Stages Of Cell Cycle Research Articles

Vascular Stem Cells and the Role of B-Raf Kinase in Survival, Proliferation, and Apoptosis.

Neovascularization is an essential process in organismal development and aging. With aging, from fetal to adult life, there is a significant reduction in neovascularization potential. However, the pathways which play a role in increased neovascularization potential during fetal life are unknown. Although several studies proposed the idea of vascular stem cells (VSCs), the identification and essential survival mechanism are still not clear. In the present study, we isolated fetal VSCs from the ovine carotid artery and identified the pathways involved in their survival. We tested the hypothesis that fetal vessels contain a population of VSCs, and that B-Raf kinase is required for their survival. We conducted viability, apoptotic, and cell cycle stage assays on fetal and adult carotid arteries and isolated cells. To determine molecular mechanisms, we conducted RNAseq, PCR, and western blot experiments to characterize them and identify pathways essential for their survival. Results: A stem cell-like population was isolated from fetal carotid arteries grown in serum-free media. The isolated fetal VSCs contained markers for endothelial, smooth muscle, and adventitial cells, and formed a de novo blood vessel ex vivo. A transcriptomic analysis that compared fetal and adult arteries identified pathway enrichment for several kinases, including B-Raf kinase in fetal arteries. Furthermore, we demonstrated that B-Raf- Signal Transducer and Activator of Transcription 3 (STAT3)-Bcl2 is critical for the survival of these cells. Fetal arteries, but not adult arteries, contain VSCs, and B-Raf-STAT3-Bcl2 plays an important role in their survival and proliferation.

Calcineurin associates with centrosomes and regulates cilia length maintenance.

Calcineurin, or protein phosphatase 2B (PP2B), the Ca2+ and calmodulin-activated phosphatase and target of immunosuppressants, has many substrates and functions that remain uncharacterized. By combining rapid proximity-dependent labeling with cell cycle synchronization, we mapped the spatial distribution of calcineurin in different cell cycle stages. While calcineurin-proximal proteins did not vary significantly between interphase and mitosis, calcineurin consistently associated with multiple centrosomal and/or ciliary proteins. These include POC5, which binds centrins in a Ca2+-dependent manner and is a component of the luminal scaffold that stabilizes centrioles. We show that POC5 contains a calcineurin substrate motif (PxIxIT type) that mediates calcineurin binding in vivo and in vitro. Using indirect immunofluorescence and ultrastructure expansion microscopy, we demonstrate that calcineurin colocalizes with POC5 at the centriole, and further show that calcineurin inhibitors alter POC5 distribution within the centriole lumen. Our discovery that calcineurin directly associates with centriolar proteins highlights a role for Ca2+ and calcineurin signaling at these organelles. Calcineurin inhibition promotes elongation of primary cilia without affecting ciliogenesis. Thus, Ca2+ signaling within cilia includes previously unknown functions for calcineurin in maintenance of cilia length, a process that is frequently disrupted in ciliopathies.

MiR-183-5p regulates ECM and EMT to promote non-small cell lung cancer progression by targeting LOXL4.

Non-small cell lung cancer (NSCLC) progression is mediated by changes in gene expression induced by microRNAs. However, the underlying mechanisms remain to be elucidated. In this study, we investigated the roles of miR-183-5p and its target gene in lung cancer development. Relative levels of miR-183-5p and lysyl oxidase-like 4 (LOXL4) expression in lung cancer cells or tissues were measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR), immunofluorescence or Western blotting as appropriate. The binding of miR-183-5p to LOXL4 sequences was verified by a dual luciferase reporter assay, and cell proliferation was assessed by Cell Counting Kit-8 (CCK-8) and Edu staining. The cell cycle stage and apoptosis were detected by flow cytometry, and Transwell assays were performed to evaluate cell migration and invasion capabilities. The tumorigenic capability of cancer cells was analyzed using a cancer cell line-based xenograft nude mouse model. miR-183-5p expression was decreased in the lung cancer tissues and cell lines and was negatively correlated with elevated LOXL4 expression. Treatment with miR-183-5p mimics suppressed LOXL4 expression, while treatment with an miR-183-5p inhibitor promoted LOXL4 expression in A549 cells. miR-183-5p was found to directly bind to the 3' UTR of the LOXL4 gene in A549 cells. Overexpression of LOXL4 enhanced cell proliferation, cell cycle progression, migration, and invasion, but repressed their apoptosis, and activated extracellular matrix (ECM) and the epithelial mesenchymal transition (EMT) process in A549 cells, while LOXL4 knockdown produced the opposite effects. Treatment with an miR-183-5P inhibitor promoted the proliferation, cell cycle progression, migration, and invasion of A549 cells but suppressed their apoptosis, and activated the ECM and EMT process, while all these effects were abrogated by LOXL4 knockdown. The tumorigenic capability of A540 cells in nude mice was greatly impaired by treatment with miR-183-5p mimics. miR-183-5p repressed the proliferation, migration, invasion, ECM formation, and EMT processes, and promoted the apoptosis of lung cancer cells by targeting LOXL4 expression.

