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Genes and Environment : the Official... Apr 2024An in vitro micronucleus assay is a standard genotoxicity test. Although the technique and interpretation of the results are simple, manual counting of the total and...
BACKGROUND
An in vitro micronucleus assay is a standard genotoxicity test. Although the technique and interpretation of the results are simple, manual counting of the total and micronucleus-containing cells in a microscopic field is tedious. To address this issue, several systems have been developed for quick and efficient micronucleus counting, including flow cytometry and automated detection based on specialized software and detection systems that analyze images.
RESULTS
Here, we present a simple and effective method for automated micronucleus counting using image recognition technology. Our process involves separating the RGB channels in a color micrograph of cells stained with acridine orange. The cell nuclei and micronuclei were detected by scaling the G image, whereas the cytoplasm was recognized from a composite image of the R and G images. Finally, we identified cells with overlapping cytoplasm and micronuclei as micronucleated cells, and the application displayed the number of micronucleated cells and the total number of cells. Our method yielded results that were comparable to manually measured values.
CONCLUSIONS
Our micronucleus detection (MN/cell detection software) system can accurately detect the total number of cells and micronucleus-forming cells in microscopic images with the same level of precision as achieved through manual counting. The accuracy of micronucleus numbers depends on the cell staining conditions; however, the software has options by which users can easily manually optimize parameters such as threshold, denoise, and binary to achieve the best results. The optimization process is easy to handle and requires less effort, making it an efficient way to obtain accurate results.
PubMed: 38659010
DOI: 10.1186/s41021-024-00305-9 -
BioMedicine 2023Gemcitabine is frequently utilized to treat pancreatic cancer. The purpose of our study was to create a gemcitabine-resistant MIA-PaCa-2 pancreatic cancer cell line...
Gemcitabine is frequently utilized to treat pancreatic cancer. The purpose of our study was to create a gemcitabine-resistant MIA-PaCa-2 pancreatic cancer cell line (MIA-GR100) and to evaluate the anti-pancreatic cancer efficacy of HMJ-38, a new quinazolinone analogue. Compared to their parental counterparts, MIA-PaCa-2, established MIA-GR100 cells were less sensitive to gemcitabine. MIA-GR100 cell viability was not affected by 10, 50 and 100 nM gemcitabine concentrations. HMJ-38 reduced MIA-GR100 cell growth and induced autophagy and apoptosis. When stained with monodansylcadaverine (MDC), acridine orange (AO), and terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL), MIA-GR100 cells shrunk, punctured their membranes, and produced autophagy vacuoles and apoptotic bodies. Combining chloroquine (CQ) and 3-methyladenine (3-MA) with HMJ-38 dramatically reduced cell viability, indicating that autophagy function as a cytoprotective mechanism. MIA-GR100 cells treated with both z-VAD-FMK and HMJ-38 were much more viable than those treated with HMJ-38 alone. HMJ-38 promotes apoptosis in MIA-GR100 cells by activating caspases. Epidermal growth factor receptor (EGFR) is one of HMJ-38's principal targets, as determined target screening with network prediction. HMJ-38 also inhibited EGFR kinase activity and EGFR-associated signaling in MIA-GR100 cells. HMJ-38 may be an effective chemotherapeutic adjuvant for gemcitabine-resistant pancreatic cancer cells, in which it induces an antitumor response.
PubMed: 38532833
DOI: 10.37796/2211-8039.1423 -
Heliyon Apr 2024Inflammation affects several aspects of lung cancer progression including cell proliferation, metastasis, apoptosis, angiogenesis, and drug resistance. Baicalin, an...
