An inducible transgene expression system for regulated phenotypic modification of human embryonic stem cells

Self-renewing pluripotent human embryonic stem (hES) cells are capable of regenerating such non-dividing cells as neurons and cardiomyocytes for therapies and can serve as an excellent experimental model for studying early human development. Both the spatial and temporal relationships of gene expression play a crucial role in determining differentiation; to obtain a better understanding of hES cell differentiation, it will be necessary to establish an inducible system in hES cells that enables specific transgene(s) to reversibly and conditionally express (1) at specific levels and (2) at particular time points during development. Using lentivirus (LV)-mediated gene transfer and a tetracycline-controlled trans-repressor (TR), we first established in hES cells a doxycycline (DOX)-inducible expression system of green fluorescent protein (GFP) to probe its reversibility and kinetics. Upon the addition of DOX, the percentage of GFP + hES cells increased time dependently: The time at which 50% of all green cells appeared (T 50 on) was 119.5 ± 3.2 h; upon DOX removal, GFP expression declined with a half-time (T 50 off) of 127.7 ± 3.9 h and became completely silenced at day 8. Both the proportion and total mean fluorescence intensity (MFI) were dose-dependent (EC 50 = 24.5 ± 2.2 ng/ml). The same system when incorporated into murine (m) ES cells similarly exhibited reversible dose-dependent responses with a similar sensitivity (EC 50 =49.5 ± 8.5 ng/ml), but the much faster kinetics (T 50 on = 35.5 ± 5.5 h, T 50 off = 71.5 ± 2.4 hours). DOX-induced expression of the Kir2.1 channels in mES and hES cells led to robust expression of the inwardly rectifying potassium (K +) current and thereby hyperpolarized the resting membrane potential (RMP). We conclude that the LV-inducible system established presents a unique tool for probing differentiation. © 2008 Mary Ann Liebert, Inc.published_or_final_versio

Distinct roles of microRNA-1 and -499 in ventricular specification and functional maturation of human embryonic stem cell-derived cardiomyocytes

BACKGROUND: MicroRNAs (miRs) negatively regulate transcription and are important determinants of normal heart development and heart failure pathogenesis. Despite the significant knowledge gained in mouse studies, their functional roles in human (h) heart remain elusive. METHODS AND RESULTS: We hypothesized that miRs that figure prominently in cardiac differentiation are differentially expressed in differentiating, developing, and terminally mature human cardiomyocytes (CMs). As a first step, we mapped the miR profiles of human (h) embryonic stem cells (ESCs), hESC-derived (hE), fetal (hF) and adult (hA) ventricular (V) CMs. 63 miRs were differentially expressed between hESCs and hE-VCMs. Of these, 29, including the miR-302 and -371/372/373 clusters, were associated with pluripotency and uniquely expressed in hESCs. Of the remaining miRs differentially expressed in hE-VCMs, 23 continued to express highly in hF- and hA-VCMs, with miR-1, -133, and -499 displaying the largest fold differences; others such as miR-let-7a, -let-7b, -26b, -125a and -143 were non-cardiac specific. Functionally, LV-miR-499 transduction of hESC-derived cardiovascular progenitors significantly increased the yield of hE-VCMs (to 72% from 48% of control; p0.05). By contrast, LV-miR-1 transduction did not bias the yield (p>0.05) but decreased APD and hyperpolarized RMP/MDP in hE-VCMs due to increased I(to), I(Ks) and I(Kr), and decreased I(f) (p<0.05) as signs of functional maturation. Also, LV-miR-1 but not -499 augmented the immature Ca(2+) transient amplitude and kinetics. Molecular pathway analyses were performed for further insights. CONCLUSION: We conclude that miR-1 and -499 play differential roles in cardiac differentiation of hESCs in a context-dependent fashion. While miR-499 promotes ventricular specification of hESCs, miR-1 serves to facilitate electrophysiological maturation.published_or_final_versio

Flavor SU(3) symmetry and QCD factorization in B→PPB \to PP and PVPV decays

Using flavor SU(3) symmetry, we perform a model-independent analysis of charmless $\bar B_{u,d} (\bar B_s) \to PP, ~PV$ decays. All the relevant topological diagrams, including the presumably subleading diagrams, such as the QCD- and EW-penguin exchange diagrams and flavor-singlet weak annihilation ones, are introduced. Indeed, the QCD-penguin exchange diagram turns out to be important in understanding the data for penguin-dominated decay modes. In this work we make efforts to bridge the (model-independent but less quantitative) topological diagram or flavor SU(3) approach and the (quantitative but somewhat model-dependent) QCD factorization (QCDF) approach in these decays, by explicitly showing how to translate each flavor SU(3) amplitude into the corresponding terms in the QCDF framework. After estimating each flavor SU(3) amplitude numerically using QCDF, we discuss various physical consequences, including SU(3) breaking effects and some useful SU(3) relations among decay amplitudes of $\bar B_s \to PV$ and $\bar B_d \to PV$.Comment: 47 pages, 3 figures, 28 table

