DCF Fluorescence Research Articles

Linolenic acid stimulates eryptosis and hemolysis through oxidative stress and CK1α/MLKL: protective role of melatonin, urea, and polyethylene glycol

Anticancer medications cause anemia in patients through ill-defined mechanisms, including hemolysis and eryptosis. Although α-linolenic acid (ALA) possesses anticancer properties against a variety of cancer cells, there is a dearth of evidence regarding how it modulates red blood cell (RBC) physiology. RBCs from healthy donors were subjected to anticancer concentrations of ALA (2.5, 5, 10, 20, 40, 80, and 100 μM) at 37 °C for 24 h, and colorimetric tests were used to determine hemolysis and acetylcholinesterase (AChE) activity. Meanwhile, flow cytometry was employed to identify eryptotic cells using annexin-V-FITC and forward scatter (FSC), Fluo4/AM to detect Ca2+, and H2DCFDA to assess oxidative stress. ALA significantly increased hemolysis and eryptosis in a concentration-dependent manner, along with elevated Fluo4 and DCF fluorescence, and erythrocyte sedimentation rate, and reduced FSC and AChE activity. Moreover, the addition of D4476, necrosulfonamide, melatonin, isosmotic urea, and polyethylene glycol 8000 – but not sucrose – significantly inhibited ALA toxicity. In conclusion, ALA stimulates hemolysis and eryptosis through Ca2+ buildup, oxidative stress, anticholinesterase activity, casein kinase 1α (CK1α), and mixed lineage kinase domain-like protein (MLKL). The anticancer activity of ALA may be potentiated by the use of Ca2+ channel blockers and chelators, antioxidants, and CK1α and MLKL inhibitors to ameliorate its toxicity to RBCs.

Improving the dichloro-dihydro-fluorescein (DCFH) assay for the assessment of intracellular reactive oxygen species formation by nanomaterials

To facilitate Safe and Sustainable by Design (SSbD) strategies during the development of nanomaterials (NMs), quick and easy in vitro assays to test for hazard potential at an early stage of NM development are essential. The formation of reactive oxygen species (ROS) and the induction of oxidative stress are considered important mechanisms that can lead to NM toxicity. In vitro assays measuring oxidative stress are therefore commonly included in NM hazard assessment strategies. The fluorescence-based dichloro-dihydro-fluorescein (DCFH) assay for cellular oxidative stress is a simple and cost-effective assay, making it a good candidate assay for SSbD hazard testing strategies. It is however subject to several pitfalls and caveats. Here, we provide further optimizations to the assay using 5-(6)-Chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA-AE, referred to as DCFH probe), known for its improved cell retention.We measured the release of metabolic products of the DCFH probe from cells to supernatant, direct reactions of CM-H2DCFDA-AE with positive controls, and compared the commonly used plate reader-based DCFH assay protocol with fluorescence microscopy and flow cytometry-based protocols. After loading cells with DCFH probe, translocation of several metabolic products of the DCFH probe to the supernatant was observed in multiple cell types. Translocated DCFH products are then able to react with test substances including positive controls. Our results also indicate that intracellularly oxidized fluorescent DCF is able to translocate from cells to the supernatant. In either way, this will lead to a fluorescent supernatant, making it difficult to discriminate between intra- and extra-cellular ROS production, risking misinterpretation of possible oxidative stress when measuring fluorescence on a plate reader.The use of flow cytometry instead of plate reader-based measurements resolved these issues, and also improved assay sensitivity. Several optimizations of the flow cytometry-based DCFH ISO standard (ISO/TS 19006:2016) were suggested, including loading cells with DCFH probe before incubation with the test materials, and applying an appropriate gating strategy including live-death staining, which was not included in the ISO standard.In conclusion, flow cytometry- and fluorescence microscopy-based read-outs are preferred over the classical plate reader-based read-out to assess the level of intracellular oxidative stress using the cellular DCFH assay.

