Cell size uniformity in healthy tissues suggests that control mechanisms might coordinate cell growth and division. We derived a method to assay whether cellular growth rates depend on cell size, by ...monitoring how variance in size changes as cells grow. Our data revealed that, twice during the cell cycle, growth rates are selectively increased in small cells and reduced in large cells, ensuring cell size uniformity. This regulation was also observed directly by monitoring nuclear growth in live cells. We also detected cell-size-dependent adjustments of G1 length, which further reduce variability. Combining our assays with chemical/genetic perturbations confirmed that cells employ two strategies, adjusting both cell cycle length and growth rate, to maintain the appropriate size. Additionally, although Rb signaling is not required for these regulatory behaviors, perturbing Cdk4 activity still influences cell size, suggesting that the Cdk4 pathway may play a role in designating the cell's target size.
While multiplexing samples using DNA barcoding revolutionized the pace of biomedical discovery, multiplexing of live imaging-based applications has been limited by the number of fluorescent proteins ...that can be deconvoluted using common microscopy equipment. To address this limitation, we develop visual barcodes that discriminate the clonal identity of single cells by different fluorescent proteins that are targeted to specific subcellular locations. We demonstrate that deconvolution of these barcodes is highly accurate and robust to many cellular perturbations. We then use visual barcodes to generate 'Signalome' cell-lines by mixing 12 clones of different live reporters into a single population, allowing simultaneous monitoring of the activity in 12 branches of signaling, at clonal resolution, over time. Using the 'Signalome' we identify two distinct clusters of signaling pathways that balance growth and proliferation, emphasizing the importance of growth homeostasis as a central organizing principle in cancer signaling. The ability to multiplex samples in live imaging applications, both in vitro and in vivo may allow better high-content characterization of complex biological systems.
Catheter ablation for atrial fibrillation (AF) has emerged as a popular procedure. The purpose of this study was to examine whether there exist differences or disparities in ablation utilization ...across gender, socioeconomic class, insurance, or race. Using the Nationwide Inpatient Sample (2000 to 2012), we identified adults hospitalized with a principal diagnosis of AF by ICD 9 code 427.31 who had catheter ablation (ICD 9 code–37.34). We stratified patients by race, insurance status, age, gender, and hospital characteristics. A hierarchical multivariate mixed-effect model was created to identify the independent predictors of AF ablation. Among an estimated total of 3,508,122 patients (extrapolated from 20% Nationwide Inpatient Sample) hospitalized with a diagnosis of AF in the United States from the year 2000 to 2012, 102,469 patients (2.9%) underwent catheter ablations. The number of ablations was increased by 940%, from 1,439 in 2000 to 15,090 in 2012. There were significant differences according to gender, race, and health insurance status, which persisted even after adjustment for other risk factors. Female gender (0.83 95% CI 0.79 to 0.87; p <0.001), black (0.49 95% CI 0.44 to 0.55; p <0.001), and Hispanic race (0.64 95% CI 0.56 to 0.72; p <0.001) were associated with lower likelihoods of undergoing an AF ablation. Medicare (0.93, 0.88 to 0.98, <0.001) or Medicaid (0.67, 0.59 to 0.76, <0.001) coverage and uninsured patients (0.55, 0.49 to 0.62, <0.001) also had lower rates of AF ablation compared to patients with private insurance. In conclusion we found differences in utilization of catheter ablation for AF based on gender, race, and insurance status that persisted over time.
Animal cells within a tissue typically display a striking regularity in their size. To date, the molecular mechanisms that control this uniformity are still unknown. We have previously shown that ...size uniformity in animal cells is promoted, in part, by size-dependent regulation of G1 length. To identify the molecular mechanisms underlying this process, we performed a large-scale small molecule screen and found that the p38 MAPK pathway is involved in coordinating cell size and cell cycle progression. Small cells display higher p38 activity and spend more time in G1 than larger cells. Inhibition of p38 MAPK leads to loss of the compensatory G1 length extension in small cells, resulting in faster proliferation, smaller cell size and increased size heterogeneity. We propose a model wherein the p38 pathway responds to changes in cell size and regulates G1 exit accordingly, to increase cell size uniformity.
Transcatheter aortic valve implantation (TAVI) is associated with a significant learning curve. There is paucity of data regarding the effect of hospital volume on outcomes after TAVI. This is a ...cross-sectional study based on Healthcare Cost and Utilization Project's Nationwide Inpatient Sample database of 2012. Subjects were identified by International Classification of Diseases, Ninth Revision, Clinical Modification procedure codes, 35.05 (Trans-femoral/Trans-aortic Replacement of Aortic Valve) and 35.06 (Trans-apical Replacement of Aortic Valve). Annual hospital TAVI volumes were calculated using unique identification numbers and then divided into quartiles. Multivariate logistic regression models were created. The primary outcome was inhospital mortality; secondary outcome was a composite of inhospital mortality and periprocedural complications. Length of stay (LOS) and cost of hospitalization were assessed. The study included 1,481 TAVIs (weighted n = 7,405). Overall inhospital mortality rate was 5.1%, postprocedural complication rate was 43.4%, median LOS was 6 days, and median cost of hospitalization was $51,975. Inhospital mortality rates decreased with increasing hospital TAVI volume with a rate of 6.4% for lowest volume hospitals (first quartile), 5.9% (second quartile), 5.2% (third quartile), and 2.8% for the highest volume TAVI hospitals (fourth quartile). Complication rates were significantly higher in hospitals with the lowest volume quartile (48.5%) compared to hospitals in the second (44.2%), third (39.7%), and fourth (41.5%) quartiles (p <0.001). Increasing hospital volume was independently predictive of shorter LOS and lower hospitalization costs. In conclusion, higher annual hospital volumes are significantly predictive of reduced postprocedural mortality, complications, shorter LOS, and lower hospitalization costs after TAVI.
