dataesr/scientific-paragraphs-categorization huggingface dataset:
There are no published data on the epidemiology of HFMD in the Lao PDR and EV-A71 cases have not been reported, although paediatricians are concerned that they have seen HFMD patients. Importantly, and in contrast with neighbouring countries, no patients with severe disease have been reported to date in Laos. This may be due do the lack of available diagnostic tests or to specific epidemiological characteristics, but also raises the issue of the possible circulation of specific low-pathogenic viral variants. To examine the latter issue, samples from children presenting at hospital with symptoms and signs of HFMD have been collected at Mahosot Hospital, Vientiane, more than 200 since June 2010, and analysed for EV detection and characterization. EV-A71 and CV-A16 were isolated from two HFMD patients, HFMD12 and HFMD4, attending Mahosot Hospital in February and May 2011, respectively.
[12]. Genomic amplification was performed using random (CV-A16) or specific (EV-A71) protocols followed by Next-Generation Sequencing using the Ion-Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA, USA). The almost complete genome sequences of EVA71/LA/ HFMD12V/2011 (GenBank KM055005); 7379 nucleotides long including the full open reading frame, 723 nucleotides of 5 0 UTR and 77 nucleotides of 3 0 UTR) and of CVA16/LA/ HFMD4V/2011 (GenBank KM055004); 6971 nucleotides long including the full open reading frame, 288 nucleotides of 5 0 UTR and 104 nucleotides of 3 0 UTR) were obtained. Both 0. 05 The VP1 sequence of HFMD12V was aligned with 44 other EV-A71 sequences from genotypes A-C, and HFMD4V was aligned with 61 CV-A16 sequences using CLUSTALX2. 1  [13]. Neighbour-joining trees were constructed using MEGA 6.
06 with the Kimura-2 distance algorithm  [14]  and bootstrap resampling with 500 replicates. The EV-A71 tree (Fig. 1 ) revealed that the HFMD12V strain belongs to genotype C4, which is the predominant genotype circulating in the Asia-Pacific region  [15]. BLAST nucleotide analysis identified 98% identity with strains HQ456308 and HQ712020 isolated in China in 2010, in Guangdon and Beijing, respectively. The CV-A16 tree (Fig. 2 ) showed that strain HFMD4V belongs to genotype B1a, with 98% and 96% identity with a strain isolated in Thailand in 2010 (GenBank JF738004) and a strain from Yunnan, China 2008 (GenBank HQ423141), respectively. AF135944_CA_1991 AF009545_US_1994 DQ846662_TW_2006 EU527983_TW_2007 FJ151500_TH_2007 FJ151599_TH_2008 AY125968_KR_2000 AY125967_KR_2000 AY125966_KR_2000 AB465408_JP_1994 AF135949_AU_1995 AF119796_TW_1998 FJ151501_TH_2008 DQ381846_AU_1999 AM939583_UK_1999 AY895133_CN_2003 DQ841966_TW_2004 AB433891_JP_2007 HFMD12V_LA_2011 FJ469159_CN_2008 EU753366_CN_2007 GU434678_CN_2009 U22521_US_1970 GQ117124_CN_2008 AB491213_NL_1966 AB491212_NL_1965 AB059813_JP_1973 AB059815_HU_1978 AF009524_US_1987 AF009540_US_1988 AY905549_MY_2003 AB177816_JP_2003 FM201328_BN_2006 FJ461788_SG_2008 AF376112_SG_2001 AF376123_SG_2000 AF376065_MY_2000 DQ341367_MY_1997 AF376094_AU_1999 DQ341368_MY_1997 U05876_CV-A16 AM939598_UK_2005 This study demonstrates the circulation in Laos of EV-A71 genotype C4, which is associated elsewhere in South-East Asia with severe disease.
