dataesr/scientific-paragraphs-categorization huggingface dataset:
Lorlatinib is Active Against ALK Resistance Mutations that Develop on Second-Generation ALK Inhibitors To investigate whether cancers that develop resistance to second-generation ALK inhibitors remain susceptible to continued ALK inhibition, we examined the activity of the thirdgeneration ALK inhibitor, lorlatinib (PF-06463922). Lorlatinib is a potent, highly selective ALK/ROS1 inhibitor that is currently being evaluated in an ongoing phase 2 clinical trial (NCT01970865). We first engineered Ba/F3 cells to express wild-type EML4-ALK (E13;A20) or EML4-ALK harboring various ALK mutations (Figure  6 ). Ba/F3 cells were treated with crizotinib, ceritinib, alectinib, brigatinib and lorlatinib. Notably, lorlatinib was the only ALK inhibitor to potently inhibit ALK phosphorylation across all single ALK secondary mutations, including ALK G1202R (IC50 49.
For whole exome sequencing, genomic DNA was extracted from formalin-fixed paraffin embedded (FFPE) samples. Whole-exome capture libraries were constructed from 100ng of extracted tumor and normal DNA. Ligated DNA was size-selected for lengths between 200-350bp and subjected to exonic hybrid capture using SureSelect v2 Exome bait (Agilent). Samples were multiplexed and sequenced on Illumina HiSeq flowcells (paired end 76bp reads) to an average on-target coverage depth ranging from 134 to 210× for all tumors and normal, respectively. Massively parallel sequencing data were processed using two consecutive pipelines as previously described  (33). Patient-derived cell lines (MGH021-5A, MGH034-2A, MGH049-1A, MGH051-2, MGH075-2E) were analyzed by NGS. RNA bait-based hybridization capture was performed to capture over 1000 known cancer genes (RightOn Cancer Sequencing Kit, developed in collaboration with Elim BioPharma), as previously described  (10).
Acknowledgments Funding Sources: This work was supported by grants from the the  National Cancer Institute  ( 5R01CA164273 , to ATS and JAE,  C06CA059267  to JFG), by a  National Foundation for Cancer Research  (to ATS), by Be a Piece of the Solution, by the  Evan Spirito Foundation , and by  LungStrong. JFG has served as a compensated consultant or received honoraria from  Bristol-Myers Squibb ,  Genentech ,  Ariad ,  Loxo ,  Novartis ,  Merck ,  Clovis ,  Boehringer Ingelheim ,  Jounce Therapeutics , and  Kyowa Hakko Kirin. SG has served as a compensated consultant for  Pfizer ,  Bristol-Myers Squibb, Ariad ,  Genentech/Roche ,  AstraZeneca , and  Boehringer Ingelheim. LVS has served as an uncompensated consultant for  Boehringer Ingelheim ,  Clovis ,  Novartis ,  Merrimack , and  Taiho Pharmaceuticals , and served as a compensated consultant or received honoraria from  AstraZeneca ,  Genentech  and  Ariad.
1% Tween and 5% milk, and then with an anti-mouse or antirabbit horseradish peroxidase-conjugated secondary antibody (1:10,000; Santa Cruz), respectively. Bound antibodies were detected by exposure to enhanced chemiluminescence reagents (ECL; GE Healthcare or Life Technologies). For quantitative detection of viral proteins, membranes were first incubated with the rabbit polyclonal antibody U2 (1:1,000) and then with an anti-rabbit infrared fluorescence (IRDye) secondary antibody and analyzed with an Odyssey imaging system and the software Image Studio (Li-Cor Biosciences). Infection assays. Mammalian cells were infected with virus at different multiplicities of infection (MOIs) in medium without FBS at 37°C for 1 h. Virus supernatant was then replaced by complete culture medium, and cultures were incubated for up to 64 h before fixation.
When the mouse monoclonal antibody 1621 (25 g • ml Ϫ1 ) was used in infection assays to neutralize DC-SIGN, UUKV-infected cells were immunostained with the rabbit polyclonal antibody U2 (1:400) and AF647-conjugated secondary anti-rabbit antibody (1:500; Life Technologies). Flow cytometry-based analysis involved the use of a FACSCalibur cytometer (Becton Dickinson) and FlowJo software (TreeStar). Fluorescence microscopy. Infected cells were fixed and permeabilized with phosphate-buffered saline (PBS) containing 0. 1% Triton X-100 (Merck Millipore), incubated with the mouse monoclonal antibody 8B11A3 (1:1,000) at room temperature for 1 h, washed, and then exposed to AF488-conjugated secondary anti-mouse (1:800; Life Technologies) at room temperature for 1 h. Nuclei were subsequently stained with Hoechst 33258 (0.
