Search Results (105)

RBL1 (p107) is a member of the retinoblastoma (RB) family of pocket proteins involved in cell cycle regulation and E2F transcriptional repression. While its promoter contains conserved E2F motifs, the integrated regulation of RBL1 by upstream tumor suppressor pathways remains incompletely understood. Here, we investigate the p53-dependent transcriptional regulation of RBL1 and dissect the contribution of its tandem E2F binding sites to this mechanism. Luciferase assays in synchronized cells demonstrated that these two conserved E2F sites are required for cell cycle-dependent activation of the RBL1 promoter. Overexpression of p53 showed that p53 represses RBL1 promoter activity in an E2F site-dependent manner. Using HCT116 p21 knockout cells, we revealed that this p53-dependent repression is mediated by p21. Chromatin immunoprecipitation confirmed dynamic in vivo binding of E2F1–3 and E2F4, while DNA pull-down assays revealed specific in vitro recruitment of RB, p107, and E2F1-4 to the two E2F sites, along with weak binding of MuvB components. Additional experiments in RB^–/– and LIN37^–/– knockouts showed that RB/E2F repressing complex plays the main role in repressing the RBL1 promoter, while E2F4, p107, and p130 can support this effect to a lesser extent. Overall, our findings demonstrate that p53 controls RBL1 expression indirectly through the p21–RB–E2F pathway by utilizing two E2F binding sites within the RBL1 promoter. Full article

Considering the world’s growing interest in health-promoting phytochemicals, the current research investigated the development of a dual-captured Ginkgo Biloba and Green Tea Extract into Collagen-Nanostructured Lipid Nanocarriers (Col-NLC-GBil-GTE) for an enhanced therapeutic efficacy against hepatic, colon or breast cancer. NLC considerably reduced cell viability; the most advanced cytotoxicity profile was determined on human colon adenocarcinoma cells (LoVo) and liver cancer cells (HepG2), e.g., tumor cell viability was 21.81% in the presence of Col-NLC-GBil-GTE, similar to that determined for Cisplatin. Col-NLC exhibited apoptosis in HepG2 and LoVo cells and no significant apoptosis induction in normal HUVECs. A 20% increase in apoptosis for HepG2 cells was registered for 100 μg/mL NLC-GBil-GTE compared to Cisplatin (Cis-Pt), e.g., a 63.4% total apoptosis for NLC-GBil-GTE versus a 52.6 apoptosis induced by 100 μg/mL of a chemotherapeutic drug. According to the cell cycle outcomes, an accumulation of hepatocyte HepG2 tumor cells in the G0/G1 phase was detected upon treatment with 100 mg/mL of NLC- and Col-NLC-GBil-GTE, simultaneously with a drastic decrease in the S phase, which may indicate a cell number reduction that enters in the division cycle. The simultaneous delivery of GBil and GTE by synchronizing their bioactivities offers several advantages; Col-NLC-GBil-GTE can be viewed as a noteworthy strategy for consideration in connection with antitumor therapeutic protocols. Full article

Gene expression measurements of tissues, tumors, or cell lines taken over multiple time points are valuable for describing dynamic biological phenomena such as the response to growth factors. However, such phenomena typically involve multiple biological processes occurring in parallel, making it difficult to identify and discern their respective contributions at any time point. Here, we demonstrate the use of unsupervised machine learning to deconvolve a series of time-dependent gene expression measurements into its underlying temporal components. We first downloaded publicly available RNAseq data obtained from synchronized HeLa cells at consecutive time points following release from cell cycle arrest. Then, we used Fourier analysis and Topic modeling to reveal three underlying components and their relative contributions at each time point. We identified two temporal components with oscillatory behavior, corresponding to the G1-S and G2-M phases of the cell cycle, and a third component with a transient expression pattern, associated with the immediate early response gene program, regulation of cell proliferation, and cervical cancer. This study demonstrates the use of unsupervised machine learning to identify hidden temporal components in biological systems, with potential applications to early detection and monitoring of diseases and recovery processes. Full article

There is a clinical need for improved antenatal diagnosis of congenital heart disease (CHD). Increasing numbers of children are born to parents undergoing fertility treatment. We asked whether time-lapse imaging of in vitro preimplantation development provides diagnostic information. We performed a retrospective multicentre analysis of morphokinetic data from patients undergoing fertility treatment. A total of 96/18,799 CHD cases were identified (rate: 0.51%). Thirty-two were included in the analysis and stratified into three cohorts: complex CHD (n = 7), mild CHD (n = 11) and murmur only (n = 14). Comparison to a large unmatched control group (n = 352) revealed no differences in time of preimplantation developmental events but suggested an increase in cleavage synchronicity during the third cell cycle of mild CHD embryos. Pairwise comparison to matched controls revealed a delay in mild CHD embryos relative to controls in reaching the morphokinetic timepoints fading of pronuclei, 2-cell stage and 4-cell stage together with a possible increase in duration of blastulation in complex CHD. Our data raises the possibility that screening of preimplantation embryos at fertility clinics could reduce the rate of CHD. However, these results are preliminary, and further work is required to confirm the findings in a larger study. Full article

