Acrydine Orange Research Articles

Enhancement of biological effects of oxidised nano- and microplastics in human professional phagocytes

Micro and nanoplastics are ubiquitous pollutants that can cause adverse health effects even in humans. Effects of virgin and oxidised (simulating the aging processes) polystyrene nano (nPS) and micro particles (mPS) with diameters of 0.1 and 1 µm were studied on human professional phagocytes (i.e., monocyte cells THP-1 and macrophage-like mTHP-1 cells). After characterization by ATR-FTIR, UV-Vis spectroscopy, SEM and dynamic light-scattering analyses, the particles were FITC functionalised to quantify cellular uptake. Changes in the cell compartments were studied by acrydine orange and the pro-oxidant, cytotoxic and genotoxic effects were assessed. Phagocytosis was dose- and time- dependent and at 24 h 52% of nPS and 58% of mPS were engulfed. Despite the high homeostasis of professional phagocytes, significant ROS increases and DNA damage were observed after exposure to oxidised particles. The results highlight that the environmental aging processes enhances the adverse health effects of micro and nanoplastics.

Comparison of Root End Sealing Ability of Three Retrograde Filling Materials in Teeth with Root Apices Resected at 90° using Dye Penetration Method under Fluorescent Microscope: An In-vitro Study

Background: Inadequate seal at the apex is the substantial cause for surgical endodontic dissatisfaction. The retrograde filling material which is used should prevent the egress of potential contaminants into periradicular tissue.
 Objectives: To evaluate the ability of MTA Angelus, Zirconomer and Bioactive bone cement to seal the root end as retrograde filling material; and to compare root end sealing ability of these three different retrograde filling materials with apices resected at 900 angles using dye penetration method under fluorescent microscope.
 Methodology: Thirty six extracted upper anterior teeth are to be cut horizontally at the CEJ. After following the standard protocols of, “cleaning, shaping and obturation” with gutta percha and “AH Plus sealer”, the samples will be resected 3mm at the apical end at 90 degree angle along the long axis of the tooth with the help of diamond disc. A root end cavity of depth 3mm will be made with Diamond coated ultrasonic surgical tip S12 90 ND. The teeth will be randomly categorized in 3 groups: Group 1: MTA angelus; Group 2: Zirconomer; Group 3: Bioactive Bone Cement. Following which the roots to be coated with nail varnish except the tip. Each material will be compressed in the root end cavity with the help of small pluggers. All the samples which are retrofilled will be kept in acrydine orange for a duration of 24 hours, following which cleaning & bucco-lingual sectioning(vertical) will be done. Fluorescent microscope will be used for observation of sectioned root samples.
 Expected Results: Bioactive bone cement is expected to have better sealing ability of the retrograde cavity preparation with minimal or no microleakage followed by MTA Angelus and then Zirconomer.
 Conclusion: If this study proves correct, this would be helpful for the clinicians to choose better and the most efficient retrograde filling material with best sealing ability and minimal microleakage in a retrograde preparation which will aid in success of the root canal treatment further resolving the infection.

TNF-α loaded on gold nanoparticles as promising drug delivery system against proliferation of breast cancer cells

Even though the significant anti tumor effect of Tumor necrosis factor (TNF-α) and the essential role in various cellular and immunological properties, the clinical use of TNF-α is delayed due to its toxicity. In this study In this study, we designed a drug delivery system by binding gold nanoparticles (GNPs) with TNF-α then investigated their anti- proliferative activity against human breast cancer AMJ13. The binding between GNPs and TNF-α was confirmed using UV-spectrum assay, and transsision electron microscopy (TEM). MTT assay and colony forming assay were used to investigate cytotoxic activity of drug delivery system against AMJ13 cell line. Apoptosis was measured by the Acrydine orange –Propodium Iodide AO/PI double staining assay. Mitochondrial membrane potential assay of AMJ13 cells after treated with GNPs-TNF was measured by flow cytometry technique. The results of this study demonstrated that the TNFα-GNPs inhibits proliferation of AMJ13 cells, resulting in apoptosis and involves mitochondrial damage. Taken together, the results of current study demonstrated that suggest that the GNPs -TNFα could be used in medical application in future and offer new drug recompense a chemotherapy drug.

