dataesr/scientific-paragraphs-categorization huggingface dataset:
For detection of CD8 + Molt 4. 8 cells in cell mixtures, an APC-conjugated anti-CD8 antibody (BD Pharmingen, Heidelberg, Germany) was used. For detection of CD19 + /CD20 + B lymphocytes, a PE-conjugated anti-CD19 antibody (DakoCytomation, Glostrup, Denmark) and a PE-Cy5conjugated anti-CD20 antibody (BD Pharmingen, Heidelberg, Germany) was applied. To identify B lymphocyte activation, PEconjugated anti-CD69 antibody (BioLegend, San Diego, USA) and PE-conjugated anti-CD71 antibody (BD Pharmingen, Heidelberg, Germany) was used. The respective isotype control antibodies were applied as controls. Data were obtained with the LSR II FACS machine (Becton Dickinson, Heidelberg, Germany) and analyzed with the FACSDiva software (Becton Dickinson, Heidelberg, Germany) or the FCS express software (De novo Software, Los Angeles, CA, USA).
This work was supported by grant  031A024C  of the  Federal Ministry for Education and Research of Germany  as part of the Ci3 Cluster and by the  LOEWE Center for Cell and Gene Therapy Frankfurt  funded by  Hessisches Ministerium fu ¨r Wissenschaft und Kunst  ( III L 4-518/17. 004[2010 ]) to CJB. QZ was supported by the  China Scholarship Council  and is an associated member of the DFG Graduiertenkolleg  GRK1172. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Susceptibility of the agsD and parental strain conidia to antifungal molecules is similar To investigate the mechanisms responsible for the in vivo growth defect, the germination of agsD mutant conidia was tested in vitro under stress conditions mimicking the in vivo environment, such as, in the presence of reactive oxidants (ROS), cationic peptides, hypoxia and depletion of iron. The agsD mutants showed similar growth rates as their parental strain in the presence of Menadione, hydrogen peroxide and LuperoxH101 with minimum inhibitory concentrations (MIC) of 30 mM, 10 mM and 2 mM, respectively (data not shown) irrespective of the pH of the medium (pH 7 or 4). The killing of resting conidia after 2-6 h of incubation with macrophages purified from uninfected p47 phox2/2 mice (depleted in ROS production) were similar to the killing by purified macrophages from uninfected wild type mice (C57BL6 H-2 b ) (Fig.
For the parental strain, force-distance curves recorded across the cell surface revealed large adhesion forces, with a magnitude of 0. 660. 039 nN as shown by the adhesion force histogram (Fig. 6A-C ). In contrast, structural changes in agsD conidia caused profound modifications of the cell surface physico-chemical properties (Fig. 6D-F , Fig. S4 ). Forcedistance curves showed the absence of adhesion forces over the entire surface of the mutant conidia. This decrease in the agsD conidial adhesion capacities indicated a modification of the cell surface hydrophobicity that could have influenced conidial phagocytosis. Further, chemical nature of the amorphous layer present on the agsD mutant conidial surface was investigated. It was not composed of polysaccharides since the labeling of b-(1,3)-glucan with the b-(1,3)-glucan receptor GNBP3, chitin with WGA, galactomannan (GM) with an anti-GM monoclonal antibody and galactosaminogalactan (GAG) with an anti-GAG monoclonal antibody were negative (data not shown).
In contrast, a strong labeling of the resting agsD conidium with ConA was observed suggesting that the surface layer was rich in glyco-conjugates (Fig. 7 ). To extract these amorphous surface materials, agsD resting conidia were incubated in 0. 5 M NaCl for 2 h and the extracted materials were positive for protein assay. As shown in the Figure  8  (and Fig. S5 ), incubation with NaCl did not release any proteins from the parental ku80 strain whereas the extracts from agsD mutant conidia contained 160 mg proteins per 10 10 conidia. It was verified that the amorphous glycoprotein layer was removed after NaCl treatment because ConA labeling on the conidia after NaCl treatment was negative (data not shown). Further, extracted protein mixture was subjected to proteomic analysis. Thirty-four proteins were identified and in-silico analysis of these proteins by SigPred (http://www.
Polysaccharide PAMPS are exposed on the surface of the swollen conidia of the triple agsD mutants Increased cytokine production seen in the macrophages over a 5 h-time period could also come from changes occurring at the surface of germinating conidia since it has been shown previously that conidia starts germinating intracellularly in the macrophage lysosome after the first 2 h of phagocytosis  [15]. In addition, Figure  2  shows that agsD conidia undergo swelling in the infected lungs before being killed. The structural changes of the early germ tubes resulting from the AGS deletion were investigated by cytochemistry. The swollen conidia of the triple agsD mutants presented an increased labeling by WGA compared to the parental strain (Fig. 10A  and data not shown). In addition, swollen agsD conidia were positive with the b-(1,3)-glucan receptor GNBP3, whereas both resting and swollen conidia of the parent strain were negative (Fig.
