Dichlorofluorescein Assay Research Articles

Biophysical insight into the binding mechanism of epigallocatechin-3-gallate and cholecalciferol to albumin and its preventive effect against AGEs formation: An in vitro and in silico approach

Advanced glycation end products (AGEs) are produced non-enzymatically through the process of glycation. Increased AGEs production has been linked to several diseases including polycystic ovary syndrome (PCOS). PCOS contributes to the development of secondary comorbidities, such as diabetes, cardiovascular complications, infertility, etc. Consequently, research is going on AGEs-inhibiting phytochemicals for their potential to remediate and impede the progression of hyperglycaemia associated disorders. In this study human serum albumin is used as a model protein, as albumin is predominantly present in follicular fluid. This article focusses on the interaction and antiglycating potential of (−)-Epigallocatechin-3-gallate (EGCG) and vitamin D in combination using various techniques. The formation of the HSA-EGCG and HSA-vitamin D complex was confirmed by UV and fluorescence spectroscopy. Thermodynamic analysis verified the spontaneity of reaction, and presence of hydrogen bonds and van der Waals interactions. FRET confirms high possibility of energy transfer. Cumulative antiglycation resulted in almost 60 % prevention in AGEs formation, decreased alterations at lysine and arginine, and reduced protein carbonylation. Secondary and tertiary structural changes were analysed by circular dichroism, Raman spectroscopy and ANS binding assay. Type and size of aggregates were confirmed by Rayleigh and dynamic light scattering, ThT fluorescence, SEM and SDS-PAGE. Effect on cellular redox status, DNA integrity and cytotoxicity was analysed in lymphocytes using dichlorofluorescein (DCFH-DA), DAPI and MTT assay which depicted an enhancement in antioxidant level by cumulative treatment. These findings indicate that EGCG and vitamin D binds strongly to HSA and have antiglycation ability which enhances upon synergism.

Melanogenic effects of 5-demethylnobiletin on mouse model of chemical-induced vitiligo

BackgroundA phytochemical 5-demethylnobiletin from Citrus reticulata Blanco has a broad spectrum of pharmacological effects on anti-inflammatory, cardiovascular and neuroprotective effects, however, its in vivo effect on depigmentation remains unknown. ObjectiveTo investigate how 5-demethylnobiletin regulates melanogenesis, both in vitro and in vivo experiments were conducted to evaluate its efficacy and mechanisms in human epidermal melanocyte and mice model of chemical-induced vitiligo. MethodsC57BL/6 mice were induced with 1% hydroquinone (HQ) to shaved dorsal skin and applied with 5-demethylnobiletin or 8-methoxypsoralen (8-MOP, positive control). Melanin contents measurement, immunohistochemistry, Fontana-Masson and HE histological analysis were used to determine melanin contents in follicles. q-PCR, Western blot, dichlorofluorescein (DCF) assay and computational docking were adopted to investigate the molecular mechanisms of 5-demethylnobiletin in the vitiligo mice model. Besides, acute dermal irritation test was conducted to evaluate the biological safety of 5-demethylnobiletin in mice. Results5-demethylnobiletin possessed in vitro anti-oxidative effect, significantly increased melanin content in hair and ameliorated hypopigmentation in mice. The expression level of melanogenesis-related genes such as MITF, tyrosinase, Trp-1 and Trp-2 were significantly upregulated in 5-demethylnobiletin-treated groups. Molecular docking results support that 5-demethylnobiletin is able to bind to tyrosinase, the key enzyme for regulating melanogenesis, suggesting the potential molecular target of 5-demethylnobiletin. Conclusions5-demethylnobiletin has potential therapeutic effect on delaying the onset of depigmentation in vivo, lessened depigmentation incidence which contributes to melanogenesis. The newly revealed biological activity indicates 5-demethylnobiletin may be a potential natural pharmaceutical for the treatment and prevention of chemical-induced depigmentation disorders, and as the components for anti-gray hair products.

Total transcriptome analysis of Candida auris planktonic cells exposed to tyrosol

