Abstract Background Oral anticoagulation (OAC) with warfarin is underused for atrial fibrillation (AF). The availability of direct oral anticoagulants (DOACs) may improve overall OAC rates in AF ...patients, but a large-scale evaluation of their effects has not been conducted. Objectives This study assessed the effect of DOAC availability on overall OAC rates for nonvalvular AF. Methods Between April 1, 2008 and September 30, 2014, we identified 655,000 patients with nonvalvular AF and a CHA2 DS2 -VASc score of >1 in the National Cardiovascular Data Registry PINNACLE registry. Temporal trends in overall OAC and individual warfarin and DOAC use were analyzed. Multivariable hierarchical logistic regression identified patient factors associated with OAC and DOAC use. Practice variation of OAC and DOAC use was also assessed. Results Overall OAC rates increased from 52.4% to 60.7% among eligible AF patients (p for trend <0.01). Warfarin use decreased from 52.4% to 34.8% (p for trend <0.01), and DOAC use increased from 0% to 25.8% (p for trend <0.01). An increasing CHA2 DS2 -VASc score was associated with higher OAC use (odds ratio OR: 1.06; 95% confidence interval CI: 1.05 to 1.07), but with lower DOAC use (OR: 0.97; 95% CI: 0.96 to 0.98). Significant practice variation was present in OAC use (median odds ratio MOR: 1.52; 95% CI: 1.45 to 1.57) and in DOAC use (MOR: 3.58; 95% CI: 3.05 to 4.13). Conclusions Introduction of DOACs in routine practice was associated with improved rates of overall OAC use for AF, but significant gaps remain. In addition, there is significant practice-level variation in OAC and DOAC use.
Fluorescence microscopy is a powerful approach for studying subcellular dynamics at high spatiotemporal resolution; however, conventional fluorescence microscopy techniques are light-intensive and ...introduce unnecessary photodamage. Light-sheet fluorescence microscopy (LSFM) mitigates these problems by selectively illuminating the focal plane of the detection objective by using orthogonal excitation. Orthogonal excitation requires geometries that physically limit the detection objective numerical aperture (NA), thereby limiting both light-gathering efficiency (brightness) and native spatial resolution. We present a novel live-cell LSFM method, lateral interference tilted excitation (LITE), in which a tilted light sheet illuminates the detection objective focal plane without a sterically limiting illumination scheme. LITE is thus compatible with any detection objective, including oil immersion, without an upper NA limit. LITE combines the low photodamage of LSFM with high resolution, high brightness, and coverslip-based objectives. We demonstrate the utility of LITE for imaging animal, fungal, and plant model organisms over many hours at high spatiotemporal resolution.
As a dividing cell exits mitosis and daughter cells enter interphase, many proteins must be dephosphorylated. The protein phosphatase 2A (PP2A) with its B55 regulatory subunit plays a crucial role in ...this transition, but the identity of its substrates and how their dephosphorylation promotes mitotic exit are largely unknown. We conducted a maternal-effect screen in
to identify genes that function with PP2A-B55/Tws in the cell cycle. We found that eggs that receive reduced levels of Tws and of components of the nuclear envelope (NE) often fail development, concomitant with NE defects following meiosis and in syncytial mitoses. Our mechanistic studies using
cells indicate that PP2A-Tws promotes nuclear envelope reformation (NER) during mitotic exit by dephosphorylating BAF and suggests that PP2A-Tws targets additional NE components, including Lamin and Nup107. This work establishes
as a powerful model to further dissect the molecular mechanisms of NER and suggests additional roles of PP2A-Tws in the completion of meiosis and mitosis.
