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  • Table 1
    Participant profile (mean ± SD)

    Preparation of alveolar macrophages from BALF

    By infusing 50 mL of sterile sodium chloride into either the middle lobe or the lingula, we obtained approximately 30 mL of bronchoalveolar lavage fluid (BALF) from each subject. BALF was centrifuged for 20 minutes at 1,500 rpm and the supernatant was discarded. The precipitate was resuspended in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum, supplemented with 100 U/mL penicillin and 100 mg/mL streptomycin. Cells were cultured on 3.5 cm plates for 3 hours, and washed three times by phosphate-buffered saline. The cells adhering to the bottom were alveolar macrophages (AMs), which were verified by cellular staining.

    Immunohistochemical analysis

    Immunohistochemistry was used to analyze the protein levels and distribution of SOCS1 in bronchial tissues of COPD and control subjects. Briefly, we embedded lung tissues (endobronchial biopsies) in paraffin and placed 5 µm-thick sections on the slides, dewaxed in xylene, and rehydrated. Endogenous peroxidases were inhibited with 0.5% hydrogen peroxide in methanol for 10 minutes, followed by overnight incubation at 4°C with a rabbit polyclonal IgG antibody against SOCS1 (Abcam, Cambridge, UK). Immunodetection was performed with diaminobenzidine, biotinylated goat anti-rabbit IgG reagent, and horseradish peroxidase reagent.

    Total RNA from AMs and bronchial tissues were extracted using an RNeasy kit (Qiagen, Valencia, CA, USA), and total RNA from RAW264.7 cells was extracted using TRIzol reagents (Thermo Fisher Scientific), according to the manufacturer’s instructions. Single-stranded cDNA was synthesized for each sample with oligo (dT) primers using RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Waltham, MA, USA), according to the manufacturer’s protocol. Total RNA (500 ng) was amplified with a 7500 Fast Real-Time PCR System (Thermo Fisher Scientific) with FastStart Universal SYBR Green (Hoffman-La Roche Ltd., Basel, Switzerland) after cDNA synthesis. The standard curve for quantification was created using a modified procedure as previously described. 23 The sequences of all primers are shown in Mens Black Sid skinny jeans River Island Free Shipping Explore tPi7o8Uugt
    . Fold change of the gene expression was calculated by 2 −Ct relative to the internal reference gene (glyceraldehyde-3-phosphate dehydrogenase, GAPDH ). The results were repeated at least three times, respectively.

    Table 2
    Primer sequences for real-time PCR

    Cell line and CS extract

    The RAW264.7 mouse macrophage cell line was obtained from the Cell Collection and Research Center of Chinese Academy of Science. CS was collected and treated with DMEM by continuous suction in a chamber connected to a vacuum. Treatment with 500 mL of DMEM required mainstream smoke from ten cigarettes and 5 minutes for each cigarette. The resultant CS extract (CSE)–DMEM was considered as 100% stock solution and ready for use after adjusting the pH to 7.4 and sterile filtration (0.2 µm). Serial dilutions of 50%, 30%, 20%, and 10% CSE solution were prepared for cell culture. DMEM only was considered as 0% CSE.

    Exposure of RAW264.7 culture to CSE and U-75302 treatment

    The most appropriate condition was determined using a combination of different concentrations of CSE stimulation at different exposure times of 3, 6, 12, 24, and 48 hours. Basically, 30% CSE was the maximal dilution without excessive cell mortality after 24 hours exposure. For treatment with U-75302, RAW264.7 cells were seeded overnight and treated with 20% CSE with 10 nmol/mL U-75302 (Enzo, Telluride, CO, USA) for 24 hours. Supernatant was then harvested and total cellular RNA and protein were extracted.

