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Methods in Molecular Biology (Clifton,... 2023The quality of chromosome preparation influences all downstream analyses and is therefore crucial. Hence, numerous protocols exist to produce microscopic slides with...
The quality of chromosome preparation influences all downstream analyses and is therefore crucial. Hence, numerous protocols exist to produce microscopic slides with mitotic chromosomes. Nevertheless, due to the high content of fibers in and around a plant cell, preparation of plant chromosomes is still far from trivial and needs to be fine-tuned for each species and tissue type. Here, we outline the "dropping method," a straightforward and efficient protocol to prepare multiple slides with uniform quality from a single chromosome preparation. In this method, nuclei are extracted and cleaned to produce a nuclei suspension. In a drop-by-drop manner, this suspension is then applied from a certain height onto the slides, causing the nuclei to rupture and the chromosomes to spread. Due to the physical forces that accompany the dropping and spreading process, this method is best suited for species with small- to medium-sized chromosomes.
Topics: Chromosomes; Cell Nucleus; Chromosomes, Plant; Metaphase
PubMed: 37335474
DOI: 10.1007/978-1-0716-3226-0_8 -
Methods in Molecular Biology (Clifton,... 2023Procedures to arrest metaphase chromosomes are used for determining chromosome numbers, chromosomal aberrations, and natural chromosome variation, as well as chromosome...
Procedures to arrest metaphase chromosomes are used for determining chromosome numbers, chromosomal aberrations, and natural chromosome variation, as well as chromosome sorting. Here is described a technique of nitrous oxide gas treatment of freshly harvested root tips that is highly effective at producing an excellent mitotic index together with well-spread chromosomes. The details of the treatment and equipment used are provided. The metaphase spreads can be used directly for determining chromosome numbers or for in situ hybridization to reveal chromosomal features.
Topics: Humans; Metaphase; Nitrous Oxide; Chromosome Aberrations; Chromosomes; In Situ Hybridization
PubMed: 37335472
DOI: 10.1007/978-1-0716-3226-0_6 -
ELife Jun 2023Human oocyte maturation arrest represents one of the severe conditions for female patients with primary infertility. However, the genetic factors underlying this human...
Human oocyte maturation arrest represents one of the severe conditions for female patients with primary infertility. However, the genetic factors underlying this human disease remain largely unknown. The spindle assembly checkpoint (SAC) is an intricate surveillance mechanism that ensures accurate segregation of chromosomes throughout cell cycles. Once the kinetochores of chromosomes are correctly attached to bipolar spindles and the SAC is satisfied, the MAD2L1BP, best known as p31, binds mitosis arrest deficient 2 (MAD2) and recruits the AAA+-ATPase TRIP13 to disassemble the mitotic checkpoint complex (MCC), leading to the cell-cycle progression. In this study, by whole-exome sequencing (WES), we identified homozygous and compound heterozygous variants in three families with female patients diagnosed with primary infertility owing to oocyte metaphase I (MI) arrest. Functional studies revealed that the protein variants resulting from the C-terminal truncation of MAD2L1BP lost their binding ability to MAD2. cRNA microinjection of full-length or truncated uncovered their discordant roles in driving the extrusion of polar body 1 (PB1) in mouse oocytes. Furthermore, the patient's oocytes carrying the mutated resumed polar body extrusion (PBE) when rescued by microinjection of full-length cRNAs. Together, our studies identified and characterized novel biallelic variants in responsible for human oocyte maturation arrest at MI, and thus prompted new therapeutic avenues for curing female primary infertility.
Topics: Animals; Female; Humans; Mice; Adaptor Proteins, Signal Transducing; Cell Cycle Checkpoints; Cell Cycle Proteins; Exome Sequencing; Infertility, Female; Mad2 Proteins; Nuclear Proteins; Oocytes; Young Adult; Adult; Meiosis
PubMed: 37334967
DOI: 10.7554/eLife.85649 -
BioRxiv : the Preprint Server For... Jun 2023Faithful segregation of chromosomes into daughter cells during mitosis requires formation of attachments between kinetochores and mitotic spindle microtubules....
