dataesr/scientific-paragraphs-categorization huggingface dataset:
I give my permission for the PRIME research team at my local centre to hold identifiable information such as my name, address, date of birth, email address, mobile phone number, NHS number or other applicable hospital identifier. I understand this may be used to collect longer term healthcare information on me from national records, such as the Office for National Statistics, NHS Digital, Public Health England, and other applicable NHS information systems, or other relevant national databases. This data may be linked to my data from the PRIME study in future research. BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) BMJ Open doi: 10. 1136/bmjopen-2022-070280 :e070280.
This would be no different than if you were not part of the study. Being involved in the study does not limit subsequent tests or treatment you may receive. If you do undergo further tests or treatment after the study is complete we may check the results of these on your records. We use the research data we have gathered from your involvement in the study to help us determine how good the diagnostic tests you have had are. We will work with other research teams to do this. We also ask your permission to use research data for teaching and education of other healthcare professionals. After completing the study, we also ask your permission to check your health through national databases. We may also contact you for further information in the future. This may include requests to complete quality of life questionnaires or for ascertaining future health status.
7. What data will be collected and use of data We will need to use information from your medical records for this research project. Your hospital will hold personal identifiable data on you. This information will include information such as age, PSA level, family history of medical conditions such as prostate cancer and examination findings. We allow the PRIME research team at your local site to hold PRIME PIS IRAS Reference number 282789 Version 3. 0 dated 08JUN2021 identifiable data on you, which will be for 10 years. Longer term data that may be requested from you include information on whether or not you have had further investigations or treatment for prostate problems and what the outcomes of those were as well as quality of life assessments. Non-identifiable data will be stored in the MARVIN database and the database will be transferred and stored at UCL within UCL's data safe haven.
You will be given a subject number and a subject identifier, and this will be used on all your study records. The code for this number will be known to the investigators at your site so that the link between your name and the data we hold on the study database is not completely broken. Any paperwork for the study will be kept in locked cupboards, staff access to these cupboards is strictly controlled. In general, UCL, as a university and a study sponsor, uses personally-identifiable information to conduct research to improve health, care and services. As a publicly-funded organisation, we have to ensure that it is in the public interest when we use personally-identifiable information from people who have agreed to take part in research. This means that when you agree to take part in a research study, we will use your data in the ways needed to conduct and analyse the research study.
samples and information collected will be deidentified to you prior to transfer to UCL, so only non-identifiable data will be transferred to UCL. This includes some pathology glass slides, which will be reviewed at Dr Alex Freeman's laboratory at University College London (UCL), for quality control. Slides sent to UCL will be PRIME PIS IRAS Reference number 282789 Version 3. 0 dated 08JUN2021 not have your name assigned. Samples will be sent using one of UCL's preferred couriers, for both pick up and return. Any data stored by the research team outside of your treating hospital will be kept at a secure location and will not contain information that can directly identify you. You will be allocated a study number, which will be used as a code to identify you on all study forms and data. The information will be linked to you so that if we did need to identify you for your safety or to clarify some information we would be able to by using a unique key, which will be known only to your local hospital team.
Your records will be available to people authorised to work on the study but may also need to be made available to people authorised by the Sponsor, which is the organisation responsible for ensuring that the study is carried out correctly. By signing the consent form, you agree to this access for the current study and any further research that may be conducted in relation to it, even if you withdraw from the current study. All will have a duty of confidentiality to you as a research participant. If you withdraw consent from further study treatment, your data and samples will remain on file and will be included in the final study analysis. In line with the regulations, at the end of the study your data will be securely archived for 20 years. Arrangements for confidential destruction will then be made.
Acknowledgements We would like to thank our patients and funders, without whom we wouldn't be able to carry out this important study:  Prostate Cancer UK , The  John Black Charitable Trust ,  European Association of Urology Research Foundation  and the  Dieckmann Foundation. We thank all the international centres taking part in PRIME. We are grateful to  EAU Research Foundation  and the  XClinical team  for their support with the MARVIN database; and  Sydney Lindner ,  Steven Lelie ,  Jessica Sternisa ,  Tyler Edwards ,  Adam Kulp ,  Jon Piper  from the  MIM Software Inc  team. We are thankful for the trial oversight provided by our sponsor,  University College London  and the  National Cancer Imaging Translational Accelerator  trials unit.