A V-shaped bis-coumarin based fluorescence probe for F- detection in tea infusions and potable water and bioimaging applications in living systems

Fluorine (F) is a pivotal element in the formation of human dental and skeletal tissues, and the consumption of water and tea constitutes a significant source of fluoride intake. However, prolonged ingestion of water and tea with excessive fluoride content can lead to fluorosis, which poses a serious health hazard. In this manuscript, a novel turn-on fluorescent probe DCF synthesized by bis-coumarin and tert-butyldiphenylsilane (TBDPS) was introduced for detecting F- in potable water and tea infusions. By leveraging the unique chemical affinity between fluoride and silicon, F- triggers the silicon-oxygen bond cleavage in DCF, culminating in a conspicuous emission of yellow fluorescence. Validated through a succession of optical tests, this probe exhibits remarkable advantages in terms of superior selectivity, a low detection limit, a large Stokes shift, and robust interference resistance when detecting inorganic fluoride. Moreover, it can serve as portable test strips for on-site real-time identification and quantitative analysis of F-. Furthermore, the application of DCF for in-situ monitoring and imaging of F- in zebrafish and soybean root tissues proved its significant value for F- detection in both animal and plant systems. This probe potentially functions as an efficient instrument for delving into the toxic mechanisms of fluoride in physiological processes.

Effect of photobiomodulation therapy on the morphology, intracellular calcium concentration, free radical generation, apoptosis and necrosis of human mesenchymal stem cells-an in vitro study.

The aim of the study was to investigate the impact of multiwave locked system (MLS M1) emitting synchronized laser radiation at 2 wavelength simultaneous (λ = 808nm, λ = 905nm) on the mesenchymal stem cells (MSCs). Human MSCs were exposed to MLS M1 system laser radiation with the power density 195-318 mW/cm2 and doses of energy 3-20J, in continuous wave emission (CW) or pulsed emission (PE). After irradiation exposure in doses of energy 3J, 10J (CW, ƒ = 1000Hz), and 20J (ƒ = 2000Hz), increased proliferation of MSCs was observed. Significant reduction of Fluo-4 Direct™ Ca2+ indicator fluorescence over controls after CW and PE with 3J, 10J, and 20J was noticed. A decrease in fluorescence intensity after the application of radiation with a frequency of 2000Hz in doses of 3J, 10J, and 20J was observed. In contrary, an increase in DCF fluorescence intensity after irradiation with laser radiation of 3J, 10J, and 20J (CW, ƒ = 1000Hz and ƒ = 2000Hz) was also shown. Laser irradiation at a dose of 20J, emitted at 1000Hz and 2000Hz, and 3J emitted at a frequency of 2000Hz caused a statistically significant loss of MSC viability. The applied photobiomodulation therapy induced a strong pro-apoptotic effect dependent on the laser irradiation exposure time, while the application of a sufficiently high-energy dose and frequency with a sufficiently long exposure time significantly increased intracellular calcium ion concentration and free radical production by MSCs.

Efficacy of Elaeagnus umbellata leaves on prevention of cadmium-induced toxicity in HepG2 cells

Elaeagnus umbellata leaves have been reported to suppress inflammation, allergic responses, lung cancer proliferation and oral bacterial growth. Cadmium (Cd) is a heavy metal that has been found to cause many toxicities, including liver toxicity. The aim of this study was to evaluate the capacity of 70% ethanol extract of E. umbellata leaves (EUL) to protect human hepatocytes from Cd toxicity. After exposure of HepG2 cells to Cd at 10 μM for 24 h, cell viability, expression levels of apoptosis- and antioxidant-related proteins, reactive oxygen species (ROS) accumulation and Cd uptake were assessed. EUL protected HepG2 cells from Cd-induced apoptosis as determined by MTT assay. A decrease in caspase-3 and p-p53 protein levels was observed in cells pretreated with EUL prior to Cd exposure. Furthermore, the Cd-induced increase in intracellular DCF fluorescence was attenuated by EUL, indicating that the Cd-induced apoptosis preventing effect was associated with the suppression of ROS accumulation. Moreover, EUL’s effects on the inhibition of p38, JNK, and AKT phosphorylation also appear to be associated with protection against Cd toxicity. Moreover, EUL upregulated Cd-depressed expression of Nrf2, HO-1, catalase, and MT-1,2 proteins, suggesting that Cd uptake-induced apoptosis in HepG2 cells may be inhibited by EUL’s antioxidative potential.

