Role of oxidative stress in the severity of SARS-COV-2 infection

Since the World Health Organization declared SARS-CoV-2 (COVID-19) a pandemic in March 2020, serious efforts have been made to understand the epidemiology, molecular mechanisms, pathology, and clinical evolution of this disease. Oxidative stress (OX-S) has been implicated in the etiologies of many diseases, including SARS-CoV-2. Recent studies suggest that superoxide radicals and the products of lipid peroxidation, such as the electrophilic aldehyde, 4-hydroxynonenal (4-HNE), are important mediators of the pathological effects of oxidative stress during microbial and viral infections. Numerous studies have confirmed that viral infections induce inflammatory responses that generate excessive amounts of reactive oxygen species and 4-HNE protein adducts in plasma and in various tissues, including alveolar epithelium and endothelium. In this book chapter, we will highlight and discuss the apparent and plausible relationships between SARS-CoV-2 virulence and oxidative stress/lipid peroxidation, which affect cellular and DNA repair mechanisms and immune response. © 2023 Elsevier Inc. All rights reserved.

Comparative Analysis of Different Systematic Odometry Error Correction Methods on a Differential Drive Indoor Service Robot

The COVID-19 pandemic has led to a surge of interest in research work involving the development of robotic systems that reduce human-to-human interaction, as such a technology can greatly benefit healthcare industries in preventing the spread of highly infectious diseases. An indoor service robot is built and equipped with wheel odometry and a 2D LiDAR. However, the presence of the systematic odometry errors is evident during field testing. Hence, the possibility of minimizing systematic odometry errors is inspected using various methods of calculation, namely: UMBmark, Lee's and Jung's. The methods all use the Bidirectional Square Path test, performed together with ROS. It is found that Jung's method is the most appropriate method showing a 20.4% improvement compared to the uncalibrated dead reckoning accuracy. Moreover, it is found that the presence of slippage, a nonsystematic error, greatly affects the return position errors of the robot. Consequently, it is recommended to improve the design of the wheelbase to minimize the effects of nonsystematic errors. © 2022 IEEE.

MicroRNAs: The Master Regulators of the Breast Cancer Tumor Microenvironment

Breast cancer is the most commonly diagnosed cancer globally and is among the leading causes of cancer deaths worldwide. Breast cancer mortality rates are increasing due to delays in diagnosis, prognosis, and treatment caused by the coronavirus disease 2019 (COVID-19) pandemic. Identification and validation of blood-based breast cancer biomarkers for early detection is a top priority worldwide. MicroRNAs (miRNAs) show the potential to serve as breast cancer biomarkers. miRNAs are small, endogenously produced RNAs that regulate growth and development. However, oncogenic miRNAs also play a major role in tumor growth and can alter the tumor microenvironment (TME) in favor of cancer metastasis. The TME represents a complex network of diverse cancerous and noncancerous cell types, secretory proteins, growth factors, and miRNAs. Complex interactions within the TME can promote cancer progression and metastasis via multiple mechanisms, including oxidative stress, hypoxia, angiogenesis, lymphangiogenesis, and cancer stem cell regulation. Here, we decipher the mechanisms of miRNA regulating the TME, intending to use that knowledge to identify miRNAs as therapeutic targets in breast cancer and use miRNAs as blood-based biomarkers. © Springer Nature Singapore Pte Ltd. 2022.

Synergistic effect of elevated glucose levels with SARS-CoV-2 spike protein induced NOX-dependent ROS production in endothelial cells.

BACKGROUND: Diabetic patients infected with coronavirus disease 2019 (COVID-19) often have a higher probability of organ failure and mortality. The potential cellular mechanisms through which blood glucose exacerbates tissue damage due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is still unclear. METHODS AND RESULTS: We cultured endothelial cells within differing glucose mediums with an increasing concentration gradient of SARS-CoV-2 Spike protein (S protein). S protein can cause the reduction of ACE2 and TMPRSS2, and activation of NOX2 and NOX4. A high glucose medium was shown to aggravate the decrease of ACE2 and activation of NOX2 and NOX4 in cultured cells, but had no effect on TMPRSS2. S protein mediated activation of the ACE2-NOX axis induced oxidative stress and apoptosis within endothelial cells, leading to cellular dysfunction via the reduction of NO and tight junction proteins which may collectively be exacerbated by elevated glucose. In addition, the glucose variability model demonstrated activation of the ACE2-NOX axis in a similar manner observed in the high glucose model in vitro. CONCLUSIONS: Our present study provides evidence for a mechanism through which hyperglycemia aggravates endothelial cell injury resulting from S protein mediated activation of the ACE2-NOX axis. Our research thus highlights the importance of strict monitoring and control of blood glucose levels within the context of COVID-19 treatment to potentially improve clinical outcomes.

