Determination Of Reactive Oxygen Species Research Articles

Comparison of Free‐Hydroxy With 3′,5′‐Di‐O‐Acetyl Ribose of 5‐Alkynyl Dicobalt Hexacarbonyl 2′‐Deoxy‐Furopyrimidines Hybrids: Synthesis, Antiproliferative Activity, and ROS Determination

ABSTRACTSynthesis of 5‐alkynyl furopyrimidine nucleoside analogs, with free‐ribose groups, was carried out, and their biological activity was evaluated. The substituents introduced at the C‐5 included propargyl and homopropargyl alcohols, 4‐methylphenyl (p‐tolyl), and 4‐pentylphenyl substituted alkynyl groups (56%–88%). Subsequently, alkyne function was coordinated to dicobalt hexacarbonyl unit, yielding the corresponding dicobalt hexacarbonyl 5‐alkynyl furopyrimidine nucleosides (51%–75%). All compounds contained 4‐pentylphenyl group attached at the C‐6 position of the bicyclic base, in line with most active antiviral structures. The antiproliferative effect of these modified nucleosides on cancer cells of different phenotypes was determined using in vitro studies. The tested compounds show antiproliferative effects with median inhibitory concentration (IC50) values of 16–23 and 9–15 μM for HeLa and K562 cells, respectively. The determination of reactive oxygen species (ROS) formation in K562 cells in the presence of modified nucleosides may suggest that the mode of action of the examined compounds may be attributed to the induction of oxidative stress.

Sunlight-mediated removal of endocrine disruptors from wastewater using trimetallic core-shell Ag-TeO2@ZnO nanocomposites

The current work emphasizes the preparation of trimetallic core-shell Ag–TeO2@ZnO nanocomposites (NCs) by thermo-mechanical method for the efficient photocatalytic degradation of 2,4-Dichlorophenol and β-naphthol pollutants. FE-SEM shows that Ag and TeO2 nanoparticles are deposited on the surface of ZnO nanotubes. The band gap of pristine ZnO NPs and 5 wt% Ag–TeO2@ZnO nanocomposites are found to be 3.16 and 2.96 eV, respectively. The calculated specific surface area (SBET) of pristine ZnO NPs and 5 wt% Ag–TeO2@ZnO nanocomposites are 40.47 and 45.66 m2 g−1 respectively, confirming that Ag and TeO2 nanoparticles contribute to increasing in surface area of pure ZnO. The synthesised nanocomposite showed excellent photocatalytic performance for the degradation of β -naphthol (95.6%) in 40 min at the concentration of (0.6 mg ml−1) and 2,4-DCP (99.6%) in 180 min (0.4 mg ml−1) under natural sunlight. Cyclic Voltammetry and Electrochemical Impedance Spectroscopy were carried out to study the electrochemical properties. The determination of reactive oxygen species (ROS) confirmed that the degradation of the pollutants by 5 wt% Ag–TeO2@ZnO NCs was due to the formation of superoxide radicals. Electron paramagnetic resonance revealed the presence of sharp signals in pure ZnO nanoparticles at g ∼1.95 and oxygen vacancy peak at g ∼2.01 in 5 wt% Ag–TeO2@ZnO NCs. To study the mechanism behind the degradation of pollutants, Scavenger test using histidine and ascorbic acid (ROS scavengers) was performed. The synthesised nanocomposites are highly stable and showed enhanced efficiency up to three cycles, confirming their reusability as a photocatalyst.

Anti-Inflammatory and Cancer-Preventive Potential of Chamomile (Matricaria chamomilla L.): A Comprehensive In Silico and In Vitro Study.

