dataesr/scientific-paragraphs-categorization huggingface dataset:
e. the monomers' stretching motions. The PES used for the accurate quantum approach calculations is based on a rigid-monomer approach for the CN -and H 2 (D 2 ) diatomic subunits (monomers) and thus no information on the stretching modes of the diatoms (ν 4 , ν 5 ) can be obtained. So far no 6D PES is available to calculate the higher energy stretching bands. As a consequence an approximate approach was used to calculate the CN -and H 2 (D 2 ) stretching vibrational bands of the complexes. The 1D potential energy of the CN -or H 2 monomers is calculated at the CCSD(T)-F12b/AV5Z level of theory using the MOLPRO program suite  [30, 31]  for a set of values of the bond length of one monomer while other coordinates are kept constant at their equilibrium values. This means that we compute a 1D cut through the 6D PES.
Acknowledgments We gratefully acknowledge the support of  Radboud University  and of  NWO  for providing the required beam time at the  FELIX Laboratory , and the skilful assistance of the FELIX staff. We thank the  Cologne Laboratory Astrophysics group  for providing the FELion ion trap instrument for the current experiments and the  Cologne Center for Terahertz Spectroscopy  funded by the  Deutsche Forschungsgemeinschaft (DFG , grant  SCHL 341/15-1 ) for supporting its operation.
M. Godet Methods A set of four methods designed for strategic foresight processes. They are methods that support the process of constructing possible scenarios and defining a strategy. Together they are designed to conduct a "strategic foresight" process  (Godet and Durance, 2011). They are based on other existing methods or techniques, to which a dynamic of consultation and interaction between experts is added, as well as certain conceptual elements from the scenario method. Each method is also associated with software for information processing and analysis. The four methods that make up this set of methods are: MIC MAC (a cross-impact matrix version), MACTOR (a stakeholder analysis version), MORPHOL (a morphological analysis version), SMIC-PROB-EXPERT (a Delphi version).
The role of external cooperation agreements signed by the Nicaraguan government that facilitated the implementation of the instruments should be highlighted, as is the case of the Treaty signed within the framework of the Bolivarian Alliance for the Peoples of Our America (ALBA), ratified in 2008, which aimed to jointly formulate and implement SSAN instruments. Within this framework, Venezuela purchased Nicaraguan products in exchange for financial support for the agricultural sector  (McBain-Haas & Wolpold-Bosien, 2008) , which was funded by the PPA until 2016, along with other funds allocated from the government budget and other donors  (Kester, 2009). The funds to finance the PINE came from the State budget, the European Union, the WFP, and other bilateral cooperation and international organizations.
It is important to remember that the target population of each program is different, so differences in the rates observed may reflect a smaller target population. In the case of Qali Warma, children are the target population, so the percentage of households without children are not covered by this program. The opposite is true for Vaso de Leche, a program with virtually no restrictions. Complementing this information, data relating to the budgets of social programs show trends in the public funds invested in each program. This gives us a better idea of their importance at State level and in the eyes of the political players concerned. Figure  10. 5 shows the total budget for social food programs. In order to fully understand the graph, it is important to remember that the national budget has increased steadily since 2004.
The changes in the total amount budgeted each year for social food programs partially coincide with the phases identified in program coverage. In the first phase, the PRONAA budget increased steadily, but a lack of comparable data prevents the identification of a clear trend for the other programs. The following phases show a stagnation in the Vaso de Leche program and a slight but steady decrease for the soup kitchens. This contrasts with a steady increase for PRONAA, carried on by Qali Warma, further accentuating the positive trend, except for a period of one year. These budget data show that the State has given priority to school-based programs in its food security strategy, while programs with a more universal approach have been downgraded over time. The level of coverage and the budgets both show that Vaso de Leche and soup kitchens were not a priority in food policy, this being illustrated by a gradual reduction in their capacity.