Supervision: CA, VK. All authors read and approved the final manuscript. Funding The PRIME trial is primarily funded by  Prostate Cancer UK  (grant number:  TLD-PF19-004 ) and The  John Black Charitable Trust   Travelling Prize Grant  (grant number:  TLD-PF19-004 ). The  EAU Research Foundation (EAU RF)  and The  Dieckmann Foundation  also supported costs for international sites. Competing interests AN is an academic clinical fellow funded by the  National Institute for Health and Care Research. PK is an academic clinical fellow funded by the  National Institute for Health and Care Research and The Urology Foundation. FG is a recipient of the  2020 Young Investigator Award  ( 20YOUN15 ) funded by the  Prostate Cancer Foundation / CRIS Cancer Foundation. SP is supported by the  National Institute of Health and Care Research (NIHR) ,  UCLH  and  UCL Biomedical Research Centre.
ME receives research support from the  National Institute of Health and Care Research (NIHR) ,  UCLH  and  UCL Biomedical Research Centre. YT is funded by a  UK NIHR   Postdoctoral Fellowship  and supported by the  NIHR Birmingham Biomedical Research Centre. CMM is an  NIHR  Research Professor, and receives grants from  MRC ,  CRUK ,  Movember , and  Prostate Cancer UK. VK is funded by  Prostate Cancer UK  and  The John Black Charitable Foundation. He receives speaker fees from the  European Association of Urology, Singapore Urology Association , The Clinical Comms Group and Got IT consulting SL. All authors declare that there are no conflicts of interest. The views expressed in this publication are those of the authors and not necessarily those of the  National Health Service , NIHR, or the  Department of Health and Social Care.
Previous studies suggest that after crush injury there may be a loss and recovery of the blood dorsal root ganglion barrier junction, associated with the expression of intercellular junctional proteins  (Hirakawa et al. 2003). Although immune cells seem to be unable to infiltrate the cell-body-rich area of the DRGs after peripheral nerve injury, there is recent evidence of accumulation of leukocytes into the dorsal root leptomeninges that recover the sensory ganglia  (Du et al. 2018). Nevertheless, additional studies will be necessary to elucidate leukocyte trafficking into these regions after peripheral nerve injury. Our study further showed that SNI-induced increase of sNAMs in the sensory ganglia is a consequence of the local proliferation of CX3CR1-resident macrophages. Our data showing an increase in the expression of Ki67, a classical marker of cell proliferation, in CX3CR1 + cells before the expansion of this population, is an important finding to support this conclusion.
The sections were washing with PBS and incubated with the appropriate secondary antibody solution for 2h at room temperature (IgG conjugated Alexa Fluor 594 and/or 647; 1:800; Invitrogen, Carlsbad, CA). The sections were washed with PBS and mounted with coverslips adding Aqueous Mounting Medium, Fluoroshield with DAPI (Sigma-Aldrich). For the evaluation of CX3CR1 and CCR2 expression, the aforementioned genetically modified Ccr2 RFP/+ : Cx3cr1 GFP/+ mice were used. The DRG sections were acquired on Zeiss LSM780 confocal microscope. Images were processed in FIJI package for ImageJ software and the quantification of macrophages was performing using the cell counter. For all measurements, 3 sections of each DRG (L4 or L5) were analyzed, and the results were averaged to generate the value for a single mouse.
Re-analysis of public scRNA-seq data The scRNA-seq data from mice DRGs was acquired from the Gene Expression Omnibus (GEO) database under the series number GSE139103  (Avraham et al. 2020). The singlecell libraries were generated using GemCode Single-Cell 3′ Gel Bead and Library Kit on the 10X Chromium system (10X Genomics). The dataset contains cells from four animals, of which two naive mice and two with injured DRG. The feature barcode matrix was analyzed using Seurat v3. The cells were filtered according to the criteria: 600-10000 total reads per cell, 500 -4000 expressed genes per cell, and mitochondrial reads <10%. Clusters were identified using shared nearest neighbor (SNN) based clustering based on the first 30 PCAs and resolution = 0. 5. The same principal components were used to generate the t-SNE projections.
Experimental study design (statistics details) The n sample was determined based on previous publications and/or internal pilot data, to be adequate for statistical analysis and ensured reproducibility. No statistical methods were used to determine the samples size. Additionalty, experimental groups blinded during qualifications. Data are reported as the means ± S. E. M. per group with 4-6 mice. Result analysis was performed by One-way ANOVA followed by the Bonferroni test (for 3 or more groups) comparing all pairs of columns. Alternatively, an unpaired Student's t-test was used to compare 2 different groups. Values of p < 0. 05 were considered statically significant. Statistical analysis was performed with GraphPad Prism 8 software.
