ROS Formation Research Articles

Uncoupling of Cytochrome P450 2B6 and stimulation of reactive oxygen species production in pharmacogenomic alleles affected by interethnic variability

Cytochrome P450 mediated substrate metabolism is generally characterized by the formation of reactive intermediates. In vitro and in vivo reaction uncoupling, results in the accumulation and dissociation of reactive intermediates, leading to increased ROS formation. The susceptibility towards uncoupling and altered metabolic activity is partly modulated by pharmacogenomic alleles resulting in amino acid substitutions. A large variability in the prevalence of these alleles has been demonstrated in CYP2B6, with some being predominantly unique to African populations.The aim of this study is to characterize the uncoupling potential of recombinant CYP2B6*1, CYP2B6*6 and CYP2B6*34 metabolism of specific substrates.Therefore, functional effects of these alterations on enzyme activity were determined by quantification of bupropion, efavirenz and ketamine biotransformation using HPLC-MS/MS. Determination of H2O2 levels was performed by the AmplexRed/horseradish peroxidase assay.Our studies of the amino acid substitutions Q172H, K262R and R487S revealed an exclusive use of the peroxide shunt for the metabolism of bupropion and ketamine by CYP2B6*K262R. Ketamine was also identified as a trigger for the peroxide shunt in CYP2B6*1 and all variants. Concurrently, ketamine acted as an uncoupler for all enzymes. We further showed that the expressed CYP2B6*34 allele results in the highest H2O2 formation.We therefore conclude that the reaction uncoupling and peroxide shunt are directly linked and can be substrate specifically induced with K262R carriers being most likely to use the peroxide shunt and R487S carrier being most prone to reaction uncoupling. This elucidates the functional diversity of pharmacogenomics in drug metabolism and safety.

Oxidative Stress-Mediated Repression of Virulence Gene Transcription and Biofilm Formation as Antibacterial Action of Cinnamomum burmannii Essential Oil on Staphylococcus aureus.

This work aimed to identify the chemical compounds of Cinnamomum burmannii leaf essential oil (CBLEO) and to unravel the antibacterial mechanism of CBLEO at the molecular level for developing antimicrobials. CBLEO had 37 volatile compounds with abundant borneol (28.40%) and showed good potential to control foodborne pathogens, of which Staphylococcus aureus had the greatest inhibition zone diameter (28.72 mm) with the lowest values of minimum inhibitory concentration (1.0 μg/mL) and bactericidal concentration (2.0 μg/mL). To unravel the antibacterial action of CBLEO on S. aureus, a dynamic exploration of antibacterial growth, material leakage, ROS formation, protein oxidation, cell morphology, and interaction with genome DNA was conducted on S. aureus exposed to CBLEO at different doses (1/2-2×MIC) and times (0-24 h), indicating that CBLEO acts as an inducer for ROS production and the oxidative stress of S. aureus. To highlight the antibacterial action of CBLEO on S. aureus at the molecular level, we performed a comparative association of ROS accumulation with some key virulence-related gene (sigB/agrA/sarA/icaA/cidA/rsbU) transcription, protease production, and biofilm formation in S. aureus subjected to CBLEO at different levels and times, revealing that CBLEO-induced oxidative stress caused transcript suppression of virulence regulators (RsbU and SigB) and its targeted genes, causing a protease level increase destined for the biofilm formation and growth inhibition of S. aureus, which may be a key bactericidal action. Our findings provide valuable information for studying the antibacterial mechanism of essential oil against pathogens.

Profiling of Petroselinum sativum (mill.) fuss phytoconstituents and assessment of their biocompatibility, antioxidant, anti-aging, wound healing, and antibacterial activities.

