dataesr/scientific-paragraphs-categorization huggingface dataset:
Dried TLC plates were exposed to a phosphor imaging screen and scanned with a Typhoon FLA 9000 (GE Healthcare). All data were obtained from three separate experiments and were fit in PRISM (GraphPad). Rates of ATP hydrolysis in units of μM/sec were determined by quantitating the amount of ATP hydrolyzed as a function of time for each different ATP concentration and the resulting data were plotted as rate of ATP hydrolysis (µM/sec) versus ATP concentration [mM]. Kinetic parameters (K M , V max and k cat ) were obtained by fitting the curves in Prism (version 6. 0) using a nonlinear regression.
DNA binding assays Srs2 898 DNA binding assays were performed at 30 °C for 10 min in 10 µl Srs2 buffer (30 mM Tris-HCL [pH 7. 6], 100 mM KCl, 2 mM MgCl 2 , 1 mM DTT, 0. 2 mg mL -1 BSA) and reactions contained increasing concentrations of Srs2 898 nM-100 nM, as indicated) together with 1. 3 µM (in nucleotides) of a 40-nt ssDNA fluorescein-labeled oligonucleotide (IDT) of the following sequence: 5'-Fluorescein-ATT AAG CTC TAA GCC ATG AAT TCA AAT GAC CTC TTA TCA A-3'. Reactions were mixed with 10 µl of Stop buffer (50% glycerol, 20 mm Tris-HCl [pH 7. 4], 2 mm EDTA) and resolved in 10% native polyacrylamide gels in TAE buffer (40 mM Tris-acetate [pH 8], 1 mm EDTA) at 4 °C for 2 h. Gels were scanned at a wavelength of 473 nm using a Typhoon FLA 9000 (GE Healthcare). The fluorescently labeled DNA bands were quantified using the open-source software package Fiji  95  and the ratio between the bound fraction to the total DNA (bound fraction plus unbound fractions) was plotted.
The fluorescently labeled DNA bands were quantified using the open-source software package Fiji  95  and the percent of the unwound dsDNA substrate was calculated as the intensity of the ssDNA band divided by the total intensity of ssDNA and dsDNA multiplied by 100. The normalized percent of unwound DNA was plotted by setting the percent of Srs2 898 at 30 min to 100% and fitting the rest of the data accordingly. All data were obtained from three separate experiments and were fit in PRISM (GraphPad).
5 mM ATP. RPA (300 nM) was then added to the reaction mix and the incubation continued for an additional 15 min. Rad54 (200 nM) and supercoiled pUC19 (NEB, Cat #N3041A) were then added to the reaction mix and the incubation continued for an additional 15 min to generate the D-loop products. Srs2 898 (40, 150 or 250 nM, as indicated) was then added to the D-loop reaction mix and incubated at 30 °C for 5, 10, 15 or 30 min, as indicated. Reactions were terminated with the addition of an equal volume of Stop buffer (20 mM EDTA, 1% SDS, and 20% glycerol). The reaction products were then de-proteinized by the addition of Proteinase K (0. 5 mg/ml, final concentration), and then were resolved on a 0. 9% agarose gel in 1xTAE buffer. Gels were scanned using a Typhoon FLA 9000 with a 635-nm laser to detect the ATTO 647 dye (GE Health Sciences) and DNA species were quantified using the open-source software package Fiji  95.
5 nM GFP-Srs2 898 injection in HR buffer (30 mM Tris-Ac [pH 7. 5], 50 mM KCl, 5 mM MgAc, 1 mM DTT, 0. 3 mg/mL BSA, and 2 mM ATP) at a frame rate of 1 frame per 10 s for a total time of approximately 20 min. Data was collected with a 100-millisecond integration time and the lasers were shuttered between images to minimizing photo-bleaching. Images were collected using Nikon software, and images were exported as individual TIFF images  74, 98. TIFF stacks were imported into ImageJ (Fiji)  95. For two-color imaging, the two channels were first corrected for stage drift and then merged into TIFF images, which were then converted to TIFF stacks. All TIFF stacks were then corrected for stage drift using the registration/translation function within Fiji  74. For each time course experiment, kymographs were generated from the TIFF image stacks by defining a 1-pixel wide region of interest (ROI) along the axis of each individual ssDNA molecule, and these ROIs were extracted from each image within the TIFF stack  74.
