dataesr/scientific-paragraphs-categorization huggingface dataset:
Three known compounds 5α,6α-epoxystigmasta-7-en-3β-ol (7), 5α,6α-epoxystigmasta-7,22-dien-3β-ol  (8) , and 5α,6α-epoxyergosta-7-en-3β-ol (9) were also isolated and characterized from the MeOH extract of I. echinata. Their structures were determined by spectral data and comparison with those reported in the literature from the marine sponge Ircinia aruensis  [12]. Evaluation of all steroids isolated from I. echinata was performed against three cancer cell lines: MCF-7 (human breast cancer cells), HepG-2 (human liver hepatocellular carcinoma cells), and Lu-1 (human lung adenocarcinoma cells). All compounds were inactive until 32 µg. mL -1 , except the two new steroids (24R)-5α,6α-epoxy-24-ethyl-cholesta-7-en-3β,9α-diol (3) and 5α,6α-epoxycholesta-7-en-3β,9α-diol (4) (Table  3 ). Compound 3 exhibited a selective inhibition towards the MCF-7 cells with an IC 50 value of 15.
Acknowledgments: We thank the  "Plateforme analytique du Muséum"  for access to the NMR and mass spectrometry facilities. The authors are grateful to  Professor Do Cong Thung  ( Institute of Marine Environment and Resources, VAST ) for the sponge identification,  T. T. H. Nguyen  for the biological assays performed in Hanoi, and  A. Marie  and  L. Dubost  ( MNHN, Paris, France ) for the MS measurements.
5 mg/mL, which was able to affect the growth after 24 h of incubation. Experiments were done in triplicate to obtain the mean and the standard deviation (SD). Statistical analysis (Two-way ANOVA) was performed by the GraphPad Prism6 software and the p value < 0. 0001 (****) showed significant difference. E. coli represented the negative control. Indeed, after 24 h of incubation, no growth inhibition was observed by testing CC or MOR/α-CD against E. coli ATCC 8739 using a dose of 0. 5 mg/mL of both CC or MOR/α-CD (Figure  2D ). Although a dose of 0. 25 mg/mL of MOR/α-CD showed a slight decrease of the E. coli growth (OD) compared to the ineffective CC antibiotic (Figure  S4 ), this result did not suggest a good antimicrobial activity. In order to compare the MOR/α-CD antimicrobial activity among the assessed strains, we analyzed the percentage (%) of the bacterial growth inhibition calculated as [(Ac -At)/Ac] × 100, where Ac was an average of three replicates of light absorption values at wavelength 600 nm of the negative controls (the OD reached by untreated bacteria after 24 h of incubation) and At was an average of the three replicates of light absorption values at a wavelength of 600 nm of the samples (the OD reached by treating bacteria after 24 h of incubation with different doses of the MOR/α-CD).
The rationale is that the MIC concentration could exert only bacteriostatic effect given the same colony count compared to the bacterial input. The use of two, four, and multiple MOR/α-CD concentrations could allow to observe bactericidal effect. Results indicated that 1 mg/mL of MOR/α-CD represented the minimum bactericidal concentration (MBC) value for each strain. Indeed, no viable bacteria (CFU/mL) have been observed after the treatment was performed by GraphPad Prism6 software to compare the most effective dose of the MOR/α-CD complex (0. 5 mg/mL) among the strains. Experiments were assessed in triplicate to obtain the mean and the standard deviation (SD). p value < 0. 0002 (***) showed a significant difference.
Each Ery/CC combination aureus ATCC BAA-977, ATCC 25923, and ATCC 6538 strains using the MIC dose (0. 5 mg/mL) of MOR/α-CD and from 2 up to 4-fold the value of MIC for each strain (0. 5, 1 and 2 mg/mL). The colony count was made after 18 h, 20 h, and 24 h of treatment. Experiments were assessed in triplicate to obtain the mean and the standard deviation (SD). A statistical analysis (Two way ANOVA) was performed by the GraphPad Prism6 software and the p value < 0. 0001 (****) showed a significant difference between the dose of 0. 5 mg/mL, 1 mg/mL, and 2 mg/mL of MOR/α-CD and the untreated bacteria (0 mg/mL) for each strain at different time points.
