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Bio-protocol Dec 2021Regulation of microtubule stability is crucial for diverse biological processes, including cell division, morphogenesis, and signaling. Various assays for microtubule...
Regulation of microtubule stability is crucial for diverse biological processes, including cell division, morphogenesis, and signaling. Various assays for microtubule stability have been developed to identify and characterize proteins involved in controlling microtubule stability. Here, we introduce a simple assay for identifying potential microtubule regulators in the wing imaginal disc of . This assay utilizes silicon rhodamine-tubulin (SiR-Tub) as a cell-permeable fluorogenic dye for labeling microtubules. In an attempt to increase the sensitivity of the screen, we designed an assay using a sensitized microtubule condition. Wing discs are treated with SiR-Tub followed by demecolcine, a microtubule inhibitor, to partially label impaired microtubules. Under this sensitized condition, we can test whether overexpression or downregulation of a gene can enhance or suppress the weakened SiR-Tub labeling. This assay allows highly sensitive detection of microtubules in developing larval tissues. Hence, it provides a useful tool for identifying new microtubule regulators in both unfixed and fixed imaginal discs in . This strategy may also be applied to characterize microtubule regulators in tissues from other model organisms. Graphic abstract: Graphical summary of microtubule stability assay using wing disc.
PubMed: 35005086
DOI: 10.21769/BioProtoc.4241 -
International Journal of Radiation... 2023The dicentric chromosome (Dic) assay, which is the gold standard for biological dose assessment in radiation emergency medicine, requires an analysis of at least 500...
PURPOSE
The dicentric chromosome (Dic) assay, which is the gold standard for biological dose assessment in radiation emergency medicine, requires an analysis of at least 500 lymphocyte metaphases or 100 Dic aberrations. Therefore, peripheral blood culture conditions able to obtain a high frequency of metaphases for efficient dose evaluation should be optimized. However, the type of blood cultures [i.e. whole blood (WB) or isolated peripheral blood mononuclear cell (PBMC)-culture] and blood volume differ between biodosimetry laboratories. The purpose of this study is to investigate the blood volume at which a high mitotic index (MI) is obtained in peripheral WB-culture and isolated PBMC-culture, and to examine the possible effect of blood volume on radiation-induced Dic frequency.
MATERIALS AND METHODS
Peripheral blood was collected from three healthy donors with their informed consent. The complete and differential blood counts were performed using an automated hematology analyzer. After blood count, peripheral blood was irradiated with 0 or 2 Gy X-ray. Blood was cultured with phytohemagglutinin (180 μg/ml) and demecolcine (0.05 μg/ml) for 48 h. The MI and Dic frequency were analyzed in 5, 10, 15, 20, 25, and 30% WB-cultures and 0.6, 1.2, 1.8, 2.4, 3.0, 3.6, and 4.2 ml WB-equivalent PBMC-cultures.
RESULTS
In WB-culture, MI showed the highest value (∼22%) in 5-15% WB-culture and then gradually decreased to ∼9% with 30% WB-culture. MI peaked at 36 and 31% in 1.8 and 2.4 ml-WB equivalent volumes for PMBC-cultures, respectively. MI progressively decreased as the amount of PBMCs increased. Although individual differences were observed in the MI values among the three subjects, all the subjects showed the same tendency and higher MI was seen in PBMC than WB-cultures. However, these factors had no significant impact on the yield of Dics. In all culture conditions, the estimated dose calculated based on the Dic frequency was equivalent to the absorbed dose of X-ray-irradiated blood.
CONCLUSION
While MI was affected by the blood culture type and the volume of cultured blood, Dic yield did not differ significantly between these conditions. These results could be used by relevant laboratories to optimize MI in certain circumstances.
Topics: Humans; Leukocytes, Mononuclear; Mitotic Index; Chromosome Aberrations; Lymphocytes; Chromosomes
PubMed: 36318780
DOI: 10.1080/09553002.2023.2142981 -
Cells Jul 2022Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the...
Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: // (), (), (), (), (), (), (), and (). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.
