dataesr/scientific-paragraphs-categorization huggingface dataset:
Part of the work was funded by the JPND EADB grant (German Federal Ministry of Education and Research (BMBF) grant: 01ED1619A). Part of the analysis was funded by the German Research Foundation (DFG) grant: RA 1971/6-1 to Alfredo Ramirez. Research of the Alzheimer Center Amsterdam is part of the neurodegeneration research program of Amsterdam Neuroscience. The Alzheimer Center Amsterdam is supported by Stichting Alzheimer Nederland and Stichting VUmc fonds. The clinical database structure was developed with funding from Stichting Dioraphte. Genotyping of the Dutch case-control samples was performed in the context of EADB (European Alzheimer DNA biobank) funded by the JPco-fuND FP-829-029 (ZonMW project number 733051061). Data collection and sharing for this project were funded by the Alzheimer's Disease Neuroimaging Initiative (ADNI) (National Institutes of Health Grant U01 AG024904) and DOD ADNI (Department of Defense award number W81XWH-12-2-0012).
3a  and Supplementary Data 2). Except for KATNBL1 and COBLL1 all uncharacterized candidates were identified to be ubiquitinated  1  and 69% contain high-ranking predicted APC/C recognition motifs  45  (Supplementary Data 1). The remaining hits included three APC/C subunits, ANAPC3, ANAPC8, ANAPC13, and the APC/C inhibitor FBXO5. The fact that verified APC/C substrates including PLK1, KIF2C, and ANLN were enriched compared to -APC/C samples, but remained below our threshold (1. 75, 1. 70, and 1. 39-fold enrichment, respectively) suggests that our dataset contains additional candidates (Supplementary Data 1). To evaluate the performance of E2~dID in identifying bonafide APC/C substrates, we generated a curated list of 53 wellcharacterized human and murine mitotic substrates with experimentally-verified APC/C recognition motifs (D box, KEN box, IR/LR tail, ABBA motif)(Supplementary Data 2).
Indeed, SGO2 and ESPL1 predominately co-precipitated with ANAPC3 in mitosis (Fig. 4d ). We determined the cell cycle stage of this interaction in more detail by synchronizing HeLa cells into all cell cycle phases (see Methods). Western blotting for cell cycle markers CCNB1 (present in HeLa cells in G2, PM, A, and S), CCNA2 (G2 and S) and CDT1 (PM, A, and G1) confirmed the identity of individual cell cycle stages (Supplementary Fig. 4a ). Immunoprecipitating ANAPC3 with a different anti-ANAPC3 antibody indicated that SGO2 and ESPL1 predominately coprecipitated with ANAPC3 from prometaphase to anaphase, thus reflecting the timing of their ubiquitination (Supplementary Fig. 4b ). Taken together our data suggest that from the selected candidates at least ESPL1, DEPDC1, and SGO2 are ubiquitinated by APC/C in vivo and that ESPL1 and SGO2 interact with APC/C in mitosis.
Compared to ubiquitin E2~dID with APC/C in human cell extracts, SUMO E2~dID with Siz1/Siz2 in S. cerevisiae extracts was less sensitive (11%) and specific (18%) in revealing experimentally validated substrates (Supplementary Data 5). We speculate this reflects the ability of Ubc9 to directly SUMOylate substrates without an E3 ligase, albeit with a comparably reduced efficiency  63. Nevertheless, we show that SUMOylation of Def1 depends on Siz1 and/or Siz2 (Fig. 6 ), suggesting that the dataset we present here likely contains additional, thus far uncharacterized Siz1/Siz2 substrates. In summary, E2~dID is a simple, robust and sensitive method for identifying substrates of specific E3 ligases. Based on the conserved enzymology E2~dID is compatible with all UBLs using RING-type E3 ligases and thus should be applicable to a multitude of experimental systems.