The development of non-toxic silver nanoparticles (AgNPs) for medical and other diverse applications is steadily increasing. However, this study specifically aims to determine the cytotoxic effects of AgNPs synthesized via a green chemistry approach using aqueous-ethanol and ethyl acetate extracts of Artemisia terrae-albae. The photophysical, morphological, and size distribution characteristics of the synthesized AgNPs are analyzed using UV-Vis spectroscopy and transmission electron microscopy (TEM). A modified Allium cepa assay is employed to evaluate biological responses, including root growth, root number, and mitotic index. In this assay, the cell cycles of onion bulbs are synchronized and pre-incubated at 4 °C for 72 h prior to treatment. This study reveals that the AgNPs synthesized from the ethanol extract exhibit notable stability and higher cytotoxicity activity, with a root length of 0.6 ± 0.13 cm, root number of 16 ± 6.88, and mitotic index of 25.0 ± 2.6. These values are significantly more cytogenotoxic than those observed for the ethyl-acetate-derived nanoparticles, which show a root length of 0.8 ± 0.17 cm, root number of 18 ± 6.27, and mitotic index of 36 ± 3.6. These findings highlight the potential of green-synthesized AgNPs as effective cytotoxic agents, especially those obtained from ethanol extract, possibly due to a greater influence of the quantity of diverse phenolic compounds present in the complex mixtures than in the ethyl acetate extract, which otherwise enhanced their morphology, shape, and size. These, overall, contributed to the biological activity. Full article

The circadian clock is a self-sustaining oscillator with a period of approximately 24 h, enabling organisms to anticipate daily recurring events, such as sunrise and sunset. Since the circadian period is not exactly 24 h and the environmental day length varies throughout the year, the clock must be periodically reset to align an organism’s physiology with the natural light/dark cycle. This synchronization, known as entrainment, is primarily regulated by nocturnal light, which can be replicated in laboratory settings using a 15 min light pulse (LP) and by assessing locomotor activity. An LP during the early part of the dark phase delays the onset of locomotor activity, resulting in a phase delay, whereas an LP in the late dark phase advances activity onset, causing a phase advance. The clock gene Period 2 (Per2) plays a key role in this process. To investigate its contributions, we examined the effects of Per2 deletion in neurons versus astrocytes using glia-specific GPer2 (Per2/GfapCre) knockout (KO) and neuronal-specific NPer2KO (Per2/NesCre) mice. All groups were subjected to Aschoff type II protocol, where an LP was applied at ZT14 or ZT22 and the animals were released into constant darkness. As control, no LP was applied. Phase shift, period, amplitude, total activity count, and rhythm instability were assessed. Our findings revealed that mice lacking Per2 in neurons (NPer2) exhibited smaller phase delays and larger phase advances compared to control animals. In contrast, mice with Per2 deletion specifically in glial cells including astrocytes (GPer2) displayed normal clock resetting. Interestingly, the absence of Per2 in either of the cell types resulted in a shorter circadian period compared to control animals. These results suggest that astrocytic Per2 is important for maintaining the circadian period but is not required for phase adaptation to light stimuli. Full article

Dictyostelium discoideum (Dicty) is a type of unicellular amoeba, but when starved, a large number of amoebas gather together to form a multicellular organism. In this review, we first introduce our cellular dynamics method for Dicty, including intracellular biochemical reactions. We then introduce a number of hidden roles of receptors revealed by our simulation studies. Of particular note is that receptor–receptor interactions are strengthened under starvation conditions, resulting in diverse dynamic functions that cannot be predicted from the action of a single receptor, such as intercellular synchronization. Furthermore, we introduce a mathematical generalization of Dicty’s receptor function and demonstrate its potential applications not only in the biological field but also in the engineering field. Full article