Acknowledgements We thank  Rodney Rothstein  and  Luke Berchowitz  for yeast strains and access to equipment. We thank members of the  Greene and Symington laboratories  for critically reading the manuscript. This research was funded by  NIH  grants  R01CA236606  and  R35GM118026  (E. C. G. ), and  R35GM126997  (L. S. S. ). We thank  Jerry Chang  and the  Precision Biomolecular Characterization Facility (PBCF) at Columbia University  for technical support and access to the CD spectrometer, and we thank  Sahiti Kuppa  for assistance with the CD measurements. The PBCF is supported by  NIH  Award  1S10OD025102.
The first wave included 1560 young adults aged between 18 and 24 years. The second wave took place a mean of five years later (2012-2014), and all young adults included in the first wave were invited for a reassessment. Only data from the second wave are described in this current study. All participants agreed to participate in the study by providing their free and written informed consent. Compensation for travel expenses was provided for the participants. This study was approved by the Research Ethics Committee of the Universidade Catoĺica de Pelotas under protocol number 2008/118. The full description of the study design is published elsewhere  54. The psychiatric diagnosis was assessed using the Mini International Neuropsychiatric Interview-Plus by trained psychologists. In addition, the severity of depressive symptoms was assessed using the MADRS.
On days 2 and 3, mice were individually placed in an arena for 10 min each. On day 4 (NORT), mice were individually placed in the arena where two identical objects were placed at equal distances from the walls and from each other, and the mice were left to explore for 10 min and put back in their home cage. Two hours later, mice were placed back in the arena where one of the two objects was replaced with a novel object, and the mice were left to explore for 10 min. A camera above the arena automatically captured the locomotor activity of each mouse, and its behavioral pattern was measured and analyzed using EthoVision XT software (Noldus) and confirmed by a blind manual measurement of the time spent sniffing the objects. To exclude a spontaneous preference towards one of the objects, several pairs of objects had previously been tested with different sets of naive young and aged animals, and objects with no preference were chosen for the experiment.
Novel object location test. Same arenas and procedure was used as in the NORT. The test took place after the NORT, so no additional habituation was performed. The objects used were different from those in the NORT, and spatial cues were provided on the walls of the arenas (sun, moon, square and triangle shaped). On the day of the test, each mouse was individually placed in the arena where two identical objects were placed at equal distances from the walls and from each other, and the mice were left to explore for 10 min and put back in their home cage. Two hours later, mice were placed back in the arena where one of the two objects was placed in a diagonal compared to its previous position, and the mice were left to explore for 10 min. A camera above the arena automatically captured the locomotor activity of each mouse, and its behavioral pattern was measured and analyzed using EthoVision XT software (Noldus) and confirmed by a blind manual measurement of the time spent sniffing the objects.
A camera above the arena automatically captured the locomotor activity of each mouse, and its behavioral pattern was measured and analyzed using EthoVision XT software (Noldus). Splash test. Mouse cages were placed in the behavior room for habituation 30 min before the experiment. Then, all mice, except for the experimental mouse, were transferred to a different cage. The experimental mouse was given a spray shot of a 10% sucrose solution on the lower back and then put back in the home cage. The viscosity of the sucrose solution dirties the fur, inducing a grooming behavior. The mouse was observed for 6 min, during which the frequency of grooming, the latency to first groom and the total grooming times were measured. After the test, the experimental mouse was placed in a different cage until all the mice of the cage had been subjected to the test.
The next morning (day 3), water and sucrose-water consumption was measured again and mice were returned to their home cages. The sucrose index we report was the average of the two measurements. Social interaction test. The same arenas as for the open field test, NORT and novel object location test were used. Mouse cages were placed in the behavior room for habituation 1 h before the experiment. Each test mouse was placed in the center of the arena. Immediately after, a stimulus mouse (intruder) was also placed in the center of the arena. Mice were left to explore the arena and interact for 5 min. A camera above the arena automatically captured the locomotor activity of the mice, and their behavioral patterns were measured and analyzed using EthoVision XT software (Noldus). Active avoidance was measured manually by a blind investigator by considering a movement of the test mouse further than 3 cm away when the intruder approaches.
Light/Dark box test. The light box (16. 5 × 22 cm) was separated from the dark box (16. 5 × 22 cm) with a dark divider with a small opening allowing the mice to go through. Time spent and frequency of entry in the light box (178 lux) were measured during 10 min. A camera above the arena automatically captured the locomotor activity of the mice, and their behavioral patterns were measured and analyzed using Etho Vision XT software (Noldus). Coat state test. The total coat state score resulted from the sum of the score of five different body parts: neck, dorsal/ventral coat, tail, forepaws and hindpaws. For each body area, a score of 0 was given for a well-groomed coat and 1 for an unkempt coat. The measurements of the coat state were done by an experimenter blind to treatments. Novelty suppressed feeding.