Petroselinum sativum, known as parsley, is a fragrant herb that possesses a rich heritage of utilization in traditional medicinal practices. In this study, we annotated the phytocontents of the aqueous and ethanolic extracts of P. sativum and investigated their antioxidant, cytoprotective, antiaging, wound healing, and antibacterial activities. LC-MS/MS analysis of both extracts revealed the presence of 47 compounds belonging to diverse groups including organic acids, phenolic acids, and flavonoids. By MTT assay, the extracts were fully biocompatible on immortalized human keratinocytes (HaCaT) while they inhibited intracellular ROS formation (DCFDA assay) and prevented GSH depletion (DTNB assay) upon UVA exposure. In addition, the extracts were potent in inhibiting the in vitro activities of skin-related enzymes mainly elastase, tyrosinase, collagenase and hyaluronidase. Using the scratch assay, P. sativum aqueous extract significantly enhanced wound closure when compared to untreated HaCaT cells. Moreover, both extracts inhibited Pseudomonas aeruginosa's growth, reduced biofilm formation, and impaired the swimming and swarming motilities. Also, the aqueous extract was able to inhibit the production of bacterial pigments on plates. These findings strongly suggest the usefulness of P. sativum as a source of phytochemicals suitable for dermo-cosmeceutical applications.

Adjuvant Activity and Toxicological Risks of Lipid Nanoparticles Contained in the COVID‑19 “mRNA Vaccines”

The LNPs reportedly used as the platform by Pfizer/BioNTech for its SARS-CoV-2 “mRNA vaccines” allegedly consist of a mixture of phospholipids, cholesterol, PEGylated lipids, and an ionizable cationic lipid. This study reviews some of the main toxicological risks and immunostimulatory properties of such nanomaterials, with particular attention to the ionizable LNPs and their adjuvant properties, inflammatory responses, stimulation of immune cells, and formation of ROS inside transfected cells. The decision not to carry out safety pharmacology, carcinogenicity, and genotoxicity tests on these nanomaterials appears unjustifiable and in contradiction with the international policies provided for novel adjuvants. Important gaps are highlighted on the activities by the relevant regulatory bodies, related to the scientific evaluation, risk management, and pharmacovigilance for new medicinal products in the EU. Given the findings discussed here, it is strongly urged that the mRNA-LNP-based “vaccines” and their boosters should be removed from the worldwide market because of unacceptable and potentially fatal safety risks.

Associations between brake and tire wear-related PM2.5 metal components, particulate oxidative stress potential, and autism spectrum disorder in Southern California

BackgroundAir pollution is a global health concern, with fine particulate matter (PM2.5) constituents posing potential risks to human health, including children's neurodevelopment. Here we investigated associations between exposure during pregnancy and infancy to specific traffic-related PM2.5 components with Autism Spectrum Disorder (ASD) diagnosis. MethodsFor exposure assessment, we estimated PM2.5 components related to traffic exposure (Barium [Ba] as a marker of brake dust and Zinc [Zn] as a tire wear marker, Black Carbon [BC]) and oxidative stress potential (OSP) markers (Hydroxyl Radical [OPOH] formation, Dithiothreitol activity [OPDTT], reactive oxygen species [ROS]) modeled with land use regression with co-kriging based on an intensive air monitoring campaign. We assigned exposures to a cohort of 444,651 children born in Southern California between 2016 and 2019, among whom 11,466 ASD cases were diagnosed between 2018 and 2022, Odds ratios (ORs) and 95% confidence intervals (CIs) were obtained with logistic regression for single pollutant and PM2.5 mass co-adjusted models, also adjusting for sociodemographic characteristics. ResultsAmong PM2.5 components, we found the strongest positive association with ASD for our brake wear marker Ba (ORper IQR = 1.29, 95 % CI: 1.24, 1.34). This was followed by an increased risk for all PM2.5 oxidative stress potential markers; the strongest association was with ROS formation (ORper IQR = 1.22, 95 % CI: 1.18, 1.25). PM2.5 mass was linked to ASD in Hispanic and Black children, but not White children, while traffic-related PM2.5 and OSP markers increased ASD risk across all groups. In neighborhoods with the lowest socioeconomic status (SES), associations with ASD were stronger for all examined pollutants compared to higher SES areas. ConclusionsOur findings suggest that brake wear-related PM2.5 and PM2.5 OSP are associated with ASD diagnosis in Southern California. These results suggest that strategies aimed at reducing the public health impacts of PM2.5 need to consider specific sources.