Chamomile tea, renowned for its exquisite taste, has been appreciated for centuries not only for its flavor but also for its myriad health benefits. In this study, we investigated the preventive potential of chamomile (Matricaria chamomilla L.) towards cancer by focusing on its anti-inflammatory activity. A virtual drug screening of 212 phytochemicals from chamomile revealed β-amyrin, β-eudesmol, β-sitosterol, apigenin, daucosterol, and myricetin as potent NF-κB inhibitors. The in silico results were verified through microscale thermophoresis, reporter cell line experiments, and flow cytometric determination of reactive oxygen species and mitochondrial membrane potential. An oncobiogram generated through comparison of 91 anticancer agents with known modes of action using the NCI tumor cell line panel revealed significant relationships of cytotoxic chamomile compounds, lupeol, and quercetin to microtubule inhibitors. This hypothesis was verified by confocal microscopy using α-tubulin-GFP-transfected U2OS cells and molecular docking of lupeol and quercetin to tubulins. Both compounds induced G2/M cell cycle arrest and necrosis rather than apoptosis. Interestingly, lupeol and quercetin were not involved in major mechanisms of resistance to established anticancer drugs (ABC transporters, TP53, or EGFR). Performing hierarchical cluster analyses of proteomic expression data of the NCI cell line panel identified two sets of 40 proteins determining sensitivity and resistance to lupeol and quercetin, further pointing to the multi-specific nature of chamomile compounds. Furthermore, lupeol, quercetin, and β-amyrin inhibited the mRNA expression of the proinflammatory cytokines IL-1β and IL6 in NF-κB reporter cells (HEK-Blue Null1). Moreover, Kaplan-Meier-based survival analyses with NF-κB as the target protein of these compounds were performed by mining the TCGA-based KM-Plotter repository with 7489 cancer patients. Renal clear cell carcinomas (grade 3, low mutational rate, low neoantigen load) were significantly associated with shorter survival of patients, indicating that these subgroups of tumors might benefit from NF-κB inhibition by chamomile compounds. This study revealed the potential of chamomile, positioning it as a promising preventive agent against inflammation and cancer. Further research and clinical studies are recommended.

Challenges in the determination of reactive oxygen species evolving during membrane water electrolysis for in situ ozone production

The treatment of ultrapure water with electrochemically produced O3 is a common means for disinfection yet leads to the formation of a variety of reactive oxygen species (ROS). The present study draws a comprehensive comparison between three commonly used photometric and fluorometric assays for ROS analysis and quantifies the individual signal responses for dissolved O3, ·OH and H2O2, respectively, to account for cross-sensitivities. By calibrating all combinations of assays and analytes, we developed a quantification procedure to reliably determine the actual ROS composition in ultrapure water environments for different operation conditions of a membrane water electrolyzer with PbO2 anodes down to concentrations of 0.97 μg L−1. While the ·OH formation rate can be described linearly over the observed current density range, substantial O3 evolution is only found for current densities of 0.75 A cm−2 and above (up to 3.7 μmol h−1 for J = 1.25 A cm−2). The formation of H2O2 is only observed when an organic carbon source is introduced into the solution. We further quantify the interference of H2O2 with the reading of the oxidation-reduction potential as a common water parameter and elaborate on its validity to monitor the peroxone process when both H2O2 and O3 are present simultaneously.

(1 1 1) Facet-dominant peracetic acid activation by octahedral CoO anchored hollow carbon microspheres for tetracycline degradation

Peracetic acid (PAA), an environmental friendly disinfectant, has attracted increasing interest in applications on advanced oxidation processes for treating emerging pollutants. Herein, octahedral cobalt oxide anchored on cation exchange resin (CER)-derived hollow carbon microspheres were fabricated via a facile strategy and employed as the heterogeneous catalyst for PAA activation to degrade tetracycline (TC) antibiotic. Calcination temperature was demonstrated to play a significant role in resulted structure and composition, physicochemical properties and catalytic activities of the cobalt-based compounds/carbon composites. With the optimal catalyst which was calcined at 600 °C, namely the Co@CER-600, complete elimination of TC was achieved within 10 min in Co@CER-600/PAA system. The determination of reactive oxygen species (ROS) indicated that both non-radical (singlet oxygen) and radical (superoxide radical) mechanisms were evidenced in Co@CER-600/PAA system. Particularly, it is notable that the extensively exposed (111) facet in octahedral CoO was confirmed to show remarkable PAA activation activity, therefore promoted the ROS generation process. Considering that facet engineering is a novel whilst important strategy for catalyst regulation, the proposed (111) facet-dominant octahedral CoO anchored hollow carbon microspheres might suggest new inspirations for catalyst design in PAA-based AOPs toward antibiotic contamination remediation.

Antibacterial mechanism of Malaysian Carey clay against food-borne Staphylococcus aureus

Depending on their chemical structure and geochemistry, clay minerals can display potent antibacterial properties against a range of bacterial pathogens. Malaysian Carey clay was evaluated for its antibacterial activity against food-borne Staphylococcus aureus ATCC 13565 strains. The minimum inhibitory concentration (MIC) and minimum bactericidal activity (MBC) of both Carey clay leachates and suspension were 125 mg/mL and 250 mg/mL, respectively. Time-kill assay revealed that 2x MIC and 4x MIC Carey clay in both leachate and suspension forms resulted in complete killing of S. aureus. Antibacterial mechanism was investigated through imaging of bacterial morphology using TEM and determination of reactive oxygen species (ROS) using NBT assay. Imaging of bacterial morphology using TEM showed abnormalities, including disrupted cell walls following exposure to Carey clay, and the antibacterial activity was associated with generation of ROS. Our study suggests that Carey clay displays promising functionality as a natural antibacterial agent in the food industry.

