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Methods in Molecular Biology (Clifton,... 2024Acridine orange is a nucleic acid binding dye that emits green fluorescence when bound to double-stranded DNA or RNA and red fluorescence when bound to single-stranded...
Acridine orange is a nucleic acid binding dye that emits green fluorescence when bound to double-stranded DNA or RNA and red fluorescence when bound to single-stranded DNA or RNA under ultraviolet light. This unique characterization allows it to be used for distinguishing or visualization of dsRNA. Here, we present a convenient and efficient protocol for detecting dsRNA in polyacrylamide gels.
Topics: Acridine Orange; RNA, Double-Stranded; Staining and Labeling; DNA, Single-Stranded; Ultraviolet Rays
PubMed: 38285384
DOI: 10.1007/978-1-0716-3702-9_2 -
Animal Reproduction Science Mar 2023Although previous studies have examined the relationship between the sperm DNA fragmentation index and fertility in stallions, other aspects of chromatin structure or...
Although previous studies have examined the relationship between the sperm DNA fragmentation index and fertility in stallions, other aspects of chromatin structure or packaging and fertility have not been explored. In the present study, relationships between fertility and DNA fragmentation index, protamine deficiency, total thiols, free thiols and disulfide bonds in stallion spermatozoa were investigated. Ejaculates (n = 36) were collected from 12 stallions and extended to prepare semen doses for insemination. One dose from each ejaculate was sent to the Swedish University of Agricultural Sciences. Aliquots of semen were stained for flow cytometry with acridine orange for the Sperm Chromatin Structure Assay (DNA fragmentation Index, %DFI), with chromomycin A3 (CMA) for protamine deficiency, and with monobromobimane (mBBr) for detection of total and free thiols and disulfide bonds. Per season pregnancy rates after insemination were obtained. Mixed linear models were used to analyze data. Negative correlations were found between pregnancy rate and %DFI (r = -0.35, P < 0.03) and pregnancy rate and free thiols (r = -0.60, P < 0.0001). Furthermore, there were positive correlations between total thiols and disulfide bonds (r = 0.95, P < 0.0001), and protamine and disulfide bonds (r = 0.4100, P < 0.01986). Since chromatin integrity, protamine deficiency and packaging were all associated with fertility, a combination of these factors could be used as a biomarker of fertility when assessing ejaculates.
Topics: Pregnancy; Female; Male; Animals; Horses; Chromatin; Semen; Biofilms; DNA Fragmentation; Bioreactors; Fertility; Spermatozoa; Protamines; Sulfhydryl Compounds; Disulfides
PubMed: 36801727
DOI: 10.1016/j.anireprosci.2023.107200 -
AAPS PharmSciTech Mar 2022The aim of this study was to fabrication PEGylated lecithin-chitosan nanoparticles (PLC-NPs) as alphα-Terpineol's (αT-PLC-NPs) delivery system and examine its...
The aim of this study was to fabrication PEGylated lecithin-chitosan nanoparticles (PLC-NPs) as alphα-Terpineol's (αT-PLC-NPs) delivery system and examine its anti-cancer effects. αT-PLC-NPs were synthesized by self-assembling method; after characterization, entrapment efficiency of α-T was measured by HPLC procedure. MTT test was conducted for cytotoxicity evaluation. Chick chorioallantoic membrane (CAM) and quantitative polymerase chain reaction (qPCR) analysis were used to determine the angiogenesis properties, and qPCR, flow cytometry, and acridine orange and propidium iodide (AO/PI) staining were used to evaluate the pro-apoptotic effects of αT-PLC-NPs. Finally, the anti-inflammatory and antibacterial activity of the αT-PLC-NPs was also evaluated. αT-PLC-NPs with a size of 220.8 nm, polydispersity index (PDI) of 0.3, zeta potential of +29.03 mV, and encapsulation efficiency of 82% showed higher inhibitory effect on MCF7 cells (IC: 750 μg/mL) compared to HFF cells (above 1000 μg/mL). Decreased angiogenesis indices and embryonic growth factors in CAM assay, decreased expression of VEGF and VEGF-R genes, and decreased cell migration showed the inhibitory effect of αT-PLC-NPs on angiogenesis. Increased expression of P53, P21, and caspase9 genes, as well as the results of AO/PI staining along with increasing the number of SubG1 phase cells in flow cytometry, confirmed the pro-apoptotic effects of αT-PLC-NPs. Also, its anti-inflammatory effects were demonstrated by inhibiting the expression of pro-inflammatory cytokines (TNF-α and IL-6). The inhibitory power of αT-PLC-NPs in suppressing gram-positive and negative bacterial strains was demonstrated by disk diffusion (DD), minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) methods. PLC-NPs are a promising carrier for α-T transfer for preclinical studies.