Tyrosol, a secondary metabolite of Candida species, regulates fungal morphogenesis, and its application may represent a novel innovative therapy against emerging multi-resistant fungal superbug such as Candida auris. In the current study, the effects of tyrosol on growth, redox homeostasis, intracellular microelement contents and activities of virulence-related enzymes released by C. auris were examined. To gain further information about the effect of tyrosol exposure, we revealed gene transcriptional changes using total transcriptome sequencing (RNA-Seq). At a concentration of 15 mM, tyrosol significantly decrease the growth of fungal cells within 2 h of its addition (5.6 × 107±1.2 × 107 and 2.5 × 107±0.6 × 107 colony forming unit/mL for control and tyrosol-treated cells, respectively). Furthermore, it enhanced the release of reactive oxygen species as confirmed by a dichlorofluorescein (DCF) assay (7.3 ± 1.8 [nmol DCF (OD640)−1] versus 16.8 ± 3.9 [nmol DCF (OD640)−1]), which was coincided with elevated superoxide dismutase, catalase and glutathione peroxidase activities. Tyrosol exerted in a 37%, 25%, 34% and 55% decrease in intracellular manganese, iron, zinc and copper contents, respectively, compared to control cells. The tyrosol treatment led to a 142 and 108 differentially transcripted genes with at least a 1.5-fold increase or decrease in transcription, respectively. Genes related to iron and fatty acid metabolism as well as nucleic acid synthesis were down-regulated, whereas those related to the antioxidative defence, adhesion and oxoacid metabolic processes were up-regulated. This study shows that tyrosol significantly influences growth, intracellular physiological processes and gene transcription in C. auris, which could highly support the development of novel treatment approaches against this important pathogen.

Shenqisherong pill ameliorates neuronal apoptosis by inhibiting the JNK/caspase-3 signaling pathway in a rat model of cervical cord compression

Ethnopharmacological relevanceThe Shenqisherong (SQSR) pill is an empirical prescription of traditional Chinese medicine (TCM), which originated from the National Chinese Medical Science Master, Shi Qi. It has been widely used in the treatment of cervical spondylotic myelopathy (CSM) and promote the recovery of spinal cord function, but underlying molecular mechanism remains unclear. Aim of the studyThe objective of this study was to confirm the neuroprotective effects of the SQSR pill. Materials and methodsA rat model of chronic compression at double-level cervical cord was used in vivo. The protective role of SQSR pill on CSM rats was measured by Basso, Beattie, and Bresnahan (BBB) locomotor scale, inclined plane test, forelimb grip strength assessment, hindlimb pain threshold assessment, and gait analysis. The levels of reactive oxygen species (ROS) were examined by Dihydroethidium (DHE) staining and 2′,7′-Dichlorofluorescein (DCF) assay, and apoptosis was detected by TdT-mediated dUTP nick-end labeling (TUNEL) assay. The expression of apoptosis proteins was evaluated by immunofluorescence staining and Western blot. ResultsSQSR pill could facilitate locomotor function recovery in rats with chronic cervical cord compression, reduce local ROS in the spinal cord and downregulate the c-Jun-N-terminal kinase (JNK)/caspase-3 signaling pathway. In addition, the SQSR pill could protect primary rat cortical neurons from glutamate-treated toxicity in vitro by reducing the ROS and downregulating the phosphorylation of JNK and its downstream factors related to neuronal apoptosis meditated by the caspase cascade. Then, the neuroprotective effect was counteracted by a JNK activator. ConclusionsTogether, SQSR pill could ameliorate neuronal apoptosis by restraining ROS accumulation and inhibiting the JNK/caspase-3 signaling pathway, indicating that SQSR pill could be a candidate drug for CSM.

Essential oil of Origanum minutiflorum exhibits anti-inflammatory and antioxidative effects in human bronchial cells and antimicrobial activity on lung pathogens

IntroductionOregano essential oil (OEO) is one of the most widely used essential oils worldwide due to its huge therapeutic benefits. Nevertheless, data on the effects of the endemic species Origanum minutiflorum, also known as wild or Turkish oregano, is scarce. On the other hand, various chronic lung diseases, characterised by persistent inflammation, oxidative stress, and common bacterial infections, do not have effective pharmacological therapy. Hence, the aim of this study is to examine the effects of wild oregano essential oil (WOEO) on human bronchial epithelial cells and lung pathogens. MethodsWe provided a detailed chemical composition of WOEO using GC-MS and GC-FID analysis. Anti-inflammatory effects of WOEO were analysed using the qRT-PCR and ELISA methods, while antioxidative properties were examined by using the dichlorofluorescein assay in BEAS-2B cells. Antibacterial activity was tested on lung pathogens by using the agar diffusion assay. ResultsThe major constituents of WOEO, analysed in this study, were carvacrol, linalool, p-cymene, γ-terpinene, and (E)-caryophyllene. We found that treatment with WOEO attenuated LPS-induced IL8 gene expression and hydrogen peroxide-induced oxidative stress in BEAS-2B cells. Moreover, WOEO showed an inhibitory effect on pathogenic bacteria Acinetobacter baumannii and highly resistant Klebsiella pneumoniae commonly seen in healthcare-associated pneumonia. ConclusionOur work presents new insights into the anti-inflammatory, antioxidative, and antimicrobial properties of WOEO which may be used as a simple and local treatment in various chronic lung diseases.