Cold Air Plasma Inhibiting Tumor-Like Biological Behavior of Rheumatoid Arthritis Fibroblast-Like Synovial Cells via G2/M Cell Cycle Arrest.

Rheumatoid arthritis fibroblast-like synovial cells (RA-FLS) have become the core effector cells for the progression of rheumatoid arthritis due to their "tumor-like cell" characteristics, such as being able to break free from growth restrictions caused by contact inhibition, promoting angiogenesis, invading surrounding tissues, and leading to uncontrolled synovial growth. In recent years, cold air plasma (CAP) has been widely recognized for its clear anticancer effect. Inspired by this, this study investigated the inhibitory effect of CAP on the tumor-like biological behavior of RA-FLS through in vitro experiments. Treatment of RA-FLS with CAP at different time doses (0s, 30s, 60s, 120s). 5-ethynyl-2'-deoxyuridine (EdU) proliferation assay was used to determine the cell viability. Analysis of cell migration and invasion was performed by wound-healing assay, transwell assay and immunofluorescent staining for f-actin, respectively. Flow cytometry technique was used for analysis of cell cycle and determination of reactive oxygen species (ROS). Hoechst staining was used for analysis of cell apoptosis. Protein expression was analyzed by Western blot analysis. Molecular and cellular level mechanisms have revealed that CAP blocks RA-FLS in the G2/M phase by increasing intracellular reactive oxygen species (ROS), leading to increased apoptosis and significantly reduced migration and invasion ability of RA-FLS. Overall, CAP has significant anti proliferative, migratory, and invasive effects on RA-FLS. This study reveals a new targeted treatment strategy for RA.

A general method for studying reactive surface species, CT-SKAn: Charge-transfer spectrokinetic analysis

A spectrokinetic methodology was developed to determine reactive surface species by combining operando ultraviolet–visible spectroscopy and charge transfer kinetic models. The methodology consisted of three general steps: 1) concomitant measurement of reaction rates and charge transfer via ultraviolet–visible spectroscopy; 2) development of rate expressions from kinetic models involving the adsorbed species of interest. These rate expressions relate reaction rates, charge transfer, and partial pressures of gas phase species; and 3) evaluation of the goodness of fit of the rate expressions to the experimental data. The species whose rate expressions show the best fit are the more likely reactive surface species for the studied reaction. An example is presented for the determination of reactive oxygen species during ethanol oxidation over Au/TiO2. The charge transfer spectrokinetic analysis showed that surface hydroperoxyl, hydroxyl, and atomic oxygen species were reactive surface intermediates, whereas surface molecular oxygen was not.

Neuroprotective and Anti-inflammatory Effects of Rubiscolin-6 Analogs with Proline Surrogates in Position 2

Naturally occurring peptides, such as rubiscolins derived from spinach leaves, have been shown to possess some interesting activities. They exerted central effects, such as antinociception, memory consolidation and anxiolytic-like activity. The fact that rubiscolins are potent even when given orally makes them very promising drug candidates. The present work tested whether rubiscolin-6 (R-6, Tyr-Pro-Leu-Asp-Leu-Phe) analogs have neuroprotective and anti-inflammatory effects. These hypotheses were tested in the 6-hydroxydopamine (6-OHDA) injury model of human neuroblastoma SH-SY5Y and lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. The determination of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), Caspase-3 activity, lipid peroxidation and nitric oxide (NO) production allowed us to determine the effects of peptides on hallmarks related to Parkinson’s Disease (PD) and inflammation. Additionally, we investigated the impact of R-6 analogs on serine-threonine kinase (also known as protein kinase B, AKT) and mammalian target of rapamycin (mTOR) activation. The treatment with analogs 3 (Tyr-Inp-Leu-Asp-Leu-Phe-OH), 5 (Dmt-Inp-Leu-Asp-Leu-Phe-OH) and 7 (Tyr-Inp-Leu-Asp-Leu-Phe-NH2) most effectively prevented neuronal death via attenuation of ROS, mitochondrial dysfunction and Caspase-3 activity. Peptides 5 and 7 significantly increased the protein expression of the phosphorylated-AKT (p-AKT) and phosphorylated-mTOR (p-mTOR). Additionally, selected analogs could also ameliorate LPS-mediated inflammation in macrophages via inhibition of intracellular generation of ROS and NO production. Our findings suggest that R-6 analogs exert protective effects, possibly related to an anti-oxidation mechanism in in vitro model of PD. The data shows that the most potent peptides can inhibit 6-OHDA injury by activating the PI3-K/AKT/mTOR pathway, thus playing a neuroprotective role and may provide a rational and robust approach in the design of new therapeutics or even functional foods.