Fast flowing populations are not well mixed

In evolutionary dynamics, well-mixed populations are almost always associated with all-to-all interactions; mathematical models are based on complete graphs. In most cases, these models do not predict fixation probabilities in groups of individuals mixed by flows. We propose an analytical description in the fast-flow limit. This approach is valid for processes with global and local selection, and accurately predicts the suppression of selection as competition becomes more local. It provides a modelling tool for biological or social systems with individuals in motion.Comment: 19 pages, 8 figure

Label-free, multi-scale imaging of ex-vivo mouse brain using spatial light interference microscopy

Brain connectivity spans over broad spatial scales, from nanometers to centimeters. In order to understand the brain at multi-scale, the neural network in wide-field has been visualized in detail by taking advantage of light microscopy. However, the process of staining or addition of fluorescent tags is commonly required, and the image contrast is insufficient for delineation of cytoarchitecture. To overcome this barrier, we use spatial light interference microscopy to investigate brain structure with high-resolution, sub-nanometer pathlength sensitivity without the use of exogenous contrast agents. Combining wide-field imaging and a mosaic algorithm developed in-house, we show the detailed architecture of cells and myelin, within coronal olfactory bulb and cortical sections, and from sagittal sections of the hippocampus and cerebellum. Our technique is well suited to identify laminar characteristics of fiber tract orientation within white matter, e.g. the corpus callosum. To further improve the macro-scale contrast of anatomical structures, and to better differentiate axons and dendrites from cell bodies, we mapped the tissue in terms of its scattering property. Based on our results, we anticipate that spatial light interference microscopy can potentially provide multiscale and multicontrast perspectives of gross and microscopic brain anatomy.ope

Epidemiological characteristics of Pandemic Influenza A (H1N1-2009) in Zhanjiang, China

Background: A novel influenza A virus strain (H1N1-2009) spread first in Mexico and the United Stated in late April 2009, leading to the first influenza pandemic of the 21st century. The objective of this study was to determine the epidemiological and virological characteristics of the pandemic influenza A (H1N1-2009) in Zhanjiang, China. Methods: The case and outbreak reports of influenza-like illness (ILI) were collected from the Chinese information system of disease control and prevention and the influenza surveillance system of Zhanjiang city. Real-time RT-PCR was conducted, and epidemic and virological characteristics of the virus were analyzed using descriptive epidemiological methods and Chi-square trend tests. Results: A total of 276 reported cases were confirmed from July 16, 2009 to June 30, 2010. The attack rate of outbreak was from 1.1% to 6.0%. The disease peak occurred in December 2009, after which the outbreak subsided gradually. The last case was confirmed in April 2010. Conclusion: The main population struck by the H1N1-2009 virus was young adults, youths and children. The outbreaks most frequently occurred in schools, and most cases were acquired locally

Observation of associated near-side and away-side long-range correlations in √sNN=5.02 TeV proton-lead collisions with the ATLAS detector

Two-particle correlations in relative azimuthal angle (Δϕ) and pseudorapidity (Δη) are measured in √sNN=5.02  TeV p+Pb collisions using the ATLAS detector at the LHC. The measurements are performed using approximately 1  μb-1 of data as a function of transverse momentum (pT) and the transverse energy (ΣETPb) summed over 3.1<η<4.9 in the direction of the Pb beam. The correlation function, constructed from charged particles, exhibits a long-range (2<|Δη|<5) “near-side” (Δϕ∼0) correlation that grows rapidly with increasing ΣETPb. A long-range “away-side” (Δϕ∼π) correlation, obtained by subtracting the expected contributions from recoiling dijets and other sources estimated using events with small ΣETPb, is found to match the near-side correlation in magnitude, shape (in Δη and Δϕ) and ΣETPb dependence. The resultant Δϕ correlation is approximately symmetric about π/2, and is consistent with a dominant cos⁡2Δϕ modulation for all ΣETPb ranges and particle pT

Search for direct pair production of the top squark in all-hadronic final states in proton-proton collisions at s√=8 TeV with the ATLAS detector

The results of a search for direct pair production of the scalar partner to the top quark using an integrated luminosity of 20.1fb−1 of proton–proton collision data at √s = 8 TeV recorded with the ATLAS detector at the LHC are reported. The top squark is assumed to decay via t˜→tχ˜01 or t˜→ bχ˜±1 →bW(∗)χ˜01 , where χ˜01 (χ˜±1 ) denotes the lightest neutralino (chargino) in supersymmetric models. The search targets a fully-hadronic final state in events with four or more jets and large missing transverse momentum. No significant excess over the Standard Model background prediction is observed, and exclusion limits are reported in terms of the top squark and neutralino masses and as a function of the branching fraction of t˜ → tχ˜01 . For a branching fraction of 100%, top squark masses in the range 270–645 GeV are excluded for χ˜01 masses below 30 GeV. For a branching fraction of 50% to either t˜ → tχ˜01 or t˜ → bχ˜±1 , and assuming the χ˜±1 mass to be twice the χ˜01 mass, top squark masses in the range 250–550 GeV are excluded for χ˜01 masses below 60 GeV