5 g • ml Ϫ1 ; Life Technologies). Infection was quantified by counting cells in three independent fields, and cells were imaged with an Olympus IX81 microscope. Statistical analysis. The data shown are representative of at least three independent experiments. Values are given as the means of triplicates Ϯ standard deviations (SD). Sequencing of the full-length M segment isolated from UUKV-infected ticks. Questing nymphs of the tick I. ricinus were collected in the region of Ramsvik and Hindens Rev (Sweden; 2013). Pools of 25 nymphs were homogenized, and the total RNA was extracted with a magnetic bead-based protocol as described elsewhere (kind gift of Janne Chirico, National Veterinary Institute, Uppsala, Sweden, and Sara Moutailler, ANSES, Maisons-Alfort, France)  (42). The cDNA corresponding to the M segment of UUKV was synthesized by RT-PCR with the reverse transcriptase Superscript III (Life Technologies) and the specific primer RT-M (Table  1 ) before amplification as a single PCR product using the Pfu DNA polymerase (Promega) and the primers UUKV-M-5NC and UUKV-M-3NC (Table  1 ).
Recovery of UUKV S23 from RNA Pol I-driven plasmid DNAs. The UUKV lab strain that we used in this study as a template for cloning purposes results from the adaptation of the prototype tick isolate strain 23 (UUKV S23) to BHK-21 cells after successive plaque purifications in CEFs  (21, 38, 39). Compared with the S, M, and L nucleotide sequences published for the original UUKV S23 that was plaque purified five times in CEFs (GenBank accession numbers NC_005221. 1, NC_005220. 1, and NC_005214. 1, respectively), we identified only a few mutations in our UUKV lab strain (Fig. 1A ). Most were in the M segment. Over the entire virus genome, only one mutation was conserved, a nonsilent substitution (A2386G) in the M transcript (43-45) (Fig. 1A ). In all further  experiments, for convenience, UUKV will refer to our current mammalian cell culture-adapted lab strain, rUUKV will refer to the same virus but rescued from cDNAs, UUKV S23 will refer to the original tick isolate plaque purified five times in CEFs, and rUUKV S23 will refer to the viral particles produced from plasmids encoding genome sequences identical to those published for UUKV S23 in the late 1980s and early 1990s  (43) (44) (45).
Flow cytometry allowed quantitative detection of infected cells. Independent of the tick cell line, nearly 50% of cells were infected at a multiplicity of infection (MOI) of 5, and about 20% were infected at an MOI as low as 1. 25 (Fig. 3A ). The sensitivity of tick cells to rUUKV S23 infection was confirmed by fluorescence microscopy after immunostaining of the newly synthesized virus nucleoprotein N (Fig. 3B ). These experiments showed that both tick cell lines support infection of rUUKV S23. We next assessed infected cells for the production of virus progeny. Although cells were infected, titration of cell-free supernatants collected from challenged tick cells indicated that the production of infectious rUUKV S23 particles was almost nonexistent within the first 24 h of infection (data not shown).
Altogether these data indicate that I. ricinus cells support persistent infection by recombinant UUKV. Mammalian cells support infection by rUUKV S23 grown in tick cells. To examine whether rUUKV S23 derived from tick cells remains able to infect mammalian cells, BHK-21 cells were exposed to the viral progeny derived from IRE/CTVM19 cells for 18 h. Infected cells were analyzed with conformation-dependent mouse monoclonal antibodies in a flow cytometry-based assay. A large proportion of the infected cells were positive for both the virus nucleoprotein N and glycoproteins G N and G C (Fig. 4A ), suggesting that infection leads to viral replication. Therefore, our phlebovirus-tick cell system appears to be an excellent model for reproducing transmission of tick-borne viruses to mammalian hosts in vitro.
7A ). The amounts of G N , G C , and N proteins incorporated into rUUKV S23 particles were determined by quantitative Western blotting using the anti-UUKV U2 and an anti-rabbit infrared fluorescence secondary antibody prior to analysis with an Odyssey imaging systems. The ratio between the amount of protein N and the number of FFU was significantly lower in tick cell-derived viruses (Fig. 7B ) while that between the amount of viral glycoproteins and number of FFU was greater (Fig. 7C ). It was also apparent that the numbers of G N and G C molecules per nucleoprotein N (Fig. 7D ) and the number of FFU per N (Fig. 7E ) were higher in virions originating from tick cells. Similar results were obtained with rUUKV (data not shown). From these results, it is most likely that the global structural organizations of viral particles exhibit differences between viruses produced in tick and mammalian cells.