Dysrangements of TLR-2 in Ph(-) myeloproliferativeneoplasm Related to Jak2 Mutations and Leukocytosis

Introduction: Chronic inflammation and oxidative stress have been considered to be the hallmarks of MPNs for the last decade. Inflammatory cytokines have been reported to be increased in murine models and in patients samples. Increased ROS results from chronic inflammation in MPN has also been well described . There are still debates whether neoplastic clone or initial inflammation were the drivers of this inflammatory changes and carcinogenesis. We have previously studied Toll-like receptors (TLRs),a group of pattern recognition receptors (PRRs) which play critical roles in the host innate immune response and in the pathogenesis of a variety of acute and chronic inflammatory conditions and carcinogenesis , and we found TLR-2 were the predominant TLR derangements in MPN ( Blood (2015) 126 (23): 5202). We now extend our studies to involve more patients and correlated TLR-2 with the other clinical features . Materials and Methods: 103 patients with MPN diseases (19 PMF, 6 post-PV-MF, 6 post-ET-MF, 51 ET, 37 PV, and 21 normal volunteer controls) were enrolled in the study. 1) TLR-2, 4quantifications were performed by immuno-staining of 1×106mononuclear cells that were incubated with fluorescence-conjugated anti-TLR-2,3, 4, 7, and 9 antibodies and assayed by flow cytometry. 2) Cell ROS ( reactive oxygen species) measurement. Cellular ROS was determined by a dichlorofluorescein (DCF) assay. PBMNCs were incubated with 10 μM 2′,7′ ichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) (Life Technologies) for 30 min at 37°C. The oxidation of H2DCFDA was measured and analyzed by flow cytometry. Results. Fig1. TLR-2 levels were significantly elevated in PV and MPN ( grouped PV , ET and MF) , while PV were significantly elevated than MF or controls and not with ET ; ET were not significantly different from MF or controls . Fig 2,TLR-4 were not significantly elevated in PV, ET or MF than controls but were elevated when they were grouped as MPN , Fig 3. MF has significantly elevated cellular ROS than PV , ET or controls while PV, ET were not different from controls and when they were grouped together as MPN , were significantly elevated than controls . Fig 4. TLR-2 were correlated to leukocytosis in MPN patients but not with anemia or thrombocytosis . Fig5, TLR2 were significantly elevated in Jak2 V617F mutation positive than controls and were more elevated than CALR mutation patients . Conclusion. From these studies , we can conclude that 1) TLR-2 were the most predominant elevated TLR in MPN patients 2) PV has significantly more elevated TLR-2 than MF or controls but not ET, and ROS were significantly elevated in MF than ET or PV , suggesting that initial inflammation was initiated by TLR2 pathway in PV or ET and then after years of chronic inflammationled to accumulation of more ROS resulting in MF transformation and JAK2 mutation, thus favoring the theory that MPN was initiated first by inflammatory response then the neoplastic clone . 3) TLR2 were more significantly elevated in JAK2 mutation and leukocytosis , and appear to be more elevated in Jak2 mutation than CALR mutation positive patients ( though not statistically significant, probably the number of patients were not enough) ; this corresponded to the clinical observation that JAK2 mutation positive status has more thrombosis than CALR mutations and correlated to leukocytosis . Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Antioxidant Potential of the Sweet Whey-Based Beverage Colada after the Digestive Process and Relationships with the Lipid and Protein Fractions.

Whey-based beverages could be an effective way of reusing a by-product of th cheese industry, mitigating environmental hazards and, at the same time, profiting a useful food with high nutritional and antioxidant properties. In this study, a traditional Ecuadorian beverage (Colada) was prepared combining sweet whey, Maracuyá and barley. Antioxidant properties before and after an in vitro digestion using the INFOGEST method were determined, and relationships with intestinal transformations of the lipid and protein fractions were analyzed. The digestive process had a positive effect on antioxidant properties based on increased values of ABTS and FRAP located in the bioaccessible fraction (BF), together with strong increments of total polyphenols. Moreover, pretreatment of Caco-2 cells with the BF of Colada significantly reduced ROS generation (p < 0.001) measured by the dichlorofluorescein assay. Substantial changes of the fatty acid profile occurred during digestion, such as a fall of saturated fatty acids and a rise of polyunsaturated. The protein profile, examined by SDS-PAGE and exclusion molecular chromatography in the BF, showed that the major part of the proteins were digested in the intestinal phase. Analysis of NanoLC-MS/MS revealed 18 antioxidant peptides originated from whey proteins, but also 16 peptides from barley with potential antioxidant properties. In conclusion, combining sweet whey with Maracuyá and barley constitutes an excellent nutritional beverage with a strong antioxidant potential.