Potential of Polyphenolic Compounds in Rosella Flowers on Reducing Oxidative Stress and Inflammation After Exercise: A Systematic Review

Study purpose. This study aims to analyze and highlight the potential of rosella flowers in reducing oxidative stress and inflammation after physical activity. Materials and methods. The study used a systematic review method by searching various journal databases such as Scopus, Web of Science, Pubmed and Embase. The inclusion criteria in this study were articles published in the last 5 years and articles discussing Rosella Flowers, Oxidative Stress, Free Radicals, Inflammation, and Exercise. The exclusion criteria in this research were articles published in disreputable journals. A total of 357 articles from the Scopus, Web of Science Pubmed and Embase databases were identified. A total of 8 articles that met the inclusion criteria were selected and analyzed for this systematic review. For standard operations, this study followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) assessment. Results. The results of this systematic review research report that the flavonoid content found in rosella flowers has anti-oxidant and anti-inflammatory properties which have the potential to reduce oxidative stress and uncontrolled inflammation caused by physical activity and intense exercise. Conclusions. Rosella flowers show their ability as an anti-oxidative by donating hydrogen atoms to free radicals through phenolic compounds, thus breaking the cycle of ROS formation. The natural active ingredients in rosella flowers are able to reduce MDA and increase GSH which contributes to reducing oxidative stress and decreasing TNF-α which provides an anti-inflammatory effect in athletes after exercise.

Free radical processes in the liver mitochondria of rats exposed to diethyl phthalate

Diethyl phthalate (DEF) is a synthetic chemical widely used as plasticizer and additive in personal care and pharmaceutical products. Low-dose exposure to this xenobiotic over a long period contributes to its intake into the human body in a high doses. To date, studies of DEF influence on free radical processes in liver cells, in which it is not only metabolized but can also have a prooxidant effect, remain limited. The aim of our research was to determine ROS formation, the intensity of oxidative modification of proteins (OMP) and proteolytic activity in the mitochondrial fraction of liver tissue of rats exposed to diethyl phtalate. The experimental white outbred rats were divided into three groups: I – intact animals (control), II and III – rats administered DEF orally for three weeks at a doses that reflect the dose levels received by humans, namely 2.5 and 5.4 mg/kg b.w. respectively. The animals were euthanized on the 14th and 21st day after xenobiotic administration. The mitochondrial fraction from the rat liver was isolated and the level of superoxide and hydroxyl radicals, protein carbonyl derivatives, SH-groups and Schiff bases was determined. Proteolytic activity was assessed in the test of hemoglobin cleavage. It was shown that administration of DEF in a dose of 2.5 mg/kg initiated ROS generation and OMP intensification in the rat liver mitochondria only with prolonged administration for 21 days, whereas its administration in a dose of 5.4 mg/kg led to intensification of these processes already on day 14th followed by further amplification on day 21st. The activity of proteolytic enzymes in the mitochondrial fraction was found to be depended on the degree of DEF–induced OMP and was increased with minor OMP intensification but decreased with significant intensification of proteins free radical oxidation. Keywords: carbonyl derivatives, diethyl phthalate, liver, mitochondrial fraction, proteolytic enzymes, ROS, SH-groups

Enhancement of the biological activity of hydroxytyrosol through its oxidation by laccase from Trametes versicolor

The laccase-catalyzed oxidation of hydroxytyrosol (HT) towards the formation of its bioactive oligomer derivatives was investigated. The biocatalytic oligomerization was catalyzed by laccase from Trametes versicolor in aqueous or various water-miscible organic solvents and deep eutectic solvent (DES)-based media. Mass Spectroscopy and Nuclear Magnetic Resonance were used for the characterization of the products. The solvent system used significantly affects the degree of HT oligomerization. The use of 50 % v/v methanol favored the production of the HT dimer, while other organic solvents as well as DESs led to the formation of hydroxytyrosol trimer and other oligomers. In vitro studies showed that the HT dimer exhibits 3- to 4-fold enhanced antibacterial activity against Gram-positive and Gram-negative bacteria compared to the parent compound. Moreover, the ability of HT dimer to inhibit the activity of soybean lipoxygenase and Candida rugosa lipase was 1.5-fold higher than HT, while molecular docking supported these results. Furthermore, HT dimer showed reduced cytotoxicity against HEK293 cells and exhibited a strong ability to inhibit ROS formation. The enhanced bioactivity of HT dimer indicates that this compound could be considered for use in cosmetics, skin-care products, and nutraceuticals.