Histone demethylase KDM4B contributes to advanced clear cell renal carcinoma and association with copy number variations and cell cycle progression

ABSTRACT Advanced renal cell carcinoma (RCC) poses a threat to patient survival. Epigenetic remodelling is the pathogenesis of renal cancer. Histone demethylase 4B (KDM4B) is overexpressed in many cancers through various pathways. However, the role of KDM4B in clear cell renal carcinoma has not yet been elucidated. The differential expression of KDM4B was first verified by analysing public databases. The expression of KDM4B in fresh tissues and pathology slides was further analysed by western blotting and immunohistochemical staining. KDM4B overexpression and knockdown cell lines were also established. Cell Counting Kit-8 (CCK-8) assay was used to detect cell growth. Transwell assays were performed to assess cell migration. Xenografts were used to evaluate tumour growth and metastasis in vivo. Finally, KDM4B expression levels associated with copy number variation (CNV) and cell cycle stage were evaluated based on single-cell RNA sequencing data. KDM4B was expressed at higher levels in tumour tissues than in the adjacent normal tissues. High levels of KDM4B are associated with worse pathological features and poorer prognosis. KDM4B also promotes cell proliferation and migration in vitro, as well as tumour growth and metastasis in vivo. Tumour cells with high KDM4B expression exhibited higher CNV levels and a greater proportion of cells in the G1/S transition phase. Our results confirm that KDM4B promotes the progression of clear cell renal carcinoma, is correlated with poor prognosis, and may be related to high levels of CNV and cell cycle progression.

The cell cycle and cell size influence the rates of global cellular translation and transcription in fission yeast

How the production of biomass is controlled as cells increase in size and proceed through the cell cycle events is important for understanding the regulation of global cellular growth. This has been studied for decades but has not yielded consistent results, probably due to perturbations induced by the synchronisation methods used in most previous studies. To avoid this problem, we have developed a system to analyse unperturbed exponentially growing populations of fission yeast cells. We generated thousands of fixed single‐cell measurements of cell size, cell cycle stage and the levels of global cellular translation and transcription. We show that translation scales with size, and additionally, increases at late S‐phase/early G2 and early in mitosis and decreases later in mitosis, suggesting that cell cycle controls are also operative over global cellular translation. Transcription increases with both size and the amount of DNA, suggesting that the level of transcription of a cell may be the result of a dynamic equilibrium between the number of RNA polymerases associating and disassociating from DNA.

CRK2 controls cytoskeleton morphogenesis in Trypanosoma brucei by phosphorylating β-tubulin to regulate microtubule dynamics.

Microtubules constitute a vital part of the cytoskeleton in eukaryotes by mediating cell morphogenesis, cell motility, cell division, and intracellular transport. The cytoskeleton of the parasite Trypanosoma brucei contains an array of subpellicular microtubules with their plus-ends positioned toward the posterior cell tip, where extensive microtubule growth and cytoskeleton remodeling take place during early cell cycle stages. However, the control mechanism underlying microtubule dynamics at the posterior cell tip remains elusive. Here, we report that the S-phase cyclin-dependent kinase-cyclin complex CRK2-CYC13 in T. brucei regulates microtubule dynamics by phosphorylating β-tubulin on multiple evolutionarily conserved serine and threonine residues to inhibit its incorporation into cytoskeletal microtubules and promote its degradation in the cytosol. Consequently, knockdown of CRK2 or CYC13 causes excessive microtubule extension and loss of microtubule convergence at the posterior cell tip, leading to cytoskeleton elongation and branching. These findings uncover a control mechanism for cytoskeletal microtubule dynamics by which CRK2 phosphorylates β-tubulin and fine-tunes cellular β-tubulin protein abundance to restrict excess microtubule extension for the maintenance of cytoskeleton architecture.

