Mitochondrial Metabolic Activity Research Articles

Biocompatible protamine sulfate@silicon nanoparticle-based gene nanocarriers featuring strong and stable fluorescence.

The development of biocompatible and fluorescent gene carriers is of particular importance in the gene-delivery field. Taking advantage of the unique optical properties (e.g., strong and robust fluorescence) of silicon nanoparticles (SiNPs), as well as the excellent biocompatibility of silicon and protamine sulfate (PS, approved by the U.S. Food and Drug Administration (FDA) for clinical use), we herein present a type of PS-modified SiNP (PS@SiNP)-based gene carrier. Plasmid DNA (pDNA) with negative charges can be effectively bound onto the surface of the as-prepared fluorescent PS@SiNP-based gene carriers via electrostatic interactions. In particular, such resultant gene carriers possess stable and high fluorescence (photoluminescent quantum yield (PLQY): ∼25%). In addition, the PS@SiNP-based gene carriers show minimal toxic effects on normal mitochondrial metabolic activity (e.g., human retinal pigment epithelial (ARPE-19) cells preserve ∼90% of their cell viability after a 48 h incubation with the resultant carriers). Based on tracking the strong and stable fluorescence signals of SiNPs, the dynamic behavior of the PS@SiNP-based gene carriers in live cells (e.g., clathrin-mediated endocytosis, lysosomal escape, pDNA release, etc.) is investigated in a long-term manner, providing valuable information for understanding the intracellular behavior of gene vectors and designing high-efficacy gene carriers.

Effect of root canal sealers on human periodontal ligament fibroblast viability: ex vivo study

The aim of the study was to compare ex vivo the toxic effects of six root canal sealers immediately after mixing or setting on human periodontal ligament fibroblasts (HPdLF). Freshly mixed (I group) or set (allowed to dry for 24 h) (II group) specimens of AH Plus Jet (AH), Apexit Plus (AP), MTA Fillapex (FL), GuttaFlow (GF), MetaSEAL Soft (META), and Tubli-Seal (TS) were prepared. HPdLF were exposed for 24 h to the specimens. 3-(4,5-dimethylthiazolo-2-yl)-2,5-diphenyltetrazolium bromide assay was used to examine the effect of the root canal sealers on mitochondrial metabolic activity. Fluorescein isothiocyanate (FITC)-annexin V (AnV) and propidium iodide staining followed by flow cytometry was used to identify the effects of the materials on cell apoptosis/necrosis. Statistical analyses were performed by one-way ANOVA followed by post hoc tests, and significance was determined at P < 0.05. Most materials from the two groups reduced the viability of the cultured cells compared with the control group (P < 0.05). Statistical analysis showed significant differences in HPdLF viability between the individual materials in each group (P < 0.001). AH and AP induced a significant increase in the percentage of apoptotic cells, while TS, FL, and META elevated the proportion of necrotic cells compared with other materials and the controls (p < 0.05). The cytotoxic effects of the tested root canal sealers (both fresh and set) on HPdLF varied. Both forms of sealers were able to cause toxic effects by inducing apoptosis and necrosis in HPdLF. The cytotoxicity of FL, META, TS was mainly associated with necrosis, while AH and AP with apoptosis.

Modulations of Keap1-Nrf2 signaling axis by TIIA ameliorated the oxidative stress-induced myocardial apoptosis.

Mounting evidence has strongly implicated oxidative stress in the development of cardiac dysfunction, and myocardial apoptosis contributes to the pathogenesis of heart failure. Quantitative cardiac proteomics data revealed that pressure load by TAC resulted in a significant decline in mitochondrial metabolic activity, where TIIA (Tanshinone IIA sulfonate) treatment reversed it in vivo, which might be mediated by Nrf2. In NRVMs, TIIA treatment ameliorated H2O2-induced caspase-3/9 activations through the suppression of p38 and mTOR signaling pathways, where caspase-mediated cleavage of YY1 and PARP resulted in the defects in mitochondrial biogenesis and DNA repair, and this event finally led to cardiomyocyte apoptosis. Mass spectrometry analysis showed that TIIA hydrophobically interacted with Keap1 (the cytoplasmic repressor of Nrf2) and induced its degradation in vitro. Site-directed mutagenesis of Keap1 identified V122/V123/I125 to be the critical residues for the TIIA-induced de-dimerization and degradation of Keap1. Besides, TIIA treatment also epigenetically up-regulated Nrf2 gene transcription, where it hypomethylated the first 5 CpGs of Nrf2 promoter. Furthermore, cardiac-specific Nrf2 knockout mice exhibited the significantly dampened anti-apoptotic effects of TIIA.

