Statistical evaluation of liquid phase sequestration of acridine orange and Cr6+ by novel mesoporous glutamic acid-g-polyacrylamide/plaster of paris/riboflavin hydrogel nanocomposite.

The adsorption of acridine orange and Cr6+ ion onto plaster of paris reinforced glutamic acid-grafted-polyacrylamide hydrogel nanocomposite modified with riboflavin, Glu-g-PAM/POP/Rb HNC was studied. The Glu-g-PAM/POP/Rb HNC was physico-chemically characterized by Fourier transform infrared spectroscopy, X-ray diffraction analysis, scanning electron microscopy coupled with energy dispersive X-ray spectroscopy, transmission electron microscopy and Brunauer-Emmett-Teller analysis. The specific surface area, pore volume and pore diameter were 15.48 m2/g, 0.015 cm3/g and 4.23 nm, respectively. Adsorption process was strategized by response surface methodology (RSM) based on a 3-level 5-factor (initial solution pH, contact time, adsorbent dose, initial adsorbate concentration and temperature) central composite design (CCD), and validity of the estimated parameters was statistically evaluated using analysis of variance (ANOVA). The optimized operating variables were: pH (AO = 10; Cr6+ = 4.15), contact time (AO = 60 min; Cr6+ = 59 min), adsorbent dose (0.8 g/L), initial adsorbate concentration (60 mg/L) and temperature (298 K). Isotherm results were coincident with Langmuir isotherm model. The experimental kinetic adsorption data was congruous with pseudo-second order model, with the uptake rate controlled by both intraparticle and liquid film diffusions. The relatively high Langmuir saturation capacity of 202.63 mg AO/g and 143.68 mg Cr6+/g, supported by the decent recyclability up to four times affirmed the promising performance of the adsorbent. The efficacy of the adsorbent for simultaneous removal of AO and Cr6+ from bi-component system was assessed. The possible adsorption mechanism mainly involved hydrogen bonding, van der Waals forces, electrostatic and π-π interactions. Adsorption of AO and Cr6+ onto Glu-g-PAM/POP/Rb HNC was feasible and exothermic as revealed by the thermodynamic parameters. The findings demonstrated superior adsorbent efficacy for the seizure of pollutants, particularly AO and Cr6+ from aqueous solution.

Hydrogel composite of lanthanum and Halorubrum ejinoor sp. cell lysate as an adsorbing material.

OBJECTIVES: Although halophilic archaea are rich in natural environments, their biotechnological applications are not as prevalent as those of other extremophiles, such as thermophiles and alkaliphiles. This study presents an simple method to prepare a hydrogel composite using crude cell lysate of a halophilic archaea, Halorubrum ejinoor sp. (H.e.) which was isolated from a saline lake in Inner Mongolia, China. Furthermore, formation mechanism and potential applications of the hydrogel as an adsorbing material are discussed. RESULTS: Halorubrum ejinoor sp. (H.e.) cell lysate was firstly prepared by adding pure water onto the H.e. cell pellet, followed by a short incubation at 60 °C. The cell lysate was injected into different metal ion (or H+) solutions to obtain the hydrogel composite. It was observed that H+, Fe3+, La3+, Cu2+, and Ca2+ induced gelation of the cell lysate, while Fe2+, Co2+, Ni2+, Mg2+, Na+, and K+ did not. DNA and extracellular polysaccharides (EPS) in the H.e. cell lysate were found to be responsible for the gelation reaction. These results suggest that DNA and EPS should be crosslinked by metal ions (or H+) and form a networked structure in which the metal ion (or H+) serves as an anchor point. Potential application of the hydrogel as an adsorbing material was explored using La3+-induced H.e. hydrogel composite. The hydrogel composite can adsorb the fluoride, phosphate and DNA-binding carcinogenic agents, such as acridine orange. CONCLUSIONS: The simplicity and cost effectiveness of the preparation method might make H.e. hydrogel a promising adsorbing material. This work is expected to expand the technical applications of haloarchaea.