2f ). Furthermore, detection of CbFic2 by its V5-tag in the same experiment confirms the association of CbFic2 with histone H3 (Fig. 2f ). While the association of GFP-CbFic2 with histone H3 after anti-H3 IP from HEK293 cells is only faint (Fig. 1f ), the association of V5-tagged CbFic2 with histone H3 in THP-1 cells is more pronounced (Fig. 2f ). To determine the target proteins of AMPylation and the effects of CbFic2 activity in MDMs, immunoprecipitation of cell lysates with an anti-AMP antibody followed by protein identification via LC-MS/MS analysis was performed. The assays were based on biological triplicates of differentiated THP-1 lysates after doxycycline-induced CbFic2 E66G or CbFic2 H205A expression for 48 h, respectively (Fig. 2a ). Based on a Student's t-test (pvalue < 0. 05), 236 proteins were identified as significantly different between both sample sets (Data are available via ProteomeXchange with identifier PXD040330).
We determined the crystal structure of wild-type CbFic2 (Fig. 3a , PDB: 8CIL). In the     52  , is the only probable assembly and is predicted to be stable. Within the dimer, the interface is composed of amino acids 20-43 aa in helix α1 within the N-terminal DUF4172 domain. According to an analysis by PDBsum  53  , residues S22 and S26 interact across the interface (Supplementary Fig. S3a, b ). Therefore, the double mutant CbFic2 S22D S26D was designed to weaken the dimer interface. The crystal structure reveals the predicted HTH domain to be a 2-stranded winged HTH domain where the tri-helical HTH motif is followed by a β-hairpin wing (Fig. 3a ). Affinity measurements of CbFic2 against ATP by fluorescence anisotropy using FAM-labeled ATP as ligand revealed a K D of CbFic2 for ATP of around 640 nM (Supplementary Fig.
50 ng H3. 1 was run on 15% glycine gels, blotted on PVDF and probed with an anti-AMP antibody. For loading controls, the WB was stripped and reprobed with anti-CbFic2 and ani-H3 antibodies. e Intact MS analysis and quantification of time-resolved TS-H3 1-36aa AMPylation by CbFic2 E66G in the absence or presence of 5 bp, 10 bp, 20 bp, 40 bp, 60 bp dsDNA. 50 µM TS-H3 1-36aa was incubated with 5 µM of CbFic2 E66G in the presence of 5 µM DNA as indicated, 2 mM ATP, 4 mM MgCl 2 at 37 °C for 22 h. AMPylation was measured by the mass increase of 329 Da, and AMPylated peaks were quantified by intensity after deconvolution. AMPylation was defined as a decrease in unAMPylated peptide over time, to reduce the complexity of multiple AMPylation. Each data point represents the mean of biological triplicates; error bars correspond to standard deviation.
See Supplementary Fig. S3d  and Supplementary Table  3. f Structural superposition of crystal structures of Z-DNA-(orange) and B-DNA-binding (red) HTH domains with that of the HTH domain of CbFic2 (cyan). B-DNA binding: human transcription factor E2F4 (PDB: 1cf7, chain C, red)  59  ; Z-DNA binding: domain hZα ADAR1 of human double-stranded RNA-specific adenosine deaminase ADAR1 (PDB: 1qbj, chain D, orange)  60. The conserved tyrosine of Z-DNAbinding domains is highlighted as stick. g, h CD measurement of 20 bp dsDNA with g 100% GC or h 40% GC content in the absence or presence of increasing amounts of CbFic2. 1 μM of dsDNA was mixed with CbFic2 to final concentrations of 1 μM ([P]/[N] = 1), 2 μM ([P]/[N] = 2).
and 4 μM ([P]/ [N] = 4). [P] and [N] stand for protein concentration and DNA concentration, respectively. Before each measurement, samples were incubated for 1 h at 25 °C. CD spectra between 230 and 320 nm were collected using a 0. 75 cm quartz cell. See Supplementary Fig. S3e, f. i-k Analysis of the binding affinity of CbFic2, CbFic2 E66G ΔHTH and CbFic2 S22D S26D against i 20 bp, j 40 bp and k 60 bp dsDNA by fluorescence anisotropy. 1 nM 5'-FITC-labeled DNA was mixed with a dilution series from 20 μM CbFic2 using a pipetting robot in a 384w format. Values were baseline corrected by anisotropy values of free DNA. Fit corresponds to "Specific binding with Hill slope" (3) (GraphPad Prism 8. 0). Data shown correspond to the mean of technical triplicates, error bars to the standard deviation.
