Optimised use of Oxford Nanopore Flowcells for Hybrid Assemblies

Hybrid assemblies are highly valuable for studies of Enterobacteriaceae due to their ability to fully resolve the structure of mobile genetic elements, such as plasmids, which are involved in the carriage of clinically important genes (e.g. those involved in antimicrobial resistance/virulence). The widespread application of this technique is currently primarily limited by cost. Recent data have suggested that non-inferior, and even superior, hybrid assemblies can be produced using a fraction of the total output from a multiplexed nanopore [Oxford Nanopore Technologies (ONT)] flowcell run. In this study we sought to determine the optimal minimal running time for flowcells when acquiring reads for hybrid assembly. We then evaluated whether the ONT wash kit might allow users to exploit shorter running times by sequencing multiple libraries per flowcell. After 24 h of sequencing, most chromosomes and plasmids had circularized and there was no benefit associated with longer running times. Quality was similar at 12 h, suggesting that shorter running times are likely to be acceptable for certain applications (e.g. plasmid genomics). The ONT wash kit was highly effective in removing DNA between libraries. Contamination between libraries did not appear to affect subsequent hybrid assemblies, even when the same barcodes were used successively on a single flowcell. Utilizing shorter run times in combination with between-library nuclease washes allows at least 36 Enterobacteriaceae isolates to be sequenced per flowcell, significantly reducing the per-isolate sequencing cost. Ultimately this will facilitate large-scale studies utilizing hybrid assembly, advancing our understanding of the genomics of key human pathogens

Kank Is an EB1 Interacting Protein that Localises to Muscle-Tendon Attachment Sites in Drosophila

Little is known about how microtubules are regulated in different cell types during development. EB1 plays a central role in the regulation of microtubule plus ends. It directly binds to microtubule plus ends and recruits proteins which regulate microtubule dynamics and behaviour. We report the identification of Kank, the sole Drosophila orthologue of human Kank proteins, as an EB1 interactor that predominantly localises to embryonic attachment sites between muscle and tendon cells. Human Kank1 was identified as a tumour suppressor and has documented roles in actin regulation and cell polarity in cultured mammalian cells. We found that Drosophila Kank binds EB1 directly and this interaction is essential for Kank localisation to microtubule plus ends in cultured cells. Kank protein is expressed throughout fly development and increases during embryogenesis. In late embryos, it accumulates to sites of attachment between muscle and epidermal cells. A kank deletion mutant was generated. We found that the mutant is viable and fertile without noticeable defects. Further analysis showed that Kank is dispensable for muscle function in larvae. This is in sharp contrast to C. elegans in which the Kank orthologue VAB-19 is required for development by stabilising attachment structures between muscle and epidermal cells

Cell Cycle-Dependent Microtubule-Based Dynamic Transport of Cytoplasmic Dynein in Mammalian Cells

BACKGROUND:Cytoplasmic dynein complex is a large multi-subunit microtubule (MT)-associated molecular motor involved in various cellular functions including organelle positioning, vesicle transport and cell division. However, regulatory mechanism of the cell-cycle dependent distribution of dynein has not fully been understood. METHODOLOGY/PRINCIPAL FINDINGS:Here we report live-cell imaging of cytoplasmic dynein in HeLa cells, by expressing multifunctional green fluorescent protein (mfGFP)-tagged 74-kDa intermediate chain (IC74). IC74-mfGFP was successfully incorporated into functional dynein complex. In interphase, dynein moved bi-directionally along with MTs, which might carry cargos such as transport vesicles. A substantial fraction of dynein moved toward cell periphery together with EB1, a member of MT plus end-tracking proteins (+TIPs), suggesting +TIPs-mediated transport of dynein. In late-interphase and prophase, dynein was localized at the centrosomes and the radial MT array. In prometaphase and metaphase, dynein was localized at spindle MTs where it frequently moved from spindle poles toward chromosomes or cell cortex. +TIPs may be involved in the transport of spindle dyneins. Possible kinetochore and cortical dyneins were also observed. CONCLUSIONS AND SIGNIFICANCE:These findings suggest that cytoplasmic dynein is transported to the site of action in preparation for the following cellular events, primarily by the MT-based transport. The MT-based transport may have greater advantage than simple diffusion of soluble dynein in rapid and efficient transport of the limited concentration of the protein

Who Cares About Being Gentle? The Impact of Social Identity and the Gender of One’s Friends on Children’s Display of Same-Gender Favoritism