Photodynamic therapy potential of cobalt phthalocyanine in triple-negative breast cancer

Triple-negative breast cancer (TNBC) has the worst prognosis among breast cancers. The deficiency of hormone receptors (ER-/PR-/HER-2-) limits treatment options. Photodynamic therapy (PDT) is a safe treatment method for target-specific applications and has no toxic side effects. In this study, the efficacy of PDT on TNBC cells after activation of phthalocyanine derivative CoPc-COOH with RED light was investigated. The cytotoxicity of CoPc-COOH in MDA-MB-231 cells was evaluated at 24, 48, and 72 h. To detect phototoxic activity, cells were exposed simultaneously to a dose range of CoPc-COOH at 48th hours and 20, 30, 40, and 50 J/cm2 RED light in a laser source with an output fluorescence of 12 mW/cm2. The apoptotic effect of single CoPc-COOH, RED light, and their combinations were examined by using the Annexin V method and the ROS generation potential using the carboxy-H2-DCFH-DA test in flow cytometry. DCF fluorescence intensity of control and dose groups were taken under the fluorescence microscope. The IC50 values of CoPc-COOH in MDA-MB-231 cells at the 24th, 48th, and 72nd hours were calculated as 131.04 µM, 66.75 µM, and 59.74 µM, respectively. In the phototoxicity test, 50 J/cm2 application increased the IC50 dose of CoPc-COOH to 73.73 µM; 20, 30, 40 J/cm2 application reduced the doses to 2.31 µM, 0.20 µM, and 28.14 µM, respectively. Compared to the untreated group, it was determined that ROS formation and apoptotic stimulation were triggered significantly in treatment groups. A combination of CoPc-COOH and 40 J/cm2 showed the highest synergistic effect.

FNDC5/irisin expression stimulates neuroprotective pathways in hippocampal neurons and associates with Alzheimer’s disease pathology

AbstractBackgroundIrisin is an exercise‐linked myokine produced by cleavage of the membrane precursor fibronectin type III domain‐containing protein 5 (FNDC5) in skeletal muscle, brain, and other tissues. Recently, irisin has been identified as a key molecule for exercise‐induced neuroprotection in mouse models of Alzheimer’s disease (AD). However, the role of FNDC5/irisin in AD pathology has not yet been comprehensively explored. Here, we investigated potential mechanisms underlying neuroprotection induced by FNDC5/irisin in AD.MethodPrimary rat hippocampal neuronal cultures were transduced with adenoviral vectors to express FNDC5 (AdFNDC5) or GFP (as a control) or exposed to recombinant irisin (25 nM) for defined timepoints. Brain‐derived neurotrophic factor (BDNF) levels were measured by ELISA; extracellular signal‐regulated kinase 1/2 (ERK 1/2) phosphorylation was determined by Western blotting; and the effects of irisin on amyloid‐β oligomers (AβO)‐induced reactive oxygen species (ROS) accumulation was determined by the DCF fluorescence. Finally, we analyzed RNA‐seq data from human postmortem hippocampi within The Aging, Dementia and Traumatic Brain Injury Study to determine potential correlations between FNDC5 gene expression and AD pathology.ResultWe found that irisin stimulates extracellular BDNF accumulation, ERK 1/2 phosphorylation (i.e. activation) and prevents AβOs‐induced ROS accumulation in primary hippocampal neurons. Analysis of RNA‐seq data indicates that hippocampal FNDC5 gene expression is reduced with aging and tau (assessed by Braak neuropathological scale and hippocampal phospho‐tau immunoreactivity), but not with amyloid pathology in humans.ConclusionThese results indicate potential mechanisms by which FNDC5/irisin signaling promotes neuroprotection and support the notion that stimulation of irisin signaling may be beneficial against neurodegeneration in AD.

Myricetin-induced suicidal erythrocyte death.

Myricetin, a type of flavonol commonly found in fruits and herbs, has demonstrated anticancer properties by triggering the process of apoptosis or programmed cell death in tumor cells. Despite the absence of mitochondria and nuclei, erythrocytes can undergo programmed cell death, also known as eryptosis.This process is characterized by cell shrinkage, externalization of phosphatidylserine (PS) on the cell membrane, and the formation of membrane blebs. The signaling of eryptosis involves Ca2+ influx, the formation of reactive oxygen species (ROS), and the accumulation of cell surface ceramide. The present study explored the effects of myricetin on eryptosis. Human erythrocytes were exposed to various concentrations of myricetin (2-8 µM) for 24h. Flow cytometry was used to assess the markers of eryptosis, including PS exposure, cellular volume, cytosolic Ca2+ concentration, and ceramide accumulation. In addition, the levels of intracellular ROS were measured using the 2',7'-dichlorofluorescin diacetate (DCFDA) assay. The myricetin-treated (8 µM) erythrocytes significantly increased Annexin-positive cells, Fluo-3 fluorescence intensity, DCF fluorescence intensity, and the accumulation of ceramide. The impact of myricetin on the binding of annexin-V was significantly reduced, but not completely eliminated, by the nominal removal of extracellular Ca2+. Myricetin triggers eryptosis, which is accompanied and, at least in part, caused by Ca2+ influx, oxidative stress and increase of ceramide abundance.