Effects of ganoderic acid A on lipopolysaccharide-induced proinflammatory cytokine release from primary mouse microglia cultures.

For several thousand years, Ganoderma lucidum (Ling-Zhi in Chinese and Reishi in Japanese) has been widely used as a traditional medication for the prevention and treatment of various diseases in Asia. Its major biologically active components, ganoderic acids (GAs), exhibit significant medicinal value due to their anti-inflammatory effects. Dysregulation of microglial function may cause seizures or promote epileptogenesis through release of proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α. At present, only little information is available on the effects of GAs on microglia-mediated inflammation in vitro and/or in vivo. The present study aimed to investigate the role of GA-A on microglia-mediated inflammation in vitro. In addition, the effect of GA-A on lipopolysaccharide (LPS)-evoked alterations in mitochondrial metabolic activity of microglia was evaluated. The results of the present study demonstrated that GA-A significantly decreased LPS-induced IL-1β, IL-6 and TNF-α release from mouse-derived primary cortical microglial cells in a concentration-dependent manner. GA-A treatment reduced LPS-induced expression of nuclear factor (NF)-κB (p65) and its inhibitor, demonstrating that non-toxic suppression of IL-1β, IL-6 and TNF-α production by GA-A is, at least in part, due to suppression of the NF-κB signaling pathway. In addition, the LPS-induced stimulation of mitochondrial activity of microglial cells was abolished by co-treatment with GA-A. Thus, GA-A treatment may be a potential therapeutic strategy for epilepsy prevention by suppressing microglia-derived proinflammatory mediators.

Antibiofilm activity of propolis extract on Fusarium species from onychomycosis.

The present study evaluated the capacity of three species of Fusarium isolated from onychomycosis to form biofilms and the antibiofilm effect of propolis extract on these biofilms. The biofilms and antibiofilm effects were evaluated by quantifying the colony-forming units, mitochondrial metabolic activity assays, total biomass by crystal violet staining and scanning electron microscopy. Propolis extract demonstrated significant antibiofilm efficiency on Fusarium spp. isolates and reduced F. solani, F. oxysporum and F. subglutinans mature biofilms. Propolis extract can be an alternative topical treatment of onychomycosis caused by Fusarium spp.

Effect of Nitric Oxide Donor on Metabolism of Apheresis Platelets.

The aim of this study is to investigate the effects of a nitric oxide (NO) donor S-nitrosoglutathione (GSNO) on themetabolism and improvement of preservation quality in apheresis platelets. A GSNO solution with a certain concentration was added into fresh apheresis platelets, and the parameters associated with platelet morphology, metabolism and function were temporally monitored for 7days. The results showed that the NO level in GSNO group were remarkably higher than those in the control group during the whole storage stage. No significant morphology or function difference was observed between the control and GSNO groups such as Platelet count, platelet distribution width, mean platelet volume and mitochondrial metabolic activity. But in metabolism there are something differences from morphology data: pCO2, pO2, cHCO3- were also found to have no clear difference between the control and GSNO groups; the lactic acid content, sugar consumption and Lactate dehydrogenase activity in the GSNO group were lower than that in the control group at some time point; and pH values in the GSNO group were higher than the control group. Our study discovered that the NO donor GSNO can reduce the metabolism and maintain the cellular characteristics of platelets in vitro during the platelet storage period.

N-Methyl-D aspartate receptor-mediated effect on glucose transporter-3 levels of high glucose exposed-SH-SY5Y dopaminergic neurons