Faithful segregation of chromosomes into daughter cells during mitosis requires formation of attachments between kinetochores and mitotic spindle microtubules. Chromosome alignment on the mitotic spindle, also referred to as congression, is facilitated by translocation of side-bound chromosomes along the microtubule surface, which allows the establishment of end-on attachment of kinetochores to microtubule plus ends. Spatial and temporal constraints hinder observation of these events in live cells. Therefore, we used our previously developed reconstitution assay to observe dynamics of kinetochores, the yeast kinesin-8, Kip3, and the microtubule polymerase, Stu2, in lysates prepared from metaphase-arrested budding yeast, . Using total internal reflection fluorescence (TIRF) microscopy to observe kinetochore translocation on the lateral microtubule surface toward the microtubule plus end, motility was shown to be dependent on both Kip3, as we reported previously, and Stu2. These proteins were shown to have distinct dynamics on the microtubule. Kip3 is highly processive and moves faster than the kinetochore. Stu2 tracks both growing and shrinking microtubule ends but also colocalizes with moving lattice-bound kinetochores. In cells, we observed that both Kip3 and Stu2 are important for establishing chromosome biorientation, Moreover, when both proteins are absent, biorientation is completely defective. All cells lacking both Kip3 and Stu2 had declustered kinetochores and about half also had at least one unattached kinetochore. Our evidence argues that despite differences in their dynamics, Kip3 and Stu2 share roles in chromosome congression to facilitate proper kinetochore-microtubule attachment.
PubMed: 37333421
DOI: 10.1101/2023.06.08.544255 -
The Plant Journal : For Cell and... Aug 2023Mitosis and cytokinesis are fundamental processes through which somatic cells increase their numbers and allow plant growth and development. Here, we analyzed the...
Mitosis and cytokinesis are fundamental processes through which somatic cells increase their numbers and allow plant growth and development. Here, we analyzed the organization and dynamics of mitotic chromosomes, nucleoli, and microtubules in living cells of barley root primary meristems using a series of newly developed stable fluorescent protein translational fusion lines and time-lapse confocal microscopy. The median duration of mitosis from prophase until the end of telophase was 65.2 and 78.2 min until the end of cytokinesis. We showed that barley chromosomes frequently start condensation before mitotic pre-prophase as defined by the organization of microtubules and maintain it even after entering into the new interphase. Furthermore, we found that the process of chromosome condensation does not finish at metaphase, but gradually continues until the end of mitosis. In summary, our study features resources for in vivo analysis of barley nuclei and chromosomes and their dynamics during mitotic cell cycle.
Topics: Hordeum; Mitosis; Chromosomes; Microtubules; Cell Nucleus; Prophase
PubMed: 37326283
DOI: 10.1111/tpj.16355 -
Journal of Cell Science Jun 2023The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to...
The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to faithfully segregate chromosomes, remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has distinct functions on MTs in each compartment. Here, we show that two proteins, Cik1 and Vik1, which form heterodimers with Kar3, regulate its localization and function within the cell, and along MTs in a cell cycle-dependent manner. Using a yeast MT dynamics reconstitution assay in lysates from cell cycle-synchronized cells, we found that Kar3-Vik1 induces MT catastrophes in S phase and metaphase, and limits MT polymerization in G1 and anaphase. In contrast, Kar3-Cik1 promotes catastrophes and pauses in G1, while increasing catastrophes in metaphase and anaphase. Adapting this assay to track MT motor protein motility, we observed that Cik1 is necessary for Kar3 to track MT plus-ends in S phase and metaphase but, surprisingly, not during anaphase. These experiments demonstrate how the binding partners of Kar3 modulate its diverse functions both spatially and temporally.
Topics: Kinesins; Saccharomyces cerevisiae; Cell Cycle; Anaphase; Metaphase
PubMed: 37305999
DOI: 10.1242/jcs.260621 -
Comparative Cytogenetics 2023To have an insight into the karyotype variation of eight Cucurbitaceae crops including Linnaeus, 1753, Linnaeus, 1753, (Thunberg, 1794) Matsumura et Nakai, 1916,...
