dataesr/scientific-paragraphs-categorization huggingface dataset:
5. Cryo protection was achieved by adding 20% (v/v) glycerol to the mother liquor condition. Crystals for the MR17-RBD complex were grown in 20% (w/v) PEG 3350 and 0. 2 M magnesium formate. Cryo protection was achieved by adding 10% (v/v) glycerol in the mother liquor condition. Crystals for the MR3-RBD complex were obtained in 9% (w/v) PEG 8000, 0. 1 M HEPES pH 7. 5, 8% (v/v) ethylene glycol, and 9. 6% (v/v) glycerol. 20% glycerol was included for cryo cooling. Crystals for the MR4-RBD complex were grown in 10% (w/v) PEG 8000, 200 mM zinc acetate, and 100 mM MES pH 6. 0. Crystals for MR17(K99Y)-RBD were grown in 0. 2 M MgCl 2 and 20% (w/v) PEG 3350. Cryo protection was performed by adding 30% (v/v) glycerol to the reservoir condition. Crystals were cryo-protected, harvested using a MitGen loop, and flash-cooled in liquid nitrogen before X-ray diffraction data collection.
02 A ̊after two times binning) in an automatic manner using SerialEM  57. Each frame was exposed for 0. 15 s at the dose rate of 8 e -(A ̊2•s) -1 and the total accumulation time was 6. 45 s, leading to a total accumulated dose of 49. 6 e -(A ̊2) -1 on the specimen. Cryo-EM 3D reconstruction. Single-particle analysis was mainly executed in Relion3. 1  58  unless otherwise specified. A total of 1753 movie stacks were aligned and summed using the MotionCor2 software  59. After contrast transfer function (CTF) parameter determination using CTFFIND4  60  , particle autopicking, manual particle checking, and reference-free two-dimensional classification, 193,764 particles remained for further processing. We used part of the data to obtain an initial model. Then all particles were refined and then re-centered against this initial model.
Plasmids encoding the mutants were generated by sitedirected mutagenesis using primers listed under "MR17 mutations" in Supplementary Data 2. The mutants were purified and characterized the same way as for MR17. Because K99Y showed higher neutralization activity than the wild type, K99W was designed for the second round. In vivo stability of sybody in mice. The female 7-week-old ICR mice weighing 27 ± 1 g were IP injected with PBS or sybodies MR3, MR3-MR3, MR3-MR3-ABD, or Fc-MR3 at 25 mg kg -1 in a final volume of 100 μL in PBS. The blood samples were collected at different time points (2 days preinjection, 6 h, 12 h, 1 day, 3 days, and 6 dpi) and subjected to neutralization assay using SARS-CoV-2 pseudotypes. Mice weights were measured till 6 dpi (n = 4). Mice were sacrificed at 1, 3, and 6 dpi; their vital organs (heart, liver, spleen, lung, kidney, and thymus) were fixed in 4% formaldehyde at 4 °C overnight and then embedded within paraffin, solidified, and cut to 15-μm thickness using a cryotome (Leica Microsystems).
Sections were stained by hematoxylin and eosin (H&E). Scale is equal to the original magnification ×100. Mice challenge experiments. C57BL/6J female mice (6-8 weeks old) were treated with adenovirus serotype 5 expressing hACE2 via the intranasal route. At 5 days post-adenovirus treatment, the mice were intranasally infected with SARS-CoV-2 strain hCoV-19/China/CAS-B001/2020 (National Microbiology Data Center NMDCN0000102-3, GISAID databases EPI_ISL_514256-7) with a high dose of 5 × 10 5 TCID 50 in a volume of 50 μL. After 12 h, the mice of MR3-MR3-ABD group (n = 3) were given 200 μL of sybody each (25 mg kg -1 body weight) by IP injection. The infection control group (n = 3) was treated with PBS buffer. Three dpi, three mice were euthanized, and the lung tissues (~1/8 of the total lungs) were fixed in 4% (v/v) paraformaldehyde for histopathological analysis using H&E staining.