FCWs have been described since the late 1970s, but thorough characterizations have only been performed in few species, such as Aspergillus fumigatus, Saccharomyces cerevisiae, Candida albicans or Cryptococcus neoformans  [4]. In these model organisms, new GT activities (i. e. , β-1,3-1,4-glucan synthase) and new structural descriptions continue to expand our knowledge of this important fungal structure  [5, 6] , but the cell wall composition and function in other fungal species remain largely unknown. The current model of cell wall polysaccharides biosynthesis in fungi proposes that it occurs at the plasma membrane, in the apical regions where the mycelium expand and in the distal regions where septa are built  [7, 8]. Among the 115 families of GTs inventoried in the carbohydrate active enzymes database (CAZy, http://www.
Among the 115 families of GTs inventoried in the carbohydrate active enzymes database (CAZy, http://www. cazy. org, August 2022  [9] , 4 families group together enzymes able to produce polysaccharides: the GT2, GT5, GT48 and GH16 families. The polysaccharide synthases in these families are iterative enzymes that catalyze the transfer of sugar residues to a carbohydrate acceptor from an activated sugar-nucleotide donor  [10]. By doing so they produce a nascent polysaccharide chain, and their supposed localization at the plasma membrane would allow the extrusion and extension of this chain into the FCW. The GT2 family contains the chitin synthases (CHS) that produce chitin, a hallmark polymer of the fungi that plays a structural role in the FCW  [11]. The other fungal GT2s have received less attention so far.
S1 ). Membrane (Mb), Oxydative (ox). (B) Predicted secondary structure of BcCps1 inside a membrane (dotted line), showing the three TM helices, the catalytic and W domains with the conserved amino-acids (capital letters) and the non-TM hydrophobic helix (M). (C) Predicted 3D model of the B. cinerea BcCps1 protein within a lipid bilayer (blue and red tilted circles). The structure was predicted using AlphaFold Monomer v2. 0 and the 3D representation was obtained using the OPM prediction tool (https://opm. phar. umich. edu/ppm_server2, 1 May 2022). The TM helices and the non-TM hydrophobic helix are colored in pink. The other protein fragments are colored following the AlphaFold confidence code (blue (high), yellow (medium), orange (low)).
Bioinformatic Analyses To construct the fungal Cps1-like protein family, the Cps1 protein of C. neoformans (CNAG_04320) was used as query in the Blastp program  [33]. One hundred and fifty-seven different proteomes available on EnsemblFungi (https://fungi. ensembl. org, 1 May 2022) or on the NCBI website (https://blast. ncbi. nlm. nih. gov, 1 May 2022) were searched. Every protein sequence with a significant blast hit and a sufficient coverage (>200 amino-acid) was considered as a Cps1-like protein. Data from the similarity search were used to select the species for the phylogenetic analyses: every Leotiomyceta species and 2-4 species for the other taxonomic subphyla or classes were chosen. For the chosen species, every Cps1-like protein was included. Amino-acid sequences were aligned using MAFFT with the L-INS-i algorithm  [34].
The mass spectrometer operated in positive ion mode at a 1. 9 kV needle voltage. Data were acquired using Xcalibur 4. 4 software in a data-dependent mode. MS scans (m/z 375-1500) were recorded at a resolution of R = 120,000 (m/z 200), a standard AGC target and an injection time in automatic mode, followed by a top speed duty cycle of up to 3 s for MS/MS acquisition. Precursor ions (2 to 7 charge states) were isolated in the quadrupole with a mass window of 1. 6 Th and fragmented with HCD@28% normalized collision energy. MS/MS data was acquired in the Orbitrap cell with a resolution of R = 30,000 (m/z 200), a standard AGC target and a maximum injection time in automatic mode. Selected precursors were excluded for 60 s. Protein identification and label-free quantification (LFQ) were performed in Proteome Discoverer 2.