Inflammation affects several aspects of lung cancer progression including cell proliferation, metastasis, apoptosis, angiogenesis, and drug resistance. Baicalin, an active component of Georgi, exhibits anticancer activity in various cancers. However, the effects of baicalin on lung cancer and the underlying molecular mechanisms remain largely unknown. This study is to explore the effect and mechanism of baicalin on lung cancer cell A549 and urethane-induced mouse lung cancer. A cell viability assay, colony formation assay, wound healing assay, acridine orange/ethidium bromide (AO/EB) staining assay, Western blot assay, urethane-induced mouse lung cancer model, hematoxylin and eosin (HE) staining, immunohistochemistry (IHC), and ELISA assay were performed to investigate the effects of baicalin on lung cancer and . Network pharmacology analysis, molecular docking, gene silencing assays, and LPS-induced inflammation model were utilized to explore the molecular mechanisms underlying the effect of baicalin on lung cancer. Baicalin showed significant anti-proliferative, anti-migratory, anti-inflammatory and pro-apoptotic effects ; it also inhibited the progression of urethane-induced mouse lung cancer . Mechanistically, suppressor of cytokine signaling 1 (SOCS1) was the key determinant for baicalin-induced inhibition of lung cancer. Baicalin increased SOCS1 expression to inactivate the NF-κB/STAT3 pathway to inhibit lung cancer and . Taken together, baicalin reduces inflammation to inhibit lung cancer via targeting SOCS1/NF-κB/STAT3 axis, providing a prospective compound and novel target for lung cancer treatment.
PubMed: 38628726
DOI: 10.1016/j.heliyon.2024.e29361 -
International Journal of Molecular... Feb 2024Spermatogenesis is the process of proliferation and differentiation of spermatogonial cells to meiotic and post-meiotic stages and sperm generation. Normal...
Spermatogenesis is the process of proliferation and differentiation of spermatogonial cells to meiotic and post-meiotic stages and sperm generation. Normal spermatogenesis occurs in vivo at 34 °C to 35 °C, and high temperatures are known to cause male infertility. The aim of the present study was to examine the effect of temperature (35 °C compared to 37 °C) on the viability/apoptosis of developed cells, on the development of different stages of spermatogenesis in 3D in vitro culture conditions, and the functionality of Sertoli cells under these conditions. We used isolated cells from seminiferous tubules of sexually immature mice. The cells were cultured in methylcellulose (as a three-dimensional (3D) in vitro culture system) and incubated in a CO incubator at 35 °C or 37 °C. After two to six weeks, the developed cells and organoids were collected and examined for cell viability and apoptosis markers. The development of different stages of spermatogenesis was evaluated by immunofluorescence staining or qPCR analysis using specific antibodies or primers, respectively, for cells at each stage. Factors that indicate the functionality of Sertoli cells were assessed by qPCR analysis. The developed organoids were examined by a confocal microscope. Our results show that the percentages and/or the expression levels of the developed pre-meiotic, meiotic, and post-meiotic cells were significantly higher at 35 °C compared to those at 37 °C, including the expression levels of the androgen receptor, the FSH receptor, transferrin, the androgen-binding protein (ABP), and the glial-derived nerve growth factor (GDNF) which were similarly significantly higher at 35 °C than at 37 °C. The percentages of apoptotic cells (according to acridine orange staining) and the expression levels of BAX, FAS, and CASPAS 3 were significantly higher in cultures incubated at 37 °C compared to those incubated at 35 °C. These findings support the in vivo results regarding the negative effect of high temperatures on the process of spermatogenesis and suggest a possible effect of high temperatures on the viability/apoptosis of spermatogenic cells. In addition, increasing the temperature in vitro also impaired the functionality of Sertoli cells. These findings may deepen our understanding of the mechanisms behind optimal conditions for normal spermatogenesis in vivo and in vitro.
Topics: Male; Mice; Animals; Sertoli Cells; Testis; Temperature; Semen; Spermatogenesis; Spermatogonia
PubMed: 38396838
DOI: 10.3390/ijms25042160 -
Orthopaedic Surgery Jun 2024The micro-nano structure of 3D-printed porous titanium (Ti) alloy with excellent performance in avoiding stress shielding and promoting bone tissue differentiation...