Antioxidant Effects of Elderberry Anthocyanins in Human Colon Carcinoma Cells: A Study on Structure-Activity Relationships.

ScopeGlycosylation is a way to increase structure‐stability of anthocyanins, yet compromises their bioactivity. The study investigates the antioxidant activity of purified cyanidin (Cy)‐based anthocyanins and respective degradation products in Caco‐2 clone C2BBe1 aiming to identify structure–activity relationships.Results and MethodsCyanidin 3‐O‐glucoside (Cy‐3‐glc) and cyanidin 3‐O‐sambubioside (Cy‐3‐sam) proved to be most potent regarding antioxidant properties and protection against hydrogen peroxide (H2O2)‐induced reactive oxygen species (ROS)‐levels measured with the dichloro‐fluorescein (DCF) assay. Cyanidin 3‐O‐sambubioside‐5‐O‐glucoside (Cy‐3‐sam‐5‐glc) and cyanidin 3‐O‐rutinoside (Cy‐3‐rut) were less efficient and not protective, reflecting potential differences in uptake and/or degradation. Following ranking in antioxidant efficiency is suggested: (concentrations ≤10 × 10−6 M) Cy‐3‐glc ≥ Cy‐3‐sam > Cy‐3‐sam‐5‐glc ≈ Cy‐3‐rut ≈ Cy; (concentrations ≥50 × 10−6 M) Cy‐3‐glc ≈ Cy‐3‐sam ≥ Cy > Cy‐3‐sam‐5‐glc ≈ Cy‐3‐rut. Cy and protocatechuic acid (PCA) reduced ROS‐levels as potent as the mono‐ and di‐glycoside, whereas phloroglucinol aldehyde (PGA) displayed pro‐oxidant properties. None of the degradation products protected from oxidative stress. Gene transcription analysis of catalase (CAT), superoxide‐dismutase (SOD), glutathione‐peroxidase (GPx), heme‐oxygenase‐1 (HO‐1), and glutamate‐cysteine‐ligase (γGCL) suggest no activation of nuclear factor erythroid 2‐related factor 2 (Nrf2).ConclusionMore complex residues and numbers of sugar moieties appear to be counterproductive for antioxidant activity. Other mechanisms than Nrf2‐activation should be considered for protective effects.

Sildenafil-evoked photoreceptor oxidative stress in vivo is unrelated to impaired visual performance in mice.

PurposeThe phosphodiesterase inhibitor sildenafil is a promising treatment for neurodegenerative disease, but it can cause oxidative stress in photoreceptors ex vivo and degrade visual performance in humans. Here, we test the hypotheses that in wildtype mice sildenafil causes i) wide-spread photoreceptor oxidative stress in vivo that is linked with ii) impaired vision.MethodsIn dark or light-adapted C57BL/6 mice ± sildenafil treatment, the presence of oxidative stress was evaluated in retina laminae in vivo by QUEnch-assiSTed (QUEST) magnetic resonance imaging, in the subretinal space in vivo by QUEST optical coherence tomography, and in freshly excised retina by a dichlorofluorescein assay. Visual performance indices were also evaluated by QUEST optokinetic tracking.ResultsIn light-adapted mice, 1 hr post-sildenafil administration, oxidative stress was most evident in the superior peripheral outer retina on both in vivo and ex vivo examinations; little evidence was noted for central retina oxidative stress in vivo and ex vivo. In dark-adapted mice 1 hr after sildenafil, no evidence for outer retina oxidative stress was found in vivo. Evidence for sildenafil-induced central retina rod cGMP accumulation was suggested as a panretinally thinner, dark-like subretinal space thickness in light-adapted mice at 1 hr but not 5 hr post-sildenafil. Cone-based visual performance was impaired by 5 hr post-sildenafil and not corrected with anti-oxidants; vision was normal at 1 hr and 24 hr post-sildenafil.ConclusionsThe sildenafil-induced spatiotemporal pattern of oxidative stress in photoreceptors dominated by rods was unrelated to impairment of cone-based visual performance in wildtype mice.

In vitro ameliorative effects of ellagic acid on vitality, motility and DNA quality in human spermatozoa.

