dataesr/scientific-paragraphs-categorization huggingface dataset:
Statistics Linear regression analyses were performed to compare insertions and deletions frequencies between the control condition (silent control variant) and the studied variant (testing variant). Mann-Whitney tests were conducted to assess the potential statistical significance of the differences between the functional scores of the different classifications of variants (benign, intermediate and deleterious). Thresholds delineating the intermediate classification were calculated following the evaluation of the standard deviation of the functional scores among the benign and deleterious variants. All statistical analyses were performed with GraphPad Prism analysis software.
All six of the clones sequenced had been edited, but only three also presented a frameshift (clones 5, 8, and 17). These three clones were pooled together to constitute the polyclonal LIG4 knock-out HAP1 cell line, to prevent side effects and putative off-target effects associated with clonal selection. In HAP1 cells, BRCA1 and BRCA2 are essential genes  [25, 26]. Genomic editing to create a functionally abnormal variant of these genes thus leads to cell death, facilitating the screening of edited cells. Moreover, edited cells with insertions or deletions instead of the variant of interest also die, due to the essential nature of the gene concerned. We checked that the absence of a variant following NGS sequencing was due to the functionality of the variant rather than a problem linked to genomic editing, by simultaneously performing a second transfection, with the same gRNA, but the insertion of a silent variant already classified as benign in databases, and therefore functionally normal, where possible (Fig.
1A ). Thus, for each variant, we designed one gRNA and two oligonucleotides (Fig. 1B, C ), one carrying the variant of interest, or testing variant, and the other carrying a silent control variant. We also added a second silent reference variant, also classified as benign where possible, located in the corresponding PAM motif or 3 nt upstream from the PAM motif. This second modification had two purposes. First, its insertion prevented from re-editing blocking gRNA association  [30]. This second variant was present in both sets of conditions and was therefore also used as reference for comparisons. Following NGS sequencing, variant frequencies were compared and functional score was calculated (Fig. 1B, C ). In our examples, the p. Pro1812Ala variant was classified as pathogenic in UMD and BRCA exchange databases and the p.
Tyr179Cys variant as benign in UMD, ClinVar, and BRCA exchange databases. When the p. Tyr179Cys variant was analyzed, the frequency of the testing variants was similar to that of the control variant (Fig. 1C ). On the contrary, for the p. Pro1812Ala variant, the frequency of the testing variant was considerably lower compared to the control variant (Fig. 1B ) due to the essentiality of BRCA1 gene. We then validated our method testing 10 variants of BRCA1 and BRCA2 already classified as benign or pathogenic in databases. We evaluated the indel frequency to estimate the editing efficiency in the "testing variant" and "control variant" conditions compared for each variant. Indeed, this indel frequency was identical for both conditions when analyzed for the 8 nt surrounding the PAM sequence (Fig.
Functional characterization of BRCA1/2 variants of unknown significance The fourth step of the guidelines, which is the application of evidence to individual variants, could then be performed. The strength of evidence was moderate for both PS3 (pathogenic) and BS3 (benign) categories with OddsPath > 4. 3 and < 0. 23 respectively. Thirteen variants of BRCA1 and BRCA2 were initially identified in our laboratory and considered as VUS based on an absence of annotation concerning their function. We then also studied other 10 variants of BRCA1 previously classified as VUS or not previously reported in the databases (ClinVar, BRCA exchange, UMD) (Table  1 ). References mentioning these variants and their potential functional impact are listed in Table  1  where available  [20, 25, [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52].
These 23 VUS and the 10 other variants we used for validating our system affected different domains of the proteins and were distributed along the entire length of these genes (Fig. 2A, B ). All reference silent variants, silent control variants, and their classifications in databases are listed in Additional file 1: Table  S3. For each variant, we checked the coverage and indel frequencies (Additional file 1: Fig. S2B  and S2C, R 2 = 0. 9328) for the testing variant and the control variant. All functional scores were then calculated from NGS frequencies (Fig. 2C  and Table  1 ), and the variants were classified according to their impact on cell survival and, consequently, their functional impact in our model. Most of the 23 VUS analyzed were functionally normal (about 65. 2%), but six were found to have an impact on edited cell survival (p.
