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Journal of Cellular Biochemistry Sep 2022Rhein is an anthraquinone found in Rheum palmatum, used in Chinese medicine. Due to potential anticancer properties, the study assessed its effect on the lysosomal...
Rhein is an anthraquinone found in Rheum palmatum, used in Chinese medicine. Due to potential anticancer properties, the study assessed its effect on the lysosomal compartment, which indirectly influences cell death. The experiment was performed on HeLa cells by treating them with rhein at concentrations of 100-300 µM. LC3-II protein and caspase 3/7 activity, level of apoptosis, the concentration of reactive oxide species (ROS), and mitochondrial potential (Δψm) were evaluated by the cytometric method. To evaluate the permeability of the lysosomal membrane (LMP), staining with acridine orange and the assessment of activity of cathepsin D and L in the lysosomal and extralysosomal fractions were used. Cell viability was assessed by -(3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) (MTT) and neutral red (NR) assays. Changes in cells were also demonstrated at the level of electron, optical, confocal, and fluorescence microscopy. Inhibition of autophagy was done using chloroquine. Rhein-induced degradation processes were confirmed by an increase in the number of primary lysosomes, autophagosomes, and autolysosomes. At high concentrations, rhein caused the generation of ROS, which induced LMP expressed by quenching of acridine orange fluorescence. These results correlated with a reduction of lysosomes, as visualized in graphical modeling, with the decreased uptake of NR by lysosomes, and increased activity of cathepsin D and L in the extralysosomal fraction. The studies also showed an increase in the activity of caspase 3/7 and a decrease in the expression of Bcl-2 protein, indicative of rhein-stimulated apoptosis. At the same time, we demonstrated that preincubation of cells with chloroquine inhibited rhein-induced autophagy and contributed to increased cytotoxicity to HeLa cells. Rhein also induced DNA damage and led to cycle arrest in the S phase. Our results indicate that rhein, by inducing changes in the lysosomal compartment, indirectly affects apoptosis of HeLa cells and in combination with autophagy inhibitors may be an effective form of anticancer therapy.
Topics: Acridine Orange; Anthraquinones; Apoptosis; Autophagy; Caspase 3; Cathepsin D; Chloroquine; HeLa Cells; Humans; Lysosomes; Neutral Red; Oxides; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species
PubMed: 35901236
DOI: 10.1002/jcb.30311 -
Heliyon Jul 2023Chemotherapy can often cause a variety of side effects including bone marrow (BM) suppression, termed as myelosuppression. Accordingly, facile and effective management...
Chemotherapy can often cause a variety of side effects including bone marrow (BM) suppression, termed as myelosuppression. Accordingly, facile and effective management of chemotherapy-induced myelosuppression is currently a pivotal task for experimental pathologists and oncologists. Here, we chose to use activated carbon (AC) with an extensive surface area for studying its possible protective effectiveness with respect to BM in doxorubicin (DOX)-treated rats. Spherical AC with an extended surface area up to 4490 m/g was prepared for (p/o) delivery, whereas for intraperitoneal (i/p) delivery we used the powdered form of AC that was derived from the aforementioned spherical AC. During the monthly treatment of animals with AC and DOX these two components were delivered alternately (not in the same day). After treatment, BM cells were isolated from femurs of sacrificed animals, stained with acridine orange (AO) and analyzed by flow cytometry. Regardless of the route of AC delivery (p/o or i/p), apparent myeloprotection with a possible regenerative effect was observed in animals that received DOX, as evidenced by recovery of the populations of total nucleated cells (TNC) and polychromatic (immature) erythrocytes accompanied by a considerable reduction of the number of apoptotic/dead cells among TNC (≤2.0%). Moreover, as a result of AC administrations, there was a significant increase of AO green and far-red fluorescence intensities in the population of TNC, which is suggestive of the ongoing quantitative and conformational changes in DNA and RNA associated with cell recovery and proliferation. Thus, AC preparations under the present experimental conditions can effectively tackle DOX-induced myelosuppression via mechanisms not necessarily associated with adsorptive detoxification.
PubMed: 37539240
DOI: 10.1016/j.heliyon.2023.e18414 -
Brain and Behavior Jun 2021Embryonic exposure to ethanol leads to a condition of physical, behavioral, and cognitive deficiencies named fetal alcohol spectrum disorders (FASD). The most severe...
INTRODUCTION
Embryonic exposure to ethanol leads to a condition of physical, behavioral, and cognitive deficiencies named fetal alcohol spectrum disorders (FASD). The most severe variations are in fetal alcohol syndrome (FAS), which is easier to diagnose and not studied in animal models. On the other side, the pFAS (partial fetal alcohol syndrome) includes cases of alcohol-related congenital disabilities and neurodevelopmental disorder with an inconclusive diagnosis. In recent years, the zebrafish has become a valuable model to study FASD and its variations.