Development, in vitro and ex vivo evaluation of bioadhesive colloidal systems for curcumin skin delivery aiming the antioxidant and photoprotective activities

This work aimed to develop bioadhesive colloidal systems containing curcumin (CUR) for antioxidant and photoprotective applications. Gels and emulgels were prepared and characterized as rheology, texture profile analysis, softness, bioadhesive properties using porcine skin, skin permeability and antioxidant activity. Moreover, the formulations were investigated for their photoprotective potential using UVB-irradiated L929 fibroblasts, analyzing cellular viability with or without UVB irradiation, determination of reactive oxygen species (ROS), DNA damage, and cellular migration. The presence of CUR in the systems did not influence their mechanical textural properties and the rheological behavior (p > 0.05), displaying shear thinning characteristics, hysteresis area and rheopexy. The formulations exhibited important antioxidant activity, by scavenging radicals DPPH•, ABTS•+, superoxide anion, and reducing ferric ions. Gel-containing CUR (GEL CUR) exhibited a less cytotoxic effect in L929 fibroblasts than free CUR, besides increasing cell viability in UVB exposure. The treatment with GEL CUR in UVB-irradiated cells attenuated oxidative stress, displaying the ability to scavenge ROS generation (31.3 %), decrease DNA damage (≈30 %), and promote cellular repopulation at wound-scratch assay (8 % after 48h), which was statistically superior in comparison to the control and showing relevant activity. These colloidal systems showed suitable properties for topical application, with good antioxidant activity, permeation ability and potential application for the prevention of UVB-induced skin photodamage.

Gut-microbiota-derived phenylacetylglutamine induces oxidative stress in endothelial cells via a NOX2-dependent pathway

Abstract Introduction The gut microbiota-derived metabolite phenylacetylglutamine (PAG) is clinically and mechanistically linked to cardiovascular disease (CVD)(1). Here, we aim to identify the effect of PAG on endothelial cells and elucidate the underlying molecular pathways. Methods We treated human aortic endothelial cells (HAEC) at a dose of 100 µM PAG (a concentration which can be observed in vivo) for 72h. As a positive control, cells were treated with H2O2 for 1h. For the determination of reactive oxygen species (ROS) formation, we used the CellROX Green assay with subsequent fluorescence microscopy. For signaling studies, protein expression levels of the NADPH oxidases NOX2 and NOX4 as well as the transcription factor NF-kB were determined by western blot analyses. Results Our studies showed that PAG treatment for 72h significantly increased the fluorescence intensity after CellROX Green staining (p<0.0001). Concomitantly, our immunoblotting results revealed that NOX2 (p<0.01) and NF-kB (p<0.001) protein levels were upregulated, whereas NOX4 remained unchanged in response to PAG. Conclusion We conclude that PAG-induced oxidative stress in endothelial cells is mediated by a NOX2 and NF-kB but not by a NOX4 dependent pathway. This study is a first step towards better understanding the molecular mechanisms of cellular responses to the gut microbiota-derived metabolite PAG.