Topics: Animals; Demecolcine; Drosophila; Drosophila Proteins; Kinesins; Kinetochores; Mammals; Microtubule-Associated Proteins; Microtubules; Mitosis; Spindle Apparatus
PubMed: 35883570
DOI: 10.3390/cells11142127 -
Foods (Basel, Switzerland) Apr 2020In this work, the phytochemical profile and the biological properties of (an unexplored Turkish cultivar belonging to Colchicaceae) have been comprehensively...
In this work, the phytochemical profile and the biological properties of (an unexplored Turkish cultivar belonging to Colchicaceae) have been comprehensively investigated for the first time. Herein, we focused on the evaluation of the in vitro antioxidant and enzyme inhibitory effects of flower, tuber, and leaf extracts, obtained using different extraction methods, namely maceration (both aqueous and methanolic), infusion, and Soxhlet. Besides, the complete phenolic and alkaloid untargeted metabolomic profiling of the different extracts was investigated. In this regard, ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) allowed us to putatively annotate 285 compounds when considering the different matrix extracts, including mainly alkaloids, flavonoids, lignans, phenolic acids, and tyrosol equivalents. The most abundant polyphenols were flavonoids (119 compounds), while colchicine, demecolcine, and lumicolchicine isomers were some of the most widespread alkaloids in each extract analyzed. In addition, our findings showed that tuber extracts were a superior source of both total alkaloids and total polyphenols, being on average 2.89 and 10.41 mg/g, respectively. Multivariate statistics following metabolomics allowed for the detection of those compounds most affected by the different extraction methods. Overall, extracts showed a strong in vitro antioxidant capacity, in terms of cupric reducing antioxidant power (CUPRAC; on average 96.45 mg Trolox Equivalents (TE)/g) and ferric reducing antioxidant power (FRAP) reducing power (on average 66.86 mg TE/g). Interestingly, each methanolic extract analyzed (i.e., from tuber, leaf, and flower) was active against the tyrosinase in terms of inhibition, recording the higher values for methanolic macerated leaves (i.e., 125.78 mg kojic acid equivalent (KAE)/g). On the other hand, moderate inhibitory activities were observed against AChE and α-amylase. Strong correlations ( < 0.01) were also observed between the phytochemical profiles and the biological activities determined. Therefore, our findings highlighted, for the first time, the potential of extracts in food and pharmaceutical applications.
PubMed: 32276367
DOI: 10.3390/foods9040457 -
Scientific Reports May 2021The knowledge of cell mechanics is required to understand cellular processes and functions, such as the movement of cells, and the development of tissue engineering in...
The knowledge of cell mechanics is required to understand cellular processes and functions, such as the movement of cells, and the development of tissue engineering in cancer therapy. Cell mechanical properties depend on a variety of factors, such as cellular environments, and may also rely on external factors, such as the ambient temperature. The impact of temperature on cell mechanics is not clearly understood. To explore the effect of temperature on cell mechanics, we employed magnetic tweezers to apply a force of 1 nN to 4.5 µm superparamagnetic beads. The beads were coated with fibronectin and coupled to human epithelial breast cancer cells, in particular MCF-7 and MDA-MB-231 cells. Cells were measured in a temperature range between 25 and 45 °C. The creep response of both cell types followed a weak power law. At all temperatures, the MDA-MB-231 cells were pronouncedly softer compared to the MCF-7 cells, whereas their fluidity was increased. However, with increasing temperature, the cells became significantly softer and more fluid. Since mechanical properties are manifested in the cell's cytoskeletal structure and the paramagnetic beads are coupled through cell surface receptors linked to cytoskeletal structures, such as actin and myosin filaments as well as microtubules, the cells were probed with pharmacological drugs impacting the actin filament polymerization, such as Latrunculin A, the myosin filaments, such as Blebbistatin, and the microtubules, such as Demecolcine, during the magnetic tweezer measurements in the specific temperature range. Irrespective of pharmacological interventions, the creep response of cells followed a weak power law at all temperatures. Inhibition of the actin polymerization resulted in increased softness in both cell types and decreased fluidity exclusively in MDA-MB-231 cells. Blebbistatin had an effect on the compliance of MDA-MB-231 cells at lower temperatures, which was minor on the compliance MCF-7 cells. Microtubule inhibition affected the fluidity of MCF-7 cells but did not have a significant effect on the compliance of MCF-7 and MDA-MB-231 cells. In summary, with increasing temperature, the cells became significant softer with specific differences between the investigated drugs and cell lines.