b Intact MS analysis and quantification of time-resolved TS-H3 1-36aa -AMP deAMPylation by CbFic2, CbFic2 S22D S26D or CbFic2 ΔHTH in the absence or presence of DNA in vitro. 50 µM TS-H3 1-36aa -AMP were incubated with 0. 5 µM CbFic2 as indicated in the presence of 5 µM DNA, 1 mM MgCl 2 at 37 °C for 24 h. DeAMPylation was measured by the mass loss of 329 Da, and peaks were quantified by intensity after deconvolution. deAMPylation was defined as an increase in unAMPylated peptide over time. Each data point represents the mean of biological triplicates; error bars correspond to standard deviation. See Supplementary Fig. S4a  and Supplementary Table  3. c WB analysis of auto-AMPylation of CbFic2 in cis/trans. 0. 3 µM CbFic2 versions as indicated were incubated alone or in the presence of another CbFic2 version, in the presence or absence of 2.
We therefore suggest that the measurable DNA-binding and DNA-stimulated enzyme activity in CbFic2 ΔHTH is the unspecific effect of a deregulated enzyme with a basic pI, whose positively charged surface interacts with negatively charged DNA, to bind a positively charged target. After all, despite its monomeric state, CbFic2 ΔHTH is not capable of either auto-(Fig. 4f ) or target-AMPylation (Fig. 3c ) if the inhibitory E66 is present. CbFic2 was injected at indicated concentrations onto a Superdex 75 pg 10/300 (Cytiva), run at 0. 5 ml min -1 in 20 mM HEPES pH 7. 5, 150 mM NaCl, 1 mM MgCl 2 , 1 mM TCEP, and protein was detected by absorbance at 280 nm. Data intensity was normalized to the internal control of vitamin B 12 (t R = 38. 5 min). Arrows indicate the gel filtration standard (BioRad) comprising bovine γ-globulin (MW 158 kDa), chicken ovalbumin (MW 44 kDa), horse myoglobin (MW 17 kDa) and vitamin B 12 (MW 1.
35 kDa). d Superimposition of CbFic2 crystal structure (lighter shades; light green dimerization interface; middle blue Fic domain; cyan HTH domain) with AlphaFold model (darker shades; dark green dimerization interface; dark blue Fic domain; turquoise HTH domain) shows structural flexibility in the HTH domain by a kink in the long connecting helix between HTH domain (turquoise) and Fic domain (blue). e Proposed model for DNA-binding induced dimerization. Superimposition of HTH domain (turquoise) of CbFic2 AlphaFold structure with human transcription factor E2F4 (PDB: 1cf7, chain C, red)  59  bound to B-DNA suggests that bound 20 bp DNA to CbFic2 might span across both monomers and thereby stimulate dimerization. f 90°turn of (e) around the vertical axis. g-i Analysis of DNA-induced dimerization by insolution FP-fusion FRET time course measurements in 20 mM HEPES pH 7.
Our data show that CbFic2 AMPylates histones in mammalian cells, but the physiological context and relevance of this modification remain unclear. Molecular biology. Unless otherwise indicated, all genes were synthesized (Integrated DNA Technologies, IDT) and codonoptimized for expression in E. coli by omitting rare codons. Plasmids were cloned by sequence-and ligation-independent cloning (SLIC)  74  using Q5® High-Fidelity DNA Polymerase (New England Biolabs (NEB)) and T4 DNA Polymerase (NEB). Point mutations, insertions and deletions were introduced by Q5® Site-Directed Mutagenesis Kit (NEB). PCRs were performed using the T100 thermal cycler (BioRad). DNA was purified from gel using the Monarch® DNA Gel Extraction Kit (NEB). Cloned constructs were transformed into chemically competent (Mix & Go, Zymo Research) Mach1 cells, or NEB® stable cells (NEB) for stable cell line generation, and positive clones were determined by complete Sanger Sequencing (GATC or Microsynth).
The histone-containing acidic supernatant was neutralized with 1/10 volume of 2 M NaOH, and protein content was determined by Bradford assay as described. Aliquots were frozen at -20 °C. Protein localization by fluorescence microscopy. Cos7 cells were seeded on 8w µ-slides (ibidi) at a density of 2-4 × 10 4 cells per cm 2 and transiently transfected with plasmid DNA using Torpedo DNA (ibidi) on the next day according to the manufacturer's instructions. Then, 24 h post-transfection cells were stained with 1 µg ml -1 Hoechst 33342 (Thermo Fisher Scientific) in PBS for 10 min to label nuclei. After staining, cells were washed three times with PBS (Sigma Aldrich) and supplemented with phenol red-free growth medium (DMEM, Sigma Aldrich) for live cell imaging. Images were acquired with a Leica DMi8 wide-field microscope (Leica microsystems) using a 100x magnification objective and the manufacturer's LAS X 2 software.
