dataesr/scientific-paragraphs-categorization huggingface dataset:
Methods This phase II study ( ClinicalTrials. gov NCT03202758) will assess the efficacy and safety of FOLFOX/D/T association in patients with mCRC (n=48). Good performance status patients (Eastern Cooperative Oncology Group <2) with untreated, RAS mutational status mCRC will be eligible. Prior adjuvant therapy is allowed provided recurrence is >6 months postcompletion. There is a safety lead in nine patients receiving FOLFOX/D/T. Assuming no safety concerns the study will go on to include 39 additional patients. Patients will receive folinic acid (400 mg/m²)/5-fluorouracil (400 mg/m² as bolus followed by 2400 mg/m 2 as a 46hour infusion)/oxaliplatin (85 mg/m 2 ) every 14 days with D (750 mg) D1 every 14 days and T (75 mg) D1 every 28 days. After six cycles of FOLFOX only D/T will continue until disease progression, death, intolerable toxicity, or patient/investigator decision to stop.
Methods study objectives Our multicentre phase I/II study aims to establish the safety and efficacy of durvalumab plus tremelimumab combined with FOLFOX in patients with metastatic CRC with MSS or MSI status of RAS mutated status. The study will be performed in two steps (figure  1 ): Step 1 will assess the safety of the combination of durvalumab 750 mg every 2 weeks + tremelimumab 75 mg every 4 weeks + FOLFOX during the first two cycles of treatment. Step 2 will assess the efficacy of the combination of durvalumab 750 mg every 2 weeks + tremelimumab 75 mg every 4 weeks + FOLFOX. The trial is registered on the ClinicalTrials. gov database NCT03202758 (NCT). study assessments and criteria for evaluation Phase Ib primary objective (step 1) To determine the safety of the combination of durvalumab (anti-PD-L1) + tremelimumab (anti-CTLA-4) + FOLFOX.
► Blood collection will be performed for analysis of peripheral T cell functions. ► Plasma collection will be performed for cytokine analysis. Safety data will also be completed during all the step 2 of the study. Step 2: efficacy assessments Efficacy analyses will be performed in modified intentto-treat (mITT) population, that is, all patients following the major inclusion criteria and with a 3-month evaluation. Analyses will be repeated in the ITT principle, that is, including all enrolled patients whatever eligibility criteria and treatment received by patients and in the per protocol population (patients who had received all the planned doses). The primary objective will be evaluated at 6 months according to the Simon's design.
overall survival OS (months) is defined as the time from the date of enrolment until death due to any cause. Any patient not known to have died at the time of analysis will be censored based on the last recorded date on which the patient was known to be alive. OS data will be collected at 12 months. Subjects who have disease control following completion of 12 months of treatment or subjects who are withdrawn from durvalumab + tremelimumab treatment for reasons other than confirmed PD will continue to have objective tumour assessments.
Fixed dosing for durvalumab and tremelimumab A population pharmacokinetic model was developed for durvalumab using monotherapy data from a phase 1 study. 31 Population PK analysis indicated only minor impact of body weight (WT) on PK of durvalumab (coefficient of ≤0. 5). The impact of body WT-based (10 mg/kg every 2 weeks) and fixed dosing (750 mg every 2 weeks) of durvalumab was evaluated by comparing predicted steady-state PK concentrations (5th, median and 95th percentiles) using the population PK model. A fixed dose of 750 mg was selected to approximate 10 mg/kg (based on median body WT of ~75 kg). A total of 1000 patients were simulated using body WT distribution of 40-120 kg. Similarly, a population PK model was developed for tremelimumab using data from phase 1 through phase 3. 32  Population PK model indicated minor impact of body WT on PK of tremelimumab (coefficient of ≤0.
statistical consideration Safety analyses will be performed on the safety-evaluable population, defined as all subjects treated with at least one dose of investigational product. *Toxicities and grades will be described at each cycle. *The following data will be given: ► The number and percentage of patients with at least one AE. ► The number and percentage of patients with at least one grade 3 or 4 AE. ► The number and percentage of patients with at least one serious AE. ► The number and percentage of patients with at least one AE leading to treatment premature stop. ► Time until grade 3-4 toxicity will be determined using the Kaplan-Meier method. Patients without toxicities will be censored. Efficacy analyses will be performed in mITT population, that is, all patients following the major inclusion criteria and with a 3-month evaluation.
