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Molecules (Basel, Switzerland) Feb 2023In this article, we used molecular dynamics (MD), one of the most common methods for simulations of membranes, to study the interaction of fluorescent membranotropic...
Insights into the Formation of Intermolecular Complexes of Fluorescent Probe 10--Nonyl Acridine Orange with Cardiolipin and Phosphatidylglycerol in Bacterial Plasma Membrane by Molecular Modeling.
In this article, we used molecular dynamics (MD), one of the most common methods for simulations of membranes, to study the interaction of fluorescent membranotropic biological probe 10--nonyl acridine orange (NAO) with the bilayer, mimicking a plasma membrane of Gram-negative bacteria. Fluorescent probes serve as an effective tool to study the localization of different components in biological membranes. Revealing the molecular details of their interaction with membrane phospholipids is important both for the interpretation of experimental results and future design of lipid-specific stains. By means of coarse-grained (CG) MD, we studied the interactions of NAO with a model membrane, imitating the plasma membrane of Gram-negative bacteria. In our simulations, we detected different NAO forms: monomers, dimers, and stacks. NAO dimers had the central cardiolipin (CL) molecule in a sandwich-like structure. The stacks were formed by NAO molecules interlayered with anionic lipids, predominantly CL. Use of the CG approach allowed to confirm the ability of NAO to bind to both major negatively charged phospholipids, phosphatidylglycerol (PG) and CL, and to shed light on the exact structure of previously proposed NAO-lipid complexes. Thus, CG modeling can be useful for the development of new effective and highly specific molecular probes.
Topics: Cardiolipins; Fluorescent Dyes; Acridine Orange; Phosphatidylglycerols; Cell Membrane; Phospholipids; Bacteria
PubMed: 36838917
DOI: 10.3390/molecules28041929 -
Genetics and Molecular Research : GMR Aug 2013Apoptosis and necrosis are among several types of cell death. We stained the nuclei of Aspergillus nidulans grown in micro-colonies with ethidium bromide and acridine...
Apoptosis and necrosis are among several types of cell death. We stained the nuclei of Aspergillus nidulans grown in micro-colonies with ethidium bromide and acridine orange to detect in situ apoptosis. Suspensions of conidia from 5-day-old colonies of the A. nidulans strains biA1methG1, G422, CLC100, and CLB3 were each put into two tubes. The suspension of one tube was irradiated with ultraviolet light for 20 s, whereas the other tube was not exposed to irradiation. The two suspensions were inoculated in complete liquid medium and 50-µL samples were placed on sterilized cover slips, spread on the surface of solid culture media on Petri dishes. After the micro-colonies were formed, the material on the cover slips was stained with ethidium bromide and acridine orange, placed on the lamina and observed under a fluorescence microscope. This staining method was efficient in discriminating normal nuclei from those going apoptosis and necrosis. Results have shown that irradiation provokes apoptosis but does not induce necrosis. There were no differences between the three strains and all data were considered to be statistically significant.
Topics: Acridine Orange; Apoptosis; Aspergillus nidulans; Cell Survival; Ethidium; Fluorescent Dyes; In Situ Hybridization, Fluorescence; Microscopy, Fluorescence; Ultraviolet Rays
PubMed: 24065645
DOI: 10.4238/2013.August.12.5 -
Journal of Clinical Microbiology Aug 1981Acridine orange, a fluorochrome strain, is potentially superior to the Gram stain in the direct microscopic examination of clinical specimens because it gives striking... (Comparative Study)
Comparative Study
Acridine orange, a fluorochrome strain, is potentially superior to the Gram stain in the direct microscopic examination of clinical specimens because it gives striking differential staining between bacteria and background cells and debris. Its value in clinical laboratories was evaluated by testing 209 cerebrospinal fluids and 288 other body fluids, tissues, and exudates by both techniques. Smears were made in duplicate, fixed with methanol, stained, and examined without knowledge of the result of the companion smear or culture. Overall, acridine orange was slightly more sensitive than the Gram stain (acridine orange, 59.9%; Gram stain, 55.8%) and equally specific in detecting microorganisms. One smear was falsely positive by the Gram stain; none was falsely positive by the acridine orange stain. We conclude that acridine orange staining is a sensitive method for screening clinical specimens and reviewing selected specimens that are purulent, but negative by the Gram stain. Bloody fluids, thick exudates, and other normally difficult-to-read specimens were easily and quickly examined. We recommend, however, that positive smears be reexamined with the Gram stain to confirm the result and determine the Gram reaction of the microorganisms.