Mesenchymal stem cells (MSCs) have a broad clinical potential, but their selection and expansion on plastic cause unknown purity and phenotypic alterations, reducing therapy efficiency. Furthermore, their behavior in non-adherent conditions during systemic transplantation remains poorly understood. The sphere formation from single cells is commonly used to assess stemness, but MSCs lack this ability, raising questions about their anchorage dependence for proliferation. We investigated whether bone marrow-derived MSCs can complete cytokinesis in non-adherent environments. Primary human and mouse bone marrow-derived MSCs were synchronized in early mitosis using nocodazole and were cultured on soft, rigid, or non-adherent surfaces. Both human and mouse MSCs displayed an ALIX (abscission licensor) recruitment to the midbody 40–90 min post-nocodazole release, regardless of the substrate adherence. Cells maintained for 4hr in the suspension remained viable, and daughter cells rapidly migrated apart upon the re-adhesion to fibronectin-coated surfaces, demonstrating cytokinesis completion in suspension. These findings distinguish MSCs from fibroblasts (which require adhesion for division), provide a more general stemness feature, and suggest that adhesion-independent cytokinesis is a trait relevant to the post-transplantation survival and tissue homing. This property may offer strategies to expand MSCs with an improved purity and functionality and to enhance engraftment by leveraging cell cycle manipulation to promote an early extracellular matrix deposition at target sites. Full article

Studying the cell cycle is essential for understanding the molecular mechanisms that regulate cell division, growth, and differentiation in living organisms. However, mitosis constitutes only a brief phase of the overall cell cycle, making its analysis challenging in asynchronous cell populations due to its transient and dynamic nature. Cell synchronization methods help to enrich populations at specific cell cycle stages, including mitosis, typically by using chemical inhibitors to arrest cells at defined checkpoints. However, many existing protocols rely on combinations of inhibitors that interfere with normal mitotic progression, disrupting dynamics and causing side effects such as chromosome non-disjunction or lagging chromosomes, which limit their applicability. In this study, we present an RO3306 block-and-release strategy to selectively enrich cell populations at defined mitotic stages without compromising cell viability or disrupting their progression to mitotic exit. This approach provides a reliable method for studying mitotic events with high temporal resolution. Furthermore, by preserving mitotic integrity, it offers a valuable framework for investigating the molecular mechanisms of cell division and the processes driving genomic instability in human cells. Full article

CRISPR–Cas9 is a powerful genome-editing technology that can precisely target and cleave DNA to induce double-strand breaks (DSBs) at almost any genomic locus. While this versatility holds tremendous therapeutic potential, the predominant cellular pathway for DSB repair—non-homologous end-joining (NHEJ)—often introduces small insertions or deletions that disrupt the target site. In contrast, homology-directed repair (HDR) utilizes exogenous donor templates to enable precise gene modifications, including targeted insertions, deletions, and substitutions. However, HDR remains relatively inefficient compared to NHEJ, especially in postmitotic cells where cell cycle constraints further limit HDR. To address this challenge, numerous methodologies have been explored, ranging from inhibiting key NHEJ factors and optimizing donor templates to synchronizing cells in HDR-permissive phases and engineering HDR-enhancing fusion proteins. These strategies collectively aim to boost HDR efficiency and expand the clinical and research utility of CRISPR–Cas9. In this review, we discuss recent advances in manipulating the balance between NHEJ and HDR, examine the trade-offs and practical considerations of these approaches, and highlight promising directions for achieving high-fidelity genome editing in diverse cell types. Full article

This paper presents a flexible and scalable battery system for maritime transportation, integrating modular converters and hybrid battery technologies that are effectively implemented in real-world scenarios. The proposed system is realized with modular DC-DC converters, which do not require complex design and control or a high number of components and combine high-power (HP) and high-energy (HE) battery cells to optimize the energy and power requirements of vessel operations without oversizing the energy storage system. Moreover, the modular design ensures flexibility and scalability, allowing for easy adaptation to varying operational demands. In particular, the system topology, control mechanisms, and communication protocols are explained in this paper. The concept has been validated through simulations and real-scale laboratory tests, demonstrating its effectiveness. Key results highlight the system’s ability to maintain the DC bus voltage while operating at high efficiency (ranging from 97% to 98%) under different load conditions, supported by reliable and demanding real-time communication using the EtherCAT standard. This real-time capability has been validated, and related results are presented in this paper, showing a synchronization accuracy below 200 ns between two modules and a stable control at a cycle time of 400 µs. This approach offers a promising solution for reducing greenhouse gas emissions in the maritime industry, aligning with global sustainability goals. Full article

