CDK1 is a nonredundant cyclin-dependent kinase (CDK) with an essential role in mitosis, but its multiple functions still are poorly understood at a molecular level. Here we identify a selective small-molecule inhibitor of CDK1 that reversibly arrests human cells at the G 2͞M border of the cell cycle and allows for effective cell synchronization in early mitosis. Inhibition of CDK1 during cell division revealed that its activity is necessary and sufficient for maintaining the mitotic state of the cells, preventing replication origin licensing and premature cytokinesis. Although CDK1 inhibition for up to 24 h is well tolerated, longer exposure to the inhibitor induces apoptosis in tumor cells, suggesting that selective CDK1 inhibitors may have utility in cancer therapy.apoptosis ͉ cancer therapy ͉ cell cycle ͉ cytokinesis ͉ replication origin T he cell cycle is a precisely controlled set of biochemical and morphological events driven by the sequential activation of cyclin-dependent kinases (CDKs) (1). Three CDKs and their activating cyclins (A, B, D, and E) play key roles in mammalian cell cycle regulation (2). It has been established that CDK4͞ cyclin D and CDK2͞cyclin E͞A promote the passage through G 1 and S phases, whereas CDK1͞cyclin B regulates the transition through late G 2 and mitosis (3). However, recent genetic and RNA interference studies in mammalian cells have revealed that CDK2 and CDK4 are not essential for cell cycle progression, thus leaving CDK1 as the only nonredundant cell cycle driver (4-6). Genetic studies in yeast and mammalian cells have established the critical role of CDK1 in mitosis, but the long list of its putative substrate proteins still is growing, and its precise functions in the context of the dividing cell are poorly understood (7,8). This deficiency is due, in part, to the lack of specific molecular tools for reversible modulation of CDK1 activity in vivo. Several potent small-molecule inhibitors have been reported, but their activity and cell cycle profiles are not consistent with specific CDK1 inhibition (9, 10). Here, we identify a selective and reversible inhibitor of the catalytic activity of human CDK1͞cyclin B1 and CDK1͞cyclin A complexes that allows synchronization of proliferating cells in the late G 2 phase and probing of CDK1 function in the cellular context. Results and DiscussionWe screened a diverse library of organic compounds for their ability to inhibit the catalytic activity of human CDK1͞cyclin B1. The hits then were tested for selectivity against CDK2͞cyclin E and CDK4͞cyclin D. A class of thiazolinone analogs emerged as a source of ATP-competitive CDK1 inhibitors that were then further optimized for potency, selectivity, and ability to modulate CDK1 in proliferating cells. One quinolinyl thiazolinone derivative, RO-3306, showed good potency, in vitro selectivity, and a cell cycle profile (G 2 ͞M arrest) consistent with CDK1 inhibition (Fig. 1A). RO-3306 inhibited CDK1͞cyclin B1 activity with K i of 35 nM, nearly 10-fold selectivity relative to CDK2͞ cyclin ...
Cities are considered major contributors to global warming, where carbon emissions are highly embedded in the overall urban metabolism. To examine urban metabolic processes and emission trajectories we developed a carbon flux model based on Network Environ Analysis (NEA). The mutual interactions and control situation within the urban ecosystem of Vienna were examined, and the system-level properties of the city's carbon metabolism were assessed. Regulatory strategies to minimize carbon emissions were identified through the tracking of the possible pathways that affect these emission trajectories. Our findings suggest that indirect flows have a strong bearing on the mutual and control relationships between urban sectors. The metabolism of a city is considered self-mutualistic and sustainable only when the local and distal environments are embraced. Energy production and construction were found to be two factors with a major impact on carbon emissions, and whose regulation is only effective via ad-hoc pathways. In comparison with the original life-cycle tracking, the application of NEA was better at revealing details from a mechanistic aspect, which is crucial for informed sustainable urban management.
