The impact of plasticizer and degree of hydrolysis on free volume of poly (vinyl alcohol) films

The effect of plasticizer species and the degree of hydrolysis (DH) on the free volume properties of poly(vinyl alcohol) (PVA) were studied using positron annihilation lifetime spectroscopy. Both glycerol and propylene glycol caused an increase in the free volume cavity radius, although exhibited distinct plasticization behavior, with glycerol capable of occupying existing free volume cavities in the PVA to some extent. The influence of water, normally present in PVA film under atmospheric conditions, was also isolated. Water added significantly to the measured free volume cavity radius in both plasticized and pure PVA matrices. Differences in plasticization behavior can be attributed to the functionality of each plasticizing additive and its hydrogen bonding capability. The increase in cavity radii upon plasticizer loading shows a qualitative link between the free volume of voids and the corresponding reduction in Tg and crystallinity. Cavity radius decreases with increasing DH, due to PVA network tightening in the absence of acetate groups. This corresponds well with the higher Tg observed in the resin with the higher DH. DH was also shown to impact the plasticization of PVA with glycerol, indicating that the larger cavities—created by the weaker hydrogen bonding acetate groups—are capable of accommodating glycerol molecules with negligible effect on the cavity dimensions

Title page Silencing the breast cancer resistance protein (BCRP) expression and function in Caco-2 cells using lentiviral vector-based shRNA

Abstract A series of stable breast cancer resistance protein (BCRP, ABCG2) knockdown cell lines were produced by transduction of Caco-2 cells with lentiviral vector-based shRNA. Caco-2 cell is a human intestinal-derived cell line widely used to study intestinal drug absorption. Caco-2 expresses three apical drug efflux transporters: BCRP, P-glycoprotein (P-gp; ABCB1) and multidrug resistance protein 2 (MRP2, ABCC2). BCRP and P-gp, in particular, play a significant role in pharmacokinetics due to their expression at several key interfaces. Over-expression of BCRP in cancer cells may also be a mechanism of tumor resistance to chemotherapeutic drugs. The goal of this study was to engineer and characterize Caco-2 cell clones with stable knockdown of BCRP expression. shRNA/BCRP lentiviral particles were used to infect a stable clone of Caco-2 cells. Expression of BCRP was monitored using quantitative PCR (qPCR), western blotting, immunofluorescence microscopy and bidirectional transport of probe substrates, estrone-3-sulfate (E3S) and pheophorbide A (PhA). Based on qPCR, expression of BCRP mRNA was knocked down in five clones with a maximum of 97% silencing in Clone D. Silencing of BCRP gene expression was maintained for at least 25 passages. Expression of BCRP protein was also reduced significantly. Functionally, BCRP knockdown was reflected in significant reduction of the efflux ratio of E3S and PhA. Clone D in particular should be a useful model for identifying and characterizing P-gp substrates and inhibitors without interference from BCRP and/or MRP2. In addition, it can be used in conjunction with wild-type or vector control Caco-2 cells to identify BCRP substrates. DMD 23309

PS1-10afx at z = 1.388: Pan-STARRS1 Discovery of a New Type of Superluminous Supernova