5. The MS Amanda 2. 0, Sequest HT and Mascot 2. 5 algorithms were used for protein identification in batch mode by searching against the ENSEMBL B. cinerea ASL83294v1 database (13,749 entries, release 53). Two missed enzyme cleavages were allowed for trypsin. Mass tolerances in MS and MS/MS were set to 10 ppm and 0. 02 DA. Oxidation (M) and acetylation (K) were searched as dynamic modifications and carbamidomethylation (C) as static modification. Peptide validation was performed using the Percolator algorithm and only "high confidence" peptides were retained, corresponding to a 1% false discovery rate at peptide level  [45]. Minora feature detector node (LFQ) was used along with the feature mapper and precursor ions quantifier. The quantification parameters were selected as follows: (1) unique peptides, (2) precursor abundance based on intensity, (3) normalization mode: total peptide amount, (4) protein abundance calculation: summed abundances, (5) protein ratio calculation: pairwise ratio based, (6) imputation mode: low-abundance resampling and (7) hypothesis test: t-test (background based).
Interestingly, an increased expression of Bccps1 was measured between 24 h, 48 h and 72 h of growth, and this suggests that BcCps1 could play an important role in the unfolding of the second phase of hyphal development in B. cinerea. As proposed by King et al. [28] , Cps1 could produce a cell wall (or extracellular matrix) polysaccharide necessary for hyphal growth on solid matrices. In A. nidulans and A. fumigatus, a decreased adherence capacity has been documented in cps1 mutants (Figure  1 ). Interestingly, young mycelia produced in 24 h by conidia of the ∆Bccps1 strain exhibited no adhesion default, while explants of the mutant mycelium showed a 40% loss in adhesion to leaves (and also to plastic surfaces, data not shown). A conserved role for Cps1 in mycelial adherence might therefore be considered.
Contributions: Conceptualization, C. B. , M. C. and M. B. ; methodology, M. B. , C. B. , C. R. , M. C. and I. R. G. ; software, I. R. G. ; validation, C. B. , M. C. , I. R. G. and N. P. ; Visualization, F. -X. G. ; investigation, M. B. , C. R. , J. -W. D. and I. R. G. ; writing-original draft preparation, M. B. and C. B. ; writing-review and editing, M. B. , M. C. , I. R. G. , N. P. and C. R. ; supervision C. B. , M. C. , N. P. and I. R. G. All authors have read and agreed to the published version of the manuscript. Funding: Matthieu Blandenet was supported by the CIFRE scholarship 2018/1683 (Convention Industrielle de Formation par la Recherche) funded by the French Ministry of Industry and Bayer SAS. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable.
Author Contributions: Conceptualization, C. B. , M. C. and M. B. ; methodology, M. B. , C. B. , C. R. , M. C. and I. R. G. ; software, I. R. G. ; validation, C. B. , M. C. , I. R. G. and N. P. ; Visualization, F. -X. G. ; investigation, M. B. , C. R. , J. -W. D. and I. R. G. ; writing-original draft preparation, M. B. and C. B. ; writing-review and editing, M. B. , M. C. , I. R. G. , N. P. and C. R. ; supervision C. B. , M. C. , N. P. and I. R. G. All authors have read and agreed to the published version of the manuscript. Funding: Matthieu Blandenet was supported by the CIFRE scholarship 2018/1683 (Convention Industrielle de Formation par la Recherche) funded by the French Ministry of Industry and Bayer SAS. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable.
In this phylum, RM systems are ubiquitous and found in 74. 2 % of the genomes. Their distribution patterns are very diverse, with more than 2040 MTases in the Restriction Enzyme database (REBASE) that were predicted from 387 sequenced mycoplasma genomes  [26] , and an average of five predicted MTases per genome. While this highlights the importance of MTases and RM systems in mycoplasmas, their role in cellular regulation and HGT has yet to be addressed. Genome methylation in bacteria is an area of great interest because of its broad implications for evolution, biology, and virulence  [27]. The development of real-time single molecule sequencing (SMRT-seq) has allowed the detection of methylated bases, whether on a plasmid or bacterial chromosome, and is particularly suited to small mycoplasma genomes.