    Cytokine enzyme-linked immunosorbent assays

    The concentrations of interleukin-8 (IL-8) and tumor necrosis factor-α (TNF-α) in the BALF, and the mouse IL-6 and TNF-α levels in the cell culture supernatant were determined using DuoSet ELISA kits (RD Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. LTB4 concentration in BALF and cell culture supernatants were measured using a specific enzyme immunoassay with a commercial kit (Cayman Chemical Co, Ann Arbor, MI, USA).

    Western blot

    Cells were lysed with 100 µL radio immunoprecipitation assay lysis buffer (Sigma-Aldrich Co., St Louis, MO, USA). Protein concentrations were measured with bicinchoninic acid assay kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Equal amounts of proteins (30 µg) were subjected to a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gels were run at 80 V for 30 minutes followed by 120 V for 1 hour before transferring onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). After blocking with phosphate-buffered saline containing 5% nonfat milk for 2 hours at room temperature, we incubated overnight at 4°C with rabbit polyclonal anti-SOCS1 antibody (1:1,000; Abcam), or a β-actin antibody (1:1,000; Cell Signaling, Danvers, MA, USA). The membrane was washed for 5 minutes with 15 mL tris-buffered saline and tween 20 three times, and then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (1:2,000; Sigma) for 1 hour at room temperature. After washing, we added 1 mL chemiluminescent substrate (Thermo Fisher Scientific) to the membrane. The signal was detected and quantified with an enhanced chemiluminescence system (ImageQuant LAS-4000 MINI; GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). All samples were normalized to β-actin.

    Statistical analysis

    Results are presented as mean ± standard error of the mean if not stated otherwise. A two tailed -test was performed in all results’ statistical analysis for comparisons of baseline characteristic. A linear regression was adopted for association between SOCS1 mRNA levels, LTB4 expression level, and FEV (forced expiratory volume in 1 second) %predicted using Spearman’s rank correlation test. Statistical analyses were performed as described in each figure legend, using GraphPad Prism. -values ≤0.05 were considered statistically significant.

    Go to:

    Immunohistochemistry staining showed the presence of SOCS1 protein mainly in the airway epithelial cells and AMs ( Figure 1 ). We found that the expression level of SOCS1 in bronchial mucosa was significantly lower in patients with COPD than in control subjects (808.17±697.06 vs 15,944±7,181.53; Figure 1A–D ).

    With the exception of F1000 Faculty Reviews , peer review takes place after publication and is driven by the authors who must suggest the referees and who decide when and how to address any criticisms raised by the referees. Communication with the referees is done by the editorial team, on behalf of the authors.

    The peer-review process is completely transparent: the referee names and their reports are published alongside the article, and the authors’ responses to the referees (or to reader comments) are also posted for readers to see.

    Revisions and updates are published as new versions, with clear explanations (in an “Amendments” section) of the changes the authors made.

    Usually, an article receives 2 or 3 referee reports. The referees choose an approval status, which contributes to determining whether the article has ‘passed peer review’ and is indexed in bibliographic databases , such as PubMed.

    Articles with 0 or 1 report have not passed peer review and are not indexed in PubMed and other bibliographic databases; if peer review is stopped in consultation with the F1000Research team , the article (which is permanently published with a DOI and cannot be removed) can be considered equivalent to a preprint and the authors may choose to submit the manuscript to a journal for peer review and publication elsewhere (it is at the discretion of the journal editors the authors are submitting to how they consider the history of the article at F1000Research ).

    Peer review of these articles can be reactivated at a later stage at the authors’ request, provided the article has not been peer reviewed and published elsewhere in the meantime.

    Publication of any material in F1000Research denotes that all its authors have agreed to its content and have ensured that F1000Research’s policies have been fully adhered to; adherence to sections 1-4 is compulsory for posters, slides and documents; sections 5 and 6 are strongly encouraged as they present good scientific practice and publishing standards.