Insufficient drug release as well as poor drug penetration are major obstacles for effective nanoparticles (NPs)-based cancer therapy. Herein, the high aqueous instability of amorphous calcium carbonate (ACC) is employed to construct doxorubicin (DOX) preloaded and monostearin (MS) coated "Pandora's box" (MS/ACC-DOX) NPs for lipase-triggered water-responsive drug release in lipase-overexpressed tumor tissue to induce a neighboring effect and enhance drug penetration. MS as a solid lipid can prevent potential drug leakage of ACC-DOX NPs during the circulatory process, while it can be readily be disintegrated in lipase-overexpressed SKOV3 cells to expose the ACC-DOX core. The high aqueous instability of ACC will lead to burst release of the encapsulated DOX to induce apoptosis and cytotoxicity to kill the tumor cells. The liberated NPs from the dead or dying cells continue to respond to the ubiquitous aqueous environment to sufficiently release DOX once unpacked, like the "Pandora's box", leading to severe cytotoxicity to neighboring cells (neighboring effect). Moreover, the continuously released free DOX molecules can readily diffused through the tumor extracellular matrix to enhance drug penetration to deep tumor tissue. Both effects contribute to achieve elevated antitumor benefits.
Catalysts are urgently needed to remove the residual CO in hydrogen feeds through selective oxidation for large-scale applications of hydrogen proton exchange membrane fuel cells. We herein propose a new methodology that anchors high concentration oxygen vacancies at interface by designing a CeO2-x/Cu hybrid catalyst with enhanced preferential CO oxidation activity. This hybrid catalyst, with more than 6.1% oxygen vacancies fixed at the favorable interfacial sites, displays nearly 100% CO conversion efficiency in H2-rich streams over a broad temperature window from 120 to 210 °C, strikingly 5-fold wider than that of conventional CeO2/Cu (i.e., CeO2 supported on Cu) catalyst. Moreover, the catalyst exhibits a highest cycling stability ever reported, showing no deterioration after five cycling tests, and a super long-time stability beyond 100 h in the simulated operation environment that involves CO2 and H2O. On the basis of an arsenal of characterization techniques, we clearly show that the anchored oxygen vacancies are generated as a consequence of electron donation from metal copper atoms to CeO2 acceptor and the subsequent reverse spillover of oxygen induced by electron transfer in well controlled nanoheterojunction. The anchored oxygen vacancies play a bridging role in electron capture or transfer and drive molecule oxygen into active oxygen species to interact with the CO molecules adsorbed at interfaces, thus leading to an excellent preferential CO oxidation performance. This study opens a window to design a vast number of high-performance metal-oxide hybrid catalysts via the concept of anchoring oxygen vacancies at interfaces.
Solid lipid nanoparticles (SLNs) have been extensively investigated and demonstrated to be a potential nanocarriers for improving oral bioavailability of many drugs. However, the molecular mechanisms related to this discovery are not yet understood. Here, the molecular transport mechanisms of the SLNs crossing simulative intestinal epithelial cell monolayers (Caco-2 cell monolayers) were studied. The cytotoxicology results of the SLNs in Caco-2 cells demonstrated that the nanoparticles had low cytotoxicity, had no effect on the integrity of the cell membrane, did not induce oxidative stress, and could significantly reduce cell membrane fluidity. The endocytosis of the SLNs was time-dependent, and their delivery was energy-dependent. For the first time, the transport of the SLNs was directly verified to be a vesicle-mediated process. The internalization of the SLNs was mediated by macropinocytosis pathway and clathrin- and caveolae (or lipid raft)-related routes. Transferrin-related endosomes, lysosomes, endoplasmic reticulum (ER), and Golgi apparatus were confirmed to be the main destinations of the SLNs in Caco-2 cells. As for the transport of the SLNs in Caco-2 cell monolayers, the results demonstrated that the SLNs transported to the basolateral side were intact, and the transport of the nanoparticles did not destroy the structure of tight junctions. The transcytosis of the SLNs across the Caco-2 cell monolayer was demonstrated to be mediated by the same routes as that in the endocytosis study. The ER, Golgi apparatus, and microtubules were confirmed to be important for the transport of the SLNs to both the basolateral and apical membrane sides. This study provides a more thoroughly understand of SLNs transportation crossing intestinal epithelial cell monolayers and could be beneficial for the fabrication of SLNs.
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