Ferroptosis and multi-organ complications in COVID-19: mechanisms and potential therapies.

COVID-19 is an infectious disease caused by SARS-CoV-2, with respiratory symptoms as primary manifestations. It can progress to severe illness, leading to respiratory failure and multiple organ dysfunction. Recovered patients may experience persistent neurological, respiratory, or cardiovascular symptoms. Mitigating the multi-organ complications of COVID-19 has been highlighted as a crucial part of fighting the epidemic. Ferroptosis is a type of cell death linked to altered iron metabolism, glutathione depletion, glutathione peroxidase 4 (GPX4) inactivation, and increased oxidative stress. Cell death can prevent virus replication, but uncontrolled cell death can also harm the body. COVID-19 patients with multi-organ complications often exhibit factors related to ferroptosis, suggesting a possible connection. Ferroptosis inhibitors can resist SARS-CoV-2 infection from damaging vital organs and potentially reduce COVID-19 complications. In this paper, we outline the molecular mechanisms of ferroptosis and, based on this, discuss multi-organ complications in COVID-19, then explore the potential of ferroptosis inhibitors as a supplementary intervention for COVID-19. This paper will provide a reference for the possible treatment of SARS-CoV-2 infected disease to reduce the severity of COVID-19 and its subsequent impact.

Transcriptome in Human Mycoses

Mycoses are infectious diseases caused by fungi, which incidence has increased in recent decades due to the increasing number of immunocompromised patients and improved diagnostic tests. As eukaryotes, fungi share many similarities with human cells, making it difficult to design drugs without side effects. Commercially available drugs act on a limited number of targets and have been reported fungal resistance to commonly used antifungal drugs. Therefore, elucidating the pathogenesis of fungal infections, the fungal strategies to overcome the hostile environment of the host, and the action of antifungal drugs is essential for developing new therapeutic approaches and diagnostic tests. Large-scale transcriptional analyses using microarrays and RNA sequencing (RNA-seq), combined with improvements in molecular biology techniques, have improved the study of fungal pathogenicity. Such techniques have provided insights into the infective process by identifying molecular strategies used by the host and pathogen during the course of human mycoses. This chapter will explore the latest discoveries regarding the transcriptome of major human fungal pathogens. Further we will highlight genes essential for host–pathogen interactions, immune response, invasion, infection, antifungal drug response, and resistance. Finally, we will discuss their importance to the discovery of new molecular targets for antifungal drugs. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2014, 2022.

Employing an in-silico approach in the evaluation of Aloe vera metabolites as inhibitors of Omicron RBD and the ROS-dependent cellular signaling processes

The pathogenesis of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is related to increased reactive oxygen species (ROS) formation. This increasing ROS formation can mediate ROS-dependent cellular signaling processes inducing cytokines and inflammations that worsen the disease. The severity of coronavirus disease 2019 (COVID-19) can progress due to the self-sustaining cycle of ROS release, inflammatory mediators, and cellular damage. For the treatment, Aloe vera is a promising plant that has the potential to be used. In this study, therefore, we identified the metabolite composition of A. vera peel and gel using liquid chromatography-mass spectrometry (LC-MS). The metabolites were molecularly docked to Omicron receptor-binding domain (RBD) and ROS-producing enzymes to obtain medicinal compounds to inhibit these targets. The LC-MS analysis revealed the peel and gel compositions are distinct, in which 13 metabolites are identified in the peel and 12 in the gel. Furthermore, these metabolites might be promising inhibitors against Omicron variant SARS-CoV-2 RBD and ROS-producing enzymes based on the docking scores and the number of bonds formed. Thus, A. vera is one promising candidate for COVID-19 treatment due to its potential to alter the RBD function of forming a complex with ACE2 and inhibit the ROS-dependent cellular signaling processes related to COVID-19 pathogenesis and disease severity progression © 2023 Billy Johnson Kepel et al. This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/)

Recent Applications of Melanin-like Nanoparticles as Antioxidant Agents.