Wash-free instant detection of giant plasma membrane vesicles.

Giant plasma membrane vesicles (GPMVs) are large extracellular vesicles produced by the exposure of cells to paraformaldehyde or other stresses, providing an experimental system to elucidate cell surface dynamics. Here we show that addition of the membrane permeable fluorescent RNA-indicators, acridine orange and thioflavin T, to GPMV-containing solutions prepared from cultured HeLa cells was sufficient for the fluorescent visualization of seemingly all GPMVs. Our findings provide a wash-free instant method using non-lipid-type fluorescent dyes for GPMV detection, which should be useful for researchers interested in studying cell membrane dynamics and biochemistry using GPMVs.

A rapid and low-cost estimation of bacteria counts in solution using fluorescence spectroscopy.

The fluorescence spectrum of bacterially bound acridine orange (AO) was investigated to evaluate its use for the rapid enumeration of bacteria. Escherichia coli ATCC 25922 samples were stained with 2 × 10-2, 2 × 10-3 or 2 × 10-4% w/v AO, followed by 3, 2 or 0 washing cycles, respectively, and fluorescence spectra were recorded using a fibre-based spectroscopic system. Independent component analysis was used to analyse the spectral datasets for each staining method. Bacterial concentration order of magnitude classification models were calculated using independent component weights. The relationship between fluorescence intensity of bound AO and bacterial concentration was not linear. However, the spectral signals collected for AO stain concentration-bacterial concentration pairs were reproducible and unique enough to enable classification of samples. When above 105 CFU ml-1, it was possible to rapidly determine what the order of magnitude of bacterial concentration of a sample was using a combination of two of the sample preparation methods. A relatively inexpensive (around US$10 per test) rapid method (within 25 min of sampling) for enumeration of bacteria by order of magnitude will reduce the time and cost of microbiological tests requiring gross concentration information. Graphical Abstract Fluorescence spectra of bacterially bound acridine orange (AO) were used for the rapid enumeration of bacteria. Order of magnitude bacterial concentration classification models were calculated using independent components analysis of these fluorescence spectra. When above 105 CFU ml-1, it was possible to rapidly determine the order of magnitude of bacterial concentration of a sample using a combination of two sample preparation methods.

Using Vital Dyes to Trace Uptake of dsRNA by Green Peach Aphid Allows Effective Assessment of Target Gene Knockdown.

RNA interference (RNAi) is an effective tool to study gene function. For in vitro studies of RNAi in insects, microinjection of double-stranded (ds)RNA may cause stress. Non-persuasive oral delivery of dsRNA to trigger RNAi is a better mode of delivery for delicate insects such as aphids because it mimics natural feeding. However, when insects feed ad libitum, some individuals may not feed. For accurate measurement of gene knockdown, analysis should only include insects that have ingested dsRNA. The suitability of eleven dyes was assessed to trace ingestion of dsRNA in an artificial feeding system for green peach aphids (GPA, Myzus persicae). Non-toxic levels of neutral red and acridine orange were suitable tracers: they were visible in the stylet and gut after feeding for 24 h, and may also attract aphids to feed. Nymphs stained with neutral red (0.02%) were analysed for target gene expression after feeding on sucrose with dsRNA (V-ATPase, vha-8). There was a greater reduction in vha-8 expression and reproduction compared to nymphs fed the diet without dye. The results confirm the importance of identifying aphids that have ingested dsRNA, and also provide evidence that the vha-8 gene is a potential target for control of GPAs.

Morphology Dependent Photocatalytic Activity of α-MoO3 Nanostructures Towards Mutagenic Acridine Orange Dye.