Patients' responses to PARP inhibitor administration and comparison with the calculated functional scores The next step was to confront our functional score to clinical data and the responses to PARP inhibitors of patients presenting alterations of BRCA1/2. The study we conducted was retrospective; hence, only 6 of the patients concerned received a PARP inhibitor treatment thus far. The mean age of these patients was 60. 5 years and all of them suffered from high-grade serous ovarian cancer with different variants of BRCA1 and BRCA2. The characteristics of their tumors are summarized in Table  2. Their responses to PARP inhibitors ranged from 3 months before resistance to 9 months and continuing. We then compared these data to the functional scores we had calculated for their variants. Five had, indeed, been classified as functionally abnormal and one with intermediate functional score (Tables  1  and 2 ).
Extension of the experimental process to the functional evaluation of POLE variants We then extended our protocol to the characterization of VUS from other tumor suppressor genes that were also essential in our model. We chose to study variants of the POLE gene because of potential interest of their functional impact for determining access to immunotherapy. We therefore selected seven POLE variants from databases, which included two classified as benign and two classified as pathogenic (Fig. 3B ). The same protocol was followed to evaluate their functional scores, and the results obtained were compared with published findings (Fig. 3A, B ). The p. Leu424Val variant, which has been classified as pathogenic in databases, was surprisingly found to have a functional score of -0. 031 rather associated with a functionally normal variant according to our experimental protocol and the cut-off we used.
Discussion Our evaluation of the functional scores of 33 variants of BRCA1/2, including 23 unreported or of uncertain significance, allows the classification of 20 of them as functionally normal, six as functionally abnormal (p. Gln210=, p. Leu291X, p. Glu1586X, p. Ala1752Thr, p. Ala1752Pro, p. Gly1770Val) and two as intermediate (p. Leu1080=  and c. 5194-2A>G) (Table  1 ). These results were consistent with databases (Clin-Var, BRCA exchange, UMD), with previous functional studies (Table  1 ), where available, and with the published saturation genome editing study of the RING and BRCT domains of BRCA1 (p. Ile31Asn, p. Ala1752Thr, p. Ala1752Pro, p. Gly1770Val and p. Pro1812Ala)  [25]. Indeed, we evaluated, as expected following their publication, variant p. Ile31Asn as functionally normal whereas the four others were functionally abnormal.
NGS sequencing results and functional scores for this mutation are associated. C Same thing with the p. Tyr179Cys variant, benign variant of the BRCA1 gene. D Insertion and deletion frequencies determined following the NGS sequencing of BRCA1/2 variants of interest, compared with those for the corresponding silent control. These means include the 8 nt surrounding the PAM sequence. E Analysis of NGS sequencing coverage with the following formula: Mutation of interest (Mutation+Reference control)/Silent control (Mutation+Reference control). The results for benign BRCA1/2 variants are compared with those obtained for pathogenic mutations (Mann-Whitney tests, p = 0. 8016) p. Gln210 = variant of BRCA1, which was also found functionally abnormal. However, the p. Gln210 = was described in databases as a variant possibly creating or strengthen a splice site of BRCA1, which is compatible with our results suggesting it is pathogenic.
One of the two intermediate variants, c. 5194-2A>G, has already Fig. 2  Functional characterization of BRCA1/2 variants of unknown significance. A Schematic representation of the introns and exons of the BRCA1 gene, showing the location of the variants selected for this analysis (http://wormw eb. org/exonintron). B Similar representation for the BRCA2 gene. C Functional score evaluation after CRISPR-Cas9 editing and NGS sequencing of BRCA1/2 variants of unknown significance and comparison with the functional scores obtained for functionally normal and abnormal mutations (Mann-Whitney tests, p = 0. 0079) Table  1  Functional scores for the evaluated BRCA1/2 variants and comparison with database annotations Variants classified as benign in databases (+) or functionally normal in our assay (Normal) are shown in green.
Variants classified as pathogenic in databases (-) or functionally abnormal in our assay (Abnormal) are shown in red. Variants with conflicting interpretation are shown in orange (+/-), unreported mutations are shown in yellow (?). Variants with an intermediate score are shown in blue. Functional scores of variants already characterized by Findlay et al. [25]  are included where available. Variants which were characterized in our laboratory are marked (X), the others were selected in the databases been reported to affect splicing and may also be functionally abnormal. Its classification as functionally intermediate might reflect the existence of a large number of BRCA1 splicing variants with a not totally deficient splicing site also called leaky splicing effect  [54] [55] [56]. The sensitivity of the patient 4, who relapsed after only 3 months on PARP inhibitor, was consistent with these hypotheses (Fig.