Wild-type UUKV originating from Swedish ticks in the 2010s. All RNA viruses are known to have high polymerase error rates. To determine whether the wild-type UUKV in vector tick populations has genetically evolved since the 1960s and, thus, whether our tick-borne virus model is still representative of the circulating virus, we analyzed 16 pools of 25 nymphs of the tick I. ricinus recently collected in the region of Ramsvik and Hindens Rev (Sweden; 2013) for the presence of the UUKV RNA segment M. The cDNA corresponding to the full-length M segment of UUKV was first synthesized by RT-PCR from vRNA extracted with magnetic beads from nymphal homogenates and then amplified by a single PCR. Out of the 16 homogenates, 4 were positive, from which it was possible to obtain the full-length nucleotide M sequence for two samples (GenBank accession numbers KX219593 and KX219594).
Full-length sequence of the viral glycoproteins G N and G C obtained from UUKV-infected field Ixodes ricinus nymphal ticks Values represent the percent identity of the sequence of interest augmented by the substitutions shown in boldface, which are assumed to have a weak impact, relative to the sequence of the precursor protein. d Total RNAs were extracted from 16 pools of 25 nymphs collected in the region of Ramsvik and Hindens Rev, Sweden, in 2013. The cDNA corresponding to the M segment of UUKV was synthesized by RT-PCR and amplified by a single PCR. Out of the 16 nymphal pools, four were positive for the virus, and from these four samples, one partial and two full-length nucleotide sequences could be obtained and. The list is shown of the amino acids found to be mutated in the full-length amino acid sequences of isolates RVS and HRS (GenBank accession numbers KX219593 and KX219594, respectively) relative to the sequence of the polypeptide precursor of the two glycoproteins G N and G C of UUKV S23 (GenBank accession number NC_005220.
ACKNOWLEDGMENTS This work was supported by grants from  CellNetworks Research Group funds , the  Natural Sciences and Engineering Research Council of Canada  (grant  419538-2012 ), and the  Banting Research Foundation  to P. -Y. L. This work was also supported by the  Swiss National Foundation and ETH Zurich. L. B. -S. is supported by  United Kingdom Biotechnology and Biological Sciences Research Council  funding (grant  BBS/E/1/00001741 ) provided to The  Pirbright Institute. We thank  Ari Helenius  for helpful discussions and support.
The semi-NMF approach, where archetypal patterns typically have entries with both positive and negative signs, and only the mixture coefficients are nonnegative, is a relaxation of K-means clustering. This variant of NMF has been shown to be more accurate than K-means on a number of datasets in retrieving known clusters  [2]. Since PosNegNMF can also be mathematically formulated as a semi-NMF of the two-way table, as will be shown later in the paper, it inherits its clustering performance. Nevertheless, both K-means and semi-NMF allow for clustering of either observations or variables, however in a non-simultaneous way, whereas our approach allows for a simultaneous clustering of observations and variables, yielding comprehensive heatmaps. We illustrate this novel clustering approach using two datasets from the research area of public health.
Data Sets Two data sets were analyzed: (i) The number of emergency hospital admissions for cardiovascular disease (CVD), myocardial infarction (MI), congestive heart failure (CHF), respiratory disease, and diabetes were collected in 26 US communities, for the years 2000-2003  [3]. (ii) The Compressed Mortality File (CMF)-a county-level national mortality and population database spanning the years 1968-2010. The table contains death counts for 13 age categories.
For example, St Louis, Los Angeles, Bakersfield and Dallas (PosNegNMF cluster 2, green crosses) appear clustered on the biplot with Kansas City, Toledo, etc. (PosNegNMF cluster 4, brown ˆsymbols) despite their relatively high level of MI, which is characteristic of cluster 2. This suggests that the biplot may be too restrictive, possibly because of being limited to the first two SVD components.
For example, St Louis, Los Angeles, Bakersfield and Dallas (PosNegNMF cluster 2, green crosses) appear clustered on the biplot with Kansas City, Toledo, etc. (PosNegNMF cluster 4, brown × symbols) despite their relatively high level of MI, which is characteristic of cluster 2. This suggests that the biplot may be too restrictive, possibly because of being limited to the first two SVD components.