Xanthohumol ameliorates drug-induced hepatic ferroptosis via activating Nrf2/xCT/GPX4 signaling pathway

BackgroundAs a canonical iron-dependent form of regulated cell death (RCD), ferroptosis plays a crucial role in chemical-induced liver injuries. Previous studies have demonstrated that xanthohumol (Xh), a natural prenylflavonoid isolated from hops, exhibits anti-inflammatory, anti-antioxidative and hepatoprotective properties. However, the regulatory effects of Xh on hepatic ferroptosis and the underlying mechanism have not yet been fully elucidated. PurposeTo investigate the hepatoprotective effects of Xh against drug-induced liver injury (DILI) and the regulatory effects of Xh on hepatic ferroptosis, as well as to reveal the underlying molecular mechanisms. Methods/study designThe hepatoprotective benefits of Xh were investigated in APAP-induced liver injury (AILI) mice and HepaRG cells. Xh was administered intraperitoneally to assess its in vivo effects. Histological and biochemical studies were carried out to evaluate liver damage. A series of ferroptosis-related markers, including intracellular Fe2+ levels, ROS and GSH levels, the levels of MDA, LPO and 4-HNE, as well as the expression levels of ferroptosis-related proteins and modulators were quantified both in vivo and in vitro. The modified peptides of Keap1 by Xh were characterized utilizing nano LC-MS/MS. ResultsXh remarkably suppresses hepatic ferroptosis and ameliorates AILI both in vitro and in vivo, via suppressing Fe2+ accumulation, ROS formation, MDA generation and GSH depletion, these observations could be considerably mitigated by the ferroptosis inhibitor ferrostatin-1 (Fer-1). Mechanistically, Xh could significantly activate the Nrf2/xCT/GPX4 signaling pathway to counteract AILI-induced hepatocyte ferroptosis. Further investigations showed that Xh could covalently modify three functional cysteine residues (cys151, 273, 288) of Keap1, which in turn, reduced the ubiquitination rates of Nrf2 and prolonged its degradation half-life. ConclusionsXh evidently suppresses hepatic ferroptosis and ameliorates AILI via covalent modifying three key cysteines of Keap1 and activating Nrf2/xCT/GPX4 signaling pathway.

Effect of Ag and Ni-Doped Cerium Oxide Nanoparticles on the Formation of ROS and Evaluation as an Alternative Physical Sunscreen Material.

CeO2 nanoparticles (nanoceria) were proposed as an alternative physical sunscreen agent with antioxidant properties and comparable UV absorption performance. Green synthesis of nanoceria with Ag and Ni dopants resulted in doped nanoceria with lower catalytic activity and biologically-safe characteristics. The doped nanoceria was characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), Rancimat Instrument, and UV-Vis Spectrophotometer for SPF (Sun Protection Factor) determination. XRD and TEM analysis showed that nanoceria had been successfully formed in nanoscale-sized with a change in crystallite size due to the crystal defect phenomenon caused by dopant addition. While the Rancimat test and band gap energy analysis were conducted to evaluate the oxidative stability and reactive oxygen species formation, it was confirmed that dopant addition could decrease catalytic activity of material, resulting in Ni-doped Ce with a longer incubation time (11.81 h) than Ag-doped Ce (10.58 h) and non-doped Ce (10.30 h). In-vitro SPF value was measured using the thin layer technique of sunscreen prototype with Virgin Coconut Oil (VCO)-based emulsion, which yielded 10.862 and 5.728 SPF values for 10% Ag-doped Ce and 10% Ni-doped Ce, respectively. The dopant addition of nanoceria could reduce catalytic activity and give a decent in vitro UV-shielding performance test; thus, Ag and Ni-doped nanoceria could be seen as promising candidates for alternative physical sunscreen agents.

Myconoside and Calceolarioside E Restrain UV-Induced Skin Photoaging by Activating NRF2-Mediated Defense Mechanisms.