0160 to 0. 0190 N/m. Force curves were analyzed in order to determine the adhesion force between the conidia and the AFM tip. These adhesions were plotted as bright pixels, brighter colors indicating larger adhesion values. For each strain, images that were obtained for at least three conidia from independent cultures and analyzed with different tips, were representative of the entire conidial population inside each mutant and parental strain. The results acquired on the spores were analyzed on JPK Data Processing software. The rodlet layer was extracted from the spore surface by incubating 10 9 dry conidia with 48% (v/v) hydrofluoric acid (HF) for 72 h at 4uC. The contents were centrifuged (10,000 rpm, 10 min) and the supernatant obtained was dried under N 2. The dried material was reconstituted in H 2 O and an aliquot was subjected to 15% (w/v) SDS-PAGE analysis and visualized by silver nitrate staining.
All experiments were performed using the agsD_5T(Figs. 1-10, Table  1 , Table  S1 ). Virulence and proteomic analyses were performed also using agsD_n8 (Figs. S1, S2, S3, S4, S5, S6, S7, S8, Table  1  and Table  S1 ). Major phenotypes and virulence data were verified with agsD_n6. 2 (Figs. S1, S5, S6, S7, S8).
(4) incubation with 1 mg/ml biotinylated affinitypurifed Ab to the N-terminal peptide of bovine IL-17A for 1 h at 37uC; (5) incubation with peroxidase-conjugated avidin (Neutravidin, Molecular Probes) diluted 1/20 000 for 30 min; (6) incubation with 3,39,5,59-tetramethylbenzidine (TMB) for ELISA (Uptima, Interchim, Montlucon, France). Optical densities at 450 nm minus the absorbance at 540 nm were measured with a Multiskan RC reader (Labsystems, Helsinki, Finland) after incubation at room temperature and addition of stop solution (1. 0 M HCl). Concentrations were calculated with the Genesis software (Labsystems). The intra-assay variability was determined by testing replicates of a few samples and of standard dilutions of recombinant IL-17A. The intra-plate and inter-plate coefficients of variations were less than 15%.
Statistical Analysis Most of the statistical analyses were performed with the StatXact software (Cytel software Corp. , Cambridge, MA, USA), using non-parametric tests. A probability level of ,0. 05 was considered statistically significant. Milk cell concentrations between groups were compared with the permutation test for independent samples (two-sided). The significance of concentration variations of cytokines and chemokines with time were tested with the Friedman test, and the significance of differences of antibody concentrations with the Mann & Whitney test. The significance of the difference of cytokine expression in mammary tissue of challenged and control quarters was tested with the permutation test for paired samples. Correlations between cell concentrations and milk concentrations of chemoattractants and cytokines were calculated with the Bravais-Pearson test (Excel software).
Antigen-induced Clinical Signs (Data Not Shown) Cows were split between 3 groups of 7 animals which received 3 subcutaneous injections of ovalbumin with two different adjuvants or received adjuvant without antigen (Figure  1 ). Following intramammary infusion of ovalbumin into one quarter of the immunized cows, local and systemic signs of inflammation developed in 9 of the 14 immunized cows but not in any of the mock-immunized animals. Elevated rectal temperatures were detected in the responsive cows at 12 hpi, peaked at 24 hpi (39. 3 to 41. 0uC) and returned to normal by 48 hpi. Local signs of inflammation in the infused quarter of responsive cows were visible at 24 hpi up to 72 hpi with some redness, a moderate swelling and some tenderness. Signs of mastitis had disappeared at 96 hpi. Milk alterations (small clots) became obvious in these quarters at 24 hpi, but greatest changes (flakes) were usually seen at 48-72 hpi.
coli cells (Invitrogen). Recombinant plasmids from positive clones were extracted, purified and sequenced by GATC Biotech (Konstanz, Germany). The obtained sequences were tested for homology with known for genes using NCBI database. Two sequences homologous to for were then identified and named variant 1 and variant 2. Primers were designed from these two sequences to amplify the corresponding full length cDNAs from freshly extracted polyA+ mRNA samples (variant 1 mRNA sense primer : ACTGTTGCTTCAGTCGCTGTTTACA, variant 2 mRNA sense primer : CAGCGTCTATCTACGTATGTGC, common antisense primer : GTAAAATCGTTGAGGCGGACAA). The absence of for homologous genes in the YR2 endosymbionts was confirmed using public annotations of genomes of the primary symbiont Buchnera aphidicola and the secondary symbiont Regiella insecticola.
