First detection of ND in the solar-mass protostar IRAS16293-2422

In the past decade, much progress has been made in characterising the processes leading to the enhanced deuterium fractionation observed in the ISM and in particular in the cold, dense parts of star forming regions such as protostellar envelopes. Very high molecular D/H ratios have been found for saturated molecules and ions. However, little is known about the deuterium fractionation in radicals, even though simple radicals often represent an intermediate stage in the formation of more complex, saturated molecules. The imidogen radical NH is such an intermediate species for the ammonia synthesis in the gas phase. Herschel/HIFI represents a unique opportunity to study the deuteration and formation mechanisms of such species, which are not observable from the ground. We searched here for the deuterated radical ND in order to determine the deuterium fractionation of imidogen and constrain the deuteration mechanism of this species. We observed the solar-mass Class 0 protostar IRAS16293-2422 with the heterodyne instrument HIFI as part of the Herschel key programme CHESS (Chemical HErschel Surveys of Star forming regions). The deuterated form of the imidogen radical ND was detected and securely identified with 2 hyperfine component groups of its fundamental transition in absorption against the continuum background emitted from the nascent protostar. The 3 groups of hyperfine components of its hydrogenated counterpart NH were also detected in absorption. We derive a very high deuterium fractionation with an [ND]/[NH] ratio of between 30 and 100%. The deuterium fractionation of imidogen is of the same order of magnitude as that in other molecules, which suggests that an efficient deuterium fractionation mechanism is at play. We discuss two possible formation pathways for ND, by means of either the reaction of N+ with HD, or deuteron/proton exchange with NH.Comment: Accepted; To appear in A&A Herschel/HIFI Special Issu

The influence of traits associated with autism spectrum disorder (ASD) on the detection of fake news.

It has been suggested that neuro-diverse individuals may be particularly good at detecting online deception (Pick 2019). A small-scale exploratory study was conducted to investigate whether individuals with traits associated with Autism Spectrum Disorder (ASD) were more or less accurate in spotting different types of fake news. A non-clinical sample of university students completed an online identification task, where both fake and real articles items were manipulated in terms of their emotive content. When individuals with low and high scores on the Autism-Spectrum Quotient (Baron-Cohen et al. 2001) were compared, there were no significant main effects on detection accuracy. However, there were two significant interactions, indicating an interesting relationship between message emotiveness, ASD and fake news detection. The results contribute to an understanding of how psychological differences, in particular ASD, may affect online judgements and will contribute to a developing body of work relating positive skills of neuro-diverse individuals to the cybersecurity industry

Rosetta FlexPepDock ab-initio: Simultaneous Folding, Docking and Refinement of Peptides onto Their Receptors

Flexible peptides that fold upon binding to another protein molecule mediate a large number of regulatory interactions in the living cell and may provide highly specific recognition modules. We present Rosetta FlexPepDock ab-initio, a protocol for simultaneous docking and de-novo folding of peptides, starting from an approximate specification of the peptide binding site. Using the Rosetta fragments library and a coarse-grained structural representation of the peptide and the receptor, FlexPepDock ab-initio samples efficiently and simultaneously the space of possible peptide backbone conformations and rigid-body orientations over the receptor surface of a given binding site. The subsequent all-atom refinement of the coarse-grained models includes full side-chain modeling of both the receptor and the peptide, resulting in high-resolution models in which key side-chain interactions are recapitulated. The protocol was applied to a benchmark in which peptides were modeled over receptors in either their bound backbone conformations or in their free, unbound form. Near-native peptide conformations were identified in 18/26 of the bound cases and 7/14 of the unbound cases. The protocol performs well on peptides from various classes of secondary structures, including coiled peptides with unusual turns and kinks. The results presented here significantly extend the scope of state-of-the-art methods for high-resolution peptide modeling, which can now be applied to a wide variety of peptide-protein interactions where no prior information about the peptide backbone conformation is available, enabling detailed structure-based studies and manipulation of those interactions

Conditional expression of RelA causes proliferation arrest in epithelial cells.

<p><b>A—C</b>. Stable HMEC (A-C) conditionally expressing Flag-tagged RelA (HRA, A), RelB (HRB, B) or cRel (HRC, C) were generated and expression of the proteins in the presence of Dox (0.1 or 1 μg/ml) was verified by immunoblot. Flag-tagged RelA and RelB runs as a slower migrating band and induction of cRel was verified using an anti-Flag antibody. The effect of expressing RelA, RelB and cRel (compare Dox- with Dox 0.1/1.0 μg/ml) on HMEC proliferation was analyzed using the MTS assay. Absorbance values at each time was normalized to that obtained at the beginning of the experiment (Day 0) and plotted as fold increase over time. <b>D.</b> Similar to A except that RelA was expressed in Fallopian tube epithelial cells (FRA).</p

RelA induced proliferation arrest is p53 independent but Rb dependent.