Chronic and excessive ultraviolet (UVA/UVB) irradiation exposure is known as a major contributor to premature skin aging, which leads to excessive reactive oxygen species generation, disturbed extracellular matrix homeostasis, DNA damage, and chronic inflammation. Sunscreen products are the major preventive option against UVR-induced photodamage, mostly counteracting the acute skin effects and only mildly counteracting accelerated aging. Therefore, novel anti-photoaging and photopreventive compounds are a subject of increased scientific interest. Our previous investigations revealed that the endemic plant Haberlea rhodopensis Friv. (HRE) activates the antioxidant defense through an NRF2-mediated mechanism in neutrophiles. In the present study, we aimed to investigate the photoprotective potential of HRE and two of its specialized compounds-the phenylethanoid glycosides myconoside (MYC) and calceolarioside E (CAL)-in UVA/UVB-stimulated human keratinocytes in an in vitro model of photoaging. The obtained data demonstrated that the application of HRE, MYC, and CAL significantly reduced intracellular ROS formation in UVR-exposed HaCaT cells. The NRF2/PGC-1α and TGF-1β/Smad/Wnt signaling pathways were pointed out as having a critical role in the observed CAL- and MYC-induced photoprotective effect. Collectively, CAL is worth further evaluation as a potent natural NRF2 activator and a promising photoprotective agent that leads to the prevention of UVA/UVB-induced premature skin aging.

Anti-dengue potential and mosquitocidal effect of marine green algae-stabilized Mn-doped superparamagnetic iron oxide nanoparticles (Mn-SPIONs): an eco-friendly approach.

Vector-borne diseases pose a significant public health challenge in economically disadvantaged nations. Malaria, dengue fever, chikungunya, Zika, yellow fever, Japanese encephalitis, and lymphatic filariasis are spread by mosquitoes. Consequently, the most effective method of preventing these diseases is to eliminate the mosquito population. Historically, the majority of control programs have depended on chemical pesticides, including organochlorines, organophosphates, carbamates, and pyrethroids. Synthetic insecticides used to eradicate pests have the potential to contaminate groundwater, surface water, beneficial soil organisms, and non-target species. Nanotechnology is an innovative technology that has the potential to be used in insect control with great precision. The goal of this study was to test the in vitro anti-dengue potential and mosquitocidal activity of Chaetomorpha aerea and C. aerea-synthesized Mn-doped superparamagnetic iron oxide nanoparticles (CA-Mn-SPIONs). The synthesis of CA-Mn-SPIONs using C. aerea extract was verified by the observable alteration in the colour of the reaction mixture, transitioning from a pale green colour to a brown. The study of UV-Vis spectra revealed absorbance peaks at approximately 290nm, which can be attributed to the surface Plasmon resonance of the CA-Mn-SPIONs. The SEM, TEM, EDX, FTIR, vibrating sample magnetometry, and XRD analyses provided evidence that confirmed the presence of CA-Mn-SPIONs. In the present study, results revealed that C. aerea aqueous extract LC50 values against Ae. aegypti ranged from 222.942 (first instar larvae) to 349.877ppm in bioassays (pupae). CA-Mn-SPIONs had LC50 ranging from 20.199 (first instar larvae) to 26.918ppm (pupae). After treatment with 40ppm CA-Mn-SPIONs and 500ppm C. aerea extract in ovicidal tests, egg hatchability was lowered by 100%. Oviposition deterrence experiments showed that in Ae. aegypti, oviposition rates were lowered by more than 66% by 100ppm of green algal extract and by more than 71% by 10ppm of CA-Mn-SPIONs (oviposition activity index values were 0.50 and 0.55, respectively). Moreover, in vitro anti-dengue activity of CA-Mn-SPIONs has good anti-viral property against dengue viral cell lines. In addition, GC-MS analysis showed that 21 intriguing chemicals were discovered. Two significant phytoconstituents in the methanol extract of C. aerea include butanoic acid and palmitic acid. These two substances were examined using an in silico methodology against the NS5 methyltransferase protein and demonstrated good glide scores and binding affinities. Finally, we looked into the morphological damage and fluorescent emission of third instar Ae. aegypti larvae treated with CA-Mn-SPIONs. Fluorescent emission is consistent with ROS formation of CA-Mn-SPIONs against Ae. aegypti larvae. The present study determines that the key variables for the successful development of new insecticidal agents are rooted in the eco-compatibility and the provision of alternative tool for the pesticide manufacturing sector.

