dataesr/scientific-paragraphs-categorization huggingface dataset:
Mycoplasma hyopneumoniae and Mycoplasma hyorhinis are two phylogenetically related species colonizing the respiratory tract of pigs but differing in pathogenicity, the basis of which is not well resolved. We hypothesize that genes belonging to the species-specific portion of the genome and being non-essential during ideal laboratory growth conditions encode possible virulent determinants and are the driver of interspecies differences. To investigate this, transposon mutant libraries were generated for both species and a transposon sequencing (Tn-seq) method for mycoplasmas was established to identify non-essential genes. Tn-seq datasets combined with bidirectional Blastp analysis revealed that 101 out of a total 678 coding sequences (CDS) are species-specific and non-essential CDS of M. hyopneumoniae strain F7.
The mean coverage was 1289×. The final assembled genome consisted of a single contig of 852 181 bp and was then circularized with to Amos to a chromosome of 840 423 bp. Both genomes were automatically annotated using a Prokka software program code 4. For the annotation of the genome of M. hyopneumoniae strain F7. 2C the genome of strain J (acc. no. NC_007295) was used as reference and for the one of M. hyorhinis strain JF5820 the genome of strain SK76 (acc. no. NC_019552) was used as a reference. Genome sequences are deposited under accession number CP034597 and CP035041, respectively.
Essentiality analysis Gene essentiality computations were performed using custom scripts located at GitHub [15] and originally developed by Turner et al. [16]. Raw sequencing data for both M. hyopneumoniae and M. hyorhinis transposon mutants were trimmed/filtered according to the presence of the primer sequences used for amplification and the transposon tag and aligned against their respective reference genomes using bowtie2  [17]. A Monte Carlo simulation method was used to simulate insertion sites across the genome and generate an expected pseudo dataset (repeated 500 times). The Monte Carlo simulations are considered as the expected values for non-essential genes. The R package EdgeR  [18]  was used to compare the differences of abundance between the real (mutant) and pseudo data sets, this package uses a negative binomial distribution to assign log fold changes.
A gene was assigned as essential in case of a negative log fold change, in other words, the number of reads over this gene in the real data were less abundant than the simulated pseudodataset counts. Log fold changes larger than -3. 69 for the Tn-seq dataset of M. hyopneumoniae and larger   than -3. 88 for the Tn-seq dataset of M. hyorhinis were assigned as non-essential CDS. For this, we refer to CDS being non-essential as CDS that are individually dispensable for the mycoplasma grown under ideal laboratory conditions. The essentiality classification based on Tnseq data sets remains on a putative level, since insertion of CDS by transposons were not analyzed for functional disruption of the gene.
A total of 5. 2 million sequences were obtained. The bioinformatics analyses were performed in Qiime  (Caporaso et al. , 2010) , following the protocols for Illumina 16S analyses available at http://qiime. org. The reads were joined into paired-end sequences, and filtered by the quality and chimeric sequences. The sequences filtered were binned into operational taxonomic units (OTU) using Sumaclust method at 97% identity  (Kopylova et al. , 2016). The taxonomic classification was based on SILVAS's ribosomal database-123  (Quast et al. , 2013). We removed the singleton and Archaea sequences. The OTU table was rarefied at 54,535 sequences/sample depth.
Statistical Analyses The statistical analyses were performed in R. 3. 4. 3  (Sasaki et al. , 2005). Firstly, we confirmed the differences in irrigation levels  by ANOVA-test followed by Tukey test (confidential level of 95%) using package 'laercio'. We evaluated the elemental (X-ray) and chemical (Mehlich III) composition of soil types (silt and sand) by principal component analysis (PCA) after the irrigation experiment. We standardized the soil types composition data by Hellinger, and the PCA was performed using the packages "FactoMineR" and "factoextra. " The results of potato experiment, such as potato production (Kg/pot), potato tuber number (unit/pot), and relative mass per potato tuber (Kg/unit), were compared by ANOVA-test and ranked by Tukey test, considering p < 0. 05). The coefficient of variation (CV) was lower than 32% indicating no transformation necessity  (Box and Cox, 1964).
