Higher ROS Production Research Articles

4-Octyl itaconate attenuates renal tubular injury in db/db mice by activating Nrf2 and promoting PGC-1α-mediated mitochondrial biogenesis

Objectives: The aim of this study was to investigate the mechanism of itaconate’s potential effect in diabetic kidney disease. Methods: Renal immune responsive gene 1 (IRG1) levels were measured in db/db mice and streptozotocin (STZ) + high-fat diet (HFD)-induced diabetic mice. Irg1 knockout mice were generated. db/db mice were treated with 4-octyl itaconate (4-OI, 50 mg/kg), a derivative of itaconate, for 4 weeks. Renal function and morphological changes were investigated. Ultrastructural alterations were determined by transmission electron microscopy. Results: Renal IRG1 levels were reduced in two diabetic models. STZ+HFD-treated Irg1 knockout mice exhibited aggravated renal tubular injury and worsened renal function. Treatment with 4-OI lowered urinary albumin-to-creatinine ratio and blood urea nitrogen levels, and restored renal histological changes in db/db mice. It improved mitochondrial damage, increased expressions of peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM) in the renal cortex of db/db mice. These were confirmed in vitro; 4-OI improved high glucose-induced abnormal mitochondrial morphology and TFAM expression in HK-2 cells, effects that were inhibited by PGC-1α silencing. Moreover, 4-OI reduced the number of apoptotic cells in the renal cortex of db/db mice. Further study showed that 4-OI increased renal Nrf2 expression and decreased oxidative stress levels in db/db mice. In HK-2 cells, 4-OI decreased high glucose-induced mitochondrial ROS production, which was reversed by Nrf2 silencing. Nrf2 depletion also inhibited 4-OI-mediated regulation of PGC-1α, TFAM, and mitochondrial apoptotic protein expressions. Conclusions: 4-OI attenuates renal tubular injury in db/db mice by activating Nrf2 and promoting PGC-1α-mediated mitochondrial biogenesis.

Investigating the Effects of Simultaneous Administration of Melatonin and Atorvastatin against High Glucose-Induced Oxidative Stress and Apoptosis in Cultured Chondrocytes.

Osteoarthritis (OA) is a prevalent joint disorder categorized into phenotypic subtypes, including those associated with age, traumatic events, and metabolic syndrome. In the aging population, type 2 diabetes mellitus (T2DM) and osteoarthritis (OA) frequently coexist. This can result in higher rates of disability and a greater financial burden. This study aimed to investigate the protective effects of melatonin and atorvastatin together against oxidative stress and apoptosis induced by high glucose in C28I2 human chondrocytes. After being pretreated for 6 hours with melatonin (10 and 100 μM) and atorvastatin (0.01 and 0.1 μM), C28I2 cells were exposed to a high concentration of D-glucose (75 mM) for 72 hours. The impact of a high D-glucose concentration (75 mM), with or without melatonin and/or atorvastatin, on cell viability, intra-cellular ROS generation, lipid peroxidation level, antioxidant activities, and the expression of proteins, including Bax, Bcl-2, and caspase-3, was analyzed. Melatonin and atorvastatin combination effectively inhibited high glucose-induced cytotoxicity, ROS production, and MDA and mitochondrial membrane potential levels. The combination of melatonin and atorvastatin was more successful in reducing ROS production compared to each of the drugs alone. Melatonin, but not atorvastatin, reversed high glucose-induced alteration in the catalase activity. Furthermore, the combination of melatonin and atorvastatin significantly enhanced the ability of each medication to lower the expression of pro-apoptotic protein Bax. The combination of melatonin and atorvastatin exerted greater protective effects against hyperglycemia-induced toxicity in chondrocytes.