DNA protein kinase promotes cellular senescence in dental follicle cells

ObjectiveShort telomeres and genomic DNA damage are causes of cellular senescence in dental follicle cells (DFCs). DesignThis study examined the role of the DNA damage response (DDR) during cellular senescence of DFCs by β-galactosidase activity and DNA damage by comet assay. Expression of genes/proteins was determined by Western Blots and reverse transcription-quantitative polymerase chain reaction, while glycolysis was enzymatically estimated. Cell cycle stages and reactive oxygen species (ROS) were investigated by flow cytometry. ResultsDuring the induction of cellular senescence gene expression of DDR genes were down-regulated, while DNA double-strand breaks occurred at the same time. Furthermore, inhibition of DNA protein kinase (DNA-PK) reduced senescence and ROS, both of which are associated with cellular senescence. In contrast, while these data suggest that inhibition of DDR is associated with the induction of cellular senescence, inhibition of DNA-PK did not result in renewal of DFCs, as inhibition resulted in typical features of depleted cells such as increased cell size and reduced cell proliferation rate. DNA-PK repression inhibited both osteogenic differentiation potential and glycolysis, which are typical features of cellular exhaustion. Moreover, DNA-PK affects cellular senescence via activation of AKT1 (protein kinase B). ConclusionOur results suggest that DNA-PK promotes cellular senescence, but DFCs may control the induction of cellular senescence via down-regulation of DDR genes. However, we also showed that inhibition of DNA-PK cannot renew senescent DFCs

Label-free multiphoton imaging flow cytometry.

Label-free imaging flow cytometry is a powerful tool for biological and medical research as it overcomes technical challenges in conventional fluorescence-based imaging flow cytometry that predominantly relies on fluorescent labeling. To date, two distinct types of label-free imaging flow cytometry have been developed, namely optofluidic time-stretch quantitative phase imaging flow cytometry and stimulated Raman scattering (SRS) imaging flow cytometry. Unfortunately, these two methods are incapable of probing some important molecules such as starch and collagen. Here, we present another type of label-free imaging flow cytometry, namely multiphoton imaging flow cytometry, for visualizing starch and collagen in live cells with high throughput. Our multiphoton imaging flow cytometer is based on nonlinear optical imaging whose image contrast is provided by two optical nonlinear effects: four-wave mixing (FWM) and second-harmonic generation (SHG). It is composed of a microfluidic chip with an acoustic focuser, a lab-made laser scanning SHG-FWM microscope, and a high-speed image acquisition circuit to simultaneously acquire FWM and SHG images of flowing cells. As a result, it acquires FWM and SHG images (100 × 100 pixels) with a spatial resolution of 500 nm and a field of view of 50 μm × 50 μm at a high event rate of four to five events per second, corresponding to a high throughput of 560-700 kb/s, where the event is defined by the passage of a cell or a cell-like particle. To show the utility of our multiphoton imaging flow cytometer, we used it to characterize Chromochloris zofingiensis (NIES-2175), a unicellular green alga that has recently attracted attention from the industrial sector for its ability to efficiently produce valuable materials for bioplastics, food, and biofuel. Our statistical image analysis found that starch was distributed at the center of the cells at the early cell cycle stage and became delocalized at the later stage. Multiphoton imaging flow cytometry is expected to be an effective tool for statistical high-content studies of biological functions and optimizing the evolution of highly productive cell strains.

Overexpressed Nup88 stabilized through interaction with Nup62 promotes NF-κB dependent pathways in cancer.