cDNAs were transcribed using the SuperScript TM II reverse transcriptase (Invitrogen) in accordance with the supplier's instructions using 1 mg of polyA+ mRNAs. The efficiency of each reverse transcription reaction was tested by amplifying the obtained cDNAs with the different primers sets used in the following qPCR reactions using the UptiTherm DNA polymerase (Uptima). The qPCR reactions were performed for each cDNA sample using the qPCR MasterMix Plus for SYBRH Green I No Rox (Eurogentec, Belgium) on a DNA Engine OpticonH2 instrument (Bio-Rad) with the following qPCR conditions : 50uC for 2 min, 95uC for 7 min, 406(95uC for 30 sec, 60uC for 30 sec, 72uC for 45 sec) and specific primers (Table  S1 ). An internal fragment of the Rpl7 ribosomal protein of A. pisum (Genbank accession NM001135898) was used as a control to normalize the for expression (RPL7-2QPCR Forward: ACTGTT-CAGATTGCGTCAGATC, RPL7 -2QPCR reverse: AGTTCCCTTACGCTCTTCAAGT).
The Acyrthosiphon pisum foraging gene To isolate A. pisum for cDNAs, we designed primers from an A. pisum EST fragment available on the Aphidbase 1. 0 database that showed unambiguous homology with the Drosophila melanogaster for gene. 3' and 5' RACE experiments resulted in the cloning of two full-length cDNA sequences we named Apfor1 (GeneBank accession number JN812212) and Apfor2 (GeneBank accession number JN812213). Thanks to the recent sequencing of the pea aphid genome, we managed to locate these two complete A. pisum for cDNAs on a genomic scaffold (Scaffold409, GeneBank accession number GL350029) and subsequently deduced that they are composed of 16 exons, exons 3 to 16 being common to Apfor1 and Apfor2 (Figure  1 ). The full size of the Apfor genomic sequence is difficult to determine because of the large size of some introns (notably intron 3 covering more than 300 kb) and of residual sequencing errors.
The functional A one-way ANOVA followed by a Fisher's PLSD test shows the statistically significant differences between groups denoted by different letters (P,0,05 or P,0,01). doi:10. 1371/journal. pone. 0065104. g002 significance of the two identified transcripts was not determined but they are full length variants which encode two putative functional proteins. These transcripts are produced by a single gene as determined by a Southern blot experiment (data not shown). The analysis of sequences from the pea aphid genome reveals a second gene coding for a cGMP-dependent protein kinase (GeneBank, accession number XM 001947008), very likely the ortholog to the dg1 gene from D. melanogaster. These two aphid genes have diverged enough (41% of similarity) not to crosshybridize using classical Southern blot techniques.
Materials and Methods Data Collection. Markets where live birds are sold are ubiquitous in Vietnam and heterogeneous in terms of the volume of poultry sold. Live bird trade is an irregular and minor activity in most markets. Therefore, sampling was conducted in a purposive manner, targeting the largest LBMs in the selected areas in terms of the amount of poultry sold. These LBMs were identified through interviews with traders. In addition, six traders were interviewed in Bac Giang province in a location outside the market system where poultry was traded. This site was identified by traders interviewed within an LBM located in its vicinity. Details on market and trader selection are provided in Fournié et al. (20). The sampling methodology may be described as a labeled star sampling approach (44): a set of markets, where traders were interviewed, allowed the identification of connections with other markets that did or did not belong to this set.
A thousand simulations were run for each combination of values of T V , with T V ∈ ð1d; 2d; 3d; 4dÞ, of P M with P M ∈ ð0; 0:1; 0:2; 0:3Þ, and of P T with P T ∈ ð0; 0:1; 0:2; 0:3Þ (SI Text). Univariable GAMs were fitted for each simulation set, with the response variable being either the susceptibility or the infectiousness and the predictor variable being the degrees, the betweenness, or the closeness. The proportion of the null deviance explained by the GAMs was used as a measure of the strength of the association between variables  (35). All analyses were run using R 2. 12. 0 (48) and the package "sna" (49). The package "tnet" (50) was used to calculate the clustering coefficient.
