dataesr/scientific-paragraphs-categorization huggingface dataset:
A conserved WxxxE motif in BtpA organizes functional surface loops BtpA and BtpB have both been shown to have immune modulatory functions in the host cell through their TIR domains by directly interfering with TLR signalling  [17] , supported by recent structural information  [25, 28, 29]. btpA and btpB are located on different genomic islands  [33]. We analysed 407 genomes in the PATRIC database (http://patricbrc. vbi. vt. edu/portal/portal/patric/Home, January 2014). Whereas the GI carrying btpB is found in the genomes of all the classical Brucella strains, the GI containing btpA shows variable distribution (Additional file 1: Figure  S1 ). The btpA and btpB genes show 11% amino acid identity, and are highly conserved throughout the genus. In addition to the well-studied TIR domain, we found that BtpA and BtpB contain a WxxxE motif (Figure  1 ) (except in all B.
Similar to observations reported by Radhakrishnan and colleagues, BtpA could prevent the microtubule destabilizing effect of nocodazole (Figure  3C, D ). Next, we analysed the subcellular localisation of different GFP-BtpA WxxxE variants and their effect on microtubule destabilisation by nocodazole. Using site-directed mutagenesis with the GFP-BtpA expression plasmid as a template, the substitutions W213A, W213F, W213S, E217D, E217A, as well as I226S, and G183A were introduced. Despite very low amino acid sequence homology between BtpA and BtpB, and members of the WxxxE GEF family, we identified a conserved isoleucine residue, 9 amino acids downstream of the WxxxE motif and included a variant in our study. Subcellular localisation of the GFP-fusion proteins was confirmed using BtpA antibodies (data not shown).
Plasmids and site-directed mutagenesis A 828 bp version of the btpA gene was cloned from B. melitensis 16M genomic DNA (BMEI1674, Genbank accession NP_540591 is the shorter annotated version) using PCR (primers BglII L1674-F 5'-GAAGATCTTATGA GTTCGTACTCTTCTAATATTG-3' , and PstIL1674-R 5'-AACTGCAGTCAGATAAGGGAATGCAGTTC-3') and cloned in frame with the GFP coding sequence in eukaryotic expression plasmid pEGFP (Clontech) using standard protocols, resulting in plasmid pIN271. A 978 version of the btpB gene was cloned from B. suis genomic DNA (BR0735, Genbank accession number AAN29664 is the shorter annotated version) using PCR (primers BglII 735-F 5'-GAAGATCTTATG ACATCTAGTCGCGACACG-3' , and PstI 735-R 5'-AACTGCAGCTAGGTGATGAGGGCGACG-3') and cloned in frame with the GFP coding sequence in pEGFP resulting in plasmid pIN292.
Immuno fluorescence microscopy was performed using a LEICA DM/IRB microscope using filter sets L5 (band pass (BP) 480/40; Beam splitter (BS) 505; emission BP527/30) and N2. 1 (515-560; BS 580; emission long pass (LP) 590), respectively. For imaging we used a Coolsnap fx (Roper Scientifique) and MetaVue software, and images were further processed using Adobe Photoshop. Confocal analysis was performed at the RIO imaging platform in Montpellier, with a Biorad MRC1024 confocal microscope.
Acknowledgements The authors thank  Marty Roop  for BtpA antibodies, and  Philippe Fort  and  Géraldine Pawlak  for helpful discussions. We thank  Montpellier RIO  imaging for confocal microscopy. U1047 received funding from  INSERM ,  Université Montpellier  1, the  Region Languedoc-Roussillon , the  Agence Nationale de Recherche (ANR-MIME ;  T4SS ,  ANR-MIE; BruCell). Work in the LT group was funded by the  ATIP-Avenir program  from the  CNRS and Ligue Contre le Cancer.
If such data indicate a lower possibility of carcinogenic or in vivo mutagenic potential, it may indicate that in vivo testing can be avoided or minimised. A workshop to address this issue was therefore hosted and sponsored by EURL ECVAM, in Ispra, Italy from 23 to 25 January 2013. Fifteen genotoxicity experts from academia, government and industry were invited to participate. Data from public databases, regulatory agencies, and company in-house archives were reviewed. The primary effort was to look at the patterns of results in the in vitro mammalian cell genotoxicity assays for: • compounds positive in the Ames test but NEGATIVE in carcinogenicity studies and, • compounds positive in the Ames test but NEGATIVE in in vivo genotoxicity assays. For comparison, the participants also looked at data from in vitro mammalian cell assays for: • compounds positive in the Ames test and POSITIVE in carcinogenicity studies and, • compounds positive in the Ames test and POSITIVE in in vivo genotoxicity assays.