    Authors of posters, slides and documents must ensure that they do not breach copyright with any content they post. Authors who wish to reproduce a figure or table from a previous copyrighted publication are responsible for obtaining the permission of copyright holders and for clearly referencing the original source. Figures that were previously published under a creative commons license may be reused under the condition of the specific license that applies to those figures.

    must ensure that they do not breach copyright

    Any poster or slide whose author’s affiliation is a recognized research or clinical institution (or an organization clearly related to the life sciences) can be posted. At least one author on the poster or slide must meet this key criterion.

    Less than a week after the premiere of In Between Dreams, on March 24, 2017, the group released their first album in over 4 years, The Ghost of Hope. The album, based around historical train wrecks from the late 19th century and early 20th century, featured guest players such as Eric Drew Feldman ("who has worked with everybody cool") and Nolan Cook. The album gained large media attention, with notable magazines like Billboard and Wired covering the album shortly before its release.

    Several months later, in late October 2017, the full In Between Dreams tour kicked off in Copenhagen, featuring the same cast and decorations as the Tokyo show, though with a substantially changed setlist that included preview tracks from an upcoming blues-styled album: "Die! Die! Die!" and "Tell Me." In Between Dreams was the first show featuring the "Real Residents"; Tyrone, the singer, Eekie, the guitarist, Erkie, the keyboardist, and a new member, Cha Cha, a percussionist (the first in the band since "Carlos'" departure.) The decoration of the show consisted of a blue and white checkered (with the iconic eyeball with the top hat breaking the pattern every so often) backdrop, several dynamic and color changing lights, and the same giant ball screen from Shadowland for displaying videos in between every couple songs. The videos consisted of various well-known figures reminiscing of dreams they had; Richard Nixon's dream about being a blues singer, John Wayne's nightmare about a lone ballerina that disappears when he attempts to approach her, Mother Theresa's dream about a train wreck, and so on. The tour ran through Europe from late October all the way to the end of November 2017, and later the United States from early April to early May 2018.

    The Residents at The Middle East (Cambridge, MA) in 2010
    The Residents at The Middle East (Cambridge, MA) in 2010

    Much of the speculation about the members' true identities swirls around its management team, known as the Cryptic Corporation . Cheap Sale Classic Buy Cheap Browse Womens Tim Ls Tee Tops French Connection Real Cheap Online jEECsJ66a
    Cryptic was formed in 1976 as a corporation in California by Jay Clem (born 1947), Homer Flynn (born April 1945), Hardy W. Fox (born 1945), and John Kennedy, all of whom denied having been band members. (Clem and Kennedy left the Corporation in 1982, much to the chagrin of some fans [17] ). The Residents members do not grant interviews, although Flynn and Fox have conducted interviews with the media.

    Due to the cover artwork of the band's debut album Meet The Residents being a parody of The Beatles 1964 North American release, Oneshoulder Asymmetric Printed Crepe Dress White Halston Heritage Buy Cheap Best Store To Get 9R42kcldZ
    there were rumours that the members of Residents were actually the Beatles, even specifically naming George Harrison .

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    README.md

    This repository contains a script to generate question/answer pairs using CNN and Daily Mail articles downloaded from the Wayback Machine.

    For a detailed description of this corpus please read: sleeveless loose fit dress Pink amp; Purple TwinSet Outlet In China Get New Wiki Sale Online 2BaXAOz
    , Hermann et al., NIPS 2015. Please cite the paper if you use this corpus in your work.

    In case the script does not work you can also download the processed data sets from [ http://cs.nyu.edu/~kcho/DMQA/ ]. This should help in situations where the underlying data is not accessible (Wayback Machine partially down).

    Python 2.7, , , , and . must be version 2.9.1. You can install from here: http://xmlsoft.org/libxslt/downloads.html

    The news article metadata is ~1 GB.

    You may need to install development packages to install :

    This will download news articles from the Wayback Machine. Some URLs may be unavailable. The script can be run again and will cache URLs that already have been downloaded. Generation of questions can run without all URLs downloaded successfully.

    Note, this will generate ~1,000,000 small files for the Daily Mail so an SSD is preferred.

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