dataesr/scientific-paragraphs-categorization huggingface dataset:
For LM immunolabeling, the sections were incubated with a rabbit polyclonal antibody against RhCG (a kind gift from Dr. Yves Colin, INSERM, Paris, France  (16, 25) ) at room temperature for 1 h after preincubation in PBS containing 0. 05 M glycine and 1% bovine serum albumin. The sections were subsequently incubated with peroxidase-conjugated secondary antibody (Dako); the peroxidase was visualized with diaminobenzidine, and the sections were counter-stained with Maier's stain for 2 min. The sections were examined in a Leica DMR microscope equipped with a Leica DFC320 camera. Images were transferred by a Leica TFC Twain 6. 1. 0 program and processed using Adobe Photoshop 8. 0. For EM immunolabeling, the sections were incubated with the primary antibody at 4 °C overnight followed by incubation at room temperature for 1 h with 10-nm gold particles coupled to anti-rabbit IgG (BioCell, Cardiff, UK).
For reverse transcription, 300 ng of RNA template were diluted in a 40-l reaction mix that contained (final concentrations) RT buffer (1ϫ), MgCl 2 (5. 5 mM), random hexamers (2. 5 M), RNase inhibitor (0. 4 unites/l), the multiscribe reverse transcriptase enzyme (1. 25 units/l), dNTP mix (500 M each), and RNase-free water. Real Time Quantitative PCR-Quantitative real time quantitative RT-PCR was performed on the ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA). Primers were chosen using the BLAST tool of Ensemble to result in amplicons no longer than 150 bp spanning intronexon boundaries to exclude genomic DNA contamination. The specificity of all primers was first tested on mRNA derived from kidney and always resulted in a single product of the expected size (data not shown).
Probes were labeled with the reporter dye 6-carboxyfluorescein at the 5Ј end and the quencher dye 5-(and 6-)-carboxytetramethylrhodamine at the 3Ј end (Microsynth, Balgach, Switzerland). The primers were designed to target the deleted sequence in Rhcg Ϫ/Ϫ animals (accession number NM_019799) with 5Ј-ATGCAGGGATGGTTCCATTA-3Ј (238 -258 bp) as the left primer located within exon 1 and 5Ј-TGGAAGAAGGTCATAATGAGCAG-3Ј (373-392 bp) as the right primer located within exon 2, and 5Ј-TTAC-TATCGCTACCCGAGCTTCCAG-3Ј as the probe (292-317 bp). Real time PCRs were performed using TaqMan Universal PCR master mix (Applied Biosystems, Foster City, CA). Briefly, 3 l of cDNA, 0. 8 l of each primer (25 M), 0. 4 l of labeled probe (5 M), 5 l of RNase-free water, and 10 l of TaqMan Universal PCR master mix reached 20 l of final reaction volume.
Reaction conditions were denaturation at 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/elongation at 60 °C for 60 s with auto ramp time. All reactions were run in triplicate. For analyzing the data, the threshold was set to 0. 2 as this value had been determined to be in the linear range of the amplification curves for all mRNAs in all experimental runs. The expression of the genes of interest was calculated in relation to hypoxanthine-guanine phosphoribosyltransferase (accession number NM_013556, forward primer, 5Ј-TTATCAGACTGAAGAGCTACTGTAAG-ATC-3Ј (442-471), reverse primer, 5Ј-TTACCAGTGTCAA-TTATATCTTCAACAATC-3Ј (539 -568), and probe, 5Ј-TGAGAGATCATCTCCACCAATAACTTTTATGTCCC-3Ј (481-515)). Relative expression ratios were calculated as R ϭ 2 ( Ct(HPRT) Ϫ Ct(test gene) , where Ct represents the cycle number at the threshold 0.
