Bacteriocins: Curial guardians of gastrointestinal tract.

The human gastrointestinal (GI) tract microbiome secretes various metabolites that play pivotal roles in maintaining host physiological balance and influencing disease progression. Among these metabolites, bacteriocins-small, heat-stable peptides synthesized by ribosomes-are notably prevalent in the GI region. Their multifaceted benefits have garnered significant interest in the scientific community. This review comprehensively explores the methods for mining bacteriocins (traditional separation and purification, bioinformatics, and artificial intelligence), their effects on the stomach and intestines, and their complex bioactive mechanisms. These mechanisms include flora regulation, biological barrier restoration, and intervention in epithelial cell pathways. By detailing each well-documented bacteriocin, we reveal the diverse ways in which bacteriocins interact with the GI environment. Moreover, the future research direction is prospected. By further studying the function and interaction of intestinal bacteriocins, we can discover new pharmacological targets and develop drugs targeting intestinal bacteriocins to regulate and improve human health. It provides innovative ideas and infinite possibilities for further exploration, development, and utilization of bacteriocins. The inevitable fact is that the continuously exploration of bacteriocins is sure to bring the promising future for demic GI health understanding and interference strategy.

The lipidation and glycosylation enabling bioactivity enhancement and structural change of antibacterial peptide G3.

Bacterial resistance has led to increased interest in the use of antibacterial peptides (AMPs), but their clinical application is limited by poor stability and solubility, as well as complex cytotoxicity. Chemical modification is a common strategy to modulate AMPs. In this study, a de novo designed AMP (G3) was modified by adding an alkyl acid at the N-terminal and a monosaccharide at the C-terminal. Bio-activity assays demonstrated that conjugation with n-caprylic acid increased the peptide's antibacterial activity and permeabilized the membrane. Attachment of glucose or galactose at the C-terminal improved its biofilm inhibitory capacity and marginally reduced cytotoxicity. The hybrid peptide, containing both n-caprylic acid and galactose, exhibited excellent antibacterial and antibiofilm activity, as well as permeabilized the outer membrane.

Exosome in Crosstalk between Inflammation and Angiogenesis: A Potential Therapeutic Strategy for Stroke.

The endothelial dysfunction, associated with inflammation and vascular permeability, remains the key event in the pathogenesis of cerebral ischemic stroke. Angiogenesis is essential for neuroprotection and neural repair following stroke. The neuroinflammatory reaction plays a vital role in stroke, and inhibition of inflammation contributes to establishing an appropriate external environment for angiogenesis. Exosomes are the heterogeneous population of extracellular vesicles which play critical roles in intercellular communication through transmitting various proteins and nucleic acids to nearby and distant recipient cells by body fluids and circulation. Recent reports have shown that exosomal therapy is a valuable and potential treatment strategy for stroke. In this review, we discussed the exosomes in complex interaction mechanisms of angiogenesis and inflammation following stroke as well as the challenges of exosomal studies such as secretion, uptake, modification, and application.

A novel bacteriocin against methicillin-resistant Staphylococcus aureus, purified from Lactiplantibacillus plantarum ZFM9.

A novel bacteriocin, plantaricin ZFM9, was purified from Lactiplantibacillus plantarum ZFM9 using a combination of ammonium sulfate precipitation, XAD-2 macroporous resin, Sephadex G-50, Sephadex LH-20, and reversed-phase high performance liquid chromatography. The molecular mass of plantaricin ZFM9 was 1151.606 Da, and the purity was 98.3%. Plantaricin ZFM9 has thermal stability (95.6% retention at 120 °C for 30 min), pH stability (pH ≤ 5), and sensitivity to the pepsin, trypsin, papain, and proteinase K. Plantaricin ZFM9 exhibited broad-spectrum antimicrobial activity and notably inhibit methicillin-resistant Staphylococcus aureus D48 (MRSA). According to the results of electron microscopy and fluorescence leakage assay, it was found that plantaricin ZFM9 caused damage to the cells membrane and leakage of the contents of S. aureus D48. In addition, Lipid II was not the anti-MRSA target of plantaricin ZFM9. This study underscores the potential of plantaricin ZFM9 for applications in the food field and biopharmaceuticals against MRSA infection.