Summaries of presented material Relevant data and analyses from the presentations given by the various participants that are pertinent to the objectives of the workshop, are summarised below. Initially, the different sources of data were reviewed intact, i. e. , knowing that there was likely to be overlap of chemicals among the various data sets, for example by using data from the same source (e. g. , the National Toxicology Program [NTP] database; International Agency for Research on Cancer [IARC] publications; Chemical Carcinogenesis Research Information System [CCRIS] database) rather than independent study data from different sources. It was also clear that some chemicals that appeared in more than one database were associated with different response patterns in the same tests. Some participants also analysed their data after omitting chemicals that were known to overlap with other databases.
Interestingly, while all analyses reviewed in vitro mammalian cell assay data obtained with Amespositive compounds, some of the analyses considered mostly data from compounds found positive in carcinogenicity studies or in vivo genotoxicity assays, while others focused on the analysis of data obtained with non-carcinogenic compounds. Each presenter used his or her own approach to the analysis of their databases. 2. 1. Review of CGX and in vivo genotoxin databases D. Kirkland presented a review of the results on in vitro mammalian cell assays available in the Carcinogenicity and Genotoxicity eXperience (CGX) database of rodent carcinogens  [9] , where 318 rodent carcinogens were found that had positive results in the Ames test. More than one third of these Ames-positive rodent carcinogens had been tested in 2 mammalian cell tests with different endpoints (gene mutation and either chromosomal aberrations or micronuclei).
Moreover, from the database of in vivo genotoxins published by Kirkland et al. [10] , 202 in vivo genotoxins were found that gave positive results in the Ames test. Almost 75% of these had been tested in 2 mammalian cell tests with different endpoints. The patterns of results in the in vitro mammalian cell genotoxicity assays were analysed in both databases. Ames-positive chemicals for which there were data from only a single in vitro mammalian cell test were not included in the analyses presented here.
Analysis of in vivo genotoxins Of 202 Ames-positive in vivo genotoxins obtained from another published database  [10] , 36 were tested for both gene mutation (Tk +/-or Hprt) and MNvit, and 34 were positive in at least one of the two in vitro mammalian cell tests. The 2 chemicals that did not give a clear positive result either for gene mutation or micronucleus induction in mammalian cells were 4-acetylaminofluorene and dimethoate. 4-Acetylaminofluorene was reported equivocal in the MLA  [17] , but unfortunately no details were given, and the original data have not been found. It was reported negative in the MNvit  [17] [18] [19] , but the studies were performed in rat hepatocytes, which is a non-standard method, and negative in the CAvit in normal and genetically engineered V79 cells. It was clearly positive in Ames strain TA98 with S9 in both preincubation and plate test protocols  [20].
Analysis of non-carcinogens In the CGX database  [9] , 170 of 183 non-carcinogens had Ames test results, and 130 of these were negative in the Ames test. Of the 40 non-carcinogens that were positive in the Ames test, 22 had been tested in mammalian cell tests covering 2 different endpoints, and 18 had given unexpected positive results in at least one of the two mammalian cell tests; 4 were negative for both endpoints. The 4 non-carcinogens found positive in the Ames test and negative in the in vitro mammalian cell assays were: C. I. Food Red 3 is an azo dye, and requires reductive metabolism to exert its genotoxic effects. For the other 3 substances, the mammalian cell studies do not meet current requirements. There are therefore as many uncertainties regarding the reliability of the negative mammalian cell results with the non-carcinogens as there are with the carcinogens and in vivo genotoxins.
These frequencies were: • 9 out of 135 (7%) Ames-positive rodent carcinogens • 6 out of 87 (7%) Ames-positive in vivo genotoxins • 4 out of 22 (18%) Ames-positive non-carcinogens. Based on the above analysis it was concluded that if a compound is positive in the Ames test, but gives negative results for 2 mammalian cell tests covering different endpoints, it is more likely to be a non-carcinogen than either a rodent carcinogen or an in vivo genotoxin (18% vs 7%), and that this is worth further investigation with a broader database of chemicals.