To have an insight into the karyotype variation of eight Cucurbitaceae crops including Linnaeus, 1753, Linnaeus, 1753, (Thunberg, 1794) Matsumura et Nakai, 1916, (Thunberg, 1784) Cogniaux, 1881, Linnaeus, 1753, (Linnaeus, 1753) Roemer, 1846, Lagenariasicerariavar.hispida (Thunberg, 1783) Hara, 1948 and Duchesne ex Poiret, 1819, well morphologically differentiated mitotic metaphase chromosomes were prepared using the enzymatic maceration and flame-drying method, and the chromosomal distribution of heterochromatin and 18S-5.8S-26S rRNA genes (45S rDNA) was investigated using sequential combined PI and DAPI (CPD) staining and fluorescence hybridization (FISH) with 45S rDNA probe. Detailed karyotypes were established using the dataset of chromosome measurements, fluorochrome bands and rDNA FISH signals. Four karyotype asymmetry indices, CV, CV, M and Stebbins' category, were measured to elucidate the karyological relationships among species. All the species studied had symmetrical karyotypes composed of metacentric and submetacentric or only metacentric chromosomes, but their karyotype structure can be discriminated by the scatter plot of M vs. CV. The karyological relationships among these species revealed by PCoA based on , 2, TCL, M, CV and CV was basically in agreement with the phylogenetic relationships revealed by DNA sequences. CPD staining revealed all 45S rDNA sites in all species, (peri)centromeric GC-rich heterochromatin in , , , and , terminal GC-rich heterochromatin in . DAPI counterstaining after FISH revealed pericentromeric DAPI heterochromatin in . rDNA FISH detected two 45S loci in five species and five 45S loci in three species. Among these 45S loci, most were located at the terminals of chromosome arms, and a few in the proximal regions. In , individual chromosomes can be precisely distinguished by the CPD band and 45S rDNA signal patterns, providing an easy method for chromosome identification of cucumber. The genome differentiation among these species was discussed in terms of genome size, heterochromatin, 45S rDNA site, and karyotype asymmetry based on the data of this study and previous reports.
PubMed: 37305810
DOI: 10.3897/compcytogen.v17.i1.99236 -
Journal of Nanobiotechnology Jun 2023Due to its complicated pathophysiology, propensity for metastasis, and poor prognosis, colon cancer is challenging to treat and must be managed with a combination of...
Due to its complicated pathophysiology, propensity for metastasis, and poor prognosis, colon cancer is challenging to treat and must be managed with a combination of therapy. Using rolling circle transcription (RCT), this work created a nanosponge therapeutic medication system (AS1411@antimiR-21@Dox). Using the AS1411 aptamer, this approach accomplished targeted delivery to cancer cells. Furthermore, analysis of cell viability, cell apoptosis, cell cycle arrest, reactive oxygen species (ROS) content, and mitochondrial membrane potential (MMP) levels revealed that functional nucleic acid nanosponge drug (FND) can kill cancer cells. Moreover, transcriptomics uncovered a putative mechanism for the FND anti-tumor effect. These pathways, which included mitotic metaphase and anaphase as well as the SMAC-mediated dissociation of the IAP: caspase complexes, were principally linked to the cell cycle and cell death. In conclusion, by triggering cell cycle arrest and apoptosis, the nano-synergistic therapeutic system allowed for the intelligent and effective targeted administration of RNA and chemotherapeutic medicines for colon cancer treatment. The system allowed for payload efficiency while being customizable, targeted, reliable, stable, and affordable.
Topics: Humans; Doxorubicin; Drug Delivery Systems; Nucleic Acids; Colonic Neoplasms; Drug Carriers; Aptamers, Nucleotide; Oligodeoxyribonucleotides; Nanoparticles; Cell Line, Tumor
PubMed: 37280622
DOI: 10.1186/s12951-023-01941-z -
Pharmaceuticals (Basel, Switzerland) Jan 2023Melanoma is considered the most aggressive form of skin cancer, showing high metastatic potential and persistent high mortality rates despite the introduction of...