OBJECTIVES
The micro-nano structure of 3D-printed porous titanium (Ti) alloy with excellent performance in avoiding stress shielding and promoting bone tissue differentiation provides a new opportunity for the development of bone implants, but it necessitates higher requirements for bone tissue differentiation and the antibacterial properties of bone implants in clinical practice.
METHODS
This study investigated the preparation, antimicrobial properties, and osteogenesis-promoting ability of the 3D printed porous Ti alloy anodic oxidized Ag-carrying (Ag@3D-TiO) scaffolds. The 3D printed porous Ti alloy (3D-Ti), anodized 3D printed porous Ti alloy (3D-TiO), and Ag@3D-TiO scaffolds were synthesized using electron beam melting. The antimicrobial properties of the scaffolds were examined using antibacterial tests and their cytocompatibility was assessed using a cell proliferation assay and acridine orange/ethidium bromide (AO/EB) staining. In vitro cellular assays were used to investigate the effects of the scaffold microstructural features on cell activity, proliferation, and osteogenesis-related genes and proteins. In vivo animal experiments were used to evaluate the anti-inflammatory and osteogenesis-promoting abilities of the scaffolds.
RESULTS
The Ag@3D-TiO scaffolds exhibited sustained anti-microbial activity over time, enhanced cell proliferation, facilitated osteogenic differentiation, and increased extracellular matrix mineralization. In addition, alkaline phosphatase (ALP), collagen type I (COL-I), and osteocalcin (OCN)-related genes and proteins were upregulated. In vivo animal implantation experiments, the anti-inflammatory effect of the Ag@3D-TiO scaffolds were observed using histology, and a large amount of fibrous connective tissue was present around it; the Ag@3D-TiO scaffolds were more bio-compatible with the surrounding tissues compared with 3D-Ti and 3D-TiO; a large amount of uniformly distributed neoplastic bone tissue existed in their pores, and the chronic systemic toxicity test showed that the 3D-Ti, 3D-TiO, and Ag@3D-TiO scaffolds are biologically safe.
CONCLUSION
The goal of this study was to create a scaffold that exhibits antimicrobial properties and can aid bone growth, making it highly suitable for use in bone tissue engineering.
Topics: Titanium; Osteogenesis; Tissue Scaffolds; Silver; Animals; Printing, Three-Dimensional; Mice; Cell Proliferation; Cell Differentiation; Anti-Bacterial Agents; Porosity
PubMed: 38706035
DOI: 10.1111/os.14081 -
International Journal of Molecular... Aug 2023Metallic nanoparticles (mNPs) are widely used as food additives and can interact with gliadin triggering an immune response, but evaluation of the effects on crypts,...
Metallic nanoparticles (mNPs) are widely used as food additives and can interact with gliadin triggering an immune response, but evaluation of the effects on crypts, hypertrophic in celiac subjects, is still lacking. This study evaluated the effects of gold and silver mNPs in combination with gliadin on crypt-like cells (HIEC-6). Transmission electron microscopy (TEM) was used to evaluate gliadin-mNP aggregates in cells. Western blot and immunofluorescence analysis assessed autophagy-related molecule levels (p62, LC3, beclin-1, EGFR). Lysosome functionality was tested with acridine orange (AO) and Magic Red assays. TEM identified an increase in autophagic vacuoles after exposure to gliadin + mNPs, as also detected by significant increments in LC3-II and p62 expression. Immunofluorescence confirmed the presence of mature autophagosomes, showing LC3 and p62 colocalization, indicating an altered autophagic flux, further assessed with EGFR degradation, AO and Magic Red assays. The results showed a significant reduction in lysosomal enzyme activity and a modest reduction in acidity. Thus, gliadin + mNPs can block the autophagic flux inducing a lysosomal defect. The alteration of this pathway, essential for cell function, can lead to cell damage and death. The potential effects of this copresence in food should be further characterized to avoid a negative impact on celiac disease subjects.