Rutin-gallium complex mediated antimicrobial photodynamic therapy: An in vitro studies against Streptococcus mutans biofilms

BackgroundDiscoloration of teeth following antimicrobial photodynamic therapy (aPDT) is a serious concern. Common photosensitizers are colored, and access to a photosensitizer that does not leave color on the teeth or is the same color as the enamel and dentin is highly demanded. The physicochemical characterization, anti-virulence, and antimicrobial effects of a novel rutin-gallium (III) (Rt-Ga) complex as novel photosensitizer are presented herein. Materials and MethodsPhotophysical properties and cytotoxicity of the Rt-Ga complex were evaluated in comparison with the parental rutin. Intracellular reactive oxygen species (ROS) generation following Rt-Ga complex-mediated aPDT was measured using the fluorescent 2′,7′-dichlorofluorescein diacetate (H2DCF-DA) method. The anti-biofilm effects of Rt-Ga complex-mediated aPDT on Streptococcus mutans were assessed using a colorimetric assay. The virulence‑associated gtfB gene expression was assessed following Rt-Ga complex-mediated aPDT by quantitative real‑time PCR. ResultsThe photophysical properties of the Rt-Ga complex revealed a significant blue-shift in absorption (60 nm shift) and increased extinction coefficient (4100 M −1 cm −1; at λmax = 450 nm). Average (± SEM) DCF fluorescence intensities in an arbitrary unit (A.U.) were 7.1 ± 0.9, 4.1 ± 0.5, and 1.7 ± 0.3 for 10.0 μM of Rt-Ga complex-mediated aPDT, 7.5 μM of Rt-Ga complex-mediated aPDT, and 10.0 μM of Rt-Ga complex alone, respectively. The corresponding DCF fluorescence intensities were 710% (P = 0.001), 410% (P = 0.001), and 170% (P = 0.02) of the untreated S. mutans as the control group (1.0 ± 0.1 A.U.), respectively. The novel Rt-Ga complex-mediated aPDT exhibited no toxicity against primary human gingival fibroblast cells, a dose dependent decrease in S. mutans biofilm cell survival and virulence were observed (91.4% and 11.7-fold, respectively). ConclusionThe Rt-Ga complex-mediated anti-virulence and antimicrobial photodynamic effects were superior to the one caused by rutin alone making the Rt-Ga complex a more promising photosensitizer than the parent material.

Продукция активных форм кислорода при действии ультрафиолетового-В излучения на клетки дрожжей

The present investigation is devoted to the study of the effect of ultraviolet-B (UV-B) radiation on the survival and production of reactive oxygen species in yeast cells. It has been determined that under the action of UV-B radiation on cells, depending on its dose, the rate of oxidation of the dye 2',7'-dichlorodihydrofluorescein diacetate (H2DCF•DA) increases and a high intensity of DCF fluorescence is observed. When cells are modified with ascorbic acid before irradiation, the rate of H2DCF oxidation and the intensity of DCF fluorescence decreases. The survival rate of modified cells is increased. Ascorbic acid reduces the amount of ROS in the suspension of irradiated cells. At high doses (4.5-10 erg/mm2) of irradiation, the antioxidant effect of ascorbic acid in yeast cells is negligible. The concentration of 2-4 dinitrophenol (DNF) 10-7 M was determined as a protector, in which the substance potentially contributes to the manifestation of the effect of "soft uncoupling" in cells and, under the influence of UV-B radiation on yeast cells, helps to reduce the production of reactive oxygen species and an increase in survival. High concentrations (10–3M–10–5 M) of DNP adversely affect the survival of yeast cells and ROS production. So, it can be concluded that under the action of UV-B rays on yeast cells, antioxidants can possibly participate in the regulation of redox homeostasis.

Zinc Oxide Nanoparticles Induce Toxicity in H9c2 Rat Cardiomyoblasts.