High glucose and insulin lead to neuronal insulin resistance. Glucose transport into the neurons is achieved by regulatory induction of surface glucose transporter-3 (GLUT3) instead of the insulin. N-methyl-D aspartate (NMDA) receptor activity increases GLUT3 expression. This study explored whether an endogenous NMDA receptor antagonist, kynurenic acid (KynA) affects the neuronal cell viability at high glucose concentrations. SH-SY5Y neuroblastoma cells were exposed to 150–250 mg/dL glucose and 40 μU/mL insulin. In KynA and N-nitro-l-arginine methyl ester (L-NAME) supplemented cultures, oxidative stress, mitochondrial metabolic activity (MTT), nitric oxide as nitrite+nitrate (NOx) and GLUT3 were determined at the end of 24 and 48-h incubation periods. Viable cells were counted by trypan blue dye. High glucose-exposed SH-SY5Y cells showed two-times more GLUT3 expression at second 24-h period. While GLUT3-stimulated glucose transport and oxidative stress was increased, total mitochondrial metabolic activity was significantly reduced. Insulin supplementation to high glucose decreased NOx synthesis and GLUT3 levels, in contrast oxidative stress increased three-fold. KynA significantly reduced oxidative stress, and increased MTT by regulating NOx production and GLUT3 expression. KynA is a noteworthy compound, as an endogenous, specific NMDA receptor antagonist; it significantly reduces oxidative stress, while increasing cell viability at high glucose and insulin concentrations.

Uptake and intracellular accumulation of diamond nanoparticles - a metabolic and cytotoxic study.

Diamond nanoparticles, known as nanodiamonds (NDs), possess several medically significant properties. Having a tailorable and easily accessible surface gives them great potential for use in sensing and imaging applications and as a component of cell growth scaffolds. In this work we investigate in vitro interactions of human osteoblast-like SAOS-2 cells with four different groups of NDs, namely high-pressure high-temperature (HPHT) NDs (diameter 18–210 nm, oxygen-terminated), photoluminescent HPHT NDs (diameter 40 nm, oxygen-terminated), detonation NDs (diameter 5 nm, H-terminated), and the same detonation NDs further oxidized by annealing at 450 °C. The influence of the NDs on cell viability and cell count was measured by the mitochondrial metabolic activity test and by counting cells with stained nuclei. The interaction of NDs with cells was monitored by phase contrast live-cell imaging in real time. For both types of oxygen-terminated HPHT NDs, the cell viability and the cell number remained almost the same for concentrations up to 100 µg/mL within the whole range of ND diameters tested. The uptake of hydrogen-terminated detonation NDs caused the viability and the cell number to decrease by 80–85%. The oxidation of the NDs hindered the decrease, but on day 7, a further decrease was observed. While the O-terminated NDs showed mechanical obstruction of cells by agglomerates preventing cell adhesion, migration and division, the H-terminated detonation NDs exhibited rapid penetration into the cells from the beginning of the cultivation period, and also rapid cell congestion and a rapid reduction in viability. These findings are discussed with reference to relevant properties of NDs such as surface chemical bonds, zeta potential and nanoparticle types.

Connexin 43 is required for the maintenance of mitochondrial integrity in brown adipose tissue

We investigated the role of connexin 43 (Cx43) in maintaining the integrity of mitochondria in brown adipose tissue (BAT). The functional effects of Cx43 were evaluated using inducible, adipocyte-specific Cx43 knockout in mice (Gja1adipoqKO) and by overexpression and knockdown of Cx43 in cultured adipocytes. Mitochondrial morphology was evaluated by electron microscopy and mitochondrial function and autophagy were assessed by immunoblotting, immunohistochemistry, and qPCR. The metabolic effects of adipocyte-specific knockout of Cx43 were assessed during cold stress and following high fat diet feeding. Cx43 expression was higher in BAT compared to white adipose tissue. Treatment with the β3-adrenergic receptor agonist CL316,243 increased Cx43 expression and mitochondrial localization. Gja1adipoqKO mice reduced mitochondrial density and increased the presence of damaged mitochondria in BAT. Moreover, metabolic activation with CL316,243 further reduced mitochondrial integrity and upregulated autophagy in the BAT of Gja1adipoqKO mice. Inhibition of Cx43 in cultured adipocytes increased the generation of reactive oxygen species and induction of autophagy during β-adrenergic stimulation. Gja1adipoqKO mice were cold intolerant, expended less energy in response to β3-adrenergic receptor activation, and were more insulin resistant after a high-fat diet challenge. Collectively, our data demonstrate that Cx43 is required for maintaining the mitochondrial integrity and metabolic activity of BAT.

Insights into the intracellular mechanisms of citronellal in Candida albicans: implications for reactive oxygen species-mediated necrosis, mitochondrial dysfunction, and DNA damage.