Role of Nrf2/GPX4 mediated ferroptosis in intestinal injury in sepsis

To investigate whether ferroptosis exists in sepsis induced intestinal injury, and to verify the association between ferroptosis in sepsis induced intestinal injury and intestinal inflammation and barrier function by stimulating and inhibiting the nuclear factor E2-related factor 2/glutathione peroxidase 4 (Nrf2/GPX4) pathway. Forty-eight SPF grade male Sprague-Darvley (SD) rats with a body weight of 220-250 g were divided into sham operation group (Sham group), sepsis group (CLP group), sepsis+iron chelating agent deferoxamine (DFO) group (CLP+DFO group) and sepsis+ferroptosis inducer Erastin group (CLP+Erastin group) using a random number table method, with 12 rats in each group. The sepsis model was established by cecal ligation and puncture (CLP). The Sham group was only performed with abdominal opening and closing operations. After modeling, the CLP+DFO group received subcutaneous injection of 20 mg/kg of DFO, the CLP+Erastin group was intraperitoneally injected with 20 mg/kg of Erastin. Each group received subcutaneous injection of 50 mg/kg physiological saline for fluid resuscitation after surgery, and the survival status of the rats was observed 24 hours after surgery. At 24 hours after model establishment, 6 rats in each group were selected. First, live small intestine tissue was taken for observation of mitochondrial morphology in smooth muscle cells under transmission electron microscopy and determination of reactive oxygen species (ROS). Then, blood was collected from the abdominal aorta and euthanized. The remaining 6 rats were sacrificed after completing blood collection from the abdominal aorta, and then small intestine tissue was taken. Western blotting was used to detect the expression of intestinal injury markers such as Claudin-1 and ferroptosis related proteins GPX4 and Nrf2. Observe the pathological changes of small intestine tissue using hematoxylin-eosin (HE) staining and complete Chiu score; Detection of tumor necrosis factor-α (TNF-α), interleukins (IL-1β, IL-6) levels in serum using enzyme-linked immunosorbent assay (ELISA). The levels of serum iron ions (Fe3+), malondialdehyde (MDA), and D-lactate dehydrogenase (D-LDH) were measured. (1) Compared with the Sham group, the 24-hour survival rate of rats in the CLP group and CLP+Erastin group significantly decreased (66.7%, 50.0% vs. 100%, both P < 0.05), while there was no significant difference in the CLP+DFO group (83.3% vs. 100%, P = 0.25). (2) Western blotting results showed that compared with the Sham group, the expressions of GPX4 and Claudin-1 in the small intestine tissue of the CLP group, CLP+DFO group, and CLP+Erastin group decreased significantly, while the expression of Nrf2 increased significantly (GPX4/β-actin: 0.56±0.02, 1.03±0.01, 0.32±0.01 vs. 1.57±0.01, Claudin-1/β-actin: 0.60±0.04, 0.96±0.07, 0.41±0.01 vs. 1.40±0.01, Nrf2/β-actin: 0.88±0.02, 0.72±0.01, 1.14±0.01 vs. 0.43±0.02, all P < 0.05). Compared with the CLP group, the expressions of GPX4 and Claudin-1 were significantly increased in the CLP+DFO group, while the expression of Nrf2 was significantly reduced. In the CLP+Erastin group, the expressions of GPX4 and Claudin-1 further decreased, while the expression of Nrf2 further increased (all P < 0.05). (3) Under the light microscope, compared with the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group showed structural disorder in the small intestinal mucosa and submucosal tissue, significant infiltration of inflammatory cells, and destruction of glandular and villous structures. The Chui score was significantly higher (3.25±0.46, 2.00±0.82, 4.50±0.55 vs. 1.25±0.45, all P < 0.05). (4) Under transmission electron microscopy, compared with the Sham group, the mitochondria in the other three groups of small intestinal smooth muscle cells showed varying degrees of volume reduction, increased membrane density, and reduced or disappeared cristae. The CLP+Erastin group showed the most significant changes, while the CLP+DFO group showed only slight changes in mitochondrial morphology. (5) Compared to the Sham group, the CLP group, CLP+DFO group, and CLP+Erastin group had serum levels of TNF-α, IL-1β, IL-6, MDA, D-LDH, and ROS in small intestine tissue were significantly increased, while the serum Fe3+ content was significantly reduced [TNF-α (ng/L): 21.49±1.41, 17.24±1.00, 28.66±2.72 vs. 14.17±1.24; IL-1β (ng/L): 108.40±3.09, 43.19±8.75, 145.70±11.00 vs. 24.50±5.55; IL-6 (ng/L): 112.50±9.76, 45.90±6.52, 151.80±9.38 vs. 12.89±6.11; MDA (μmol/L): 5.61±0.49, 3.89±0.28, 8.56±1.17 vs. 1.86±0.41; D-LDH (kU/L): 39.39±3.22, 25.38±2.34, 53.29±10.53 vs. 10.79±0.52; ROS (fluorescence intensity): 90 712±6 436, 73 278±4 775, 110 913±9 287 vs. 54 318±2 226; Fe3+ (μmol/L): 22.19±1.34, 34.05±1.94, 12.99±1.08 vs. 51.74±11.07; all P < 0.05]. Compared with CLP group, the levels of TNF-α, IL-1β, IL-6, MDA, D-LDH and ROS in CLP+Erastin group were further increased, and the content of Fe3+ was further decreased, the CLP+DFO group was the opposite (all P < 0.05). Ferroptosis exists in the intestinal injury of septic rats, and stimulating or inhibiting ferroptosis through the Nrf2/GPX4 pathway can effectively intervene in the inflammatory state and intestinal mechanical barrier of the body.

Cell-type-specific determination of reactive oxygen species by flow cytometry.