METHODS
This study characterizes the zebrafish embryonic and larval development after low and moderate ethanol concentration exposure. Fish eggs were exposed to 0.0%, 0.25%, 0.5%, and 1.0% ethanol at 24 hr postfertilization, and embryonic development was observed every 8 hr up to 120 hpf. It evaluated movements, phenotypic abnormalities, hatching, cardiac function and heartbeat frequency, larvae length at 120 hpf, and the apoptotic cells' fluorescence stained with acridine orange.
RESULTS
Embryonic exposure to 0.5% and 1% ethanol presented reduced body size, decreased heartbeat rate, higher numbers of apoptotic cells, and hatching time differences.
CONCLUSIONS
Our results suggest any ethanol exposure during embryogenesis can be harmful and reinforces zebrafish as a suitable model for fetal alcohol spectrum disorders (FASD).
Topics: Animals; Disease Models, Animal; Ethanol; Female; Fetal Alcohol Spectrum Disorders; Larva; Pregnancy; Zebrafish
PubMed: 33939334
DOI: 10.1002/brb3.2062 -
Comparative Biochemistry and... Sep 2023The zebrafish is a prominent vertebrate model popularly used for toxicity testing because of its rapid development and transparent embryos. Fluchloralin, a...
The zebrafish is a prominent vertebrate model popularly used for toxicity testing because of its rapid development and transparent embryos. Fluchloralin, a dinitroaniline herbicide used to control weeds, inhibits microtubule formation and cell division. The structurally homologous substances ethalfluralin and pendimethalin, which belong to the dinitroaniline family, were found to be genotoxic and to exert developmental toxicity via mitochondrial dysfunction in a zebrafish model. To date, developmental toxicity of fluchloralin in zebrafish has not been reported. In the present study, we identified morphological changes in developing zebrafish, including decreased survival rate and body length, and increased yolk sac edema. In dose-dependent response to fluchloralin exposure, inhibition of neurogenesis in the spinal cord and motor neuron defects were observed in transgenic zebrafish models (olig2:dsRed). Zebrafish exposed to fluchloralin also displayed organ dysfunction in the heart, liver, and pancreas in cmlc2:dsRed and lfabp:dsRed;elastase:GFP transgenic models. Fluchloralin increased cell death in the brain by promoting apoptosis, visualized via acridine orange staining, and by activating apoptosis signaling proteins, including cytochrome c1, zBax, and Bcl-XL. This study provides novel evidence supporting the necessity of controlling pollutants in aquatic environments.
Topics: Animals; Zebrafish; Embryo, Nonmammalian; Liver; Nervous System; Embryonic Development; Water Pollutants, Chemical
PubMed: 37290698
DOI: 10.1016/j.cbpc.2023.109679 -
Methods in Molecular Biology (Clifton,... Mar 2024Leishmaniasis is a neglected tropical disease caused by numerous species of Leishmania parasites, including Leishmania major. The parasite is transmitted by several...
Leishmaniasis is a neglected tropical disease caused by numerous species of Leishmania parasites, including Leishmania major. The parasite is transmitted by several species of sandfly vectors and infects myeloid cells leading to a myriad of inflammatory responses, immune dysregulations, and disease manifestations. Every cell undergoes autophagy, a self-regulated degradative process that permits the cells to recycle damaged or worn-out organelles in order to maintain cellular health and homeostasis. Studies have shown that Leishmania modulates their host cell autophagic machinery and there are indications that the parasite-specific autophagic processes may be valuable for parasite virulence and survival. However, the role of autophagy in Leishmania is inconclusive because of the limited tools available to study the Leishmania-specific autophagic machinery. Here, we describe methods to study and definitively confirm autophagy in Leishmania major. Transmission electron microscopy (TEM) allowed us to visualize Leishmania autophagosomes, especially those containing damaged mitochondrial content, as well as dividing mitochondria with ongoing fusion/fission processes. Flow cytometry enabled us to identify the amount of acridine orange dye accumulating in the acidic vacuolar compartments in Leishmania major by detecting fluorescence in the red laser when autophagic inhibitors or enhancers were included. These methods will advance studies that aim to understand autophagic regulation in Leishmania parasites that could provide insights into developing improved therapeutic targets against leishmaniasis.
PubMed: 38441724
DOI: 10.1007/7651_2024_517 -
Pakistan Journal of Pharmaceutical... Mar 2023Plasmid borne antibiotics resistance is the global threat to healthcare facilities. Such antibiotics resistance is inherited stably within the same bacterial generations...