Phenyl lactic acid alleviates Helicobacter pylori infection in C57BL/6 mice.

Helicobacter pylori is a gastric pathogen that colonizes approximately 50% of the world's population. Infection with H. pylori causes chronic inflammation and significantly increases the risk of developing duodenal and gastric ulcer disease and gastric cancer. In the present study, we found that phenyl lactic acid (PLA) derived from Lactobacillus plantarum ZJ316 (L. plantarum ZJ316) can directly inhibit the growth and urease activity of H. pylori in vitro with a minimum inhibitory concentration (MIC) of 2.5 mg mL-1. Moreover, PLA also caused a dramatic morphological transformation from a spiral to a coccoid form in H. pylori. In this work, we also analyzed the beneficial effects of PLA in mice. The results showed that PLA administration ameliorated H. pylori-induced gastric mucosal damage and significantly decreased lymphocyte infiltration and inflammatory cytokines, including interleukin-1ß (IL-1ß), interleukin 6 (IL-6), and interferon-γ (IFN-γ) by 59.93%, 63.95%, and 48.05%, respectively, but elevated the interleukin-10 (IL-10) and glutathione (GSH) levels. Furthermore, PLA administration improved microbiota diversity with increased Bacteroidetes abundance and decreased Proteobacteria abundance by 46.39% and 24.05%, respectively. PLA also significantly reduced the abundance of H. pylori but increased the relative abundances of potential beneficial bacteria, such as Faecalibacterium, Bifidobacterium, and Lactobacillus. These results demonstrated that PLA can ameliorate H. pylori-induced inflammation and support beneficial gut bacteria, providing a new perspective against H. pylori infection.

Current evidence, clinical applications, and future directions of transcranial magnetic stimulation as a treatment for ischemic stroke.

Transcranial magnetic stimulation (TMS) is a non-invasive brain neurostimulation technique that can be used as one of the adjunctive treatment techniques for neurological recovery after stroke. Animal studies have shown that TMS treatment of rats with middle cerebral artery occlusion (MCAO) model reduced cerebral infarct volume and improved neurological dysfunction in model rats. In addition, clinical case reports have also shown that TMS treatment has positive neuroprotective effects in stroke patients, improving a variety of post-stroke neurological deficits such as motor function, swallowing, cognitive function, speech function, central post-stroke pain, spasticity, and other post-stroke sequelae. However, even though numerous studies have shown a neuroprotective effect of TMS in stroke patients, its possible neuroprotective mechanism is not clear. Therefore, in this review, we describe the potential mechanisms of TMS to improve neurological function in terms of neurogenesis, angiogenesis, anti-inflammation, antioxidant, and anti-apoptosis, and provide insight into the current clinical application of TMS in multiple neurological dysfunctions in stroke. Finally, some of the current challenges faced by TMS are summarized and some suggestions for its future research directions are made.

The Effect of Indole-3-Lactic Acid from Lactiplantibacillus plantarum ZJ316 on Human Intestinal Microbiota In Vitro.

Microbiota-derived tryptophan metabolites are essential signals for maintaining gut homeostasis, yet the potential contribution to modulating gut microbiota has been rarely investigated. In this study, Lactiplantibacillus plantarum ZJ316 (CCTCC No. M 208077) with a high production (43.14 µg/mL) of indole-3-lactic acid (ILA) was screened. ILA with 99.00% purity was prepared by macroporous resin, Sephadex G-25 and reversed-phase high-performance liquid chromatography. Purified ILA can effectively inhibit foodborne pathogens such as Salmonella spp., Staphylococcus spp., Escherichia coli and Listeria monocytogenes. In an in vitro model of the human gut microbiota, a medium-dose ILA (172 mg/L) intervention increased the average relative abundance of phyla Firmicutes and Bacteroidota by 9.27% and 15.38%, respectively, while Proteobacteria decreased by 14.36% after 24 h fermentation. At the genus level, the relative abundance of Bifidobacterium and Faecalibacterium significantly increased to 5.36 ± 2.31% and 2.19 ± 0.77% (p < 0.01), respectively. Escherichia and Phascolarctobacterium decreased to 16.41 ± 4.81% (p < 0.05) and 2.84 ± 1.02% (p < 0.05), respectively. Intestinal short-chain fatty acids, especially butyric acid, were significantly increased (2.98 ± 0.72 µmol/mL, p < 0.05) and positively correlated with Oscillospira and Collinsella. Overall, ILA has the potential to regulate the gut microbiota, and an in-depth understanding of the relationship between tryptophan metabolites and gut microbiota is needed in the future.