Topics: Humans; Gold; Glutens; Silver; Gliadin; Autophagy; Acridine Orange; Nanoparticles; ErbB Receptors
PubMed: 37685847
DOI: 10.3390/ijms241713040 -
Heliyon May 2024Tracing individual cell pathways among the whole population is crucial for understanding their behavior, cell communication, migration dynamics, and fate. Optical...
Tracing individual cell pathways among the whole population is crucial for understanding their behavior, cell communication, migration dynamics, and fate. Optical labeling is one approach for tracing individual cells, but it typically requires genetic modification to induce the generation of photoconvertible proteins. Nevertheless, this approach has limitations and is not applicable to certain cell types. For instance, genetic modification often leads to the death of macrophages. This study aims to develop an alternative method for labeling macrophages by utilizing photoconvertible micron-sized capsules capable of easy internalization and prolonged retention within cells. Thermal treatment in a polyvinyl alcohol gel medium is employed for the scalable synthesis of capsules with a wide range of fluorescent dyes, including rhodamine 6G, pyronin B, fluorescein, acridine yellow, acridine orange, thiazine red, and previously reported rhodamine B. The fluorescence brightness, photostability, and photoconversion ability of the capsules are evaluated using confocal laser scanning microscopy. Viability, uptake, mobility, and photoconversion studies are conducted on RAW 264.7 and bone marrow-derived macrophages, serving as model cell lines. The production yield of the capsules is increased due to the use of polyvinyl alcohol gel, eliminating the need for conventional filtration steps. Capsules entrapping rhodamine B and rhodamine 6G meet all requirements for intracellular use in individual cell tracking. Mass spectrometry analysis reveals a sequence of deethylation steps that result in blue shifts in the dye spectra upon irradiation. Cellular studies on macrophages demonstrate robust uptake of the capsules. The capsules exhibit minimal cytotoxicity and have a negligible impact on cell motility. The successful photoconversion of RhB-containing capsules within cells highlights their potential as alternatives to photoconvertible proteins for individual cell labeling, with promising applications in personalized medicine.
PubMed: 38813172
DOI: 10.1016/j.heliyon.2024.e30680 -
Gels (Basel, Switzerland) Jan 2024Microneedle patches are attractive drug delivery systems that give hope for treating skin disorders. In this study, to first fabricate a chitosan-based low-cost...
Microneedle patches are attractive drug delivery systems that give hope for treating skin disorders. In this study, to first fabricate a chitosan-based low-cost microneedle patch (MNP) using a CO laser cutter for in vitro purposes was tried and then the delivery and impact of Glycyrrhiza glabra extract (GgE) on the cell population by this microneedle was evaluated. Microscopic analysis, swelling, penetration, degradation, biocompatibility, and drug delivery were carried out to assess the patch's performance. DAPI staining and acridine orange (AO) staining were performed to evaluate cell numbers. Based on the results, the MNs were conical and sharp enough (diameter: 400-500 μm, height: 700-900 μm). They showed notable swelling (2 folds) during 5 min and good degradability during 30 min, which can be considered a burst release. The MNP showed no cytotoxicity against fibroblast cell line L929. It also demonstrated good potential for GgE delivery. The results from AO and DAPI staining approved the reduction in the cell population after GgE delivery. To sum up, the fabricated MNP can be a useful recommendation for lab-scale studies. In addition, a GgE-loaded MNP can be a good remedy for skin disorders in which cell proliferation needs to be controlled.
PubMed: 38391417
DOI: 10.3390/gels10020087 -
Asian Pacific Journal of Cancer... Jan 2024Clerodendrum infortunatum L. has long been used in traditional medicine in Sri Lanka for tumours, cancer, and certain skin diseases. The present study aimed to assess...
OBJECTIVE
Clerodendrum infortunatum L. has long been used in traditional medicine in Sri Lanka for tumours, cancer, and certain skin diseases. The present study aimed to assess the anticancer properties of the aqueous extract of C. infortunatum L. root (AECIR) through the activation of the apoptotic pathway on hepatocellular carcinoma (HepG2) and thus give it a scientific validation. Further, the contribution of polyphenols in antioxidant activity and cell cytotoxicity was investigated.