Bidirectional nucleo-cytoplasmic transport, regulating several vital cellular processes, is mediated by the Nuclear Pore Complex (NPC) comprising the nucleoporin (Nup) proteins. Nup88, a constituent nucleoporin, is overexpressed in many cancers, and a positive correlation exists between progressive stages of cancer and Nup88 levels. While a significant link of Nup88 overexpression in head and neck cancer exists but mechanistic details of Nup88 roles in tumorigenesis are sparse. Here, we report that Nup88 and Nup62 levels are significantly elevated in head and neck cancer patient samples and cell lines. We demonstrate that the elevated levels of Nup88 or Nup62 impart proliferation and migration advantages to cells. Interestingly, Nup88-Nup62 engage in a strong interaction independent of Nup-glycosylation status and cell-cycle stages. We report that the interaction with Nup62 stabilizes Nup88 by inhibiting the proteasome-mediated degradation of overexpressed Nup88. Overexpressed Nup88 stabilized by interaction with Nup62 can interact with NF-κB (p65) and sequesters p65 partly into nucleus of unstimulated cells. NF-κB targets like Akt, c-myc, IL-6 and BIRC3 promoting proliferation and growth are induced under Nup88 overexpression conditions. In conclusion, our data indicates that simultaneous overexpression of Nup62 and Nup88 in head and neck cancer stabilizes Nup88. Stabilized Nup88 interacts and activates p65 pathway, which perhaps is the underlying mechanism in Nup88 overexpressing tumors.

Efficient Production of Doubled Haploids as Isogenic Line Founders via Double Heat Shock Treatment in Willow Gudgeon (Gnathopogon caerulescens)

Doubled haploids (DHs) are essential founders for breeding via isogenic line production. We determined the optimal timing of the first and second (HST1 and HST2) double heat shock treatment for DH production in the willow gudgeon (Gnathopogon caerulescens). Gynogenesis was induced by fertilisation with UV-irradiated sperm (20 mJ/cm2). Each HST was performed at 40.5°C for 1 min. In experiment I, HST1 was applied 23–31 min after the induction of gynogenetic development (incubated at 20°C). HST2 was performed 15 min after HST1 treatment. When HST1 was started 25–26 min after activation, normally hatched larvae appeared relatively frequently (35.6–100%), and most were DHs. In experiment II, HST1 was started 26 min after activation, and HST2 was started 10–25 min after HST1. Starting HST2 10 min after HST1 resulted in 33% more normally hatched larvae than starting 22.5–25 min after. DHs were prevalent among hatched larvae (45.1% and 20.8%, respectively) when performing HST2 10–12.5 min after HST1. Under appropriate HST1 and HST2 timing, gynogens were at the zygote stage, early prophase, and prophase or early prometaphase of the first cell cycle. These results establish the appropriate timing of HST1 and HST2 for the induction of willow gudgeon DHs.

Vast heterogeneity in cytoplasmic diffusion rates revealed by nanorheology and Doppelgänger simulations.

The cytoplasm is a complex, crowded, actively-driven environment whose biophysical characteristics modulate critical cellular processes such as cytoskeletal dynamics, phase separation, and stem-cell fate. Little is known about the variance in these cytoplasmic properties. We employed particle-tracking nano-rheology on genetically-encoded multimeric 40-nm nanoparticles (GEMs) to measure diffusion within the cytoplasm of the fission yeast Schizosaccharomyces pombe. We found that the apparent diffusion coefficients of individual GEM particles varied over a 400-fold range, while the average particle diffusivity for each individual cell spanned a 10-fold range. To determine the origin of this heterogeneity, we developed a Doppelgänger Simulation approach that uses stochastic simulations of GEM diffusion that replicate the experimental statistics on a particle-by-particle basis, such that each experimental track and cell had a one-to-one correspondence with their simulated counterpart. These simulations showed that the large intra- and inter-cellular variations in diffusivity could not be explained by experimental variability but could only be reproduced with stochastic models that assume an equally wide intra- and inter-cellular variation in cytoplasmic viscosity. Through experimental perturbations, we found that the variance in diffusivity was largely independent of factors such as temperature, cytoskeleton, cell cycle stage, and spatial locations, but was magnified by hyperosmotic shocks. The model additionally predicts diffusion through a heterogeneous cytoplasm should be weakly non-ergodic at short time scales, consistent with the experimental data. Taken together, our results provide a striking demonstration that the cytoplasm is not “well-mixed” but represents a highly heterogeneous environment in which subcellular components at the 40-nm size-scale experience dramatically different effective viscosities within an individual cell, as well as in different cells in the population. These findings carry significant implications for the origins and regulation of biological noise at cellular and subcellular levels.

Decoding chromatin organization in the nuclear environment.

