.80.09Male56.25%63.16%0.677†sO2 (mean ± SD)82.1±18.776.1±18.6–20.5 to 8.40.40Hypertension12.5%42.1%0.053†Diabetes25%75%0.08†Cardiovascular disease0%15.8%0.10†Cancer0%10.5%0.18†Bronchial asthma6.25%10.5%0.65†ACE inhibitors0%7.14%0.47†ARBs9.09%7.14%0.85†calcium channel blocker14.28%7.14%0.60†Beta blockers0%7.14%0.47†Diuretics0%7.14%0.47†Sulphonylurea14.28%21.43%0.69†Other oral hypoglycemic0%21.43%0.18†Insulin42.85%21.43%0.30†Anticoagulant57.14%50.0%0.76†Steroids71.42%64.28%0.74†Hydroxychloro-quine14.28%7.14%0.60†IL-6 receptor antibody28.57%21.42%0.72†Proton-pump inhibitor28.57%42.85%0.52†Azithromycin14.28%28.57%0.47†Cephalosporin42.85%21.43%0.30†Carbapenem42.85%42.85%1.0†Oxazolidinone42.85%28.57%0.51†Fluoro-quinolone42.85%21.43%0.30†Nitrofuran14.28%0%0.15†Remdesivir14.28%28.57%0.47†Ivermectin0%28.57%0.11† sO2, blood oxygen saturation level; ACE, angiotensin-converting enzyme; ARB, angiotensin II receptor blocker; IL-6, interleukin-6. † p values obtained through Pearson's χ2 test. Table 2 Laboratory parameters of the current study patients. WBC, white blood cell; INR, international normalized ratio; CRP, high-sensitivity C-reactive protein; ICU,intensive care unit; PLT, platelet; ALT, alanine transaminase; AST, aspartate transaminase. The Tukey'scalculated p-values as well as upper and lower 95% confidence levels for the Sev-R vs. Sev-D means'comparisons are given. Sev-R (mean ± SD)Sev-D (mean ± SD)Tukey 95% CIpWBCs (×103 /ml)10.6±4.013.9±8.0–1.47 to 8.060.17Platelets (×106 /ml)260±75.5213.7±115.8–116.7 to 24.20.19INR1.29±0.561.23±0.23–0.38 to 0.260.69CRP (mg/L)51.19±54.4103.77±86.3–2.35 to 107.50.06D-dimer (mg/ml)1.47±1.93.17±3.56–0.55 to 3.960.13IL-6 (pg/ml)314.1±527325.3±619–591 to 6140.97Ferritin922.6±5651078±578–281 to 5940.47Albumin (g/ml)31.47±7.9526.97±5.1–8.7 to –0.260.038Hemoglobin (g/dl)12.26±2.012.16±2.0–1.53 to –1.330.97ALT (U/L)33.64±24.1546.5±37.2–10.37 to 36.13 ALT, alanine transaminase; AST, aspartate transaminase; CRP, high-sensitivity C-reactive protein; ICU,intensive care unit; PLT, platelet; INR, international normalized ratio; WBC, white blood cell. The Tukey'scalculated p-values as well as upper and lower 95% confidence levels for the Sev-R vs. Sev-D means'comparisons are given. Neutrophils are a major source of reactive oxygen species It has been recently proposed that the high neutrophil-to-lymphocyte ratio (NLR) observed in critically ill COVID-19 patients may tip the redox homeostasis due to increased reactive oxygen species (ROS) production (Laforge et al., 2020). Our hypothesis implicates elevated oxidative stress as a major cause of HSA damage in severe COVID-19 patients. As a result, we started by following the dependence of clinical outcomes and mortality on ROS levels in blood cells. First, we used flow cytometry to assess percentages of neutrophils, lymphocytes, and platelets in all patients as described in Materials and methods (Figure 2A). Furthermore, we used the ROS-sensitive DCF dye to probe intracellular ROS levels in various cell populations in whole blood from all groups. Figure 2 shows that while lymphocyte counts decrease, a parallel dramatic increase in neutrophil counts (% total) was observable when going from Control (40.78±14.0, n=9) to Sev-R (64.0±20.0, n=10) to Sev-D (76.4± 6.8, n=11) groups (overall ANOVA p=3.9×10–5). Similar trend was clearly seen in the heat map depicting parameters for all patients analyzed by flow cytometry (Figure 2B). It is also clear from Figure 2B&C that changes in DCF-positive neutrophils follow similar trend observed for neutrophil counts. To confirm this relation, we compared neutrophil counts with DCF-positive neutrophil counts and found that the two parameters were strongly correlated (Pearson's r=0.8, p=3×10–7; Figure 2C). Moreover, both parameters individually showed statistically significant increases in both of the studied COVID-19 groups when compared with the control group (Figure 2D&E). These results suggest that neutrophils are major sources of