Seminal leukocyte-generated reactive oxygen species may have a significant impact on sperm intracellular reactive oxygen species levels, therefore contributing to oxidative damage and consequent functional impairment of spermatozoa. This relationship may be utilized for male urogenital inflammation-driven oxidative stress diagnostics. To obtain seminal cell-specific, reactive oxygen species-related fluorescence intensity cut-off values to differentiate leukocytospermic samples displaying reactive oxygen species overproduction (oxidative burst) from normozoospermic seminal samples. Ejaculates gained by masturbation were obtained from patients in the framework of andrology consultations. The results published in this paper were generated from samples for which the attending physician requested spermatograms and seminal reactive oxygen species laboratory tests. Routine seminal analyses were performed according to World Health Organization guidelines. Samples were divided into normozoospermic "non-inflamed," and leukocytospermic groups. The semen was stained by 2',7'-dichlorodihydrofluorescein diacetate and the reactive oxygen species-related fluorescence signal and the percentage of reactive oxygen species-positive spermatozoa within the living population were quantified by flow cytometry. Reactive oxygen species-related mean fluorescence intensity was higher in both spermatozoa and leukocytes from leukocytospermic samples than in those from normozoospermic samples. Mean fluorescence intensity in spermatozoa was positively and linearly correlated with mean fluorescence intensity measured in leukocytes in both groups. The capacity of spermatozoa to generate reactive oxygen species is at least three log lower than that of granulocytes. The question is whether the reactive oxygen species-producing machinery of spermatozoa is capable of causing autologous oxidative stress or whether leukocytes are the predominant source of seminal oxidative stress. Based on our observations, the reactive oxygen species production of leukocytes may have a significant impact on the overall reactive oxygen species levels measured in spermatozoa. Reactive oxygen species-overproducing leukocytospermic and normozoospermic seminal samples can reliably be differentiated based on reactive oxygen species mean fluorescence intensity measurement.

Residue Lys219 of CpxR is critical in the regulation of the antibiotic resistance of Escherichia coli.

CpxR is a critical regulator in bacterial adaptation to various harmful stresses, and is known to regulate bacterial resistance to commonly used antibiotics, such as aminoglycosides, β-lactams and polypeptides. However, the detailed study of functional residues of CpxR remains insufficient. To investigate the contribution of Lys219 to CpxR's function in regulating antibiotic resistance of Escherichia coli. We performed sequence alignment and conservative analysis of the CpxR protein and constructed mutant strains. We then performed electrophoretic mobility shift assay, real-time quantitative PCR assay, determination of reactive oxygen species (ROS) levels, molecular dynamics simulation, conformational analysis and circular dichroism. All mutant proteins (K219Q, K219A and K219R) lost the cpxP DNA-binding ability. Additionally, the three complemented strains eK219A, eK219Q, and eK219R exhibited lower resistance to copper toxicity and alkaline pH toxicity than eWT. Molecular dynamics analysis revealed that mutation of Lys219 leads to looser and more unstable conformation of CpxR, leading to its decreased binding affinity with downstream genes. Moreover, the Lys219 mutation resulted in the down-regulation of efflux pump genes (acrD, tolC, mdtB and mdtA), leading to the accumulation of antibiotics inside the cells and an increase in ROS production, which significantly reduces antibiotic resistance. The mutation of the key residue Lys219 causes a conformational change that results in the loss of regulatory ability of CpxR, which may potentially reduce to antibiotic resistance. Therefore, this study suggests that targeting the highly conserved sequence of CpxR could be a promising strategy for the development of new antibacterial drugs.

Synergistic effect of manganese (II) phosphate & diamond nanoparticles in electrochemical sensors for reactive oxygen species determination in seminal plasma

In this work, we explore the ability of manganese (II) phosphate (MnP) as a catalytic element for the determination of reactive oxygen species (ROS) in seminal plasma, when MnP is employed as modifier of a glassy carbon electrode. The electrochemical response of the manganese (II) phosphate-modified electrode shows a wave at around +0.65 V due to the oxidation of Mn2+ to MnO2+, which is clearly enhanced after addition of superoxide, the molecule considered as the mother of ROS. Once proved the suitability of manganese (II) phosphate as catalyst, we evaluate the effect of including a 0D (diamond nanoparticles) or a 2D (ReS2) nanomaterial in the sensor design. The system consisting of manganese (II) phosphate and diamond nanoparticles yielded the largest improvement of the response. The morphological characterization of the sensor surface was performed by scanning electron microscopy and atomic force microscopy, while cyclic and differential pulse voltammetry were employed for the electrochemical characterization of the sensor. After optimizing the sensor construction, calibration procedures by chronoamperometry were performed, leading to a linear relation between peak intensity and the superoxide concentration in the range of 1.1 10−4 M − 1.0 10−3 M with a limit of detection of 3.2 10−5 M. Seminal plasma samples were analysed by the standard addition method. Moreover, the analysis of samples fortified with superoxide at the μM level leads to recoveries of 95%.