A cell state-specific metabolic vulnerability to GPX4-dependent ferroptosis in glioblastoma

Glioma cells hijack developmental programs to control cell state. Here, we uncover a glioma cell state-specific metabolic liability that can be therapeutically targeted. To model cell conditions at brain tumor inception, we generated genetically engineered murine gliomas, with deletion of p53 alone (p53) or with constitutively active Notch signaling (N1IC), a pathway critical in controlling astrocyte differentiation during brain development. N1IC tumors harbored quiescent astrocyte-like transformed cell populations while p53 tumors were predominantly comprised of proliferating progenitor-like cell states. Further, N1IC transformed cells exhibited increased mitochondrial lipid peroxidation, high ROS production and depletion of reduced glutathione. This altered mitochondrial phenotype rendered the astrocyte-like, quiescent populations more sensitive to pharmacologic or genetic inhibition of the lipid hydroperoxidase GPX4 and induction of ferroptosis. Treatment of patient-derived early-passage cell lines and glioma slice cultures generated from surgical samples with a GPX4 inhibitor induced selective depletion of quiescent astrocyte-like glioma cell populations with similar metabolic profiles. Collectively, these findings reveal a specific therapeutic vulnerability to ferroptosis linked to mitochondrial redox imbalance in a subpopulation of quiescent astrocyte-like glioma cells resistant to standard forms of treatment.

Nano-enabled thermo-responsive local delivery of tamoxifen and carvacrol for enhanced anticancer action

Tamoxifen (TMX) is widely employed in the treatment of breast cancer. However, owing to its poor solubility (BCS class II drug), rapid first-pass metabolism and free radical generation leading to hepatotoxicity, a better drug delivery platform is required. A sufficient concentration of TMX must be achieved while avoiding harm to healthy cells in the target for effective therapy. Carvacrol (CV), a natural monoterpene, has reported antitumor activity and cell growth inhibition effects on MCF-7 and MDA-MB-231 breast cancer cells. In the current study, TMX and CVR-loaded nano-emulsion was prepared and incorporated into thermosensitive poloxamer gel to achieve enhanced anticancer action locally. Box-Behnken Design was employed to extensively optimize the prepared formulation wherein the droplet size obtained was 97.53 ± 1.80 nm and PDI was 0.195 ± 0.0026. The developed thermogel was characterized for gelation temperature, rheology, hemolysis and drug release. The gelation and higher viscosity were attended closer to body temperature (37 ± 0.5 °C), and showed retarded drug release (∼60 % after 5 days). The developed thermogel showed 16.02 % hemolysis as compared to 71.89 % from free TMX. Further, In-vitro cell culture studies of the developed formulation revealed significantly enhanced cytotoxicity and almost 6-fold higher cellular internalization. In addition, dose-dependent lowering of mitochondrial membrane potential and 5.9-fold higher ROS production by TMX-CVR-NE was observed. Therefore, an in-situ thermo-gel depot of TMX and CVR imparted an attractive way for the controlled delivery and enhanced local activity of TMX in breast cancer.

Epigenetic reprogramming of mtDNA and its etiology in mitochondrial diseases.

Mitochondrial functionality and its regulation are tightly controlled through a balanced crosstalk between the nuclear and mitochondrial DNA interactions. Epigenetic signatures like methylation, hydroxymethylation and miRNAs have been reported in mitochondria. In addition, epigenetic signatures encoded by nuclear DNA are also imported to mitochondria and regulate the gene expression dynamics of the mitochondrial genome. Alteration in the interplay of these epigenetic modifications results in the pathogenesis of various disorders like neurodegenerative, cardiovascular, metabolic disorders, cancer, aging and senescence. These modifications result in higher ROS production, increased mitochondrial copy number and disruption in the replication process. In addition, various miRNAs are associated with regulating and expressing important mitochondrial gene families like COX, OXPHOS, ND and DNMT. Epigenetic changes are reversible and therefore therapeutic interventions like changing the target modifications can be utilized to repair or prevent mitochondrial insufficiency by reversing the changed gene expression. Identifying these mitochondrial-specific epigenetic signatures has the potential for early diagnosis and treatment responses for many diseases caused by mitochondrial dysfunction. In the present review, different mitoepigenetic modifications have been discussed in association with the development of various diseases by focusing on alteration in gene expression and dysregulation of specific signaling pathways. However, this area is still in its infancy and future research is warranted to draw better conclusions.

The level of blood neutrophils primary reactive oxygen species production in Graves’ disease patients undergoing conservative therapy with thiamazole.