The rest of the lungs were weighed and homogenized for RNA extraction and virus titration by qRT-PCR using a kit (Mabsky Biotech Co. , Ltd. ) following the manufacturer's protocols. Mean and standard error of the mean of all three individual data points were reported. The raw data were log-transformed  64  to approximate normal distribution for statistical analysis using the two-tailed, unpaired Student's t test. Animal procedures SARS-CoV-2 (hamsters). Female Syrian golden hamsters (Mesocricetus auratus; strain RjHan:AURA, purpose bred from Janvier, France) were allowed to acclimatize and aged 6 weeks at the start; for procedures, they were briefly anesthetized by chamber induction (5 L 100% O 2 /min and 3-5% isoflurane); 6-h prior to inoculation with the virus, groups of 5 animals were treated with 2.
Acknowledgements We thank the staff members of the  NCPSS Large-scale Protein Preparation System  and the  Electron Microscopy facility  for equipment maintenance and management and staff scientists at the SSRF-BL19U1 beamline at  National Facility for Protein Science (Shanghai) , and the staff of BSL-3 in the  Institute of Microbiology, Chinese Academy of Sciences  for technical support and assistance. We thank  Dr. Zhipu Luo  at  Soochow University (China)  for helpful discussions regarding data processing. This work has been supported by the  Strategic Priority Research Program  of  CAS  ( XDB37020204 ,  D. Li ;  XDB29010102 , Y. B. ),  Key Program of CAS Frontier Science (QYZDB-SSW-SMC037 ,  D. Li ),  CAS Facility-based Open Research Program , the  National Natural Science Foundation of China  ( 31870726 ,  D.
Data availability The structure factors and coordinates are available through the protein data bank (PDB) under accession codes 7C8V (SR4-RBD), 7C8W (MR17-RBD), and 7CAN (MR17-K99Y in complex with the RBD). The cryo-EM map of MR3-Spike has been deposited to EMDB with accession ID of EMD-31328. Source data are provided with this paper.
90-2. 94 No. of reflections 25,148 13,256 11,264 R work /R free 0. 1836/ 0. 2029/ 0. 2149/0. 2676 0. 2239 0. 2659 No. of atoms 2810 2536 2509 Protein 2510 2482 2465 Ligands 62 54 44 Water 238 0 0 No. of residues 322 315 312 B-factors (Å 2 ) 35. 13 73. 28 79. 38 Protein 34. 16 72. 27 78. 53 Ligand/ion 55. 11 119. 79 126. 90 Water 40. 22 R. m. s. deviations Bond lengths (Å) 0. 007 0. 011 0. 004 Bond angles (°) 0. 850 1. 10 0. 68 Ramachandran Favored (%) 98. 06 96. 12 96. 08 Allowed (%) 1. 94 3. 88 3. 59 Outlier (%) 0 0 0. 33 PDB ID 7C8V 7C8W 7CAN a Highest resolution shell is shown in parenthesis. b CC Ã ¼ ffiffiffiffiffiffiffiffiffi ffi q 2CC 1=2 1þCC 1=2 :.
Crystallogenesis and X-ray crystallography Concentrated complexes (7 mg/ml) were crystallized by vapor diffusion at 20 • C using a 96-well sitting drop plate (SWISSCI 3 Lens Crystallization Microplate). Crystals grew spontaneously within 48 h by equilibrating 300 nl of protein with 100 nl of 0. 1 M Tris-HCl (pH 8. 0), 0. 1 M NaCl, 8% (w/v) of PEG 8000 as F5 solution of the ProPlex HT-96 screen (Molecular Dimensions). Crystals were cryo-protected with reservoir solution with 20% PEG 200 before flash freezing in liquid nitrogen. X-ray diffraction was performed on the beamline Proxima1 at the Soleil synchrotron. Data from crystals were collected at = 1. 28242 Å. Datasets were processed individually and analysed with the autoPROC toolbox  (31). A weak anomalous signal around 4 Å was detected for four crystals.