Zinc finger peptide based optic sensor for detection of zinc ions

In the present work, polyacrylamide gel has been used as a matrix for the immobilization of zinc finger peptide and fluorescent dye acrydine orange on the micro well plate to fabricate the fluorescence based biosensor for the detection of zinc ions in milk samples. The fluorescent dye moves in the hydrophobic groove formed after folding of the peptide in the presence of zinc ions. Under optimized conditions, linear range was observed between 0.001µg/l to 10µg/l of Zinc ions, with a lowest detection limit of 0.001µg/l and response time of 5min. Presented biosensor has shown 20% decrease in fluorescent intensity values after 5 regenerations and stable for more than one month, stored at 4°C. Interference study with other metal ions like lead, cadmium and copper showed a negligible change in fluorescence intensity in comparison to zinc ions. Developed bio sensing system was found to be novel, quick, reliable, miniaturized, stable, reproducible and repeatable and specific for zinc ion, which has been applied to various milk samples.

Comparison of the Root End Sealing Ability of Four Different Retrograde Filling Materials in Teeth with Root Apices Resected at Different Angles - An Invitro Study.

Insufficient apical seal is the significant reason for surgical endodontic disappointment. The root-end filling material utilized should avoid egress of potential contaminants into periapical tissue. The aim of this study was to compare the sealing ability of four root-end filling materials MTA, Portland cement, IRM, RMGIC in teeth with root apices resected at 0 and 45 angle using dye penetration method under fluorescent microscope. Hundred extracted human maxillary anterior teeth were sectioned horizontally at the cement-enamel junction. After cleaning, shaping and obturation with gutta-percha and AH Plus sealer, the tooth samples were randomly divided in two groups (the root apices resected at 0° and 45° to the long axis of the root). The root resections were carried out by removing 2 mm and 1 mm in both the groups. Following which 3 mm deep root-end cavities were prepared at the apices and the root were coated with nail varnish except the tip. The teeth in both the group were randomly divided into four subgroups each (Pro root MTA, Portland cement, IRM and Light cure nano GIC Ketac N-100). All the retrofilled samples were stored in acrydine orange for 24 hours after which they were cleaned and vertically sectioned buccolingually. The sectioned root samples were observed under fluorescent microscope. The root apex sealing ability of Mineral Trioxide Aggregate (MTA) was superior to Portland cement, Intermediate Restorative Material (IRM) and LC GIC. IRM demonstrated the maximum apical leakage value among all the materials. Portland cement and LC GIC showed comparable sealing ability. The angulation whether 0° or 45° angle did not affect the sealing ability of all the four materials used, MTA proved to be one of the superior materials for root-end filling.

Cytotoxic activity and apoptosis induction of 8-hydroxyisocapnolactone-2´,3´-diol and its combination with Doxorubicin on MCF-7 and T47D cells

The cytotoxic activity and apoptosis induction of 8-hydroxyisocapnolactone-2΄,3΄-diol (HICD) and its combination with doxorubicin (Doxo) on MCF-7 and T47D cells have been evaluated. The cytotoxic assay was performed using MTT method. The IC50 was used to express the cytotoxic potency, while the combination index (CI) was calculated to determine the effect of combination. The apoptotic assay was carried out using acrydine orange - ethidium bromide double staining method, while Bcl-2 and Bax expression were investigated by immunocytochemistry. The HICD exhibited cytotoxic activity on MCF-7 and T47D cells with the IC50 of 8 µg/ml (21.2 µM) and 4 µg/ml (10.6 µM), respectively. The combination of HICD with Doxo showed synergistic effect and increased apoptosis induction on both cell lines. The HICD did not affect Bcl-2 but increased Bax expression on MCF-7 cells, while on T47D cells it suppressed Bcl-2 expression but did not modulate Bax expression