Background. Conservative therapy with thiamazole in Graves’ disease helps to reduce the level of oxidative stress by restoring euthyroidism and the total reactive antioxidant potential of plasma. However, until now, in patients with Graves’ disease, the radical-producing ability of neutrophils remains unexplored at the initial stage of free radical conversion at different durations of drug-induced euthyroidism, which seems significant in revealing the mechanisms of disease recurrence. The aim is to study the level of production of primary reactive oxygen species in peripheral blood neutrophils depending on the duration of drug — induced euthyroidism in patients with Graves’ disease. Materials and methods. A single-center, prospective, controlled study was conducted with the participation of patients with laboratory-confirmed Graves’ disease. Subgroups of patients with Graves’ disease were formed depending on the duration of achieved drug-induced euthyroidism with continuous conservative therapy with thiamazole. The level of ROS production in peripheral blood neutrophils was determined by chemiluminescence. The assessment of spontaneous and zymosan-induced chemiluminescence was carried out for 90 minutes on a 36-channel chemiluminescence analyzer BLM-3607 (Medbiotech LLC, Krasnoyarsk). Results. The study included 102 Graves’ disease women with an average age of 47.54 ± 15.47 years. Of these, 75 (73.5 %) with relapse and 27 (26.47 %) with newly diagnosed disease. The total synthesis of (S) primary ROS increased statistically significantly relative to the control values in the examined patients with a duration of the euthyroid state of 5–8 and 9–12 months. It was found that in patients with Graves’ disease with stable drug-induced euthyroidism for 5–8 and 9–12 months, the S index of zymosan-induced lucigenin-dependent chemiluminescence of neutrophils increased statistically significantly, respectively, by 10.7 and 7.4 times relative to the control values. Conclusion. The restoration and maintenance of drug-induced euthyroidism for more than 12 months against the background of conservative thiamazole therapy in patients with Graves’ disease reduces the load of systemic oxidative stress on the immune system. However, the high total production of primary ROS in neutrophils of patients with Graves’ disease in a state of drug-induced euthyroidism with conservative thiamazole therapy may play a significant role in disconnecting the functioning of the NADPH oxidase complex and determine the functional capabilities of neutrophils in case of disease recurrence.

Design, synthesis, and biological evaluation of eugenol-isoxazoline hybrid derivatives as potential anti-leishmanial agents

Leishmaniasis is a group of protozoan parasitic diseases caused by various Leishmania species. The clinical manifestations range from skin abrasions to lethal infection in the visceral organs such as the liver and spleen. The emergence of drug resistance, long hospitalization periods, and various adverse effects of the current therapeutic regimen have incited the quest for exploring novel molecules as efficient treatment regimens. Eugenol is the major component of clove essential oil and has significant biological potential with anti-microbial properties. Moreover, natural product derivatives having the isoxazoline group have shown promising pharmacological applications. Thus, various analogs were synthesized and characterized to explore the anti-leishmanial potency of eugenol and isoxazoline hybrid. Results from the in vitro assay against Leishmania donovani parasites identified three potent derivatives (7e, 8a, and 8b) with IC50 values in the range of 7–17 µM. Cytotoxicity analysis against the J774A.1 macrophage cell line confirmed the satisfactory safety index of these potent hits and ex vivo infectivity assay established the role of these hits in reducing intracellular amastigote burden. Next, various mechanistic studies, including SEM analysis, higher ROS production, reduced intracellular ATP levels, mitochondrial dysfunction, formation of intracellular lipid bodies, and acidic vesicular organelles, confirmed the role of these potent hits in parasite cell death. According to the molecular docking analysis results, the most potent derivative, 8b, had strong binding interactions with residues Ile296, Pro299, and Phe354 of sterol 14α-demethylase protein of the Leishmania parasite. Entirely the study highlights the role of eugenol-isoxazoline hybrid derivatives as potential leishmanicidal agents.