The length of the open and closed arms was of 37. 5 cm and the height 53 cm. To measure stress and anxiety, each mouse was placed in the center of the EPM and left to explore the arms for 6 min. A camera above the apparatus automatically captured the activity of the mouse and its behavioral pattern, as well as the time spent in the open and closed arms. Measurements and analysis were done using EthoVision XT software (Noldus). Nest building test. Each mouse was housed individually starting in the evening of the testing day. Two intact cotton pellets (2 × 2 cm; 2. 5 g) were placed in the cage and mice were left undisturbed overnight. The next morning each nest was assessed for two parameters: the height of the nest and the nest quality. The latter was given a score based on percentage of the nest height compared to mouse size and shape (0: material untouched; 5: cloud-like shape, material finely shredded).
RNA-seq of primary hippocampal neurons RNA isolation. Total RNA was isolated from samples using Trizol (Life Technologies) according to the manufacturer's instructions. After adding chloroform, the samples were centrifuged at maximum speed, and the upper phase was then used for RNA cleanup using RNAeasy (Qiagen) minicolumns following the manufacturer's instructions. Briefly, an equal volume of 70% ethanol was added to the upper phase and processed through the columns. After two washes, the RNA was eluted with water. RNA-seq. RNA-seq and bioinformatic data analysis were performed by NovoGene. Briefly, messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by second strand cDNA synthesis.
The libraries were checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on an Illumina platform, and paired-end reads were generated. Raw reads were filtered to remove those containing adapters, poly-N and low-quality reads from raw data, and Q20, Q30 and guanine-cytosine (GC) were calculated. All downstream analyses were performed on clean data with high quality. Paired-end reads were aligned to the GRCm38/mm10 reference genome using Hisat2 v2. 0. 5, and feature-Counts v1. 5. 0-p3 was used to count the read numbers mapped to each gene, normalized by FPKM. Differential expression analysis. Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2 R package (1.
20. 0). The resulting P values were adjusted using the Benjamini and Hochberg's approach, and genes with an adjusted P value ≤ 0. 05 found by DESeq2 were assigned as differentially expressed. Enrichment analysis of differentially expressed genes. Gene Ontology enrichment analysis of differentially expressed genes was done using the clusterProfiler R package for which gene length bias was corrected. Genes were ranked according to the degree of differential expression, and then predefined gene sets were tested to using a local version of the GSEA analysis tool (http://www. broadinstitute. org/ gsea/index. jsp).
Acknowledgements We thank  G. Lepousez ,  C. Mazo  and  F. Sonthonnax  for advice on the miniosmotic pumps;  M. Pende  for discussions and sharing mTORpathway antibodies;  S. Pons  and  N. Lemière  for sharing equipment;  J. Carbonne  for technical help with the in vitro experiments on neurons; and  B. Charbit  for advice and assistance with the Seahorse apparatus. L. K. acknowledges support by the  AGEMED INSERM program on Aging (Programme Transversal sur le Vieillissement   2016-2022 ) and  ADPS Recherche sur la Longévité. L. K. and A. C. received a fellowship from  LabEx/Revive. The Lledo lab was supported by  Institut Pasteur ,  Centre National pour la Recherche Scientifique , the life insurance company ' AG2R-La-Mondiale ',  Agence Nationale de la Recherche  ( ANR-15-CE37-0004-01 ),  National Institutes of Health   US-French Research Proposal Grants   1R01DC015137-01  and  ANR-15-NEUC-0004  (Circuit-OPL) and the  Laboratory for Excellence Programme 'Revive' (Investissement d'Avenir ,  ANR-10-LABX-73 ).
Acknowledgements We acknowledge support from the  Swiss National Science Foundation  grant number  212723  and the  Ecole Polytechnique Fédérale de Lausanne (EPFL). We thank  Farzaneh Fadaei-Tirani  for important contributions to the X-ray single crystal structure analyses. We thank  Dr A. Sienkiewicz  for EPR data collections. L. M. is a senior member of the  Institut Universitaire de France. CalMip  is acknowledged for a generous grant of computing time.