Topics: Acridine Orange; Bacteria; Body Fluids; Cerebrospinal Fluid; Evaluation Studies as Topic; Exudates and Transudates; False Positive Reactions; Haemophilus influenzae; Humans; Staining and Labeling
PubMed: 6168652
DOI: 10.1128/jcm.14.2.201-205.1981 -
Scientific Reports Nov 2018Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells...
Automated blood cell counters can distinguish cells based on their size and the presence or absence of a nucleus. However, most vertebrates have nucleated blood cells that cannot be counted automatically. We established an alternative automatic method for counting peripheral blood cells by staining cells with the fluorescent dye acridine orange (AO) and analysing cell populations using flow cytometry (FCM). As promising new animal models, we chose Xenopus laevis and three inbred strains of X. tropicalis. We compared the haematological phenotypes, including blood cell types, cell sizes, cellular structure, and erythrocyte lifespans/turnover rate among X. laevis and the three inbred strains of X. tropicalis. Each cell type from X. laevis was sorted according to six parameters: forward- and side-scattered light emission, AO red and green fluorescence intensity, and cellular red and green fluorescence. Remarkably, the erythrocyte count was the highest in the Golden line, suggesting that genetic factors were associated with the blood cells. Furthermore, immature erythrocytes in anaemic X. laevis could be separated from normal blood cells based on red fluorescence intensity. These results show that FCM with AO staining allows for an accurate analysis of peripheral blood cells from various species.
Topics: Acridine Orange; Animals; Animals, Inbred Strains; Animals, Wild; Blood Cell Count; Blood Cells; Cell Separation; Flow Cytometry; Fluorescent Dyes; Models, Animal; Species Specificity; Staining and Labeling; Xenopus laevis
PubMed: 30390005
DOI: 10.1038/s41598-018-34631-0 -
International Journal of Pharmaceutics Aug 2020Manufacturing of liposomal nanomedicines (e.g. Doxil®/Caelyx®) is a challenging and slow process based on multiple-vessel and batch processing techniques. As a result,... (Comparative Study)
Comparative Study
Manufacturing of liposomal nanomedicines (e.g. Doxil®/Caelyx®) is a challenging and slow process based on multiple-vessel and batch processing techniques. As a result, the translation of these nanomedicines from bench to bedside has been limited. Microfluidic-based manufacturing offers the opportunity to address this issue, and de-risk the wider adoption of nanomedicines. Here we demonstrate the applicability of microfluidics for continuous manufacturing of PEGylated liposomes encapsulating ammonium sulfate (250 mM). Doxorubicin was subsequently active-loaded into these pre-formed liposomes. Critical process parameters and material considerations demonstrated to influence the liposomal product attributes included solvent selection and lipid concentration, flow rate ratio, and temperature and duration used for drug loading. However, the total flow rate did not affect the liposome product characteristics, allowing high production speeds to be adopted. The final liposomal product comprised of 80-100 nm vesicles (PDI < 0.2) encapsulating ≥ 90% doxorubicin, with matching release profiles to the innovator product and is stable for at least 6 months. Additionally, vincristine and acridine orange were active-loaded into these PEGylated liposomes (≥ 90% and ~100 nm in size) using the same process. These results demonstrate the ability to produce active-loaded PEGylated liposomes with high encapsulation efficiencies and particle sizes which support tumour targeting.
Topics: Acridine Orange; Ammonium Sulfate; Antibiotics, Antineoplastic; Doxorubicin; Drug Liberation; Drug Stability; Drug Storage; Lipids; Liposomes; Microfluidics; Nanoparticles; Particle Size; Polyethylene Glycols; Solvents; Vincristine
PubMed: 32622812
DOI: 10.1016/j.ijpharm.2020.119566 -
Memorias Do Instituto Oswaldo Cruz 1992Accurate diagnosis of Babesia bigemina infection, an economically important tick-transmitted protozoan parasite of cattle, is essential in the management of disease... (Review)
Review
Accurate diagnosis of Babesia bigemina infection, an economically important tick-transmitted protozoan parasite of cattle, is essential in the management of disease control and in epidemiological studies. The currently used methods of diagnosis are blood smear examination and serological tests which include agglutination and immunofluorescence tests. These tests have been used in the fild but because they lack sensitivity and specificity, newer and improved methods of diagnosis are being developed. The quantitative buffy coat (QBC) method, using microhaematocrit tubes and acridine orange staining allows rapid and quicker diagnosis of B. bigemina and other blood parasites compared to light microscopic examination of stained smears. Parasite specific monoclonal antibodies have been used in antigen/antibody capture enzymelinked immunosorbent assays with greater sensitivity and specificity than previously described serological tests. Similarly, DNA probes, derived from a repetitive sequence of the B. bigemina genome, offer a method of detecting very small numbers of parasites which are undetectable by conventional microscopy. An extrachromosomal DNA element, present in all the tick-borne protozoan parasites so far tested, provides an accurate means of differentiating mixed parasite populations in infected animals. These improved methods will greatly facilitate epidemiological studies.