Topics: Chitosan; Humans; Lecithins; MCF-7 Cells; Nanoparticles; Polyethylene Glycols
PubMed: 35314914
DOI: 10.1208/s12249-022-02245-5 -
Methods and Protocols Aug 2023Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of...
Loss of lysosomal membrane integrity results in leakage of lysosomal hydrolases to the cytosol which might harm cell function and induce cell death. Destabilization of lysosomes often precede apoptotic or necrotic cell death and occur during both physiological and pathological conditions. The weak base acridine orange readily enters cells and accumulates in the acidic environment of lysosomes. Vital staining with acridine orange is a well-proven technique to observe lysosomal destabilization using fluorescence microscopy and flow cytometry. These analyses are, however, time consuming and only adapted for discrete time points, which make them unsuitable for large-scale approaches. Therefore, we have developed a time-saving, high-throughput microplate reader-based method to follow destabilization of the lysosomal membrane in real-time using acridine orange. This protocol can easily be adopted for patient samples since the number of cells per sample is low and the time for analysis is short.
PubMed: 37623923
DOI: 10.3390/mps6040072 -
Indian Journal of Microbiology Sep 2022This study demonstrates the therapeutic potential of indole-3-butanoyl-polyethylenimine (IBP) nanostructures formed via self-assembly in aqueous system. Dynamic light...
UNLABELLED
This study demonstrates the therapeutic potential of indole-3-butanoyl-polyethylenimine (IBP) nanostructures formed via self-assembly in aqueous system. Dynamic light scattering (DLS) analysis confirmed the formation of the nanostructures in the size range of ~ 194-331 nm. These nanostructures showed commendable antimicrobial activity against wide range of microbes including multi-drug resistant bacteria. Besides, appreciable antioxidant and anti-inflammatory activities were also observed. Results of cytotoxicity studies, performed on normal transformed human embryonic kidney (HEK 293) cells and human red blood cells (hRBCs), revealed almost non-toxic behavior of these nanostructures, however, remarkable toxicity on human breast cancer cells (MCF-7), human osteosarcoma cells (Mg63) and human liver cancer cells (HepG2) was observed. The pre-apoptotic and anti-proliferative activity of IBP nanostructures were confirmed by acridine orange/propidium iodide dual staining assay followed by confocal microscopy and scratch assay on Mg63 cells. Taken together, these results advocate the promising potential of the synthesized IBP nanostructures in the therapeutic applications.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s12088-022-01015-y.
PubMed: 35974923
DOI: 10.1007/s12088-022-01015-y -
Combinatorial Chemistry & High... 2021Electroanalytical methods are very functional to detect drugs in pharmaceuticals (tablets, syrups, suppositories, creams, and ointments) and biological samples.
BACKGROUND
Electroanalytical methods are very functional to detect drugs in pharmaceuticals (tablets, syrups, suppositories, creams, and ointments) and biological samples.
OBJECTIVE
This study is aimed to make selective, sensitive, simple, fast, and low cost electrochemical analysis of expectorant drug guaifenesin in pharmaceuticals and serum samples.