As expected, in the presence of MOR/α-CD, no bacterial growth was observed (Figure  7  and Figure  S7 ). Moreover, no CFU was counted by seeding 10 μL from each antibiotic combined well, after 24 h of MOR/α-CD treatment (data not shown). The experiment was performed using the doses of CC that naturally exerted antimicrobial activity (1, 0. 5, 0. 25, 0. 125, 0. 06 mg/mL) combined with different doses of Ery (from 2 mg/mL to 0. 06 mg/mL) against BAA-977. (B) The same experiment was done using the ATCC 25923 strain, lacking the resistance inducible gene. A statistical analysis (Two way ANOVA) was performed by the GraphPad Prism6 software and the p value < 0. 0001 (****) showed a significant difference between the treatment with 1 mg/mL of MOR/α-CD of the resistant strain in each combined well and the untreated bacteria (0 mg/mL).
In order to evaluate the antimicrobial activity of MOR/α-CD against the CC-resistant BAA-977 strain, we performed a similar combined assay, adding 1 mg/mL of MOR/α-CD on each well of the microplates with the Ery/CC combinations. As expected, in the presence of MOR/α-CD, no bacterial growth was observed (Figures  7  and S7 ). Moreover, no CFU was counted by seeding 10 µL from each antibiotic combined well, after 24 h of MOR/α-CD treatment (data not shown). The experiments were assessed in triplicate to obtain the mean and the standard deviation (SD). A statistical analysis (Two way ANOVA) was performed by the GraphPad Prism6 software and the p value < 0. 0001 (****) showed a significant difference between the treatment with 1 mg/mL of MOR/α-CD of the resistant strain in each combined well and the untreated bacteria (0 mg/mL).
5, 0. 25, 0. 125, 0. 06 mg/mL) and Ery (from 2 mg/mL to 0. 002 mg/mL). The MOR/α-CD inhibition activity was evaluated by measuring the OD reached after 24 h of treatment. The experiments were assessed in triplicate to obtain the mean and the standard deviation (SD). A statistical analysis (Two way ANOVA) was performed by the GraphPad Prism6 software and the p value < 0. 0001 (****) showed a significant difference between the treatment with 1 mg/mL of MOR/α-CD of the resistant strain in each combined well and the untreated bacteria (0 mg/mL).
FUNDiNg This work was supported by the  Centre National de la Recherche Scientifique (CNRS) , the  University of Lille-Sciences and Technologies , the  Hauts-de-France region , the  CPER "Photonics for Society , " the  Agence Nationale de la Recherche (ANR) , the  EU  (grant number  FLAG-ERA JTC 2015-Graphtivity ), and the  Marie Sklodowska-Curie action  (grant number  H2020-MSCA-RISE-2015  and grant number  PANG-690836 ).
Quantification of BC-like structures. BC formed in HepaRG cell cultures appear as characteristic refractive structures, corresponding to bright/white objects on phase-contrast photographs, as previously reported  21  , whereas cells appear as dense/black elements, containing nuclei recognizable by their round/delimited shape and differential density. After capturing images using an Axiovert 40 C phase contrast microscope (Carl Zeiss, Le Pecq, France) and adjusting brightness parameters to optimally distinguish between white and black densities, bright objects reflecting BC as well as round/delimited nuclei-related objects were segmented by adjusting the shape and area parameters to exclude non-corresponding objects, using a dedicated in-house macro-program based on ImageJ 1. 48 software  22.
Primers were specific ready-to-use KiCqStart ® gene primers (from Sigma Aldrich) or in-house custom primers, whose sequences are indicated in Supplementary Table  S1. Amplification curves of the PCR products were analyzed with the CFX Manager software (Bio-Rad), using the comparative cycle threshold method. Relative quantification of the steady-state target mRNA levels was calculated after normalization to the 18S rRNA level, used here as an internal control. Data were expressed as % of expression levels found in isolated human hepatocytes, arbitrarily set at 100%, or as fold changes relative to mRNA levels in control cells.
Introduction Postmarketing pharmacovigilance reports have raised concerns about non-bleeding adverse events associated with direct oral anticoagulants (DOACs), but only limited results are available from large claims databases. Objective The aim of this study was to assess the potential association between DOAC initiation and the onset of four types of non-bleeding adverse events by sequence symmetry analysis (SSA). Methods SSA was performed using nationwide data from the French National Healthcare databases (Regime General, 50 million beneficiaries) to assess a cohort of 386,081 DOAC new users for the first occurrence of four types of non-bleeding outcomes: renal, hepatic, skin outcomes identified by using hospitalization discharge diagnoses, and gastrointestinal outcomes by using medication.