Topics: Acridine Orange; Animals; Antibodies, Protozoan; Antigens, Protozoan; Babesia; Babesiosis; Cattle; Cattle Diseases; DNA, Protozoan; Enzyme-Linked Immunosorbent Assay; Extrachromosomal Inheritance; Fluorescent Dyes; Microscopy, Fluorescence; Repetitive Sequences, Nucleic Acid; Sensitivity and Specificity; Serology
PubMed: 1343691
DOI: 10.1590/s0074-02761992000700033 -
Indian Journal of Medical Microbiology 2010The purpose of this study was to ascertain if the simple practice of Gram stain, acridine orange stain and direct sensitivity determination of positive blood culture... (Comparative Study)
Comparative Study
PURPOSE
The purpose of this study was to ascertain if the simple practice of Gram stain, acridine orange stain and direct sensitivity determination of positive blood culture bottles could be used to guide early and appropriate treatment in trauma patients with clinical suspicion of sepsis. The study also aimed to evaluate the error in interpreting antimicrobial sensitivity by direct method when compared to standard method and find out if specific antibiotic-organism combination had more discrepancies. Findings from consecutive episodes of blood stream infection at an Apex Trauma centre over a 12-month period are summarized.
MATERIALS AND METHODS
A total of 509 consecutive positive blood cultures were subjected to Gram staining. AO staining was done in BacT/ALERT-positive Gram-stain negative blood cultures. Direct sensitivity was performed from 369 blood culture broths, showing single type of growth in Gram and acridine orange staining. Results of direct sensitivity were compared to conventional sensitivity for errors.
RESULTS
No 'very major' discrepancy was found in this study. About 5.2 and 1.8% minor error rates were noted in gram-positive and gram-negative bacteria, respectively, while comparing the two methods. Most of the discrepancies in gram-negative bacteria were noted in beta lactam - beta lactamase inhibitor combinations. Direct sensitivity testing was not reliable for reporting of methicillin and vancomycin resistance in Staphylococci.
CONCLUSIONS
Gram stain result together with direct sensitivity testing is required for optimizing initial antimicrobial therapy in trauma patients with clinical suspicion of sepsis. Gram staining and AO staining proved particularly helpful in the early detection of candidaemia.
Topics: Acridine Orange; Anti-Bacterial Agents; Bacteremia; Bacteria; Blood; Coloring Agents; Diagnostic Errors; Gentian Violet; Humans; Microbial Sensitivity Tests; Phenazines; Sensitivity and Specificity; Staining and Labeling; Wounds and Injuries
PubMed: 20404460
DOI: 10.4103/0255-0857.62491 -
Clinical and Experimental Immunology Jan 1980Fluorescent probes can monitor events in lymphocytes stimulated by mitogens and antigens. Early activation is associated with conformational changes in membrane... (Review)
Review
Fluorescent probes can monitor events in lymphocytes stimulated by mitogens and antigens. Early activation is associated with conformational changes in membrane macromolecules, and has been studied by measurement of fluorescence intensity or polarization of the membrane-localizing probes ANS, NPN, DPH and TMRITC. Subsequent changes in cytoplasmic macromolecules have been detected by altered fluorescence polarization of intracellular fluorescein. Altered metabolic activity in the activated lymphocyte is also revealed by fluorescent probes: the increased red fluorescence of lysosomes seen by AO staining, is attributable to altered lysosome membrane permeability. AO fluorescence has also detected early changes in the nuclear nucleoprotein complex. The later synthesis of new DNA is readily demonstrated by increased staining with the nuclear probes AO, ethidium bromide, propidium iodide, mithramycin and the Hoechst dyes. Adaptation of fluorescent probe analyses to the now rapidly developing flow microfluorimeters is providing rapid and sensitive assays of lymphocyte stimulation. Such methods will permit routine detection of lymphocyte response to particular antigens or mitogens, as well as identification of antigenic substances by their stimulation of known reactive lymphocytes. Last but not least, fluorescent probes are providing new understanding of the cellular events and regulatory mechanisms associated with lymphocyte activation.