All statistical analyses will be performed with Stata V. 11 or SAS V. 9. 3. The efficacy will be determined using a Simon 2 step phase II design. The hypotheses are the following: 1. A PFS of 3 months is not considered of interest. 2. A PFS of 6 months is expected. This is equivalent to assume that a PFS of 50% at 3 months is insufficient and a PFS of 70. 7% is expected. With α=10%, β=10% (90% power), 10% of non-evaluable patients, 48 patients are needed including patients. dIsCussIon Immune checkpoint inhibitors have recently changed the management of several types of cancer. In CRC, results remain modest except in MSI tumour. Preclinical data have shown that some chemotherapies such as oxaliplatin and 5-FU could generate an increase of immunogenicity of tumour and improve the efficacy of immune checkpoint inhibitors.
Statistical analysis Mann-Whitney U tests were used to compare two groups of samples after testing for parametric distribution. To assess the diagnostic accuracy of CSF aSyn in the discrimination of sCJD from non-CJD cases, receiver operating characteristic curve analyses were carried out, and areas under the curve with 95% confidence intervals were calculated using GraphPad Prism 6. 01. The best cutoff value was then estimated based on the Youden index (sensitivity 1 specificity 2 1). Spearman rank correlation coefficients were used to assess associations between continuous biomarker levels. Agreement between MSD assays and between two different lots was investigated through a Passing-Bablok regression  [22] , using the Meth-Comp package in R  [23]. ROC curve for aSyn in the comparative analysis between non-sCJD cases and sCJD cases.
Acknowledgments The authors thank  Silja Koechy  for excellent technical assistance. Funding sources: This study was funded by the  Spanish Ministry of Health-Instituto Carlos III  ( Miguel Servet  programme:  CP16/00041 ) to F. L. and by the  Robert Koch Institute  through funds from the  Federal Ministry of Health  (grant no. :  1369-341 ) to I. Z. A. V-P was a recipient of a  Postdoctoral Dorothea Schl€ ozer scholarship (Georg-August-Universit€ at G€ ottingen ).
For a subset of compounds, we showed that the rates agree well with GSH consumption t 1/2 (Table  S1  in the Supporting Information). Several results arise from the analysis of the kinetic data from the entire library. First, the most reactive fragments were labeled only ∼100-fold faster than the least reactive fragments; the majority of library fragments (78%) falls within only a 30-fold difference. Second, the chloroacetamides are clearly more reactive (show faster kinetics) than the acrylamides (p = 4. 6 × 10 -38 ; two-sided t test), in accordance with previous experiments for a handful of fragments. 51 Finally, for reference, we measured the reactivity of iodoacetamide, a generally used, nonselective thiol alkylator, and found it to be 21-fold more reactive than the average chloroacetamide and 26-fold more reactive than the median choloroacetamide (Data set 1 in the Supporting Information).
org/10. 5281/zenodo. 1244111). We have synthesized, on the basis of one of the initial hits, a series of diphenylacetamide analogues and have soaked them into NUDT7 crystals, which yielded a structure in complex with compound NUDT7-REV-1 (Figure  5A ), A comparison of this structure with the covalent NUDT7/PCM-0102716 structure reveals an almost perfect overlap of one of the phenyl rings (Figure  5E ), suggesting a clear strategy for fragment merging. The merged compound, NUDT7-COV-1 (see chemical synthesis in File S1 in the Supporting Information), combines the key features of both fragments and has much improved properties. In the cocrystal structure, the merged compound adopts exactly the predicted pose (root-mean-square deviation of 0. 4 Å over the shared atoms; Figure  5F ), and at 5 μM, NUDT7-COV-1 labels NUDT7 to 95% in 15 min (2 μM protein).