Methods Molecular cloning. Constructs for bacterial and mammalian expression were generated by standard molecular biological techniques using the templates, primers and restriction sites indicated in Supplementary Data 6. Inserts of all constructs were verified by sequencing. Cell culture and cell synchronization. Cell lines were cultured according to standard mammalian tissue culture protocol and sterile technique at 37 °C in 5% CO 2 and tested in regular intervals for mycoplasma contamination. hTERT RPE-1, HeLa K, and HeLa FRT/TO-3xFlag-FZR1 ala cells were a kind gift from Jonathon Pines (ICR, London, UK). HeLa FRT/TO-3xFlag-Venus-SBP-ANAPC4 + TIR1 + mAID-vhhGFP4 cells were described previously  49. hTERT RPE-1 cells were cultured in DMEM/F12 (Sigma Aldrich) supplemented with 10% (v/v) FBS (Gibco), 1% (v/v) penicillin-streptomycin (Sigma-Aldrich), 1% (v/v) Glutamax (Gibco), 0.
Labeled peptides were mixed, dried in a SpeedVac and fractionated on a U3000 HPLC system (Thermo Fisher) using an XBridge BEH C18 column (2. 1 mm id × 15 cm, 130 Å, 3. 5 µm, Waters) at pH = 10, and a flow rate at 200 µl/min in 30 min linear gradient from 5-35% acetonitrile/NH 4 OH. The fractions were collected at every 30 sec into a 96-well plate by columns, concatenated by rows to 8 pooled fractions and dried in the SpeedVac. Co-immunoprecipitation and western blotting. APC/C was immunoprecipitated (IP) from extracts using custom mouse monoclonal ANAPC3 (Supplementary Figure  5b ) or commercial (Bethyl) rabbit polyclonal anti-ANAPC3 antibodies (Fig. 4d ). Control (ctr) immunoprecipitations (IP) were performed with mouse monoclonal anti-FLAG or non-specific rabbit IgG antibodies (see Supplementary Data 7 for dilutions and catalogue numbers).
Western blotting was performed in 20% ethanol/MOPS buffer (all Thermo Fisher Scientific) in a Mini Trans-Blot electrophoretic cell (Bio-Rad), and detected with the indicated antibodies (see Supplementary Data 7 for dilutions and catalogue numbers). Uncropped scans of blots presented in the main figures are provided in Supplementary Figure  6  within the Supplementary Information. Yeast methods and Def1-HA immunoprecipitation. Yeast Media and genetic manipulations were done using standard methods. The Def1-HA strain has the S288C background. Anti-HA immunoprecipitations were performed using glassbead lysates from 100 OD yeast cells grown in YPD, in a buffer containing 50 mM TrisCl pH 7. 6. 150 mM NaCl, 1 mM MgCl 2 , 1 mM EGTA, supplemented with 1 mM PMSF, yeast protease inhibitor cocktail (Sigma P8215)) and 25 mM NEM for 2 h at 4 °C using the mouse anti-HA antibody (Sigma HA-7, H3663) and Panmouse Dynabeads (Invitrogen).
Both instrument and columns used below are from Thermo Fisher. The peptides were first loaded to a PepMap C18 trap (100 µm i. d. × 20 mm, 100 Å, 5 µm) for 10 min at 10 µl/min with 0. 1% FA/ H 2 O, then separated on a PepMap C18 column (75 µm i. d. × 500 mm, 100 Å, 2 µm) at 300 nl/min and a linear gradient of 4-28% ACN/0. 1%FA in 180 min/cycle at 210 min for each fraction. The data acquisition used the SPS10-MS3 method with Top Speed at 3 s per cycle time. The full MS scans (m/z 380-1500) were acquired at 120,000 resolution at m/z 200 with a lock mass at 445. 12003, and the AGC was set at 4e5 with 50 ms maximum injection time. Then the most abundant multiplycharge ions (z = 2-6, above 5000 counts) were subjected to MS/MS fragmentation by CID (35% CE) and detected in ion trap for peptide identification.