<p><b>A.</b> HRA cells constitutively expressing a short dominant negative form of p53 (DDp53, HRAD cells) was generated and expression of DDp53 was verified by the stabilization of endogenous p53. Proliferation assay was performed using HRAD cells in the presence or absence of Dox (0.1μg/ml or 1μg/ml) using the MTS assay. MTS values at each time-point was normalized to MTS values corresponding to initial amount of cells plated and plotted as fold increase in growth over time. <b>B.</b> HRA or HRAD cells were mixed with methocult medium supplemented with B27 and cultured in the absence and presence of Dox (1μg/ml) for 2 weeks. Representative images of colonies from a triplicate experiment are shown. <b>C.</b> HRA cells constitutively expressing SV40 Large T antigen (HRA-SV40LT) were generated and verified by immunoblot. HRA and HRA-SV40LT cells were plated in triplicates and cultured in the presence or absence of Dox (1μg/ml) for 3 days. Amount of cells under each condition was estimated using the MTS assay and normalized to values obtained in the un-induced samples. Rescue of RelA-induced proliferation arrest by SV40LT was statistically significant (p-value: 8.5 E -14). <b>D.</b> Double-conditional HRA cells expressing both RelA and a miR-shRNA targeting Rb (HRA-kd-Rb) were generated and verified by immunoblot. HRA and HRA-kd-Rb cells were plated in triplicates and proliferation of the cells was analyzed as in <b>C</b>. Rescue of RelA-induced proliferation arrest by knocking down Rb was statistically significant (p-value: 6.8 E -8). <b>E.</b> Scatter plot showing the correlation between expression of <i>RelA</i> and <i>NFKBIA</i> (IkB-α) in ER+/HER2- tumors with > 75% purity within the TCGA cohort. The Kendall-Tau correlation co-efficient and the associated p-values are shown. The linear regression of the correlation is indicated by the red line. <b>F.</b> Similar to the panel <b>E</b> except that the correlation is between AURKA, a marker for proliferation and NFKBIA.</p

RelA levels vary widely in breast cancer and negatively correlate with proliferation in certain subtypes.

<p><b>A.</b> Representative images of breast tumors with each RelA staining profile: cytoplasm low/high; nuclear positive/negative. Red arrows indicate positive nuclei and scale bar = 100μM. <b>B.</b> The table shows number of tumors (divided by ER and HER2 based subtypes) within each category of RelA staining pattern in the Boston-cohort (n = 105) and the Croatia-cohort (n = 198). The Fishers exact test (FET) p-values for the distribution is indicated. <b>C.</b> Box plot showing the distribution of the percentage of Ki67 positive nuclei within each category of breast tumors based on RelA staining. Nuclear negative/cytoplasm high (nNcH), nuclear negative/cytoplasm low (nNcL) and nuclear positive/cytoplasm high (nPcH). The Wilcoxon rank sum test was used to estimate the significance of the association and the p-values are indicated.</p

RelA induction down-regulates CDK4 resulting in Rb hypo-phosphorylation and cell cycle arrest.

<p><b>A.</b> HRA cells were cultured in the presence and absence of Dox (1μg/ml) for 48 hours and changes in indicated proteins was monitored every 12 hours by immunoblot. <b>B.</b> HRA cells were cultured in the presence and absence of Dox (1μg/ml) for 48 hours and cytoplasmic (C) and nuclear (N) proteins were extracted. The samples were analyzed by immunoblot. HSP90 was used as a marker for purity of the nuclear fraction. <b>C.</b> HRA cells were co-transfected with an NF-kB reporter Firefly luciferase and background control Renilla luciferase plasmids. Transfected cells were trypnized, plated in triplicates and cultured in the presence or absence of Dox (1μg/ml) for 48 hours. Subsequently, expression of Firefly and Renilla luciferase was estimated. Activity of Firefly luciferase in the Dox+ and Dox– samples were normalized to the respective activity of Renilla luciferase. Activity values in the Dox+ sample was normalized to the Dox- sample to obtain the bar plot. Increase in Firefly Luciferase activity following RelA induction was statistically significant (p-value: 1.3 E -7). <b>D.</b> HRA cells were cultured in the absence (No Dox) or presence of Dox (1μg/ml) for 72 hours (72+). Parallel HRA cells cultured in the presence of Dox for 48 or 24 hours and then switched to medium devoid of Dox for 24 (48+, 24-) or 48 (24+, 48-) hours were fixed and stained using Propidum Iodite. Distribution of cells in the G2, S and G1 phases of the cell cycle was analyzed using flow cytometry. <b>E.</b> HRA cells were treated according to the scheme in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140243#pone.0140243.s004" target="_blank">S4A Fig</a>. Total RNA was extracted from the samples and global gene expression levels was determined using the Affymetrix Primeview array. Differentially expressed genes among the four conditions were identified (FDR < 0.05) and hierarchical clustering of the genes and samples was performed using the differentially expressed genes. <b>F.</b> Expression levels of the core G1/S transition genes including cyclins, CDKs and CDK inhibitors in 24+, 72+ and DW samples were normalized to the expression level in the ND samples. The bar plot indicates differentially expressed genes (p-value <0.05, fold change > 2) from the core G1/S transition gene list. <b>G.</b> HRA cells were treated with dox according to the scheme in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140243#pone.0140243.s004" target="_blank">S4C Fig</a>. Whole cell lysates were analyzed using indicated antibodies, including antibodies specific to phosphorylated (S536 and S468) and acetylated (K310) forms of RelA. <b>H</b>. Differential expression (p-value < 0.05, fold change > 4) of feedback negative regulators of RelA including IkB, CYLD and TNFAIP3 were identified. The bar plot shows expression changes relative to the ND sample.</p