Purification and characterization of bacteriocin produced by a strain of Lacticaseibacillus rhamnosus ZFM216.

The recent surge in demand for natural preservatives has ushered in a new era of research into novel bacteriocins capable of effectively combating food-borne infections. In this study, the bacteriocin from Lacticaseibacillus rhamnosus ZFM216, which has a molecular mass of 11851.9 Da, was purified using macroporous resin, gel chromatography, and reversed-phase high performance liquid chromatography. This bacteriocin could inhibit both Gram-positive and Gram-negative bacteria. It had a strong inhibitory effect on Staphylococcus aureus D48 with minimum inhibitory concentration values of 1.75 µM. Bacteriocin ZFM216 was heat stable and showed pH stability under weakly acidic conditions. It was sensitive to pepsin, proteinase K and trypsin. Electron microscopy results showed that when treated with bacteriocin ZFM216, S. aureus D48 was severely deformed, the cell structure was obviously changed, and the intracellular electrolyte leaked to the outside of the cell. Bacteriocin ZFM216 caused the ATP level of the indicator to decrease, the conductivity to sharply increase, and the transmembrane potential difference (ΔΨ) to instantaneously decrease. This research formed the basis for further development and utilization of bacteriocin ZFM216 which has potential in the food industry.

The roles, mechanism, and mobilization strategy of endogenous neural stem cells in brain injury.

Brain injury poses a heavy disease burden in the world, resulting in chronic deficits. Therapies for brain injuries have been focused on pharmacologic, small molecule, endocrine and cell-based therapies. Endogenous neural stem cells (eNSCs) are a group of stem cells which can be activated in vivo by damage, neurotrophic factors, physical factor stimulation, and physical exercise. The activated eNSCs can proliferate, migrate and differentiate into neuron, oligodendrocyte and astrocyte, and play an important role in brain injury repair and neural plasticity. The roles of eNSCs in the repair of brain injury include but are not limited to ameliorating cognitive function, improving learning and memory function, and promoting functional gait behaviors. The activation and mobilization of eNSCs is important to the repair of injured brain. In this review we describe the current knowledge of the common character of brain injury, the roles and mechanism of eNSCs in brain injury. And then we discuss the current mobilization strategy of eNSCs following brain injury. We hope that a comprehensive awareness of the roles and mobilization strategy of eNSCs in the repair of cerebral ischemia may help to find some new therapeutic targets and strategy for treatment of stroke.

Therapeutic effect of AiWalker on balance and walking ability in patients with stroke: A pilot study.

BACKGROUND: AiWalker is a newly developed robot-assisted gait training system, which features over-ground walking paradigm and somatosensory stimulation during training compared to commonly-used robot-assisted gait training devices (e.g. Lokomat). However, no study has examined its true therapeutic effect and possible mechanism or mediating factor(s). OBJECTIVES: To investigate 1) the therapeutic effect of AiWalker on the balance and walking ability in patients with stroke, and 2) whether the improvement in somatosensory function represents one of the possible mediating factors for such effect. METHODS: Three patients with impaired balance and walking ability due to stroke were recruited. Two patients received AiWalker training plus conventional training; while the other one only experienced conventional training. Standing balance and walking ability were assessed before and after all the training, which were represented by 6 variables. Lower limb somatosensory function was examined using Fugl-Meyer Assessment Scale. RESULTS: Five out of the 6 variables showed greater changes in patients who received AiWalker training compared to the one who only experienced conventional training. Greater improvement in lower limb somatosensory function was observed in one patient who received AiWalker training compared to the one who only experienced conventional training. CONCLUSION: The novel robot-assisted gait training system may elicit greater improvement of balance and walking ability in patients with stroke compared to conventional interventions. Lower limb somatosensory function may be improved by AiWalker, and its improvement might represent one of the possible mediating factors for the therapeutic effect of AiWalker on balance and walking ability.