METHODS
Differential pulse adsorptive stripping voltammetric method for determination of guaifenesin on a poly(acridine orange) modified glassy carbon electrode has been developed. Glassy carbon electrode was modified with electropolymerization of the acridine orange monomer for the sensitive determination of guaifenesin. Guaifenesin provided highly reproducible and welldefined irreversible oxidation peaks at +1.125 V and +1.128 V (vs. Ag/AgCl) in the selected supporting electrolyte and human serum samples, respectively.
RESULTS
Under optimized conditions, linear response of peak current on the concentration of guaifenesin has been obtained in the ranges of 2.00×10-7 to 1.00×10-4 M in Britton Robinson buffer solution at pH 7.0 and 4.00×10-7 to 1.00×10-4 M in serum samples. The precision of the method was detected by intraday and inter-day repeatability studies in the supporting electrolyte and serum samples media.
CONCLUSION
The analytical applicability of the proposed method exhibited satisfying determination results for guaifenesin from pharmaceutical dosage forms (syrup) and human serum samples without any pre-separation procedures.
Topics: Acridine Orange; Carbon; Drug Compounding; Electrochemical Techniques; Electrodes; Guaifenesin; Healthy Volunteers; Humans; Molecular Structure; Pharmaceutical Preparations; Polymers
PubMed: 32646355
DOI: 10.2174/1386207323666200709170450 -
Journal of Orthodontic Science 2022To assess the chemical composition and oral biofilm formation on different types of commercially available clear orthodontic retainer materials (CORM).
AIM
To assess the chemical composition and oral biofilm formation on different types of commercially available clear orthodontic retainer materials (CORM).
MATERIALS AND METHODS
Four types of CORM commercially available were used (Clear advantage series I (CAS1), Clear advantage series II (CAS2), Endure (ES), and CENTRI FORM-clear rigid material (CFCRM)). Circular samples (12 mm diameter) of each CORM were prepared for (n = 40). Unstimulated saliva from twenty volunteers was collected. Fourier Transformation Infrared Spectroscopy (FTIR) was used for the evaluation of the chemical composition of CORM. For the quantitative assessment of oral biofilm formation, samples of each CORM were incubated for twenty-four hours, and crystal violet assay (CVA) was utilized. The degree of absorbance was measured using a spectrophotometer at 570 nm. For qualitative evaluation of oral formation, the samples of each CORM were incubated for 24 hours, and viable biofilm cells stained by acridine orange were examined under a fluorescent microscope.
RESULTS
FTIR findings showed that CAS2 was made of polypropylene and ES is made of polyvinyl chloride, while others were made of co-polyester. CVA results confirmed that CAS2 showed the lowest biofilm formation, which differs significantly compared to CAS1, CFCRM, and ES. No significant difference in biofilm formation was detected between CAS1, CFCRM, and ES. Viable biofilm cells staining by acridine orange showed that CAS2 demonstrated smaller microcolonies of viable biofilm cells compared with CAS1, CFCRM, and ES, which confirmed the result obtained by CVA.
CONCLUSIONS
CAS2 showed anti-microbial activities with a decrease the biofilm formation, which may be related to its chemical composition.
PubMed: 36188210
DOI: 10.4103/jos.jos_7_22 -
In Vivo (Athens, Greece) 2022Gadolinium has been reported to cause liver lobular necrosis and nephrogenic systemic fibrosis. However, its toxicity to the skin remains unknown. This study aimed to...
BACKGROUND/AIM
Gadolinium has been reported to cause liver lobular necrosis and nephrogenic systemic fibrosis. However, its toxicity to the skin remains unknown. This study aimed to investigate the effect of a high dose of gadolinium-based contrast agent gadodiamide on the human keratinocyte HaCaT cell line.
MATERIALS AND METHODS
Cell viability was assessed using MTT assay, and autophagy was assessed using acridine orange and LysoTracker Red staining. Western blotting was performed to verify the changes in Bcl2 and Bax levels.
RESULTS
The viability of HaCaT cells was significantly suppressed after gadodiamide treatment. Interestingly, gadodiamide caused autophagic vacuoles, whereas the autophagy inhibitors 3-methyladenine and chloroquine significantly alleviated autophagic cell death. Simultaneously, gadodiamide induced apoptosis, which was reduced by caspase inhibitors. Gadodiamide also inhibited Bcl-2 expression and promoted Bax expression.