Strategically tailored double S-scheme heterojunction in h-MoO3 doped Bi7O9I3 decorated with Cr–CdS quantum dots for efficient photocatalytic degradation of phenolics

Phenolic compounds are widely used in various industrial processes, but they can be harmful to the environment if released without proper treatment. In this study, we developed a ternary nanocomposite (NCs) consisting of molybdenum trioxide, bismuth oxoiodide, and chromium-doped cadmium sulphide quantum dots h-MoO3/Bi9O7I3/Cr–CdS QDs (MBC) nanocomposite (NCs) to enhance the photocatalytic degradation of p-chlorophenol (p-CP). The nanomaterial morphology, distribution of depositing material, and heterojunction formation were investigated using SEM, TEM, SAED, and elemental mapping. Further, the structural characterization was done by XRD, XPS, and Raman spectroscopy which confirmed its purity, chemical states, bonding nature, and the presence of oxygen vacancies (OVs) and sulphur defects. The 30-MBC NCs exhibited the highest surface area of 354.5 m2/g and pore volume of 0.76 cm3/g, with the H3 hysteresis loop indicating their mesoporous nature. The Kubelka-Munk plot determined the band gap of the NCs to be 2.22 eV and ensured successful visible-light sensitization. PL and ESR studies revealed that 30-MBC NCs had the lowest charge carrier recombination and highest ROS production. The photocatalytic degradation of p-CP by 30-MBC NCs achieved 94% in 300 min. The highest efficiency was observed at 75 mg/L NCs, 150 mg/L p-CP, and pH 6. Additionally, NCs retained activity in the presence of different ions and degraded 82% p-CP even after six consecutive cycles. •O2− was the dominant ROS involved in the degradation process, while OVs and sulphur defects played a crucial role in ameliorating charge transfer in the 30-MBC NCs. GC-MS/MS analysis identified the possible degradation pathways of p-CP, and ECOSAR showed that less toxic by/end products were produced. Overall, this study highlights the promising potential of 30-MBC NCs for extensive wastewater treatment.

Female rats present higher oxidative damage and inflammation during goitrogenesis.

Thyroid disorders affect more women than men, but the underlying mechanisms contributing to this disparity remain incompletely understood. Thyrotropin (TSH), the primary regulator of thyroid oxidative hormonogenesis, has been implicated as a risk factor for proliferative thyroid diseases and a predictor of malignancy. In this study, we aimed to evaluate the impact of sustained elevated TSH levels on thyroid redox homeostasis, inflammatory markers, and DNA damage response in both male and female rats. Rats were treated with methimazole for 7 or 21 days, and hormonal measurements were conducted. H2O2 levels were evaluated in thyroid membrane fractions, while enzymatic activities were assessed in total thyroid homogenates. Sex-specific differences emerged, with females displaying higher reactive oxygen species levels - increased transiently NOX and sustained DUOX activities. Lipid peroxidation marker 4-hydroxynonenal (4-HNE) was elevated in females at both time points, contrasting with males just at 21 days. Sexual dimorphism was observed in DNA damage response, with females showing higher γH2AX levels at 21 days. Elevated IL-1β, TNF-α, CD11b mRNA, and phospho-NF-κB levels at 7 days indicated a distinct inflammatory profile in females. Notably, both sexes exhibited upregulated antioxidant enzymes. Our data suggest that females are more susceptible to oxidative damage and inflammation in our goiter model, which may be associated with higher ROS production and a less-efficient antioxidant defense system. These findings provide insights into the sex-specific mechanisms underlying thyroid dysfunction and highlight the importance of considering sex disparities in thyroid disorder research.

Mixed-ligand copper(ii)-diimine complexes of 3-formylchromone-N 4-phenyl thiosemicarbazone: 5,6-dmp co-ligand confers enhanced cytotoxicity.