Nanosized antioxidants are highly advantageous in terms of versatility and pharmacokinetics, with respect to conventional molecular ones. Melanin-like materials, artificial species inspired by natural melanin, combine recognized antioxidant (AOX) activity with a unique versatility of preparation and modification. Due to this versatility and documented biocompatibility, artificial melanin has been incorporated into a variety of nanoparticles (NP) in order to give new platforms for nanomedicine with enhanced AOX activity. In this review article, we first discuss the chemical mechanisms behind the AOX activity of materials in the context of the inhibition of the radical chain reaction responsible for the peroxidation of biomolecules. We also focus briefly on the AOX properties of melanin-like NP, considering the effect of parameters such as size, preparation methods and surface functionalization on them. Then, we consider the most recent and relevant applications of AOX melanin-like NPs that are able to counteract ferroptosis and be involved in the treatment of important diseases that affect, e.g., the cardiovascular and nervous systems, as well as the kidneys, liver and articulations. A specific section will be dedicated to cancer treatment, since the role of melanin in this context is still very debated. Finally, we propose future strategies in AOX development for a better chemical understanding of melanin-like materials. In particular, the composition and structure of these materials are still debated, and they present a high level of variability. Thus, a better understanding of the mechanism behind the interaction of melanin-like nanostructures with different radicals and highly reactive species would be highly advantageous for the design of more effective and specific AOX nano-agents.

Neurological damages in COVID-19 patients: Mechanisms and preventive interventions.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus, causes coronavirus disease 2019 (COVID-19) which led to neurological damage and increased mortality worldwide in its second and third waves. It is associated with systemic inflammation, myocardial infarction, neurological illness including ischemic strokes (e.g., cardiac and cerebral ischemia), and even death through multi-organ failure. At the early stage, the virus infects the lung epithelial cells and is slowly transmitted to the other organs including the gastrointestinal tract, blood vessels, kidneys, heart, and brain. The neurological effect of the virus is mainly due to hypoxia-driven reactive oxygen species (ROS) and generated cytokine storm. Internalization of SARS-CoV-2 triggers ROS production and modulation of the immunological cascade which ultimately initiates the hypercoagulable state and vascular thrombosis. Suppression of immunological machinery and inhibition of ROS play an important role in neurological disturbances. So, COVID-19 associated damage to the central nervous system, patients need special care to prevent multi-organ failure at later stages of disease progression. Here in this review, we are selectively discussing these issues and possible antioxidant-based prevention therapies for COVID-19-associated neurological damage that leads to multi-organ failure.

Functional Role of Natural Antioxidants in Controlling Oxidative Stress Associated with SARS-CoV-2 Infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a pathogenic coronavirus that emerged in late 2019, resulting in coronavirus disease (COVID-19). COVID-19 can be potentially fatal among a certain group of patients. Older age and underlying medical illness are the major risk factors for COVID-19-related fatal respiratory dysfunction. The reason for the pathogenicity of COVID-19 in the older age group remains unclear. Factors, such as coagulopathy, cytokine storm, metabolic disrup-tion, and impaired T cell function, may worsen the symptoms of the disease. Recent literature has indicat-ed that viral infections are particularly associated with a high degree of oxidative stress and an imbalance of antioxidant response. Although pharmacological management has taken its place in reducing the severity of COVID-19, the antioxidants can serve as an adjunct therapy to protect an individual from oxidative damage triggered by SARS-CoV-2 infection. In general, antioxidant enzymes counteract free radicals and prevent their formation. The exact functional role of antioxidant supplements in reducing disease symptoms of SARS-CoV-2 infection remains mostly unknown. In this review, the functional role of natural antioxidants in SARS-CoV-2 infection management is discussed in brief.Copyright © 2022 Bentham Science Publishers.

Autonomous Medical Assistive Robot for Medical Camp

The outbreak of the Covid-19 epidemic has devastated the generation and impacted multiple layers of the healthcare sector. Resulting from this kind of exceptionally contagious virus and a shortfall of medical workers in the hospitals, front-line health workers, and patients are at risk. Thus, with an aim to diminish the risk of infections, a mobile robotic system is proposed that can autonomously ensure safety and protection in the hospital. The system can monitor the patients by moving autonomously and sanitizing the floor throughout the hospital, which is implemented by Robot Operating System (ROS), SLAM (Simultaneous Localization and Mapping) algorithm, and A∗ search algorithm, and then it uses the MobileNetV2 algorithm for safety mask detection and giving voice alert. The system also offers AI voice communication to assist and diagnose the patients, which can lessen person-to-person contact. The system has anticipated 89% accuracy for AI custom dataset, whereas the validation accuracy for face mask detection is 95%. © 2022 IEEE.