The morphological evolutions of orthorhombic molybdenum oxide nanostructures with high crystalline nature have been successfully synthesized by combining low-temperature sol-gel and annealing processes. Strong influence of gelation temperature is a factor facilitated to control the material morphology. Morphological transformations like nanospheres, nanoplatelets, mixtures of hexagonal platelets, and one-dimensional nanobars were obtained. The possible morphological formation mechanism has been proposed as a self-assemble process of nucleation and a mechanism for particle growth by Ostwald ripening. The as-prepared nanostructures were recognized as photocatalysts for the degradation of Acridine Orange under Ultra Violet light. The obtained mixed morphology (hexagonal nanoplatelets and nanobars) showed a high photocatalytic property to degrade mutagenic Acridine Orange dye. Moreover, they could be easily recycled without changing the photocatalytic activity due to their 1-Dimensional and 2-Dimensional nanostructure property.

Watermelon rinds as cost-efficient adsorbent for acridine orange: a response surface methodological approach.

In the current investigation, watermelon rinds (WMR) have been utilized as an eco-friendly and cost-efficient adsorbent for acridine orange (AO) from contaminated water samples. Adsorption of AO onto raw (RWM) and thermally treated rinds (TTWM250 and TTWM500) has been studied. The adsorption efficiency of the three adsorbents was evaluated by measuring the % removal (%R) of AO and the adsorption capacity (qe, mg/g). Dependent variables (%R and qe) were optimized as a function of four factors: pH, sorbent dosage (AD), the concentration of AO (DC), and contact time (ST). Box-Behnken (BB) design has been utilized to obtain the optimum adsorption conditions. Prepared adsorbents have been characterized using scanning electron microscopy (SEM), Fourier-transform infrared (FT-IR), and Raman spectroscopies. The surface area of RWM, TTWM250, and TTWM500, as per the Brunauer-Emmett-Teller (BET) analysis, was 2.66, 2.93, and 5.03 m2/g, respectively. Equilibrium investigations suggest that Freundlich model was perfectly fit for adsorption of AO onto TTWM500. Maximum adsorption capacity (qmax) of 69.44 mg/g was obtained using the Langmuir equation. Adsorption kinetics could be best described by the pseudo-second-order (PSO) model. The multi-cycle sorption-desorption study showed that TTWM500 could be regenerated with the adsorption efficiency being preserved up to 87% after six cycles.

The use of acridine orange and glutaraldehyde-fixed chicken red blood cells for absolute counting of residual white blood cells in leuco-depleted packed red blood cells.

BACKGROUND: We have previously developed an affordable flow cytometric method for absolute cell count using glutaraldehyde-fixed chicken red blood cells. However, its use is limited to CD4+ T cells. In the current investigation, we studied the potential use of glutaraldehyde-fixed chicken RBCs to determine the number of residual white blood cells (rWBCs) in WBC-reduced blood component. METHODS: Acridine orange (AO) was used to identify leucocytes in serial diluted blood samples ranging from 0.65 to 1,000 cells/microL. The absolute number of AO stained leucocytes were determined by using a known number of glutaraldehyde-fixed chicken RBCs on flow cytometer. The results were compared with the expected value and the absolute count determined by BD Leucocount (Becton Dickinson Bioscience). In addition, the stability of AO stained leucocytes and sample stability at various time points were measured. Reproducibility of the assay method was also addressed. RESULTS: There was a good correlation in the number of leucocytes between our new method and the expected numbers from serially diluted blood samples (r2 = 0.99, y = 1.04x + 0.50, p < 0.001). Furthermore, absolute leucocyte counts determined by the new method correlated well with those obtained from BD Leucocount (r2 = 0.99, y = 1.31 x - 6.37, p < 0.001). FL-1 intensity and the absolute number of AO stained leucocytes were stable for at least 24 hours after staining. Samples stored at 4 degrees C were stable for 48 hours and CV of the assay was at an acceptable level. CONCLUSION: This flow cytometric method for absolute leucocyte counts using AO and glutaraldehyde-fixed chicken RBCs is a simple, rapid, reliable and inexpensive method for routine monitoring of low levels of leucocytes in blood products.

Environmentally relevant concentrations of F-53B induce eye development disorders-mediated locomotor behavior in zebrafish larvae.