ACKNOWLEDGMENTS. The authors are grateful to the study participants and the interviewers. They also express their thanks to  Rowland Kao ,  James Wood ,  Richard Kock ,  Angel Ortiz-Pelaez , and  Thibaud Porphyre  for their suggestions, which helped improve the analysis;  Anna Dean  for her constructive comments on the manuscript;  Raphaëlle Métras  and  Kim Stevens  for providing maps;  Jeff Gilbert  and  Andrew Bisson  for their support in the implementation of the field survey in Vietnam; and two anonymous reviewers for their constructive comments. G. F. thanks the  Bloomsbury Consortium  and the  University of London Central Research Fund  for their support. A. C. G. acknowledges support from the  UK Medical Research Council.
Light emission was measured using a TD 20/20 luminometer by adding a 100-l subsample to a 10-l bovine serum albumin (BSA) (Sigma Co. ) (2%)-aldehyde (n-decanal) (Sigma Co. ) (0. 2%) solution. Strains with a positive response during 8 h in three replicate experiments were explored further. When the response to water activity and osmotic stress was tested, media with double the amount of the final concentration of NaCl were mixed 1:1 with the growing cell culture. Except in the screening in which luciferase expression is reported as relative light units (RLU)/ml/min, all of the study results are reported as RLU/ml/min/OD at 595 nm (OD 595 ). Concentrations mimicking a reduction in water activity were calculated from empirically obtained data presented by  Leistner and Rodel (47)  and by Brown  (48).
EcoRI-digested, digoxigenin (DIG)-labeled pRL1063a served as the probe (Boehringer Mannheim, Richfield, CT). The insertion site was determined by sequencing outward from the insertion sequence using primers as previously described by Milcamps et al. (41)  and comparing the sequences to the S. meliloti 1021 database (http://iant. toulouse. inra. fr/bacteria /annotation/cgi/rhime. cgi/). The Tn5luxAB insertions were transduced using ⌽M12 to reconstruct the same mutation in a new genetic background to reduce the possibility of secondary mutations  (40). Transductants were selected on GTS with kanamycin and streptomycin, and the copy number of Tn5luxAB was determined as described above. Molecular sequence analysis. The amino acid sequences from the loci tagged in strain Sce10 and Sce11 were searched using BLAST and the cDart and the conserved domain database at NCBI (http://www.
RESULTS Identification of a NaCl-responsive putative operon. The goal of this work was to identify genetic loci in S. meliloti 1021 involved in survival during desiccation. Previous studies have shown that sur-vival during desiccation of S. meliloti increases in the presence of NaCl  (10, 13). Therefore, a S. meliloti Tn5luxAB transcriptional fusion mutant bank  (41)  was screened for an increase in luciferase expression during exposure to 400 mM NaCl followed by a screen for loci affecting the desiccation phenotype of mutant strains. After 8 h of exposure to NaCl, 12 mutants whose expression was significantly (P Ͻ 0. 001) induced in the presence of NaCl were identified (Fig. 1 ) that had a single copy of Tn5luxAB integrated (data not shown). These strains were annotated sodium chloride expressed-strains, abbreviated as Sce1 to Sce12.
Except for Sce5 and Sce12, all strains showed a significant increase in luciferase expression after 3 h of incubation in the presence of 400 mM NaCl (fold increase Ն 2) (P Ͻ 0. 001; n Ն 6). The genomic sequence of regions immediately adjacent to Tn5luxAB was determined for 10 strains and was used to identify the tagged loci in S. meliloti 1021 (Table  1 ). Expression of Sce10 (asnO) in response to NaCl was confirmed using reverse transcription-PCR (RT-PCR) (data not shown). Four of the 10 Tn5luxAB-tagged loci identified in strains Sce2, Sce3, Sce10, and Sce11 mapped to the same part of the genome and had inserted into what appears to be one operon (Fig. 2A ). The first four genes are involved in dipeptide uptake, the fifth is a potential regulator of this operon, and the last two are hypothetically involved in the synthesis of NAGGN, a compatible solute known to result in growth at increased osmolarity upon osmotic shock  (52, 53).
To determine if loci asnO and ngg also affect the ability of S. meliloti 1021 to survive desiccation, strain Sce10 and Sce11 were dried at 22% RH for 3 days in PMM in the presence of 400 mM NaCl. As controls, the survival rates of S. meliloti 1021 and strain Sce3 were also determined. As the data indicate, strains Sce3 and Sce10 were able to survive desiccation to a degree similar to that seen with the reference strain (P Ͼ 0. 05), while survival of strain Sce11 was 15. 4-fold reduced relative to that of the reference strain (P ϭ 3. 5 ϫ 10 Ϫ3 ; Fig. 3B ). The ability of strains Sce3 and Sce10 to survive desiccation shows that the presence of the transposon, the expression of luciferase in the presence of NaCl, the ability to grow in the presence of NaCl, the presence of NAGGN, or polar effects cannot account for the reduced ability of strain Sce11 to survive desiccation.