Citronellal (Cit) possesses antifungal activity and has possible implications for reactive oxygen species (ROS) generation in Candida albicans. In this study, the effects of Cit on ROS generation and the mechanisms by which Cit exerts anti-Candida effects were examined. A 2',7'-dichlorodihydrofluorescein diacetate assay was used to assess oxidative damage. Cell necrosis was determined by flow cytometry after FITC-Annexin V staining. Mitochondrial function was studied based on mitochondrial potential, metabolic activity (MTT assay), and phenotypic susceptibility on a non-fermentable carbon source. Membrane intactness and DNA damage were estimated by a propidium iodide (PI) uptake assay and 4',6-diamidino-2-phenylindole (DAPI) staining. ROS generation was enhanced in response to Cit, leading to necrosis (2%). Additional hallmarks of cell death in response to Cit, such as mitochondrial membrane depolarization and DNA damage, were also observed. Cit treatment resulted in dysfunctional mitochondria, as evidenced by poor labeling with the mitochondrial membrane potential-sensitive probe rhodamine B, reduced metabolic activity (61.5%), and inhibited growth on a non-fermentable carbon source. Furthermore, Cit induced DNA damage based on DAPI staining. These phenotypes were reinforced by RT-PCR showing differences in gene expression (30-60%) between control and Cit-treated cells. Finally, PI uptake in the presence of sodium azide confirmed non-intact membranes and suggested that Cit activity is independent of the energy status of the cell. Cit possesses dual anticandidal mechanisms, including membrane-disruptive and oxidative damage. Taken together, our data demonstrated that cit could be used as a prominent antifungal drug.

Improvement of mitochondrial function by celastrol in palmitate-treated C2C12 myotubes via activation of PI3K-Akt signaling pathway

Compelling evidences posited that high level of saturated fatty acid gives rise to mitochondrial dysfunction and inflammation in the development of insulin resistance in skeletal muscle. Celastrol is a pentacyclic triterpenoid derived from the root extracts of Tripterygium wilfordii that possesses potent anti-inflammatory properties in a number of animal models with metabolic diseases. However, the cellular mechanistic action of celastrol in alleviating obesity-induced insulin resistance in skeletal muscle remains largely unknown. Therefore, the present investigation evaluated the attributive properties of celastrol at different concentrations (10, 20, 30 and 40nM) on insulin resistance in C2C12 myotubes evoked by palmitate. We demonstrated that celastrol improved mitochondrial functions through significant enhancement of intracellular ATP content, mitochondrial membrane potential, citrate synthase activity and decrease of mitochondrial superoxide productions. Meanwhile, augmented mitochondrial DNA (mtDNA) content with suppressed DNA oxidative damage were observed following celastrol treatment. Celastrol significantly enhanced fatty acid oxidation rate and increased the level of tricarboxylic acid (TCA) cycle intermediates in palmitate-treated cells. Further analysis revealed that the improvement of glucose uptake activity in palmitate-loaded myotubes was partly mediated by celastrol via activation of PI3K-Akt insulin signaling pathway. Collectively, these findings provided evidence for the first time that the protection from palmitate-mediated insulin resistance in C2C12 myotubes by celastrol is likely associated with the improvement of mitochondrial functions-related metabolic activities.

Anticancer activity of some polyamine derivatives on human prostate and breast cancer cell lines.

The aim of this study was to expand our knowledge about anticancer activity of some polyamine derivatives with quinoline or chromane as terminal moieties. Tested compounds were evaluated in vitro towards metastatic human prostate adenocarcinoma (PC3), human carcinoma (DU145) and mammary gland adenocarcinoma (MCF7) cell lines. Cell viability was estimated on the basis of mitochondrial metabolic activity using water-soluble tetrazolium WST1 to establish effective concentrations of the tested compounds under experimental conditions. Cytotoxic potential of polyamine derivatives was determined by the measurement of lactate dehydrogenase activity released from damaged cells, changes in mitochondrial membrane potential, the cell cycle distribution analysis and apoptosis assay. It was revealed that the tested polyamine derivatives differed markedly in their antiproliferative activity. Bischromane derivative 5a exhibited a rather cytostatic than cytotoxic effect on the tested cells, whereas quinoline derivative 3a caused changes in cell membrane integrity, inhibited cell cycle progression, as well as induced apoptosis of prostate and breast cancer cells which suggest its potential application in cancer therapy.