Topics: Actins; Biomechanical Phenomena; Breast Neoplasms; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Demecolcine; Female; Fibronectins; Heterocyclic Compounds, 4 or More Rings; Humans; MCF-7 Cells; Magnetic Iron Oxide Nanoparticles; Microtubules; Temperature; Thiazolidines
PubMed: 34031462
DOI: 10.1038/s41598-021-90173-y -
Acta Histochemica Apr 2023Maximising the number of cells arrested at metaphase and their resolution is fundamentally important for molecular cytogenetic investigations, particularly in fish,...
Maximising the number of cells arrested at metaphase and their resolution is fundamentally important for molecular cytogenetic investigations, particularly in fish, which typically yield low mitotic index and have highly condensed chromosomes. To overcome these limitations, fish were injected with a mitotic stimulator (the yeast, Saccharomyces cerevisiae) to improve the mitotic index, and the intercalating agent ethidium bromide to produce elongated chromosomes. Specifically, adults were injected with activated yeast and then Colcemid (0.025 µg/µl solution, 10 µl per 1 g of body weight) at 24-96 h post yeast injections, followed by chromosome preparations from multiple tissues. Results showed that gill tissue had the highest number of dividing cells at 72 h post yeast exposure with no significant (p > 0.05) differences between the sexes. Nonetheless, sex-specific differences in the mitotic index were observed in spleen, kidney, and liver, which may be attributed to sex-specific differences in immune responses. For elongation of mitotic chromosomes, individuals (both sexes) were first injected with activated yeast and after 48 h with ethidium bromide (2 or 4 µg/ml) and Colcemid (0.05 µg/µl solution, 10 µl per 1 g of body weight). Following which, animals were sampled at three time points (1, 4 and 8 h) for chromosome preparations. The results show that the optimum elongation of metaphase chromosomes of males and females was achieved by using 2 µg/ml and 4 µg/ml, respectively, for 1 h. Interestingly, the average mitotic chromosome length (μm) of males and females post-ethidium bromide exposure was significantly different (p < 0.05) for both concentrations, except at 1 h exposure for 2 µg/ml EtBr. Such differences can be attributed to overall chromosomal condensation differences between sexes. Regardless, the increased mitotic index and chromosome resolution could benefit cytogenetic studies in other fish species.
Topics: Male; Animals; Female; Ethidium; Saccharomyces cerevisiae; Demecolcine; Chromosomes; Cytogenetic Analysis; Cyprinodontiformes; Body Weight
PubMed: 37062122
DOI: 10.1016/j.acthis.2023.152029 -
BMC Biology Dec 2021The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as...
BACKGROUND
The integrity of microtubule filament networks is essential for the roles in diverse cellular functions, and disruption of its structure or dynamics has been explored as a therapeutic approach to tackle diseases such as cancer. Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt microtubules. However, due to associated side effects on healthy cells, there is a need to develop safer drug regimens that still retain clinical efficacy. Currently, many questions remain open regarding the extent of effects on cellular physiology of microtubule-interacting drugs at clinically relevant and low doses. Here, we use super-resolution microscopies (single-molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid.
RESULTS
We identify previously undetected microtubule (MT) damage caused by clinically relevant doses of colcemid. Short exposure to 30-80 nM colcemid results in aberrant microtubule curvature, with a trend of increased curvature associated to increased doses, and curvatures greater than 2 rad/μm, a value associated with MT breakage. Microtubule fragmentation was detected upon treatment with ≥ 100 nM colcemid. Remarkably, lower doses (< 20 nM after 5 h) led to subtle but significant microtubule architecture remodelling characterized by increased curvature and suppression of microtubule dynamics.
CONCLUSIONS
Our results support the emerging hypothesis that microtubule-interacting drugs induce non-mitotic effects in cells, and establish a multi-modal imaging assay for detecting and measuring nanoscale microtubule dysfunction. The sub-diffraction visualization of these less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs offers potential for improved understanding and design of anticancer agents.