Zinc oxide nanoparticles (ZnO NPs) are widely used in the cosmetic industry. They are nano-optical and nano-electrical devices, and their antimicrobial properties are applied in food packaging and medicine. ZnO NPs penetrate the body through inhalation, oral, and dermal exposure and spread through circulation to various systems and organs. Since the cardiovascular system is one of the most vulnerable systems, in this work, we studied ZnO NPs toxicity in H9c2 rat cardiomyoblasts. Cardiac cells were exposed to different concentrations of ZnO NPs, and then the morphology, proliferation, viability, mitochondrial membrane potential (ΔΨm), redox state, and protein expression were measured. Transmission electron microscopy (TEM) and hematoxylin-eosin (HE) staining showed strong morphological damage. ZnO NPs were not observed inside cells, suggesting that Zn2+ ions were internalized, causing the damage. ZnO NPs strongly inhibited cell proliferation and MTT reduction at 10 and 20 μg/cm2 after 72 h of treatment. ZnO NPs at 20 μg/cm2 elevated DCF fluorescence, indicating alterations in the cellular redox state associated with changes in ΔΨm and cell death. ZnO NPs also reduced the intracellular expression of troponin I and atrial natriuretic peptide. ZnO NPs are toxic for cardiac cells; therefore, consumption of products containing them could cause heart damage and the development of cardiovascular diseases.

Oxidative Crosstalk with Venular Capillary Mitochondria Mediates Barrier Failure in Acute Lung Injury

OBJECTIVE. To elucidate mechanisms underlying alveolar‐capillary crosstalk in the context of lipopolysaccharide (LPS)‐induced Acute Lung Injury (ALI), which causes The Acute Respiratory Distress Syndrome (ARDS).HYPOTHESISOxidative crosstalk between alveolar epithelium and adjacent endothelium causes mitochondrial injury and barrier failure in Acute Lung InjuryMETHODS. We instilled LPS (5mg/kg) by the intranasal (i.n.) route in anesthetized mice. We evaluated microvascular endothelia of isolated blood‐perfused mouse lungs by confocal microscopy, and ALI by bronchoalveolar lavage (BAL) 48h later. For microscopy, we gave the potentiometric mitochondrial dye MTDR, and the reactive oxygen species (ROS) indicator DCF by intravascular infusion. We determined the role of endothelial uncoupling protein 2 (UCP2) by (1) UCP2 knockdown in endothelia by vascular siRNA injection, and (2) endothelial‐specific knockout of UCP2. We transfected alveolar epithelium by i.n. instillation of catalase‐expressing plasmid, or depleted neutrophils by intraperitoneal injection of anti‐Gr1 antibody, 24 hours before i.n. LPS. We quantified barrier permeability through BAL protein content.RESULTS. Confocal imaging indicated that baseline fluorescence intensity of both endothelial MTDR and DCF was steady, indicating that endothelial mitochondrial potential and endothelial ROS were stable. However, 48 hours after LPS instillation, MTDR fluorescence decreased in venular capillaries more than in alveolar capillaries (n=4, p<0.05), indicating endothelial mitochondrial depolarization. Simultaneously, DCF fluorescence increased in venular capillaries more than in alveolar capillaries (n=4, p<0.05), indicating an increase in endothelial ROS. LPS instillation increased BAL protein (n=4, p<0.05). Knockdown of endothelial UCP2 expression, or expression of transfected catalase in the alveolar epithelium blocked LPS‐induced endothelial mitochondrial depolarization and BAL protein increase (p<0.05). Endothelial‐specific knockout of UCP2, but not neutrophil depletion, blocked LPS‐induced BAL protein increase (p<0.05).CONCLUSIONS. We show here for the first time that the hyper‐permeable responses of LPS‐induced ALI result from mitochondrial depolarization in venular capillaries. Transfer of H2O2 from alveoli, not neutrophils activated UCP2, causing proton influx into the mitochondrial matrix. Our findings reveal a novel mechanism of LPS‐induced ALI, implicating mitochondria‐oxidant coupling in barrier failure. Thus, UCP2 presents a potential therapeutic target for ARDS.