Plasmid borne antibiotics resistance is the global threat to healthcare facilities. Such antibiotics resistance is inherited stably within the same bacterial generations and transmitted horizontally to other species of bacteria. The elimination of such resistance plasmid is of great importance to contain dispersal of antibiotics resistance. E. coli strains were identified, screened for the presence of antibiotics resistance by disc diffusion method, and cured by sub-lethal concentrations of Ethidium bromide and Acridine orange. After curing, again antibiotic resistance was determined. Before and after curing, plasmids were extracted by column spin Kit and subjected to 1% agarose gel electrophoresis and antibiotic resistance genes were identified by PCR. The Ethidium bromide was more effective than Acridine orange in eliminating antibiotics resistance and resistance genes bearing plasmids (4, 5, 6, 8, 9, 10 and <10kb). The most frequently eliminated antibiotic resistance was against Imipenem and Meropenem followed by Cefoperazone-sulbactam, Amikacin and cephalosporins in sequence. The loss of antibiotic resistance was associated with the elimination of plasmid-borne antibiotic resistance genes; bla-TEM, bla-SHV, bla-CTX-M, qnrA, qnrB, qnrC and qnrD. Some E. coli strains did not show the removal of antibiotics resistance and plasmids, suggesting the presence of resistance genes on main chromosome and or non-curable plasmids.
Topics: Anti-Bacterial Agents; Escherichia coli; Ethidium; Acridine Orange; Microbial Sensitivity Tests; Plasmids; Drug Resistance, Microbial; beta-Lactamases
PubMed: 37548194
DOI: No ID Found -
Chemical Biology & Drug Design Mar 2020Monoamine oxidase (MAO) is an important drug target as the MAO isoforms play key roles in neurodegenerative disorders such as Alzheimer's disease and Parkinson's...
Monoamine oxidase (MAO) is an important drug target as the MAO isoforms play key roles in neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease, as well as in neuropsychiatric diseases such as depression. Methylene blue is an inhibitor of MAO-A, while azure B, the major metabolite of methylene blue, and various other structural analogues retain the ability to inhibit MAO-A. Based on this, the present study evaluated 22 dyes, many of which are structurally related to methylene blue, as potential inhibitors of human MAO-A and MAO-B. The results highlighted three dye compounds as good potency competitive and reversible MAO inhibitors, and which exhibit higher MAO inhibition than methylene blue: acridine orange, oxazine 170 and Darrow red. Acridine orange was found to be a MAO-A specific inhibitor (IC = 0.017 μM), whereas oxazine 170 is a MAO-B specific inhibitor (IC = 0.0065 μM). Darrow red was found to be a non-specific MAO inhibitor (MAO-A, IC = 0.059 μM; MAO-B, IC = 0.065 μM). These compounds may be advanced for further testing and preclinical development, or be used as possible lead compounds for the future design of MAO inhibitors.
Topics: Acridine Orange; Anthraquinones; Azure Stains; Coloring Agents; Drug Design; Humans; Methylene Blue; Monoamine Oxidase; Monoamine Oxidase Inhibitors; Neurodegenerative Diseases; Neuroprotective Agents; Oxazines; Structure-Activity Relationship
PubMed: 31834986
DOI: 10.1111/cbdd.13654 -
Animal Biotechnology Dec 2023Theileriosis is a hemoprotozoan illness of cattle in tropical regions that poses a severe economic loss to dairy farmers in the form of production loss and mortality. We...
Theileriosis is a hemoprotozoan illness of cattle in tropical regions that poses a severe economic loss to dairy farmers in the form of production loss and mortality. We designed and optimized a multiplex real-time PCR by using Taq-Man probe for detection and quantification of and simultaneously by targeting 18 s rRna and MPSP (surface merozoite protein) genes, respectively. Fifty-five EDTA blood samples from clinically -suspected cows of three -endemic districts of Odisha were processed using acridine dye based fluorescent microscopy, Giemsa staining, and PCR. PCR revealed and in 11/42 (26.11%) and 24/42 (57.14%) cases, respectively. Mixed infection due to both the spp. was recorded in 7/42 (16.66%). On comparison with gold standard test (PCR), the accuracy, sensitivity, and specificity were 92.72, 95.12, and 85.71% for Giemsa staining and 96.36, 97.56, and 92.85% for acridine orange dye. Multiplex real time PCR using Taq-Man probe detected two species of and simultaneously. Acridine dye based fluorescent microscopy is comparatively easy and rapid method in detection of spp.
Topics: Humans; Female; Cattle; Animals; Theileriasis; Theileria annulata; Cattle Diseases; RNA, Ribosomal; Membrane Proteins; Acridines
PubMed: 36695009
DOI: 10.1080/10495398.2023.2168197 -
European Journal of Obstetrics,... Dec 2021Cryopreservation refers to the cooling of cells and tissues to sub-zero temperatures in order to stop all biologic activity and preserve them for future use. Human sperm...