We performed the bacterial functionality prediction using the method and package 'Tax4Fun'  (Aßhauer et al. , 2015). The Tax4fun produced the KO table (KO codes per samples), which we standardized by percentage of row. Using the package "PathView" and KO table, we evaluated the relative abundance of each gene predicted across nitrogen metabolism (KEGG map 00910), sulfur metabolism (KEGG map 00920), methane metabolism (KEGG map 00680), and carbon fixation pathways in prokaryotes (KEEG map 00720). The microbial association network using the package "SpiecEasi"  (Kurtz et al. , 2015). Microbial co-occurrence patterns were inferred using the covariance selection method and Stability Approach to Regularization Selection (StARS) as bootstrap. The microbial association network was based on the minimum occurrence of 40% of treatment samples, as indicated by  Faust and Raes (2012).
The SPIEC-EASI package produced the nodes and edges list, which were used in Gephi 0. 9. 2  (Bastian et al. , 2009)  to build the network plots. The topological properties of each network analysis, such as the number of interactions, the minimum distance between nodes  (Newman, 2003) , modularity  (Newman, 2006) , and Positive/Negative ratio  (Ma, 2018) , were performed using the package "igraph"  (Csardi and Nepusz, 2006). Positive/Negative ratio (P/N) is obtained by the Equation (  2 ), where P/N higher than 1 indicates low competition, and lower than 1 indicates high competition of microbial community  (Ma, 2018). P N ratio = Number of positive edges Number of negative edges (2) The first step of correlation analyses between irrigation levels, soil type and potato variety on the total structure of the soil bacterial community (OTU table) were performed using 'vegan' package.
Also, we verified the soil bacterial alpha diversity (by Shannon index). As the values of the alpha diversity from -45 kPa did not follow a normal distribution (Shapiro-Wilk test, p < 0. 01) we transformed the data by interquartile range. The alpha diversity indicated unexpected results for each irrigation level (Figure  4 ). The irrigation levels with higher potato production (-15 and -25 kPa) showed the lowest alpha diversity. On the other hand, irrigation levels of -30 and -45 kPa showed high alpha diversity. We tested four metabolic paths, those of nitrogen, sulfur, methane, and carbon fixation for prokaryotes, aiming to verify the impacts of irrigation levels on the potential functionality of soil bacterial community. The results obtained for each metabolic paths are indicated in the Supplemental Material (Figures  S3-6 ).
For this fact, we propose a similar explanation used for the potential microbial interactions, where the excess or deficit of water may increase the connections of soil bacterial community, producing secondary metabolites and also opening different niches in the system. Thus, the opening of new niches would be a collateral effect of the microbial community interactions aiming to tolerate the water stress. This unexpected absence of linkage between alpha diversity and productivity might be more important in the field (e. g. , the impact on long-term productivity), which also could include other variables such as microbial a OTU table that assigned using SILVAS's ribosomal database-123  (Quast et al. , 2013) , and rarified at 54,535 sequences/sample depth; b KO table containing the functionality prediction composition obtained by the method and package "Tax4Fun"  (Aßhauer et al.
ACKNOWLEDGMENTS This work was supported by  NSERC (Natural Sciences and Engineering Research Council of Canada ). We are grateful to Silvio's team ( Laval University/Department of Soils and Agri-Food Engineering ) for providing the soil samples and the metadata. We thank  Charles Goulet  and  Marie-Claire Goulet  for all the support with molecular analyses. We also acknowledge the comments and discussion provided by  Annelise Mendes Nascimento  and  Ademir Durrer.