A Novel Bacteriocin From Lactobacillus Pentosus ZFM94 and Its Antibacterial Mode of Action.

Bacteriocins are bioactive antimicrobial peptides synthesized in the ribosome of numerous bacteria and released extracellularly. Pentocin ZFM94 produced by Lactobacillus pentosus (L. pentosus) ZFM94, isolated from infant feces with strong antibacterial activity, was purified by ammonium sulfate precipitation, dextran gel chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The molecular mass of the purified bacteriocin was 3,547.74 Da determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Pentocin ZFM94 exhibited broad-spectrum antimicrobial activity against tested Gram-positive and Gram-negative bacteria, and the minimal inhibitory concentrations (MICs) of Micrococcus luteus (M. luteus) 10,209, Staphylococcus aureus (S. aureus) D48, and Escherichia coli (E. coli) DH5α were 1.75, 2.00, and 2.50 µm, respectively. Pentocin ZFM94 was heat-stable (30 min at 80°C) and showed inhibitory activity over a wide pH range (5.00-7.00). It could be degraded by trypsin and pepsin, but not by amylase, lysozyme, lipase, and ribonuclease A. Fluorescence leakage assay showed that pentocin ZFM94 induced disruption of the cell membrane and caused leakage of cellular content. Furthermore, lipid II was not an antibacterial target of pentocin ZFM94. This study laid the foundation for further development and utilization of L. pentosus ZFM94 and its bacteriocin.

Agrin Involvement in Synaptogenesis Induced by Exercise in a Rat Model of Experimental Stroke.

BACKGROUND: Agrin is a proteoglycan that aggregates nicotinic acetylcholine receptors (AChRs) on neuromuscular junctions and takes part in synaptogenesis in the development of the central nervous system. However, its effects on neural repair and synaptogenesis after stroke are still unclear. OBJECTIVE: This study aimed to investigate the effects of agrin on neural repair and synaptogenesis after stroke and the effects of exercise on this process in vivo and in vitro. METHODS: Exercise with gradually increased intensity was initiated at 1 day after middle cerebral artery occlusion (MCAO) for a maximum of 14 days. Neurological deficit scores and foot fault tests were used to assess the behavioral recovery. Western blotting, immunofluorescence, and electron microscopic images were used to detect the expression of agrin, synaptogenesis-related proteins, and synaptic density in vivo. In vitro, the ischemic neuron model was established via oxygen-glucose deprivation (OGD). The lentivirus overexpressed agrin and CREB inhibitor were used to investigate the mechanism by which agrin promoted synaptogenesis. RESULTS: Exercise promoted behavioral recovery and this beneficial role was linked to the upregulated expression of agrin and increased synaptic density. Overexpressed agrin promoted synaptogenesis in OGD neuron, CREB inhibitor downregulated the expression of agrin and hampered synaptogenesis in cultured neurons. CONCLUSIONS: These results indicated that exercise poststroke improved the recovery of behavioral function after stroke. Synaptogenesis was an important and beneficial factor, and agrin played a critical role in this process and could be a potential therapeutic target for the treatment of stroke and other nervous system diseases.

Isolation and functional identification of a novel human hepatic growth factor: hepatopoietin Cn.