This research assessed children’s same-gender favoritism by examining whether children value traits descriptive of their own gender more than traits descriptive of the other gender. We also investigated whether children’s proportion of same-gender friends relates to their same-gender favoritism. Eighty-one third and fourth grade children from the Midwest and West Coast of the U.S. rated how well 19 personality traits describe boys and girls, and how important each trait is for their gender to possess. Results replicate and extend past trait assignment research by demonstrating that both genders valued same-gender traits significantly more than other-gender traits. Results also indicated that boys with many same-gender friends derogated feminine-stereotyped traits, which has implications for research on masculinity norms within male-dominated peer groups

Effects of PPARγ ligands on TGF-β1-induced epithelial-mesenchymal transition in alveolar epithelial cells

BACKGROUND: Transforming growth factor beta1 (TGF-beta1)-mediated epithelial mesenchymal transition (EMT) of alveolar epithelial cells (AEC) may contribute to lung fibrosis. Since PPAR gamma ligands have been shown to inhibit fibroblast activation by TGF-beta1, we assessed the ability of the thiazolidinediones rosiglitazone (RGZ) and ciglitazone (CGZ) to regulate TGF-beta1-mediated EMT of A549 cells, assessing changes in cell morphology, and expression of cell adhesion molecules E-cadherin (epithelial cell marker) and N-cadherin (mesenchymal cell marker), and collagen 1 alpha 1 (COL1A1), CTGF and MMP-2 mRNA. METHODS: Serum-deprived A549 cells (human AEC cell line) were pre-incubated with RGZ and CGZ (1 - 30 microM) in the absence or presence of the PPAR gamma antagonist GW9662 (10 microM) before TGFbeta-1 (0.075-7.5 ng/ml) treatment for up to 72 hrs. Changes in E-cadherin, N-cadherin and phosphorylated Smad2 and Smad3 levels were analysed by Western blot, and changes in mRNA levels including COL1A1 assessed by RT-PCR. RESULTS: TGFbeta-1 (2.5 ng/ml)-induced reductions in E-cadherin expression were associated with a loss of epithelial morphology and cell-cell contact. Concomitant increases in N-cadherin, MMP-2, CTGF and COL1A1 were evident in predominantly elongated fibroblast-like cells. Neither RGZ nor CGZ prevented TGF beta 1-induced changes in cell morphology, and PPAR gamma-dependent inhibitory effects of both ligands on changes in E-cadherin were only evident at submaximal TGF-beta1 (0.25 ng/ml). However, both RGZ and CGZ inhibited the marked elevation of N-cadherin and COL1A1 induced by TGF-beta1 (2.5 ng/ml), with effects on COL1A1 prevented by GW9662. Phosphorylation of Smad2 and Smad3 by TGF-beta1 was not inhibited by RGZ or CGZ. CONCLUSIONS: RGZ and CGZ inhibited profibrotic changes in TGF-beta1-stimulated A549 cells independently of inhibition of Smad phosphorylation. Their inhibitory effects on changes in collagen I and E-cadherin, but not N-cadherin or CTGF, appeared to be PPAR gamma-dependent. Further studies are required to unravel additional mechanisms of inhibition of TGF-beta1 signalling by thiazolidinediones and their implications for the contribution of EMT to lung fibrosis

X-ray Absorption and Reflection in Active Galactic Nuclei

X-ray spectroscopy offers an opportunity to study the complex mixture of emitting and absorbing components in the circumnuclear regions of active galactic nuclei, and to learn about the accretion process that fuels AGN and the feedback of material to their host galaxies. We describe the spectral signatures that may be studied and review the X-ray spectra and spectral variability of active galaxies, concentrating on progress from recent Chandra, XMM-Newton and Suzaku data for local type 1 AGN. We describe the evidence for absorption covering a wide range of column densities, ionization and dynamics, and discuss the growing evidence for partial-covering absorption from data at energies > 10 keV. Such absorption can also explain the observed X-ray spectral curvature and variability in AGN at lower energies and is likely an important factor in shaping the observed properties of this class of source. Consideration of self-consistent models for local AGN indicates that X-ray spectra likely comprise a combination of absorption and reflection effects from material originating within a few light days of the black hole as well as on larger scales. It is likely that AGN X-ray spectra may be strongly affected by the presence of disk-wind outflows that are expected in systems with high accretion rates, and we describe models that attempt to predict the effects of radiative transfer through such winds, and discuss the prospects for new data to test and address these ideas.Comment: Accepted for publication in the Astronomy and Astrophysics Review. 58 pages, 9 figures. V2 has fixed an error in footnote