As control, dsDNA was measured in 5 mM HEPES, pH 7. 0, 4 M NaCl, and 75% ethanol. Before each measurement, samples were incubated for 1 h at 21 °C. CD spectra were recorded at 21 °C on a Chirascan CD Spectrometer (Applied Photophysics, Leatherhead, Surrey, UK) between 230 and 320 nm using a 0. 5 cm quartz cuvette. Machine settings were as follows: 1 nm bandwidth, 1 s response and 1 nm data pitch. Spectra were background subtracted and visualized using GraphPad Prism 8. 0. Each curve represents the mean of three separate measurements. Fluorescence anisotropy. Concentrations of all proteins were determined by Bradford as described. Per measurement, all data points were pipetted in technical triplicates into black low-binding, flat-bottom 384w plates (Greiner Bio-One) using the epMotion 5075t pipetting robot (Eppendorf).
Per well, 20 µl of 1 nM labeled ligand in 20 mM HEPES pH 7. 5, 150 mM NaCl, 1 mM MgCl 2 , 1 mM TCEP were pipetted. 20 µM or 50 µM of the indicated CbFic2 version in the same buffer served as the starting point of a dilution series by a factor of 0. 75 over 31 data points. The last data point corresponds to buffer without protein. For each triplicate, 10 µl of the dilution series were added to the ligand. The plate was incubated at 25 °C for 10 min before fluorescence anisotropy was measured in a Spark plate reader (Tecan) after 10 s of orbital shaking with excitation at 485 nm, bandwidth 20 nm and emission at 535 nm, bandwidth 25 nm, gain 70 at 25 °C. Technical triplicates were averaged and the delta anisotropy was calculated by subtracting protein-free anisotropy from the anisotropy at highest protein concentration.
The standard deviation was mapped as error bars. Data were blotted and fitted using the "Specific binding with Hill slope" model (3) in GraphPad Prism 8. 0: Y ¼ B max X h K D h þ X h ð3Þ with B max as the maximum of specific binding in the same unit as Y, K D as the concentration of the ligand at half-maximum binding in the same unit as X, h as the hill slope and indicator of cooperativity. If DNA was used as ligand, 5'-fluoresceine isothiocyanate (FITC) fluorescent oligonucleotides were hybridized with their respective non-fluorescent reverse complement oligonucleotide (Supplementary Table  5 ) as described. For ATP binding measurements, N6-(6-Aminohexyl)-ATP-5-FAM (Jena Bioscience) was applied as ligand. Solution based FP-FRET. Time course measurements were performed using a Jasco FP-8300 Spectrofluorometer.
Measurements were performed at 25 °C, with an excitation wavelength of 405 nm, bandwidth 2. 5 nm, and an emission wavelength of 530 nm, bandwidth 5 nm, a response time of 1 s, a data interval of 1 s and medium sensitivity (with the exception of low sensitivity for the control measurement at 2 µM). Data were collected with the Spectra Time Course Measurement and smoothed by the means-movement method using a convolution width of 11 with Spectra Analysis Version 2. 15. 18 within the Spectra Manager Version 2. 15. 01 (JASCO Corporation). Intensities were normalized to the value at 760 s corresponding to the endpoint intensity of donor addition and visualized using GraphPad Prism 8. 0. Analytical size exclusion chromatography. In 100 μl, CbFic2, CbFic2 ΔHTH , CbFic2 S22D S26D , CbFic2 S22D S26D ΔHTH or CbFic2 E66G were diluted to 40 µM, 20 µM, 10 µM, 5 µM or 2.
The option to use calibrated charge-dependent ETD parameters was enabled and the maximum injection time was set to 40 ms (target value 5•10 4 ). Data were acquired using Xcalibur software version 3. 0sp2 (Thermo Scientific). The MS raw file was analyzed with MaxQuant software (version 1. 5. 3. 8)  84  and the peptide sequence ARTKQTARKSTGGKAPRKQL used for the implemented Andromeda search engine. LC-MS/MS analysis of anti-AMP IP: After anti-AMP IP from cell lysates, all samples were dissolved in 0. 1 M TEAB with 1% SDC and heated for 5 min at 95 °C for protein denaturation. Disulfide bonds were reduced, using 10 mM DTT for 30 min at 60 °C. Alkylation of free thiol groups was achieved with 20 mM iodoacetamide (IAA) for 30 min at 37 °C in the dark. For tryptic digestion, 250 ng protein was used.