The suprachiasmatic nucleus (SCN) is the master regulator of the circadian system, modulating the daily timing of physiological and behavioral processes in mammals. While SCN synchronization is primarily driven by environmental light signals, sex hormones, particularly androgens, have a crucial role in regulating behavioral and reproductive processes to align with daily or seasonal cycles. SCN cell populations express receptors for sex steroid hormones, contributing to circadian synchronization mechanisms. Specifically, the activation of androgen receptors in the SCN has been shown to modulate clock gene expression and influence circadian rhythms. Rabbits, widely used in experimental research, exhibit unique behavioral patterns, including plasticity in circadian typology and seasonal variations in testosterone secretion. In this study, we explored, in male rabbits, the effect of castration on the daily pattern of locomotor activity and the expression of the clock protein PERIOD 1 (PER1) in the SCN. Our results show that castration significantly reduces daily locomotor activity and PER1 expression in the SCN. Moreover, a 4 h delay in the acrophase of PER1 expression was observed. We conclude that androgens have an important role in SCN synchronization mechanisms, contributing to the organization of physiological and behavioral events in this species. Full article

Our investigation into Tisochrysis lutea’s cell cycle regulation involved natural and chemical synchronization methods to maximize their proportion at the division phase (G_2/M). Hence, cultures were grown under different light/dark cycles (24:0, 12:12, and 8:16 h) to assess the impact of extended dark periods on cell division. Flow cytometry analyses of the cell cycle revealed that extending the dark phase resulted in a higher number of cells entering G_2/M. However, this remained a minority within the overall culture (peaking at 19.36% ± 0.17 under an 8:16 h L/D cycle). To further enhance synchronization, chemical agents (nocodazole, hydroxyurea, and aphidicolin) were tested for their efficacy in blocking specific cell cycle stages. Only aphidicolin successfully induced significant G_2/M accumulation (>90%). The commitment point for cell division was examined by exposing cultures to varying light durations (0 to 8 h) and measuring cell concentration and size distribution every 4 h. Our findings identified a critical minimum cell size (“sizer”) of approximately 56.2 ± 0.6 µm³ and a required minimal light exposure (“timer”) of 4 h to reliably trigger cell division. These findings highlight key conditions needed for optimal division of Tisochrysis lutea, offering more controlled and efficient cultivation strategies for future biotechnological applications. Full article

Dinoflagellate birefringent chromosomes (BfCs) contain some of the largest known genomes, yet they lack typical nucleosomal micrococcal-nuclease protection patterns despite containing variant core histones. One BfC end interacts with extranuclear mitotic microtubules at the nuclear envelope (NE), which remains intact throughout the cell cycle. Ultrastructural studies, polarized light and fluorescence microscopy, and micrococcal nuclease-resistant profiles (MNRPs) revealed that NE-associated chromosome ends persisted post-mitosis. Histone H3K9me3 inhibition caused S-G₂ delay in synchronous cells, without any effects at G₁. Differential labeling and nuclear envelope swelling upon decompaction indicate an extension of the inner compartment into telosomal anchorages (TAs). Additionally, limited effects of low-concentration sirtinol on bulk BfCs, coupled with distinct mobility patterns in MNase-digested and psoralen-crosslinked nuclei observed on 2D gels, suggest that telomeric nucleosomes (TNs) are the primary histone structures. The absence of a nucleosomal ladder with cDNA probes, the presence of histone H2A and telomere-enriched H3.3 variants, along with the immuno-localization of H3 variants mainly at the NE further reinforce telomeric regions as the main nucleosomal domains. Cumulative biochemical and molecular analyses suggest that telomeric repeats constitute the major octameric MNRPs that provision chromosomal anchorage at the NE. Full article

As the most promising high energy density technology, lithium metal batteries are associated with serious interfacial challenges because the electrolytes employed are unable to meet the requirements of both electrodes simultaneously, namely, the systems that work for Li metal are highly likely to be unsuitable for the cathode, and vice versa. In this study, we investigate the synergistic effects of lithium bis (oxalate) borate (LiBOB), fluoroethylene carbonate (FEC) and adiponitrile (ADN) to develop a formula that is compatible with both elements in the battery. The solid–electrolyte interphase (SEI) multi-layer generated from LiBOB and FEC successfully protects the electrolyte from the lithium and suppresses the decomposition of ADN on lithium, identified by the tiny amounts of isonitriles on the surface of the anode. Simultaneously, most of the ADN molecules remain and protect the cathode particles via the absorption layer of the nitrile groups, in the same way that this process works in commercial lithium-ion batteries. Benefiting from the stable interfacial films formed synchronously on the anode and cathode, the Li/LiNi_0.8Co_0.1Mn_0.1O₂ cells with an area capacity of ~3 mAh cm⁻² operate stably beyond 250 cycles and target the accumulated capacity to levels as high as ~653.4 mAh cm⁻². Our approach demonstrates that electrolyte engineering with known additives is a practical strategy for addressing the challenges of lithium batteries. Full article