The promising biological applications of thiosemicarbazone derivatives have inspired the design, synthesis, and study of their Cu(ii) complexes for anticancer therapeutic applications. Herein, we have evaluated the DNA/protein binding, DNA cleaving, and cytotoxic properties of four mixed-ligand Cu(ii) complexes of the type [Cu(L)(diimine)](NO3) 1-4, where HL is 4-oxo-4H-chromene-3-carbaldehyde-4(N)-phenylthiosemicarbazone and diimine is 2,2'-bipyridine (bpy, 1) 1,10-phenanthroline (phen, 2), 5,6-dimethyl-1,10-phenanthroline (5,6-dmp, 3), or dipyrido-[3,2-f:2',3'-h]-quinoxaline (dpq, 4). Interestingly, complex 3 with higher lipophilicity shows stronger DNA binding and oxidative DNA cleavage, higher ROS production, and more reversible redox behaviour, resulting in its remarkable cytotoxicity (IC50, 1.26 μM) against HeLa cervical cancer cells, and rendering it 5 times more potent than the widely used drug cisplatin. The same complex induces enhanced apoptotic cell death on HeLa cells but lower toxicity towards the non-cancerous PBMC cells. Molecular docking studies suggest that all the complexes bind in the minor groove of DNA and subdomain II of HSA, which is in close agreement with the experimental results. Also, 3 shows cytotoxicity higher than the analogous mixed ligand Cu(ii) complexes, reported already, emphasizing the importance of co-ligand in tuning the anticancer activity.

Myocardial ischemia-reperfusion injury; Molecular mechanisms and prevention.

Cardiovascular diseases are one of the leading causes of mortality in developed countries. Among cardiovascular disorders, myocardial infarction remains a life-threatening problem predisposing to the development and progression of ischemic heart failure. Ischemia/reperfusion (I/R) injury is a critical cause of myocardial injury. In recent decades, many efforts have been made to find the molecular and cellular mechanisms underlying the development of myocardial I/R injury and post-ischemic remodeling. Some of these mechanisms are mitochondrial dysfunction, metabolic alterations, inflammation, high production of ROS, and autophagy deregulation. Despite continuous efforts, myocardial I/R injury remains a major challenge in medical treatments of thrombolytic therapy, heart disease, primary percutaneous coronary intervention, and coronary arterial bypass grafting. The development of effective therapeutic strategies to reduce or prevent myocardial I/R injury is of great clinical significance.

The Impact of Semicarbazide Sensitive Amine Oxidase Activity on Rat Aortic Vascular Smooth Muscle Cells.

Semicarbazide-sensitive amine oxidase (SSAO) is both a soluble- and membrane-bound transmembrane protein expressed in the vascular endothelial and in smooth muscle cells. In vascular endothelial cells, SSAO contributes to the development of atherosclerosis by mediating a leukocyte adhesion cascade; however, its contributory role in the development of atherosclerosis in VSMCs has not yet been fully explored. This study investigates SSAO enzymatic activity in VSMCs using methylamine and aminoacetone as model substrates. The study also addresses the mechanism by which SSAO catalytic activity causes vascular damage, and further evaluates the contribution of SSAO in oxidative stress formation in the vascular wall. SSAO demonstrated higher affinity for aminoacetone when compared to methylamine (Km = 12.08 µM vs. 65.35 µM). Aminoacetone- and methylamine-induced VSMCs death at concentrations of 50 & 1000 µM, and their cytotoxic effect, was reversed with 100 µM of the irreversible SSAO inhibitor MDL72527, which completely abolished cell death. Cytotoxic effects were also observed after 24 h of exposure to formaldehyde, methylglyoxal and H2O2. Enhanced cytotoxicity was detected after the simultaneous addition of formaldehyde and H2O2, as well as methylglyoxal and H2O2. The highest ROS production was observed in aminoacetone- and benzylamine-treated cells. MDL72527 abolished ROS in benzylamine-, methylamine- and aminoacetone-treated cells (**** p < 0.0001), while βAPN demonstrated inhibitory potential only in benzylamine-treated cells (* p < 0.05). Treatment with benzylamine, methylamine and aminoacetone reduced the total GSH levels (**** p < 0.0001); the addition of MDL72527 and βAPN failed to reverse this effect. Overall, a cytotoxic consequence of SSAO catalytic activity was observed in cultured VSMCs where SSAO was identified as a key mediator in ROS formation. These findings could potentially associate SSAO activity with the early developing stages of atherosclerosis through oxidative stress formation and vascular damage.

Expression of Concern: bFGF-Regulating MAPKs Are Involved in High Glucose-Mediated ROS Production and Delay of Vascular Endothelial Cell Migration.