Oxidative stress (OS) plays a significant role in the etiology of male infertility, resulting in the impairment of male reproduction. This condition, characterized by an imbalance in the levels of oxidizing and antioxidant species in the seminal fluid, has a harmful impact on sperm functions and DNA integrity. The present study aimed to evaluate the anti-genotoxic action of ellagic acid, a polyphenolic molecule of natural origin having a powerful antigenotoxic, anti-inflammatory and antiproliferative role. An OS condition was induced in vitro by incubating normozoospermic human semen samples in benzene for 45, 60 and 90 min. DNA integrity was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling assay, RAPD-PCR was performed to calculate the genome template stability, while the percentage of intracellular reactive oxygen species (ROS) was assessed by the 2', 7'-dichlorofluorescein assay. Our results showed that ellagic acid has a consistent protective effect on DNA integrity, as well as on sperm vitality and motility, by counteracting generation of intracellular ROS. The results of this study suggest ellagic acid as a suitable molecule to protect sperm DNA from oxidative stress, with a potentially significant translational impact on the management of the male infertility.

Repressive effect of Rhus coriaria L. fruit extracts on microglial cells-mediated inflammatory and oxidative stress responses

Ethnopharmacological relevanceRhus coriaria L. represents a herbal shrub that is used widely in traditional medicine in the Middle East region to treat different diseases including inflammation-related disorders. R. coriaria extracts have been well characterized in terms of their biological activities, pharmacological potential and phytochemical components. However, the effect of R. coriaria on neuro-inflammation has not been studied previously in detail. Aim of the studyIn the present study, we performed a qualitative phytochemical analysis and investigated the antioxidant and anti-neuro-inflammatory potential of R. coriaria extracts on BV-2 microglial cells. Materials and MethodsR. coriaria extracts were prepared using two different solvents: distilled water and ethanol. Phytochemical screening was performed to determine the principal bioactive components. The radical scavenging activity was assessed by DPPH method (2,2-diphenyl-1-picrylhydrazyl). The effect of R. coriaria on neuro-inflammation was studied upon measuring the production of oxidative stress and inflammatory factors using DCF (2′,7′-dichlorofluorescein) and Nitric oxide (NO) assays respectively, and by analyzing the mRNA (TNFα, IL-10, iNOS and COX-2) and protein (NFκβ) levels of genes involved BV-2 microglia cells-mediated inflammation using quantitative Real Time PCR and Western blot, respectively. ResultsWe found that R. coriaria extracts contain high phenolic and flavonoid contents. Interestingly, the ethanolic extract exerted a potent anti-inflammatory potential on insulted BV-2 cells manifested by: i) inhibition of Reactive Oxygen species (ROS) production and nitric oxide (NO) release; ii) suppressing TNFα, iNOS and COX-2 mRNA levels; iii) reducing NFκβ activation; and iiii) enhancing IL-10 transcription levels. ConclusionOur results indicate that the neuro-inflammation inhibitory activity of R. coriaria extracts involves the inhibition of NF-κB signaling pathway. These findings suggest that R. coriaria might carry therapeutic potential against neurodegenerative diseases.

Glutathione Improves the Antioxidant Activity of Vitamin C in Human Lens and Retinal Epithelial Cells: Implications for Vitreous Substitutes

ABSTRACT Purpose Tissues in the eye are particularly susceptible to oxidative damage due to light exposure. While vitamin C (ascorbic acid) has been noted as a vital antioxidant in the vitreous humor, its physiological concentration (1–2 mM) has been shown to be toxic to retinal and lens epithelial cells in in vitro cell culture. We have explored adding vitamin C to hydrogel vitreous substitutes as a potential therapeutic to prevent oxidative damage to intraocular tissues after vitrectomy. However, vitamin C degrades rapidly even when loaded at high concentrations, limiting its long-term effectiveness. Glutathione, another antioxidant found abundantly in the lens at concentrations of 2–10 mM, was proposed to be used in conjunction with vitamin C. Methods Cell viability and reactive oxygen species activity of human retinal and lens epithelial cells treated with various combinations of vitamin C, glutathione, hydrogen peroxide, and a hydrogel vitreous substitute were determined using CellTiter-Glo luminescent cell viability assay and dichlorofluorescein assay, respectively. The vitamin C remaining in hydrogel vitreous substitute or glutathione-vitamin C solutions was determined using a microplate reader at 265 nm wavelength, compared against standard solutions with known concentrations. Results Glutathione protected the lens and retinal cells from the negative effect of vitamin C on cell viability and prolonged the antioxidant effect of vitamin C in vitro. While the detected reading of pure vitamin C solution decreased rapidly from 100% to 10% by 3 days, glutathione provided a significant extension to vitamin C stability, with 70% remaining after 14 days when the glutathione was used at physiological concentrations found in the lens (2–10 mM). Conclusions These results indicate glutathione might be an effective addition to vitamin C in intraocular implants, including potential vitreous substitutes, and warrants additional studies on the effectiveness of the vitamin C – glutathione combination in preventing oxidative stress post-vitrectomy.