The lag-1 autocorrelation over the full period, equal to 0. 45, was used as a measure of the temporal structure of the annual rainfall series. The evolution of the land cover on the site was assessed by  Trichon et al (2018) , from field surveys over a transect  (since 1985) , and aerial and satellite images. This dataset describes the evolution of the perennial woody cover, the areas where seasonal herbaceous vegetation can develop, and the runoff-prone bare soil areas (supplementary section S1 available online at stacks. iop. org/ERL/14/105005/mmedia). Those variables evolve over years, with a negligible intra-seasonal cycle. The well-developed woody vegetation bands have progressively shrunk since 1955, especially since the drought. In 1955, the structure of vegetated/bare areas and the near-absence of rills suggested that the thickets completely infiltrated runoff from bare soil patches, and that hillslope-scale runoff was very low, as expected for a healthy tiger bush.
The 1000 parameter sets giving the lowest RMSE values were selected as the members of the simulation ensemble. Each member selected by this calibration procedure was considered as an equally plausible model of the system. As estimation errors were not provided for B in  Trichon et al (2018) , the calibration was performed without accounting for the uncertainties on W and B, and the impact of this assumption was evaluated (supplementary section S8). The model was implemented under the R environment (R/3. 4. 3), using the 'deSolve' package  (Soetaert et al 2010)  for solving differential equations and 'SAFER' for latin hypercube sampling  (Gollini et al 2015 , Pianosi et al 2015). The code was parallelized using the 'parallel' package (R Core Team 2018) and run on a 28-core node on the MESO@LR-Montpellier University computing facility.
Acknowledgments This study was funded by  IRD  through a post-doctoral fellowship contract. It has been realized with the support of the  High Performance Computing Platform MESO@LR , financed by the  Occitanie/Pyrénées-Méditerranée Region ,  Montpellier Mediterranean Metropole  and the  University of Montpellier (France). The authors are grateful to the  Malian Meteorological Service  for the historical rainfall datasets. Recent observations were provided by the AMMA-CATCH observatory www. amma-catch. org. The authors thank  V Dakos  and  S Kéfi  for fruitful discussions about this work, and the two anonymous reviewers for their input, which improved the manuscript.
Data availability statements The data that support the findings of this study are presented in  Trichon et al (2018). The land cover dataset, derived from  Trichon et al (2018) , is given in supplementary section S1 (table  S1 ). The yearly rainfall series at the Hombori meteorological station, from the Malian Meteorological Service and the AMMA-CATCH Observatory (Galle et al 2018), is not publicly available for legal reasons, but it is available from the corresponding author upon reasonable request.
The experimental data of Figure  1  were well represented (dashed lines) by the Benoit-Doty expression (see Section III. 5. 1. in the Supporting Information (SI)) using l p as a single adjustable parameter. For all fractions, the experimental R g values were well superimposed into two master curves representing both limiting rod and coiled behaviors in Figure  1c, d. For the rod-like regime, the scaling R g ∼ L 1 was tentatively observed in the low L/l p limit, while a flexible coil, R g ∼ L 1/2 , was obeyed at high L/l p (>50). The distinct crossover from the rod-like to the flexible coil chain conformation according to the Benoit-Doty master plots (Figure  1d ) occurred at higher L (or N w ) with increasing dendron generation, as revealed in Figure  1a, b. b Obtained from SEC-MALLS data fitting using the Benoit-Doty equation (eq 1 in the SI).
MR766 has undergone extensive passaging in mouse brains and cell cultures leading to virus variants that have been well documented  [17]. Some variants of MR766 lack of glycosylation due to a deletion spanning the EDI-GL or mutation which causes a loss of the carbohydrate attachment site on residue Asn154. Among the deposited MR766 sequences, we decided to select the MR766-NIID variant (accession number LC002520), which contains a residue Ile at position E-156 leading to non-glycosylated E protein. The construction of BR15 MC was based on the deposited sequence of epidemic Brazilian ZIKV strain BeH819015 (accession number KU365778) which was isolated in Brazil in 2015  [16]. Utilizing the ISA method, we constructed a molecular clone CHIM (here referred to as ZIKBeHMR-1) derived from MR766 MC in which the region coding for C, prM and the E ectodomain (E-1 to E-436) from MR766 was replaced with the one of BeH819015  [16].