We determined the k inact /K I value of NUDT7-COV-1 to be 757 M -1 s -1 (k inact = 0. 01 s -1 , K I = 13. 21 μM; Figure  S27  in the Supporting Information), in comparison to k inact /K I = 233. 1 M -1 s -1 for the best primary hit from the screen, PCM-0102558, for which we were not able to determine the k inact and K I values separately. We evaluated NUDT7 cellular target engagement by NUDT7-COV-1 via a cellular thermal shift assay (CETSA) in intact HEK293 cells. Indeed, NUDT7-COV-1 showed significant stabilization of FLAG-tagged NUDT7 in comparison to DMSO, confirming that the compound binds the target in living cells (Figure  5H ). Finally, we performed an isoTOP-ABPP experiment, in which we profiled the cellular targets of NUDT7-COV-1 (10 μM; 2 h incubation; HEK293T cells; Data set 7 and Figure  S22B  in the Supporting Information).
The absorbances were acquired every 15 min for 7 h. The assay was performed in a 384-well plate using a Tecan Spark10 M plate reader. The background absorbance of compounds was subtracted by measuring the absorbance at 412 nm of each compound under the same conditions without DTNB. Compounds were measured in triplicate. The data were fitted to a second-order reaction equation such that the rate constant k is the slope of ln( [A][B 0 ]/[B][A 0 ] ), where [A 0 ] and [B 0 ] are the initial concentrations of the compound (200 μM) and TNB 2-(100 μM), respectively, and [A] and [B] are the remaining concentrations as a function of time as deduced from the spectrometric measurement. Linear regression using Prism was performed to fit the rate against the first 4 h of measurements. GSH Reactivity Assay.
4 mL/min. The gradient used for BSA was 20% B for 2 min, increasing linearly to 60% B for 4 min, holding at 60% B for 2 min, changing to 0% B in 0. 1 min, and holding at 0% for 1. 9 min. The gradient for the other proteins was 20% B for 2 min, increasing linearly to 60% B for 3 min, holding at 60% B for 1. 5 min, changing to 0% B in 0. 1 min, and holding at 0% for 1. 4 min. The mass data were collected on a Waters SQD2 detector with an m/z range of 2-3071. 98 at a range of 750-1550 m/z for NV3CP, K-Ras G12C , and NUDT7, 700-1300 m/z for QSOX1, PBP3 R504C , USP8, and PCAF and 1000-2000 m/z for BSA. The desolvation temperature was 500 °C with a flow rate of 1000 L/h. The voltages used were 0. 69 kV for the capillary and 46 V for the cone. Raw data were processed using openLYNX and deconvoluted using MaxEnt.
All of the compounds from bad wells were assigned as no available data in Data set 3 in the Supporting Information. Quantitative Processing of Mass Spectrometry Data. A Python script for processing the MaxEnt deconvoluted spectra is supplied as File 2 in the Supporting Information. Below we briefly outline the logic of the processing. We first identify the 10 highest peaks for each well and discard the rest. The peaks are then normalized from ion counts to percentages, where the highest peak is defined as 100%. The unlabeled protein mass is deduced from a reference well that contains just the protein. Up to four noncompound "adducts" are also assigned from that well (buffer adducts, protein oxidations, etc. ); these often keep their proportion when a compound labels the protein. We discard peaks lower than 10% of the maximum peak.