The perfluorooctane sulfonate alternative, F-53B, induces multiple physiological defects but whether it can disrupt eye development is unknown. We exposed zebrafish to F-53B at four different concentrations (0, 0.15, 1.5, and 15 µg/L) for 120 h post-fertilization (hpf). Locomotor behavior, neurotransmitters content, histopathological alterations, morphological changes, cell apoptosis, and retinoic acid signaling were studied. Histology and morphological analyses showed that F-53B induced pathological changes in lens and retina of larvae and eye size were significantly reduced as compared to control. Acridine orange (AO) staining revealed a dose-dependent increase in early apoptosis, accompanied by upregulation of p53, casp-9 and casp-3 genes. Genes related to retinoic acid signaling (aldh1a2), lens developmental (cryaa, crybb, crygn, and mipa) and retinal development (pax6, rx1, gant1, rho, opn1sw and opn1lw) were significantly downregulated. In addition, behavioral responses (swimming speed) were significantly increased, while no significant changes in the neurotransmitters (dopamine and acetylcholine) level were observed. Therefore, in this study we observed that exposure to F-53B inflicted histological and morphological changes in zebrafish larvae eye, induced visual motor dysfunctions, perturbed retinoid signaling and retinal development and ultimately triggering apoptosis.

NPC-16, a novel naphthalimide-polyamine conjugate, induced apoptosis and autophagy in human hepatoma HepG2 cells and Bel-7402 cells.

The antitumor effects and molecular mechanism of NPC-16, a novel naphthalimide-polyamine conjugate, were evaluated in HepG2 cells and Bel-7402 cells. Apoptosis and necrosis were evaluated by Annexin V-FITC detection kit, and autophagy by acridine orange and Lyso-Tracker Red staining. The change of mitochondrial transmembrane potential was measured using rhodamine 123 staining. The protein expression of Beclin 1, LC3 II and mTOR, p70S6 K, 14-3-3, caspase, and Bcl-2 family members was detected by immunofluorescence assays and Western Blot. Here, we elucidated the nature of cellular response of HepG2 cells and Bel-7402 cells to NPC-16 at IC(50). NPC-16 induced caspase-dependent apoptosis via the mitochondrial pathway and death receptor pathway in Bel-7402 cells. Differently, NPC-16 triggered HepG2 cells both apoptosis and autophagy, further autophagy facilitated cellular apoptosis. Furthermore, mTOR signal pathway was involved in NPC-16-mediated autophagy in HepG2 cells. Thus, NPC-16 may be useful as a potential template for investigation the molecular mechanism of naphthalimide-polyamine conjugate against hepatocellular carcinoma.

[Regulatory volume increase (RVI) and apoptotic volume decrease (AVD) in U937 cells placed into hyperosmotic medium].

Time course of changes in intracellular water, K+ and Na+ of U937 cells incubated in hyperosmolar medium with addition of 200 mM sucrose was studied. Ouabain-sensitive and ouabain-resistant Rb+ (K+) influxes were measured during regulatory cell volume increase (RVI) and apoptotic volume decrease (AVD). Microscopy of cells stained by Acrydine orange, Ethydium bromide, APOPercenrage Dye and polycaspase marker FLICA was performed. We found that initial osmotic cell shrinkage induced both RVI and AVD responses. RVI dominated at the early stage whereas AVD prevailed at the later stage. In view of the data obtained in U937 cells the current opinion that RVI "dysfunction" is a prerequisite for apoptosis and AVD (Subramanyam et al., 2010) should be revised. U937 cells are capable to trigger of apoptosis and AVD in spite of the unimpaired RVI response. It is concluded that AVD plays a significant role in preventing osmotic lysis of apoptotic cells rather than in the initiation of apoptosis.

[Fluorescence of acridine orange in the composition of blood and tissues].

The interaction of acridine orange with blood albumins and tissue cells from different organs of white mouse has been studied by the spectral luminescence method. It was shown that acridine orange, by penetrating cells or orangelles, is able to intercalate between base pairs in the DNA molecule. It was found that the application of acridine orange as a fluorescent probe can influence the metabolic activity of organs.