Melanoma is considered the most aggressive form of skin cancer, showing high metastatic potential and persistent high mortality rates despite the introduction of immunotherapy and targeted therapies. Thus, it is important to identify new drug candidates for melanoma. The design of hybrid molecules, with different pharmacophore fragments combined in the same scaffold, is an interesting strategy for obtaining new multi-target and more effective anticancer drugs. We designed nine hybrid compounds bearing piperine and chlorogenic acid pharmacophoric groups and evaluated their antitumoral potential on melanoma cells with distinct mutational profiles SK-MEL-147, CHL-1 and WM1366. We identified the compound named PQM-277 () to be the most cytotoxic one, inhibiting mitosis progression and promoting an accumulation of cells in pro-metaphase and metaphase by altering the expression of genes that govern G2/M transition and mitosis onset. Compound downregulated , , , , , and , and upregulated . Molecular docking showed that could interact with the CUL1-RBX1 complex, which activity is necessary to trigger molecular events essential for FOXM1 transactivation and, in turn, G2/M gene expression. In addition, compound effectively induced apoptosis by increasing ratio. Our findings demonstrate that is an important antitumor candidate prototype and support further investigations to evaluate its potential for melanoma treatment, especially for refractory cases to BRAF/MEK inhibitors.
PubMed: 37259298
DOI: 10.3390/ph16020145 -
Journal of Obstetrics and Gynaecology :... Dec 2023FAM64A is a mitotic regulator which promotes cell metaphase-anaphase transition and is highly expressed in a cell-cycle-dependent manner. In this study, we examined the...
FAM64A is a mitotic regulator which promotes cell metaphase-anaphase transition and is highly expressed in a cell-cycle-dependent manner. In this study, we examined the clinicopathological and prognostic significance of mRNA expression in gynecological cancers. We conducted a bioinformatics analysis of mRNA expression using Gene Expression Omnibus (GEO), The Cancer Genome Atlas (TCGA), xiantao, The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN), and Kaplan-Meier (KM) plotter databases. expression was elevated in breast, cervical, endometrial, and ovarian cancers when compared with normal tissue. Expression was positively correlated with white race, low T stages, infiltrating ductal carcinoma, or favourable PAM50 classification in breast cancer patients, and with clinical stage, histological grade and TP53 mutation, and endometrial cancer serous subtype. expression was negatively associated with overall and/or recurrence-free survival rates in breast and endometrial cancer patients, while the opposite was observed in cervical and ovarian cancer patients. functioned as an independent predictor of overall and disease-specific survival in breast cancer patients. -correlated genes were involved in ligand-receptor interactions, and chromosomal, cell cycle, and DNA replication processes in breast, cervical, endometrial and ovarian cancers. Top hub genes primarily included cell cycle-related proteins in breast cancer, mucins and acetylgalactosaminyl transferases in cervical cancer, kinesin family members in endometrial cancer, and synovial sarcoma X and the cancer/testis antigen in ovarian cancer. mRNA expression was positively related to Th2 cell infiltration, but negatively associated with neutrophil and Th17 cell infiltration in breast, cervical, endometrial, and ovarian cancers. expression may be considered a potential biomarker reflecting carcinogenesis, histogenesis, aggressive behaviour, and prognosis in gynecological cancers.Impact statement FAM64A is located in cell nucleolar and nucleoplasmic regions, and during mitosis it putatively controls metaphase-to-anaphase transition. FAM64A appears to regulate different physiological processes, including apoptosis, tumorigenesis, neural differentiation, stress responses, and the cell cycle. expression was up-regulated in breast, cervical, endometrial, and ovarian cancers, and positively correlated with white race, low T stages, infiltrating ductal carcinoma, or favourable PAM50 classification in breast cancer patients, and with clinical stage, histological grade, and TP53 mutation, and a serous subtype in endometrial cancer. expression was negatively associated with overall and/or recurrence-free survival rates in breast and endometrial cancer patients, while the opposite was observed in cervical and ovarian cancer patients. functioned as an independent predictor of overall and disease-specific survival in breast cancer. -correlated genes were involved in ligand-receptor interactions, chromosomal, cell cycle, and DNA replication processes, while mRNA expression was positively related to Th2 cell infiltration but negatively correlated with neutrophil and Th17 cell infiltration in four gynecological cancers. In the future, abnormal mRNA expression may serve as a biomarker of carcinogenesis, histogenesis, aggressiveness, and prognosis in gynecological malignancies.
Topics: Female; Humans; Male; Breast Neoplasms; Carcinogenesis; Carcinoma, Ductal; Computational Biology; Endometrial Neoplasms; Gene Expression Regulation, Neoplastic; Ligands; Ovarian Neoplasms; Prognosis; RNA, Messenger
PubMed: 37227120
DOI: 10.1080/01443615.2023.2216280