Comparison of oxidative stress response of in vitro retinal cells exposed to blue light from emissive versus reflective displays

During COVID-19, there was increased use of handheld displays in educational settings. There is growing concern that eye health may be affected by prolonged exposure to the light-emitting diodes used as frontlights or backlights in handheld displays. The potential impact of light exposure from tablet-sized devices with different display technologies and various spectral outputs was assessed in an in vitro model using human retinal epithelial (ARPE-19) cells. Cellular response was quantified by measuring reactive oxidative species (ROS) and by analyzing mitochondrial morphology. Control experiments established a baseline ROS response to hazardous blue light exposure and also that red light resulted in no detectable ROS response. Under identical conditions, ROS response increased with time for all devices. However, different device spectra caused ROS to accumulate at different rates. When operating the devices in the same mode (day or night), cells accumulated ROS two to three times more slowly on exposure to frontlit electronic paper displays compared to backlit liquid crystal displays. With increasing ROS accumulation, mitochondrial morphology shifted from elongate interconnected features typically observed under normal conditions to rounded disconnected features associated with oxidative stress response. © 2023 E Ink Corporation. Journal of the Society for Information Display published by Wiley Periodicals LLC on behalf of Society for Information Display.

Cerium Oxide Nanoparticles Applications in the Field of Cardiovascular Diseases

Cardiovascular disease (CVD) is the sixth mos common human disease that causes about 17.3 million deaths yearly Advancement in nanomedicine enables the usage of nanomaterials fo treating cardiac disorders. Among these, Cerium Oxide (CeO2 nanoparticle is gaining attention due to their irreversible oxidativ states (Ce3+ and Ce4+), which provide them with many unique physiologically important traits like antioxidant property, Anti inflammatory property, free radicle scavenging potential etc. All thes features make CeO2 an excellent agent for treating many cardia related disorders. We have used the search engines PubMed an Google scholar to identify the relevant papers in last 5 years on thi topic. Some old classical articles were also identified, and the review was written based on these relevant papers. Cerium nanoparticle (CeNPs) are being used in the therapy of CVD mainly for reducin oxidative stress, inflammation, and damage caused by free radicals CeO2 can also protect endothelial cells (ECs) from apoptosis. It can b used as a wound-healing agent during cardiac surgeries. This i because of its potential to enhance endothelialization. In this article we reviewed various applications of CeO2 in the field of CVD.Copyright © 2023 Society of Pharmaceutical Sciences and Research. All rights reserved.

Is copper beneficial for COVID-19 patients?

Copper (Cu) is an essential micronutrient for both pathogens and the hosts during viral infection. Cu is involved in the functions of critical immune cells such as T helper cells, B cells, neutrophils natural killer (NK) cells, and macrophages. These blood cells are involved in the killing of infectious microbes, in cell-mediated immunity and the production of specific antibodies against the pathogens. Cu-deficient humans show an exceptional susceptibility to infections due to the decreased number and function of these blood cells. Besides, Cu can kill several infectious viruses such as bronchitis virus, poliovirus, human immunodeficiency virus type 1(HIV-1), other enveloped or nonenveloped, single- or double-stranded DNA and RNA viruses. Moreover, Cu has the potent capacity of contact killing of several viruses, including SARS-CoV-2. Since the current outbreak of the COVID-19 continues to develop, and there is no vaccine or drugs are currently available, the critical option is now to make the immune system competent to fight against the SARS-CoV-2. Based on available data, we hypothesize that enrichment of plasma copper levels will boost both the innate and adaptive immunity in people. Moreover, owing to its potent antiviral activities, Cu may also act as a preventive and therapeutic regime against COVID-19.

Reactive oxygen species are associated with the inhibitory effect of N-(4-hydroxyphenyl)-retinamide on the entry of the severe acute respiratory syndrome-coronavirus 2.