Chromatin structure tightly controls genome function within the nucleus. The nuclear environment is complex, featuring tension exerted by various force-generating proteins and molecular crowding modulated by different ionic concentrations. Disentangling these factors to understand how they affect chromatin structure would reveal the mechanism underlying the accessibility and organization of chromatin with an important connection to transcriptional activity and epigenetic regulation of genes. Nevertheless, it is unclear how chromatin adapts its structure to different nuclear environments. Here, we computationally characterize chromatin structures under tension, crowding, and ionic environments using a near-atomistic resolution chromatin model. Our simulations show that DNA unwrapping causes chromatin to break into a series of trimeric and tetrameric nucleosome clutches under tension. This irregularity further facilitates the formation of interdigitated chromatin structures via the trans interactions among multiple chains under a crowded environment, such as interphase heterochromatin and mitotic chromatin. Finally, our simulation shows that monovalent and divalent ions can further modulate chromatin structures, consistent with experiments. Since the nucleus features various environments at different cell cycle stages, our collective work can facilitate understanding the package of genetic materials in various cellular contexts and the genomic adaptation to an ever-changing external environment.

The potential role of N6-methyladenosine modification of LncRNAs in contributing to the pathogenesis of chronic glomerulonephritis.

Increasing evidence indicates that N6-methyladenosine (m6A) modification of mRNAs has been shown to play a critical role in the occurrence and development of many diseases, while little is known about m6A modification in long non-coding RNAs (LncRNAs). Our study aims to investigate the potential functions of LncRNA m6A modifications in lipopolysaccharide (LPS)-induced mouse mesangial cells (MMCs), providing us with a new perspective on the molecular mechanisms of chronic glomerulonephritis (CGN) pathogenesis. Differentially methylated LncRNAs were identified by Methylated RNA immunoprecipitation sequencing (MeRIP-seq). LncRNA-mRNA and LncRNA-associated LncRNA-miRNA-mRNA (CeRNA) networks were constructed by bioinformatics analysis. Furthermore, we utilized gene ontology (GO) and pathway enrichment analyses (KEGG) to explore target genes from co-expression networks. In addition, the total level of m6A RNA methylation and expression of methyltransferase and pro-inflammatory cytokines were detected by the colorimetric quantification method and western blot, respectively. Cell viability and cell cycle stage were detected by cell counting kit-8 (CCK-8) and flow cytometry. In total, 1141 differentially m6A-methylated LncRNAs, including 529 hypermethylated LncRNAs and 612 hypomethylated LncRNAs, were determined by MeRIP-seq. The results of GO and KEGG analysis revealed that the target mRNAs were mainly enriched in signal pathways, such as the NF-kappa B signaling pathway, MAPK signaling pathway, Toll-like receptor signaling pathway, and apoptosis signaling pathway. In addition, higher METTL3 expression was found in CGN kidney tissues using the GEO database. METTL3 knockdown in MMC cells drastically reduced the levels of m6A RNA methylation, pro-inflammatory cytokines IL6 and TNF-α, and inhibited cell proliferation and cycle progression. Our findings provide a basis and novel insight for further investigations of m6A modifications in LncRNAs for the pathogenesis of CGN.

Unraveling the effect of the inflammatory microenvironment in spermatogenesis progression.

Experimental autoimmune orchitis (EAO) is a chronic inflammatory disorder that causes progressive spermatogenic impairment. EAO is characterized by high intratesticular levels of nitric oxide (NO) and tumor necrosis factor alpha (TNFα) causing germ cell apoptosis and Sertoli cell dysfunction. However, the impact of this inflammatory milieu on the spermatogenic wave is unknown. Therefore, we studied the effect of inflammation on spermatogonia and preleptotene spermatocyte cell cycle progression in an EAO context and through the intratesticular DETA-NO and TNFα injection in the normal rat testes. In EAO, premeiotic germ cell proliferation is limited as a consequence of the undifferentiated spermatogonia (CD9+) cell cycle arrest in G2/M and the reduced number of differentiated spermatogonia (c-kit+) and preleptotene spermatocytes that enter in the meiotic S-phase. Although inflammation disrupts spermatogenesis in EAO, it is maintained in some seminiferous tubules at XIV and VII-VIII stages of the epithelial cell cycle, thereby guaranteeing sperm production. We found that DETA-NO (2mM) injected in normal testes arrests spermatogonia and preleptotene spermatocyte cell cycle; this effect reduces the number of proliferative spermatogonia and the number of preleptotene spermatocytes in meiosis S-phase (36h after). The temporal inhibition of spermatogonia clonal amplification delayed progression of the spermatogenic wave (5days after) finally altering spermatogenesis. TNFα (0.5 and 1µg) exposure did not affect premeiotic germ cell cycle or spermatogenic wave. Our results show that in EAO the inflammatory microenvironment altered spermatogenesis kinetics through premeiotic germ cell cycle arrest and that NO is a sufficient factor contributing to this phenomenon.