P 100 - Isothiocyanates trigger early disruption of mitochondrial function in cells overexpressing Bcl-2

Epidemiological evidence indicates that increased consumption of isothiocyanates is associated with reduced incidence of cancer. One mechanism to explain this is the ability of isothiocyanates to trigger apoptosis, including in cells that overexpress the anti-apoptotic protein Bcl-2, which is overexpressed in a number of cancers. This study aimed to examine the mechanisms by which phenethyl isothiocyanate (PEITC) induces apoptosis in Bcl-2 overexpressing (Bcl-2 OE) cells. Bcl-2 OE cells were more sensitive than wild-type cells to PEITC (LC50=8 μM vs. 12.5 μM). Bax-/-Bak-/-and Bim-/-Bid-/- cells were resistant to PEITC, indicating a classical apoptotic cell death. Cell death occurred more rapidly in the Bcl-2 OE cells, and increased levels of caspase activity and earlier activation were observed. PEITC had a greater effect on mitochondrial metabolic activity in the Bcl-2 OE cells; they had higher basal mitochondrial respiration, however, this was susceptible to inhibition by PEITC. There was also a significant decline in mitochondrial spare respiratory capacity. These effects occurred prior to caspase activation and cell death. Together, these results suggest that isothiocyanates bypass the action of Bcl-2 by acting directly on mitochondria to promote Bax/Bak-dependent apoptosis. This information may be valuable for designing novel isothiocyanates to target Bcl-2 OE cancer cells.

Toward greener electrochemical synthesis of composition-tunable luminescent CdX-based (X = Te, Se, S) quantum dots for bioimaging cancer cells

A novel versatile and clean colloidal processing route was developed for synthesizing CdX (X=Te, Se or S) quantum dots (QDs) based on the simultaneous production of the respective precursors via cadmium sacrificial anode electrochemical methodology. The CdX QDs stabilized by 3-mercaptopropionic acid (MPA) were synthesized in one-pot electrochemical process using aqueous medium at room temperature with full reaction time of approximately 16min. The CdX quantum dots conjugates were extensively characterized by UV–vis, PL, FTIR, TEM-EDS, XRD, DLS, XPS, and Zeta potential, evidencing that very stable colloidal quantum dots were produced with uniform narrow-size distributions and average nanoparticle diameter of approximately 3.0nm and quantum yield (QY) of approximately 10%. The cell viability response of HeLa cells toward the CdX-MPA conjugates clearly demonstrated that they are non-toxic based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) mitochondrial metabolic activity assay for 24h of incubation with HeLa cervical cancer cells. Moreover, they showed effective fluorescent activity with time-dependent intensities for evaluating continuous endocytosis of cancer cells, thus, offering innumerous possibilities to be used as fluorescent nanoprobes for bioimaging and biosensing of cancer cells.

Xenotransplantation of human mesenchymal stem cells alters host gene expression following prolonged high fat feeding

BackgroundInitially identified as plastic‐adherent cells, human mesenchymal stem cells (hMSCs) are non‐hematopoietic stem cells which are capable of differentiating into a multitude of cell lineages. Over the past decade the number of clinical trials involving hMSCs has steadily increased, resulting in hMSCs being the most commonly used cells in tissue engineering and regenerative medicine. Known to preferentially home to sites of injury and inflammation, administered hMSCs have exhibited promotion of host tissue repair through donor cell engraftment, release of cell signaling factors, and transfer of healthy organelles. As such, enhanced hMSC engraftment to host tissues and improved therapeutic potential may be induced by a state of low‐grade inflammation. Here we induce low‐grade inflammation through excessive nutrient consumption, achieved through prolonged high fat feeding.PurposeTo examine the impact of hMSC treatment on the liver tissue of mice following a prolonged high fat feeding regiment.MethodsAt 4 weeks of age, mice were placed on a high fat diet (60% kcal fat) for 20 weeks in order to elicit an obese phenotype. Following dietary manipulation, mice were randomly divided into two groups: hMSC treatment (HFM; n = 8) or saline control (HFS; n = 8). Mitochondrial metabolism, microarray gene expression profiling, and metabolomic profiling of host liver tissue were specifically highlighted for their important role in the obese phenotype.ResultsAs a measure of mitochondrial metabolic activity, maximal complex I oxygen consumption (supported by the addition of ADP) was higher in HFM animals (p&lt;0.05). The marker of mitochondrial abundance, citrate synthase, was also elevated following hMSC administration (p&lt;0.05). Microarray gene expression profiling identified 226 genes with significant differential expression between groups ‐ fold change (±1.2) and p‐value (&lt;0.01). Specific gene networks and pathway analysis commonly identified cellular signalling and actin cytoskeleton regulation as upregulated processes in HFM mice (p&lt;0.01).ConclusionsOur data indicate that xenogeneic hMSC treatment improves metabolic capacity of host liver and results in widespread host gene shifting. The upregulation of cell signaling and actin cytoskeleton regulation may provide mechanistic insight into how hMSCs impart their ability to migrate to areas of inflammation and transfer organelles to host tissues.Support or Funding InformationThis study was supported by NSERC (JS). This research was supported by PhD funding to CN from MitoCanada and Alberta Innovates – Health Solutions.