Lutein inhibits breast cancer cell growth by suppressing antioxidant and cell survival signals and induces apoptosis.

Reduced risk of breast cancer upon intake of lutein-rich food supplements creates an interest to investigate the molecular mechanism underlying the growth inhibitory potential of lutein in MCF-7 and MDA-MB-231 cells. Lutein purified from Spinacia oleracea was identified by high-performance liquid chromatography and liquid chromatography-mass spectrometry. The cell viability was measured by water-soluble tetrazolium-1 assay. The intracellular reactive oxygen species level was examined by 2',7'-dichlorofluorescein assay. The protein expression of the markers of antioxidant defense, cell survival, and apoptosis was analyzed by western blot analysis. The induction of apoptosis by lutein was measured by 4',6-diamidino-2-phenylindole staining and caspase-3 activity assay. The purified lutein inhibited the viability of MCF-7 and MDA-MB-231 cells. The growth inhibitory effect of lutein was associated with suppressed protein expression of superoxide dismutase-2 and heme oxygenase-1, and its transcription factor nuclear factor erythroid 2-related factor-2. Lutein treatment subsequently blocked the expression of intracellular cell survival proteins, phosphorylated protein kinase B, phosphorylated extracellular-regulated kinase 1/2, and nuclear factor-kB. Suppression of antioxidant defense and cell survival markers by lutein was further linked to apoptosis induction with elevated caspase-3 activity and downregulated expression of Bcl-2 and poly-ADP ribose polymerase. Our results emphasize a significant role of lutein as an effective inhibitor of human breast cancer cell growth that activates cell death partly through the modulation of antioxidant defense response-linked cell survival signaling markers.

In vitro and in vivo Effect of Exogenous Farnesol Exposure Against Candida auris.

The spreading of multidrug-resistant Candida auris is considered as an emerging global health threat. The number of effective therapeutic regimens is strongly limited; therefore, development of novel strategies is needed. Farnesol is a quorum-sensing molecule with a potential antifungal and/or adjuvant effect; it may be a promising candidate in alternative treatment against Candida species including C. auris. To examine the effect of farnesol on C. auris, we performed experiments focusing on growth, biofilm production ability, production of enzymes related to oxidative stress, triazole susceptibility and virulence. Concentrations ranging from 100 to 300 μM farnesol caused a significant growth inhibition against C. auris planktonic cells for 24 h (p < 0.01–0.05). Farnesol treatment showed a concentration dependent inhibition in terms of biofilm forming ability of C. auris; however, it did not inhibit significantly the biofilm development at 24 h. Nevertheless, the metabolic activity of adhered farnesol pre-exposed cells (75 μM) was significantly diminished at 24 h depending on farnesol treatment during biofilm formation (p < 0.001–0.05). Moreover, 300 μM farnesol exerted a marked decrease in metabolic activity against one-day-old biofilms between 2 and 24 h (p < 0.001). Farnesol increased the production of reactive species remarkably, as revealed by 2′,7′-dichlorofluorescein (DCF) assay {3.96 ± 0.89 [nmol DCF (OD640)–1] and 23.54 ± 4.51 [nmol DCF (OD640)–1] for untreated cells and farnesol exposed cells, respectively; p < 0.001}. This was in line with increased superoxide dismutase level {85.69 ± 5.42 [munit (mg protein)–1] and 170.11 ± 17.37 [munit (mg protein)–1] for untreated cells and farnesol exposed cells, respectively; p < 0.001}, but the catalase level remained statistically comparable between treated and untreated cells (p > 0.05). Concerning virulence-related enzymes, exposure to 75 μM farnesol did not influence phospholipase or aspartic proteinase activity (p > 0.05). The interaction between fluconazole, itraconazole, voriconazole, posaconazole, isavuconazole and farnesol showed clear synergism (FICI ranges from 0.038 to 0.375) against one-day-old biofilms. Regarding in vivo experiments, daily 75 μM farnesol treatment decreased the fungal burden in an immunocompromised murine model of disseminated candidiasis, especially in case of inocula pre-exposed to farnesol (p < 0.01). In summary, farnesol shows a promising therapeutic or adjuvant potential in traditional or alternative therapies such as catheter lock therapy.

Pyrocatalysis-The DCF assay as a pH-robust tool to determine the oxidation capability of thermally excited pyroelectric powders.