Compressed Mortality File We have presented an example where the PosNegNMF method outperforms the original NMF or CA/SVD methods. Nevertheless, whenever the studied matrix can be approximated well by a rank-1 bilinear form, using this new approach is not recommended. The Compressed Mortality File (CMF)-a county-level national mortality and population database spanning the years 1968-2010-will help illustrate this important point. The table contains death counts for males for 13 age categories. The analysis of the normalized matrix of contingency ratios shows that the second SVD component contributes only 6% of the overall variance. Thus, rows and columns can be reordered through the rank-1 bilinear approximation given by the first SVD component, revealing well-known trends in the pivot period marking the end of 20th century (Figure  7 ), such as more deaths being observed in the age category 85+, and less deaths are observed in younger age categories.
Compressed Mortality File We have presented an example where the PosNegNMF method outperforms the original NMF or CA/SVD methods. Nevertheless, whenever the studied matrix can be approximated well by a rank-1 bilinear form, using this new approach is not recommended. The Compressed Mortality File (CMF)-a county-level national mortality and population database spanning the years 1968-2010will help illustrate this important point. The table contains death counts for males for 13 age categories. The analysis of the normalized matrix of contingency ratios shows that the second SVD component contributes only 6% of the overall variance. Thus, rows and columns can be reordered through the rank-1 bilinear approximation given by the first SVD component, revealing well-known trends in the pivot period marking the end of 20th century (Figure  7 ), such as more deaths being observed in the age category 85+, and less deaths are observed in younger age categories.
Applying the same operation to separate the elements of h into m maximally homogeneous clusters does the same for the columns, while separating the elements of both the row and column markers will cluster the matrix V into km internally homogeneous blocks. This approach was used in Liu et al. [11]  where a robust incomplete-data implementation of SVD has been used to obtain the approximants w and h. The segmentation of the row and column markers was carried out using the dynamic programming optimization method from Hawkins  [12]. It may not always be sensible to permute the rows or columns of the matrix, for instance when one considers time series or geographic data, which are naturally ordered. In this setting a separation into rectangular blocks can still be performed by partitioning the array into homogenous.
Uncropped blots are presented in Supplementary Fig. 12. Cell death assays. Cell death was measured by assessing lactate dehydrogenase release using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega) according to manufacturer's instructions. ASC speck imaging. Live imaging of ASC speck formation was performed using iBMDMs transfected to stably express ASC conjugated to mCherry protein. Mouse ASC (Accession Number NM_023258) was amplified by PCR using primers flanked by gateway recombination sequences (forward primer: 5 0 -ATGGGGCGGGCACG AGATGCC-3 0 , reverse primer: 5 0 -GCTCTGCTCCAGGTCCATCAC-3 0 ). A third generation 'pLNT' lentiviral transfer system was used to express N-terminally mCherry-tagged amplified ASC gene from a constitutive ubiquitin-ligase C promoter  42. Stably transduced cells were plated overnight at 5 Â 10 5 cells per ml.
2 mm 2 ) using ImageJ. Cysteine modification and NMR. Flufenamic acid or MNS (0. 05 mmol) was dissolved in DMSO-d 6 (400 ml) in an NMR tube. N-Acetyl-L-cysteine methyl ester (17. 7 mg, 0. 10 mmol) solubilized in DMSO-d 6 (100 ml) was added and an NMR spectrum was recorded every 10 min after the addition for 2 h and then every 1 h until 24 h. A Bruker Avance 400 spectrometer was used to record 1 H spectra at 300. 1 MHz. Chemical shifts are defined in parts per million and referenced against tetramethylsilane (d ¼ 0). NMR analysis was conducted using Bruker's TopSpin software package. Electrophysiological recordings. Membrane currents were measured using the whole-cell configuration of the patch-clamp technique. An EPC-10 patch-clamp amplifier (HEKA) was interfaced to a computer for pulse application and data recording using the programme PatchMaster (HEKA).
After washing with extracellular solution, glass coverslips were mounted in a chamber on an inverted Olympus IX50 microscope equipped with a Â 40 water immersion objective UApo/340 (Olympus Optical, Co. ). The fluorescence imaging system consisted of a monochromator, a charge-coupled device (CCD) camera and a Windows 7 based image processing software (Till Photonics). Cells were exposed to alternating 340±5 and 380±5 nm wavelengths of UV light and emission light was passed through a 400 nm dichroic mirror and a 420 nm long pass emission filter (both Olympus) before image acquisition by the CCD camera. Images were collected every 20 s. Cells were continuously superfused with Na þ /K þ -containing extracellular solution using a four-barrel microperfusion pipette positioned in close proximity to the viewing field.