Cells, Viruses, and Reagents Vero cells (ATCC, CCL-81) were cultured at 37 • C under a 5% CO 2 atmosphere in MEM medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) and 0. 002 M L-Glutamine, 0. 001 M sodium pyruvate, 100 U/mL of penicillin, 0. 1 mg/mL of streptomycin and 0. 5 µg/mL of fungizone (PAN Biotech, Aidenbach, Germany). The ZIKV strain PF-25013-18 isolated in French Polynesia in 2013 has been previously described  [23, 24]. The molecular clones MR766 MC derived from historical African strain MR766 (Genbank access n • LC002520) and BR15 MC derived from epidemic Brazilian strain BeH819015 (Genbank access n • KU365778) were previously described by Gadea et al. [15]  and Bos et al. [16]. ZIKV BR15 virus is available at BEI Resources (Manassas, VA) under the catalog number NR-51129 (www.
Immunodetection of Viral Proteins For immunofluorescence assay, cells were fixed with 3. 7% paraformaldehyde in PBS and permeabilized with 0. 15% Triton X-100 in PBS. ZIKV E protein was detected with the anti-pan flavivirus E mAb 4G2 (1:500), followed by incubation with AlexaFluor488-conjugated secondary antibody (1:500) and DAPI for the staining of nucleus (100 ng/mL). The slides were examined using a fluorescent Nikon Eclipse E2000-U microscope (Nikon, Tokyo, Japan). Images were obtained using a Hamamatsu ORCA-ER camera and the imaging software NIS-Element AR (Nikon). Western blot assay was essentially performed as previously described  [23, 25]. Briefly, cells were lyzed with RIPA lysis buffer (Sigma, Lyon, France) and concentration of proteins in the cell lysates was quantified using the BCA protein assay.
Proteins in samples were separated on 4-12% PAGE-SDS gels and transferred to nitrocellulose membranes. The blots were reacted with mouse serum (1:500) and then horseradish peroxidase (HRP)-conjugated goat anti-mouse antibodies  (1:2000). Revelation was done using Amersham ECL prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA) and exposed on an Amersham imager 600 (GE Healthcare). All Western-blot data are representative of three independent experiments.
Indirect ELISA Indirect ELISA measured the production of anti-ZIKV IgGs in immunized mice. UV-inactivated ZIKBeHMR-2 samples in MEM/2%FBS were coated on 96-well ELISA plates at the concentration of equivalent 5 log PFU per well in 0. 1 mL PBS overnight. After washing 3 times with PBS-0. 05% Tween, 0. 1 mL of PBS-milk 3% was added as a blocking agent. Serum samples were two-fold serial diluted in 3% milk in PBS-tween 0. 05% with a starting dilution of 1:50. Plates were incubated with mouse serum samples and then incubated in the presence of HRP-conjugated anti-mouse IgG antibody  (1:2000). Plates were incubated with TMB substrate (eBioscience) and then stopped with 1N HCl. Absorbance was measured using microplate reader at 450 nm. To measure the production of anti-ZIKV EDIII IgGs in immunized mice, indirect ELISA was performed using recombinant Domain III of the E protein (rEDIII) from epidemic ZIKV strain PF-25013-18 (GenBank number KJ776791) as previously described  [6].
Statistical Analysis Data obtained between different treatments were compared by one-way or two-way ANOVA tests, as appropriate. Values of p < 0. 05 were considered statistically significant for a post-hoc Tukey's test. All statistical tests were done using the software Graph-Pad Prism version 6. 01. Degrees of significance are indicated in the as follow: * p < 0. 05; ** p < 0. 01; *** p < 0. 001, **** p < 0. 0001, ns = not significant.