Secreted Wnt proteins regulate development and adult tissue homeostasis by binding and activating cell-surface Frizzled receptors and co-receptors including LRP5/6. The hydrophobicity of Wnt proteins ...has complicated their purification and limited their use in basic research and as therapeutics. We describe modular tetravalent antibodies that can recruit Frizzled and LRP5/6 in a manner that phenocopies the activities of Wnts both in vitro and in vivo. The modular nature of these synthetic Frizzled and LRP5/6 Agonists, called FLAgs, enables tailored engineering of specificity for one, two or multiple members of the Frizzled family. We show that FLAgs underlie differentiation of pluripotent stem cells, sustain organoid growth, and activate stem cells in vivo. Activation of Wnt signaling circuits with tailored FLAgs will enable precise delineation of functional outcomes directed by distinct receptor combinations and could provide a new class of therapeutics to unlock the promise of regenerative medicine.
GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but ...the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.
Humanized monoclonal antibodies (mAb) against HER2 are being engineered to treat cancer. We utilized phage-display technology to generate a novel anti-HER2 mAb (named 73JIgG) that binds an epitope of ...HER2 distinct from that of trastuzumab. Although these mAbs bind to the same cell surface receptor, they have different cell distribution profiles. After 3h of incubation, almost 10% of the total 73JIgG reaches the lysosome compared to less than 3% of trastuzumab. Interestingly, 73JIgG disassociates from HER2 whereas trastuzumab remains bound to the receptor. Importantly, HER2 distribution is not affected by the antibody binding epitope, thus negating this mechanism as the reason for the difference in intracellular trafficking of 73JIgG versus trastuzumab. Each of trastuzumab and 73JIgG was chemically-modified with either a small molecule or polymeric nanoparticle to better understand the influence of conjugation on cellular localization. Relative to antibody alone, antibody–nanoparticle conjugates resulted in a higher concentration of antibodies in the lysosome whereas antibody–small molecule conjugates did not affect cell trafficking to the lysosome. Given the importance of lysosomal targeting, these results demonstrate the importance of understanding the influence of the antibody-conjugate on cell trafficking for ultimate optimization of treatment selection.
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Abstract Since the introduction of new antiplatelet and anticoagulant agents in the last decade, large-scale data studying gastrointestinal bleeding (GIB) in patients undergoing percutaneous coronary ...intervention (PCI) are lacking. Using the Nationwide Inpatient Sample, we identified all hospitalizations from 2006 to 2012 that required PCI. Temporal trends in the incidence and multivariate predictors of GIB associated with PCI were analyzed. A total of 4,376,950 patients underwent PCI in the United States during the study period. The incidence of GIB was 1.1%. Mortality in the GIB group was significantly higher (9.71% vs. 1.1%, p < 0.0001). Although the incidence of GIB remained stable during the study period (0.97% in 2006 to 1.19% in 2012), in-hospital mortality increased significantly from 7.9% in 2006 to 10.78% in 2012, with a peak of 12% in 2010. The GIB group had a longer median length of stay (5.80 vs. 1.57 days) as well as an increased median cost of hospitalization ($26,564 vs. $16,879). The predictors of GIB included cardiovascular comorbidities such as acute myocardial infarction, cardiogenic shock, atrial fibrillation, congestive heart failure, valvular heart diseases and history of transient ischemic attack/stroke. Gastrointestinal comorbidities including diverticulosis, esophageal cancer, stomach cancer, small intestine cancer, large intestine cancer, recto-sigmoid cancer, gastrointestinal ulcer and liver disease were predictors of GIB. Interestingly, a lower risk of GIB was associated with obese patients and patients with private insurance. A higher risk of GIB was noted in urgent vs. elective admissions and weekend vs. weekday admissions. In conclusion, the incidence of GIB in patients who underwent PCI remained stable from 2006 to 2012, however the in-hospital mortality increased significantly. Identifying patients at higher risk for GIB is critically important to develop preventive strategies to reduce morbidity and mortality.
Paclitaxel has emerged as a front line treatment for aggressive malignancies of the breast, lung, and ovary. Successful therapy of cancer is frequently undermined by the development of paclitaxel ...resistance. There is a growing need to find other therapeutic targets to facilitate treatment of drug-resistant cancers. Using a proteomics approach, elevated levels of Prohibitin1 (PHB1) and GSTπ were found associated with paclitaxel resistance in discrete subcellular fractions of two drug-resistant sublines relative to their sensitive sublines. Immunofluorescence staining and fractionation studies revealed increased levels of PHB1 on the surface of resistant cell lines. Transiently silencing either PHB1 or GSTπ gene expression using siRNA in the paclitaxel-resistant cancer cell sublines partially sensitized these cells toward paclitaxel. Intriguingly, silencing PHB1 but not GSTπ resulted in activation of the intrinsic apoptosis pathway in response to paclitaxel. Similarly, stably silencing either PHB1 or GSTπ significantly improved paclitaxel sensitivity in A549TR cells both in vitro and in vivo. Our results indicate that PHB1 is a mediator of paclitaxel resistance and that this resistance may depend on the cellular localization of the protein. We suggest PHB1 as a potential target for therapeutic strategies for the treatment of drug-resistant tumors.
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