Dataset merging with AIMLESS allowed enriching the anomalous signal at low resolution corresponding to the S scatter  (32). The structure was solved by combining the molecular replacement and anomalous signal methods, using PHASER  (33). The original placement of a VHH structure homologue (PDB: 5IMO presenting 70% sequence identity) allowed obtaining a partial map and the anomalous difference map helped to calculate the initial electron density map and align the sequence that identifies the MTase. Density was modified with PARROT  (34) , and auto-building with BUCCANNEER (35) allowed extending the model that was then manually built with Coot (36) and refined with BUSTER  (37). The final model had a R work = 19. 6% and a R free = 23. 4%, and its good stereochemistry was confirmed with MolProbity  (38).
Comparison of domains, modeling of missing loops and structural analysis Homologous structures were searched with DALI (39) and 3D-fold  (40)  search to retrieve MTase structures. Comparison with the VSV and hMPV MTase structures (PDB: 4UCZ and 5A22, respectively) allowed confirming the coiled structure that corresponds to the missing loop. To ensure the complete continuity of the main chain and the proper surface analysis, the two missing loops (regions 1764-1775 and 1795-1808) were modelled. The structural analysis and reference files for modelling were prepared with CHIMERA  (41)  and the missing loops were modelled with MODELLER 9. 23  (42). Surface electrostatics was calculated with APBS  (43). Sequences and interface were analysed with ENDSCRIPT  (44).
After two washes with 10 mM ammonium formate pH 8. 0, two washes with water, and a last wash with ethanol, filters were soaked with liquid scintillation fluid to measure the 3 H-methyl transfer to the RNA substrates using a Wallac MicroBeta TriLux Liquid Scintillation Counter13. For statistical analysis, it was assumed that the different experimental groups were independent, and data followed a Gaussian distribution with the same variance. Two-way ANOVA and multiple comparisons Dunnett test were used (Prism) to evaluate differences between groups. The level of significance for ␣ = 0. 05 is indicated as follows: *P < 0. 05, **P < 0. 01, ***P < 0. 001.
FUNDING Délégation Générale pour l'Armement (DGA)  [ 2009. 34. 0038 ]; Aix-Marseille Université PhD fellowship  (to B. M. ); C. V. was funded by the  National Research Agency ANR  under the program  ANR Rab-cap  [ ANR-16 CE11 0031 01 ];  French Infrastructure for Integrated Structural Biology (FRISBI)  [ ANR-10-INSB-05-01 ]; C. V. , B. M. , B. Co. and E. D. designed and performed experiments, using material prepared by F. D. and J-J. V. ; F. F. solved the crystal structure; V. Z. participated in protein purification and crystallogenesis; A. D. identified nanobodies against SUDV MTase; C. V. , B. M. ,  B. Ca , F. F. , B. Co. and E. D. analysed the data and wrote the paper. Funding for open access charge:  ANR  [ ANR-16 CE11 0031 01 ].
1782 (0. 2455) R free - 0. 2009 (0. 2663) No. of non-H atoms - 3571 -Protein - 3070 -Ligand - 7 3 -Water - 428 R. m. s. deviations -Bonds ( Å) -Angles ( • ) -- 0. 01 1. 51 Average B-factors ( Å2 ) - 39. 00 -Protein - 36. 5. -Ligand - 56. 05 -Water - 54. 12 Ramachandran Plot -Favoured (%) - 97. 92 -Allowed (%) - 1. 82 -Outliers (%) - 0. 26 Clashcore - 5. 15 PDB code - 6 Y U 8.