We also analyzed three POLE VUS (Fig. 3A, B ). Prediction algorithms in databases were conflicted concerning the consequences of p. Arg1826Trp variant for protein function: SIFT (deleterious) and PolyPhen-2 (probably damaging). The functional score of this variant was consistent with it being a functionally abnormal variant. Moreover, two VUS were classified as intermediate (p. Asn363Asp and p. Asp301Gly), but the cut-off used here should be refined by the evaluation of more wellknown variants. Both these intermediate variants are located in the exonuclease domain and may therefore affect the proofreading activity of the protein, leading to a hypermutation phenotype  [64]. The amino acids Asp301 and Asn363 are highly conserved between species. Both are predicted to affect function, but no functional tests have been performed.
Baseline tumor characteristics of patients who were treated with PARP inhibitors Extension of the experimental process to the functional evaluation of POLE variants. A Functional score evaluation following the CRISPR-Cas9 editing and NGS sequencing of POLE variants of unknown significance, and comparison with the scores obtained for two benign and two pathogenic mutations. B Table comparing the calculated functional scores of POLE variants and ClinVar and OncoKb databases classifications: mutations classified as benign in databases or functionally normal in our assay are shown in green, unreported mutations are shown in yellow, mutations classified as pathogenic in databases or functionally abnormal in our assay are shown in red and intermediate mutations are shown in blue.
Comparative Gene Expression Analysis With E. coli MGH1655 Exposed to Individual Antibiotics A comparison of our transcriptional data with the global transcriptome profiling of E. coli MGH1665 K12 strain exposed to 37 antibiotics was carried out. Only those genes that gave statistically significant differential expression patterns were selected from both datasets. Our gene expression dataset was compared to each of the 37 E. coli transcriptional profiles and genes that had similar expression patterns (both up-and downregulation) were counted.
Validation of RNA-Seq Data by Quantitative RT-PCR Analysis RNA isolated from the samples were converted to cDNA by using high-capacity RNA to cDNA kit (Thermo Fisher Scientific, Ireland). Primers based on the selected genes of interest were designed with 6-FAM/ZEN/IBFQ double-quenched probes and synthesized commercially by Integrated DNA Technologies (IDT, Belgium) (Supplementary Table  2 ). The cDNA was used as a template and analysis was done by the addition of PrimeTime Gene Expression Master Mix. qRT-PCR was performed in an Eppendorf Mastercycler realplex ep gradient S (Eppendorf, United Kingdom). This analysis was carried out in two biological replicates each along with three technical replicates. The foldchange in the expression of the genes of interest was determined by the method of  Livak and Schmittgen (2001) , i.
Statistical Analysis The results for the biochemical assay were analyzed using unpaired Student t-test as appropriate for the dataset. The qRT-PCR measurements data were statistically analyzed using Prism software (v. 8. 0 GraphPad Software) following the twoway analysis of variance. Boneferroni method was used to analyze the multiple comparisons. The symbol "ns" used in the graphs corresponds to statistically non-significant with p > 0. 05. The asterisk symbols in the graphs correspond to * p ≤ 0. 05, * * p ≤ 0. 01, and * * * p ≤ 0. 001. All the data points represent the mean of two independent measurements. The uncertainties were represented as standard deviations. In RNAseq results, NSE represents non-significant expression.
, 2012; Anes et al. , 2019). The VOOM function in the limma package  (Ritchie et al. , 2015)    was used to identify differentially regulated genes. We obtained statistically significant data p ≤ 0. 05 for a total of 3,760 and 3,945 genes following 5 and 30 min exposure, respectively (Figure  4A  and Supplementary Dataset WS1). Statistically significant genes with a log 2 fold change expression of ≥1. 0 and ≤-1. 0 (exposed vs. unexposed cells) were considered to be up-and down-regulated, respectively  (Dash et al. , 2018; Anes et al. , 2019). For the post-5 min exposure, 1,008 genes were found to be upregulated and 1,063 genes were downregulated. At 30 min post exposure, 1,205 genes were found to be upregulated and 1,246 genes were downregulated (Figure  4B ). Among all the upregulated genes at the 5-and 30-min, 705 genes were found to be commonly upregulated, whereas 303 and 500 genes were uniquely upregulated at 5 and 30 min, respectively.