UNLABELLED: Hepatic stimulating substance (HSS) was first isolated from weanling rat liver in 1975 and found to stimulate hepatic DNA synthesis both in vitro and in vivo. Since then, mammalian and human HSS have been investigated for their potential to treat hepatic diseases. However, the essential nature in composition and structure of HSS remain puzzling because HSS has not been completely purified. Heating, ethanol precipitation, and ion-exchange chromatographies had been carried out to isolate the protein with specific stimulating activity from newborn calf liver, and [(3)H]thymidine deoxyribose (TdR)/bromodeoxyuridine (BrdU) incorporation and carboxyfluorescein diacetate succinimidyl ester (CFSE)-based proliferation assay to determine the bioactivity in vitro and in vivo. We report the purification of a novel 30-kDa protein from a crude extract of calf liver HSS. This protein is a member of the leucine-rich acidic nuclear protein family (LANP) and has been named hepatopoietin Cn (HPPCn). Studies of partially hepatectomized (PH) mice show that levels of HPPCn messenger RNA (mRNA) increase after liver injury. Furthermore, the recombinant human protein (rhHPPCn) was shown to stimulate hepatic DNA synthesis and activate signaling pathways involved in hepatocyte proliferation in vitro and in vivo. CONCLUSION: HPPCn is a novel hepatic growth factor that plays a role in liver regeneration.

Treatment of chronical myocardial ischemia by adenovirus-mediated hepatocyte growth factor gene transfer in minipigs.

Growth factor gene transfer-induced therapeutic angiogenesis has become a novel approach for the treatment of myocardial ischemia. In order to provide a basis for the clinical application of an adenovirus with hepatocyte growth factor gene (Ad-HGF) in the treatment of myocardial ischemia, we established a minipig model of chronically ischemic myocardium in which an Ameroid constrictor was placed around the left circumflex branch of the coronary artery (LCX). A total of 18 minipigs were randomly divided into 3 groups: a surgery control group, a model group and an Ad-HGF treatment group implanted with Ameroid constrictor. Ad-HGF or the control agent was injected directly into the ischemic myocardium, and an improvement in heart function and blood supply were evaluated. The results showed that myocardial perfusion remarkably improved in the Ad-HGF group compared with that in both the control and model groups. Four weeks after the treatment, the density of newly formed blood vessels was higher and the number of collateral blood vessels was greater in the Ad-HGF group than in the model group. The area of myocardial ischemia reduced evidently and the left ventricular ejection fraction improved significantly in the Ad-HGF group. These results suggest that HGF gene therapy may become a novel approach in the treatment of chronically ischemic myocardium.

Fatty Acid Oxidation-Driven Src Links Mitochondrial Energy Reprogramming and Oncogenic Properties in Triple-Negative Breast Cancer.

Transmitochondrial cybrids and multiple OMICs approaches were used to understand mitochondrial reprogramming and mitochondria-regulated cancer pathways in triple-negative breast cancer (TNBC). Analysis of cybrids and established breast cancer (BC) cell lines showed that metastatic TNBC maintains high levels of ATP through fatty acid ß oxidation (FAO) and activates Src oncoprotein through autophosphorylation at Y419. Manipulation of FAO including the knocking down of carnitine palmitoyltransferase-1A (CPT1) and 2 (CPT2), the rate-limiting proteins of FAO, and analysis of patient-derived xenograft models confirmed the role of mitochondrial FAO in Src activation and metastasis. Analysis of TCGA and other independent BC clinical data further reaffirmed the role of mitochondrial FAO and CPT genes in Src regulation and their significance in BC metastasis.

Rescuing lymphocytes from HLA-G immunosuppressive effects mediated by the tumor microenvironment.

Several studies have demonstrated that the antitumor activities of both T and natural killer (NK) effector populations are limited by the immunosuppressive strategies of tumors. In several malignant transformations, the expression of HLA-G by tumor cells rises dramatically, rendering them strongly immunosuppressive. In this study, we postulated that the absence of HLA-G receptors would prevent the immunosuppressive effects of both soluble and membrane-bound HLA-G. Thus, we investigated the therapeutic potential of effector NK cells genetically modified to downregulate the expression of ILT2 (HLA-G receptor) on their cell surfaces. We have shown that the proliferation of modified NK is still dependent on stimulation signals (no malignant transformation). ILT2- NK cells proliferate, migrate, and eliminate HLA-G negative targets cells to the same extent parental NK cells do. However, in the presence of HLA-G positive tumors, ILT2- NK cells exhibit superior proliferation, conjugate formation, degranulation, and killing activities compared to parent NK cells. We tested the effectiveness of ILT2- NK cells in vivo using a xenograft cancer model and found that silencing ILT2 rescued their anti-tumor activity.We believe that combining ILT2- NK cells with existing therapeutic strategies will strengthen the antitumor response in cancer patients.

