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Understanding the molecular mechanisms underlying neutrophil dynamics during COVID−19 disease progression is essential for managing severe inflammatory conditions. We investigated whether alterations in neutrophil mitochondrial function and calcium handling are associated with disrupted neutrophil homeostasis in critically ill COVID−19 patients. We analyzed neutrophil counts, phenotypes, and apoptotic profiles in critically ill COVID−19 survivors (ICU-S) and non-survivors (ICU-NS) compared with healthy controls. Flow cytometry, metabolic profiling, immunofluorescence imaging, and small RNA sequencing (miRNA-seq) were used to characterize neutrophil apoptosis-related pathways and mitochondrial function in freshly isolated neutrophils. Critically ill COVID−19 patients showed marked neutrophilia and a higher proportion of immature CD16low neutrophils relative to controls. Both ICU-S and ICU-NS groups exhibited reduced neutrophil apoptosis, as evidenced by fewer annexin V+ cells and lower cleaved caspase−3 signal compared with healthy controls. Although exploratory miRNA-seq in a small subset of ICU patients identified differentially expressed miRNAs with predicted enrichment in apoptosis- and calcium-related pathways, these mortality-associated miRNA signatures were not corroborated by functional apoptosis readouts (cleaved caspase−3 and annexin V) between ICU-S and ICU-NS. Neutrophils from ICU patients also demonstrated altered calcium handling, hyperpolarized mitochondrial membrane potential, increased complex II–linked respiration, and elevated mitochondrial ROS relative to controls. Neutrophils from critically ill COVID−19 patients display coordinated alterations in calcium handling, mitochondrial activity, and apoptosis consistent with impaired neutrophil clearance and disrupted homeostasis. These findings are observational and do not establish causality; the miRNA results should be interpreted as hypothesis-generating rather than validated mortality biomarkers.

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 H 2 O 2 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 [H 2 O 2 ]>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)/[H 2 O 2 ]) 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 χ 2 =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.

Roles of platelets during infections surpass the classical thrombus function and are now known to modulate innate immune cells. Leukocyte-platelet aggregations and activation-induced secretome are among factors recently gaining interest but little is known about their interplay with severity and mortality during the course of SARS-Cov-2 infection. The aim of the present work is to follow platelets’ bioenergetics, redox balance, and calcium homeostasis as regulators of leukocyte-platelet interactions in a cohort of COVID-19 patients with variable clinical severity and mortality outcomes. We investigated COVID-19 infection-related changes in platelet counts, activation, morphology (by flow cytometry and electron microscopy), bioenergetics (by Seahorse analyzer), mitochondria function (by high resolution respirometry), intracellular calcium (by flow cytometry), reactive oxygen species (ROS, by flow cytometry), and leukocyte-platelet aggregates (by flow cytometry) in non-intensive care unit (ICU) hospitalized COVID-19 patients (Non-ICU, n=15), ICU-survivors of severe COVID-19 (ICU-S, n=35), non-survivors of severe COVID-19 (ICU-NS, n=60) relative to control subjects (n=31). Additionally, molecular studies were carried out to follow gene and protein expressions of mitochondrial electron transport chain complexes (ETC) in representative samples of isolated platelets from the studied groups. Our results revealed that COVID-19 infection leads to global metabolic depression especially in severe patients despite the lack of significant impacts on levels of mitochondrial ETC genes and proteins. We also report that severe patients’ platelets exhibit hyperpolarized mitochondria and significantly lowered intracellular calcium, concomitantly with increased aggregations with neutrophil. These changes were associated with increased populations of giant platelets and morphological transformations usually correlated with platelets activation and inflammatory signatures, but with impaired exocytosis. Our data suggest that hyperactive platelets with impaired exocytosis may be integral parts in the pathophysiology dictating severity and mortality in COVID-19 patients.

Impaired depolarizing-to-hyperpolarizing (D/H) switch of gamma-aminobutyric acid (GABA) is reported during brain development in rodent valproate-model of autism spectrum disorder (VPA-ASD). We hypothesize that this impairment triggers NADPH oxidases (NOXs)-induced reactive oxygen species (ROS) overproduction. Here, we followed the impact of prenatal exposure to VPA on the synaptic protein expression of potassium chloride cotransporter 2 (KCC2), sodium potassium chloride cotransporter 1 (NKCC1) and, in brains of male and female Wistar rats during infantile (P15), juvenile (P30) and adult (P60) stages. We also assessed alterations in synaptic NOX isoforms 2 and 4 (NOX2 and NOX4) activities and expressions in developing rat brains. Our findings revealed a significant reduction in KCC2 expression and a concomitant increase in NOX activity and NOX4 expression in synaptosomes of VPA-exposed rats, particularly at P15 and P30. Prenatal exposure to shikonin, (10 mg/kg/day, intraperitoneal (i.p.) into pregnant dam, daily from G12.5 until birth), ameliorated these effects by reducing synaptic protein expression of NOX4, generally quenched synaptic NOX activity and enhanced synaptic protein expression of KCC2. Indeed, shikonin reversed VPA-induced sociability deficits in ASD rats. These results suggest that targeting the NOX-ROS pathway may be a potential therapeutic strategy for ASD.