When luciferase induction levels were measured during the course of growth, differential responses of asnO::Tn5luxAB and ngg:: Tn5luxAB were found during the lag, exponential, and stationary phases. Expression of asnO::Tn5luxAB drastically decreased during exponential growth of strain Sce10 and increased again in the stationary phase. If the organism was responsive at all, expression of ngg::Tn5luxAB slightly increased very early during exponential growth; however, expression of asnO::Tn5luxAB showed a decrease in luciferase activity in the early stages of growth, while activity increased again during the stationary phase. The domain structures of AsnO and Ngg relate to antibiotic resistance. A BLAST analysis of the amino acid sequence against the cDart and the conserved domain database at NCBI revealed that AsnO S.
meliloti 1021 contains a GnAT domain between amino acids 2 and 224 (Evalue ϭ 6e Ϫ32 ) and is associated with a conserved Asn synthase domain located between amino acids 246 and 524 (Evalue ϭ 1. 7e Ϫ55 ) (Fig. 2B ). AsnO S. meliloti 1021 is in a class of sequences most closely related to AsnB E. coli (Evalue ϭ 5. 6e Ϫ118 ), a glutamine-hydrolyzing asparagine synthase, as reported earlier  (53, 55, 56). More diverse members of this group of proteins include AsnO Bacillus subtilis and LtsA Corynebacterium glutamicum  (55). Although previously not appreciated, AsnO S. meliloti also has similarity to ␤-LS Streptomyces clavuligerus , a protein with the ability to synthesize clavulanic acid, an inhibitor of ␤-lactamases  (57). An amino acid sequence comparison of Ngg to entries in cDart and the conserved domain databases revealed two conserved domains.
ACKNOWLEDGMENTS This work was partially funded by  LiphaTech, Inc. (grant  74576  to F. J. D. B. ) and by the  USDA (CSREES  grant  MAS 00087  to K. N. ). We thank  Peter Wolk  ( MSU-PRL-DOE ) for use of the photonic camera,  Pamela Green  ( MSU-PRL-DOE ) for use of the luminometer,  Christine White-Zeigler  ( Smith College ) for assistance with RT-PCR, and, last, two anonymous reviewers who gave excellent recommendations to increase the quality of the manuscript.
(Middle) Quantitative RT-PCR measurements of Clk mRNA levels in head extracts of flies collected at CT2. Mean values +/2 s. e. m. from at least three independent experiments are shown with the per 0 tim 0 control set to 100. (Right) CLK protein/Clk mRNA ratio calculated with mean values of Western blot quantification and quantitative RT-PCR data at CT2. Ratios were normalized to the control (per 0 tim 0 ) set to 100. (C) Cycloheximide chase of CLK degradation in the presence of CK2a overexpression. per and tim dsRNA was applied to S2 cells prior to transfection. We transfected 1 mg pAc-Clk-V5/ His6 with or without 3 mg of the FMO02931 CK2a expression vector. Transfections were split in four equal volumes for the degradation assay. ''h in CHX'' indicates the hours for which respective cells were incubated in cycloheximide to stop protein synthesis.
Behavioral Analysis Behavioral assays for locomotor activity rhythms were carried out with 1-to 5-d-old males at 25uC in Drosophila activity monitors (TriKinetics). Illumination was provided by standard white fluorescent low-energy bulbs. Light intensity at fly level was in the range of 300-1000 mW/cm 2. Flies were first entrained to 12 h:12 h light-dark (LD) cycles for 4 d and then transferred to constant darkness (DD). Activity data were analyzed from the second to the ninth day in DD. Data analysis was done with the FaasX 1. 16 software that is derived from the Brandeis Rhythm Package (see  [65] ) and is freely available upon request (Apple Mac OSX only). Rhythmic flies were defined by x2 periodogram analysis of an 8-d dataset with the following criteria (filter ON): power $20, width $1.
In the case of anti-CK2b, TrueBlot ULTRA Anti-Mouse IgG-HRP (eBioscience, 1:2,000) was used as a secondary antibody to circumvent problems resulting from primary antibody light chain detection after immunoprecipitation. Blots were revealed with the Amersham ECL Plus reagent (GE Healthcare). SimplyBlue SafeStain (Life Technologies) was used to stain membranes after blotting. Images were quantified with the NIH ImageJ (1. 43 k) software after background subtraction. Calculations were done and histograms were generated with Microsoft Excel.