Constriction kinetics of the cytokinetic ring are expected to depend on dynamic adjustment of contractile ring composition, but the impact of ring component abundance dynamics on ring constriction is ...understudied. Computational models generally assume that contractile networks maintain constant total amounts of components, which is not always true. To test how compositional dynamics affect constriction kinetics, we first measured F-actin, non-muscle myosin II, septin, and anillin during Caenorhabditis elegans zygotic mitosis. A custom microfluidic device that positioned the cell with the division plane parallel to a light sheet allowed even illumination of the cytokinetic ring. Measured component abundances were implemented in a three-dimensional agent-based model of a membrane-associated contractile ring. With constant network component amounts, constriction completed with biologically unrealistic kinetics. However, imposing the measured changes in component quantities allowed this model to elicit realistic constriction kinetics. Simulated networks were more sensitive to changes in motor and filament amounts than those of crosslinkers and tethers. Our findings highlight the importance of network composition for actomyosin contraction kinetics.
Successive cell divisions during embryonic cleavage create increasingly smaller cells, so intracellular structures must adapt accordingly. Mitotic spindle size correlates with cell size, but the ...mechanisms for this scaling remain unclear. Using live cell imaging, we analyzed spindle scaling during embryo cleavage in the nematode Caenorhabditis elegans and sea urchin Paracentrotus lividus. We reveal a common scaling mechanism, where the growth rate of spindle microtubules scales with cell volume, which explains spindle shortening. Spindle assembly timing is, however, constant throughout successive divisions. Analyses in silico suggest that controlling the microtubule growth rate is sufficient to scale spindle length and maintain a constant assembly timing. We tested our in silico predictions to demonstrate that modulating cell volume or microtubule growth rate in vivo induces a proportional spindle size change. Our results suggest that scalability of the microtubule growth rate when cell size varies adapts spindle length to cell volume.
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•Microtubule dynamics scales with cell size during embryonic development•Microtubule growth rate controls spindle length adaptation to cell size•Timing of spindle assembly is constant and independent of spindle and cell size•Microtubule growth rate regulates spindle length and assembly timing
During early embryonic development, the mitotic spindle scales with decreasing cell size across successive reductive divisions. Lacroix et al. uncover, using in vivo and in silico analyses, a common scaling mechanism whereby spindle microtubule growth rate is coupled to cell volume for size scaling while maintaining constant spindle assembly timing.
Epigenetic mechanisms regulate genome activation in diverse events, including normal development and cancerous transformation. Centromeres are epigenetically designated chromosomal regions that ...maintain genomic stability by directing chromosome segregation during cell division. The histone H3 variant CENP-A resides specifically at centromeres, is fundamental to centromere function and is thought to act as the epigenetic mark defining centromere loci. Mechanisms directing assembly of CENP-A nucleosomes have recently emerged, but how CENP-A is maintained after assembly is unknown. Here, we show that a small GTPase switch functions to maintain newly assembled CENP-A nucleosomes. Using functional proteomics, we found that MgcRacGAP (a Rho family GTPase activating protein) interacts with the CENP-A licensing factor HsKNL2. High-resolution live-cell imaging assays, designed in this study, demonstrated that MgcRacGAP, the Rho family guanine nucleotide exchange factor (GEF) Ect2, and the small GTPases Cdc42 and Rac, are required for stability of newly incorporated CENP-A at centromeres. Thus, a small GTPase switch ensures epigenetic centromere maintenance after loading of new CENP-A.
Under replete growth conditions, abundant nutrient uptake leads to the systemic activation of insulin/IGF-1 signalling (IIS) and the promotion of stem cell growth/proliferation. Activated IIS can ...stimulate the ERK/MAPK pathway, the activation of which also supports optimal stem cell proliferation in various systems. Stem cell proliferation rates can further be locally refined to meet the resident tissue's need for differentiated progeny. We have recently shown that the accumulation of mature oocytes in the C. elegans germ line, through DAF-18/PTEN, inhibits adult germline stem cell (GSC) proliferation, despite high systemic IIS activation. We show here that this feedback occurs through a novel cryptic signalling pathway that requires PAR-4/LKB1, AAK-1/AMPK and PAR-5/14-3-3 to inhibit the activity of MPK-1/MAPK, antagonize IIS, and inhibit both GSC proliferation and the production of additional oocytes. Interestingly, our results imply that DAF-18/PTEN, through PAR-4/LKB1, can activate AAK-1/AMPK in the absence of apparent energy stress. As all components are conserved, similar signalling cascades may regulate stem cell activities in other organisms and be widely implicated in cancer.