Review of NTP database The NTP database was assembled by E. Zeiger from NTPcontracted test results between 1979 and 2013. The advantage of this database is that the in vitro mammalian cell assays were performed by standard protocols in only 5 laboratories, with little variation over the years (with the exception of S9 concentrations), and the data were evaluated by standardised criteria. Also, all of the test data are publicly available through the web at http://tools. niehs. nih. gov/ntp tox/index. cfm?fuseaction= ntpsearch. searchhome&showMessage=true. The following analyses were performed considering only the clear positive and negative results. Chemicals that had equivocal results in the genetic toxicity assays or in the rodent cancer assay were not included in the analyses, although their presence in the database is noted.
Only 2 chemicals (calcium cyanamide and dioxathion), were negative in both mammalian cell tests. Moreover, there were MNviv test data for 8 of these noncarcinogens; 5 (2-chloroethanol, 2-chloromethylpyridine HCl, 2,4dimethoxyaniline HCl, glutaraldehyde and 8-hydroxyquinoline) were negative in MNviv and 2 (methyl methacrylate and 4-nitroo-pheylenediamine) were judged equivocal. Therefore 88% (15/17) of the Ames-positive non-carcinogens found in the NTP database were also positive in at least one in vitro mammalian cell assay, and no non-carcinogens were negative in both in vitro assays. The NTP database contains 9 phenylenediamines and Nsubstituted phenylenediamines that were tested for rodent carcinogenicity and in the CAvit and/or MLA tests. Four (2chloro-p-phenylenediamine sulfate, 4-nitro-o-phenylenediamine, HC Blue 2 and p-phenylenediamine HCl) were non-carcinogens and two of the three were positive in both CAvit and MLA.
The 3 carcinogens (2,6-dichloro-p-phenylenediamine, HC Blue 1 and 2nitro-p-phenylenediamine) that were tested in CAvit and MLA were positive in both tests. The contrasting results for the carcinogen HC Blue 1, which was positive in both CAvit and MLA, and the structurally analogous non-carcinogen HC Blue 2, which was negative in both CAvit and MLA, are notable and interesting. As was noted previously  [6] , a large proportion of the Amespositive non-carcinogens in the NTP database are benzenamines or N-substituted benzenamines. Of the 17 non-carcinogens tested in both in vitro mammalian cell tests, 9 (41%) are in this structural class. With the exception of 4-nitro-o-phenylenediamine and HC Blue 2, the chemicals in this class of non-carcinogens were positive in all in vitro mammalian cell tests.
Of these 36 chemicals, 25 (including CI Basic Red 9 HCl) were negative or equivocal in MNviv. When the MNviv results were examined to determine if they could distinguish the Ames-positive carcinogens from the noncarcinogens, 31/33 (94%) carcinogens were positive; a negative response in MNviv was not predictive for carcinogenicity, i. e. , 16% (8/51) were non-carcinogenic. These results show that the responses in the in vitro mammalian cell tests, or the combination of in vitro and MNviv, cannot be used to distinguish Ames-positive carcinogens from Ames-positive noncarcinogens. It is also clear from this database that the use of MNviv provides no added value to the predictivity for cancer of the in vitro mammalian cell results.
Review of Japanese CSCL and ISHL databases T. Morita reported on data from the Ames test and CAvit obtained from the Japanese Chemical Substances Control Law (CSCL) and the Industrial Safety and Health Law (ISHL) databases. The CSCL database (its other name is Japan Existing Chemical Data Base)  [32]  includes 277 chemicals with results from the Ames and in vitro CA test with CHL cells (as of January 2012). The ISHL database consists of 5 data books, "Mutagenicity Test Data of Existing Chemical Substances", i. e. , Data book, 1996  [33] ; Suppl. 1997  [34] ; Suppl. 2, 2000  [35] ; Suppl. 3, 2005  [36] ; and Suppl. 4, 2008  [37]. The ISHL database includes 412 chemicals with results from Ames test and CAvit with CHL cells. Both tests in the two databases were conducted in accordance with Good Laboratory Practices (GLP) and Japanese and/or OECD Test Guidelines.
For further genotoxicity data searches, the following documents or databases were used: OECD Screening information data set (OECD SIDS)  [38] , EU risk assessment reports (EU RAR) [39], monographs from IARC  [40] , NTP database [25], and a publication from  Morita et al. (1997)   [41]. For carcinogenicity data searches, the following databases were used; IARC  [42] , Carcinogenicity potency database (CPDB)  [43] , EU Classification, Labelling and Packaging (EU CLP) list  [44] , "Maximale Arbeitsplatz-Konzentration" (MAK) list  [45] , NTP database [25], and Japanese Ministry of Health and Labour and Welfare (MHLW) database  [46]. The results of the analyses were as follows.