N-(4-hydroxyphenyl)-retinamide (4-HPR) inhibits the dihydroceramide Δ4-desaturase 1 (DEGS1) enzymatic activity. We previously reported that 4-HPR suppresses the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) spike protein-mediated membrane fusion through a decrease in membrane fluidity in a DEGS1-independent manner. However, the precise mechanism underlying the inhibition of viral entry by 4-HPR remains unclear. In this study, we examined the role of reactive oxygen species (ROS) in the inhibition of membrane fusion by 4-HPR because 4-HPR is a well-known ROS-inducing agent. Intracellular ROS generation was found to be increased in the target cells in a cell-cell fusion assay after 4-HPR treatment, which was attenuated by the addition of the antioxidant, α-tocopherol (TCP). The reduction in membrane fusion susceptibility by 4-HPR treatment in the cell-cell fusion assay was alleviated by TCP addition. Furthermore, fluorescence recovery after photobleaching analysis showed that the lateral diffusion of glycosylphosphatidylinositol-anchored protein and SARS CoV-2 receptor was reduced by 4-HPR treatment and restored by TCP addition. These results indicate that the decrease in SARS-CoV-2 spike protein-mediated membrane fusion and membrane fluidity by 4-HPR was due to ROS generation. Taken together, these results demonstrate that ROS production is associated with the 4-HPR inhibitory effect on SARS-CoV-2 entry.

Direct Application of 3-Maleimido-PROXYL for Proving Hypoalbuminemia in Cases of SARS-CoV-2 Infection: The Potential Diagnostic Method of Determining Albumin Instability and Oxidized Protein Level in Severe COVID-19.

Oxidative stress and the albumin oxidized form can lead to hypoalbuminemia, which is a predisposing factor for reduced treatment effectiveness and an increased mortality rate in severe COVID-19 patients. The aim of the study is to evaluate the application of free radical 3-Maleimido-PROXYL and SDSL-EPR spectroscopy in the in vitro determination of ox/red HSA in serum samples from patients with SARS-CoV-2 infection. Venous blood was collected from patients intubated (pO2 < 90%) with a positive PCR test for SARS-CoV-2 and controls. At the 120th minute after the incubation of the serum samples from both groups with the 3-Maleimido-PROXYL, the EPR measurement was started. The high levels of free radicals were determined through the nitroxide radical TEMPOL, which probably led to increased oxidation of HSA and hypoalbuminemia in severe COVID-19. The double-integrated spectra of 3-Maleimido-PROXYL radical showed a low degree of connectivity due to high levels of oxidized albumin in COVID-19 patients. The low concentrations of reduced albumin in serum samples partially inhibit spin-label rotation, with Amax values and ΔH0 spectral parameters comparable to those of 3-Maleimido-PROXYL/DMSO. Based on the obtained results, we suggest that the stable nitroxide radical 3-Maleimido-PROXYL can be successfully used as a marker to study oxidized albumin levels in COVID-19.

Susceptibility of SARS COV-2 nucleocapsid and spike proteins to reactive oxygen species and role in inflammation.

Chemiluminescence was used to test the susceptibility of the SARS-CoV-2 N and S proteins to oxidation by reactive oxygen species (ROS) at pH 7.4 and pH 8.5. The Fenton's system generates various ROS (H2O2, OH, -OH, OOH). All proteins were found to significantly suppress oxidation (the viral proteins exhibited 25-60% effect compared to albumin). In the second system, H2O2 was used both as a strong oxidant and as a ROS. A similar effect was observed (30-70%); N protein approached the effect of albumin at physiological pH (∼45%). In the O2.--generation system, albumin was most effective in the suppression of generated radicals (75%, pH 7.4). The viral proteins were more susceptible to oxidation (inhibition effect no more than 20%, compared to albumin). The standard antioxidant assay confirmed the strong antioxidant capacity of both viral proteins (1.5-1.7 fold higher than albumin). These results demonstrate the effective and significant inhibition of ROS-induced oxidation by the proteins. Obviously, the viral proteins could not be involved in the oxidative stress reactions during the course of the infection. They even suppress the metabolites involved in its progression. These results can be explained by their structure. Probably, an evolutionary self-defense mechanism of the virus has been developed.

Multi-omics of NET formation and correlations with CNDP1, PSPB, and L-cystine levels in severe and mild COVID-19 infections.