Three authentic contigs were eventually defined by extensive restriction double digest mapping, subcloning, limited sequencing after PCR, and comparison to genomic DNA by Southern blot (Figure  S1 ). One of these contigs corresponded to cluster I (the chicken MHC, or BF-BL region) which we had already shown contained a BG gene provisionally named the 8. 5 gene (later renamed BG1). We fully sequenced the 8. 5 gene (accession number KC963427,  [59] ), and later the whole of the cluster I contig (accession number AL023516,  [73] ). The other two contigs (named cluster V and VI) each contained six BG genes, which were given a variety of provisional names (now renamed BG2 through BG13). In addition, a related region without BG genes was found in each cluster. Three representative cosmids covering most of clusters V and VI were fully sequenced by standard shotgun techniques, confirming the presence of the six BG genes in each cluster along with a small region containing genes for a kinesin motor, a C-type lectin-like receptor and an unidentified protein (Figure  1 , Figure  S2 , accession number KC955130).
We were concerned whether we had cloned all of the BG genes from the CB chicken. Screening revealed one additional B12 BG sequence (accession number KC955131) from one of our caecal tonsil cDNA libraries, which was called CTBG (and which we will now rename BG0). BLAST analysis of the chicken WGS sequence (www. ensembl. org, release 2. 1) showed that this gene is present on chromosome 2 (positions 100590000-100600000), a different chromosome from the chicken MHC on chromosome 16 (Figure  S3 ). Using the partial sequences of all the genes identified at the time, we had designed potentially universal primers, and performed RT-PCR on a variety of cells and tissues (Figure  2 , Figure  S4 ). We found all of the genes from the cosmids expressed (except one, BG2, which we later realised had a single nucleotide change compared to the 39 end of one of the primers).
Each BG gene has very specific cell and tissue expression, with one group expressed in haemopoietic cells and another group expressed in tissues As mentioned above, we performed reverse transcriptase-PCR (RT-PCR) on a variety of cells and tissues with what we thought at the time were universal primers (which however turn out not to amplify BG0 or BG2). For each cell and tissue, we cloned the PCR products and counted the number of clones from several independent amplifications, a method used successfully for assessing the relative expression of MHC genes  [76, 77]. With this simple assay, we found truly striking patterns of expression for each of the analysed genes, with only a few genes expressed in each cell type and restricted patterns even for tissues (Figure  2 ). To provide additional support for this approach, we developed specific RT-qPCR assays for two haemopoietic and two tissue BG genes, and found that the results with spleen, bone marrow, liver and duodenum confirm our expectations based on the data from the approach of amplifying, cloning and sequencing (Figure  S6 ).
Some cDNAs show unspliced (or retained) introns for which the sequence remains in frame, and we suggested  [78]  that these extra stretches coding for protein have the potential for interesting functions. The original cDNAs for BG1 (8. 5) have a long stretch of contiguous C-terminal sequence (accession numbers KC955132 to KC955136,  [59] ), but our analysis shows that this region is in fact due to what was originally an unspliced intron, because the flanking 21 nucleotide repeat exons can be identified which have apparently reasonable splice sites (Figure  2 , Figure  S11 ). Interestingly, this region has recently been identified as containing a functional immunoreceptor tyrosine-based inhibitory motif (ITIM)  [71] , fulfilling our original prediction. We examined all of the sequences for the possibility of unspliced introns with in-frame sequence, and found between one and five per gene (Figure  S11 ).
The 14 BG genes of the B12 haplotype (as in Figure  1 ) are depicted with coloured boxes illustrating presumed origin (as in Figure  5 ). See Figure  S11  for an alternative view. doi:10. 1371/journal. pgen. 1004417. g008 Figure  9. Comparison of cosmid cluster VI from the B12 haplotype with the BQ haplotype from a red junglefowl, showing regions of virtual identity separated by two large indels, one in the middle of the sequences and the other where the red junglefowl haplotype (but not the B12 haplotype) continues into the TRIM region. Genomic organisation on bottom line is from cluster VI of this paper (accession number KC955130) to two sequences from the BQ haplotype, middle line from the WGS sequence assembly (nucleotides 166492-252491 on chromosome 16) and top line from the sequence of a BAC from the same individual chicken (accession number AB268588.