Total database including overlapping chemicals Among 689 chemicals from the CSCL and ISHL databases, a total of 64 chemicals were found which were Ames-positive and had results from CAvit. About half  (29)  of them also had data from another mammalian cell test(s) (MLA or Hprt mutation). Of these 64 chemicals, 49 had been tested for carcinogenicity, and 15 had been tested for in vivo genotoxicity but not tested for carcinogenicity. Of the 49 Ames-positive chemicals tested for carcinogenicity there were: • 23 carcinogens that had data in 2 mammalian cell tests (CAvit or MNvit plus Tk +/-or Hprt gene mutation); • 15 carcinogens that had data in only 1 mammalian cell test (CAvit); • 5 non-carcinogens that had data in 2 mammalian cell tests (CAvit or MNvit plus Tk +/-or Hprt gene mutation); • 6 non-carcinogens that had data in only 1 mammalian cell test (CAvit).
Of the 15 Ames-positive chemicals with in vivo genotoxicity data, but not tested for carcinogenicity, there were: The results for these 64 chemicals are summarised in Table  3. It can be seen that almost all (97%, 37/38) Ames-positive carcinogens were positive in at least 1 mammalian cell test. The database of chemicals tested for in vivo genotoxicity was much smaller, but nonetheless 4/5 (80%) Ames-positive in vivo genotoxic chemicals were also positive in at least 1 mammalian cell test. • 1 chemical.
Database with some overlapping chemicals removed T. Morita then excluded the 15 chemicals that overlapped with the CGX and in vivo genotoxin databases discussed above (see Section 2. 1). This left 49 chemicals which were Ames-positive and had data from in vitro mammalian cell tests. The 15 chemicals that overlapped had all been tested for carcinogenicity, and so this left 34 that had been tested for carcinogenicity, together with the 15 (discussed above) that had been tested for in vivo genotoxicity but not for carcinogenicity. Of the remaining 34 Ames-positive chemicals tested for carcinogenicity there were: • 8 carcinogens that had data in 2 mammalian cell tests (CAvit or MNvit plus Tk +/-or Hprt gene mutation); • 15 carcinogens that had data in only 1 mammalian cell test (CAvit); • 5 non-carcinogens that had data in 2 mammalian cell tests (CAvit or MNvit plus Tk +/-or Hprt gene mutation); • 6 non-carcinogens that had data in only 1 mammalian cell test (CAvit).
Review of European Pesticide Peer Review database J. M. Parra Morte presented an analysis of data collected from 186 pesticide peer-reviewed active substances at the European Union level  [47, 48]. Only 11 were positive in the Ames test. Data from Mechanistic data were available for some pesticide active substances in order to demonstrate a non-genotoxic mode of action for carcinogenicity. However, these data were not collected and were not taken into account in the analysis. Results were categorised as positive, negative or equivocal on a weight of evidence approach since it might be that more than one study was available for some endpoints. Results are detailed in Table  4. The 7 out of 10 Ames-positive substances that were found positive in at least one mammalian cell test were either rodent carcinogens or genotoxic in vivo.
Analysis of data from SCCS V. Rogiers presented data that had been collected at the Vrije Universiteit Brussel (VUB) by herself, Tatyana Y. Doktorova and Gamze Ates, and has been published  [49]. For this survey, the VUB's Cosmetics ingredients DataBase (CosDB) was used, which contains toxicological information on cosmetic ingredients that are present in the Annexes of the Cosmetic Directive 76/768/EEC (preservatives, UV-filters, colorants and specially studied substances with a potential risk for human health such as hair dyes). The data was extracted from the opinions generated in the period between 2000 and 2012 by the Scientific Committee on Cosmetic Products and non-food products (SCC(NF)P) and Scientific Committee on Consumer Safety (SCCS)  [50]. For the purpose of the workshop, only the genotoxicity test results were evaluated, and in particular the substances showing a positive Ames test were selected.
Review of ECHA database At the time of the workshop, data on industrial chemicals submitted to the European Chemicals Agency (ECHA) under the REACH legislation were not available. However, lists of chemicals for which carcinogenicity in vivo, and in vitro genotoxicity data were retrieved from the ECHA's registration database by P. Karamertzanis and F. Le Curieux, and were subsequently analysed by Ph. Vanparys on behalf of EURL ECVAM. The dossiers considered for the current evaluation were retrieved from ECHA's registration database (http://echa. europa. eu/information-on-chemicals/registered-substances; last accessed 22 July 2014) by applying a set of filters. The first filter that was applied was to select lead and individual dossiers that fulfilled the information requirements of REACH Annex IX or X and were submitted prior to the 2010 registration deadline.