The detailed mechanisms of COVID-19 infection pathology remain poorly understood. To improve our understanding of SARS-CoV-2 pathology, we performed a multi-omics and correlative analysis of an immunologically naïve SARS-CoV-2 clinical cohort from blood plasma of uninfected controls, mild, and severe infections. Consistent with previous observations, severe patient populations showed an elevation of pulmonary surfactant levels. Intriguingly, mild patients showed a statistically significant elevation in the carnosine dipeptidase modifying enzyme (CNDP1). Mild and severe patient populations showed a strong elevation in the metabolite L-cystine (oxidized form of the amino acid cysteine) and enzymes with roles in glutathione metabolism. Neutrophil extracellular traps (NETs) were observed in both mild and severe populations, and NET formation was higher in severe vs. mild samples. Our correlative analysis suggests a potential protective role for CNDP1 in suppressing PSPB release from the pulmonary space whereas NET formation correlates with increased PSPB levels and disease severity. In our discussion we put forward a possible model where NET formation drives pulmonary occlusions and CNDP1 promotes antioxidation, pleiotropic immune responses, and vasodilation by accelerating histamine synthesis.

Anti-Staphylococcal Activities of Rosmarinus officinalis and Myrtus communis Essential Oils through ROS-Mediated Oxidative Stress.

Rosmarinus officinalis and Myrtus communis essential oils (EOs) are well-known for their ethno-pharmaceutical properties. In the present study, we have analyzed the chemical composition of both EOs by gas chromatography-mass spectrometry. Then we assessed their antibacterial, antibiofilm, and anti-virulence actions against the opportunistic pathogen Staphylococcus aureus. The cytotoxic effect of agents tested against this bacterium was investigated by monitoring reactive oxygen-species (ROS) generation and antioxidant-enzyme (catalase) production. Regarding the antistaphylococcal effects, our results showed antibacterial efficacy of both Eos and their combination, where the minimum inhibitory concentrations ranged between 0.7 and 11.25 mg/mL. A combination of tested agents showed the highest anti-hemolytic and anti-protease effects. Additionally, association between EOs displayed more potency against the development of biofilm performed by S. aureus, with percentage of removal reaching 74%. The inhibitory impacts of EOs on S. aureus virulence factors were discovered to be concentration-dependent. Furthermore, our results provide insight on the abilities of R. officinalis and M. communis EOs, as well as their potential in combination, to generate ROS and affect oxidative stress enzyme catalase in S. aureus, leading to their antagonistic effect against this pathogen.

Elevated neutrophil extracellular traps by HBV-mediated S100A9-TLR4/RAGE-ROS cascade facilitate the growth and metastasis of hepatocellular carcinoma.

BACKGROUND: Neutrophil extracellular traps (NETs) are considered significant contributors to cancer progression, especially metastasis. However, it is still unclear whether NETs are involved in hepatitis B virus (HBV)-related hepatocarcinogenesis and have potential clinical significance during evaluation and management for hepatocellular carcinoma (HCC). In this study, we aimed to investigate the functional mechanism of NETs in HBV-related hepatocarcinogenesis and their clinical significance. METHODS: A total of 175 HCC patients with and without HBV infection and 58 healthy controls were enrolled in this study. NETs were measured in tissue specimens, freshly isolated neutrophils and blood serum from these patients, and the correlation of circulating serum NETs levels with malignancy was evaluated. The mechanism by which HBV modulates NETs formation was explored using cell-based studies. In addition, in vitro and in vivo experiments were further performed to clarify the functional mechanism of NETs on the growth and metastasis of HCC. RESULTS: We observed an elevated level of NETs in blood serum and tissue specimens from HCC patients, especially those infected with HBV. NETs facilitated the growth and metastasis of HCC both in vitro and in vivo, which were mainly dominated by increased angiogenesis, epithelial-mesenchymal transition (EMT)-related cell migration, matrix metalloproteinases (MMPs)-induced extracellular matrix (ECM) degradation and NETs-mediated cell trapping. Inhibition of NETs generation by DNase 1 effectively abrogated the NETs-aroused HCC growth and metastasis. In addition, HBV-induced S100A9 accelerated the generation of NETs, which was mediated by activation of toll-like receptor (TLR4)/receptor for advanced glycation end products (RAGE)-reactive oxygen species (ROS) signaling. Further, circulatory NETs were found to correlate with viral load, TNM stage and metastasis status in HBV-related HCC, and the identified NETs could predict extrahepatic metastasis, with an area under the ROC curve (AUC) of 0.83 and 90.3% sensitivity and 62.8% specificity at a cutoff value of 0.32. CONCLUSIONS: Our findings indicated that activation of RAGE/TLR4-ROS signaling by HBV-induced S100A9 resulted in abundant NETs formation, which subsequently facilitated the growth and metastasis of HCC cells. More importantly, the identified circulatory NETs exhibited potential as an alternative biomarker for predicting extrahepatic metastasis in HBV-related HCC.