Data are presented as mean ± SEM of three independent experiments. Statistical significance between ZIKBeHMR-1 and ZIKBeHMR2 values is indicated. To compare the growth of viral clones ZIKBeHMR-1 and ZIKBeHMR-2, progeny virus productions in ZIKV-infected Vero cells were examined at 24, 48, and 72 h post-infection using a conventional plaque-forming assay (Figure  2C ). We noted that ZIKBeHMR-1 and ZIKBeHMR-2 had comparable viral growths and virus progeny productions. Thus, a loss of N-linked glycosylation in the E protein did not cause obvious changes in the biological characteristics of ZIKBeHMR-2 in Vero In (A), immunoblot assay on RIPA cell lysates using anti-E mAb 4G2. In (B), immunofluorescence assay using mAb 4G2. Magnification: 200×. In (C), virus progeny production was determined by plaque-forming assay on Vero cells.
Virus titers are presented as the mean (± SEM) from three independent experiments. In (D), the loss of viability of ZIKV-infected cells was determined using a MTT assay. Viability was expressed as the percentage of cell metabolic activity in ZIKV-infected cells relative to those in mock-infected cells. Data are presented as mean ± SEM of three independent experiments. Statistical significance between ZIKBeHMR-1 and ZIKBeHMR2 values is indicated. To compare the growth of viral clones ZIKBeHMR-1 and ZIKBeHMR-2, progeny virus productions in ZIKV-infected Vero cells were examined at 24, 48, and 72 h post-infection using a conventional plaque-forming assay (Figure  2C ). We noted that ZIKBeHMR-1 and ZIKBeHMR-2 had comparable viral growths and virus progeny productions. Thus, a loss of N-linked glycosylation in the E protein did not cause obvious changes in the biological characteristics of ZIKBeHMR-2 in Vero cells.
Vaccines 2019, 7 , 17 Figure 3. 7173 Figure 3. Reactivity of mouse anti-ZIKBeHMR-2 immune sera on ZIKV antigens. In (A), indirect ELISA on pooled immune sera (n = 5) of adult BALB/c mice that received one (1st dose) or two (2nd dose) doses of ZIKBeHMR-2 (5 log PFU) with an interval of 6 weeks. Inactivated ZIKBeHMR-2 virus particles served as capture viral antigen. Data are presented as mean ± SEM of three independent experiments. Statistical significance between 1st dose and 2nd dose values is indicated. In (B), Vero cells were infected during 24 h with ZIKBeHMR-2 (ZIKV) at m. o. i. of 1 or mock-infected (m. i. ) and then lysed with RIPA lysis buffer. Immunoblot assay on cell lysates was performed with pooled immune sera of mice (ZIKV antisera) that received one or two doses of 5 log PFU of ZIKBeHMR-2 with an interval of 6 weeks.
In four samples of A. vespertilionis from Hungary, the same Rickettsia genotype was identified, which was reported from bat soft ticks collected in France (GenBank: JN038177, see Table  2 )  [12]. More importantly, in one A. vespertilionis from Hungary rickettsial DNA was detected, which in the amplified part of the gltA gene had 99. 9-100% sequence identity (depending on the nucleotide at position 679: C or T) to sequences of R. africae from Ethiopia (GenBank: CP001612) and from migratory bird fleas reported in neighboring Slovakia (GenBank: HM538186)  [13]. Two other markers were also successfully amplified from this sample: the 17 kDa gene sequence was identical with that of several Rickettsia species, whereas the OmpA sequence showed 2 bp differences from that of R. africae (Table  2 ).
