A Highly Sensitive Assay for Monitoring the Secretory Pathway and ER Stress

Background: The secretory pathway is a critical index of the capacity of cells to incorporate proteins into cellular membranes and secrete proteins into the extracellular space. Importantly it is disrupted in response to stress to the endoplasmic reticulum that can be induced by a variety of factors, including expression of mutant proteins and physiologic stress. Activation of the ER stress response is critical in the etiology of a number of diseases, such as diabetes and neurodegeneration, as well as cancer. We have developed a highly sensitive assay to monitor processing of proteins through the secretory pathway and endoplasmic reticulum (ER) stress in real-time based on the naturally secreted Gaussia luciferase (Gluc). Methodology/Principle Findings: An expression cassette for Gluc was delivered to cells, and its secretion was monitored by measuring luciferase activity in the conditioned medium. Gluc secretion was decreased down to 90% when these cells were treated with drugs that interfere with the secretory pathway at different steps. Fusing Gluc to a fluorescent protein allowed quantitation and visualization of the secretory pathway in real-time. Expression of this reporter protein did not itself elicit an ER stress response in cells; however, Gluc proved very sensitive at sensing this type of stress, which is associated with a temporary decrease in processing of proteins through the secretory pathway. The Gluc secretion assay was over 20,000-fold more sensitive as compared to the secreted alkaline phosphatase (SEAP), a well established assay for monitoring of protein processing and ER stress in mammalian cells. Conclusions/Significance: The Gluc assay provides a fast, quantitative and sensitive technique to monitor the secretory pathway and ER stress and its compatibility with high throughput screening will allow discovery of drugs for treatment of conditions in which the ER stress is generally induced

The Sloan Digital Sky Survey Quasar Catalog V. Seventh Data Release

We present the fifth edition of the Sloan Digital Sky Survey (SDSS) Quasar Catalog, which is based upon the SDSS Seventh Data Release. The catalog, which contains 105,783 spectroscopically confirmed quasars, represents the conclusion of the SDSS-I and SDSS-II quasar survey. The catalog consists of the SDSS objects that have luminosities larger than M_i = -22.0 (in a cosmology with H_0 = 70 km/s/Mpc Omega_M = 0.3, and Omega_Lambda = 0.7) have at least one emission line with FWHM larger than 1000 km/s or have interesting/complex absorption features, are fainter than i > 15.0 and have highly reliable redshifts. The catalog covers an area of 9380 deg^2. The quasar redshifts range from 0.065 to 5.46, with a median value of 1.49; the catalog includes 1248 quasars at redshifts greater than four, of which 56 are at redshifts greater than five. The catalog contains 9210 quasars with i < 18; slightly over half of the entries have i< 19. For each object the catalog presents positions accurate to better than 0.1" rms per coordinate, five-band (ugriz) CCD-based photometry with typical accuracy of 0.03 mag, and information on the morphology and selection method. The catalog also contains radio, near-infrared, and X-ray emission properties of the quasars, when available, from other large-area surveys. The calibrated digital spectra cover the wavelength region 3800-9200 Ang. at a spectral resolution R = 2000 the spectra can be retrieved from the SDSS public database using the information provided in the catalog. Over 96% of the objects in the catalog were discovered by the SDSS. We also include a supplemental list of an additional 207 quasars with SDSS spectra whose archive photometric information is incomplete.Comment: Accepted, to appear in AJ, 7 figures, electronic version of Table 2 is available, see http://www.sdss.org/dr7/products/value_added/qsocat_dr7.htm

HST/WFC3 grism observations of z∼1z\sim1 clusters: evidence for evolution in the mass-size relation of quiescent galaxies from poststarburst galaxies

Minor mergers have been proposed as the driving mechanism for the size growth of quiescent galaxies with decreasing redshift. The process whereby large star-forming galaxies quench and join the quiescent population at the large size end has also been suggested as an explanation for this size growth. Given the clear association of quenching with clusters, we explore this mechanism by studying the structural properties of 23 spectroscopically identified recently quenched (or "poststarburst" (PSB)) cluster galaxies at $z\sim1$. Despite clear PSB spectral signatures implying rapid and violent quenching, 87\% of these galaxies have symmetric, undisturbed morphologies in the stellar continuum. Remarkably, they follow a mass-size relation lying midway between the star-forming and quiescent field relations, with sizes $0.1$ dex smaller than $z\sim1$ star-forming galaxies at log$(M_{*}/M_{\odot})=10.5$. This implies a rapid change in the light profile without directly effecting the stellar distribution, suggesting changes in the mass-to-light ratio gradients across the galaxy are responsible. We develop fading toy models to explore how star-forming galaxies move across the mass-size plane as their stellar populations fade to match those of the PSBs. "Outside-in" fading has the potential to reproduce the contraction in size and increase in bulge-dominance observed between star-forming and PSB cluster galaxies. Since cluster PSBs lie on the large size end of the quiescent mass-size relation, and our previous work shows cluster galaxies are smaller than field galaxies, the sizes of quiescent galaxies must grow both from the quenching of star-forming galaxies and dry minor mergers.Comment: 25 pages, 10 figures, accepted for publication in MNRA

Treatment After Anterior Cruciate Ligament Injury: Panther Symposium ACL Treatment Consensus Group