The total ammonium concentration ([Am]) is equal to the sum of the concentrations of the two species NH 3 and NH 4 ϩ and is the quantity actually measured by the microfluorimetric assay. The equilibrium between the two species is defined by the Henderson-Hasselbalch Equation  4 , pH ϭ pK a ϩ log͓͑NH 3 ͔/͑NH 4 ϩ ͒͒ (Eq. 4) The pK a equals 9. 03 at physiological pH and temperature. Knowing the values for pH and [Am], the values for [NH 3 ] and [NH 4 ϩ ] may be determined simultaneously. Statistics-Data are expressed as means Ϯ S. E. Statistical comparisons were tested by analysis of variance and Student's t test using the Graphpad Prism software (GraphPad). p values Ͻ 0. 05 were considered as statistically significant.
Thus, RhCG sustains both apical and basolateral transport of NH3 in CD cells, in agreement Kidneys from all three genotypes were dissected after 4 days of HCl load. Cortex (C), outer medulla (OM), and inner medulla (IM) were separated, and tissue ammonium content was measured. In wild type and heterozygous mice, a gradient in renal tissue ammonia content from cortex to inner medulla was observed. In Rhcg Ϫ/Ϫ mice, the ammonia content increased from cortex to outer medulla, but ammonium content was lower in the inner medulla than in mice from the other two genotypes. (n ϭ 5-8 mice/genotype), * statistically different from Rhcg ϩ/ϩ mice (p Ͻ 0. 05). NS, not significant. with structural data and reconstituted RhCG suggesting that the protein forms a NH 3 -permeable channel  (39, 40). Compensatory Adaptations to the Loss of Rhcg-We finally examined whether Rhcg deletion affects mechanisms involved in renal ammoniagenesis and ammonium excretion.
(1992). The Statistical Package for Social Sciences (Version 17. 0 for Windows, SPSS Inc. ) was used to perform ANOVA and Tukey tests (p ≤ 0. 05). Then the overall coefficients of variation (OCVs) were calculated as follows: (standard deviation/average) × 100. In this formula, to calculate the standard deviation and average of the five treatments, we considered the five average values. Therefore, the higher the difference between the five treatments compared, the higher the OCV.
Acknowledgments This research was supported by the  National Secretary of Superior Education, Science and Technology of Ecuador (SENESCYT) ; the  Polytechnic Agriculture and Cattle Husbandry University of Manabi Manuel Félix López (ESPAM-MFL ); and the  Bioplant Center (University of Ciego de Avila, Cuba). We are grateful to  Mrs. Julia Martínez  and  Mrs. Alitza Iglesias  for their excellent technical assistance.
This strain was mostly identified in Egypt before 2005,  7  but has since spread rapidly throughout Africa and the Middle East. The slight decrease in isolation rates for this strain in 2011 probably resulted from the "Arab Spring", which may have discouraged travel to the area in which this strain is endemic. Thus, 150 Salmonella Kentucky CIP R isolates were obtained at the FNRC-Salm in 2012 (data not shown), a number similar to that obtained in 2010. The Salmonella Kentucky CIP R strain was first identified in the Indian subcontinent in 2009, and a pattern of current spread across Asia is also suggested by the isolation of two Salmonella Kentucky CIP R isolates from French patients reporting travel to Vietnam and Indonesia in 2012 (data not shown). As the geographic spread of this strain has been predicted by a French surveillance system, it may be partially biased by the preferred destinations of French travellers and particular migrant populations with historical links to France.
Reverse transcriptase-polymerase chain reaction (RT-PCR) Cells were harvested at confluence and total RNA (1. 5 lg), extracted with the Qiamp RNA blood mini-kit (Qiagen), was used to prepare cDNA using the Advantage RT-for-PCR kit (Clontech). PCR on 5 ll cDNA using specific pairs of primers for the mucin genes and analyses of PCR products were performed as previously described  [23, 28, 29]. Primers used to study the expression of IL-1b, IL-6, IL-8, TNF-a, and NF-jB p65 subunit are indicated in Table  1. The 18S rRNA subunit was used as an internal control. The gene of interest/18S ratio was calculated by densitometric analysis of the DNA bands using the Gel Analyst Gel Smart software (ClaraVision, Paris, France). All PCR studies were performed in triplicate on samples from three different sets of experiments.