Abstract 19061: Kca3.1 Regulates Monocytosis and Monocyte Adherence in Atherosclerosis

Introduction: Atherosclerosis is associated with a monocytosis of an inflammatory subset (Ly-6Chi in mice), which is preferentially recruited to lesions and gives rise to pro-atherosclerotic macrophages (MΦ). We have shown that the intermediate-conductance Ca2+-activated K+ channel KCa3.1 is predominantly expressed in blood Ly-6Chi monocytes (MONO) and lesion MΦ, and KCa3.1 blockade or knockout reduces the development of atherosclerosis in mice. To further study the function of KCa3.1 in atherogenesis, we investigated its role in monocytosis and in MONO adherence to the endothelium in vivo. Methods and Results: Flow cytometric analyses of peripheral blood and bone marrow from Apoe-/- and Apoe-/- KCa3.1-/- mice showed that KCa3.1 deficiency decreased circulating MONO (8.8±1.5% of leukocytes, p<0.05 vs. 15.4±2.1%; n=7-8) and Ly6Chi MONO (4.2±0.7% of leukocytes, p<0.05 vs. 9.8±1.5%, n=7-8), with unchanged Ly6Clo MONO (4.6±1.3% of leukocytes, p=ns vs. 5.6±0.7%) and polymorphonuclar (11.7±1.4% of cells, p=ns vs. 12.5±1.2%; n=7-8). Apoe-/- KCa3.1-/- mice had reduced hematopoietic stem and multipotential progenitor cells (HSPCs) (2.4±0.5% of lineage negative cells, p<0.05 vs. 3.0±0.5%, n=7). In vivo confocal endomicroscopic analysis of leukocyte adherence was performed in anesthetized mice 4 hours after topical application of 0.5 μg TNF-α and 0.125 μg IL-1β onto the right external carotid artery where an imaging probe (Cellvizio®, field of view=8x105 μm2) was placed. Blood and circulating leukocytes were stained by intravenous injection of 200 μg FITC-dextran and 0.02% acrydine orange, respectively. KCa3.1 deficiency markedly inhibited leukocyte rolling (4±2 vs. 52±15 cells/min, contact time: 1.6±0.5 vs. 2.7±0.2 sec, distance: 78±11 vs. 165±5 μm, and velocity: 145±24 vs. 119±5 μm/sec) and adhesion (0.5±0.3 vs. 2.0±0.6 cells/min, p<0.05 for all data, n=6). Immunohistochemistry of lesions showed that Apoe-/- KCa3.1-/- mice had less CD68-positive MONO/MΦ compared to Apoe-/- mice (616±101, p<0.05 vs. 937±90 cells/mm2, n=8-9). Conclusion: KCa3.1 modulates monocytosis and plays a major role in MONO adherence to the endothelium in vivo. Thus KCa3.1 constitutes a promising therapeutic target for the prevention of atherosclerosis.

Antiproliferative Effect of Chloroform Extract of Cangkring Leaves (Erythrina fusca Lour.) and Its Isolates against HeLa Cells

Cervical cancer is a type of cancer possessing the 3rd highest incidence among women worldwide. Today’s therapies against cancer are still ineffective, thus people are seeking for alternative method. Erythrina fusca Lour. has been used traditionally as anticancer. This experiment is conducted to study the ability of chloroform extract of E. fusca Lour. leaves and its isolates on HeLa cervical cancer cells. The cytotoxicity assay and doubling time assay were done using direct counting method, while the DNA staining assay was done using acrydine orange. The chloroform extract and isolate # 30 showed cytotoxic activity with IC50 of 16 and 5 µg/ml, respectively, while isolate # 2 and isolate # 8 didn’t. The effluent # 30 could not affect HeLa cells growth at the given concentration, but it might promote DNA fragmentation indicating apoptosis induction. The molecular mechanisms underlying these effects still need to be further explored.Keywords: Erythrina fusca Lour., isolate and effluent of chloroform extract, antiproliferative, cervical cancer