Expression of Concern: bFGF-Regulating MAPKs Are Involved in High Glucose-Mediated ROS Production and Delay of Vascular Endothelial Cell Migration.

Characterization of the Dynamic Gastrointestinal Digests of the Preserved Eggs and Their Effect and Mechanism on HepG2 Cells.

Preserved eggs, an alkaline-fermented food, have been widely searched for their anti-inflammatory activity. Their digestive characteristics in the human gastrointestinal tract and anti-cancer mechanism have not been well explained. In this study, we investigated the digestive characteristics and anti-tumor mechanisms of preserved eggs using an in vitro dynamic human gastrointestinal-IV (DHGI-IV) model. During digestion, the sample pH dynamically changed from 7.01 to 8.39. The samples were largely emptied in the stomach with a lag time of 45 min after 2 h. Protein and fat were significantly hydrolyzed with 90% and 87% digestibility, respectively. Moreover, preserved eggs digests (PED) significantly increased the free radical scavenging activity of ABTS, DPPH, FRAP and hydroxyl groups by 15, 14, 10 and 8 times more than the control group, respectively. PED significantly inhibited the growth, cloning and migration of HepG2 cells at concentrations of 250-1000 μg/mL. Meanwhile, it induced apoptosis by up/down-regulating the expression of the pro-apoptotic factor Bak and the anti-apoptotic gene Bcl-2 in the mitochondrial pathway. PED (1000 μg/mL) treatment resulted in 55% higher ROS production than the control, which also led to apoptosis. Furthermore, PED down-regulated the expression of the pro-angiogenic genes HIF-1α and VEGF. These findings provided a reliable scientific reference for the study of the anti-tumor activity of preserved eggs.

Biological mechanism of cell oxidative stress and death during short-term exposure to nano CuO

It is well known that copper oxide nanoparticles (CuO NPs) are heavily toxic on in vitro systems. In human alveolar epithelial cells, the mechanism of toxicity is mostly related to oxidative insults, coming from intracellularly dissolved copper ions, finally leading to apoptotic or autophagic cell death. Our hypothesis is based on possible early oxidative events coming from specific NP surface reactivity able to undermine the cell integrity and to drive cell to death, independently from Lysosomal-Enhanced Trojan Horse mechanism. Two types of CuO NPs, with different oxidative potential, were selected and tested on A549 cells for 1 h and 3 h at 10, 25, 50 and 100 µg/ml. Cells were then analyzed for viability and oxidative change of the proteome. Oxidative by-products were localized by immunocytochemistry and cell-NP interactions characterized by confocal and electron microscopy techniques. The results show that CuO NPs induced oxidative changes soon after 1 h exposure as revealed by the increase in protein carbonylation and reduced-protein-thiol oxidation. In parallel, cell viability significantly decreased, as shown by MTT assay. Such effects were higher for CuO NPs with more crystalline defects and with higher ROS production than for fully crystalline NPs. At these exposure times, although NPs efficiently interacted with cell surface and were taken up by small endocytic vesicles, no ion dissolution was visible inside the lysosomal compartment and no effects were produced by extracellularly dissolved copper ions. In conclusion, a specific NP surface-dependent oxidative cell injury was demonstrated. More detailed studies are required to understand which targets precociously react with CuO NPs, but these results introduce new paradigms for the toxicity of the metal-based NPs, beyond the Lysosomal-Enhanced Trojan horse-related mechanism, and open-up new opportunities to investigate the interactions and effects at the bio-interface for designing safer as well as more effective CuO-based biocides.

