Quantitative optical mapping of two-dimensional materials

The pace of two-dimensional materials (2DM) research has been greatly accelerated by the ability to identify exfoliated thicknesses down to a monolayer from their optical contrast. Since this process requires time-consuming and error-prone manual assignment to avoid false-positives from image features with similar contrast, efforts towards fast and reliable automated assignments schemes is essential. We show that by modelling the expected 2DM contrast in digitally captured images, we can automatically identify candidate regions of 2DM. More importantly, we show a computationally-light machine vision strategy for eliminating false-positives from this set of 2DM candidates through the combined use of binary thresholding, opening and closing filters, and shape-analysis from edge detection. Calculation of data pyramids for arbitrarily high-resolution optical coverage maps of two-dimensional materials produced in this way allows the real-time presentation and processing of this image data in a zoomable interface, enabling large datasets to be explored and analysed with ease. The result is that a standard optical microscope with CCD camera can be used as an analysis tool able to accurately determine the coverage, residue/contamination concentration, and layer number for a wide range of presented 2DMs

<i>NuSTAR </i>reveals the extreme properties of the super-Eddington accreting supermassive black hole in PG 1247+267

PG1247+267 is one of the most luminous known quasars at z similar to 2 and is a strongly super-Eddington accreting supermassive black hole (SMBH) candidate. We obtained NuSTAR data of this intriguing source in December 2014 with the aim of studying its high-energy emission, leveraging the broad band covered by the new NuSTAR and the archival XMM-Newton data. Several measurements are in agreement with the super-Eddington scenario for PG1247+267: the soft power law (Gamma = 2.3 +/- 0.1); the weak ionized Fe emission line; and a hint of the presence of outflowing ionized gas surrounding the SMBH. The presence of an extreme reflection component is instead at odds with the high accretion rate proposed for this quasar. This can be explained with three different scenarios; all of them are in good agreement with the existing data, but imply very different conclusions: i) a variable primary power law observed in a low state, superimposed on a reflection component echoing a past, higher flux state; ii) a power law continuum obscured by an ionized, Compton thick, partial covering absorber; and iii) a relativistic disk reflector in a lamp-post geometry, with low coronal height and high BH spin. The first model is able to explain the high reflection component in terms of variability. The second does not require any reflection to reproduce the hard emission, while a rather low high-energy cutoff of similar to 100 keV is detected for the first time in such a high redshift source. The third model require a face-on geometry, which may affect the SMBH mass and Eddington ratio measurements. Deeper X-ray broad-band data are required in order to distinguish between these possibilities

The Intracellular Transport and Secretion of Calumenin-1/2 in Living Cells

Calumenin isoforms 1 and 2 (calu-1/2), encoded by the CALU gene, belong to the CREC protein family. Calu-1/2 proteins are secreted into the extracellular space, but the secretory process and regulatory mechanism are largely unknown. Here, using a time-lapse imaging system, we visualized the intracellular transport and secretory process of calu-1/2-EGFP after their translocation into the ER lumen. Interestingly, we observed that an abundance of calu-1/2-EGFP accumulated in cellular processes before being released into the extracellular space, while only part of calu-1/2-EGFP proteins were secreted directly after attaching to the cell periphery. Moreover, we found the secretion of calu-1/2-EGFP required microtubule integrity, and that calu-1/2-EGFP-containing vesicles were transported by the motor proteins Kif5b and cytoplasmic dynein. Finally, we determined the export signal of calu-1/2-EGFP (amino acid positions 20–46) and provided evidence that the asparagine at site 131 was indispensable for calu-1/2-EGFP stabilization. Taken together, we provide a detailed picture of the intracellular transport of calu-1/2-EGFP, which facilitates our understanding of the secretory mechanism of calu-1/2

Silkworm Coatomers and Their Role in Tube Expansion of Posterior Silkgland

Background: Coat protein complex I (COPI) vesicles, coated by seven coatomer subunits, are mainly responsible for Golgito-ER transport. Silkworm posterior silkgland (PSG), a highly differentiated secretory tissue, secretes fibroin for silk production, but many physiological processes in the PSG cells await further investigation. Methodology/Principal Findings: Here, to investigate the role of silkworm COPI, we cloned six silkworm COPI subunits (a,b,b9, d, e, and f-COP), determined their peak expression in day 2 in fifth-instar PSG, and visualized the localization of COPI, as a coat complex, with cis-Golgi. By dsRNA injection into silkworm larvae, we suppressed the expression of a-, b9- and c-COP, and demonstrated that COPI subunits were required for PSG tube expansion. Knockdown of a-COP disrupted the integrity of Golgi apparatus and led to a narrower glandular lumen of the PSG, suggesting that silkworm COPI is essential for PSG tube expansion. Conclusions/Significance: The initial characterization reveals the essential roles of silkworm COPI in PSG. Although silkworm COPI resembles the previously characterized coatomers in other organisms, some surprising findings require further investigation. Therefore, our results suggest the silkworm as a model for studying intracellular transport, and woul