Isolation and sequencing of BG genes (Figures 1, S1 and S2) Standard molecular biology techniques used in this paper are described in detail  [91, 92] , which typically are referenced along with modifications in the accompanying citations given below. Southern blotting using BG cDNA probes  [78]  identified the transcribed fragment 8. 5 from various cosmid clones of the line CB (B12) chicken  [72]  as a BG gene (subsequently named BG1). A 6. 5 kB fragment from cosmid cb12  [72]  cut with Nru I and Hind III was isolated and cloned into Bluescript (cb12NK6. 5BS2a clone), subclones were sequenced as described  [93]  by dideoxynucleotide technology using 32 P-labelled ATP, some portions after isolation of single strands, and the sequence was deposited in Genbank (accession number KC963427). A cosmid library from a line CB (B12) chicken  [72]  was screened with cDNA clones G1, G5, G7 and G8  [53]  labelled with 32 P by nick-translation.
The 50 positive plaques were picked and grown up for isolation of DNA, which was analysed by Southern blot using various BG cDNA probes isolated from a H. B19 (B19) chicken spleen library  [78]. Double-digestion restriction maps and limited sequencing were used to group the cosmids into clusters  [59] , with extraneous sequence of chimeric inserts determined by genomic Southern blots. DNA from cosmids cG43, cG3 and cG24 was sheared, cloned and sequenced using commercial fluorescent dye reaction kits followed by capillary electrophoresis at the Sanger Institute. The clusters were linked using the data from Figure  S5C  in Cambridge, and the whole sequence deposited in Genbank (accession number KC955130).
BG cDNA was amplified using a T3 thermocycler, a commercial Taq polymerase kit (Roche), with an annealing temperature of 45uC. BG1 cDNAs were amplified using reverse primer 2773 containing a Not I site (ATATATgcggccgcCTYTGGTTT-CTTTCTTCCAATTGG) based on the cDNA clones G3 and G8, and a series of forward primers each containing a Sal I site, 2960 (TATATgtcgacTGGCAGAATTACTGGTGCC), 2961 (TATATgtcgacCTGGTGATAGCAGAGACCC) and 2962 (TA-TATgtcgacGGTAGAAGCTGGGC) designed based on the genomic sequence (accession number KC963427) to establish the 59 end of the BG1 transcript. BG10 and BG11 cDNAs were amplified with forward primer 8081 containing an Nru I site (CACGTtcgcgaCCATGSNSTTYNYATYRRGMTGC) and reverse primer 2774 (sequence above). Amplified fragments were cut with appropriate restriction enzymes, gel-purified and cloned into Bluescript plasmid, with BG1 clones PCRX1, 3 and 5 from transfectant 1.
Forward and reverse primers for qPCR were TGTG-CTGTGCAAGATGAT and TTCCAGGGATGGATGATG for BG4, TGTGTTGTGCAAGATGAC and GAAAAGCAAT-GATGACAA for BG7, TGTGCTGTGCAAGATGGT and ACGATCTGGGAAAAGGGG for BG10, and GGAC-GATCTGGGAAAAGA and TATGCAGAAGCTGTGGTGA for BG11. Targets were amplified over 40 cycles (initial enzyme activation 15 min 95uC, then 40 cycles of 15 s at 94uC, 30 s at 60uC and 30 s at 72uC) using 10 pmol each primer in AbsoluteBlue qPCR Mix (Thermo Scientific). Samples were compared to a 5-point standard curve, and normalised to cyclophilin A and a reference spleen sample. Fluorescence data were collected and analysed using an iCycler (BioRad) with subsequent analyses preformed in Microsoft Excel.
