Open science in archaeology

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Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Relative metabolomic outputs of U87MG cells subjected to UPR stress compared to unstressed cells.

<p>U87 cells were grown in Knockout DMEM medium with serum replacement as described above. Cells were harvested, washed, and replated in the same (fresh) medium with or without 1 mM DTT, and with 5 mM ¹³C-glucose, for 4 hrs prior to cell and media harvest and PCA extraction as described in Materials and Methods. ¹H-, ³¹P-, and ¹³C-NMR spectra were obtained and quantified; data analyses were conducted as described. Graphs compare metabolite components from untreated cells (set to 100%) vs treated cells; error bars show standard deviation, and * = p< 0.05 derived from Student’s <i>t</i> test comparing treated to untreated in averages of 3 separate experiments. (<b>A</b>) displays data for soluble metabolites. Cho = choline; Cr = creatine GSH = glutathione; Lac = lactate. (<b>B</b>) displays data for high energy phosphates and [1-¹³C] glucose uptake. P-Choline = phosphocholine; GP-Choline = glycerophosphocholine; PME = phosphomonoesters; PDE = phosphodiesters; PC = phosphocholine; GPC = glycerophosphocholine; P-Creatine = phosphocreatine; UDPG = uridine diphosphoglucose; Glc = glucose; Lac = lactate. (<b>C</b>) shows data for lipid compounds. MUFA = monounsaturated fatty acids; TAG = triacylglycerols; Glycerol-Plipids = glycerol phospholipids; PtdCholine = phosphatidylcholine; PtdEthanolamine = phosphatidylethanolamine; PUFA = polyunsaturated fatty acids; FA = fatty acids.</p

Identification of UPR signaling response patterns in high-grade glioma xenografts and cell lines.

<p>Human glioma xenografts grown in <i>nu/nu</i> mice were derived from U87MG, and U87+EGFR (wild type) (cell lines described in the text and Materials and Methods). (<b>A</b>) Northern blots of 10 µg total RNA from replicate tumors (n=3) and normal brain from <i>nu/nu</i> mice; 10 µg total RNA from U87 tissue culture cells (“cells”) treated with the reducing agent DTT ([+]) lanes) to induce the UPR. Note transcriptional upregulation of UPR-induced mRNAs for ER chaperones (GRP94, BiP/GRP78) and UPR signaling components (XBP-1, CHOP, ATF4, ATF6). Quantification of BiP/GRP78 (<b>B</b>) and GRP94 (<b>C</b>) mRNA expression compared to mean level of expression in normal murine brain (dotted line). (<b>D</b>) U87MG, U87+EGFR, and U87+EGFRvIII (U87 cells transfected with the tumor-specific EGFR mutant variant III [in-frame deletion of exons 2-7]) cells show greater UPR inducibility with 1 mM DTT (determined by Northern blotting for XBP-1 and CHOP messages) than do HeLa cells. (<b>E</b>) Human glioma xenografts were derived from U87MG, U87+EGFR, and from D245MG, from a patient-derived Duke high grade glioma (from the Duke Brain Tumor BioRepository). Immunoblot of replicate tumors (n=3) from xenograft glioma models and normal brain from <i>nu/nu</i> mice. Note upregulation of ER chaperones in tumor lysates vs brain lysates: GRP170/ORP150, GRP94, calnexin (CNX), ERp72, protein disulfide isomerase (PDI), calreticulin (CRT), homocysteine-induced ER protein (HERP), and ER membrane markers (Sec61α and translocon associated protein, TRAPα) relative to loading control (β-actin). GRP78/BiP protein expression was variable in our Western blot assays. Blots probing for actin as loading controls are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073267#pone.0073267.s001" target="_blank">Supplemental Figure S1</a>. Blots for GRPs 170 and 78, for ERp72 and TRAPα were replicate blots. Blots for GRP94, CNX, CRT, HERP, and Sec61α were stripped and reprobed for actin.</p

Primary tissue culture cells from newly-resected gliomas also display inducible elements of the UPR.

<p>Dissociated cell cultured from freshly-resected GBMs were grown under serum-free conditions and were treated (or not, “Cont”) with 1 mM DTT (“+DTT”) for 4 hrs. Cell cultures were harvested, and cells lysed described. Proteins were separated on SDS-PAGE and Western blotted and probed with the antibodies listed. Upregulation of some of the UPR components is evident. Actin probe is used as a loading control, from the stripped CRT blot. Other actin blots to verify loading are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073267#pone.0073267.s003" target="_blank">Supplemental Figure S3</a>.</p

Immunohistochemistry and Western blots of patient brain tumors reveal high expression of UPR-related proteins.

<p>A Cybrdi “brain glioblastoma” tissue microarray (TMA) was probed for GRP94 by immunohistochemistry (IHC); (<b>A</b>) Scores were derived as describe in Materials and Methods, and show that high grade tumors such as glioblastoma multiforme (GBM, WHO grade IV) and anaplastic astrocytomas (WHO grade III—AA3) express significantly higher levels of GRP94 than do lower grade tumors (grade II anaplastic astrocytomas, AA2), anaplastic hyperplasia (Hyp) or normal brain (<b>*</b>, p < 0.05 by ANOVA comparing high grade gliomas vs the rest of the samples). Examples of the IHC staining are shown for 3 GBMs and normal brain (<b>B</b>). (<b>C</b>) Grade IV (GBM) tumor lysates (3 different tumors, not the same as those in <b>B</b>) and normal brain (cortex) lysates were separated by SDS-PAGE and electroblotted for Western blotting. Blots were probed with the antibodies listed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073267#pone-0073267-g002" target="_blank">Figures 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073267#pone-0073267-g003" target="_blank">3</a>; “FASN” = Fatty Acid Synthase; “p90/p50/p36” = full length and cleaved forms of ATF6 “spliced/unspliced” = spliced or unspliced protein product of XBP1. Molecular weight markers are listed at left. Actin blot shown as loading control was a replicate for GRP78 and CRT.</p