During meiosis, each chromosome must selectively pair and synapse with its own unique homolog to enable crossover formation and subsequent segregation. How homolog pairing is maintained in early ...meiosis to ensure synapsis occurs exclusively between homologs is unknown. We aimed to further understand this process by examining the meiotic defects of a unique Drosophila mutant, Mcm5A7. We found that Mcm5A7 mutants are proficient in homolog pairing at meiotic onset yet fail to maintain pairing as meiotic synapsis ensues, causing seemingly normal synapsis between non-homologous loci. This pairing defect corresponds with a reduction of SMC1-dependent centromere clustering at meiotic onset. Overexpressing SMC1 in this mutant significantly restores centromere clustering, homolog pairing, and crossover formation. These data indicate that the initial meiotic pairing of homologs is not sufficient to yield synapsis exclusively between homologs and provide a model in which meiotic homolog pairing must be stabilized by centromeric SMC1 to ensure proper synapsis.
Centromeres provide a robust model for epigenetic inheritance as they are specified by sequence-independent mechanisms involving the histone H3-variant centromere protein A (CENP-A). Prevailing ...models indicate that the high intrinsic stability of CENP-A nucleosomes maintains centromere identity indefinitely. Here, we demonstrate that CENP-A is not stable at centromeres but is instead gradually and continuously incorporated in quiescent cells including G0-arrested tissue culture cells and prophase I-arrested oocytes. Quiescent CENP-A incorporation involves the canonical CENP-A deposition machinery but displays distinct requirements from cell cycle-dependent deposition. We demonstrate that Plk1 is required specifically for G1 CENP-A deposition, whereas transcription promotes CENP-A incorporation in quiescent oocytes. Preventing CENP-A deposition during quiescence results in significantly reduced CENP-A levels and perturbs chromosome segregation following the resumption of cell division. In contrast to quiescent cells, terminally differentiated cells fail to maintain CENP-A levels. Our work reveals that quiescent cells actively maintain centromere identity providing an indicator of proliferative potential.
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•CENP-A nucleosomes are gradually incorporated in quiescent cells and oocytes•CENP-A deposition during quiescence is required for future chromosome segregation•RNA Polymerase transcription at centromeres promotes gradual CENP-A exchange•Terminally differentiated muscle cells fail to retain CENP-A nucleosomes
Epigenetic marks must be retained during extended periods of quiescence to ensure the proper function of genomic loci. Although centromeric CENP-A nucleosomes were thought to be immobile, Swartz et al. identify gradual CENP-A deposition in quiescent cells and oocytes. CENP-A exchange is essential for faithful cell division following long arrests.
Multicellular development requires that cells reduce in size as a result of consecutive cell divisions without increase in embryo volume. To maintain cellular integrity, organelle size adapts to cell ...size throughout development. During mitosis, the longest chromosome arm must be shorter than half of the mitotic spindle for proper chromosome segregation. Using high-resolution time-lapse microscopy of living Caenorhabditis elegans embryos, we have quantified the relation between cell size and chromosome length. In control embryos, chromosome length scaled to cell size. Artificial reduction of cell size resulted in a shortening of chromosome length, following a trend predicted by measurements from control embryos. Disturbing the RAN (Ras-related nuclear protein)-GTP gradient decoupled nuclear size from cell size and resulted in chromosome scaling to nuclear size rather than cell size; smaller nuclei contained shorter chromosomes independent of cell size. In sum, quantitative analysis relating cell, nuclear, and chromosome size predicts two levels of chromosome length regulation: one through cell size and a second in response to nuclear size.
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