Interestingly, the OmpA sequence from this A. vespertilionis was identical with that of the Rickettsia strain "Atlantic rainforest" (GenBank: MF536975  [14] ) and Rickettsia sp. "Atlantic rainforest Aa46" (GenBank: KY113110  [15] ), which represent a genetic variant of the human pathogen R. parkeri  [14, 15]  detected so far only in the New World. Nevertheless, we consider the species detected in A. vespertilionis to belong to R. africae because of the following four reasons: (i) the gltA gene is a reliable genetic marker for species identification and phylogenetic comparison of rickettsiae  [13, 16] ; (ii) R. africae was identified based on this gene in previous studies (e. g. [13] ); (iii) the gltA phylogenetic analysis confirmed that the rickettsial genotype from A. vespertilionis collected in Hungary clustered with R.
africae, but apart from R. parkeri (Fig. 1 ); and (iv) the OmpA gene of the type strain of R. parkeri (GenBank: U43802) was only 98. 3% (469/477 bp) identical with the OmpA sequence obtained here. The soft tick containing the R. africae-like DNA was collected from Myotis dasycneme, which occurs north of the Mediterranean Basin and is a facultative, middle distance migrant bat species, not known to move between Europe and Africa  [1]. Therefore, this result implies the autochthonous occurrence of a R. africae-like genotype in Europe. In the phylogenetic analysis, this genotype was clearly separated (with moderate, 72% bootstrap support value) from the Rickettsia sp. from A. vespertilionis reported in France (Fig. 1 ). In addition, R. hoogstraalii was identified in a soft tick from South Africa (Table  2 ).
This rickettsia has only been reported from Europe and North America  [17] , therefore its occurrence in Africa is new. Similarly, R. lusitaniae was formerly only reported in Europe (Portugal)  [18]  and Central America (Mexico)  [19] , the latter being confirmed in the present study (Table  2 ). However, a gltA genotype highly similar to R. lusitaniae (1 bp difference from JQ771933, i. e. 99. 9% identity) was also shown here, for the first time, to occur in Asia (China) (Table  2 ). The level of OmpA sequence divergence of this Chinese isolate (MH383149) was the same (3 bp) from R. lusitaniae in Portugal (JQ771935) and from R. lusitaniae in Mexico (GenBank: KX377432). In summary, bat soft ticks contained the DNA of three Rickettsia species from the spotted fever group (SFG), and two further ones from the Rickettsia felis group (RFG) (Fig.
1 ). Anaplasma phagocytophilum DNA was detected here in the hard tick species, I. simplex, in both Hungary and Romania. Previously, Anaplasma sp. DNA was also shown to be present in bat feces in Hungary (GenBank: KP862895). This low prevalence in bat ticks, suggests that bats may be susceptible to this pathogen, but most likely play a subordinate (if any) role in the epidemiology of granulocytic anaplasmosis in the evaluated region. Bartonellae associated with bat ectoparasites, including ticks, have been reported for the first time in Hungary  [10]. Based on high Ct values of the majority of bartonella-positive samples here, sequencing was only possible from two hard ticks (one I. ariadnae and one I. vespertilionis; Table  2 ). Based on two genetic markers (gltA and ITS), Bartonella sp. "Ia23" from I.
ariadnae was relatively (Table  2 : 98. 2-98. 7%) similar to Bartonella sp. isolates detected in bats (My. emarginatus) in Georgia, Caucasus  [20, 21]. In I. vespertilionis, known to feed on humans  [3] , Bartonella sp. "Iv76" was shown to be present (Table  2 ). The gltA sequence of this genotype was 100% (317/317 bp) identical to "Candidatus Bartonella hemsundetiensis", reported from Finland  [22]  (GenBank: KR822802, Table  2 ), but only 99. 7% (316/317 bp) identical to Bartonella sp. isolates (GenBank: KX300127, KX300131, KX300136) detected in bats (My. blythii) in Georgia, Caucasus  [20]. The ITS sequence of Bartonella sp. "Iv76" was 95. 1% (291/306 bp) and 93. 8% (287/306 bp) identical to Bartonella sp. isolates (Gen-Bank: MF288124 and KX420717, respectively) from bats (My. blythii and My.