Pengaruh Frekuensi Latihan Fisik Submaksimal Terhadap Kadar Serum Glutamic Pyruvic Transaminase (SGPT) Tikus Putih (Rattus norvegicus) Betina Galur Wistar

Abstract
 According to WHO data (2020), individuals with inadequate physical exercise develop a mortality risk of 20-30% compared to individuals with adequate physical exercise. Regular physical exercise has a positive impact in maintaining a healthy lifestyle, whereas acute intense physical exercise such as in untrained individuals can have a negative impact through the occurrence of oxidative stress. Physical exercise with repetition of exercise can form an adaptation mechanism thereby increasing the work of endogenous antioxidants and inhibiting the formation of ROS. Meanwhile, acute physical exercise without repetition of exercise can cause a decrease in endogenous antioxidant levels and an increase in ROS. Increased ROS induces oxidative stress, which then plays a role in liver damage. This study aims to determine the effect of submaximal physical exercise frequency on SGPT levels, an indicator of liver damage. This research was a laboratory experimental study with a post-test only control group design using 27 Wistar female white rats which were divided into 3 groups; K1 was given standard feed for 4 weeks, K2 was given standard feed for 4 weeks, submaximal physical exercise on days 1 to 14 and 21 of the study, and K3 was given standard feed for 4 weeks and submaximal physical exercise on day 21 of the study. After intervention, all groups of rats were taken for blood to calculate SGPT levels and then the results of the data were analyzed statistically. The results of the Kruskal-Wallis test showed a significance of 0.479 (p>α) with α=0.05, which means that there was no significant difference in the frequency of submaximal physical exercise on SGPT levels in female Wistar rats (Rattus norvegicus). The conclusion of this study showed that there was no effect of the frequency of submaximal physical exercise on SGPT levels in female Wistar rats (Rattus norvegicus).

Lipid Peroxidation as the Mechanism Underlying Polycyclic Aromatic Hydrocarbons and Sunlight Synergistic Toxicity in Dermal Fibroblasts.

Light and atmospheric pollution are both independently implicated in cancer induction and premature aging. Evidence has been growing more recently on the toxic synergy between light and pollutants. Polycyclic aromatic hydrocarbons (PAHs) originate from the incomplete combustion of organic matter. Some PAHs, such as the Benzo[a]pyrene (BaP), absorb ultraviolet A (UVA) wavelengths and can act as exogenous chromophores, leading to synergistic toxicity through DNA damage and cytotoxicity concomitant to ROS formation. In this study, we shed light on the mechanism underlying the toxic synergy between PAHs and UVA. Using dermal fibroblasts co-exposed to UVA and BaP, we have demonstrated that the photosensitization reaction causes mortality, which is most likely caused by ROS accumulation. We have shown that these ROS are concentrated in the lipids, which causes an important induction of lipid peroxidation and malondialdehyde, by-products of lipid peroxidation. We have also shown the accumulation of bulky DNA damage, most likely generated by these by-products of lipid peroxidation. To our knowledge, this study represents the first one depicting the molecular effects of photo-pollution on dermal skin.

Modulation in growth, oxidative stress, photosynthesis, and morphology reveals higher toxicity of alpha-cypermethrin than chlorpyrifos towards a non-target green alga at high doses

Considering the frequent detection of pesticides in the aquatic environment, the ecotoxicological effects of Chlorpyrifos (CHP), an organophosphate, and alpha-cypermethrin (ACM), a pyrethroid, on freshwater microalgae were compared for the first time in this study. High concentrations of both CHP and ACM significantly suppressed the growth of test microalga Graesiella emersonii (p < 0.05). The 96-h EC50 of CHP and ACM were 54.42 mg L−1 and 29.40 mg L−1, respectively. Sub-inhibitory doses of both pesticides increased ROS formation in a concentration-dependent manner, which was accompanied by changes in antioxidant enzymes activities, lipid peroxidation, and variations in photosynthetic pigment concentration. Furthermore, both pesticides influenced photosystem II performance, oxygen-evolving complex efficiency and, intracellular ATP levels. Scanning electron microscopy analysis revealed that high concentrations of both CHP and ACM caused considerable morphological changes in the microalga. In comparison, CHP was more toxic than ACM at low concentrations, whereas ACM was more toxic at high concentrations.