Peptide ions with charge at 3-6 were selected for HCD fragmentation at 38% collision energy. The isolation width was set 0. 7 Da. The fragment ions were detected in Orbitrap with 30 K resolution, and the AGC was set 50,000 with maximum injection time at 100 ms. Data analysis. The LC-MS/MS data were processed in Proteome Discoverer 2. 1 (Thermo Fisher Scientific) using the SequestHT search engine to search against the reviewed Uniprot protein database of Homo sapiens (20,238 entries, Swiss-prot), or Saccharomyces cerevisiae (7904 entries, Swiss-Prot), plus the in-house contaminate database. The precursor mass tolerance was set at 20 ppm and the fragment ion mass tolerance was set at 0. 5 Da. Spectra were searched for fully tryptic peptides with maximum 2 miss-cleavages. Carbamidomethyl at Cys was set as static modifications, and the dynamic modifications included N-acetylation (Protein Nterminus), Deamidation (N, Q), Oxidation (M), TMT6plex (Peptide N-terminus, K), and GGTMT6plex ( + 343.
2059) (K). Note, we used a SMT3 KGG mutant  13  to obtain a shorter SUMO remnant. Peptides were validated by Percolator with qvalue set at 0. 05 for the Decoy database search. The search result was filtered by the Consensus step where the protein FDR was set at 0. 01 (strict) and 0. 05 (relaxed). The TMT10plex reporter ion quantifier used 20 ppm integration tolerance on the most confident centroid peak at the MS3 level. Both unique and razor peptides were used for quantification. Peptides with average reported S/N > 3 were used for protein quantification. Only master proteins were reported (Supplementary Data 1, UBE2C E2~dID and Supplementary Data 4, Ubc9 E2~dID). Scaled TMT abundances were used to generate heat maps (Morpheus, Broad Institute, USA) and calculate thresholds as indicated in the main text and the legends of Supplementary Data 1 and 4.
Acknowledgements J. M. is supported by the  German Research Foundation (DFG)  ( Emmy Noether ;  MA 5831/1-1 ) and receives funding from the  European Research Council (ERC)  under the European Union's  Horizon 2020 research and innovation program  (grant agreement no. 680042 ). G. B. and I. A. G were members of the  Dresden International Graduate School for Biomedicine and Bioengineering (DIGS-BB ) PhD program, G. B. received a wrap up DIGS-BB Postdoc fellowship and I. A. G. was funded by a  DIGS-BB  PhD fellowship. J. S. C, L. Y. , and T. I. R. were funded by grant from the  Wellcome Trust  ( WT098051 ). We are grateful to  Magdalena Gonciarz  and  Doris Müller  for technical support,  Frauke Melchior  for anti-SMT3 antibodies,  Nils Kröger  for excellent technical input,  Sonja Lorenz  for critically reading the manuscript and  Olaf Stemmann  for SGO2 and ESPL1 antibodies and the sharing of unpublished results.
In addition, six subjects (7. 7%) had a homozygous loss-of-function variant in CFAP44 (Table  2 ). These two CFAP genes (for cilia and flagella associated protein) were reported in public expression databases to be strongly expressed in the testis and to be connected with cilia and flagella structure and/or functions  7. Quantitative real-time reverse transcription PCR (RT-qPCR) experiments performed in human and mouse tissue panels confirmed that CFAP43 and CFAP44 mRNA in testis was predominant and very significantly higher than in the other tested tissues (Supplementary Fig. 1 ). Taking into account the high number of mutated patients and the specific expression pattern of the two genes, we focused on these two genes, which appeared as the best candidates to explain the primary infertility observed for these individuals.