elevated oxidative stress in critically ill patients. Note that the observed trends in platelets, lymphocyte, neutrophils, and NLR are similar to reported values (Sun et al., 2020; Yang et al., 2020). Figure 2 Download asset Open asset Hematologic cellular counts and neutrophil-ROS levels reflect severity and mortality in COVID-19 patients. (A) Representative flow cytometric diagrams comparing morphologic, hematologic, and ROS levels in control (representative of n=9; upper row), Sev-R (representative of n=10; middle row), and Sev-D (representative of n=11; lower row) groups. (B) Heat diagram comparing lymphocyte, neutrophils, platelets, and DCF-positive neutrophil counts as the percentage of total cell counts in all of the studied subjects. Yellow areas are either group separators or missing data due to insufficient sample size or processing errors. (C) A diagram showing statistically positive correlation between neutrophil count and count of neutrophils stained positive for DCF dye in all groups (black dots denote controls; blue are Sev-R; and red represent Sev-D patients). (D) When neutrophil counts were compared for all groups, both Sev-R and Sev-D groups showed statistically significant neutrophilia relative to control groups. However, only a weak trend has been observed when comparing the two groups with COVID-19. (E) DCF staining revealed increased levels of ROS in Sev-R and Sev-D groups relative to control neutrophils. Sev-D showed a trend of increased ROS level relative to Sev-R group. Multiple comparisons were carried out using ANOVA followed by Tukey test and p values are given. ROS, reactive oxygen species. Figure 2—source data 1 Raw source data for Figure 2B-E. https://cdn.elifesciences.org/articles/69417/elife-69417-fig2-data1-v2.xlsx Download elife-69417-fig2-data1-v2.xlsx Hydrogen peroxide levels in plasma correlate with mortality Next, we reasoned that elevated oxidative stress in both groups with critical COVID-19 infection would be echoed in plasma levels of hydrogen peroxide. Hydrogen peroxide is the most stable ROS and is highly stable under prolonged storage at low temperatures. We used a highly specific catalase-based assay that we developed and verified in our laboratory to quantify [H2O2] in plasma samples of all groups. The assay relies on high-resolution detection and quantification of released oxygen due to hydrogen peroxide decompostion by catalase (Figure 3A&B). We constructed a calibration curve to confirm the catalase-mediated H2O2 to O2 stoichiometric conversion (Figure 3B). A linear relation was obtained with zero intercept and slope of 0.47±0.03 which closely matches the theoretically expected value of 0.5 (95% confidence interval [CI]: 0.37‒0.56, p=5.6×10–4, Pearson's r=0.994). Indeed, we detected striking differences between groups even with relatively small sample sizes (Mean ± SD, Control, n=11: 2.95±0.77, Sev-R, n=16: 7.21±2.4, Sev-D, n=23: 9.67±2.0; overall ANOVA p=2.6×10–11; Figure 3C). The differences between groups have reached statistical significance (Sev-R vs. Control, 95% CI: 2.37‒6.13, p=4.9×10–6; Sev-D vs. Control, 95% CI: 4.95‒8.48, p=0.0; Sev-D vs. Sev-R, 95% CI: 0.90‒4.03, p=0.001). It appears from these results that a measure of oxidative stress, that is, [H2O2] in plasma, is doubled in survivors and tripled in deceased COVID-19 patients relative to controls' plasma average levels. Figure 3 Download asset Open asset Hydrogen peroxide levels in plasma and neutrophils reflect mortality in COVID-19 patients. Catalase was used to specifically and quantitatively determine levels of hydrogen peroxide in identical plasma volumes collected from control (n=11), Sev-R (n=16), and Sev-D (n=23) groups. (A) Oxygen levels are monitored and recorded while 50 μl batches of plasma from control, Sev-R, and Sev-D subjects are sequentially infused into tightly air-controlled O2k chamber containing catalase (315 units/ml) in deoxygenated buffer. In addition to the initial rise due to residual oxygen in