Antiproliferative Effect and Apoptosis Induced in Human Cell Lines by Bruguiera gymnorhiza Barks Methanol Extract

The Antiproliferative effects of methanol extract from Bruguiera gymnorhiza (B. gymnorhiza) barks were tested in vitro against three human cell lines: Hela, Raji and Myeloma cells. The extract was found to have antiproliferative effects against Hela, Raji and Myeloma cells with an IC50 value of 133, 504 and 384 µg/mL, respectively. The antiproliferative test was then performed on Hela, Raji, and Myeloma cells. Cytotoxicity assay of the extract was then determined using MTT method. There were some indications of apoptosis, such DNA fragmentation, as assessed by acrydine orange- ethidium bromide staining. These results indicate that extract from B. gymnorhiza barks can induce apoptosis in human cell lines.Keywords: Antiproliferative, Apoptosis, B. gymnorhiza

Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells in hypertonic medium

Changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium supplemented with 200 mM sucrose have been studied. Cells were stained with acrydine orange, ethydium bromide, APOPercentage dye, which marks the phosphatidyl serine distribution on the plasma membrane; and FLICA polycaspase fluorescent dye. It was found that cell shrinkage produced by direct osmotic effect induced both a regulatory volume increase and apoptotic volume decrease. The regulatory volume increase dominated at the early stage, whereas apoptotic volume decrease prevailed at the later stage. Therefore, U937 cells were capable of triggering apoptosis and apoptotic volume decrease, despite the unimpaired regulatory volume increase response, and the current opinion that the dysfunction of the regulatory volume increase is a prerequisite for apoptosis and apoptotic volume decrease (Subramanyam et al., 2010) should be revised. It is concluded that the apoptotic volume decrease plays a significant role in preventing osmotic lysis in apoptotic cells, rather than in initiating apoptosis.

Screening of high cytotoxic tumor killer cells using a sensitive adherent target detachment assay

Screening of high cytotoxic tumor killer cells is of great importance in adoptive immunotherapy. Here, we describe a more sensitive assay, as compared to traditional 51Cr- or Calcein-release assay, to measure cytolytic activity of killer cells. This adherent target detachment (ATD) assay is carried out in microwells of Terasaki tissue culture trays using adherent tumor cells as targets. Target tumor cells are seeded at the concentration of 300–400 cells/well and incubated overnight to allow for the adhesion of cells to the plastic surface of the wells. Effector cells were added at various effector: target (E:T) ratios, and incubated for 24 h. During incubation, dead target cells became nonadherent and together with the added effector cells were removed by washing. The remaining viable adherent target cells were stained with acrydine orange contained in the quencher and optically counted by microcomputer. A notable dose-dependent killing on target tumor cells was reproducibly obtained by tested effector cells. Cytotoxic activities of most effector cells were significantly higher in the 24-h ATD assay than those of concordant 4-h target cell lysis test. Chloroquine (chq) inhibition test in the 24-h ATD assay showed negative or weak inhibition on cytotoxicity against adherent targets. Linear regression analysis also manifested the lack of a close correlation between 4- and 24-h target cell lysis tests, indicating these two assays measured different killing activities of the same effector cells. Because of its high sensitivity and reproducibility, this semiautomatic 24-h assay with computerized fluorescence measurement system could serve as a sensitive screening assay to select high cytotoxic tumor killer cells in adoptive immunotherapy.

Unexpected photoinduced phenomena in chiral?photochromic cholesteric copolymers with a triplet sensitizer.