Study design and population This was a retrospective, multicenter study that included patients over 18 years of age with histologically confirmed locally advanced or metastatic PA who were treated with at least three cycles of firstline FOLFIRINOX between January 2011 and December 2018 and whose tumor response was evaluable. Patients with known dihydropyrimidine dehydrogenase or UDP-glycosyltransferase 1 polypeptide A1 (UGT1A1) deficiency or who objected to the use of their medical data were excluded from the study. Six French centers (three university hospitals, two cancer centers, one general hospital) participated in the study, and the data were retrospectively collected from the electronic medical records.
We estimated the cumulative probability of any kind of grade III or IV toxicity using the Kaplan-Meier method from the start of treatment (1-Kaplan-Meier curve). Patients who did not develop toxicity were censored on the date of reassessment. The association between cmRDI and the risk of toxicity was assessed using the Cox model, adjusting for PS and center. For patients who experienced toxicity, the cmRDI was recalculated up to the date of toxicity onset. All estimates are presented with a 95% confidence interval (CI), and the tests were performed at a two-tailed significance level of 5%. All statistical analyses were performed using Stata ® software (version 15. 0; StataCorp LLC, College Station, TX).
Oral iron supplementation was not able to increase serum iron in PAH patients. 14 Such negative results were already reported in trials on chronic left HF  17  where they are explained by hepcidin-related absorption defect. 1  Indeed, although oral iron supplementation is convenient, readily available and inexpensive, oral iron is not effectively absorbed, particularly in HF and PAH patients due to the resulted effects on the gastrointestinal tract and the elevated hepcidin levels. 18, 19 Despite these valuable insights from the left heart, an ongoing open-label phase III clinical trial is currently studying the effects of oral iron supplementation in a larger cohort of PAH patients (clinicaltrials. gov; ID: NCT03371173). On the other hand, intravenous iron supplementation improved quality of life and skeletal muscle exercise capacity, due to better peripheral oxygen supply by increased hemoglobin levels.
14, 15 In addition, a randomized, placebo-controlled crossover study is currently investigating the effect of intravenous iron supplementation on cardiopulmonary hemodynamics, exercise capacity, and quality of life in iron-deficient PAH patients (clinicaltrials. gov; ID: NCT01447628). Reassessment and correction of iron status may become part of the regular follow-up of PAH patients, but the longterm benefit remains to be determined, since the duration of follow-up was limited to 4-12 weeks in all studies. 14, 15 oreover, the available findings cannot answer the question of whether iron deficiency is a cause of more advanced symptoms with possible correction by iron supplementation, or if it corresponds to a marker of PAH severity. A very recent study suggested that iron deficiency is likely a secondary marker for the progression of PAH.
In multiple myeloma diseases, monoclonal immunoglobulin light chains (LCs) are abundantly produced, with, as a consequence in some cases, the formation of deposits affecting various organs, such as the kidney, while in other cases remaining soluble up to concentrations of several g•L -1 in plasma. The exact factors crucial for the solubility of LCs are poorly understood, but it can be hypothesized that their amino acid sequence plays an important role. Determining the precise sequences of patient-derived LCs is therefore highly desirable. We establish here a novel de novo sequencing workflow for patient-derived LCs, based on the combination of bottom-up and top-down proteomics without database search. PEAKS is used for the de novo sequencing of peptides that are further assembled into full length LC sequences using ALPS.
[10] [11] [12] [13]  Unfortunately no clear-cut conclusion could be drawn. To achieve this goal, a database including the biophysical properties of LCs and their sequence is essential. Sequencing monoclonal LCs is challenging, because they all have a unique amino acid sequence determined by somatic recombination and various mutations. 14, 15 This sequence diversity translates into a diverse clinical picture,  16  and thus, the mechanisms behind a particular disease are hard to decipher. LCs consist of a N-terminal variable region (v-region), which is capable of recognizing the antigen, and a C-terminal constant region (c-region), which specifies the effector function of the molecule. 17 Important for the antigen binding site are three hypervariable loops, so-called CDRs (complementarity-determining regions), present in the variable region.