Electrochemical approaches based on micro- and nanomaterials for diagnosing oxidative stress.

This review article comprehensively discusses the various electrochemical approaches for measuring and detecting oxidative stress biomarkers and enzymes, particularly reactive oxygen/nitrogen species, highly reactive chemical molecules, which are the byproducts of normal aerobic metabolism and can oxidize cellular components such asDNA, lipids, and proteins. First, we address the latest research on the electrochemical determination of reactive oxygen species generating enzymes, followed by detection of oxidative stress biomarkers, and final determination of total antioxidant activity (endogenous and exogenous). Most electrochemical sensing platforms exploited the unique properties of micro- and nanomaterials such as carbon nanomaterials, metal or metal oxide nanoparticles (NPs), conductive polymers and metal-nano compounds, which have been mainly used for enhancing the electrocatalytic response of sensors/biosensors. The performance of the electroanalytical devices commonly measured by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) in terms of detection limit, sensitivity, and linear range of detection is also discussed. This article provides a comprehensive review of electrode fabrication, characterization and evaluation of their performances, which are assisting to design and manufacture an appropriate electrochemical (bio)sensor for medical and clinical applications. The key points such as accessibility, affordability, rapidity, low cost, and high sensitivity of the electrochemical sensing devices are also highlighted for the diagnosis of oxidative stress. Overall, this review brings a timely discussion on past and current approaches for developing electrochemical sensors and biosensors mainly based on micro and nanomaterials for the diagnosis of oxidative stress.

Antioxidant and anti-aging effects of polysaccharide LDP-1 from wild &lt;em&gt;Lactarius deliciosus&lt;/em&gt; on &lt;em&gt;Caenorhabditis elegans&lt;/em&gt;

Background: Edible fungi (mushrooms) have attracted much more concerns due to their abundant nutritions and functional bioactive substances like polysaccharides.&#x0D; Objective: In this study, the anti-oxidation and anti-aging activities of polysaccharide fraction (LDP-1) from the wild Lactarius deliciosus fruiting bodies were systematically evaluated using Caenorhabditis elegans (C. elegans) as a model.&#x0D; Methods: Lifetime determination of C. elegans (survival status) was observed via microscope. Effects of LDP-1 on C. elegans induced by heat and oxidative stress were, respectively, performed in an artificial climate chamber and Juglone solution. Determination of lipofuscin levels in C. elegans was observed by laser confocal scanning microscopy. Determination of reactive oxygen species in C. elegans in vivo was conducted via fluorescence spectrophotometer reader.&#x0D; Results: The results revealed that LDP-1 could significantly extend the lifespan of C. elegans, and the lifetime of C. elegans treated with 1,000 μg/mL LDP-1 could be prolonged by 32.8% compared with the control. The survival rates of the experimental C. elegans under heat shock and oxidative stress conditions were clearly improved after treated with 1,000 μg/mL LDP-1 (40 and 19.8%, respectively), while under the same circumstances all the C. elegans in the blank group died. Fluorescence microscopy analysis confirmed that LDP-1 could effectively reduce the accumulation of lipofuscin and reactive oxygen free radicals in C. elegans, where the respective maximum reduction reached 22.8 and 42.7% compared with the control.&#x0D; Conclusion: These results indicate that LDP-1 had favorable antioxidant and anti-aging effects, which could be explored as potential dietary additives to renovate oxidative damage and slow down aging process.

Successive mycelial subculturing decreased lignocellulase activity and increased ROS accumulation in Volvariella volvacea.

Strain degradation is a common problem in many artificially-cultivated edible mushrooms. As a fungus with poor tolerance to low-temperature, Volvariella volvacea cannot delay its degradation by long-term low temperature storage like other fungi, so its degradation is particularly severe, which hinders industrial applications. Periodic mycelial subculture is a common storage method for V. volvacea, but excessive subculturing can also lead to strain degeneration. After 20 months of continuous subculturing every 3 days, V. volvacea strains S1–S20 were obtained, and their characteristics throughout the subculture process were analyzed. With increasing number of subculture, the growth rate, mycelial biomass, the number of fruiting bodies and biological efficiency gradually decreased while the production cycle and the time to primordium formation was lengthened. Strains S13–S20, obtained after 13–20 months of mycelial subculturing, also lacked the ability to produce fruiting bodies during cultivation experiments. Determination of reactive oxygen species (ROS) content as well as enzyme activity showed that decreased lignocellulase activity, along with excessive accumulation of ROS, was concomitant with the subculture-associated degeneration of V. volvacea. Reverse transcription polymerase chain reaction (RT-PCR) was eventually used to analyze the gene expression for lignocellulase and antioxidant enzymes in subcultured V. volvacea strains, with the results found to be consistent with prior observations regarding enzyme activities. These findings could form the basis of further studies on the degeneration mechanism of V. volvacea and other fungi.