A human anti-matriptase-2 antibody limits iron overload, α-globin aggregates, and splenomegaly in β-thalassemic mice

AbstractIron plays a major role in the deterioration of β-thalassemia. Indeed, the high levels of transferrin saturation and iron delivered to erythroid progenitors are associated with production of α-globin precipitates that negatively affect erythropoiesis. Matriptase-2/TMPRSS6, a membrane-bound serine protease expressed in hepatocytes, negatively modulates hepcidin production and thus is a key target to prevent iron overload in β-thalassemia. To address safety concerns raised by the suppression of Tmprss6 by antisense oligonucleotides or small interfering RNA, we tested a fully human anti-matriptase-2 antibody, RLYB331, which blocks the protease activity of matriptase-2. When administered weekly to Hbbth3/+ mice, RLYB331 induced hepcidin expression, reduced iron loading, prevented the formation of toxic α-chain/heme aggregates, reduced ros oxygen species formation, and improved reticulocytosis and splenomegaly. To increase the effectiveness of RLYB331 in β-thalassemia treatment even further, we administered RLYB331 in combination with RAP-536L, a ligand-trapping protein that contains the extracellular domain of activin receptor type IIB and alleviates anemia by promoting differentiation of late-stage erythroid precursors. RAP-536L alone did not prevent iron overload but significantly reduced apoptosis in the erythroid populations of the bone marrow, normalized red blood cell counts, and improved hemoglobin and hematocrit levels. Interestingly, the association of RLYB331 with RAP-536L entirely reversed the β-thalassemia phenotype in Hbbth3/+ mice and simultaneously corrected iron overload, ineffective erythropoiesis, splenomegaly, and hematological parameters, suggesting that a multifunctional molecule consisting of the fusion of RLYB331 with luspatercept (human version of RAP-536L) would allow administration of a single medication addressing simultaneously the different pathophysiological aspects of β-thalassemia.

Organic Extract of Streptomyces Induces Apoptosis, Anti-Proliferation and Cell-Cycle Arrest in Colon Cancer Cells: Caco-2 and HCT116 Cell Line as Surrogate Model

Background: Colorectal cancer (CRC) is a prevalent and very lethal malignancy, resulting in more than one million deaths and nearly two million newly diagnosed cases in the year 2018. Streptomyces bacteria are a potential source of conventional compounds, as their extract contains bioactive chemicals with antiproliferative effects in cell culture settings. The aim of this research was to investigate the antiproliferative effects of the Streptomyces ethyl acetate fraction against the Caco-2 and HCT116 cell line in addition to its fundamental mechanisms. Methods: We examined the effects of Streptomyces extract on colon cancer cell lines Caco-2 and HT-116 by incubating the cells with and without the extract. Antiproliferative effects are analyzed using the MTT cell proliferation assay, cell cycle analysis and flow cytometric detection of Annexin V/PI apoptosis. The formation of intracellular ROS was measured, in addition to Western Blot for Bax, Bcl2 and caspase-3 activation. Result: Our findings showed the dose-dependent inhibition of cell growth by streptomyces extract. Both Caco-2 and HT-116 cells treated with streptomyces extract displayed the beginning of apoptotic events and G1 cell-cycle arrest. Additionally, treat with the streptomyces extract significantly increased (P less than 0.05) ROS production, Bax and cleaved-caspase-3 were increased, while Bcl-2 was downregulated, as determined by western blotting.

Ineffectiveness of mitoTEMPO on Cardiomyocyte S-phase Activity in TNNI3K-expressing Mice