Microscopical Detection of Cell Death Processes During Scots Pine Zygotic Embryogenesis.

Programmed cell death (PCD) processes are essential in the plant embryogenesis. To understand how PCD operates in a developing seed, the dying cells need to be identified in relation to their surviving neighbors. This can be accomplished by the means of in situ visualization of fragmented DNA-a well-known hallmark of PCD. In the developing Scots pine (Pinus sylvestris L.) seed, several tissues die via morphologically different PCD processes during the embryogenesis. Here, we describe the protocols for the characterization of Scots pine seeds at the early and late developmental stages and, further, the localization of nucleic acids and DNA fragmentation by the acridine orange staining and TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling) assay in the dying seed tissues.

Haemocyte lysosomal fragility facing an environmental reality: a toxicological perspective with atrazine and Lymnaea stagnalis (Gastropoda, Pulmonata) as a test case.

The present study investigates the potential use of haemocyte lysosomal fragility as a reliable indicator of toxic injury in snails subjected to herbicide atrazine. Two cytochemical assays were used. The first one was based on microscopic observations of neutral red dye, and the second one on a fluorescence spectroscopy approach using acridine orange, which had the advantage of being more reliable in assessing minor cell damage. Two sets of experiments were carried out in controlled laboratory conditions (temperature, photoperiod) and in outdoor microcosms, which produce more realistic ecological conditions. An unequivocal relationship was shown, linking atrazine exposure to Lymnaea stagnalis haemocytes lysosomal permeability. High temperatures also had a negative effect on the lysosomal membrane. In the microcosms, the response was affected by seasonal factors, and led to an integrated cell response. It was concluded that lysosomal sensitivity reflects environmental changes. Moreover, the technique using acridine orange was shown to be sufficiently sensitive and reliable to serve as a lysosomal marker in a controlled environment.

[AO distribution and fluorescence spectra in myoblasts and single muscle fibres].

Using spectral scanning regime of Leica TCS SL confocal microscope, acridine orange (AO) fluorescence spectra in nuclei and cytoplasms of living myoblasts L6J1 and frog single muscle fibres have been studied. AO fluorescence spectra in salt solutions dependent on free AO concentrations and in AO complexes with DNA have also been obtained for comparison. Myoblasts nuclei fluoresced in green spectral region with maximum at approximately 530 nm (corresponding AO monomers fluorescence), nucleoli fluoresced most brightly. Nuclear chromatin fluoresced not uniformly in these cells. We saw similar to myoblasts AO emission in nucleoli and nuclei of frog single muscle fibres. The uniformed weak green fluorescence was observed for myoblast cytoplasm. As to the muscle fibres sarcoplasm, we saw also AO green fluorescence in A-discs. In myoblasts and muscle fibre cytoplasm we saw the fluorescent red, yellow and green granules which were acidic organelles. The comparison of AO fluorescence spectra in living cells with fluorescence spectra of different AO concentrations and complexes of AO with DNA in buffer solutions allows estimation of AO concentration in acidic granules which is of interest in the investigation of cellular organelles functions in the processes of intracellular transport, adaptation, apoptosis and a number of pathological conditions.

[Neutrophil extracellular traps: method of detection and assessment of bacterial trapping efficacy].

AIM: To develop express method for detection of neutrophil extracellular traps represented by extracellular strands of neutrophils' DNA. MATERIALS AND METHODS: Acridine orange staining was used for visualization of neutrophil exracellular traps, which allows to quickly reveal the forming structures and to quantify this phenomenon. RESULTS: Performed studies showed that after 30-min of in vitro interaction of Escherichia coli, Staphylococcus aureus, lactobacteria, bifidobacteria and latex particles with neutrophils, the latter produces extracellular net-like structures, which were well visualized after staining of fixed preparations with acridine orange. Such structures are able to trap latex particles and bacteria more efficiently than live cell. Additionally, representatives of normal flora (bifidobacteria) actively stimulate production of traps. CONCLUSION: Production of neutrophil extracellular traps could be one of the main and effective mechanisms in antimicrobial defense of mucosal surfaces.