Linearolactone Induces Necrotic-like Death in Giardia intestinalis Trophozoites: Prediction of a Likely Target.

Linearolactone (LL) is a neo-clerodane type diterpene that has been shown to exert giardicidal effects; however, its mechanism of action is unknown. This work analyzes the cytotoxic effect of LL on Giardia intestinalis trophozoites and identifies proteins that could be targeted by this active natural product. Increasing concentrations of LL and albendazole (ABZ) were used as test and reference drugs, respectively. Cell cycle progression, determination of reactive oxygen species (ROS) and apoptosis/necrosis events were evaluated by flow cytometry (FCM). Ultrastructural alterations were analyzed by transmission electron microscopy (TEM). Ligand–protein docking analyses were carried out using the LL structure raised from a drug library and the crystal structure of an aldose reductase homologue (GdAldRed) from G. intestinalis. LL induced partial arrest at the S phase of trophozoite cell cycle without evidence of ROS production. LL induced pronecrotic death in addition to inducing ultrastructural alterations as changes in vacuole abundances, appearance of perinuclear and periplasmic spaces, and deposition of glycogen granules. On the other hand, the in silico study predicted that GdAldRed is a likely target of LL because it showed a favored change in Gibbs free energy for this complex.

Apoptotic and autophagic death union by Thuja occidentalis homeopathic drug in cervical cancer cells with thujone as the bioactive principle

Objective“Multi-targeting” drugs can prove fruitful to combat drug-resistance of multifactorial disease—cervical cancer. This study envisioned to reveal if Thuja homeopathic mother tincture (MT) and its bioactive component could combat human papillomavirus (HPV)-16-infected SiHa cervical cancer cells since it is globally acclaimed for HPV-mediated warts. MethodsThuja MT was studied for its antiproliferative and antimigratory properties in SiHa cells followed by microscopic determination of reactive oxygen species (ROS) generation by 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) staining and loss in mitochondrial membrane potential (MtMP) by rhodamine 123 (Rh123) staining. Apoptosis and autophagy inductions were studied by acridine orange/ethidium bromide (AO/EB) staining and immunoblot analyses of marker proteins. The bioactive component of Thuja MT detected by gas chromatography-mass spectrometry was studied for antiproliferative and antimigratory properties along with in silico prediction of its cellular targets by molecular docking and oral drug forming competency. ResultsThuja MT showed significant antiproliferative and antimigratory potential in SiHa cells at a 50% inhibitory concentration (IC50) of 17.3 µL/mL. An increase in DCFDA fluorescence and loss in Rh123 fluorescence prove that Thuja MT acted through the burst of ROS and loss in MtMP respectively. AO/EB-stained cells under the microscope and immunoblot analyses supported Thuja-induced cellular demise via dual pathways—apoptosis and autophagy. Immunoblots showed cleavage of caspase-3 and poly(adenosine diphosphate-ribose) polymerase-1 (PARP-1) along with upregulation of Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B)-II, and p62 proteins. Hence, the apoptotic cascade followed a caspase-3-dependent pathway supported by PARP-1 cleavage, while autophagic death was Beclin-1-dependent and mediated by accumulation of LC3BII and p62 proteins. Thujone, detected as the bioactive principle of Thuja MT, showed greater anti-proliferative and anti-migratory potential at an IC50 of 77 µg/mL, along with excellent oral drug competency with the ability for gastrointestinal absorption and blood–brain-barrier permeation with nil toxicity. Molecular docking depicted thujone with the strongest affinity for mammalian target of rapamycin, phosphoinositide 3-kinase, and protein kinase B followed by B-cell lymphoma 2, murine double minute 2 and adenosine monophosphate-activated protein kinase, which might act as upstream triggers of apoptotic-autophagic crosstalk. ConclusionRobust “multi-targeting” anticancer potential of Thuja drug and thujone for HPV-infected cervical cancer ascertained its therapeutic efficacy for HPV infections.