OBJECTIVE
Cryopreservation refers to the cooling of cells and tissues to sub-zero temperatures in order to stop all biologic activity and preserve them for future use. Human sperm cryopreservation is an important tool for assisted reproductive technology and male fertility preservation. However, cryopreservation significantly reduces the quality of spermatozoa. The antioxidant effects of curcumin on different cells have been widely reported. This study was aimed to evaluate changes in post-thaw viability, morphology, motility, chromatin condensation and DNA integrity in response to the addition of curcumin to human sperm freezing extender.
MATERIALS AND METHODS
Semen of 23 normozoospermic men was collected and each sample was divided into three equal aliquots: Control, DMSO, Curcumin. The samples were analyzed freshly for viability (Eosin Y), morphology (Diff-Quick), motility (following WHO standarts), sperm chromatin packaging (aniline blue) and DNA integrity (acridine orange). The control group remained untreated and was mixed with cryopreservation medium (in-house 1:1). The DMSO group was mixed with cryopreservation medium containing 0.1% DMSO. The curcumin group was mixed with cryopreservation medium containing 10 µM curcumin. Samples stained with Diff-Quick and aniline blue were examined under light microscope, samples stained with Eosin Y were examined under phase-contrast microscope and samples stained with acridine orange were examined under fluorescence microscope. Ten days after cryopreservation, samples were thawed and pre-freeze analyses repeated.
RESULTS
Obtained results showed that cryopreservation significantly (P < 0.001) reduces sperm parameters. In Curcumin group, progressive motility, sperm chromatin condensation and DNA integrity significantly (P < 0.001) increased after the thawing process, as compared with the control and the DMSO group.
CONCLUSION
These results suggest that the addition of curcumin to cryopreservation medium improves post-thaw progressive motility, sperm chromatin condensation and DNA integrity. It seems that curcumin ameliorates detrimental effects of cryopreservation on human spermatozoa. Further research is needed on the use of curcumin and other antioxidant substances in sperm cryopreservation.
Topics: Cryopreservation; Curcumin; Humans; Male; Semen Preservation; Sperm Motility; Spermatozoa
PubMed: 34773879
DOI: 10.1016/j.ejogrb.2021.10.027 -
Heliyon Dec 2019This study aims to evaluate the use of fluorescent dye Dil and super vital dye acridine orange (AO) tracking of labeled in the fibroblast cells.
BACKGROUND
This study aims to evaluate the use of fluorescent dye Dil and super vital dye acridine orange (AO) tracking of labeled in the fibroblast cells.
METHODS
Dil crystal and AO were used to stain in a co-culture of the fibroblasts with the parasite. AO staining solution was added to 1 × 10 parasites. After 10 min, the stained parasites were centrifuged and washed seven times with phosphate buffered saline (PBS). The stained promastigote was incubated with fibroblasts for 6-8 h. The presence of stained parasites with AO in the fibroblast was assessed using a fluorescence microscope. 1 × 10/mL promastigote of was gently suspended and mixed by Dil staining solution with an ultimate concentration of 0.002 μg/mL and it was kept for 20 min at the room temperature. Subsequently, after washing it in PBS for seven times, it was centrifuged at 3000 rpm for 10 min. The supernatant was removed and the precipitate containing stained promastigote was suspended in fresh DMEM F12 with fibroblasts at 37 °C for 6 h. The presence of stained parasites with Dil in fibroblast was assessed using a fluorescence microscope. Fibroblast characterization was undertaken by a real-time polymerase chain reaction (PCR).
RESULTS
Acridine orange staining assisted in detection of the live parasite in the fibroblast cells. Free promastigote looked green before entering into the fibroblasts after 12 h culture. The parasite entered the cytoplasm of fibroblasts at the beginning of the exposure and gradually entered the nucleus of the fibroblast. The fibroblast nucleus was entirely stained green by AO. The appeared green under the fluorescent microscope. Dil staining revealed that the internalized parasites with red/orange color were localized within the cytoplasm after 6-hours and the nucleus of the fibroblasts after 72-hours following culture. Human fibroblasts were positive at the expression of CD10, CD26, matrix metalloproteinase-1 (MMP-1) and matrix metalloproteinase-3 (MMP-3) and negative for CD106 and integrin alpha 11.
CONCLUSION
The fluorescent dye Dil staining is a safe, easy to use, inexpensive and fast method for labeling of the parasite in the fibroblast cells. Acridine orange staining could be useful for tracing the parasites in the fibroblasts too. In this study, both Dil and AO were compared and considered as suitable vital dyes for identifying labeled in the fibroblast , but Dil was superior to AO with its feature does not transfer from the labeled to unlabeled cells.
PubMed: 31890980
DOI: 10.1016/j.heliyon.2019.e03073