All individuals with CFAP43 or CFAP44 mutations were unrelated; to our knowledge, none carried rare variants in genes previously reported to be associated with male infertility. CFAP43 and CFAP44 encoded proteins that belong to the WDR protein family and are both composed of nine WD repeats  8. CFAP43 (also known as WDR96, NM_025145) is localized on chromosome 10 and contains 38 exons encoding a predicted 1,665-amino-acid protein (Q8NDM7). We identified 9 different pathogenic variants in CFAP43 in 10 unrelated individuals from the cohort. The splicing variant c. 3541-2A>C was identified in two unrelated individuals and affects a consensus splice acceptor site of CFAP43 intron 27 (Table  2 , Fig. 1b ). This variant was absent from the 60,706 unrelated individuals aggregated in the ExAC database (http://exac.
broadinstitute. org), consistent with a deleterious effect on protein function. Three other variants correspond to stop-gain mutations identified in three subjects: c. 2658G>A and c. 2680C>T are located in exon 21 and c. 3352C>T in the exon 26 (Fig. 1b ). These three variants were found in the ExAC database with very low allele frequency ranging from 8. 24e -06 to 9. 89e -05. Two others mutations were small frameshift indels not listed in ExAC: c. 1240_1241delGT (found in two unrelated patients) and c. 3882delA (Table  2 , Fig. 1b ). All these mutations generate premature stop codons and are predicted to produce no protein or truncated non-functional proteins. Apart from these obvious harmful mutations found in eight patients, we also identified two patients harboring likely deleterious mutations in CFAP43.
The last mutation, c. 1890 +1C>T, affects exon 15 consensus splice donor site. All these mutations are predicted to generate a premature stop codon. None of these variants were listed in the ExAC database. The presence of all variants was verified by bidirectional Sanger sequencing (Supplementary Fig. 2 ). The detailed sperm parameters of the patients carrying DNAH1, CFAP43, or CFAP44 mutations were compared with each other. There was no significant difference between the three groups (Table  1 ) as illustrated by the similar morphologies of sperm from patient P 43 -8 and P 44 -3, mutated for CFAP43 and CFAP44, respectively (Fig. 1a ). Data from all these groups were therefore pooled and compared with data from the patients with no identified mutation. Patients with a mutation had a significantly higher rate of spermatozoa with short or absent flagella and presented a significantly lower motility rate compared to patients with no identified mutations.
brucei genome database  23  using human CFAP43 and CFAP44 sequences identified T. brucei orthologs Tb927. 7. 3560 (named TbCFAP44 in this study) and Tb927. 4. 5380 (named TbCFAP43 in this study), respectively. The sequence identity, calculated with clustal omega  24  , between the four proteins shows that CFAP44 is closest to TbCFAP44 with 25. 6% sequence identity and CFAP43 closest to TbCFAP43 with 20. 8% sequence identity. Previous functional genomics and proteomic studies identified TbCFAP44 and TbCFAP43 as flagellar proteins  25, 26. In addition, TbCFAP44 is the Chlamydomonas FAP44 ortholog, and is involved in flagellar motility (also referred to as T. brucei components of motile flagella 7 (TbCMF7 in ref. 27  ). However, the function of TbCFAP44 and TbCFAP43 are currently unknown. To confirm that both Trypanosoma proteins are the orthologs of the mouse and human proteins, we compared their secondary structure.
Interestingly among CFAP43-mutated patients, only one (P43-9) carried a missense variant, with potentially a milder effect (Val347Ala). This patient has the lowest percentage of sperm with short flagella (22%) with 10% of morphologically normal spermatozoa (versus 0% for the other CFAP43-mutated subjects) and therefore presents a milder phenotype compared to the others with homozygous truncating mutations. These data support the existence of a genotype-phenotype correlation as had been demonstrated for DNAH1-mutated subjects  5, 6. We have previously demonstrated that MMAF patients with DNAH1 mutations had a good prognosis using ICSI  37  , but it remains difficult to predict the success rate for the other MMAF genotypes. Additional correlation studies should now be performed to take into account the individual genotypes in the counseling of MMAF patients.