Statistics Statistical analyses were carried out using both Excel and GraphPad 4. 0 software (GraphPad Software, La Jolla, CA, USA). Differences in the means of samples were analyzed using Student's t test or one-way analysis of variance (ANOVA) test with selected comparison using Tukey post hoc test with differences less than 0. 05 considered as statistically significant (*) (**p \ 0. 01, ***p \ 0. 001).
Effects of Hp infection on cytokines and NF-jB expression in KATO-III and AGS cells To correlate mucin gene-altered expression after gastric cell infection by Hp and virulence factors to known proinflammatory cytokines and signaling pathways regulated by Hp, the expression levels of IL-8, IL-1b, IL-6, TNF-a, and NF-jB mRNA were measured. Our results indicate that IL-8 and TNF-a mRNA levels were increased in infected KATO-III cells as early as 1 h incubation (Fig. 5a ). IL-6 mRNA was neither expressed nor upregulated in these cells (data not shown). IL-8 induction by N6 wild-type strain seemed to be mediated by the urease factor because the activation was decreased when cells were infected with the UreB -mutant. TNF-a mRNA was strongly induced by wild-type strains as early as 1 h infection.
Acknowledgments We thank  Daniele Petitprez  for her excellent technical help and  Dr. Agnes Labigne  for the gift of the Helicobacter pylori mutated strains ( Institut Pasteur, Pathogenie bacterienne des muqueuses, Paris ). This work was supported by the  European Regional Development Fund (I. Van Seuningen). This work received the  Janssen-Cylen Award  at the  GEFH (Groupe d'Etude Francais des Helicobacter) Helicobacter pylori Day, Institut Pasteur, Paris, France.
280G > A and g. 359A > C, denoted as ACSL4I3B280R and ACSL4I3B359M , respectively; the allele "1" of the two SNPs represents the nucleotide "G" for ACSL4I3B280R or "A" for ACSL4I3B359M; GenBank No. AJ785784) and a 14-bp deletion mutation in the SLC25A5 gene (intron2:g. 103-116del 14, denoted as SLC25A5I2B102DE; the allele "1" represents the nondeleted allele, GenBank No. AM746979). The 22 markers are all located in the chromosome X-specific region, i. e. non-pseudoautosomal region.
05). In addition, to test whether a trait was affected by 2 QTL, the LRT of 2-QTL vs. 1-QTL hypotheses were estimated using the QTLMap software (http://dga7. jouy. inra. fr/qtlmap/; [25] ). There were no significant evidence for additional QTL influencing these traits (data not shown), except for IMF. Two QTL for IMF were revealed only in the INRA population (the maximum LRT 2vs1 is equal to 57. 40); they were located at 87 cM and 92 cM respectively, and exhibited opposite effects (1. 95% and -0. 06%, respectively) on the trait. As distinguishing the effects of linked positions requires both informative markers and informative meiosis  [26] , it is usually unlikely to find close significant linked QTL in linkage analyses, or the risk of pointing out statistical artifacts is high. In the present design, two genetic markers were genotyped between position 87 cM and position 92 cM, with 2/3 of the dams being heterozygous for the first one, and all for the second.
Animals and traits Data from the INRA and JXAU population were used. Details about raising and management of the two populations and the traits recorded were previously described elsewhere  [1, 4, 9, 28]. Briefly, the INRA population was created by the cross between 6 Large White boars and 6 Meishan sows. The 488 F 2 castrated males generated from 6 F 1 boars and 23 F 1 sows were slaughtered at approximately 180 days of age and submitted to a standardized cutting of the carcass. The JXAU population was established by crossing 2 White Duroc boars to 17 Erhualian sows, from which 9 F 1 boars and 59 F 1 sows were used to produce 1912 F 2 progeny. At 240 ± 3 days of age, 548 F 2 castrated males and 481 F 2 females were slaughtered and phenotyped for carcass composition traits. In the INRA experiment, carcass weight (CW), two backfat thicknesses (BFT1 and BFT2), ham weight (HW) and loin eye area (LEA) were obtained after slaughter and analyzed in the current study.