Pyrocatalysis uses thermally excited pyroelectric materials for the generation of reactive oxygen species in water. This unique feature allows it to harvest energy in the form of natural temperature gradients or waste heat from industrial processes in order to degrade organic pollutants at low costs. Its further development into an advanced oxidation process for water remediation is dependent on the availability of pH-robust and nonspecific redox assays for the determination of its oxidation capability. Nevertheless, previous studies neglected the influence of pH changes and they were focused mainly on the degradation of one organic compound or specific chemical dosimetries. In this study, a pH-robust and nonspecific reaction protocol of the dichlorofluorescein assay was established for the investigation of the oxidation capability of the pyrocatalytic process. This reaction protocol was tested on three pyroelectric powders (LiNbO3, LiTaO3, BaTiO3) in different amounts and it overcomes major constraints of a previously used dichlorodihydrofluorescein diacetate-based reaction protocol. Instead of its diacetate, dichlorodihydrofluorescein was used as fluorogenic probe and its concentration was drastically reduced to 1 μM. For the first time, these changes enable the determination and comparison of the oxidation capability independently of pH-rising processes, which are present for all investigated pyroelectric powders up to a pH of 11. Additionally, the precision of the dichlorofluorescein assay was drastically increased and the determination and consideration of autoxidation processes was enabled. Of all three pyroelectric powders, BaTiO3 exhibited the highest oxidation capability with a linear increase with respect to the powder amount.

In vitro effects of vasoconstrictive retraction agents on primary human gingival fibroblasts.

The biological activity of chemical retraction/displacement agents in surrounding periodontal tissues is of unquestionable importance, but the activity of these agents has not been completely elucidated. In the present study, we aimed to evaluate the in vitro effects of vasoconstrictive retraction agents on primary human gingival fibroblasts (HGFs). A total of six commercial adrenergic solutions (0.05 and 0.01% HCl-epinephrine, two based on 0.05% HCl-tetrahydrozoline, 0.05% HCl-oxymetazoline, and 10% HCl-phenylephrine) and three experimental gel formulations (EG-1, EG-2, and EG-3) were used to treat primary HGFs. The biological effect of the retraction treatment on the expression of collagen types I and III was detected by performing immunocytochemical analysis. The generation of reactive oxygen species triggered by the retraction agents were evaluated by using the dichlorofluorescein (DCF) fluorescent probe. The effect of retraction agents on the expression of fibronectin was visualized by confocal laser scanning microscopy. According to the results, experimental retraction gels did not limit the expression of collagen types I and III. EG-3 even induced the synthesis of both types of collagen. The DCF assay indicated oxidative stress similar to the control cells for most of the selected retraction agents. Experimental gels did not cause degradation of the cellular shape and morphology of the primary HGFs. The proposed experimental retraction gels in the present study demonstrated higher biocompatibility with primary HGFs, suggesting their use as clinical vasoconstrictive agents for the application of gingival retraction with minimal damage to periodontal tissues.

Implications of dichlorofluorescein photoinstability for detection of UVA-induced oxidative stress in fibroblasts and keratinocyte cells.

Although the dichlorofluorescein (DCF) assay is widely used to detect the production of UVA-induced ROS, the photostability and phototoxicity of the probe after UVA irradiation remains controversial and the experimental conditions often vary across studies, making it difficult to compare results from different studies. This study aimed to evaluate the suitability of the DCF assay for detection of UVA-induced ROS in human cells after UVA irradiation. Human primary fibroblasts (HPF) and HaCaT cells were loaded with 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) (2, 10, and 50 μM) for 10 and 30 min, before and after exposure to UVA radiation (5-50 J cm-2). Fluorescence was recorded immediately or 30 min after irradiation using three different techniques: microplate reading, flow cytometry, and confocal scanning microscopy. Cell viability was assessed by flow cytometry before and after UVA exposure. A UVA-dose-dependent increase in ROS was observed at 5-50 μM DCFDA, and the magnitude of the fluorescent signal was affected by RPMI medium, as well as DCFDA loading concentration and incubation period. However, higher concentrations of DCFDA compromised the viability of both HaCaT and HPF cells after UVA irradiation. The most sensitive and reliable combination for the ROS assay was pre-incubation with 10 μM DCFDA for 30 min in PBS. Reading the fluorescence 30 min after UVA irradiation diminished the emission signal, as did the DCFDA post-incubation. In conclusion, this single-point DCF assay allowed reproducible and sensitive UVA-induced ROS detection in HaCaT and HPF cells without compromising the cell viability or morphology.

A Novel Role of Ascorbic Acid in Anti-Inflammatory Pathway and ROS Generation in HEMA Treated Dental Pulp Stem Cells.