BUP can provide high sequence coverage confirming the sequence of recombinant antibodies and the presence of expected PTMs in particular when using a combination of enzymes. 24 6] [27]  Several papers have shown that a combination of BUP and intact mass profiling can be of great help. 28, 29 For LCs, which are in the 25 kDa range, the use of top-down proteomics (TDP) based on the fragmentation of intact proteins is also possible. 30, 31 For instance, this approach has been used by Marshall and co-workers to classify plasma cell disorders (including amyloidosis) from the analysis of monoclonal immunoglobulin LCs in human serum. 32 A 21 T FT-ICR mass spectrometer was employed in combination with a database-aided de novo MS/MS algorithm. 4] [35]  In Marshall's paper, only 70% sequence coverage was obtained on LCs.
32 A solution is to combine BUP and TDP and use dedicated software tools such as TBnovo. 36 However, sequence gaps often remain because of the lack of fragment ions both in BUP and TDP. We, therefore, developed here a complete de novo sequencing workflow for the characterization of patient-derived LC proteoforms based on a combination of BUP and TDP with specific data analysis. PEAKS is used for the de novo sequencing of peptides that are further assembled into full length LC sequences using ALPS. TDP provides the molecular masses of proteoforms and allows the exact determination of the amino acid sequence including all PTMs. The characterization of 10 different clinical samples covers the amino acid sequence with in most of the cases I/L distinction, all PTMs (including disulfide bonds), and the ratio between the monomeric and dimeric proteoforms in the clinical samples.
For isoleucine/leucine discrimination, trypsin and chymotrypsin LC digests were mixed (1:1 ratio) and analyzed by LC-MS/MS on a Fusion Lumos mass spectrometer. Ions corresponding to peptides containing one or more leucine or isoleucine were added to an inclusion list to be fragmented by EThcD (MS 2 ) or HCD (MS 4 ) as previously described. 38, 39 e Novo Peptide Sequencing and Concatenation. PEAKS Studio X was used for peptide de novo sequencing. The data were refined using precursor mass correction only and the chimera scan option activated. De novo searches were performed with 2 ppm error for precursor mass, 0. 01 Da for the fragment ions with fixed carbamidomethylation (Cys) and variable oxidation (Met) as PTMs. Enzyme rules were specified for each sample, with no rules for the Nepenthes digestive fluid and pepsin digestions.
De novo sequencing results were exported and sequences, local confidence, and area were used in ALPS  29  to concatenate the overlapping peptides. Although other values were tested, kmer from 7 to 9 were finally used to generate putative LC sequences. The theoretical masses of these sequences were compared to those obtained from the intact mass measurement of LCs to ensure correct concatenation and select the appropriate sequences. BUP Data Analysis. The raw files obtained for the tryptic digests of each LC sample were searched with MaxQuant (parameters described in the SI). Draw Map from MSTools Web applications was used for visualization of protein sequence coverage. 40 For disulfide bridge localization, data obtained from nonreduced nonalkylated tryptic digests were searched against the corresponding LC sequences with Mass Spec Studio 41 using the CRIMP workflow.
A loss of two hydrogens (-2. 0156 Da) was used as a virtual cross-linker mass modification. The search parameters are provided in the SI. The most intense cross-linked peptides identified were used to assign disulfide bridges. LC-MS Analysis of Intact LCs (TDP). Intact LCs were analyzed on an Orbitrap Fusion Lumos mass spectrometer with or without reduction/alkylation. Both MS and targeted MS/MS experiments were undertaken. To maximize sequence coverage, 12 different fragmentation conditions were used. All details are given in the SI. TDP Data Analysis. Intact protein mass spectra were deconvoluted using Protein Deconvolution v3. 0 software (Thermo-Scientific) either with the Xtract algorithm for isotopically resolved mass spectra or with the ReSpect algorithm for unresolved mass spectra. MS/MS spectra were deconvoluted in FreeStyle 1.
