Different populations of RNA polymerase II in living mammalian cells

RNA polymerase II is responsible for transcription of most eukaryotic genes, but, despite exhaustive analysis, little is known about how it transcribes natural templates in vivo. We studied polymerase dynamics in living Chinese hamster ovary cells using an established line that expresses the largest (catalytic) subunit of the polymerase (RPB1) tagged with the green fluorescent protein (GFP). Genetic complementation has shown this tagged polymerase to be fully functional. Fluorescence loss in photobleaching (FLIP) reveals the existence of at least three kinetic populations of tagged polymerase: a large rapidly-exchanging population, a small fraction resistant to 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) but sensitive to a different inhibitor of transcription (i.e. heat shock), and a third fraction sensitive to both inhibitors. Quantitative immunoblotting shows the largest fraction to be the inactive hypophosphorylated form of the polymerase (i.e. IIA). Results are consistent with the second (DRB-insensitive but heat-shock-sensitive) fraction being bound but not engaged, while the third (sensitive to both DRB and heat shock) is the elongating hyperphosphorylated form (i.e. IIO)

Synchronizing Cardiac Cycle Phase with Foot Strike to Optimize Cardiac Performance in Patients with Chronic Systolic Heart Failure and Cardiac Resynchronization Therapy (CRT)

Despite advances in medical and Cardiac Resynchronization Therapy (CRT), patients with chronic systolic heart failure (HF) have persistent symptoms including dyspnea on exertion and exercise intolerance. Novel strategies to improve exercise performance in these patients, such as optimizing cardio-locomotor coupling, could be particularly beneficial to improve functional capacity. For example, runners display a lower heart rate and higher oxygen pulse, suggestive of a higher stroke volume (SV), when foot strike occurs in diastole. Whether patients with HF undergoing CRT can similarly increase SV is unknown. PURPOSE: To compare the effects of diastolic versus systolic foot strike timing on exercise hemodynamics in patients with HF and CRT. METHODS: Ten patients (Age: 58 ± 17 years, 40% Female) with HF and previously implanted CRT pacemakers completed repeated 5-minute bouts of walking on a treadmill at a fixed but individualized speed (range: 1.5-3mph). Participants were randomized to walking to an auditory tone to synchronize their foot strike to either the systolic (ECG R-wave; 0 or 100%±15% or R-R interval) or diastolic phase (45±15% of the R-R interval) of their cardiac cycle. Participants were included if ≥50% of their steps were valid (i.e. in time). Patients wore a chest strap with an attached ECG sensor and accelerometer (CounterpaceR). Foot strike timing and associated valid step counts were assessed via CounterpaceR or post-hoc analysis of foot strike waveforms. Cardiopulmonary parameters were measured breath by breath via indirect calorimetry and cardiac output was measured via acetylene rebreathing, with SV calculated as the quotient of cardiac output and heart rate. RESULTS: There was no difference in oxygen uptake between conditions (1.02 ± 0.44 vs. 1.04 ± 0.44 L/min, P=0.298). When compared to systolic walking, stepping in diastole was associated with higher SV (Diastolic: 80 ± 28 vs. Systolic: 74 ± 26 ml, P=0.003) and cardiac output (8.3 ± 3.5 vs. 7.9 ± 3.4 L/min, P=0.004); heart rate (paced) was not different between conditions (101 ± 15 vs. 103 ± 14 bpm, P=0.300). Mean arterial pressure was significantly lower during diastolic walking (85 ± 12 vs. 98 ± 20 mmHg, P=0.007). CONCLUSION: In patients with HF and previous CRT, synchronizing foot strike with diastole during walking increased SV and cardiac output and reduced arterial pressure. Our findings indicate that in such paced hearts, diastolic stepping increases oxygen delivery and decreases afterload, which may facilitate increased exercise capacity. Therefore, if added to pacemakers, this cardio-locomotor coupling technology may maximize CRT efficiency and increase exercise participation and quality of life in patients with HF

Search for solar axions in XMASS, a large liquid-xenon detector

XMASS, a low-background, large liquid-xenon detector, was used to search for solar axions that would be produced by bremsstrahlung and Compton effects in the Sun. With an exposure of 5.6ton days of liquid xenon, the model-independent limit on the coupling for mass $\ll$ 1keV is $|g_{aee}|< 5.4\times 10^{-11}$ (90% C.L.), which is a factor of two stronger than the existing experimental limit. The bounds on the axion masses for the DFSZ and KSVZ axion models are 1.9 and 250eV, respectively. In the mass range of 10-40keV, this study produced the most stringent limit, which is better than that previously derived from astrophysical arguments regarding the Sun to date

The global oscillation network group site survey. II. Results

The Global Oscillation Network Group (GONG) Project will place a network of instruments around the world to observe solar oscillations as continuously as possible for three years. The Project has now chosen the six network sites based on analysis of survey data from fifteen sites around the world. The chosen sites are: Big Bear Solar Observatory, California; Mauna Loa Solar Observatory, Hawaii; Learmonth Solar Observatory, Australia; Udaipur Solar Observatory, India; Observatorio del Teide, Tenerife; and Cerro Tololo Interamerican Observatory, Chile. Total solar intensity at each site yields information on local cloud cover, extinction coefficient, and transparency fluctuations. In addition, the performance of 192 reasonable components analysis. An accompanying paper describes the analysis methods in detail; here we present the results of both the network and individual site analyses. The selected network has a duty cycle of 93.3%, in good agreement with numerical simulations. The power spectrum of the network observing window shows a first diurnal sidelobe height of 3 × 10⁻⁴ with respect to the central component, an improvement of a factor of 1300 over a single site. The background level of the network spectrum is lower by a factor of 50 compared to a single-site spectrum

Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation

Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications. © 2013 Goh et al