Considering the important clinical role of oral anticoagulants (OACs), the declining use of VKAs, and the increasing prevalence of AF with ageing of the population, DOAC prescription is therefore expected to continue to increase over the next years  [8]. Continuous and careful monitoring of their safety profile is therefore needed. Large administrative databases provide a timely opportunity to perform signal detection and investigate adverse reactions. The relative safety of DOACs versus warfarin in nonvalvular AF patients  [9] [10] [11] , or versus heparin in the treatment and prevention of recurrence of venous thromboembolism  [12] [13] [14] , has been demonstrated in large-scale randomized clinical trials (RCTs), and has been confirmed globally by observational studies  [15, 16] ; however, both clinical trials and observational studies have mainly focussed on bleeding adverse events to date  [17].
Postmarketing data, including pharmacovigilance reports  [18]  and case reports, have raised concerns about other, albeit rarer, non-bleeding adverse events that could not be detected by RCTs due to intrinsic limitations, such as small sample size and short follow-up. Renal and hepatic adverse events are among the emerging DOAC safety issues  [17]. Rare but severe skin adverse events, such as vasculitis, have also been reported in dabigatran-treated patients  [19] [20] [21]. Furthermore, nonbleeding adverse events, including gastrointestinal symptoms such as dyspepsia, reported in dabigatran-treated patients  [22] [23] [24] , have been found to be one of the main reasons for permanent DOAC discontinuation. At the request of the pharmacovigilance department of the French Medicines Agency (ANSM), we investigated a possible association between DOAC (dabigatran, rivaroxaban, apixaban) use and these potential non-bleeding adverse events by using data from the French Healthcare databases  [25].
Data Sources French national health insurance (Assurance Maladie) covers the entire French population and is divided into several specific schemes according to beneficiaries' occupational sector, with the largest scheme being 'Regime general' (approximately 50 million beneficiaries). This study was conducted using data from the French health insurance system database (SNIIRAM, Systeme national d'information inter-regimes de l'assurance maladie) linked to the French hospital discharge database (PMSI, Programme de medicalisation des systemes d'information). The SNIIRAM database contains individualised, anonymous, and comprehensive data on health spending reimbursements. Demographic data include date of birth, sex, and vital status. Drugs are coded according to the Anatomical Therapeutic Chemical (ATC) classification and packaging of each product is identified by means of a national specific pack identifier code providing information on the name of the product, active ingredient and dose in each pill, number of pills, and route of administration, but not the prescribed dose.
The PMSI database provides detailed medical information on all hospitalizations in France. The medical indication for drug reimbursements and the results of medical procedures or laboratory tests are not available in these databases. However, medical diagnosis information is available from two independent sources encoded according to the International Classification of Diseases, 10th edition (ICD-10): (1) diagnoses corresponding to patient eligibility for 100% reimbursement of severe and costly long-term diseases (LTDs) and disability, such as AF, coronary heart disease, certain debilitating diseases (such as multiple sclerosis, inflammatory bowel disease or rheumatoid arthritis), HIV infection, cancer, etc. ; and (2) discharge diagnoses from hospitalization data. The SNIIRAM-PMSI databases also indicate medical procedures performed in the ambulatory setting or during a hospital stay.
Rationale SSA is a case-only design based on the rationale that if a medication causes an adverse event, this medication will be prescribed more often before than after occurrence of this event  [26, 27]. Asymmetry in the distribution of this outcome of interest before and after initiation of a tested medication is therefore used to assess the association between this medication and an outcome of interest. Outcomes can be identified either by medication prescription/reimbursement or hospitalization in healthcare databases. The advantages and pitfalls of this method have been recently described in detail  [32].
Data Analysis A separate SSA was performed for each definition of the outcome of interest. Patients were classified according to the temporal sequence (outcome ? DOAC or DOAC ? outcome) between the DOAC first reimbursement and the date of occurrence of the first outcome of interest during the time window considered. Patients who experienced the outcome of interest on the same day as the prescription of DOAC therapy were excluded from the analysis. Three time windows (on either side of the index date) were tested to search for the adverse event: 3 months (90 days), 6 months (180 days), and 12 months (360 days)  [27]. The asymmetry of sequences was measured by calculating a crude sequence ratio (cSR) as the ratio of the number of patients who initiated DOAC therapy before the outcome over the number of patients for whom the outcome occurred before the initiation of DOAC therapy.