5 mM MgCl 2 ), 1. 5% polyethylene glycol and 0. 04 mM of each dNTP. After initial denaturation, there were 5 cycles of 0. 75 min at 95uC, 0. 5 min at 59. 8uC and 1. 5 min at 72uC, followed by 30 cycles of 0. 75 min at 95uC, 1 sec at 59. 8uC and 1. 5 min at 72uC, followed by a final extension step. The amplicons were cloned using the CloneJET kit (Fermentas) following manufacturer's instructions, and sequenced using commercial fluorescent dye kits followed by capillary electrophoresis at the DNA Sequencing Facility of the Department of Biochemistry, University of Cambridge (www2. bioc. cam. ac. uk/ ,pflgroup/DNA_Facility). Sequence data was analyzed using CLC DNA workbench (version 5. 7. 1, www. clcbio. com/). Alignments were performed using ClustalX (www. clustal. org/) and MAFFT (http://mafft.
cbrc. jp/alignment/software/). Some phylogenetic trees were created using the neighbour joining (NJ) method implemented by ClustalX with 1000 bootstrap seeds. Other trees were created using a Bayesian approach implemented by MrBayes  [104]  (version 3. 1. 2, http://mrbayes. sourceforge. net/), with the GTR substitution model used with gamma-distributed rate variation across sites, and with the MCMC analysis using one chain, 20000 generations and sampling every 100 generations. Still other trees were created maximum parsimony (MP) phylogenies, using PAUP 4. 0b10 for Unix  [105]  (www. paup. csit. fsu. edu/). AU and SH tests  [106]  on MP trees were performed in CONSEL version 0. 2 (www. is. titech. ac. jp/,shimo/prog/consel/). Finally, other trees were created using neighbour network analysis implemented by SplitsTree4  [107, 108]  (http://www.
splitstree. org/), and tested for significance using a Phi test for recombination  [109]. Phylogenetic trees were visualized using Dendroscope (http://ab. inf. uni-tuebingen. de/software/dendroscope/). After removal of gaps in the alignment of all 14 BG genes by G-blocks using default stringent parameters  [110, 111] , an automated partitioning analysis performed using SAGUARO  [112]  (www. sourceforge. net/projects/saguarogw/) gave cacti which were handled with PHYLIP version 1:3. 68-2  [113]  from the Ubuntu repositories (www. launchpad. net/ubuntu/lucid/+source/phylip/1:3. 68-2). Dotplots were created with a wordsize of 150, using dottup from the EMBOSS package (version 6. 1. 0-5, http://emboss. sourceforge. net/). Helical wheels were created using pepwheel from the EMBOSS package (version 6.
Acknowledgments We thank  Christian Do  ¨hring and  Elizabeth Langley  for technical assistance, and  Karel Hala  from  Innsbruck  for three CB chicks so long ago. We thank two anonymous reviewers for their reasonable comments and questions. We acknowledge the guest editor and one anonymous reviewer for requiring the analyses for Figures  S6 ,  S12 ,  S13 ,  S14  and  S15 , as well as the extensive discussion of the data in the light of current evolutionary theory. We also acknowledge a new reviewer in the second round of review for requiring an explanation of blood vessels in tissues. Finally, we honour the memory of  Morten Simonsen , who started us off many decades ago on the work that finally resulted in this story.
6E-3); furthermore, there is no significant difference between RBF and RBFD253A protein expression levels (ANOVA, p = 0. 48). (C) Quantification of rbf mRNA by RT-qPCR in wing imaginal discs. Fifty wing imaginal discs per genotype were dissected on ice. Total RNAs were extracted from each sample using the RNeasy Mini kit (QIAGEN), RT was performed on each sample using random primer oligonucleotides (Invitrogen) with Recombinant Taq DNA Polymerase (Invitrogen). Real-time PCR analysis was performed using the C1000 Touch TM Thermal cycler (Biorad). Data are normalized against rp49 and correspond to the mean of three independent experiments. Error bars are the S. E. M. Asterisks indicate statistical significant difference between two genotypes (Student test, p,0,05).