Angiotensinogen uptake and stimulation of oxidative stress in human pigment retinal epithelial cells

We previously reported that isolated proximal tubules (PT) internalize the precursor protein angiotensinogen and that the 125Iodine-labeled protein accumulated in the nuclear and mitochondrial fractions of the PT cells; however, whether internalization of angiotensinogen occurs in non-renal epithelial cells is unknown. Therefore, the present study assessed the cellular uptake of 125I-angiotensinogen in human retinal pigment ARPE-19 epithelial cells, a widely utilized cell model for the assessment of retinal injury, inflammation and oxidative stress. ARPE-19 cells, maintained in serum-free media to remove extracellular sources of bovine serum angiotensinogen and renin, were incubated with 125Iodine-angiotensinogen at 37 °C and revealed the time-dependent uptake of angiotensinogen over 24 h. In contrast, incubation with labelled Ang II, Ang-(1−7) or Ang I revealed minimal cellular uptake. Subcellular fractionation following a 4-hour uptake of 125I-angiotensinogen revealed that the majority of the labeled protein localized to the nuclear fraction with lower accumulation in the mitochondrial and cytosolic fractions. Finally, we show that addition of angiotensinogen (2 nM) to the ARPE-19 cells increased oxidative stress as assessed by DCF fluorescence that was blocked by pretreatment of the cells with either the NADPH oxidase 1/4 inhibitor GKT137831, apocynin or atorvastatin, but not the AT1 receptor antagonist losartan. In contrast, treatment of the cells with Angiotensin II at an equivalent dose to angiotensinogen failed to stimulate oxidative stress. We conclude that human retinal pigment cells internalize angiotensinogen to elicit an increase in oxidative stress through a pathway that appears distinct from the Ang II-AT1 receptor axis.

Sonosensitized Aggregation-Induced Emission Dots with Capacities of Immunogenic Cell Death Induction and Multivalent Blocking of Programmed Cell Death-Ligand 1 for Amplified Antitumor Immunotherapy

Sonosensitized Aggregation-Induced Emission Dots with Capacities of Immunogenic Cell Death Induction and Multivalent Blocking of Programmed Cell Death-Ligand 1 for Amplified Antitumor Immunotherapy

Redox-modulation, Suppression of “Oncogenic” Superoxide and Induction of Apoptosis in Burkitt’s Lymphoma Cells Using Geum urbanum L. Extracts

Burkitt’s lymphoma is a highly aggressive type of non-Hodgkin’s lymphoma, linked to the Epstein-Barr virus, which induces oxidative stress and DNA damage in the infected cells. We investigated the cytotoxicity and redox-modulating ability of ethyl acetate (EtOAc) and n-butanol (n-BuOH) extracts from Geum urbanum L. roots and aerial parts on Burkitt`s lymphoma cells (BLC), to elucidate their impact on oxidative stress and cell survival. BLC Raji was treated with EtOAc and n-BuOH extracts to analyze: cell viability; induction of apoptosis; hydroperoxides and reactive nitrogen species (RNS) by 2’,7’-dichlorodihydrofluorescein assay; superoxide by dihydroethidium assay; total antioxidant capacity by TAC assay. All extracts suppressed cell growth and induce apoptosis. n-BuOH extracts possessed higher cytotoxicity and pro-apoptotic activity compared to EtOAc. The fractions decreased the hydroperoxides and RNS levels. There was no correlation between the DCF fluorescence in the treated cells and their viability (R = -0.3722; p > 0.05). Root extracts decreased the superoxide level, while the leaf extracts did not. There was a good correlation between the dihydroethidium fluorescence in the treated cells and their viability (R = 0.9843; p < 0.01). All extracts increased the TAC of BLC. G. urbanum extracts serve as redox-modulators and anti-inflammatory compounds, decreasing the intracellular level of “oncogenic” superoxide and cell proliferation.