Resin (co)monomers issued from restorative dental materials are able to distribute in the dental pulp or the gingiva, to get to the saliva and to the flowing blood. Many authors have recently shown that methacrylate-based resins, in particular 2-hydroxyethylmethacrylate (HEMA), are responsible of inflammatory and autophagic processes in human dental pulp stem cells (hDPSCs) while ascorbic acid (AS), an antioxidant molecule, can assume a protective role in cell homeostasis. The purpose of the current work was to study if 50 µg/mL AS can affect the inflammatory status induced by 2 mM HEMA in hDPSCs, a tissue–specific cell population. Cell proliferation, cytokine release, morphological arrangement and reactive oxygen species (ROS) formation were determined respectively by MTT, ELISA, morphological analysis and dichlorofluorescein assay. The hDPSCs exposed to HEMA let to an increment of ROS formation and in the expression of high levels of inflammatory mediators such as nuclear factor-κB (NFkB), inflammatory cytokines such as interleukin IL6, IL8, interferon (IFN)ɣ and monocyte chemoattractant protein (MCP)1. Moreover, HEMA induced the up-regulation of pospho-extracellular signal–regulated kinases (pERK)/ERK signaling pathway associated to the nuclear translocation. AS treatment significantly down-regulated the levels of pro-inflammatory mediators. Then, the natural product AS reduced the detrimental result promoted by methacrylates in clinical dentistry, in fact restore cell proliferation, reduce the pro-inflammatory cytokine, downregulate ROS production and of NFkB/pERK/ERK signaling path. In synthesis, AS, could improve the quality of dental care and play a strategic role as innovative endodontic compound easy to use and with reasonable cost.

Oxidative Potential Associated with Urban Aerosol Deposited into the Respiratory System and Relevant Elemental and Ionic Fraction Contributions

Size-segregated aerosol measurements were carried out at an urban and at an industrial site. Soluble and insoluble fractions of elements and inorganic ions were determined. Oxidative potential (OP) was assessed on the soluble fraction of Particulate Matter (PM) by ascorbic acid (AA), dichlorofluorescein (DCFH) and dithiothreitol (DTT) assays. Size resolved elemental, ion and OP doses in the head (H), tracheobronchial (TB) and alveolar (Al) regions were estimated using the Multiple-Path Particle Dosimetry (MPPD) model. The total aerosol respiratory doses due to brake and soil resuspension emissions were higher at the urban than at the industrial site. On the contrary, the doses of anthropic combustion tracers were generally higher at the industrial site. In general, the insoluble fraction was more abundantly distributed in the coarse than in the fine mode and vice versa for the soluble fraction. Consequently, for the latter, the percent of the total respiratory dose deposited in TB and Al regions increased. Oxidative potential assay (OPAA) doses were distributed in the coarse region; therefore, their major contribution was in the H region. The contribution in the TB and Al regions increased for OPDTT and OPDCFH.

Lutein reverses hyperglycemia-mediated blockage of Nrf2 translocation by modulating the activation of intracellular protein kinases in retinal pigment epithelial (ARPE-19) cells.

Diabetic retinopathy (DR) is a major cause of acquired blindness among working adults. The retinal pigment epithelium (RPE), constitutes an outer blood-retinal barrier, is vastly affected in diabetic humans and animals. Lower levels of lutein in the serum and retina of diabetic population, and beneficial effects of carotenoids supplementation in diabetic retinopathy patients created an interest to examine the protective effect of lutein on hyperglycemia-mediated changes in oxidative stress and antioxidant defense system in ARPE-19 cells. The WST-1 assay was performed to analyze the impact of glucose, and lutein on the viability of ARPE-19. The intracellular oxidative stress was measured by a DCF (dichlorofluorescein) assay, mitochondrial membrane potential (MMP) was monitored using a JC-10 MMP assay kit and GSH level was examined using GSH/GSSG ratio detection kit. The oxidative stress markers, protein carbonyl and malondialdehyde were spectrophotometrically measured using 2,4-dinitrophenylhydrazine and 2-thiobarbituric acid, respectively. The expression of endogenous antioxidant enzymes and regulatory proteins in ARPE-19 was quantified by western blotting. The localization of Nrf2 protein was examined by immunofluorescent staining. The results show that lutein (up to 1.0μM) did not affect the viability of ARPE-19 grown in both normal and high-glucose conditions. Lutein treatment blocked high glucose-mediated elevation of intracellular ROS, protein carbonyl and malondialdehyde content in ARPE-19 cells. The decreased MMP and GSH levels observed in ARPE-19 grown under high-glucose condition were rescued by lutein treatment. Further, lutein protected high glucose-mediated down-regulation of a redox-sensitive transcription factor, Nrf2, and antioxidant enzymes, SOD2, HO-1, and catalase. This protective effect of lutein was linked with activated nuclear translocation of Nrf2, which was associated with increased activation of regulatory proteins such as Erk and AKT. Our study indicates that improving the concentration of lutein in the retina could protect RPE from diabetes-associated damage.