the added plasma samples, the decomposition of hydrogen peroxide in these samples produces oxygen quantitatively. (B) To verify the assay we measured the released oxygen upon adding an increasing volume of standard hydrogen peroxide solution in PBS buffer with 0.2, 0.8, 1.2, and 1.6 μM final concentrations; inset. Linear fitting of the plotted [O2] versus [H2O2] relation yielded a slope=0.47±0.03 (Pearson's r=0.994, p=5.6×10–4), which is very close to the theoretically expected value of 0.5 as the catalase-mediated decomposition of one mole of H2O2 produces ½-mole O2. (C) Plasma contents of H2O2 in plasma significantly increased in the order Sev-D>Sev-R>Cont using ANOVA followed by Tukey test applied on n=11, 16, and 23 for control, survivors, and non-survivors, respectively. (D) Fluorescence imaging was used to assess levels of ROS in freshly isolated neutrophils using DCF (2,7-Dichlorodihydrofluorescein diacetate, green) staining in all groups. Hoechst binds strongly to adenine–thymine-rich regions in DNA thus mapping nuclei through emitting blue fluorescence. Merged DCF and Hoechst images are shown in the third column. Images were acquired using Cytation 5 Cell Imaging Multi-Mode Reader (Agilent) and analyzed using Gen5 Software package 3.08. Scale bar: 100 µm. Figure 3—source data 1 Raw polarographic data for released oxygen (A), calibration curve (B), and calculated plasma hydrogen peroxide levels in all groups (C). https://cdn.elifesciences.org/articles/69417/elife-69417-fig3-data1-v2.xlsx Download elife-69417-fig3-data1-v2.xlsx To confirm this finding, we performed DCF fluorescence imaging on freshly isolated neutrophils of representative group of individuals from each group (Figure 3D). We simultaneously stained neutrophils' nuclei with Hoechst 33342 (blue stain) to follow nuclear morphologic changes and DNA diffusion in all groups. Although requiring more detailed studies, a closer look at the acquired images of Hoechst-stained neutrophils from a survivor patient showed significantly reduced average neutrophil size DNA vs. Sev-R DNA two samples with more more nuclei and However, neutrophils from a non-survivor and in a DNA than normal and times that of Sev-R neutrophils vs. both control and Sev-R of the DCF fluorescence images indicated that the neutrophils populations of appears to be and that are highly of mean DCF fluorescence cell confirmed results obtained by flow cytometry and catalase assay increased levels of ROS in the order ± Control Sev-R Sev-D two samples for all parameters reflecting albumin changes are of COVID-19 mortality It has been previously shown that non-survivor COVID-19 patients relative to survivors et al., 2020). We started by albumin levels in the studied cohort of subjects to confirm follow similar We found that in plasma in the order (Mean ± SD, Control, Sev-R, n=16: Sev-D, n=23: overall ANOVA Figure We detected statistically significant decrease in in plasma of Sev-R and Sev-D groups (Sev-R vs. Control, 95% CI: to Sev-D vs. Control, 95% CI: to Sev-D vs. Sev-D, 95% CI: to The of HSA in serum is approximately we and groups found that COVID-19 mortality with et al., 2020). However, high of in numerous and the of this protein its diagnostic and As a result, we parameters to HSA protein in whole blood and plasma of all groups as of this critical protein functions. Figure Download asset Open asset EPR of binding strong dependence of binding on mortality in COVID-19 patients. (A) Albumin level in plasma of control Sev-R (n=16), and Sev-D groups showed that both survivors and COVID-19 patients statistically significant between representative spectra showing changes in that are to mobility and of HSA-bound 5-DSA (B) and 16-DSA (C) in whole blood of a control (black a Sev-R (blue and a Sev-D patients. parameters including order rotational correlation and the ratio between strongly bound to bound spin labels as in Figure 1 and described in Materials and methods comparisons by ANOVA followed by Tukey tests