45 and quantified. Conditioned Sequencing Primer V2 was annealed to the size selected SMRTCell library. The annealed library was then loaded on four SMRTCells using P6-C4 chemistry on the PacBio RSII sequencer with 360 min movies. Sequencing reads were de novo assembled using the Hierarchical Genome Assembly Process (HGAP) protocol RS_Assembly v. 3 implemented in SMRT Analysis software v. 2. 3 with default parameters (https://smrt-analysis. readthedocs. io/en/latest/SMRT-Pipe-Reference-Guide-v2. 3. 0/). An average of 1×10 5 reads per strain were obtained, corresponding to an average of 100X coverage. Open reading frame (ORF) prediction and automatic annotation were performed using Rapid Annotation using Subsystem Technology (RAST)  [31]. GenBank editing and manual expert inspection were performed using Artemis 16.
Counterparts of contig extremities in one genome were 100 % methylated in the other and vice versa, confirming the technical detection bias. This was possible because the organisation and sequence of the two genomes were very similar. The 5632 methylome was evaluated after 24 or 48 h of growth to define the impact of the growth phase on the methylation status. SMRT analysis detected the same methylated motifs in the exponential phase (24 h) and the stationary phase (48 h), with a decrease of approximately 20 % of the m6A methylated sites at 24 h and 40 % for m4C (data not shown). Illumina sequencing of bisulphite-treated chromosomal DNA (termed BS-seq) was performed to reliably detect 5-methylcytosine (m5C). Two reference strains 5632 and PG2 were used for BS-seq, and the 5632-like strains 13377, 4025, 14668, and 4055 were chosen based on Dcm encoding gene detection by blast analyses against the Type II m5C methyltransferase genes databases (dcm database) available on REBASE (Table  S1 )  [26].
BS-seq was performed at Novogene (UK) and included DNA fragmentation into 200-400 bp using Covaris S220, DNA libraries, and bisulphite treatments (EZ DNA Methylation Gold Kit, Zymo Research, CA, USA). Libraries were sequenced using a HiSeq 2500 platform, which enables paired-end sequencing and generates 125 bp long reads. The BS-seq raw data sets were analysed with Bismark software (v0. 22. 1) to detect m5C modifications and identify corresponding methylation motifs. Raw reads were mapped on the finished reference genomes using Bismark Bisulfite mapper v0. 22. 1 (https://www. bioinformatics. babraham. ac. uk/projects/bismark/) in non-directional mode. The methylated cytosines were extracted with bismark_methylation_extractor using --comprehensive, --merge_non_CpG, and --CX options. BedGraph and cytosine_report outputs were generated.
The sequence context was analysed by extracting ±10 bp around each identified methylated cytosine. The MEME-ChIP suite (Motif Analysis of Large Nucleotide Datasets, v5. 1. 0; http://meme-suite. org/) was used to determine a context consensus at methylated positions, and Artemis 16. 0. 0 [32]  was used for visual inspection. RM system-encoding genes and methylation motif(s) co-occurrence were addressed by comparative genome analysis. All identified ORFs were searched for similarity to known RM systems using BLASTP and the 5632 and PG2 reference genomes and the REBASE database (http://rebase. neb. com/rebase/rebase. html) [26]. Significant BLASTP hits were selected using a cut-off e-value of <0. 001 and exhibiting over 30 % similarity across at least 80 % of the sequence length. The active MTases were also analysed by BLASTP on NCBI (https://blast.