METHODS
Powdered plant material was boiled with water (100°C) to obtained AECIR. The DPPH assay was used to determine the antioxidant potential. The activity of AECIR on HepG2 and normal rat fibroblast (CC1) cell growth was determined using MTT assay. The morphological changes related to apoptotic pathway was examined by Ethidium Bromide/Acridine Orange (EB/AO), Rhodamine 123 (Rh123) and DNA fragmentation assay.
RESULTS
The AECIR demonstrated antioxidant potential with an EC50 of 350.2 ± 1.5 ug/mL for DPPH assay. The HO•, H2O2 and •NO free radical scavenging activity was observed with EC50 of 19.7 ± 2.3, 11.7 ± 0.1 and 273.1 ± 0.9 ug/mL, respectively. The antiproliferative effect of AECIR on HepG2 cells was observed in a time and dose dependent manner with an EC50 of 239.1 ± 1.3 μg/mL while CC1 cells showed a nontoxic effect with an EC50 1062.7 ± 3.4 μg/mL after 24hrs treatment. A significant decrease in antioxidant activity (p<0.001) and 90% HepG2 cell viability was observed with polyphenol removed AECIR compared to the polyphenol present AECIR. The EB/AO uptake, depletion of mitochondrial transmembrane potential, and DNA fragmentation assay results revealed that the apoptosis was induced by AECIR.
CONCLUSION
The obtained result of the present study demonstrates that the antioxidant potential and antiproliferative activity of AECIR is attributed to the presence of polyphenols. Furthermore, the findings provide the scientific base for anti-cancer potential of AECIR.
Topics: Animals; Rats; Humans; Carcinoma, Hepatocellular; Polyphenols; Antioxidants; Hep G2 Cells; Clerodendrum; Hydrogen Peroxide; Plant Extracts; Liver Neoplasms; Cell Proliferation; Apoptosis
PubMed: 38285803
DOI: 10.31557/APJCP.2024.25.1.351 -
BMC Complementary Medicine and Therapies Nov 2023Screening of herbal plants for various therapeutic properties is the hour as it shows promising activity. Scientific evidence of the pharmacological activity of the...
BACKGROUND
Screening of herbal plants for various therapeutic properties is the hour as it shows promising activity. Scientific evidence of the pharmacological activity of the plant strengthens the traditional application of plants.
METHODS
Rose flowers (Rosa chinensis) were procured and grounded into a coarse powder. The DNA was isolated from rose flower and molecular identification was performed by rbcL-BF and rbcL-724R primers. Antibacterial activity was evaluated by using disc and agar diffusion methods and the anti-cancer effect of the rose flower extract (RE) was examined using MTT assay in lung cancer cell line. The mechanism of cell death induced by RE was qualitatively measured using Acridine orange/Ethidium bromide staining and Hoechst staining. GC-MS analysis was performed using GC-MS-5975C.
RESULT
The RE showed potent antimicrobial activity against various ATCC cultures. The rose extract strongly inhibits the growth of ESBL resistant organism along with inhibition of biofilm formation in the ESBL resistant organism. The extract caused apoptotic and necrotic cell death in lung cancer cells. GC-MS analysis demonstrated the presence of several biologically active compounds such as Clindamycin, Phytol, Octanoic acid, and Stigmasterol which might be the reason for the therapeutic properties of the plant.
CONCLUSION
This study shows the antimicrobial and biofilm inhibition activity against the clinical isolates of Klebsiella pneumonia. The study shows the cytotoxic and apoptotic activity in A549 cancer cell line. Thus, the plant may act as a potent antimicrobial drug against resistant strains.
Topics: Humans; Rosa; Lung Neoplasms; Plant Extracts; Acetone; Anti-Infective Agents; A549 Cells
PubMed: 37950173
DOI: 10.1186/s12906-023-04222-2