Sports Post‐Concussion Syndrome (PCS) Model in Drosophila melanogaster

AbstractBackgroundScientific and societal interest is growing in understanding links between sports concussion and neurodegeneration. When multiple symptoms, such as memory loss, anxiety and headaches, persist after head impact(s), this is defined as post‐concussion syndrome (PCS). Aetiology of PCS is poorly understood and there is no specific pharmacological cure. This study aimed to generate a novel sports PCS model in Drosophila melanogaster to facilitate further understanding of the syndrome.MethodsThe HIT device was constructed as per prior methods1, but with protractor added and impact forces measured with a pancake load cell for repeatable trauma application to fly subgroups at 0° (control), 30°, 45° or 60° release angles, measured as 0N, 1.1N, 1.8N and 2.1N respectively. Hits were repeated five times with 48‐72 hour intervals to model multiple traumas contact‐sports players experience during their careers. To evaluate effects of impacts, motor ability, survival and reactive oxygen nitrogen species (RONS) levels were measured. Motor ability was reported as a percentage of the number of flies that climbed above 5.5cm in 5.5 seconds. Flies were frozen at 21‐22 days post‐eclosure (middle‐age), heads and bodies separated and homogenised, then post‐centrifugation supernatant was assayed with DCF‐DA: condensed into highly fluorescent DCF in the presence of RONS, quantified by fluorescence spectroscopy.ResultsAs with most human mild head trauma cases, fly motor ability was not significantly different acutely or long‐term following HIT device impacts. Similarly, with 1.8N impacts, lifespan was not significantly different compared to non‐trauma controls. Meanwhile, RONS levels increased in both fly heads (brains) and bodies (periphery) with five 1.8N impacts, representative of physiological stress in contact sportspeople with PCS.ConclusionsFive 1.8N hits (at 45° release angle) produced the most suitable Drosophila sports PCS model. This novel model offers a rapid, inexpensive drug screening approach for prospective PCS treatments, and shows promise in investigating the oxidative stress mechanisms underlying PCS. Additional analyses, including neurodegeneration biomarker quantification and behavioural monitoring, are currently being explored to validate this model.Reference: 1 Katzenberger RJ et al. (2013) PNAS 110, 4152‐4159.

Abstract 10163: Ca 2+ Within the Mitochondria Can Affect the Occurrence of Triggered Arrhythmias in Rats with Right Ventricular Hypertrophy

Background: Ca 2+ within the sarcoplasmic reticulum affects the propagation of intracellular Ca 2+ (Ca i ) waves, and the velocity of Ca i waves play important roles in the occurrence of triggered arrhythmias. It is still unknown, however, whether Ca 2+ within the mitochondria (Ca m ) affects the velocity of Ca i wave and the occurrence of arrhythmias in right ventricular (RV) hypertrophy. Objective: To know the roles of Ca m in the velocity of Ca i waves and the occurrence of triggered arrhythmias in rats with RV hypertrophy. Methods: Rats were subcutaneously injected with 60 mg/kg monocrotaline (MCT-rats) or solvent (Ctr-rats). Four weeks after the injection, trabeculae were dissected from the RVs. The force was measured using a silicon strain gauge, Ca m using rhod-2, Ca i using microinjected fura-2 and a CCD camera, and production of reactive oxygen species (ROS) from the slope DCF fluorescence during electrical stimulation. Ca 2+ waves and arrhythmias were induced by electrical stimulation (24°C). Results: MCT-rats showed a higher RV pressure, a larger weight ratio of RV free wall to left ventricle (RV/LV), and faster and larger Ca i waves than Ctr-rats (p<0.01). Among MCT-rats, Ru360 (10 μM), a mitochondrial calcium uniporter (MCU) inhibitor, decreased the occurrence of arrhythmias in trabeculae from rats with a higher RV pressure and a larger RV/LV (n=19). Ru360 decreased rhod-2 signal within trabeculae depending on the RV/LV (r=-0.85, p<0.01, n=10) without changes in Ca i transients, suggesting that inhibition of MCU decreases Ca m in MCT-rats with more severe RV hypertrophy. In addition, Ru360 decreased the velocity of Ca i waves depending on systolic RV pressure (r=-0.60, n=6) and the RV/LV (r=-0.64, n= 7), suggesting that inhibition of MCU decreased the velocity of Ca i waves in MCT-rats with higher RV pressure and more severe RV hypertrophy. Ru360 decreased the slope of DCF fluorescence (p<0.01, n=13). Conclusions: In the hearts with severe RV hypertrophy, inhibition of MCU can cause larger reduction of Ca m , decrease ROS production, and decrease the velocity of Ca i waves and arrhythmias. Thus, in the hearts with severe RV hypertrophy, more Ca 2+ may enter the mitochondria through the MCU, and higher Ca m may be involved in the occurrence of arrhythmias.

Effect of butyrate, a bacterial by-product, on the viability and ICAM-1 expression/production of human vascular endothelial cells: Role in infectious pulpal/periapical diseases.