Glutamate-mediated effects of caffeine and interferon-γ on mercury-induced toxicity

The molecular mechanisms mediating mercury-induced neurotoxicity are not yet completely understood. Thus, the aim of this study was to investigate whether the severity of MeHg- and HgCl2-mediated cytotoxicity to SH-SY5Y human dopaminergic neurons can be attenuated by regulating glutamate-mediated signal-transmission through caffeine and interferon-γ (IFN-γ). The SH-SY5Y cells were exposed to 1, 2 and 5 μM of either MeHgCl2 or HgCl2 in the presence or absence of L-glutamine. To examine the effect of adenosine receptor antagonist, the cells were treated with 10 and 20 μM caffeine. The total mitochondrial metabolic activity and oxidative stress intensity coefficient were determined in the 1 ng/ml IFN-γ- and glutamate-stimulated SH-SY5Y cells. Following exposure to mercury, the concentration-dependent decrease in mitochondrial metabolic activity inversely correlated with oxidative stress intensity. MeHg was more toxic than HgCl2. Mercury-induced neuronal death was dependent on glutamate-mediated excitotoxicity. Caffeine reduced the mercury-induced oxidative stress in glutamine-containing medium. IFN-γ treatment decreased cell viability and increased oxidative stress in glutamine-free medium, despite caffeine supplementation. Although caffeine exerted a protective effect against MeHg-induced toxicity with glutamate transmission, under co-stimulation with glutamine and IFN-γ, caffeine decreased the MeHg-induced average oxidative stress only by half. Thereby, our data indicate that the IFN-γ stimulation of mercury-exposed dopaminergic neurons in neuroinflammatory diseases may diminish the neuroprotective effects of caffeine.

Mitochondrial Function and Cell Size: An Allometric Relationship.

Allometric scaling of metabolic rate results in lower total mitochondrial oxygen consumption with increasing organismal size. This is considered a universal law in biology. Here, we discuss how allometric laws impose size-dependent limits to mitochondrial activity at the cellular level. This cell-size-dependent mitochondrial metabolic activity results in nonlinear scaling of metabolism in proliferating cells, which can explain size homeostasis. The allometry in mitochondrial activity can be controlled through mitochondrial fusion and fission machinery, suggesting that mitochondrial connectivity can bypass transport limitations, the presumed biophysical basis for allometry. As physical size affects cellular functionality, cell-size-dependent metabolism becomes directly relevant for development, metabolic diseases, and aging.

Treatment of Adult mdx Mice with Phosphorodiamidate Morpholino Oligomer Restores Cardiac Mitochondrial Energetics and Prevents the Dystrophic Cardiomyopathy