Background and Hypothesis: The limited regenerative capacity of the mammalian adult myocardium is a significant roadblock for therapeutic approaches in cardiovascular disease. Cell cycle arrest following S-phase is widely considered a primary contributor to the reduced proliferative capacity of adult cardiomyocytes. Recently, expression of troponin I-interacting kinase (Tnni3k) was shown to increase cardiomyocyte S-phase activity in mice. Tnni3k was previously shown to enhance ROS formation and adverse cardiac remodeling following injury. Our primary hypothesis was that TNNI3K-induced cardiomyocyte DNA synthesis resulted from enhanced ROS signaling. To test this, cardiomyocyte S-phase activity in TNNI3K-expressing mice was compared between those treated with the ROS scavenging agent mitoTEMPO and untreated mice.&#x0D; Project Methods: Transgenic mice expressing TNNI3K were subjected to 14 days infusion with mitoTEMPO (experimental group) or vehicle (control group). The mice were also subjected to 14 days infusion with bromodeoxyuridine (BrdU) to identify DNA synthesis during S-phase (all mice carried a cardiomyocyte-restricted nuclear-localized transgenic reporter to aid in cardiomyocyte nuclei identification). The proportion of cardiomyocytes in S-phase was determined and mean S-phase activity was compared between treatment groups. Ploidy analysis was also conducted to determine if cardiomyocytes completing S-phase progressed through karyokinesis.&#x0D; Results: The percentage of cardiomyocytes in S-phase in the control and mitoTEMPO treated group were 0.819% ± 0.163% and 0.855% ± 0.138%, respectively (mean ± SEM, p=0.873). Ploidy analysis revealed no overt difference in DNA content in S-phase-positive cardiomyocyte nuclei between the groups. Hence, we have shown that there is no appreciable difference in cell cycle induction or progression in cardiomyocytes from control vs. mitoTEMPO treated mice expressing TNNI3K.&#x0D; Conclusion and Potential Impact: These data suggest that (a) TNNI3K-induced cardiomyocyte S-phase activity is not secondary to elevated ROS activity, and (b) reduction of ROS activity does not relax the cell cycle block between S-phase and karyokinesis in adultcardiomyocytes.

Oxidative stress and senescence in aging kidneys: the protective role of SIRT1.

Aging leads to a gradual decline in kidney function, making the kidneys increasingly vulnerable to various diseases. Oxidative stress, together with cellular senescence, has been established as paramount in promoting the aging process of the kidney. Oxidative stress, defined as an imbalance between ROS formation and antioxidant defense mechanisms, has been implicated in the kidney's cellular injury, inflammation, and premature senescence. Concurrently, the accumulation of SCs in the kidney also exacerbates oxidative stress via the secretion of pro-inflammatory and tissue-damaging factors as the senescence-associated secretory phenotype (SASP). Recently, SIRT1, a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase, has been pivotal in combating oxidative stress and cellular senescence in the aging kidney. SIRT1 acts as a potential antioxidant molecule through myriad pathways that influence diverse transcription factors and enzymes essential in maintaining redox homeostasis. SIRT1 promotes longevity and renal health by modulating the acetylation of cell cycle and senescence pathways. This review covers the complex relationship between oxidative stress and cellular senescence in the aging kidney, emphasizing the protective role of SIRT1. See also the graphical abstract(Fig. 1).

Exploring the cellular antioxidant mechanism against cytotoxic silver nanoparticles: a Raman spectroscopic analysis.

Silver nanoparticles (AgNPs) hold great promise for several different applications, from colorimetric sensors to antimicrobial agents. Despite their widespread incorporation in consumer products, limited understanding of the detrimental effects and cellular antioxidant responses associated with AgNPs at sublethal concentrations persists, raising concerns for human and ecological well-being. To address this gap, we synthesized AgNPs of varying sizes and evaluated their cytotoxicity against human dermal fibroblasts (HDF). Our study revealed that toxicity of AgNPs is a time- and size-dependent process, even at low exposure levels. AgNPs exhibited low short-term cytotoxicity but high long-term impact, particularly for the smallest NPs tested. Raman microspectroscopy was employed for in-time investigations of intracellular molecular variations during the first 24 h of exposure to AgNPs of 35 nm. Subtle protein and lipid degradations were detected, but no discernible damage to the DNA was observed. Signals associated with antioxidant proteins, such as superoxide dismutase (SOD), catalase (CAT) and metallothioneins (MTs), increased over time, reflecting the heightened production of these defense agents. Fluorescence microscopy further confirmed the efficacy of overexpressed antioxidant proteins in mitigating ROS formation during short-term exposure to AgNPs. This work provides valuable insights into the molecular changes and remedial strategies within the cellular environment, utilizing Raman microspectroscopy as an advanced analytical technique. These findings offer a novel perspective on the cytotoxicity mechanism of AgNPs, contributing to the development of safer materials and advice on regulatory guidelines for their biomedical applications.