Utilization of Sequence Symmetry Analysis on the French Healthcare Database for Monitoring Direct Oral Anticoagulant Adverse Events This analysis, based on reimbursement data for approximately 390,000 DOAC new users and 310,000 VKA new users in 2013-2015, showed an association between OAC therapies and the well-known and specific increased bleeding risk, as well as an association between these cardiovascular drugs and depression, in line with the initial paper describing this method  [26]. This suggests that this method can be used to monitor OAC adverse events in the French healthcare databases. However, if no association was observed between DOAC initiation and hospitalization for glaucoma, a negative control outcome, VKA therapy was associated with a tendency for reduction of hospitalization for glaucoma, and this association was significant when considering the 3-month time window (40% decreased risk of hospitalization for glaucoma).
Data on the risk of DILI with DOACs are mostly derived from postmarketing experience, including case reports/case series  [17, [47] [48] [49]  and a pharmacovigilance study  [50]. In a recent cohort study on the Mar-ketScan Commercial and Medicare databases, dabigatran was associated with the lowest risk of liver injury compared with rivaroxaban and apixaban new users  [51]. The results of this study are therefore consistent with the postmarketing literature  [50, 52] , which suggests that DOACs, especially rivaroxaban, can cause DILI. Overall, our results and the published literature advocate the monitoring of patients' liver function in the first months following DOAC initiation, especially in the presence of preexisting liver conditions or concomitant intake of hepatotoxic agents. DOAC therapy was associated with the initiation of drugs for functional gastrointestinal disorders when drugs for acid-related disorders were excluded from the outcome definition.
Strengths and Limitations In France, National Health Insurance covers the entire population and most people also subscribe to a private complementary health insurance plan. Selection bias related to the access of patients to more expensive DOAC therapy is therefore not an issue when using the French healthcare databases  [31, 61]. In addition to being based on large nationwide healthcare databases, a major strength of this study is that SSA is able to overcome some of the pitfalls that can threaten the validity of other observational designs. The SSA design inherently controls for timeconstant, patient-specific measured and unmeasured confounders, including sociodemographic characteristics, comorbidities, genetic and environmental factors, and healthcare-related behaviors  [32]. The aSRs were corrected for time trends in the occurrence of exposure and events.
For instance, for apixaban, the nSR estimates were smaller than 1, and this deviation grew with increasing length of the time window. In particular, this reflects the trend in apixaban prescribing because, in France, the indication of apixaban was extended to stroke prevention in January 2014, with a corresponding sharp increase in apixaban prescription during the period 2014 and 2015 of the inclusion period. However, for all DOACs, the nSR was close to 1 for most outcome definitions. The definitions of the investigated outcomes were not strictly validated in our databases. However, recent studies assessing the quality of PMSI data, including ICD-10 codes, to identify healthcare outcomes have shown these data to be reliable for epidemiological purposes  [62, 63]. Positive and negative control outcomes, and the use of the cohort of VKA new users as an active comparator, were used to guide interpretation of the results observed with DOACs in an attempt to identify results that could have been affected by bias, which is clearly a strength of this study.
Paired images were collected every 10-30 sec during 20 min and analyzed with Metafluor software. Basal fluorescent level is recorded for 100 sec, afterwards the coverslips is available for treatments. At the end of the experiment, ionomycine (5 µM) was used as positive control. Fluorescence changes were expressed as the ratio F340/F380 normalized to basal value 1. Moreover, for CF macrophages experiments, maximal level obtained by ionomycin were normalized to value 4 (mean maximal level in CF macrophages). Area under curve (AUC) is determined between signal and basal line (Y = 1) after 100 sec. At 100 sec, treatment was added and AUC was measured between 100 sec until 1250 sec. Average AUC from multiple independent coverslips were used to calculate the sample mean ± s. e. m obtained after analysis of at least 10 cells.