Conclusion: Oral switch was non-inferior to intravenous standard therapy in participants with lowrisk SAB. However, a careful assessment of patients for signs and symptoms of complicated SAB at time of presentation and thereafter is necessary before considering early oral switch therapy. The trial was registered as NCT01792804 in ClinicalTrials. gov, as DRKS00004741 in the German Clinical trials register, and as EudraCT 2013-000577-77.
TRIAL DESIGN AND OVERSIGHT We conducted a randomized, parallel-group, open-label, non-inferiority trial. In-patients with lowrisk SAB were recruited in 31 sites in four European countries from 20 December 2013 to 22 December 2019 (Figure  1  and Supplementary Table  S1 ). The study protocol was published and approved by the Ethics Committees of all participating centers and the respective competent authorities. 22, 23  All participants gave written informed consent before enrolment. An independent data monitoring committee periodically assessed the safety of the trial participants. The trial was registered as NCT01792804 in ClinicalTrials. gov, as DRKS00004741 in the German clinical trials register, and as EudraCT 2013-000577-77.
The intention-to-treat population included all randomly assigned participants. The modified intention-to-treat population comprised participants that were randomized AND received any study drug, excluding participants in whom an inclusion criterion incompatible with low-risk SAB was violated before starting study drug (e. g. , persistent positive blood cultures). In both intention-totreat populations, data were analyzed as assigned with indeterminate and missing outcomes counted as failures. The clinically evaluable population included all study subjects who were treated according to protocol with study drug, were observed until end-of-follow-up or reached the primary endpoint and did not receive antimicrobial therapy during follow-up that could have masked the primary endpoint. Study drug was considered according to protocol when the total duration of antimicrobial therapy was between 12 and 16 days with at least 5 days of initial intravenous therapy.
A masked, independent Clinical Review Committee carefully evaluated study outcomes and treatment. The safety population included all study subjects who received any study drug. Specifically, participants who ever received an oral antimicrobial agent were compared to participants who never received an oral antimicrobial agent. A Kaplan-Meier analysis was performed to assess differences in time to SAB-related complications. Hazard ratios were estimated from Cox regression and compared with an unstratified log-rank test. Competing risks were calculated according to Fine et al considering non-attributable mortality as competing event. 29  Statistical calculations were done with the software SAS 9. 4 (SAS Inc. , Cary, NC, USA) and Stata 17. 0 (Stata/SE, College Station, TX, USA).
Acknowledgements The trial was funded by the  Deutsche Forschungsgemeinschaft  (project number  217917502  to A. J. K. ). We thank participants and staff from all sites who contributed to the trial (listed in the Supplementary Material). We thank  Vance G. Fowler ,  Stephan Harbarth , and  Guy Thwaites  for expert opinion in the Trial Steering Committee,  Vincent LeMoing ,  Miquel Pujol Rojo , and  Estée Török  for participation in the Clinical Review Committee, and  Walter E. Häfeli ,  Alexandra Heininger ,  Geraldine Rauch  for participation in the Data Monitoring Committee.
We focused our study on TEAD1 and TEAD4, as they were the most abundant protein isoforms by LC/MS in the NCI-H2030 cell line (TEAD1 = 1. 5 fmol, TEAD4 = 0. 7 fmol, data not shown). TOR demonstrated specific binding of compounds to their target protein; 90% of TEAD1 and 50% of TEAD4 were occupied by K-975, while 80% of KRAS G12C was occupied by adagrasib (Supplementary Figure  S1A ). Data were comparable for each target in the single and the combination treatments. Next, we characterized target pathway-specific biomarker modulation for K-975 and adagrasib. We introduced a gene-reporter element carrying the TEAD-responsive promoter and a luciferase gene reporter (TEAD-Luc) to NCI-H2030 and HOP 62 cell lines. We used this gene reporter as a biomarker for YAP1-TEAD activity. As expected, we detected potent inhibition of TEAD-Luc by K-975 (IC 50 of 10-20 nM) in NCI-H2030 (TEAD-Luc) and HOP 62 (TEAD-Luc) cells and observed no effect for adagrasib (Supplementary Figure  S1B ).
These data were finally crossed to see the direct target of our combination. (B) Representative GSEA of hallmark gene sets comparing all the treatments versus DMSO. The first three most modulated signatures are shown. Genes positively regulated are in red, while genes negatively regulated are in blue. For graphical matters, we cut the panels to show only the first set of 35-40 genes as representative figures FDR q value < 0. 1. (C) Volcano plots showing proteomic data. Proteins modulated in the single or combination treatments with K-975 and/or adagrasib versus DMSO are shown. Downregulated proteins are represented as green dots and upregulated proteins as red dots. Highlighted in dark green and indicated by the arrows are PLK1, CDC20, DIAPH3, and BUB1B. FC > 2, p value < 0. 01. (D) GO and pathway map in heatmap representation was performed from proteomic data and using Metacore.