Marker genotyping and linkage map construction Primer sequences for 13 newly developed markers (Additional file 1: Table  S1 ) were designed using Primer3 software (http://primer3. sourceforge. net/). To confirm the locations of all used markers, they were mapped onto the INRA-University of Minnesota porcine radiation hybrid (IMpRH) panel using IMpRH sever  [30, 31]. PCR were typically performed in a 10 μl reaction volume containing 20-25 ng of template DNA, 1 × PCR buffer, 200 uM each dNTP, 0. 25 uM each of forward and reverse primer (forward primer were labeled with fluorescent tags), and 0. 25 U Taq polymerase (AmpliTaq Gold DNA polymerase; Applied Biosystems). For some forward primers without fluorescent tags, an M13 adaptor (5'-GTTTTCCCAGTCACGACGTTG-3') was added to their 5' ends, as described by Schuelke  [32].
In this case, the concentrations of forward, reverse and M13 primers in PCR reaction were adjusted to 0. 1, 0. 15 and 0. 15 uM, respectively. The typical PCR profiles included an initial denaturation at 94°C for 5 min followed by 35-45 cycles of 94°C for 30 sec, annealing temperatures (50-60°C) for 30 sec and 72°C for 30 sec, with a final extension at 72°C for 20 min. PCR products of microsatellites were analyzed for fragment length using ABI3130 or ABI3730 sequencers and GeneMapper 3. 7 software (ABI, Foster City, USA). The ACSL4I3B280R and ACSL4I3B359M were genotyped by PCR-RFLP, after digestion by enzyme MspI and Tsp509I, respectively. Length polymorphism of SLC25A5I2B102DE was assessed by agarose gel (3%) electrophoresis. Female-specific linkage map was constructed using CRIMAP 2. 4  [33] , with the 22 markers' genotypes in both populations.
Haplotype analyses of F 1 sows The phase of the paternally and maternally inherited chromosomes for F 1 sows were constructed using the Gemma software (https://www-lgc. toulouse. inra. fr/internet/index. php/Tools/Gemma. html). We previously reported that there was a significant heterogeneity in the recombination rate among the F 1 sows in the region SW1426-SW1943 within QTL interval, and inferred that this heterogeneity was associated with maternal haplotypes of Chinese origin  [24]. In the INRA population, three F 1 full-sisters (910002, 910009 and 910010) inheriting the same maternal (MS) haplotype showed significantly higher recombination rates in the region, compared with their other full-sister 910013 who carried an alternative maternal haplotype. Meanwhile, two related F1 sows 910018 and 910097 shared the same maternal haplotype over most of the QTL region with the 910013.
Acknowledgements This work was funded by  INRA , the  Major State Basic Research Development Program of China  ( 973 program ,  2012CB124702 ) and the  Natural Science Foundation of Jiangxi Province  ( 2010GZN0048 ). JM is supported by a PhD scholarship from the  INRA Animal Genetics Division. Genotyping for INRA population were performed on the  Genomic Platform of Genotoul  (http://genomique. genotoul. fr/).
The HvNTR2 transcript level was reduced in aleurone layers treated with GA for up to 18 h  (Shahpiri et al. , 2008). Quantitative PCR  (Kirkensgaard, 2011)  showed a slight downregulation of HvNTR1, and a more than twofold upregulation of HvNTR2 by ≥100 nM GA after 24 h. The expression level of HvNTR2 was therefore confirmed to be 10-40 times higher than HvNTR1, suggesting that HvNTR2 is the most important isoform in aleurone layers subjected to GA. The levels for both HvTrxh transcripts were around five times higher than even the highest level of HvNTR2. Overall, the data suggest that the activity level of the NTR/Trx system in barley grain tissues is determined by transcriptional regulation of NTR genes, coupled with post-translational regulation of HvTrxh protein levels. In this context it is relevant to point out that barley microarray analysis has shown that loss of dormancy leads to increased expression of HvNTR1, HvNTR2, and HvTrxh1 in embryos of imbibed grains  (Barrero et al.