Finally, cells were washed three times and co-stained by 10 min-incubation in a blocking solution containing Hoechst to dye nuclei and WGA to dye membrane. After washing, coverslips were mounted with mounting solution. Fluorescent-labeled cells were captured using a confocal Leica SP8 microscope with the Leica LASAF software. When lipids rafts were analyzed, coverslips were stained using a blocking solution (PBS-1% FCS) containing cholera toxin subunit B (CTX-B) coupled to red-fluorescent Alexa Fluor 647 (Molecular Probes, Life Technologies) for 30 min at 4 °C before fixation. Fluorescence quantification and co-staining was examined using FIJI software. For fluorescence quantification, data were obtained after passed the images in a macro. The TRPV2 membrane versus cytosolic fluorescence intensity was obtained after particles identification using the control WGA (wheat germ agglutinin) fluorescence (Supplementary Fig.
The amplification curves of the PCR products were analyzed with the ABI Prism SDS software using the comparative cycle threshold (CT) method. Relative gene expression was calculated by comparing the number of thermal cycles that were necessary to generate threshold amounts of product (CT). The CT was calculated for each gene and for the housekeeping gene 18 S. For each cDNA sample, the 18 S CT was subtracted from the CT for each gene to yield the ΔCT, thus normalizing the initial amount of RNA used. The amount of mRNA was calculated as 2 -ΔΔCT , where the ΔΔCT is the difference between the ΔCT of the two cDNA samples to be compared. The data from CF samples are expressed relative to the mRNA level found in non-CF samples.
Data analysis Results were obtained using a scanned image of the bottom of the 96-well plate (CanoScan 4400 F, Canon France) at a resolution of 600 dots per inch (DPI), to monitor the color of each well. We used ImageJ V R software to record a 14,329-pixel circle which made it possible to quantify for each pixel of the selected area, the intensity of red, green and blue colors, and automatically give means and standard deviation values for each color  (Schindelin et al. 2015). For the purpose of our study we used the mean intensities for blue, as the differences between the positive (red well) and negative (white well) controls were the greatest for this color (see Figure  1 ). After that, the mean intensities obtained for blue were subtracted from 255, which is the maximum value measured by this software.
LC-(ESI)-HRMS/MS analysis We solubilized 75 mg of plant extract using an ultrasonic bath (FisherBrand, France) in an 80/20 ethanol/water solution. The solutions were then centrifuged to collect the supernatant which contained soluble metabolites. All extracts were profiled using a UHPLC-LTQ-Orbitrap XL instrument (Ultimate 3000, Thermo Fisher Scientific, Hemel Hempstead, UK). Mass detection was performed using an electrospray source in positive and negative ionization (ESI) modes at 15,000 resolving power. The mass scanning range was m/z 100-1500 Da. The capillary temperature was 300 C and ISpray voltage was fixed at 4. 2 (positive mode) and 3. 0 kV (negative mode). Mass measurement was externally calibrated before starting the experiment. Each full MS scan was followed by data dependent MS/MS on the four most intense peaks using stepped collision-induced dissociation (35% normalized collision energy, isolation width 2 Da).
Furthermore, we used in silico MS/MS fragmentation for compound annotation. Firstly, in-house databases were interrogated based on genera Zanthoxylum and Calotropis, then family databases were screened (Rutaceae and Apocynaceae, respectively). Finally, natural product databases focused on plants were selected: Universal Natural Products Database (UNPD), KNApSAck, PlantCyc, Dictionary of Natural Products (DNP, CRC press, v25:2) and CheBI. The results were presented as a list of compounds sorted according to the closeness of the match materialized by a score value which encompassed uncertainty on accurate mass, the isotopic pattern score and the experimental MS/MS fragmentation mirrored to in-silico matches. Only structures with a total score above 7. 5 and with a structure in agreement between databases were retained (Table  1 ).
Mass Error was also calculated for each compound using the finding formula on MS-FINDER. Identified peaks present in our in-house plant family databases are represented in blue, whereas identified peaks present in homemade plant genera databases are represented in green. assay does not work with patients who have just received a blood transfusion; these patients were excluded from the experiment. Treatment with hydroxyurea, which induces the production of Hb F, could also cause some variations in the microbead assay. Hydroxyurea is able to change the percentage of Hb S in RBC, leading to difficulties in obtaining sickled RBC with sodium metabisulfite. This bias could be moderated by using RBC from the same patient for each test. Moreover, deoxygenation of RBC modifies the color of the sample, giving a mauve color instead of a red color.