COVID-19 severity and high in-hospital mortality are often associated with severe hypoxemia, hyperlactatemia, and acidosis. Since neutrophil numbers in severe COVID-19 can exceed 80% of the total circulating leukocytes and that they are massively recruited to infected lungs, we investigated whether metabolic acidosis mediated by the glycolytic neutrophils is associated with lung damage and impaired oxygen delivery in critically ill patients. Based on prospective mortality outcome, 102 critically ill-hospitalized COVID-19 patients were divided into two groups: ICU-Survivors (ICU-S, n=36) and ICU-Non-survivors (ICU-NS, n=66). Blood samples were collected from patients and control subjects to explore correlations between neutrophil counts, lung damage, glycolysis, blood lactate, blood pH, hemoglobin oxygen saturation, and mortality outcome. We also interrogated isolated neutrophils for glycolytic activities and for apoptosis using high-throughput fluorescence imaging complemented with transcriptomic analyses. Stratified survival analyses were conducted to estimate mortality risk associated with higher lactate among predefined subgroups. Neutrophil counts were consistently higher in critically ill patients while exhibiting remarkably lower apoptosis. Transcriptomic analysis revealed miRNAs associated with downregulation of genes involved in neutrophils apoptosis. Both CT lung damage scores and neutrophil counts predicted mortality. Severinghaus fitting of hemoglobin oxygen saturation curve revealed a right-shift indicating lower oxygen capacity in non-survivors, which is consistent with lower blood-pH observed in the same group. Levels of blood lactate were increased in patients but significantly more in the ICU-NS relative to the control group. ROC analysis followed by Kaplan-Meyer survival analysis stratified to the obtained cut-off values showed that CT damage scores, neutrophil counts, and lactate levels are predictors of mortality within 15 days following blood collection. The current results implicate neutrophilia as a potential player in metabolic acidosis and deranged oxygen delivery associating SARS-CoV-2 infection thus contributing to mortality outcome.

Valorization of poultry waste is a significant challenge addressed in this study, which aimed to produce cost-effective and sustainable peptones from poultry waste. The isolation process yielded the highly potent proteolytic B.subtilis isolate P6, identified through 16S rRNA gene sequencing to share 94% similarity with the B.subtilis strain KEMET024 (GenBank accession number PP694485.1) and deposited in MIRCEN culture collection, Cairo, Egypt as EMCC 998871. It reached optimal production levels during 24 h of incubation, with biomass at 2.5 g/L, protease activity at 455 U/mL, and total amino acid (TAA) concentration at 208 mg/mL. For screening the most significant factors for peptone production, the Plackett–Burman design identified meat and bone meal concentration as the main significant factor influencing total amino acid reaching 420 mg/mL. BOX-Behnken design optimized peptone production increasing its production level by twofold to reach 2850 U/mL of protease activity and 580 mg/mL of total amino acids. The produced peptone demonstrated a superior amino acid profile compared to commercial peptones, with a remarkably higher total amino acid content of 621.556 mg/g and elevated levels of essential amino acids like aspartic acid (37.745%), glutamic acid (90.876%), glycine (117.272%), and alanine (50.373%). Characterization revealed optimal pH and temperature conditions of around pH 8 and 50–60°C, respectively, for the proteolytic activity. The Michaelis–Menten and Lineweaver–Burk plots determined a Km of 0.5 mg/mL and Vmax of 174.08 U/mL suggesting cooperative substrate binding and providing insights into the enzyme’s maximum rate and affinity. The produced peptone exhibited minimal cytotoxicity at lower concentrations (≤ 1 mg/mL), with cell viability exceeding 94% against normal human skin fibroblast (HSF) cells. However, higher concentrations (≥ 3 mg/mL) displayed increased cytotoxic effects. Moreover, the results strongly indicate that the produced peptone, particularly at 0.5% concentration, is an effective nitrogen source for B. subtilis cultivation, demonstrating its potential for biotechnological applications. This study successfully valorized poultry waste by developing a sustainable and cost-effective alternative to commercial peptones, contributing to waste valorization and sustainable biotechnological processes. Graphical abstract