We present the Pan-STARRS1 discovery of PS1-10afx, a unique hydrogen-deficient superluminous supernova (SLSN) at redshift z = 1.388. The light curve peaked at z P1 = 21.7 mag, making PS1-10afx comparable to the most luminous known SNe, with Mu = –22.3 mag. Our extensive optical and near-infrared observations indicate that the bolometric light curve of PS1-10afx rose on the unusually fast timescale of ~12 days to the extraordinary peak luminosity of 4.1 × 1044 erg s–1 (M bol = –22.8 mag) and subsequently faded rapidly. Equally important, the spectral energy distribution is unusually red for an SLSN, with a color temperature of ~6800 K near maximum light, in contrast to previous hydrogen-poor SLSNe, which are bright in the ultraviolet (UV). The spectra more closely resemble those of a normal SN Ic than any known SLSN, with a photospheric velocity of ~11, 000 km s–1 and evidence for line blanketing in the rest-frame UV. Despite the fast rise, these parameters imply a very large emitting radius (gsim 5 × 1015 cm). We demonstrate that no existing theoretical model can satisfactorily explain this combination of properties: (1) a nickel-powered light curve cannot match the combination of high peak luminosity with the fast timescale; (2) models powered by the spindown energy of a rapidly rotating magnetar predict significantly hotter and faster ejecta; and (3) models invoking shock breakout through a dense circumstellar medium cannot explain the observed spectra or color evolution. The host galaxy is well detected in pre-explosion imaging with a luminosity near L*, a star formation rate of ~15 M ☉ yr–1, and is fairly massive (~2 × 1010 M ☉), with a stellar population age of ~108 yr, also in contrast to the young dwarf hosts of known hydrogen-poor SLSNe. PS1-10afx is distinct from known examples of SLSNe in its spectra, colors, light-curve shape, and host galaxy properties, suggesting that it resulted from a different channel than other hydrogen-poor SLSNe.Astronom

The Ultraviolet-bright, Slowly Declining Transient PS1-11af as a Partial Tidal Disruption Event

We present the Pan-STARRS1 discovery of the long-lived and blue transient PS1-11af, which was also detected by Galaxy Evolution Explorer with coordinated observations in the near-ultraviolet (NUV) band. PS1-11af is associated with the nucleus of an early type galaxy at redshift z = 0.4046 that exhibits no evidence for star formation or active galactic nucleus activity. Four epochs of spectroscopy reveal a pair of transient broad absorption features in the UV on otherwise featureless spectra. Despite the superficial similarity of these features to P-Cygni absorptions of supernovae (SNe), we conclude that PS1-11af is not consistent with the properties of known types of SNe. Blackbody fits to the spectral energy distribution are inconsistent with the cooling, expanding ejecta of a SN, and the velocities of the absorption features are too high to represent material in homologous expansion near a SN photosphere. However, the constant blue colors and slow evolution of the luminosity are similar to previous optically selected tidal disruption events (TDEs). The shape of the optical light curve is consistent with models for TDEs, but the minimum accreted mass necessary to power the observed luminosity is only ~0.002 M ☉, which points to a partial disruption model. A full disruption model predicts higher bolometric luminosities, which would require most of the radiation to be emitted in a separate component at high energies where we lack observations. In addition, the observed temperature is lower than that predicted by pure accretion disk models for TDEs and requires reprocessing to a constant, lower temperature. Three deep non-detections in the radio with the Very Large Array over the first two years after the event set strict limits on the production of any relativistic outflow comparable to Swift J1644+57, even if off-axis

Additional Serine/Threonine Phosphorylation Reduces Binding Affinity but Preserves Interface Topography of Substrate Proteins to the c-Cbl TKB Domain

The E3-ubiquitin ligase, c-Cbl, is a multi-functional scaffolding protein that plays a pivotal role in controlling cell phenotype. As part of the ubiquitination and downregulation process, c-Cbl recognizes targets, such as tyrosine kinases and the Sprouty proteins, by binding to a conserved (NX/R)pY(S/T)XXP motif via its uniquely embedded SH2 domain (TKB domain). We previously outlined the mode of binding between the TKB domain and various substrate peptide motifs, including epidermal growth factor receptor (EGFR) and Sprouty2 (Spry2), and demonstrated that an intrapetidyl hydrogen bond forms between the (pY-1) arginine or (pY-2) asparagine and the phosphorylated tyrosine, which is crucial for binding. Recent reports demonstrated that, under certain types of stimulation, the serine/threonine residues at the pY+1 and/or pY+2 positions within this recognition motif of EGFR and Sprouty2 may be endogenously phosphorylated. Using structural and binding studies, we sought to determine whether this additional phosphorylation could affect the binding of the TKB domain to these peptides and consequently, whether the type of stimulation can dictate the degree to which substrates bind to c-Cbl. Here, we show that additional phosphorylation significantly reduces the binding affinity between the TKB domain and its target proteins, EGFR and Sprouty2, as compared to peptides bearing a single tyrosine phosphorylation. The crystal structure indicates that this is accomplished with minimal changes to the essential intrapeptidyl bond and that the reduced strength of the interaction is due to the charge repulsion between c-Cbl and the additional phosphate group. This obvious reduction in binding affinity, however, indicates that Cbl's interactions with its TKB-centered binding partners may be more favorable in the absence of Ser/Thr phosphorylation, which is stimulation and context specific in vivo. These results demonstrate the importance of understanding the environment in which certain residues are phosphorylated, and the necessity of including this in structural investigations

Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens

The use of next-generation sequencing technologies to produce genomic copy number data has recently been described. Most approaches, however, reply on optimal starting DNA, and are therefore unsuitable for the analysis of formalin-fixed paraffin-embedded (FFPE) samples, which largely precludes the analysis of many tumour series. We have sought to challenge the limits of this technique with regards to quality and quantity of starting material and the depth of sequencing required. We confirm that the technique can be used to interrogate DNA from cell lines, fresh frozen material and FFPE samples to assess copy number variation. We show that as little as 5 ng of DNA is needed to generate a copy number karyogram, and follow this up with data from a series of FFPE biopsies and surgical samples. We have used various levels of sample multiplexing to demonstrate the adjustable resolution of the methodology, depending on the number of samples and available resources. We also demonstrate reproducibility by use of replicate samples and comparison with microarray-based comparative genomic hybridization (aCGH) and digital PCR. This technique can be valuable in both the analysis of routine diagnostic samples and in examining large repositories of fixed archival material

Spatial Organization and Molecular Correlation of Tumor-Infiltrating Lymphocytes Using Deep Learning on Pathology Images

Beyond sample curation and basic pathologic characterization, the digitized H&E-stained images of TCGA samples remain underutilized. To highlight this resource, we present mappings of tumorinfiltrating lymphocytes (TILs) based on H&E images from 13 TCGA tumor types. These TIL maps are derived through computational staining using a convolutional neural network trained to classify patches of images. Affinity propagation revealed local spatial structure in TIL patterns and correlation with overall survival. TIL map structural patterns were grouped using standard histopathological parameters. These patterns are enriched in particular T cell subpopulations derived from molecular measures. TIL densities and spatial structure were differentially enriched among tumor types, immune subtypes, and tumor molecular subtypes, implying that spatial infiltrate state could reflect particular tumor cell aberration states. Obtaining spatial lymphocytic patterns linked to the rich genomic characterization of TCGA samples demonstrates one use for the TCGA image archives with insights into the tumor-immune microenvironment

Pan-Cancer Analysis of lncRNA Regulation Supports Their Targeting of Cancer Genes in Each Tumor Context

Long noncoding RNAs (lncRNAs) are commonly dys-regulated in tumors, but only a handful are known toplay pathophysiological roles in cancer. We inferredlncRNAs that dysregulate cancer pathways, onco-genes, and tumor suppressors (cancer genes) bymodeling their effects on the activity of transcriptionfactors, RNA-binding proteins, and microRNAs in5,185 TCGA tumors and 1,019 ENCODE assays.Our predictions included hundreds of candidateonco- and tumor-suppressor lncRNAs (cancerlncRNAs) whose somatic alterations account for thedysregulation of dozens of cancer genes and path-ways in each of 14 tumor contexts. To demonstrateproof of concept, we showed that perturbations tar-geting OIP5-AS1 (an inferred tumor suppressor) andTUG1 and WT1-AS (inferred onco-lncRNAs) dysre-gulated cancer genes and altered proliferation ofbreast and gynecologic cancer cells. Our analysis in-dicates that, although most lncRNAs are dysregu-lated in a tumor-specific manner, some, includingOIP5-AS1, TUG1, NEAT1, MEG3, and TSIX, synergis-tically dysregulate cancer pathways in multiple tumorcontexts

Genomic, Pathway Network, and Immunologic Features Distinguishing Squamous Carcinomas

This integrated, multiplatform PanCancer Atlas study co-mapped and identified distinguishing molecular features of squamous cell carcinomas (SCCs) from five sites associated with smokin