High-throughput quantitative detection of basal autophagy and autophagic flux using image cytometry.

Quantitative assessment of changes in macro-autophagy is often performed through manual quantification of the number of LC3B foci in immunofluorescence microscopy images. This method is highly laborious, subject to image-field selection and foci-counting bias, and is not sensitive for analyzing changes in basal autophagy. Alternative methods such as flow cytometry and transmission electron microscopy require highly specialized, expensive instruments and time-consuming sample preparation. Immunoblots are prone to exposure-related variations and noise that prevents accurate quantification. We report a high-throughput, inexpensive, reliable and objective method for studying basal level and flux changes in late-stage autophagy using image cytometry and acridine orange staining.

[Spectrophotometric determination of deoxyribonucleic acid by its quenching effect on acridine orange].

Based on the reaction of acridine orange with deoxyribonucleic acid (DNA), and under the optimized condition, a novel method to determine the DNA was developed with the spectrophotometry. The absorbance of acridine orange at the maximum absorption wavelength of 444 nm responded to the concentration of DNA negatively with excellent linearity. It has an upper linear limit concentration of 8.0 microg x mL(-1) and a detection limit of 0.12 microg x mL(-1) with the correlation coefficient of 0.999 8. The method is simple, rapid and sensitive, and could be applied to sample assay satisfactorily. Finally, the reaction mechanism is discussed.

Fluorescent reversible regulation based on photoinduced electron transfer from DNA to quantum dots and intercalation binding of DNA intercalator to DNA.

Based on the fluorescent reversible regulation, a novel sensor platform was designed for the detection of DNA intercalators utilizing the intercalation binding of DNA intercalators to DNA as an inherent exhibition and the fluorescence change of quantum dots (QDs) as an external manifestation. To prove its feasibility, acridine orange (AO) was chosen as an example of DNA intercalator. When different concentrations of herring sperm DNA (hsDNA) were added to cysteamine (CA)-capped ZnSe QDs solution, the hsDNA bound with the QDs through electrostatic interaction due to the photoinduced electron transfer from hsDNA to QDs and formed QDs-hsDNA complexes with 1:1 ratio, leading to the fluorescence quenching of the QDs; and upon addition of different concentrations of AO to the QDs-hsDNA complex system, the AO first caused the release of the hsDNA from the complexes and concomitantly bound with them through intercalation binding and formed AO-hsDNA complexes with 1:3 ratio on account of the fact that the intercalation binding constant between AO and hsDNA (1.932 × 105 L/mol) was greater than the electrostatic interaction constant between QDs and hsDNA (7.874 × 104 L/mol), resulting in the fluorescence recovery of the QDs. Therefore, the detection of AO could be achieved through the relationship between the fluorescence recovery yield of the QDs and the concentration of AO added. The results illustrated that the fluorescence recovery yield of the QDs-hsDNA system was linearly dependent to the concentration of AO in the range of 5.0-75.0 × 10-5 mol/L with a detection limit (3σ/K) of 1.5 × 10-5 mol/L. This dual-directional fluorescent regulation provided a novel method for the detection of DNA intercalators such as polycyclic aromatic hydrocarbons and drugs interfering with DNA-synthesis and possessed some potential applications in the investigation of the interactions between DNA intercalators and DNA.

Flow Cytometry and Fluorescence Microscopy as Tools for Structural and Functional Analysis of Vacuoles Isolated from Yeast and Plant Cells.

A series of optimized protocols to isolate vacuoles from both yeast and plant cells, and to characterize the purified organelles at a functional and structural level, are described. For this purpose, we took advantage of the combined use of cell fractionation techniques with different fluorescence-based approaches namely flow cytometry, fluorescence microscopy and spectrofluorimetry. These protocols altogether constitute valuable tools for the study of vacuole structure and function, as well as for the high-throughput screening of drug libraries to identify new molecules that target the vacuole.