For downregulated genes, 810 genes were found to be commonly downregulated at both time points, whereas 253 and 436 genes were specifically downregulated at 5 and 30 min (Figure  4C ). We selected representative genes related to the bacterial defense system (soxS), transcriptional regulator (ramA), outer membrane porin proteins (ompC), and virulence (rfaH) to validate our transcriptomic data using qRT-PCR. Gene expression analysis reflected a similar pattern of results when compared to RNA-seq data (Supplementary Figure  4 ).
, 2015). In L-Ag NP exposed K. pneumoniae MGH78578, soxS was highly upregulated (300and 88-fold in 5 and 30 min) and in contrast oxyR was moderately upregulated (approximately 5-fold in both time points). This suggested that the oxidative stress regulon was active throughout the 30 min of L-Ag NP exposure time, particularly for the soxS regulon. To confirm this, we compared our RNA-seq dataset to the K. pneumoniae MGH78578 oxidative soxS regulon generated earlier  (Anes et al. , 2020). We found that of the 254 genes belonging to the oxidative soxS regulon, 129 (51%) had similar expression patterns in both datasets, confirming that the soxS regulon is activated in L-Ag NP exposed K. pneumoniae (Supplementary Dataset A typical example of soxS induction can be visualized in the expression of fpr (ferredoxin-NADP + reductase), which was induced at 22-and 9-fold at 5 and 30 min.
, 2020). This global transcriptional profile of E. coli exposed to 37 antibiotics generated a list of 447 genes whose signature expression pattern characterized the mechanism of action associated with each antibiotic, leading the authors to hypothesize that the antibacterial action mechanism of any unknown/uncharacterized compounds can be deduced by comparing the transcriptional profile of a compound of interest with that of these 447 E. coli MG1665 K12 genes. We compared our exposed K. pneumoniae MGH78578 transcriptional data set with each individual dataset obtained from E. coli global transcriptional profile. Our comparative analysis showed that the highest number of similarly expressed gene pairs were obtained from the triclosan exposure dataset, suggesting that exposure to L-Ag NPs induced a triclosan-like exposure response in K.
pneumoniae MGH78578. Some 76% of the gene pairs (73/96) at 5 min L-Ag NP exposure (Supplementary Datasets WS3, WS4) and 74% (87/117) at 30 min exposure (Supplementary Datasets WS5, WS6) had similar expression patterns compared with the E. coli triclosan dataset. Triclosan is a broad-spectrum antimicrobial compound that acts by inhibiting FabI (a NADH-dependent enoylacyl carrier), a protein belonging to the type II fatty acid biosynthesis, part of a well-conserved pathway that is essential for bacterial survival. Inhibition of fatty acid biosynthesis compromises the bacterial cell membrane. During exposure to L-Ag NPs, some K. pneumoniae MGH78578 genes associated with type II fatty acid biosynthesis including fabA/H/D/G/F/B were significantly downregulated particularly during the adaptive response at 30 min.
coli genes that respond to triclosan exposure  (Yasir et al. , 2020). In comparison with these data, we identified genes that were both enriched in the E. coli TraDIS-Xpress dataset and that were found to be differentially expressed in our K. pneumoniae MGH78578 dataset. Common genes like purL, purH, waeL, wzx were downregulated at either one or both time points while metB was upregulated. Similarly, K. pneumoniae MGH78578 genes like trkA, pcnB, infB, and ubiB/F were downregulated at least in one or both time points, while lonH was upregulated (Supplementary Dataset WS1). TraDIS-Xpress selected rbs in their triclosan exposure screen. In E. coli, RbsABC forms the ABC-type high-affinity D-ribose transporter, while RbsD/K phosphorylates D-ribose to D-ribose 5-phosphate  (Shimada et al. , 2013).
The violent paroxysm of Mount Etna on 23 November 2013 was considered as a test case. Satellite retrievals by the Ozone Mapping and Profiler Suite (OMPS) sensor and the Visible Infrared Imaging Radiometer Suite (VIIRS) sensor both onboard the Suomi National Polar-orbiting Partnership (NPP) spacecraft, and the Meteosat Second Generation (MSG) Spinning Enhanced Visible and Infrared Imager (SEVIRI) images data were used to validate the outputs of the different setup configurations. This work represents the preliminary step towards the forecast simulations of meteorology and ash dispersion of Mount Etna. This would be achievable in near real-time, assimilating into WRF-Chem model the input data from the VOLDORAD-2B monitoring system, aside from the end-member eruption scenarios used nowadays (one-weak long-lived emission similar to 2002-2003 and one strong short-lived eruption similar to 5 January 1990).