For the first time the possibility of energy transfer from a triplet photosensitizer to chiral–photochromic fragments in photoactive cholesteric systems was demonstrated. For this purpose we prepared mixtures containing chiral–photochromic cholesteric copolymers with a triplet sensitizer—acrydine orange (AO). Chiral–photochromic groups in copolymers contain a C=C bond capable for undergoing E–Z isomerization during UV irradiation. All polymer mixtures form a chiral nematic phase displaying selective light reflection with λmax~650–1,000 nm depending on the structure and concentration of the chiral groups. Irradiation of mixtures by visible light (λ>450 nm) leads to a shift of the selective light reflection peak to a long-wavelength spectral region. This effect is associated with a decrease of anisometry of chiral–photochromic fragments in copolymers during their E–Z isomerization. It is important to emphasize, that chiral–photochromic side groups of copolymers do not absorb visible light themselves; therefore, the previously mentioned changes can be explained by the energy transfer from photoexcited AO molecules in the triplet state to isomerizable fragments. The study of the kinetics of this process revealed a rather unexpected phenomenon: after the first 60–80-min irradiation, the helix pitch of the supramolecular structure of the mixtures increases, but after successive irradiation the helix pitch decreases. The possible explanations of this phenomenon were suggested. It was demonstrated that these mixtures may be used for irreversible recording of optical information.

Mutation Technique for Increasing the Production of Antibacteria Lactobacillus plantarum TGR-2

Production of antibacteria of Lactobacillus plantarum TGR-2 was carried out by mutation technique using UV radiation and chemical mutagenic agent (acrydine orange (AO), ethyl methane sulfonate (EMS), and N-methyl-N-nitro sulfonate nitrosoguanidine (NTG). As a marker was applied chloramphenicol for 10 ppm. Total 214 mutants were obtained from all of the treatments and then screened based on their activities against Staphylococcus aureus FNCC 0047 as bacterial indicator, using agar diffusion and turbidimetric assay techniques. Results showed that 24 mutants could produce higher amount of antibacteria and two of them, NTG and acrydine orange treatments, had higher stability than others. Characterization of two mutants, determination of biochemistry traits and optimization of antibacterial (bacteriocin) production were done. The product showed that UV-15, EMS-10, AO-6 and NTG-21 mutants could produce antibacterial substance 3,90, 4.40, 3.40 and 4.17 AU/ml for 16, 12, 12, and 16 hours incubation, respectively, compared to Lactobacillus plantarum TGR-2 which produced 1.07 UA/ ml for 24 hours incubation.

Small nuclear RNAs and small nuclear ribonucleoprotein complexes purified from the feline placenta.

Small nuclear RNAs (snRNAs) and small nuclear ribonucleoprotein particles (snRNPs) were purified from the cat placenta as an initial step in the analysis of the regulatory mechanism(s) of gene expression in the placenta. For visual observations, snRNA species including 7S, U2, U1, U4, X-I, X-II, 5S, U5, U6 and X-III were stained with acrydine orange, following the extraction of RNAs with phenol from cat placental cell nuclei and their fractionation by electrophoresis on a 10% polyacrylamide gel containing 7 M urea. The small nuclear RNA U1 from the cat placenta showed approximately the same mobility as that of mouse (129/sv) liver used as a reference on the gel. The logarithms of the nucleotide lengths of cat placenta snRNAs were inversely related to their mobilities. The approximate number of snRNA molecules in the cat placenta cell nucleus was also calculated. Highly purified U1-snRNP which comprises U1 snRNA and eight polypeptide components ranging in molecular weight from 68, 000 to 9, 500 was immunochemically purified using systemic lupus erythematosus-anti(U1)RNP antibody column chromatography. Any polypeptide component involved in the U1-snRNP did not immunoreact with normal human IgG. No immunoreactive materials with anti(U1)RNP antibodies could be detected in cat amniotic fluid. The U1-snRNP and U2-snRNP rich fractions were separated biochemically on a DEAE Sepharose column.