Acknowledgments We are grateful to  S. Gaumer  and  V. Rincheval  for their critical reading of the manuscript. We wish to thank  M. N. Soler , for expert support with confocal microscopy and  N. Boggetto  for expert support with flow cytometry. The Imaging and  Cell Biology  facility of the  IFR87  ( FR-W2251 ) '' La plante et son environnement '' is supported by  Action de Soutien ala Technologie et la Recherche en Essonne ,  Conseil de l'Essonne. The flow cytometry facility of the  Jacques Monod Institute  is supported by the  Ligue Nationale contre le Cancer (ComiteIle de France)  (# R03/75-79 ).
Overall, the data obtained from in vitro and in vivo experiments indicate that some dermaseptin derivatives have a variety of potential medical and antimicrobial applications  (30). However, no work on the antibiofilm activity of dermaseptins has been performed to date. Accordingly, the present study was undertaken to investigate the feasibility and gain effects of using dermaseptins against biofilms formed by important biofilm-forming pathogens, including Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. It particularly focused on evaluation of the antibiofilm potential of dermaseptin S 4 derivatives. A number of chemically modified peptides were synthesized and investigated for antibiofilm activity. HeLa cultures were used to evaluate the safety and toxicity of the peptides, and fluorescent binding and confocal microscopy assays were performed to assess the speed and reversibility of their antibiofilm activities.
The percentage of cell survival was expressed as (live cell number in the test group/live cell number in the control group) ϫ 100. Statistical analysis. Normality in this study was assessed using the Proc Univariate procedure in SAS software  (34). All data were statistically analyzed using the least-squares method with the GLM (general linear model) procedure. Effects were treatment, bacteria, and treatment-bacterium interaction. The Student-Newman-Keuls multivariate test was used to compare means for bacteria, and Dunnett's test was used to compare each experimental treatment (peptide) mean with the control mean.
aureus. However, truncation of the N-terminal carboxyl in S 4 , which potentially could result in a less toxic peptide  (31) , did not affect peptide potency. Effects of dermaseptin S 4 derivatives on in vitro static biofilms. We first assessed the activities of dermaseptin S 4 and its derivatives against mature bacterial biofilms using biofilms formed in microtiter PVC plates. While exposure of bacterial biofilms to the peptides at concentrations of 2ϫ MIC did not dissolve the biofilms, as measured by crystal violet staining (data not shown), survival of biofilm cells was reduced (Fig. 1 ). K 4 S 4 and K 4 K 20 S 4 were the most active peptides against all strains tested, whereas D 4 D 20 S 4 was the least active. However, K 4 S 4 (1-16) and S 4 (5-28), which strongly affected E. coli and P.
ANIMALS AS TOOLS Historically, biology has greatly benefited and is still benefiting from the study of animals. A quick search of the PubMed database for "animal models" yields some 41,000 studies, and many others will come. Once regarded as the subject of biological studies [Darwin's finches are a wellknown example  (Peterson, 2008) ], animals have become tools to seize and solve the genetic secrets that underlie most of the biological aspects of life. Like in developmental or cancer biology, studies of bacterial infections and more general studies on the interactions involved in hostparasite relationships have been greatly boosted by the availability of animal models that can recapitulate such complex events. Looking backward for the root of this transition, the work of the English scientist Edward Jenner can be observed as an important turning point.
Finally, it has been shown  (Abnave et al. , 2014)  that planarians are highly resistant to infections by bacteria that are highly pathogenic to humans, C. elegans and D. melanogaster, and planarian display a genuine immune response involving at least three genes with orthologs in humans, MORN2 (Membrane Occupation and Recognition Nexus-2 protein), DUSP19 (Dual-Specificity phosphatase enzyme), and PAQR3 (progestin/adipoQ receptor-3). Particularly, they demonstrated for the first time that MORN2 has a role in LC3-associated phagocytosis (LAP), and it is essential in eliminating bacterial pathogens by human macrophages as well as by flatworms. Thus, the dataset collected by P. Abnave and colleagues highlights, for the first time, a major interest in studying planarian defense mechanisms to identify conserved immune factors.