Synergism and phytotoxicity: The effects of tank‐mix additives on the biological efficacy of Cu2+ against Venturia inaequalis and Podosphaera leucotricha

AbstractThe wetting behaviour of the spray and the biological efficacy of Cu2+ active ingredients in agrochemical formulations may be enhanced by tank‐mix additives. We investigated how three BREAK‐THRU® additives (BT301: biodegradable, BT133 and BT420: bio‐based and biodegradable) tank‐mixed with commercial copper preparations influenced the spray distribution, leaf uptake and biological efficacy of copper additive mixtures against apple scab and apple powdery mildew under controlled conditions. We quantified the synergetic effects of these additives in foliar applications. In addition, we determined the phytotoxic potential and evaluated their impacts on photosynthetic activity, non‐photochemical quenching and ROS activity. The additives BT301 and BT420 strongly reduced surface tension and contact angle of copper treatments. The fluorescence observations revealed that BT301 achieved better spreading of copper formulation with more complete coverage of the leaf surface than BT420 and BT133, whereas ‘coffee‐ring’ spreading was observed with BT133. The additive BT301 showed an increase in relative fluorescence area, indicating higher ROS production as a signal of intra‐cellular tissue activity. The photochemical efficiency of photosystem II (Fv/Fm) was not negatively influenced by copper or additive treatment. Thus, we observed no phytotoxic effects of copper‐additive mixtures on apple leaves at treatment doses of 4 g Cu2+ L−1. All copper treatments reduced apple scab infections significantly, by 53%–76%. Interestingly, addition of BT301 to copper preparations showed the strongest biological efficacy (83% reduction) against Venturia inaequalis, whereas addition of BT420 showed the strongest effect against Podosphaera leucotricha (89% infection reduction). The synergetic effects of additives on the biological efficacy without phytotoxic effects on plants may have potential for reducing copper loads in horticultural production systems.

Evaluation of Oxidative Stress and Metabolic Profile in a Preclinical Kidney Transplantation Model According to Different Preservation Modalities.

This study addresses a joint nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy approach to provide a platform for dynamic assessment of kidney viability and metabolism. On porcine kidney models, ROS production, oxidative damage kinetics, and metabolic changes occurring both during the period between organ retrieval and implantation and after kidney graft were examined. The 1H-NMR metabolic profile-valine, alanine, acetate, trimetylamine-N-oxide, glutathione, lactate, and the EPR oxidative stress-resulting from ischemia/reperfusion injury after preservation (8 h) by static cold storage (SCS) and ex vivo machine perfusion (HMP) methods were monitored. The functional recovery after transplantation (14 days) was evaluated by serum creatinine (SCr), oxidative stress (ROS), and damage (thiobarbituric-acid-reactive substances and protein carbonyl enzymatic) assessments. At 8 h of preservation storage, a significantly (p &lt; 0.0001) higher ROS production was measured in the SCS vs. HMP group. Significantly higher concentration data (p &lt; 0.05-0.0001) in HMP vs. SCS for all the monitored metabolites were found as well. The HMP group showed a better function recovery. The comparison of the areas under the SCr curves (AUC) returned a significantly smaller (-12.5 %) AUC in the HMP vs. SCS. EPR-ROS concentration (μmol·g-1) from bioptic kidney tissue samples were significantly lower in HMP vs. SCS. The same result was found for the NMR monitored metabolites: lactate: -59.76%, alanine: -43.17%; valine: -58.56%; and TMAO: -77.96%. No changes were observed in either group under light microscopy. In conclusion, a better and more rapid normalization of oxidative stress and functional recovery after transplantation were observed by HMP utilization.

Short-Term In Vitro ROS Detection and Oxidative Stress Regulators in Epiretinal Membranes and Vitreous from Idiopathic Vitreoretinal Diseases.

A plethora of inflammatory, angiogenic, and tissue remodeling factors has been reported in idiopathic epiretinal membranes (ERMs). Herein we focused on the expression of a few mediators (oxidative, inflammatory, and angiogenic/vascular factors) by means of short-term vitreal cell cultures and biomolecular analysis. Thirty-nine (39) ERMs and vitreal samples were collected at the time of vitreoretinal surgery and biomolecular analyses were performed in clear vitreous, vitreal cell pellets, and ERMs. ROS products and iNOS were investigated in adherent vitreal cells and/or ERMs, and iNOS, VEGF, Ang-2, IFNγ, IL18, and IL22 were quantified in vitreous (ELISA/Ella, IF/WB); transcripts specific for iNOS, p65NFkB, KEAP1, NRF2, and NOX1/NOX4 were detected in ERMs (PCR). Biomolecular changes were analyzed and correlated with disease severity. The higher ROS production was observed in vitreal cells at stage 4, and iNOS was found in ERMs and increased in the vitreous as early as at stage 3. Both iNOS and NOX4 were upregulated at all stages, while p65NFkB was increased at stage 3. iNOS and NOX1 were positively and inversely related with p65NFkB. While NOX4 transcripts were always upregulated, NRF2 was upregulated at stage 3 and inverted at stage 4. No significant changes occurred in the release of angiogenic (VEGF, Ang-2) and proinflammatory (IL18, IL22 and IFNγ) mediators between all stages investigated. ROS production was strictly associated with iNOS and NOX4 overexpression and increased depending on ERM stadiation. The higher iNOS expression occurred as early as stage 3, with respect to p65NFkB and NRF2. These last mediators might have potential prognostic values in ERMs as representative of an underneath retinal damage.