Topics: Acridine Orange; Anilino Naphthalenesulfonates; Animals; Cell Membrane; Cytoplasm; DNA; Diphenylhexatriene; Fluoresceins; Fluorescence Polarization; Fluorescent Dyes; Humans; In Vitro Techniques; Lymphocyte Activation; Membrane Fluidity; Microscopy, Fluorescence; Molecular Conformation; RNA
PubMed: 6156040
DOI: No ID Found -
International Journal of Molecular... Mar 2023Photodynamic therapy is a minimally invasive procedure used in the treatment of several diseases, including some types of cancer. It is based on photosensitizer...
Photodynamic therapy is a minimally invasive procedure used in the treatment of several diseases, including some types of cancer. It is based on photosensitizer molecules, which, in the presence of oxygen and light, lead to the formation of reactive oxygen species (ROS) and consequent cell death. The selection of the photosensitizer molecule is important for the therapy efficiency; therefore, many molecules such as dyes, natural products and metallic complexes have been investigated regarding their photosensitizing potential. In this work, the phototoxic potential of the DNA-intercalating molecules-the dyes methylene blue (MB), acridine orange (AO) and gentian violet (GV); the natural products curcumin (CUR), quercetin (QT) and epigallocatechin gallate (EGCG); and the chelating compounds neocuproine (NEO), 1,10-phenanthroline (PHE) and 2,2'-bipyridyl (BIPY)-were analyzed. The cytotoxicity of these chemicals was tested in vitro in non-cancer keratinocytes (HaCaT) and squamous cell carcinoma (MET1) cell lines. A phototoxicity assay and the detection of intracellular ROS were performed in MET1 cells. Results revealed that the IC values of the dyes and curcumin in MET1 cells were lower than 30 µM, while the values for the natural products QT and EGCG and the chelating agents BIPY and PHE were higher than 100 µM. The IC of MB and AO was greatly affected by irradiation when submitted to 640 nm and 457 nm light sources, respectively. ROS detection was more evident for cells treated with AO at low concentrations. In studies with the melanoma cell line WM983b, cells were more resistant to MB and AO and presented slightly higher IC values, in line with the results of the phototoxicity assays. This study reveals that many molecules can act as photosensitizers, but the effect depends on the cell line and the concentration of the chemical. Finally, significant photosensitizing activity of acridine orange at low concentrations and moderate light doses was demonstrated.
Topics: Humans; Photosensitizing Agents; Intercalating Agents; Reactive Oxygen Species; Curcumin; Acridine Orange; Cell Line, Tumor; Early Detection of Cancer; Photochemotherapy; Skin Neoplasms; Dermatitis, Phototoxic; Coloring Agents
PubMed: 36982675
DOI: 10.3390/ijms24065602 -
Cell Communication and Signaling : CCS Oct 2022Frameshift mutations in LRPAP1 are responsible for autosomal recessive high myopia in human beings but its underlying mechanism remains elusive. This study aims to...
BACKGROUND
Frameshift mutations in LRPAP1 are responsible for autosomal recessive high myopia in human beings but its underlying mechanism remains elusive. This study aims to investigate the effect of LRPAP1 defect on ocular refractive development and its involved mechanism.
METHODS
A lrpap1 mutant zebrafish line with homozygous frameshift mutation was generated by CRISPR/Cas9 technology and confirmed by Sanger sequencing. The ocular refractive phenotype was analyzed by calculating the relative refractive error (RRE) with vivo photography and histological analysis at different development stages, together with examining ocular structure change via transmission electron microscopy. Further, RNA sequencing and bioinformatics analysis were performed. The potentially involved signaling pathway as well as the interacted protein were investigated in vivo.
RESULTS
The lrpap1 homozygous mutant zebrafish line showed myopic phenotype. Specifically, the mutant lines showed larger eye axial length-to-body length in one-month old individuals and a myopic shift with an RRE that changed after two months. Collagen fibers became thinning and disordered in the sclera. Further, RNA sequencing and bioinformatics analysis indicated that apoptosis signaling was activated in mutant line; this was further confirmed by acridine orange and TUNEL staining. Moreover, the expression of TGF-β protein was elevated in the mutant lines. Finally, the treatment of wild-type embryos with a TGF-β agonist aggravated the degree of eyeball apoptosis; conversely, the use of a TGF-β inhibitor mitigated apoptosis in mutant embryos.
CONCLUSION
The study provides functional evidence of a link between lrpap1 and myopia, suggesting that lrpap1 deficiency could lead to myopia through TGF-β-induced apoptosis signaling. Video abstract.
Topics: Animals; Humans; Acridine Orange; Apoptosis; Collagen; Myopia; Sclera; Transforming Growth Factor beta; Zebrafish; LDL-Receptor Related Protein-Associated Protein; Zebrafish Proteins
PubMed: 36261846
DOI: 10.1186/s12964-022-00970-9