02 Da) following the S-S bond reduction (+2. 02 Da) and indicates other cysteine modifications. De Novo Peptide Sequencing and Assembly. We first performed a digestion using four different enzymes (trypsin, Lys-C, pepsin, and Nepenthes digestive fluid) to maximize the generation of overlapping peptides. The LC-MS/MS method was optimized to obtain the high-quality data required for de novo sequencing (see the Experimental Section). For instance, the number of microscans was increased compared to a regular proteomics analysis. The data generated were analyzed with PEAKS to obtain de novo sequenced peptides. 43 These peptides were further  assembled using ALPS, with the intact mass previously measured used as a constraint with 1 Da tolerance. The ALPS kmer parameter, which represents the number of overlapping amino acids required to concatenate two peptides, was found to be optimal between 7 and 9, depending on the LC sample.
* sı Supporting Information The Supporting Information is available free of charge at https://pubs. acs. org/doi/10. 1021/acs. analchem. 1c01955. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD025884. Fragmentation maps obtained for P15 in TDP (Figure  S1 ), sequence coverage maps obtained in BUP (tryptic digests) (Figure  S2 ), the HCD MS/MS spectrum of the N-terminal tryptic peptide of P15 (Figure  S3 ), sequence coverage map obtained for P15 digested by the four enzymes (Figure  S4 ), the HCD MS/MS spectrum of the C134-C194 disulfide-linked peptide of P15 (Figure  S5 ), spectra showing I/L differentiation (Figure  S6 ), mass spectra of intact proteins under various conditions (Figure  S7 ), fragmentation maps obtained for all LCs in TDP (Figure  S8 ), and sequence identity (%) between LCs (Figure  S9 ) (PDF) LC samples studied in this work (Table  S1 ), detailed information on all LC proteoforms (Table  S2 ), sequence coverages obtained in TDP and BUP for the tryptic digests (Table  S3 ), and peptides and type of experiment used for the I/L discrimination (Table  S4 ) (XLSX) Mass Spec Studio search parameters for S-S bond assignment (TXT).
Contours comparison method The variability and the differences in the delineated volumes were quantified using Artiview ™ v2. 8. 2 software (Aquilab, Loos Les Lille, France). The comparative analysis was performed by using specific metrics (Additional file 1: Figure  S1 )  [17] [18] [19] [20] [21]. The standard deviation was calculated. If Cn refers to the contour determined by the observer n and CR the reference contour, then these two contours can be compared using the following criteria: Overlap ratio (OV) is defined as the ratio between the intersection of Cn with CR and their union  [22]. (Optimal value = 1) Dice similarity coefficient or Kappa Index (KI) or Cohen's Kappa was used to determine the agreement between the reference contour and the user's contour, as described elsewhere  [23].
Taking into account the inter-individual variability linked to the software In order to study the human factor separately, it appeared necessary to evaluate three types of contouring uncertainties that could be linked to the technology with a second study: (1) inaccuracy of the manual contouring tools OV = Cn ∩ CR Cn ∪ CR VR = Cn CR VCC = Cn ∩ CR CR VSC = Cn ∩ CR Cn of the TPS used by the observers, (2) imprecision of the thresholding automatic when used to contour, (3) uncertainties related to the import-export of structures from the local TPS to the centralized analyze interface. To address these three points, the experts delineated the contralateral (right) structures. AQUILAB then sent back the observers the reference contrast-enhanced CT scans as well as a RT-STRUCT file containing the right OARs and additional structures to be contoured.
Results The database was frozen on April 2018. Professionals from sixteen centers uploaded their sets of structures representing overall 57 professionals: 33 senior radiation oncologists (including 11 ANOCEF members), 18 residents training in radiation oncology (out of whom one-AQ21-was excluded due to an aberrant contour set) and 6 "specialized" RTT (Table  1 ). Two datasets were not exploitable (AQ23 and 29) as they did not refer to the right exam and therefore appeared shifted. Eight centers were represented by more than 2 professionals and have been analyzed for their own account.