Oroxylum indicum Vent Root Bark Extract Inhibits the Proliferation of Cancer Cells and Induce Apoptotic Cell Death

Oroxylum indicum Vent is a medium-sized deciduous tree that belongs to the family Bignoniaceae. The roots of this tree are used as one of the ten ingredients to prepare the Dasamula formulation in the Ayurvedic system of medicine in India. Although traditional uses are attributed to the medicinal plant, there are limited scientific data on its potential. The present study thus analyzed the cytotoxic and apoptotic potential of the plant against different cancer cells. MTT assay was used to determine cytotoxicity using HeLa, HCT 15, and MDA-MB-231 cells, with the IC50 values, revealed at concentrations of 92.43, 133.0, and 112.84 µg/mL respectively. However, the extract was less toxic to non-cancer cells. HeLa cells further treated with OIM were subjected to flow cytometric analysis for studying the cell cycle stages. When untreated cells at G1 phase were found at a relative percentage of 71.9%, it increased to 79.3 and 86% with OIM treatment at concentrations of 50 and 100 µg/mL; cells in the S phase decreased from 10.3 to 8.2 and 7.5%, concluding the arrest of the cell cycle at G1 phase. With further study of apoptotic morphology with dual acridine orange–ethidium bromide staining and Annexin–Hoechst staining, cells at early and late apoptotic stages were observed with OIM treatment at 100 µg/mL concentration. Although such effects were noticed with OIM treatment, it could not be concluded that the extract had remarkable anti-proliferative effects, since the small changes noticed in cell cycle arrest and apoptotic induction were attained at a high concentration of OIM 100 µg/mL. The biological activities of plants and their extracts are attributed to the presence of multifarious compounds present in them. LC-MS Q-TOF analysis confirmed the presence of biochanin A and baicalein in OIM. HPLC-based quantification of baicalein and chrysin was shown to be 3.36 and 1.11 mg/gram dry weight. To conclude, the above results suggest that the root bark of O. indicum has a broad spectrum of biological activities, including anticancer and apoptotic properties.

Visualizing Chromosome Territories and Nuclear Architecture of Large Plant Genomes Using Alien Introgressions.

Visualization of chromosome territories is a challenging task in plant genomes due to the lack of chromosome-specific probes, especially in species with large genomes. On the other hand, combination of flow sorting, genomic in situ hybridization (GISH), confocal microscopy, and employment of software for 3D modeling enables to visualize and characterize chromosome territories (CT) in interspecific hybrids. Here, we describe the protocol for the analysis of CTs in wheat-rye and wheat-barley hybrids, including amphiploids and introgression forms, where a pair of chromosomes or chromosome arms from one species is introgressed into the genome of another species. In this way, the architecture and dynamics of CTs in various tissues and different stages of cell cycle can be analyzed.

Exploring cyclin-dependent kinase inhibitors: a comprehensive study in search of CDK-6 inhibitors using a pharmacophore modelling and dynamics approach.