Briefly, for each variant, independent calls are made on each strand, and only positions where both calls agree are retained. The most promising candidate variants were identified using an in-house bioinformatics pipeline, as follows. Variants with a minor allele frequency >5% in the NHLBI ESP6500 (Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA) or in 1,000 Genomes Project phase 1 datasets  54  , or >1% in ExAC 55 , were discarded. We also compared these variants to an in-house database of 94 control exomes obtained from subjects mainly originated from North Africa and Middle East corresponding to the geographical origin of most patients from this study and which is under-represented in SNP public databases. All variants present in homozygous state in this database were excluded.
We used Variant Effect Predictor (VEP version 81  56  ) to predict the impact of the selected variants. We only retained variants impacting splice donor/ acceptor or causing frameshift, inframe insertions/deletions, stop-gain, stop loss, or missense variants, except those scored as "tolerated" by SIFT  57  (sift. jcvi. org) and as "benign" by Polyphen-2  58  (genetics. bwh. harvard. edu/pph2). Finally, identified mutations were validated by Sanger sequencing. PCR primers and protocols used for each patient are listed in the Supplementary Table  3. Sequencing reactions were carried out with BigDye Terminator v3. 1 (Applied Biosystems). Sequences analysis were carried out on ABI 3130XL (Applied Biosystems). Sequences were analyzed using SeqScape software (Applied Biosystems). RT-qPCR analysis.
RT-qPCR data were normalized using the reference housekeeping gene ACTB for human and mouse with the -ΔΔCt method  59. The 2-ΔΔCt value was set at 0 in brain cells, resulting in an arbitrary expression of 1. Primers sequences and RT-qPCR conditions are indicated in the Supplementary Table  4. The efficacy of primers was checked using a standard curve. Melting curve analysis was used to confirm the presence of a single PCR product. Statistics were performed using a two-tailedt test on the Prism 4. 0 software (GraphPad, San Diego, CA, USA) to compare the relative expression of CFAP43 and CFAP44 transcripts in several organs. Statistical tests with a two-tailedP values ≤0. 05 were considered significant. Immunostaining in human and mouse sperm cells. We performed IF staining on human and mouse spermatozoa as described by our laboratory  60.
Nuclei and kinetoplasts were stained with DAPI (4',6-diamidino-2-phenylindole; 10 μg/ml). Images were acquired on a Zeiss Imager Z1 microscope, using a Photometrics Coolsnap HQ2 camera, with Zeiss ×100 or ×63 objectives (NA 1. 4) using the Metamorph software (Molecular Devices), and processed with ImageJ. For STED analysis on whole cells, cells were collected, washed, and loaded on poly-L-lysine-coated coverslips and air-dried. After a 5 min rehydration step in PBS, cells were fixed in methanol at -20 °C for at least 30 min. Samples were incubated with antibodies in four steps: (1) with the primary antibody anti-TY1 (BB2)  32  , a mouse IgG1 monoclonal, (2) followed by the secondary antibody Alexa-594 coupled anti-mouse IgG1, (3) anti-tubulin (TAT1)  64  , a mouse IgG2a monoclonal together with anti-PFR2 (rabbit polyclonal), (4) Alexa-488-coupledanti-mouse IgG2a and ATTO647N-coupled anti-rabbit secondary antibodies.
70. The motility parameters measured were curvilinear velocity, average path velocity (VAP), and straight line velocity (VSL). At least 100 motile sperm were analyzed for each assay. Motile sperm and progressive sperm were characterized by VAP >1 μm/s, by average path velocity >30 μm/s and straightness (VSL/VAP) >70%, respectively. For each genotype, five mice were used. Statistical analyses were performed with the SigmaPlot software. Unpaired t test was used to compare the different genotypes. Statistical tests with a two-tailedP values ≤0. 05 were considered significant. RNA expression analysis. Total RNA was isolated from 10 8 cells of parental, noninduced, and tetracycline-induced RNAi cells using the TRIzol reagent according to the manufacturer's instructions (Life Technologies). The constitutively expressed housekeeper telomerase reverse transcriptase was used as an internal control  65.