Ca 2 þ and Na þ influx was induced by extracellular solution containing 5 mM ATP in the presence or absence of mefenamic acid. DMSO was used as vehicle control in all experiments. For each individual cell, mean intensity values from background subtracted pictures were determined and ratios F340/F380 calculated, accordingly. Data are presented as mean values ± s. e. m. Regulatory volume decrease. THP-1 cells were adjusted to a density of 1 Â 10 6 cells per ml in RPMI media (10% FBS, 1% P/S, 1% Glutamine) and primed with LPS (1 mg ml À 1 , 4 h). A 300 mOsm isotonic buffer was prepared consisting of 147 mM NaCl, 10 mM HEPES, 13 mM glucose, 2 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , adjusted to pH 7. 4 using NaOH. From this a 90 mOsm hypotonic buffer was achieved by diluting the isotonic buffer 1:4 with sterile water.
Lavage was collected from each animal, passed through 100 mm cell strainers before analysis by ELISA as above or by flow cytometry. For the flow cytometry cells were adjusted to a density of 5 Â 10 6 cells per ml in ice-cold PBS, 1% BSA, 5 mM EDTA before plating out on a clear V-bottomed 96-well plate (Thermo Scientific) at 200 ml per well. Cells were stained with antibodies (Anti-CD45 conjugated to FITC at 1 ng ml À 1 , anti-Ly6G conjugated to APC at 1 ng ml À 1 and anti-F4/80 conjugated to PE at 2. 5 ng ml À 1 , all Tonbo Biosciences) for 45 min on ice in the dark before washing twice and fixing in paraformaldehyde (3. 7-4. 1% in PBS, 200 ml) for 15 min at room temperature. The following day, cells were analysed on a FACSVerse flow cytometer (BD Biosciences) with BD FACSuite software. Peritoneal inflammation model.
Images were overlaid and stacked using Fiji Image J  49. Statistical analyses. Data are presented as mean values þ standard error of the mean (s. e. m). Statistical analyses performed were one-sample t-tests, one-way analysis of variance (ANOVA) and two-way ANOVA tests with Sidak corrected post hoc. Equal variance and normality were assessed with the Levene's test and the Shapiro-wilk test, respectively, and appropriate transformations were applied when necessary. Accepted levels of significance were *Po0. 05, **Po0. 01, ***Po0. 001. Statistical analyses were carried out using GraphPad Prism or SPSS. Images were processed using Fiji ImageJ  49  and analysed by manual counting with experimenter blinded to image identity throughout. Flow cytometry data were analysed and populations quantified using FlowJo V10.
Acknowledgements We are grateful to  Dr Vishva Dixit  ( Genentech ) for providing the NLRP3 KO mice and to  Prof Clare Bryant  ( University of Cambridge ) for providing the immortalized BMDMs. We thank  Mr Neil O'Hara  ( UoM ) for assistance with NMR kinetic profiling. We are also grateful to  Prof Nancy Rothwell  ( UoM ) for providing critical feedback on the manuscript. We are also grateful for the technical support of the  Bioimaging facility (UoM). MJDD is funded by a  MRC   DTP studentship  ( MR/K501311/1 ). C. B. L. , J. R. A. and D. B. are funded by the  Alzheimer's Society  ( 211 ( AS-PG-2013-2007 )). P. P. holds a  BBSRC David Phillips Research Fellowship  ( BB/I017976/1 ). This work also supported by  BBSRC  grant  BB/K003097/1. The work leading to these results has received funding from the  European Union Seventh Framework Programme  ( FP7/2012-2017 ) under grant agreement no  305564.
Behavioural task The computerized learning task was programmed using visual basic (REALbasic, 2009, release 2. 1) software as reported elsewhere (e. g. ,  Msetfi, Wade, & Murphy, 2013). Participants were required to learn the relation between pressing a key on the keyboard (the spacebar) and the occurrence of an auditory cue presented through the computer speakers. During each trial, a simulated button appeared on the screen for 3 s during which time participants chose whether or not to press the button using the keyboard. The auditory outcome was programmed to occur on the basis of probability and it did so at the end of a 3 s limited hold period (for a total of 2 s), or else no sound was presented for 2 s before the start of the next trial. Each of 40 trials was separated from the next by a 15 s inter-trial-interval (ITI).
Control ratings In addition to responding, participants provided ratings of perceived control of the action and the context at producing the outcome. We analysed the ratings data using a mixed factorial analysis of variance. Judgement (response or context) was the repeated measures factor, and between subjects factors were drug treatment (escitalopram 10 mg, Placebo), BDI (low, high), gender (male, female) and outcome density (low, high). In this analysis, in order to control for any variations in experienced contingency across participants, the individual experienced contingency for each participant was included in the model as a covariate (M = À. 002, SE =. 014). Initial analyses showed there were no significant effects or interactions involving any of the experimental manipulations on this particular variable, all ps >.