Importance of Intracellular pH in Determining the Uptake and Efficacy of the Weakly Basic Chemotherapeutic Drug, Doxorubicin

Low extracellular pH (pHe), that is characteristic of many tumours, tends to reduce the uptake of weakly basic drugs, such as doxorubicin, thereby conferring a degree of physiological resistance to chemotherapy. It has been assumed, from pH-partition theory, that the effect of intracellular pH (pHi) is symmetrically opposite, although this has not been tested experimentally. Doxorubicin uptake into colon HCT116 cells was measured using the drug's intrinsic fluorescence under conditions that alter pHi and pHe or pHi alone. Acutely, doxorubicin influx across the cell-membrane correlates with the trans-membrane pH-gradient (facilitated at alkaline pHe and acidic pHi). However, the protonated molecule is not completely membrane-impermeant and, therefore, overall drug uptake is less pHe-sensitive than expected from pH-partitioning. Once inside cells, doxorubicin associates with slowly-releasing nuclear binding sites. The occupancy of these sites increases with pHi, such that steady-state drug uptake can be greater with alkaline cytoplasm, in contradiction to pH-partition theory. Measurements of cell proliferation demonstrate that doxorubicin efficacy is enhanced at alkaline pHi and that pH-partition theory is inadequate to account for this. The limitations in the predictive power of pH-partition theory arise because it only accounts for the pHi/pHe-sensitivity of drug entry into cells but not the drug's subsequent interactions that, independently, show pHi-dependence. In summary, doxorubicin uptake into cells is favoured by high pHe and high pHi. This modified formalism should be taken into account when designing manoeuvres aimed at increasing doxorubicin efficacy

Measurement of the Forward-Backward Asymmetry in the B -> K(*) mu+ mu- Decay and First Observation of the Bs -> phi mu+ mu- Decay

We reconstruct the rare decays $B^+ \to K^+\mu^+\mu^-$, $B^0 \to K^{*}(892)^0\mu^+\mu^-$, and $B^0_s \to \phi(1020)\mu^+\mu^-$ in a data sample corresponding to $4.4 {\rm fb^{-1}}$ collected in $p\bar{p}$ collisions at $\sqrt{s}=1.96 {\rm TeV}$ by the CDF II detector at the Fermilab Tevatron Collider. Using $121 \pm 16$ $B^+ \to K^+\mu^+\mu^-$ and $101 \pm 12$ $B^0 \to K^{*0}\mu^+\mu^-$ decays we report the branching ratios. In addition, we report the measurement of the differential branching ratio and the muon forward-backward asymmetry in the $B^+$ and $B^0$ decay modes, and the $K^{*0}$ longitudinal polarization in the $B^0$ decay mode with respect to the squared dimuon mass. These are consistent with the theoretical prediction from the standard model, and most recent determinations from other experiments and of comparable accuracy. We also report the first observation of the $B^0_s \to \phi\mu^+\mu^- decay and measure its branching ratio${\mathcal{B}}(B^0_s \to \phi\mu^+\mu^-) = [1.44 \pm 0.33 \pm 0.46] \times 10^{-6}$using$27 \pm 6$signal events. This is currently the most rare$B^0_s$ decay observed.Comment: 7 pages, 2 figures, 3 tables. Submitted to Phys. Rev. Let

Search for a New Heavy Gauge Boson Wprime with Electron + missing ET Event Signature in ppbar collisions at sqrt(s)=1.96 TeV

We present a search for a new heavy charged vector boson $W^\prime$ decaying to an electron-neutrino pair in $p\bar{p}$ collisions at a center-of-mass energy of 1.96\unit{TeV}. The data were collected with the CDF II detector and correspond to an integrated luminosity of 5.3\unit{fb}^{-1}. No significant excess above the standard model expectation is observed and we set upper limits on $\sigma\cdot{\cal B}(W^\prime\to e\nu)$. Assuming standard model couplings to fermions and the neutrino from the $W^\prime$ boson decay to be light, we exclude a $W^\prime$ boson with mass less than 1.12\unit{TeV/}c^2 at the 95\unit{%} confidence level.Comment: 7 pages, 2 figures Submitted to PR