Cancer prevalence and resistance issues in cancer treatment are a significant public health concern globally. Among the existing strategies in cancer therapy, targeting cyclin-dependent kinases (CDKs), especially CDK-6 is found to be one of the most promising targets, as this enzyme plays a pivotal role in cell cycle stages and cell proliferation. Cell proliferation is the characteristic feature of cancer giving rise to solid tumours. Our research focuses on creating novel compounds, specifically, pyrazolopyrimidine fused azetidinones, using a groundbreaking molecular hybridization approach to target CDK-6. Through computational investigations, ligand-based pharmacophore modelling, pharmacokinetic studies (ADMET), molecular docking, and dynamics simulations, we identified 18 promising compounds. The pharmacophore model featured one aromatic hydrophobic centre (F1: Aro/Hyd) and two H-bond acceptors (F2 and F3: Acc). Molecular docking results showed favourable binding energies (-6.5 to -8.0 kcal mol-1) and effective hydrogen bonds and hydrophobic interactions. The designed compounds demonstrated good ADMET profiles. Specifically, B6 and B18 showed low energy conformation (-7.8 kcal and -7.6 kcal), providing insights into target inhibition compared to the standard drug Palbociclib. Extensive molecular dynamics simulations confirmed the stability of these derivatives. Throughout the 100 ns simulation, the ligand-protein complexes maintained structural stability, with acceptable RMSD values. These compounds hold promise as potential leads in cancer therapy.

Real-time fluorescence imaging flow cytometry enabled by motion deblurring and deep learning algorithms.

Fluorescence imaging flow cytometry (IFC) has been demonstrated as a crucial biomedical technique for analyzing specific cell subpopulations from heterogeneous cellular populations. However, the high-speed flow of fluorescent cells leads to motion blur in cell images, making it challenging to identify cell types from the raw images. In this study, we present a real-time single-cell imaging and classification system based on a fluorescence microscope and deep learning algorithm, which is able to directly identify cell types from motion-blur images. To obtain annotated datasets of blurred images for deep learning model training, we developed a motion deblurring algorithm for the reconstruction of blur-free images. To demonstrate the ability of this system, deblurred images of HeLa cells with various fluorescent labels and HeLa cells at different cell cycle stages were acquired. The trained ResNet achieved a high accuracy of 96.6% for single-cell classification of HeLa cells in three different mitotic stages, with a short processing time of only 2 ms. This technology provides a simple way to realize single-cell fluorescence IFC and real-time cell classification, offering significant potential in various biological and medical applications.

Xenopus cell-free extracts and their applications in cell biology study.

Xenopus has proven to be a remarkably versatile model organism in the realm of biological research for numerous years, owing to its straightforward maintenance in laboratory settings and its abundant provision of ample-sized oocytes, eggs, and embryos. The cell cycle of these oocytes, eggs, and early embryos exhibits synchrony, and extracts derived from these cells serve various research purposes. Many fundamental concepts in biochemistry, cell biology, and development have been elucidated through the use of cell-free extracts derived from Xenopus cells. Over the past few decades, a wide array of cell-free extracts has been prepared from oocytes, eggs, and early embryos of different Xenopus species at varying cell cycle stages. Each of these extracts possesses distinct characteristics. This review provides a concise overview of the Xenopus species employed in laboratory research, the diverse types of cell-free extracts available, and their respective properties. Furthermore, this review delves into the extensive investigation of spindle assembly in Xenopus egg extracts, underscoring the versatility and potency of these cell-free systems in the realm of cell biology.

Full confluency, serum starvation, and roscovitine for inducing arrest in the G0/G1 phase of the cell cycle in puma skin-derived fibroblast lines

The puma population is constantly decreasing, and cloning by somatic cell nuclear transfer can be used to conserve the species. One of the factors determining the success of the development of cloned embryos is the cell cycle stage of the donor cells. We evaluated the effects of full confluency (~100%), serum starvation (0.5% serum), and roscovitine (15 µM) treatments on the cell cycle synchronization in G0/G1 of puma skin-derived fibroblasts by flow cytometric analysis. Also, we assessed the effects of these synchronization methods on morphology, viability, and apoptosis levels using microscopy tools. The results showed that culturing the cells to confluence for 24 h (84.0%), 48 h (84.6%), and 72 h (84.2%) and serum starvation for 96 h (85.4%) yielded a significantly higher percentage of cells arrested in the G0/G1 (P 0.05) phase than cells not subjected to any cell cycle synchronization method (73.9%). Nevertheless, while serum starvation reduced the percentage of viable cells, no difference was observed for the full confluence and roscovitine treatments (P 0.05). Moreover, roscovitine for 12 h (78.6%) and 24 h (82.1%) was unable to synchronize cells in G0/G1 (P 0.05). In summary, full confluency induces puma fibroblast cell cycle synchronization at the G0/G1 stage without affecting cell viability. These outcomes may be valuable for planning donor cells for somatic cell nuclear transfer in pumas.