Acknowledgements We thank  Anne Bertrand  from the  GIN electron microscopy platform  and the IAB microscopy platform and  Alexei Grichine  and  Jacques Mazzega  for their technical help. We thank  Klaus Ersfeld  ( Bayreuth University ) for the anti-myc antibody,  Nicolas Biteau  ( Bordeaux University ) for the anti-PFR2 antibody,  Keith Gull  ( Oxford University ) for the TAT1 antibody,  Philippe Bastin  ( Institut Pasteur, Paris ) for the anti-TY1 antibody, and  Vanderperre Benoît  and  Martinou Jean Claude  ( UNIGE university, Geneva, CH ) for the Mpcl1l antibody. We thank  Denise Escalier  and  Gérard Gascon  for discussions and expertise on the topic. The  STED microscopy was done in the Bordeaux Imaging Centre a service unit of the CNRS-INSERM and Bordeaux University , member of the national infrastructure  France BioImaging  supported by the  French National Research Agency  ( ANR-10-INBS-04 ).
| Telemetric electroencephalographic recording Under isoflurane anesthesia (induction 5%, maintenance 2%), rats were placed in a stereotaxic frame (David Kopf Instruments). Two or four holes were drilled through the skull above the somatosensory cortex (Figure  2A4 ) for electrode implantation using coordinates relative to the bregma (anteroposterior, -0. 26; lateral, ±3. 5). A reference electrode was placed on the cerebellum (anteroposterior, -12. 3; lateral, ±3. 0). Electrodes were secured with screws and mounted with dental acrylic. A subcutaneous pocket was created to insert the telemetry transmitter (CTA-F40 or EET-F40; DSI) on the left dorsal side of the rat. Electroencephalographic (EEG) signals were digitized at 500 Hz and stored. Spike-and-wave discharges (SWDs) were identified offline by an automatic seizure detection module in NeuroScore (DSI), blind to the status of animals, which is usually revealed after histological analysis.
Greenhalgh et al have pointed to the lack of theory-driven approaches used to evaluate the use of PRO measures in routine clinical practice and propose that the mechanisms by which the proposed intervention is hypothesized to affect patient outcomes be clearly delineated  [15]. Others like Basch et al have argued that the proliferation of robust survey methods coupled with computerized technologies has provided a potentially viable means of collecting this information and ideally integrating it into electronic medical records (EMR)  [16]. Computerized and touch-screen technology can substantially facilitate data collection compared with paper forms, eliminating data-entry and scoring time, and therefore decrease staff burden. Yet, often EMRs and clinical research database systems have been designed to allow for data entry from study staff, making the collection of PROs challenging in some care or research settings  [17].
In the context of HIV cohort research, The University of Washington HIV Cohort was among the first to experiment with the routine computerized collection of patient-based measures. Crane et al described efforts to institute the routine collection of electronic PROs (ePROs) in HIV care, concluding that it was both promising for research and clinical care  [18]. Whereas Kozak et al highlighted some of the challenges of capturing high-quality data in routine care and the limitations of data recorded in patients' paper medical or EMR  [19]. They note that the demands of clinical care and patients' willingness to disclose sensitive information may compromise the comprehensiveness and the quality of data captured via EMRs. In their 2012 study, conducted at the University of Alabama at Birmingham 1917 HIV/AIDS clinic, they compared self-reported and EMR data and looked at the association between substance abuse, depression, and poor ART adherence in PLWH.
Objectives This paper outlines the formative research protocol being undertaken to develop a Web-based system to collect ePROs linked to the existing data capture infrastructure for those in HIV care in southwestern France. The first aim of the ePRO system is to expand and improve the data collection for the ANRS CO3 Aquitaine Cohort of PLWH being followed up in the 13 public hospitals in the region. The second aim is to make this information available to clinicians in a convenient format together with patients' locally developed, HIV-specific EMR. We describe the sequential process planned to ensure that the proposed ePRO system is ready for formal pilot testing, referred to herein as phases 1a and 1b. We also describe the planned pilot testing designed to evaluate the acceptability and use of the ePRO system from the perspective of patients (phase 2).