539 research outputs found
Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission.
Proper completion of mitosis requires the concerted effort of multiple RhoGEFs. Here we show that leukemia-associated RhoGEF (LARG), a RhoA-specific RGS-RhoGEF, is required for abscission, the final stage of cytokinesis, in which the intercellular membrane is cleaved between daughter cells. LARG colocalizes with α-tubulin at the spindle poles before localizing to the central spindle. During cytokinesis, LARG is condensed in the midbody, where it colocalizes with RhoA. HeLa cells depleted of LARG display apoptosis during cytokinesis with unresolved intercellular bridges, and rescue experiments show that expression of small interfering RNA-resistant LARG prevents this apoptosis. Moreover, live cell imaging of LARG-depleted cells reveals greatly delayed fission kinetics in abscission in which a population of cells with persistent bridges undergoes apoptosis; however, the delayed fission kinetics is rescued by Aurora-B inhibition. The formation of a Flemming body and thinning of microtubules in the intercellular bridge of cells depleted of LARG is consistent with a defect in late cytokinesis, just before the abscission event. In contrast to studies of other RhoGEFs, particularly Ect2 and GEF-H1, LARG depletion does not result in cytokinetic furrow regression nor does it affect internal mitotic timing. These results show that LARG is a novel and temporally distinct RhoGEF required for completion of abscission
Autonomous inâsitu calibration of ionâsensitive field effect transistor pH sensors
Ionâsensitive field effect transistorâbased pH sensors have been shown to perform well in high frequency and longâterm ocean sampling regimes. The Honeywell Durafet is widely used due to its stability, fast response, and characterization over a large range of oceanic conditions. However, potentiometric pH monitoring is inherently complicated by the fact that the sensors require careful calibration. Offsets in calibration coefficients have been observed when comparing laboratory to fieldâbased calibrations and prior work has led to the recommendation that an inâsitu calibration be performed based on comparison to discrete samples. Here, we describe our work toward a selfâcalibration apparatus integrated into a SeapHOx pH, dissolved oxygen, and CTD sensor package. This SelfâCalibrating SeapHOx is capable of autonomously recording calibration values from a high quality, traceable, primary reference standard: equimolar tris buffer. The SelfâCalibrating SeapHOx\u27s functionality was demonstrated in a 6âd test in a seawater tank at Scripps Institution of Oceanography (La Jolla, California, U.S.A.) and was successfully deployed for 2âweeks on a shallow, coral reef flat (Lizard Island, Australia). During the latter deployment, the trisâbased selfâcalibration using 15 onâboard samples exhibited superior reproducibility to the standard spectrophotometric pHâbased calibration using \u3eâ100 discrete samples. Standard deviations of calibration pH using tris ranged from 0.002 to 0.005 whereas they ranged from 0.006 to 0.009 for the standard spectrophotometric pHâbased method; the two independent calibration methods resulted in a mean pH difference of 0.008. We anticipate that the SelfâCalibrating SeapHOx will be capable of autonomously providing climate quality pH data, directly linked to a primary seawater pH standard, and with improvements over standard calibration techniques
Collective and single cell behavior in epithelial contact inhibition
Control of cell proliferation is a fundamental aspect of tissue physiology
central to morphogenesis, wound healing and cancer. Although many of the
molecular genetic factors are now known, the system level regulation of growth
is still poorly understood. A simple form of inhibition of cell proliferation
is encountered in vitro in normally differentiating epithelial cell cultures
and is known as "contact inhibition". The study presented here provides a
quantitative characterization of contact inhibition dynamics on tissue-wide and
single cell levels. Using long-term tracking of cultured MDCK cells we
demonstrate that inhibition of cell division in a confluent monolayer follows
inhibition of cell motility and sets in when mechanical constraint on local
expansion causes divisions to reduce cell area. We quantify cell motility and
cell cycle statistics in the low density confluent regime and their change
across the transition to epithelial morphology which occurs with increasing
cell density. We then study the dynamics of cell area distribution arising
through reductive division, determine the average mitotic rate as a function of
cell size and demonstrate that complete arrest of mitosis occurs when cell area
falls below a critical value. We also present a simple computational model of
growth mechanics which captures all aspects of the observed behavior. Our
measurements and analysis show that contact inhibition is a consequence of
mechanical interaction and constraint rather than interfacial contact alone,
and define quantitative phenotypes that can guide future studies of molecular
mechanisms underlying contact inhibition
Pharmacokinetics of Clindamycin in Obese and Nonobese Children
ABSTRACT Although obesity is prevalent among children in the United States, pharmacokinetic (PK) data for obese children are limited. Clindamycin is a commonly used antibiotic that may require dose adjustment in obese children due to its lipophilic properties. We performed a clindamycin population PK analysis using data from three separate trials. A total of 420 samples from 220 children, 76 of whom had a body mass index greater than or equal to the 95th percentile for age, were included in the analysis. Compared to other metrics, total body weight (TBW) was the most robust measure of body size. The final model included TBW and a sigmoidal maturation relationship between postmenstrual age (PMA) and clearance (CL): CL (liters/hour) = 13.8 Ă (TBW/70) 0.75 Ă [PMA 2.83 /(39.5 2.83 +PMA 2.83 )]; volume of distribution ( V ) was associated with TBW, albumin (ALB), and alpha-1 acid glycoprotein (AAG): V (liters) = 63.6 Ă (TBW/70) Ă (ALB/3.3) â0.83 Ă (AAG/2.4) â0.25 . After accounting for differences in TBW, obesity status did not explain additional interindividual variability in model parameters. Our findings support TBW-based dosing for obese and nonobese children
Systematic identification of signaling pathways with potential to confer anticancer drug resistance
Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)âmTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a Îł-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF[superscript V600E] melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts.National Institutes of Health (U.S.) (Grant CA103866)National Institutes of Health (U.S.) (Grant AI07389
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