Duchenne muscular dystrophy (DMD) occurs due to the absence of dystrophin, and is associated with a fatal dilated cardiomyopathy. DMD-associated cardiomyopathy is underpinned by disrupted cytoskeletal architecture and poor energy metabolism. The L-type Ca2+ channel (LTCC) plays a critical role facilitating Ca2+ influx required for contraction, but also regulates mitochondrial function. This crosstalk is mediated via the cytoskeleton, and is disrupted in a mouse model of DMD (mdx). We previously demonstrated that treatment of pre-cardiomyopathic mdx mice with 10mg/kg/wk phosphorodiamidate morpholino oligomer (PMO) to induce skipping of exon 23 (M23D) for 24wks partially restored increases in mitochondrial membrane potential (Ψm) and metabolic activity in response to activation of LTCC in myocytes isolated from pre-cardiomyopathic mice. Here we investigated the effect of a short-term, but high PMO treatment dose, namely 120mg/kg/wk for 3wks, on alterations in Ψm and metabolic activity. Treatment resulted in partial restoration of Ψm and metabolic activity. Based on these findings we investigated whether a long-term and high PMO treatment dose would more effectively restore Ψm and metabolic activity to wt levels. 24wk old pre-cardiomyopathic mdx mice were treated with 120mg/kg/wk for 19wks. Positive exon skipping was verified via RT-PCR, and immunohistochemistry confirmed the presence of dystrophin positive fibers within mdx heart tissue. PMO treatment completely restored Ψm to wt levels (wt untreated: 30.9±4.2%, n=11; mdx untreated: 1.6±0.8%, n=8; mdx treated: 31.6±2.6%, n=7). PMO treatment also completely restored metabolic activity (assessed as formation of formazan from tetrazolium salt) (wt untreated: 48.9±6.4%, n=15; mdx untreated: 3.0±1.3%, n=11; mdx treated: 64.9±14.0%, n=26). Furthermore, PMO treatment prevented development of the mdx cardiomyopathy assessed using serial echocardiography. These data indicate that treatment with M23D restores cardiac mitochondrial energetics in adult mdx mice. We propose that restoring mitochondrial function may play a vital role in mediating PMO-induced improvements in contractility.

Negligible cytotoxicity induced by different titanium dioxide nanoparticles in fish cell lines

Titanium dioxide nanoparticles (TiO2-NPs) have a wide number of applications in cosmetic, solar and paint industries due to their photocatalyst and ultraviolet blocking properties. The continuous increase in the production of TiO2-NPs enhances the risk for this manufactured nanomaterial to enter water bodies through treated effluents or agricultural amendments. TiO2-NPs have shown very low toxicity in a number of aquatic organisms. However, there are no conclusive data about their deleterious effects and on their possible mechanisms of toxic action. At this level, in vitro cell culture systems are a useful tool to gain insight about processes underlying the toxicity of a wide variety of substances, including nanomaterials. Differences in the physiology of different taxa make advisable the use of cells coming from the taxon of interest, but collecting data from a variety of cellular types allows a better understanding of the studied processes. Taking all this into account, the aim of the present study was to assess the toxicity of three types of TiO2-NP, rutile hydrophobic (NM-103), rutile hydrophilic (NM-104) and rutile-anatase (NM-105), obtained from the EU Joint Research Centre (JRC) repository, using various fish cell lines (RTG-2, PLHC-1, RTH-149, RTL-W1) and rainbow trout primary hepatocytes. For comparative purposes, the effect of different dispersion protocols, end-point assays and extended exposure time was studied in a fish cell line (RTG-2) and in the rat hepatoma cell line (H4IIE). TiO2-NPs dispersions showed a variable degree of aggregation in cell culture media. Disruption of mitochondrial metabolic activity, plasma membrane integrity and lysosome function was not detected in any cell line after exposure to TiO2-NPs at any time and concentration ranges tested. These results are indicative of a low toxicity of the TiO2-NPs tested and show the usefulness of fish cells maintained in vitro as high throughput screening methods that can facilitate further testing in the framework of integrated testing strategies.

Cytotoxicity assessment of three endodontic sealing cements used in periapical surgery. In vitro study

Introduction: Presently there are many retrofilling materials in the market, nevertheless, little is known about their toxicity on gingival fibroblasts. Objective: To assess cytotoxicity of three materials to human gingival fibroblasts and L929 mouse fibroblasts cell line. Material and methods: EndoSequence® BC RRMTM (ERRM; root repair material), white MTA Angelus® (MTA) and intermediate restoration material (IRM®) conditioned media were obtained when materials were freshly mixed, at setting time and after 1, 24 and 72 hours of setting time. Cell morphology was assessed with light microscopy and cell viability was assessed through mitochondrial metabolic activity with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Statistical analysis was conducted with ANOVA. Results: We found that ERRM material did not exhibit cytotoxic effects on used fibroblast, nevertheless, MTA and IRM® respectively exhibited moderate and severe cytotoxicity, thus indicating the materials were not fully harmless. Conclusion: Bioceramic cements like ERRM could be considered the most compatible retrofilling-materials.