The immunoprecipitation with phosphotyrosine (pY) assay revealed seven phosphorylated targets specific to the combinations. Beta-tubulin showed the strongest dephosphorylation that was combination specific. This dephosphorylation was seen with both KRAS inhibitors tested (Supplementary Table  S2 ). By Immobilized Metal Affinity Chromatography (IMAC) assay, we detected 25 phosphorylated peptides in the combinations. Most were implicated in ubiquitination and sumoylation processes involved in the transition G2/M or in the correct kinetochore assembly (Supplementary Table  S2 ). These data confirmed the impact of the combination on the G1 and G2/M phases previously observed.
Cell Lines Culture Conditions Cell lines were maintained at Sanofi and grown at 37 • C under 5% CO 2 , except for SW1573, which was at 0% CO 2. A short tandem repeat assay authenticated all cell lines at the Microsynth AG (Balgach, Switzerland). PCR using the Venor ® GeM kit excluded mycoplasma infection (Biovalley, MB minerva biolabs, Skillman, NJ, USA). HOP 62 (#CRL-11350) RRID: CVCL_1285, NCI-H2030 (#CRL-5914) RRID: CVCL_1517, NCI-H1792 (#CRL-5895) RRID: CVCL_1495, NCI-H23 (#CRL-5800) RRID: CVCL_1547, NCI-H358 (#CRL-5807) RRID: CVCL_1559, NCI-H1373 (#CRL-5866) RRID: CVCL_1465, NCI-H2122 (#CRL-5985) RRID: CVCL_1531, SW1573 (#CRL-2170) RRID: CVCL_1720 were purchased from the American Type Culture Collection (ATCC), while LU99A was purchased from the Japanese Collection of Research Bioresources Cell Bank (JCRB0044) and cultured according to supplier's recommendation.
Luminescence was detected using the TECAN SPARK plate readers. To characterize the interactions between adagrasib and K-975 on NCI-H2030 and HOP 62 cells, the IC50 of adagrasib alone and with K-975 were estimated from ranges of adagrasib between 0. 02 and 10,000 nM alone and mixed with eleven constant concentrations of K-975 between 0. 5 and 3333 nM. The ratio R = IC 50adagrasib with K-975 /IC 50adagrasib alone was calculated with its confidence interval with SAS ® version 9. 4. If R is significantly lower than 1 (i. e. , the lower bound of its confidence interval is lower than 1), the IC 50 of K-975 with adagrasib is significantly lower than the IC 50 of adagrasib alone. Moreover, if K-975 is nonactive, a potentiation can be concluded. Three independent experiments were conducted to obtain six replicates of the wells with single compounds and triplicates of the wells with combinations.
0 (Omicsoft, Qiagen). Following raw-read QC, the last few bases were trimmed and mapped to the reference genome Human. GRCh38. The read count data were generated using GeneModel RefGene20200204. The resulting data were normalized by the DESeq package, transformed to a log2 value, and used for pathway prediction and one-way ANOVA analyses. For pathway enrichment, GSEA was performed using the Hallmark gene set of Molecular Signatures Database v7. 4  [20]. To estimate the TEAD-dependent transcription rate and to evaluate TEAD inhibitors' pharmacodynamics, we used a transcriptional signature of TEAD activity and a scoring method described elsewhere  [37].
2% formic acid. Samples were spiked with six heavy isotopic AQUA standards peptides at 10 fmol each (Thermo Fisher Scientific). All samples were separated into two aliquots, one analyzed in Data Dependent Acquisition (DDA) mode LC/MS analysis for global proteome differential analysis and the other one subjected to targeted LC/MS analysis for TEAD1, TEAD2, TEAD3, and TEAD4 expression level determination and target occupancy ratio (TOR) measurement. Analyses were performed using a nano-ACQUITY Ultra-Performance LC system (Waters, Milford, MA, USA) coupled to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). LC separation was performed with a trapping column (nano-Acquity Symmetry C18, 100 Å, 5 µm, 180 µm × 20 mm) at 15 µL/min flow rate and an analytical column (nano-Acquity BEH C18, 130 Å, 1.