All X-ray diffraction data were collected on the beamline I04-1 at the Diamond Light Source (Harwell, U. K. ) unless stated otherwise. OTUB2 Structure Determination. Diffraction data were automatically processed by software pipelines at the Diamond Light Source. 91 Initial refinement and map calculation was carried out with DIMPLE. 92 PanDDA 93 was used for hit identification. Further refinement and model building was performed with REFMAC  94  and COOT,  95  respectively. Coordinates and structure factors for all data sets are deposited in the RCSB Protein Data Bank under PDB IDs 5QIO, 5QIP, 5QIQ, 5QIR, 5QIS, 5QIT, 5QIU, 5QIV, 5QIW, 5QIX, 5QIY, and 5QIZ. Data collection and refinement statistics are available from the PDB pages. OTUB2 Inhibition Assays. The assays were performed in "nonbinding surface flat bottom low flange" black 384-well plates (Corning) at room temperature in a buffer containing 50 mM Tris•HCl, 100 mM NaCl, pH 7.
All volumes were equalized to 400 nL by means of a DMSO back-fill. A 15 μL portion of a 533 nM Ub-Rho solution was added to each well using a Biotek Multiflow FX liquid dispenser, and the plate was shaken vigorously for 10 s and then centrifuged for 1 min at 1000 rpm. Next, 5 μL of a 100 nM OTUB2 solution was added to each well, the plate was quickly loaded into a BMG Clariostar plate reader, and the increase in fluorescence (excitation 487 nm, emission 535 nm) over time was recorded immediately. All data fitting and calculations were done using GraphPad Prism 7 software. The fluorescence intensities were plotted against time (in seconds) after a baseline correction using the DMSO control for each inhibitor concentration. The data were fitted to the equation - [ - ] - vi k e FI 1 k t obs obs NUDT7 Crystallization.
5, 500 mM NaCl, and 5% glycerol with 30 nL of 20 mM compound in DMSO in sitting-drop crystallization plates containing 0. 1 M BisTris pH 5. 5, 0. 1 M ammonium acetate, and 4-16% (w/v) PEG 10000 in the reservoir at 20 °C. After overnight incubation of protein and compound, 100 nL of reservoir solution and 30 nL of a crystal seed solution obtained from a previous crystallization experiment were added to the drop. Hexagonshaped crystals appeared within several days. Prior to data collection, all crystals were transferred to a solution consisting of the precipitation buffer supplemented with 25% ethylene glycol and subsequently flashcooled in liquid nitrogen. All X-ray diffraction data were collected on beamline I04-1 and beamline I03 at the Diamond Light Source (Harwell, U. K. ). NUDT7 Structure Determination.
Diffraction data were automatically processed by software pipelines at the Diamond Light Source. 91 Initial refinement and map calculation was carried out with DIMPLE. 92 PanDDA 93 was used for hit identification, and further refinement and model building was performed with REFMAC  94  and COOT,  95  respectively. All structure determination steps were performed within the XChemExplorer 97 data management and workflow tool. Coordinates and structure factors for all data sets are deposited in the Protein Data Bank under group deposition ID G_1002045. Data collection and refinement statistics are summarized in Table  5  in the Supporting Information. The complete PanDDA analysis and all processed data from the NUDT7 fragment campaign (including information about soaked compounds) can be accessed via the ZENODO data repository under DOI 10.
5281/zenodo. 1244111. NUDT7 Activity Assay. Mass spectrometry assays monitoring acetyl-CoA hydrolysis by NUDT7 were performed on a Agilent 6530 RapidFire QTOF mass spectrometer in a 384-well plate format using polypropylene plates (Greiner, code 781280) and an assay buffer containing 20 mM HEPES pH 7. 5, 200 mM NaCl, and 5 mM MgCl 2. All bulk liquid handling steps were performed using a multidrop combi reagent dispenser (Thermo Scientific, Code 5840300) equipped with a small tube plastic tip dispensing cassette (Thermo Scientific, Code 24073290). For inhibitor IC 50 determinations an 11-point and 2-fold serial dilution was prepared from a 50 mM stock solution in DMSO, which was transferred to give four replicates using an ECHO 550 acoustic dispenser (Labcyte). The transferred volume was 400 nL, giving a final DMSO concentration of 0.