Topics: Cytoskeleton; Demecolcine; Microscopy, Fluorescence; Microtubules
PubMed: 34895240
DOI: 10.1186/s12915-021-01164-4 -
Journal of Biomolecular Structure &... Sep 2022Sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate (S1P) signaling regulates numerous diseases such as cancer, diabetes, and inflammation-related ailments,...
Sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate (S1P) signaling regulates numerous diseases such as cancer, diabetes, and inflammation-related ailments, rheumatoid arthritis, atherosclerosis, and multiple sclerosis. The importance of SphK1 in chemo-resistance has been extensively explored in breast, lung, colon, and hepatocellular carcinomas. SphK1 is considered an attractive drug target for the development of anticancer therapy. New drug molecules targeting the S1P signaling are required owing to its pleiotropic nature and association with multiple downstream targets. Here, we have investigated the binding affinity and SphK1 inhibitory potential of cinchonine and colcemid using a combined molecular docking and simulation studies followed by experimental analysis. These compounds bind to SphK1 with a significantly high affinity and subsequently inhibit kinase activity (IC 7-9 μM). Further, MD simulation studies revealed that both cinchonine and colcemid bind to the residues at the active site pocket of SphK1 with several non-covalent interactions, which may be responsible for inhibiting its kinase activity. Besides, the binding of cinchonine and colcemid causes substantial conformational changes in the structure of SphK1. Taken together, cinchonine and colcemid may be implicated in designing potential drug molecules with improved affinity and specificity for SphK1 targeting anticancer therapy.Communicated by Ramaswamy H. Sarma.
Topics: Cinchona Alkaloids; Demecolcine; Molecular Docking Simulation; Phosphotransferases (Alcohol Group Acceptor)
PubMed: 33565370
DOI: 10.1080/07391102.2021.1882341 -
Cell Biochemistry and Biophysics Dec 2022The dicentric chromosome assay (DCA), is considered the 'gold standard' for radiation biodosimetry. Yet, DCA, as currently implemented, may be impractical for emergency...
The dicentric chromosome assay (DCA), is considered the 'gold standard' for radiation biodosimetry. Yet, DCA, as currently implemented, may be impractical for emergency response applications, especially when time is of the essence, owing to its labor-intensive and time-consuming nature. The growth of a primary lymphocyte culture for 48 h in vitro is required for DCA, and manual scoring of dicentric chromosomes (DCs) requires an additional 24-48 h, resulting in an overall processing time of 72-96 h for dose estimation. In order to improve this timing. we introduce a protocol that will detect the metaphase cells in a population of cells, and then will harvest only those metaphase cells. Our metaphase enrichment approach is based on fixed human lymphocytes incubated with monoclonal, anti-phosphorylated H3 histone (ser 10). Antibodies against this histone have been shown to be specific for mitotic cells. Colcemid is used to arrest the mitotic cells in metaphase. Following that, a flow-cytometric sorting apparatus isolates the mitotic fraction from a large population of cells, in a few minutes. These mitotic cells are then spread onto a slide and treated with our C-Banding procedure [Gonen et al. 2022], to visualize the centromeres with DAPI. This reduces the chemical processing time to ~2 h. This reduces the time required for the DCA and makes it practical for a much wider set of applications, such as emergency response following exposure of a large population to ionizing radiation.
Topics: Chromosome Aberrations; Chromosomes, Human; Demecolcine; Dose-Response Relationship, Radiation; Histones; Humans; Lymphocytes; Metaphase; Radiometry
PubMed: 36216973
DOI: 10.1007/s12013-022-01106-z -
Bioinformation 2020It is known that beta-catenin is associated with fibromatosis, sarcoma and mesenchymal tumor. Therefore, it is of interest to design an effective inhibtitor to the...
It is known that beta-catenin is associated with fibromatosis, sarcoma and mesenchymal tumor. Therefore, it is of interest to design an effective inhibtitor to the target protein beta-catenin. In this study, we report the molecular docking analysis of alkaloid compounds (aristolochicacid, cryptopleurine, demecolcine, fagaronine and thalicarpine) with beta-catenin for further consideration towards the design and development of potential inhintors for the treatmnet of colon cancer.
PubMed: 32308271
DOI: 10.6026/97320630016283