CONCLUSION
Gadodiamide induced both autophagy and apoptosis in HaCaT cells. Physicians should carefully assess the gadodiamide dosage used clinically.
Topics: Apoptosis; Autophagy; Gadolinium DTPA; Humans; Keratinocytes
PubMed: 35241512
DOI: 10.21873/invivo.12743 -
Journal of Agricultural and Food... Nov 2023Sodium sulfite is a widely used preservative in the food industry. Ferroptosis has been a newly discovered form of iron-dependent oxidative cell death in recent years....
Sodium sulfite is a widely used preservative in the food industry. Ferroptosis has been a newly discovered form of iron-dependent oxidative cell death in recent years. However, the potential connection between sodium sulfite and ferroptosis has not been explored. In our study, we observed the abnormal expression of ferroptosis marker protein , suggesting that sodium sulfite caused ferroptosis . Next, our study revealed that sodium sulfite caused the overproduction of mitochondrial reactive oxygen species (mtROS) in the AML-12 cells. It is well established that reactive oxygen species (ROS) can induce lysosomal membrane permeabilization. After lysosomal membrane permeabilization occurs, the outflow of Fe in lysosomes triggers the Fenton reaction and subsequently results in the increase of intracellular ROS level, which is closely related to ferroptosis. As speculated, acridine orange (AO) staining and LysoTracker red staining showed that sodium sulfite-induced lysosomal membrane permeabilization could be alleviated by mtROS scavenger TEMPO. In addition, TEMPO, lysosomal stabilizer mannose, and lysosomal iron chelator deferoxamine (DFO) inhibited sodium sulfite-induced ferroptosis. Overall, the results showed that sodium sulfite induced lysosomal iron efflux through the mtROS-lysosomal membrane permeabilization pathway and eventually led to ferroptosis. Our study might provide a new mechanism for the hepatotoxicity of sodium sulfite and a theoretical basis for the risk assessment of sodium sulfite as a food additive.
Topics: Ferroptosis; Reactive Oxygen Species; Iron; Hepatocytes; Lysosomes
PubMed: 37871339
DOI: 10.1021/acs.jafc.3c06085 -
Journal of Equine Veterinary Science Dec 2021Flow cytometry procedures can be used for evaluation of both spermatogenic efficiency and diagnose disorders of stallion spermatogenesis. Aims of this study were to...
Flow cytometry procedures can be used for evaluation of both spermatogenic efficiency and diagnose disorders of stallion spermatogenesis. Aims of this study were to compare two testicular sample acquisition techniques (needle aspirate-N and tissue wedge-T) and results when using flow cytometry and histology procedures. Testicular cell types were stained with acridine orange, and nine regions (R2 to R10) were identified and enumerated following acquisition by either N or T. Testes were also grouped and analyzed by size and sexual maturity (Small [immature] compared with Large [mature]) and used to determine if flow cytometry procedures could be used to detect differences. For both N and T, percentages of 2n cell types were greater in the Small than Large testes, whereas percentages of 1n cell types in N were greater in the Large than Small testes (P < .05). Testicular cell types in N regions were correlated to similar T regions (r between 0.51 and 0.99; P < .05) in both groups. Flow cytometry and histology scores were correlated in both groups (r between -0.95 and 0.93, P < .05). There were small differences in number of testicular cell types from N and T. With both sample acquisition methods, there was discrimination between the Small and Large testes, therefore, evaluation of testicular cell types using flow cytometry procedures might have clinical applications. Results with comparison of flow cytometry to histology procedures indicate that flow cytometry can be applied clinically to identify changes in testicular cell types of stallions using a needle aspirate.
Topics: Animals; Flow Cytometry; Horses; Male; Spermatogenesis; Testis
PubMed: 34802628
DOI: 10.1016/j.jevs.2021.103778