Effect of melatonin in different extenders on the quality of frozen semen of goats

The objective of this study was to investigate the effect of melatonin supplement in different extenders on the quality of cryopreserved goat semen. Six semen pools were cryopreserved in skim milk (SM - Experiment 1) or tris-egg yolk (TEY- Experiment 2) based extenders supplemented with melatonin (0, 0.5, 1, 2 and 4 mM). After thawing, the kinetic sperm parameters [total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight-line velocity (VSL), linearity (LIN), straightness (STR), wobble (WOB)] and viability parameters [plasma and acrosomal membrane integrity (iMPA), high mitochondrial membrane potential (hPMM) and intracellular ROS production (iROS)] were observed at time 0 hours and after 2 hours of incubation (37 °C). In the experiment I, only for the hMMP variable (p <0.05), a melatonin vs. time interaction was observed. Was noted a negatively influenced in the variables over the incubation period, except for the STR, iMPA, hMMP and iROS parameters. The treatment with melatonin had different effects in the variables VCL, VSL, WOB, hPMM, and iROS, however, in the variables TM, PM, LIN, STR and iMPA there was no significant difference regarding the different melatonin concentrations. In the experiment II, no interaction was observed, melatonin vs. time, for the analyzed variables. Was noted a negatively influenced (p <0.05) in all variables throughout the incubation period, with the exception of the STR, iMPA and hMMP parameters. The treatment with melatonin showed different effects in the variables TM, PM, VSL, LIN, STR, WOB, iMPA, hPMM and iROS, however, in the variable VCL there was no significant difference regarding the different concentrations of melatonin. In conclusion, melatonin does not improve the post-thawing quality of goat semen cryopreserved in SM or TEY based extenders and may be deleterious when supplemented at concentration of 4 mM.

Heterosigma akashiwo in Patagonian Fjords: Genetics, Growth, Pigment Signature and Role of PUFA and ROS in Ichthyotoxicity.

Heterosigma akashiwo is the only raphidophyte described for Chilean waters. A recent 2021 fish-killing bloom event of this raphidophyte ignited scientific research, but the ichthyotoxic mechanism and environmental conditions that promote its growth are still unclear. This is the first study confirming the occurrence of H. akashiwo in Chilean waters on the basis of the region D1/D2 of the 28S ribosomal gene. The pigment signature of the CREAN_HA03 strain revealed chlorophyll-a, fucoxanthin, and violaxanthin as the most abundant pigments, but profiles were variable depending on culture and field conditions. A factorial temperature–salinity growth experiment showed a maximal growth rate of 0.48 d−1 at 17 °C and 35 in salinity, but reached a maximal cell abundance of ~50,000 cells mL−1 at 12 °C and 25 in salinity. The fatty acid profile included high levels of saturated (16:0) and polyunsaturated (18:4 ω3; 20:5 ω3) fatty acids, but superoxide production in this strain was low (~0.3 pmol O2– cell−1 h−1). The RTgill-W1 bioassay showed that the H. akashiwo strain was cytotoxic only at high cell concentrations (>47,000 cells mL−1) and after cell rupture. In conclusion, salmon mortality during H. akashiwo bloom events in Patagonian fjords is likely explained by the high production of long-chain PUFAs at high cell densities, but only in the presence of high ROS production.