Myocardin-A enhances expression of promyogenic genes without depressing telomerase activity in adipose tissue-derived mesenchymal stem cells.

BACKGROUND: The stromal compartment of adult adipose tissue consists of mesenchymal stem cells (MSCs) characterized by co-expression of myocardin-A (McA) and telomerase reverse transcriptase (TERT). OBJECTIVE: This study aims at testing the growth and myogenic regenerative properties of adipose tissue-derived MSCs, and the outcome of McA and TERT overexpression or suppression. METHODS AND RESULTS: TERT and/or McA in murine MSCs derived from adult adipose tissue were transduced with cDNA and co-immunoprecipitated with specific antibodies. The MSCs with TERT or TERT plus McA overexpressed were highly proliferative. Bioluminescence resonance energy transfer assay evidenced close interactions between TERT and McA in MSCs co-transfected with TERT and McA, which expressed the stem cell oncogene Oct-4, and promyogenic genes GATA-4, Nkx2.5, MLC2v, Mef2c, DTEF and McA. The increased myogenic gene expression depended on TERT and McA expression as siRNAs for TERT and McA diminished the TERT activities. The co-transfected MSCs also developed a stronger activity of serum response factor, a key factor for expression of cardiomyogenic genes. McA overexpression did not interfere with TERT-mediated proliferative effects, and rather slightly increased proliferation of MSCs. CONCLUSIONS: McA and TERT may interplay in promoting promyogenic gene expression and maintaining growth capacity of MSCs.

Blocking KCa3.1 channels increases tumor cell killing by a subpopulation of human natural killer lymphocytes.

Natural killer (NK) cells are large granular lymphocytes that participate in both innate and adaptive immune responses against tumors and pathogens. They are also involved in other conditions, including organ rejection, graft-versus-host disease, recurrent spontaneous abortions, and autoimmune diseases such as multiple sclerosis. We demonstrate that human NK cells express the potassium channels Kv1.3 and KCa3.1. Expression of these channels does not vary with expression levels of maturation markers but varies between adherent and non-adherent NK cell subpopulations. Upon activation by mitogens or tumor cells, adherent NK (A-NK) cells preferentially up-regulate KCa3.1 and non-adherent (NA-NK) cells preferentially up-regulate Kv1.3. Consistent with this different phenotype, A-NK and NA-NK do not display the same sensitivity to the selective KCa3.1 blockers TRAM-34 and NS6180 and to the selective Kv1.3 blockers ShK-186 and PAP-1 in functional assays. Kv1.3 block inhibits the proliferation and degranulation of NA-NK cells with minimal effects on A-NK cells. In contrast, blocking KCa3.1 increases the degranulation and cytotoxicity of A-NK cells, but not of NA-NK cells. TRAM-34, however, does not affect their ability to form conjugates with target tumor cells, to migrate, or to express chemokine receptors. TRAM-34 and NS6180 also increase the proliferation of both A-NK and NA-NK cells. This results in a TRAM-34-induced increased ability of A-NK cells to reduce in vivo tumor growth. Taken together, our results suggest that targeting KCa3.1 on NK cells with selective blockers may be beneficial in cancer immunotherapy.

Induction and testing of hypoxia in cell culture.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers, rheumatoid arthritis, atherosclerosis etc… Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia. In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Production and detection of reactive oxygen species (ROS) in cancers.

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H(2;)O(2;) (hydrogen peroxide), NO (nitric oxide), O(2;)(-) (oxide anion), peroxynitrite (ONOO(-)), hydrochlorous acid (HOCl), and hydroxyl radical (OH(-)). Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases, autoimmune diseases , etc…) but also during physiological (non-pathological) situations such as cellular metabolism. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation, migration etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants). Free radicals are beneficial in low amounts. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise. Several publications have also demonstrated that ROS are involved in insulin sensitivity. Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.