© The Author(s) 2020. Treatment strategies for anterior cruciate ligament (ACL) injuries continue to evolve. Evidence supporting best-practice guidelines for the management of ACL injury is to a large extent based on studies with low-level evidence. An international consensus group of experts was convened to collaboratively advance toward consensus opinions regarding the best available evidence on operative versus nonoperative treatment for ACL injury. The purpose of this study was to report the consensus statements on operative versus nonoperative treatment of ACL injuries developed at the ACL Consensus Meeting Panther Symposium 2019. There were 66 international experts on the management of ACL injuries, representing 18 countries, who were convened and participated in a process based on the Delphi method of achieving consensus. Proposed consensus statements were drafted by the scientific organizing committee and session chairs for the 3 working groups. Panel participants reviewed preliminary statements before the meeting and provided initial agreement and comments on the statement via online survey. During the meeting, discussion and debate occurred for each statement, after which a final vote was then held. Ultimately, 80% agreement was defined a priori as consensus. A total of 11 of 13 statements on operative versus nonoperative treatment of ACL injury reached consensus during the symposium. Overall, 9 statements achieved unanimous support, 2 reached strong consensus, 1 did not achieve consensus, and 1 was removed because of redundancy in the information provided. In highly active patients engaged in jumping, cutting, and pivoting sports, early anatomic ACL reconstruction is recommended because of the high risk of secondary meniscal and cartilage injuries with delayed surgery, although a period of progressive rehabilitation to resolve impairments and improve neuromuscular function is recommended. For patients who seek to return to straight-plane activities, nonoperative treatment with structured, progressive rehabilitation is an acceptable treatment option. However, with persistent functional instability, or when episodes of giving way occur, anatomic ACL reconstruction is indicated. The consensus statements derived from international leaders in the field will assist clinicians in deciding between operative and nonoperative treatment with patients after an ACL injury

The Ninth Data Release of the Sloan Digital Sky Survey: First Spectroscopic Data from the SDSS-III Baryon Oscillation Spectroscopic Survey

The Sloan Digital Sky Survey III (SDSS-III) presents the first spectroscopic data from the Baryon Oscillation Spectroscopic Survey (BOSS). This ninth data release (DR9) of the SDSS project includes 535,995 new galaxy spectra (median z=0.52), 102,100 new quasar spectra (median z=2.32), and 90,897 new stellar spectra, along with the data presented in previous data releases. These spectra were obtained with the new BOSS spectrograph and were taken between 2009 December and 2011 July. In addition, the stellar parameters pipeline, which determines radial velocities, surface temperatures, surface gravities, and metallicities of stars, has been updated and refined with improvements in temperature estimates for stars with T_eff<5000 K and in metallicity estimates for stars with [Fe/H]>-0.5. DR9 includes new stellar parameters for all stars presented in DR8, including stars from SDSS-I and II, as well as those observed as part of the SDSS-III Sloan Extension for Galactic Understanding and Exploration-2 (SEGUE-2). The astrometry error introduced in the DR8 imaging catalogs has been corrected in the DR9 data products. The next data release for SDSS-III will be in Summer 2013, which will present the first data from the Apache Point Observatory Galactic Evolution Experiment (APOGEE) along with another year of data from BOSS, followed by the final SDSS-III data release in December 2014.Comment: 9 figures; 2 tables. Submitted to ApJS. DR9 is available at http://www.sdss3.org/dr

Expert Opinion on Laparoscopic Surgery for Colorectal Cancer Parallels Evidence from a Cumulative Meta-Analysis of Randomized Controlled Trials

PMID: 22532846 [PubMed - indexed for MEDLINE] PMCID: PMC3332109 Free PMC ArticlePeer reviewedPublisher PD

Gluc as a reporter in mammalian cells.

<p>(A) Schematic representation of the expression cassettes for Gluc-IRES-CFP cloned in the CSCW lentivirus vector. (B) High infection rate of cells with lentivirus vectors (M.O.I. = 30) as monitored by cerulean fluorescence. Scale bar, 100 µm. (C) Levels of Gluc activity in cells vs. medium vs. cells+medium. 293T cells were infected with the lentivirus vector carrying the expression cassette for Gluc-IRES-CFP and 20,000 cells were plated in wells of a 96-well plate. 48 hrs post-infection, new medium was added to the wells and Gluc activity was measured 24 hrs later in conditioned medium, viable washed cells or cells+conditioned medium after adding 2.5 µM coelenterazine.</p

Monitoring of ER stress with Gluc.

<p>293T cells expressing Gluc were subjected to no or different concentrations of DTT (A–D) or thapsigargin (E–F) to induce ER stress. (A) Real-time RT-PCR for spliced XBP-1 mRNA which increased in response to >1 mM DTT 4 hrs after treatment. Fold induction is calculated with respect to the control non-infected/non-treated cells. (B&F) Western blot analysis showing upregulation of BiP and/or phosphorylated eIF2alpha levels in response to ER stress 24 hrs after treatment. Blot is also probed with Gluc antibody as well as beta-tubulin antibody for equal loading. (C&E) Conditioned medium assayed for Gluc activity 4 hrs after DTT or thapsigargin treatment showing that Gluc secretion is decreased in response to ER stress. (D) Untreated or treated cells with 1 mM DTT were monitored overtime for the level of Gluc secretion by assaying an aliquot of the conditioned medium for Gluc bioluminescence. The mean ± S.E.M. is presented on the graphs (n = 3), with *p≤0.01 as calculated by the student's t-Test.</p

Gluc-YFP fusion to visualize secretory pathway in real-time.

<p>(A) Schematic representation of the Gluc-YFP fusion cloned in the lentivirus vector. (B) Uninfected 293T cells or cells infected with a lentivirus vector expressing Gluc-YFP fusion were lysed and analyzed by western blotting with anti-Gluc antibody. (C) Cells expressing Gluc-YFP fusion were treated with BFA or nocodazole and their conditioned medium were assayed for Gluc activity 24 hrs later. *p≤0.01 as predicted by student T-test. (D) Fluorescence microscopy of a single live cell expressing Gluc-YFP and either untreated or treated with BFA showing that this fusion is trapped in the ER upon BFA treatment. Scale bar, 10 µm.</p