The enzyme reaction was stopped by addition of 10 μL of 50 mM EDTA, and the plate was transferred to a RapidFire RF360 highthroughput sampling robot. Samples were aspirated under vacuum and loaded onto a C4 solid-phase extraction (SPE) cartridge equilibrated and washed for 5. 5 s with 1 mM octylammonium acetate in LCMS grade water to remove nonvolatile buffer components. After the aqueous wash, analytes of interest were eluted from the C4 SPE onto an Agilent 6530 accurate mass Q-TOF in an organic elution step (85% acetonitrile in LC-MS grade water). Ion data for the acetyl-CoA and hydrolyzed product were extracted and peak area data integrated using RapidFire integrator software (Agilent). Percent conversion of substrate to product was calculated in Excel, and IC 50 curves were generated using Graphpad prism version 7.
Measurements were performed using StepOnePlus rtPCR from 25 to 95 °C with 0. 3 °C steps. T m was determined using StepOne Software v2. 3. The reported T m was calculated as the average of three triplicates for each compound. NUDT7 Cellular Thermal Shift Assay. HEK293 cells were cultured at 37 °C in a humidified 5% CO 2 atmosphere in DMEM supplemented with GlutaMAX and 10% FBS. Cells were grown in T175 flasks until around 70% confluence and transfected with Flag-NUDT7 using Lipofectamine2000 transfection reagent. Twenty-four hours post-transfection, cells were detached, and 13 × 10 6 cells were seeded in T75 flasks for treatment and control samples, respectively. After 24 h cells were treated with either DMSO or 20 μM NUDT7-COV-1 for 30 min at 37 °C. Cells were harvested, washed with PBS, and aliquoted into PCR tubes.
Luminescence was measured using the luminescence module of a PheraStar FS plate reader (BMG Labtech). Data analysis was performed using GeneData 12 analytic software. NNMT Activity Assay. Compounds were transferred into black microplates (Greiner 784900) using Labcyte Echo acoustic dispensing. Assay ready plates were then sealed with heat seals. If they were not used immediately, plates were frozen at -20 °C and held in polypropylene boxes with silica gel desiccant. Reagents were obtained as follows: nicotinamide (Sigma 47865-U), SAM (Sigma A7007), SAH-FITC (Axis Shield, RPBB350), Anti-SAH (Axis Shield, RPBB278). All liquid handling was done with a GNF Washer/dispenser II. All reagents were prepared in 20 mM phosphate buffer, pH = 7. 6. A 6 μL portion of 3X NNMT (120 nM) was added to assay plates and incubated for 10 min at room temperature.
A 6 μL portion of 3X substrate mixture (120 mM Nicotinamide, 6 μM SAM) was added and incubated for 1 h at 30 °C. A 6 μL portion of 3X detection mixture (150 ng/mL SAH-FITC and 30 μg/mL anti-SAH antibody) was prepared and incubated for 30 min at room temperature before adding to reaction mixtures. Plates were further incubated for another 2 h at 30 °C, protected from light. Fluorescence polarization reactions were read with a BMG Pherastar FS instrument using a 485/ 520/520 nm module. Data were normalized to DMSO (100%) and no enzyme (0%) controls using Genedata Screener software. Profiling of OTUB2 and NUDT7 Reactive Cysteines by Competitive isoTOP-ABPP. The profiling was performed according to a slightly modified competitive isoTOP-ABPP protocol. 74 Briefly, HEK-293T cells were cultured at 37 °C under 5% CO 2 atmosphere in DMEM culture medium supplemented with 10% FBS and 1% PS.