These strategies allowed us to identify promoters for each sigma factor (Table  S6 , S7 and S8). We identified about 40 promoters likely to be utilized by RNA polymerase associated to one of the forespore sigma factors (s F or s G ) (Table  S6 ). The consensus elements recognized by s F in C. difficile were determined by using 10 mapped promoters of genes specifically controlled by s F in transcriptome (downregulated in a sigF mutant but not in the sigE, sigG or sigK mutant) (Table  S6 ). The conserved motifs for s Gdependent promoters were defined from a dataset of 30 promoters located upstream of genes specifically controlled by s G in transcriptome (downregulated in a sigG mutant but not in a sigK mutant) (Table  S6 ). The in silico analysis identified 9 of 10 s F promoters and 23 of 30 s G promoters.
To test the possible role of SpoIIID in the mother cell regulatory network in C. difficile, we constructed a spoIIID mutant (Fig. S2 ) and compared the expression profiles of the 630Derm strain and of the spoIIID mutant after 15 h of growth in SM medium. We found 96 genes differentially expressed with a p value ,0. 05 between the 630Derm strain and the spoIIID mutant (Table  S10 ). 12 and 84 genes were down and upregulated in the spoIIID mutant, respectively. We then performed qRT-PCR on a subset of genes regulated by SpoIIID in our transcriptome. The qRT-PCR results confirmed the microarrays data for the tested genes (Table  S5 ). First, 47 genes that are not under the control of sporulation sigma factors in our transcriptome analyses were regulated by SpoIIID either positivelly (10 genes) or negativelly (37 genes) (Table  S10 ).
Construction of C. difficile strains The ClosTron gene knockout system  [87]  was used to inactivate the spoIIID (CD0126), spoVT (CD3499) and spoIIR (CD3564) genes giving strains CDIP224 (630Derm CD0126::erm), CDIP227 (630Derm CD3499::erm) and CDIP238 (630Derm CD3564::erm). Primers to retarget the group II intron of pMTL007 to these genes (Table  S12 ) were designed by the Targetron design software (http://www. sigmaaldrich. com). The PCR primer sets were used with the EBS universal primer and intron template DNA to generate by overlap extension PCR, a 353-bp product that would facilitate intron retargeting to CD0126, CD3499 or CD3564. The PCR products were cloned between the HindIII and BsrGI sites of pMTL007 to yield pDIA6123 (pMTL007::Cdi-CD0216-39s), pDIA6120 (pMTL007::Cdi-CD3499-157a) and pMS459 (pMTL007::Cdi-CD3564-38a).
The data were visualized at a strand-specific manner using COV2HTML (http://mmonot. eu/COV2HTML/). All TSS detected by RNAseq were inspected manually. Deep sequencing data are available at https://mmonot. eu/COV2HTML/visualisation. php?str_id = -14. To perform transcriptional analysis for each sigma factor, we first tested the impact of their inactivation on the expression of two target genes at different times to optimize conditions. For this purpose, we used bona fide targets of these sigma factors in B. subtilis  [16] : gpr and spoIIR for s F , spoIIIAA and spoIVA for s E and sspA and sspB for s G. For s K , we used cotCD and cotA encoding recently described C. difficile spore coat proteins  [41]. This preliminary test allowed us to define a time for maximal differential expression for these targets between wild-type and mutant strains for each sigma factor.