Remote-Sensing Data 2. 2. 1. VOLDORAD-2B Doppler Radar System Etna tephra emissions have been monitored since 2009 using a unique volcano Doppler radar, VOLDORAD-2B, operated jointly by OPGC and INGV-OE from the Montagnola station, 3 km south of the New SE Crater. During the 2011-2015 period, the radar has successfully recorded data from about 50 paroxysms of tephra plumes fed by lava fountains, available from an open-access database of the Observatoire de Physique du Globe de Clermont-Ferrand (http://voldorad. opgc. fr/bddtr. php (accessed on 17 May 2021);  [37] ). Using a fixed-pointing antenna beam, this 23. 5 cm-wavelength pulsed radar sounds 13 atmospheric volumes, 120 m-deep, right above the summit craters. The echo power and radial velocities (level 1 data) are computed from the raw Doppler spectra recorded at intervals of 0.
Suomi NPP (OMPS/VIIRS) Data The Ozone Mapping and Profiler Suite (OMPS) is onboard the joint National Aeronautics and Space Administration/National Oceanic and Atmospheric Administration (NASA/NOAA) Suomi National Polar-orbiting Partnership (Suomi NPP) satellite, launched in October 2011. In particular, the OMPS-NPP L2 NM Aerosol Index swath orbital product provides aerosol index values from the OMPS Nadir-Mapper (NM) instrument. This is actually the official NASA aerosol index product, which replaces the aerosol index found in the OMPS-NPP L2 NM Total Ozone product. The aerosol index is derived from normalized radiances using two wavelength pairs at 340 and 378. 5 nm in the ultra-violet spectrum. The dataset is accessible through the NASA Earthdata Search web portal (https://search. earthdata.
nasa. gov/search (accessed on 5 May 2021). The Visible Infrared Imaging Radiometer Suite (VIIRS) is onboard the Suomi NPP and the Joint Polar Satellite System (JPSS) spacecrafts. The VIIRS instrument has 22-spectral bands, covering the spectrum between 0. 412 and 12. 01 µm, including 16 moderate resolution bands (M-bands) with a spatial resolution of 750 m at nadir, and 5 imaging resolution bands (I-bands) with a 375 m resolution at nadir. The brightness temperature was downloaded from the NOAA-CLASS web portal at https://www. avl. class. noaa. gov/saa/ products/welcome (accessed on 6 July 2021) by selecting the VIIRS data records from the Suomi NPP products. The brightness temperature of the thermal infrared channels has been widely used in dust storm monitoring  [40]  but also to detect volcanic plumes  [41].
It uses on-site automated ingestion of MSG-SEVIRI geostationary data for direct dissemination on a full web-GIS interface of ready-to-use products characterizing volcanic ash, sulphur dioxide, and lava flow emissions from spectral band combinations. Satellite products are delivered in the form of geo-referenced images (geotiff) tiled on a background map and time series (csv) through an open-access database. Although several elaborated ash products (ash plume altitude, mass, contour) have been made available recently from combinations of thermal infrared channel radiance data, for the 23 November 2013 ash and SO 2 plume, we were able to use only ash RGB composite images from the HOTVOLC archive data combining IR8. 7, IR10. 8, and IR12. 0 channels.
Multi-Satellite Volcanic Sulfur Dioxide Long-Term Global Database The input parameters for SO 2 were obtained from the Multi-Satellite Volcanic Sulfur Dioxide L4 Long-Term Global Database in its Version 4 (MSVOLSO2L4), which is provided as an ASCII file and available for download at NASA Goddard Earth Sciences Data and Information Services Center (GES DISC) (10. 5067/MEASURES/SO2/DATA405). Version 4  [10]  is the current version of the dataset and older versions are no longer available. The data represent the best estimates of the volcanic and anthropogenic contribution to global atmospheric SO 2 concentrations. It consists of a long-term database of volcanic SO 2 emission derived from ultraviolet satellite measurements from 31 October 1978 to present, including data from Total Ozone Mapping Spectrometer (TOMS), Ozone Monitoring Instrument (OMI), Ozone Mapping and Profiler Suite (OMPS), and TROPOspheric Monitoring Instrument (TROPOMI) sensors (https://disc.