The two samples were mixed, resuspended in 1. 2% SDS/PBS, and diluted to 0. 2% SDS/PBS for streptavidin enrichment and subsequent trypsin and TEV digestion. The resulting TEV digests were analyzed by LC-MS/MS on a Q-Exactive Orbitrap instrument, and the MS data were searched and quantified by the Openpfind algorithm. 98 ASSOCIATED CONTENT * S Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10. 1021/jacs. 9b02822. Additional information on the distribution of the electrophile-fragment library properties including MW distribution of the adduct form and diversity of substructures, experimental data for thiol-reactivity assay in different TCEP concentrations, reactivity rates for resourced compounds and comparison with GSH t 1/2 , correlations between reactivity rates and goodness of fit to the second-order reaction equation, between reactivity and occurrences of double labeling, between reactivity and overall labeling, between reactivity and cellular toxicity, and between reactivity of unsubstituted and methyl-substituted chloroacetamides, comparison of labeling with single compound vs compounds in pools of five, labeling of OTUB2 with selected compounds, identification of cysteine modification sites for sample compounds for USP8 and NUDT7, structures of unique hits, promiscuous compounds, compounds containing aminothiazoles, and second-generation OTUB2 binders, cocrystal structures of OTUB2 with two compounds containing the acethydrazide motif and with OTUB2-COV-1 and crystallographic statistics, experimental data of jump dilution experiment for OTUB2 with OTUB2-COV-1, gel-based ABPP selectivity of OTUB2 inhibitors in lysates and in cells, stabilization of NUDT7 by covalent fragments, and enzymatic inhibition of NUDT7, K inact /K i determination and competitive isoTOP-ABPP for both OTUB2-COV-1 and NUDT7-COV-1 (PDF) Data for thiol-reactivity assay, LC/MS/MS of NUDT7 and USP8 with a selection of compounds, isoTOP-ABPP ratios for all peptides, cellular viability and NNMT enzymatic inhibition, labeling data for all fragments in the electrophile library against 10 proteins, double-labeling occurrence, and comparison between labeling of one compound vs pool of five compounds (XLSX) Detailed synthetic procedures and 1 H and  13  C NMR spectra and LC/MS for synthesized compounds (PDF) Labeling quantification script from raw LC/MS data (PDF).
115766 ),  Janssen ,  Merck KGaA  ( Darmstadt, Germany ),  MSD ,  Novartis Pharma AG , the  Ontario Ministry of Research, Innovation and Science (MRIS) ,  Pfizer ,  Sao Paulo Research Foundation-FAPESP, Takeda , and  Wellcome  ( 106169/Z/14/ Z ). Supported by a research grant from the  Nancy and Stephen Grand Israel National Center for Personalized Medicine. We thank  Ilana Berlin  for providing the GFP-OTUB2 plasmids. We thank  Prof. Deborah Fass  for the generous gift of QSOX1 protein.
Associations between study-specific factors with SF-36, COF, FES, or SAFFE were analyzed using multiple linear regression. To determine the relative importance (RI) of variables  (29, 30) , we used the Linderman, Merenda and Gold (LMG) method with the R-package RELAIMPO  (31). This method allows the contribution of different correlated regressors to be differentiated in a multiple linear regression model. This metric is based on unweighted averages over sequential R 2 's of each variable in all permutation of available regressors to avoid regressors' order effects. The total R 2 is split into one non-negative R 2 share per regressor, all of which sum to the total explained R 2. This is done by calculating the contribution of each predictor at all possible points of entry into the model, and taking the average of those contributions.
Adiabatic labelling pulses were applied through a plane positioned perpendicular to the common carotid artery, 13 mm posterior to the imaging slice. Single slice, single shot, echo planar images (EPI) were then collected with TR/TE = 3500/11 ms, 1 mm coronal slice thickness, and 250 μm nominal in-plane resolution. Quantification of resting perfusion was achieved via multi-post label delay (PLD) pCASL imaging; thirty-two labelled-unlabelled EPI pairs were acquired for each of nine PLDs (0, 50, 150, 250, 350, 500, 750, 1000, and 2000 ms in randomized order). Voxelwise resting CBF and arterial transit time (ATT) were then computed in absolute units by modelling the mean pCASL signal vs. PLD, using an in-house Python implementation (Python Software Foundation, https://www. python. org/) of the single-compartment model  [24].