Microarray design, DNA-array hybridization and transcriptome analysis The microarray of C. difficile 630 genome (GEO database accession number GPL10556) was designed as previously described  [27]. Transcriptome was performed using for each condition four (630Derm compared to the mutant inactivated for each sigma factor) or two different RNA preparations (630Derm compared to spoIIID). Labeled DNA hybridization to microarrays and array scanning were done as previously described  [27]. The slides were analyzed using R and limma software (Linear Model for Microarray Data) from Bioconductor (www. bioconductor. org). We corrected background with the 'normexp' method, resulting in strictly positive values and reducing variability in the log ratios for genes with low levels of hybridization signal.
Then, we normalized each slide with the 'loess' method  [93]. To test for differential expression, we used the bayesian adjusted t-statistics and we performed a multiple testing correction of Benjamini and Hochberg based on the false discovery rate (FDR)  [94]. A gene was considered as differentially expressed when the p-value is ,0. 05. The complete data set was deposited in the GEO database with a series record accession number GSE43202.
5), k max and k min are the maximal and minimal observed spacer length, respectively, and M(k) is the count of spacer length k. The necessary procedures were implemented in SignalX  [95]. These profiles determined with the training set were further used to score all identified TSS. Candidate promoters scoring higher than the lowestscoring promoter in the training set were retained for further analysis (Table  S8 ). A powerful approach, as for transcription factors, is to apply comparative techniques based on the assumptions that functional regulatory sites should be conserved in related species. Genome sequences from C. difficile six closest relatives from the Clostridium genus (C. saccharolyticum, C. thermocellum, C. tetani, C. cellulolyticum, C. ljungdahlii, C. kluyveri) and B. subtilis 168 were downloaded from GenBank.
We searched the intergenic regions of the six Clostridia using the profiles defined for C. difficile (Table  S8 ). As the exact TSS positions in these species are unknown, we analyzed regions (2100 +10) relative to annotated start codons. The data on B. subtilis promoters were obtained from DBTBS  [96]. Finally, for each candidate promoter, we calculated the number of genomes also having a candidate promoter upstream of an orthologous gene. Orthologs were defined using the bidirectional best hit criterion implemented in GenomeExplorer  [95]. Purification of s F -His 6 and s E -His 6 for antibody production The coding sequences of sigF and sigE were amplified by PCR using primer pairs CDsigF-pET28aFw/CDsigF-pET28aRev and CDsigE-pET28aFw/CDsigE-pET28aRev, respectively. The resulting fragments were introduced between the NcoI and XhoI sites of pET28a to produce pFT35 (s F -His 6 ) and pFT34 (s E -His 6 ).
Cells were mounted on 1. 7% agarose coated glass slides and imaged in a Leica DM6000B microscope as previously described  [64]. Fluorescent signals were visualized with a phase contrast Uplan F1 1006 objective and captured with a CCD Andor Ixon EM camera (Andor Technologies). Images were acquired and analyzed using the Metamorph software suite version 5. 8 (Universal Imaging). and C. difficile are aligned. The amino acids conserved in these four sequences are indicated by a star. The region 4. 2, which may interact with the 235 regions of their cognate promoters is underlined. In B. subtilis, the specificity of interaction of these sigma factors with the 235 region sequences (a T for s E and a C for s K ) is associated with the presence of a glutamine at position 217 of s E and of an arginine in s K  [32].
(PDF) Table  S5  Validation of microarrays data by qRT-PCR on selected genes. qRT-PCR experiments were performed on two different RNA preparations for each mutant. The results presented corresponded to the mean of at least two independent experiments. (PDF) Table  S6  Identification of promoters recognized by s F and s G. The transcriptional start sites and the 210 and 235 boxes are indicated in red and blue, respectively. The position of 59 start was identified by RNA-seq analysis with indicated score corresponding to the read length (51 bases) coverage ratio for TAP+ and TAP2 samples. For s F and s G , the genes underlined are those validated by in silico analysis (see Materials and Methods) and listed in Table  S8. TSS can be visualized at https://mmonot. eu/COV2HTML/ visualisation. php?str_id = -14.