). The algorithm did not attempt to recognise weight of evidence in Ames, i. e. one reliable positive result was sufficient to select the dossier, regardless of the outcome of other Ames studies. At this stage, study results based on read-across were also included. This approach led to approximately 400 dossiers with evidence of positive Ames results that were then used to contrast the positive Ames outcome with in vivo mutagenicity and carcinogenicity data in the dossier. Because identifying cancer evidence in carcinogenicity studies is not straightforward, the algorithm used the carcinogenicity classification. In some cases a harmonised classification for carcinogenicity was available, (i. e. Globally Harmonized System of Classification and Labeling of Chemicals (GHS) categories C1A, C1B or C2), in others the substances had been self-classified by the registrants  [51].
List 3 contained 280 dossiers related to 266 unique substances. • Ames positive chemicals that were positive for in vivo genotoxicity (list 4): at least one reliable in vivo genotoxicity study, and at least one positive result in a reliable in vivo genotoxicity, or the availability of a harmonised or a self-classification as germ cell mutagen M1A, M1B or M2  [51]. List 4 contained 240 dossiers related to 226 unique substances. There were dossiers that could be found in more than one of these lists. After this automated procedure the lists were manually examined and post-processed. In particular, the following substances were removed: petroleum derivatives (which accounted for the majority of the substances), substances duplicated within the other databases considered for the overall analysis (9 chemicals overlapped with NTP, 11 with the Japanese CSCL Database, 11 with CGX  [9] , 1 with DG SANCO SSCS and Cosmetics Industry DB), polymers and inorganic compounds with no structure and/or no molecular weight, and mixtures.
This resulted in a final list of 73 registered dossiers corresponding to 73 unique substances. These 73 selected dossiers were downloaded from the ECHA dissemination website and reviewed. At this stage, read-across data were not considered. Tests that were reviewed for the purpose of this analysis were: Ames test, CAvit, MNvit, MLA, Hprt, CAviv, MNviv, UDSviv, in vivo comet, and transgenic mouse models. For carcinogenicity the study reports were reviewed and the classification (harmonized or self-) was reported when available. A positive, negative, inconclusive or equivocal call was given for each test result. Since for some chemicals more than one study per test was available, a total of 1062 studies were reviewed. Among these studies some were considered not informative for the purpose of this exercise (e.
g. read-across). Subsequently, for each test method an overall call was given following the same criteria as those used in the construction of a consolidated database (see  [52] ). The review of the ECHA dossiers relied entirely on the information submitted by the applicant. In several cases the information reported was not sufficient to allow a firm overall conclusion for a certain test in accordance with the criteria defined. This was the case with negative or unclear results, where no firm conclusion could be made in terms of requirements specified in current OECD Test Guidelines or recommended best practices (e. g. studies only without S9 or only with S9, abnormal pH levels or osmolality, not the standard set of bacterial strains, no adequate levels of toxicity, no proof of target cell exposure in vivo, etc.
). Often the submitted studies reported only the conclusions and omitted the original data; in other cases, references were made to reports which were not accessible or where references were not available. Thus, for the above reasons, despite the massive review work, several studies were given an overall "inconclusive" call and were as such not considered informative. The re-evaluation of the 73 compounds resulted in 29 Amespositive chemicals, 12 Ames-equivocal, 16 Ames-inconclusive, and 16 Ames-negative. These data were considered insufficient to allow a meaningful analysis of the ECHA results in isolation. However, it was decided to add these data to a new consolidated database  [52] , but it was necessary to conduct a further search of literature data with the aim of clarifying some of the inconclusive or equivocal calls.
Such compounds are seldom assessed in vivo for genotoxicity or carcinogenicity, and there are only 11 examples of APIs in the GSK database with positive Ames results and additional in vivo genotoxicity and/or carcinogenicity data (Table  5 ). One reason for this is the positive impact of quantitative structural activity relationship (QSAR) assessment on drug design where careful consideration of chemical structure during leaddrug optimisation avoids the synthesis of API with overt mutagenic structures. Reactive chemistry is still the mainstay of small molecule drug synthesis and the generation of the final API can often require several intermediate steps. The control of drug synthesis intermediates and impurities in the final API is therefore an important consideration for worker safety and for patients, respectively.