To investigate the effects of butyric acid (BA), a metabolic product generated by pulp and root canal pathogens, on the viability and intercellular adhesion molecule-1 (ICAM-1) production of endothelial cells, which are crucial to angiogenesis and pulpal/periapical wound healing. Endothelial cells were exposed to butyrate with/without inhibitors. Cell viability, apoptosis and reactive oxygen species (ROS) were evaluated using an MTT assay, PI/annexin V and DCF fluorescence flow cytometry respectively. RNA and protein expression was determined using a polymerase chain reaction assay and Western blotting or immunofluorescent staining. Soluble ICAM-1 (sICAM-1) was measured using an enzyme-linked immunosorbent assay. The quantitative results were expressed as mean±standard error (SE) of the mean. The data were analysed using a paired Student's t-test where necessary. A p-value ≤0.05 was considered to indicate a statistically significant difference between groups. Butyrate (>4mM) inhibited cell viability and induced cellular apoptosis and necrosis. It inhibited cyclin B1 but stimulated p21 and p27 expression. Butyrate stimulated ROS production and hemeoxygenase-1 (HO-1) expression as well as activated the Ac-H3, p-ATM, p-ATR, p-Chk1, p-Chk2, p-p38 and p-Akt expression of endothelial cells. Butyrate stimulated ICAM-1mRNA/protein expression and significant sICAM-1 production (p<.05). Superoxide dismutase, 5z-7oxozeaenol, SB203580 and compound C (p<.05), but not ZnPP, CGK733, AZD7762 or LY294002, attenuated butyrate cytotoxicity to endothelial cells. Notably, little effect on butyrate-stimulated sICAM-1secretion was found. Valproic acid, phenylbutyrate and trichostatin (three histone deacetylase inhibitors) significantly induced sICAM-1 production (p<.05). Butyric acid inhibited proliferation, induced apoptosis, stimulated ROS and HO-1 production and increased ICAM-1mRNA expression and protein synthesis in endothelial cells. Cell viability affected by BA was diminished by some inhibitors; however, the increased sICAM-1secretion by BA was not affected by any of the tested inhibitors. These results facilitate understanding of the pathogenesis, prevention and treatment of pulpal/periapical diseases.

Low Concentration Ethanol Prevents Oxidative Stress- Induced Cardiac Cells Injury Under HyperglycemicConditions by Inhibiting the SAPK/JNK Signaling Pathway and ROS Generation

The purpose of this study was to determine the effects and mechanisms of ethanol on oxidative stress-induced cardiac H9c2 cells mitochondrial injury under hyperglycemic conditions. Under hyperglycemic conditions, ethanol pretreatment (10-100 μM) prevented H2O2-induced mitochondria swelling, as well as decreased cell viability and Respiratory Control Ratio (RCR) in the H9c2 cells. It also prevented TMRE fluorescence intensity loss and DCF fluorescence intensity increase under hyperglycemic conditions. These effects of ethanol were reversed by the SAPK/JNK agonist, anisomycin. Finally, treatment of H9c2 cells with 33mM glucose significantly enhanced Akt and ERK phosphorylation, which was not affected by ethanol. However, ethanol decreased the phosphorylation of SAPK/JNK under hyperglycemic conditions. Collectively, these findings indicate that under hyperglycemic conditions, that ethanol prevents oxidative stress-induced mitochondrial injury in cardiac H9c2 cells by preventing ROS generation via inhibiting the SAPK/JNK signaling pathway.

Lipid biosynthesis mediated by the cyclic adenosine monophosphate (cAMP) signaling pathway in Chlorella pyrenoidosa under salt-induced osmotic stress

An increase in lipid accumulation in algal cells often occurs under salt-induced osmotic stress, but the link between the two is unclear. In this study, the effects of osmotic stress as a kind of biomechanical stress on lipid biosynthesis and the cAMP signaling pathway were investigated using Chlorella pyrenoidosa, and a plausible and novel mechanism by which cAMP regulates lipid accumulation in C. pyrenoidosa under osmotic stress conditions was proposed. The maximal lipid content in C. pyrenoidosa exposed to osmotic stress (453 mOsm/kg) (38.31 ± 0.99%) was increased by 63% compared to that in the control group (32 mOsm/kg) (23.45 ± 0.61%). Under osmotic stress, the levels of lipogenic genes, cAMP (3.24 ± 0.09 nM), and reactive oxygen species (ROS) (285.46 ± 26.51 DCF fluorescence intensity) and the total antioxidant capacity (T-AOC) (12.14 ± 1.03 U mgprot−1) increased. Activated cAMP downregulated Snf1 levels, resulting in enhanced expression of accD. As a result, the metabolic flow of carbon in C. pyrenoidosa was shifted to lipid biosynthesis, resulting in an improvement in biodiesel quality. This study provides novel insight into the role of signaling pathways in modulating lipid accumulation in algal cells under osmotic stress.