were used for means' comparisons and revealed decrease in the binding and packing of the local the spin calculated parameters with p values are given in the into acids binding are followed by with which into the EPR Representative EPR of 16-DSA in whole blood of control and Sev-D are showing the of 16-DSA and 5-DSA bound to HSA by in whole blood. are shown as the percentage of the of the middle samples spin and 3 and measured at and of the EPR the of the spin EPR, electron paramagnetic resonance; HSA, human serum albumin. Figure data 1 and EPR parameters used for statistical Download reported that changes in HSA may be to reflect critical functional changes in albumin and diagnostic and prognostic values of these changes in (Haeri et al., et al., 2006). It has also been found that long-chain fatty acids binding the binding of in the two major drug binding sites of HSA et al., We employed EPR spectroscopy to probe binding and protein in all groups as detailed in the Materials and methods of the 5-DSA and 16-DSA EPR spectra revealed changes in between control and COVID-19 groups (Figure and Figure Figure 4, 16-DSA in whole the in Figure and used to the protein packing order is to effects as interactions, on the spin probe that is in one of the HSA fatty acids binding the S/W ratio Figure to strongly bound populations of 16-DSA spin probe may reflect changes in protein that can Furthermore, the rotational correlation which is a measure of the spin probe rotational mobility is also Figure 5 Download asset Open asset of HSA transport function of HSA is assessed through the of fatty by following the rise in both strongly (A) and (B) bound SLFA with blood from representative subjects for each These results the fatty by HSA of critically ill patients relative to To the function of HSA, increasing volumes of were added to identical of all groups and the EPR spectra were acquired (C) to follow (D) and strongly (E) bound populations of of the fatty populations are through and increased of free fatty This is remarkably in critically ill patients reflecting and of fatty acids from HSA in those patients. HSA, human serum spin-labeled fatty acid. EPR parameters for all groups are in 1 including ANOVA and Tukey test p values with the of subjects 5-DSA and 16-DSA were used to probe the of local in sites the SLFA is in the protein mobility, and closer to the mobility due to with and and Indeed, 5-DSA reflected significantly values relative to 16-DSA both in plasma and in whole blood (Figure However, of the spin probe and both in plasma and whole blood samples, the order has been lower in Sev-R which was in Sev-D patients relative to the control group (Figure In whole similar results that showed more statistically robust differences have been of as described in Materials and methods showed rotational mobility of 16-DSA is significantly faster when bound with HSA from COVID-19 patients relative to that from control subjects in plasma or whole blood. similar trends have been observed for the S/W which of the strongly and bound of 16-DSA in different fatty acids these results that COVID-19 pathology is with structural changes in the HSA protein that the of of this critical into in whole blood of COVID-19 patients We followed the spin labels through analysis of the EPR by the et al., Figure experimental 16-DSA and in whole blood of control subjects remarkably faster when compared with both Sev-R and Sev-D group, and of the EPR of COVID-19 patients by the of the spin within the It is clear from these data that the HSA of COVID-19 patients is relative to control However, the of the HSA of both COVID-19 groups was not significantly different in of transport function of HSA in critically ill COVID-19 patients in severe patients relative to (Figure with the observed significant changes in the are to functional damage and 1990; et al., et al., et al., et al., In this changes in the are to functional changes in the solution of albumin due to of paramagnetic in