DNA immunization using LL-FnBPA+ BLG or LL-BLG strains elicits different BLG-specific primary immune responses In order to know if the production of FnBPA at the surface of L. lactis could influence its immunomodulatory properties, mice were orally or intranasally administered with LL-wt (control), LL-BLG or LL-FnBPA+ BLG strains and the BLG specific primary immune response was monitored. No BLG specific IgG1, IgG2a or IgE could be detected in mice sera regardless of administration route (data not shown). Splenocytes from orally and intranasally administered mice were reactivated with BLG and secreted IFN-c, IL-4 and IL-5 cytokines were assayed in the medium. Splenocytes of mice intranasally (Fig. 1 A ) or orally immunized (Fig. 1 B ) with LL-BLG strain secreted only IFN-c, IL-4 and IL-5 were detected in medium of BLG-reactivated splenocytes from mice receiving LL-FnBPA+ BLG (Fig.
Acknowledgments Antibodies and reagents were kindly provided by  Karine Adel Patient and Jean-Michel Wal (INRA ,  UR496 ,  United'Immuno-Allergie Alimentaire , F-78352 Jouy-en-Josas, France;  CEA ,  Institut de Biologie et de Technologie de Saclay, iBiTeC-S, Laboratoire d'Etudes et de Recherches en Immunoanalyse, F-91191 Gif-sur-Yvette, France ).
Silvia Innocentin  was a recipient of a  European Marie Curie Ph. D grant  from the  LABHEALTH program  ( MEST-CT-2004-514428 ). Daniela Pontes  was a recipient of grants from the  French-Brazilian CAPES   COFECUB  project  nu539/06  and from  Region Ile de France DIM ASTREA. The research leading to these results has received funding from the  European Community 's  Seventh Framework Programme  ( FP7/2007-2013)  under grant agreement  nu215553-2. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
3 (rs79464052, P=5. 57×10  -8  ) in ARAP3 (Supplementary Figure  4 ), and one at 3q13. 33 (rs2681416), although the latter SNP did not replicate in stage 2 or 3. Within the HLA region, rs2523607 (P=3. 35×10 -  ) was carried forward for replication in stage 3. This SNP, localized at 6p21 in HLA-B, reached a combined P=2. 40×10 -10 in a meta-analysis of all three stages (Table  1 ; Figure  1d ). To further evaluate the association of HLA variants with DLBCL risk, we imputed classical HLA alleles at six loci (HLA-A, B, C, DRB1, DQA1, and DQB1) in the four GWAS datasets from stages 1-2 and conducted a meta-analysis (Online Methods). The imputation accuracy of HLA types was high (>95. 2%) when compared to HLA sequencing (four-digit resolution) previously performed on a subset of the NCI samples  14  scanned as part of this study in stage 1 (Online Methods).
Of all SNPs and classical HLA alleles tested across the MHC, only the SNP rs2523607 (OR=1. 34, P=3. 3×10  -9  in stages 1 and 2) and the classical allele HLA-B*08:01 (OR=1. 30, P=3. 16×10 -8 in stages 1 and 2) reached genome-wide significance (Supplementary Table  9 ). These markers were in very high LD (r 2 =0. 91), and after adjusting for the effect of HLA-B*08:01, the association of rs2523607 was greatly weakened (P=5. 5×10 -  ). To gain additional insight into potential biological mechanisms, expression quantitative trait loci (eQTL) analyses were performed in two datasets consisting of lymphoblastoid cell lines (Online Methods). In one of the datasets, significant associations were observed for rs116446171 with HIST1H3F and rs2523607 with HCG27 (Supplementary Table  10 ), while in the other dataset significant associations (FDR<0.