Intrauterine Growth Restriction Is a Direct Consequence of Localized Maternal Uropathogenic Escherichia coli Cystitis

Despite the continually increasing rates of adverse perinatal outcomes across the globe, the molecular mechanisms that underlie adverse perinatal outcomes are not completely understood. Clinical studies report that 10% of pregnant women will experience a urinary tract infection (UTI) and there is an association of UTIs with adverse perinatal outcomes. We introduced bacterial cystitis into successfully outbred female mice at gestational day 14 to follow pregnancy outcomes and immunological responses to determine the mechanisms that underlie UTI-mediated adverse outcomes. Outbred fetuses from mothers experiencing localized cystitis displayed intrauterine growth restriction (20–80%) as early as 48 hours post-infection and throughout the remainder of normal gestation. Robust infiltration of cellular innate immune effectors was observed in the uteroplacental tissue following introduction of UTI despite absence of viable bacteria. The magnitude of serum proinflammatory cytokines is elevated in the maternal serum during UTI. This study demonstrates that a localized infection can dramatically impact the immunological status as well as the function of non-infected distal organs and tissues. This model can be used as a platform to determine the mechanism(s) by which proinflammatory changes occur between non-contiguous genitourinary organ

Aberrant community architecture and attenuated persistence of uropathogenic Escherichia coli in the absence of individual IHF subunits.

Uropathogenic Escherichia coli (UPEC) utilizes a complex community-based developmental pathway for growth within superficial epithelial cells of the bladder during cystitis. Extracellular DNA (eDNA) is a common matrix component of organized bacterial communities. Integration host factor (IHF) is a heterodimeric protein that binds to double-stranded DNA and produces a hairpin bend. IHF-dependent DNA architectural changes act both intrabacterially and extrabacterially to regulate gene expression and community stability, respectively. We demonstrate that both IHF subunits are required for efficient colonization of the bladder, but are dispensable for early colonization of the kidney. The community architecture of the intracellular bacterial communities (IBCs) is quantitatively different in the absence of either IhfA or IhfB in the murine model for human urinary tract infection (UTI). Restoration of Type 1 pili by ectopic production does not restore colonization in the absence of IhfA, but partially compensates in the absence of IhfB. Furthermore, we describe a binding site for IHF that is upstream of the operon that encodes for the P-pilus. Taken together, these data suggest that both IHF and its constituent subunits (independent of the heterodimer), are able to participate in multiple aspects of the UPEC pathogenic lifestyle, and may have utility as a target for treatment of bacterial cystitis

IHF binds the upstream region of <i>papB</i>.

<p>A) Lane 1–2: 0 & 50 nM IHF were incubated with labeled probe and subjected to DNase I digestion. The sequence protected by IHF is marked with the dashed line on the gel and the actual sequence is shown on the right side of the figure. The solid line on the sequence and gel represent the IHF recognition site, bold bases indicate the hypersensitive sites (< on the gel) and lower case letters are not protected. The numbers indicate the position from the transcription start (+1) of <i>papB</i>. B) Organization of the divergent <i>papB</i> and <i>papI</i> promoter region with the IHF protected region indicated by the dashed line.</p

Type 1 piliation does not restore bladder colonization in the absence of IhfA.

<p>Each symbol represents single infected murine bladder or combined kidney pair. Female mice were infected with wild type UTI89/pMMB66 (black filled squares), ROL745/pMMB66 (UTI89 <i>ihfA11</i>; gray filled circles), or ROL603/pMMB66 (UTI89 <i>ΔihfB;</i> gray filled triangles) for 6 hours post inoculation. Bacterial burden of bladders and kidneys was enumerated as colony forming units ( CFUs). Statistical significance determined using non-parametric Mann Whitney (**<0.002, *** = 0.0006).</p

Community architecture is restored upon complementation in trans.

<p>Female mice were infected with UTI89, ROL745/ pHNá (UTI89 <i>ihfA11</i>), or ROL603/ pHNâ (UTI89 <i>ΔihfB</i>) for 6 hours. Bladders were prepared for visualization by immune-fluorescent microscopy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048349#pone.0048349-Justice3" target="_blank">[41]</a>. Images were taken as an optical section. The strains are indicated above each panel. Scale bar  = 10 µm and scale is unchanged between images.</p

Architecture of Intracellular Bacteria.

<p>Female mice were infected with UTI89/pANT4, ROL745/pANT4 (UTI89 <i>ihfA11</i>), ROL603/pANT4 (UTI89 <i>ΔihfB</i>), SJ1000/pANT4 (UTI89 <i>surA</i>), or UTI89 <i>Δkps</i>/pANT4 for 6 hours. Bladders were prepared for visualization by fluorescent microscopy <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048349#pone.0048349-Justice3" target="_blank">[41]</a>. The intracellular characteristics of each of the strains indicated were visualized using strains that constitutively produce green fluorescent protein. Images were taken as an optical section (upper panel UPEC) or as total fluorescence of entire community (all other panels). The strains are indicated above each panel. Scale bar  = 10 µm and scale is unchanged between images.</p