M. agalactiae transformation was performed as previously described, with some minor modifications  [36]. Briefly, a culture containing 10 8 c. f. u. ml -1 was centrifuged at 10 000 g at 4 °C for 20 min. The pellet was washed twice with sterile cold DPBS (Dulbecco's phosphate-buffered saline, Sigma-Aldrich, MO, USA) 1X and centrifuged at 10 000 g at 4 °C for 10 min. After centrifugation, cells were resuspended in 375 µl of cold 0. 1 M CaCl 2 and incubated on ice for 30 min. Cold CaCl 2 -incubated cells (100 µl) were gently mixed with 10 µg of yeast tRNA (Life Technologies, CA, USA) and 3 µg of pMT85Gm MTase gene-harbouring plasmids or the pMT85Pur plasmid, which carries a puromycin resistance pac gene (GenBank: ACR82772. 1). One millilitre of 50 % PEG 8000 (Sigma-Aldrich) was then applied for 1 min, and the reaction was stopped by the addition of 5 ml SP4 liquid medium.
Correlation analyses between HGT and RM systems in M. agalactiae Mobile elements present in M. agalactiae genomes were identified after annotation using the RAST server  [31]. CDS annotated 'mobile elements' by RAST but also integrases, transposases, and prophage elements were counted, as well as genes identified as ICE by blast analyses against 5632 ICEA (e-value less than to 10 -3 ). ICEs were considered when at least three of their essential components that are conserved across mycoplasma ICEs (CD5, CDS17, and CDS22) were present within a 30 kb DNA fragment. These were CDS5 and CDS17 encoding the conjugation factors TraE and TraG and CDS22 that encodes the DDE transposase  [12]. Correlation analyses were performed with GraphPad Prism version 6. 00 (GraphPad Software, San Diego, CA, USA).
‡This m4C fraction was only detected with SMRT sequencing, which explains why it is lower than those obtained with the same motifs detected by BS-seq (see above). §Type III MTase WT, Wild-Type; Fract, fraction nDetect: number of detected methylated motifs nGenome: number of motifs present in the genome for strain 13377. One particular feature of this group was the presence of Type II m5C MTase genes, as revealed by blast analyses against the dcm REBASE database  [26] , with that of 4055 being truncated (Table  S1 ). In agreement with this observation, m5C methylations were detected in all 5632-like strains except 4055 and PG2-like strains (Fig. 1 ).
The analysis involved 27 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 36 positions in the final dataset. Evolutionary analyses were conducted in mega7  [67]. Finally, hsdR was disrupted in PG2, as well as in the 4021, 4210, 5276, and 14634 genomes (Fig. 2b ). This was due to the insertion of two nucleotides in a poly(A) region located in the middle of the gene that resulted in a premature stop codon (  [19]  and this study) and a severely truncated product. This implies that hsdR is not required for Type I methylation to occur in M. agalactiae. Furthermore, a complex of HsdS and HsdM was shown to be sufficient in E. coli for the preferential methylation of the target sequence at adenine residues, while the three Hsd subunits, M, S, and R, were required for DNA cleavage  [39].
Notably, 5′-GA m6 NTC-3′ and 5′-GA m6 TC-3′ were the most prevalent methylated motifs found among the PacBio SMRT sequenced Mycoplasma sp. of the REBASE Genomes database. This supports that these epigenetic modifications play an important biological role in mycoplasmas. A third Type II m6 methylated site, the 5′-GA m6 TGC-3′ motif, was detected in two strains, 4025 and 14668, and blast against the REBASE database retrieved a cognate MTase gene only in the genome of these two strains (see Fig. 3 ). These MTase coding sequences displayed 96 % identity with a putative Type II N6-adenine DNA methyltransferase of M. bovis PG45 (MBOVPG45_0722), which recognised the 5′-GCA m6 TC-3′ motif, the reverse complement of GATGC, and corresponded to the SfaNI prototype of Streptococcus faecalis (renamed Enterococcus faecalis) (REBASE database  [26] ).
This motif was associated with the MAGa3950 MTase of 5632, homologs of which were encoded in 13377, 4025, and 14668 but was truncated in 4055 (Fig. 3 ). The three other cytosine-methylated sites, 5′-C m5 G-3′, 5′-C m5 ATG-3, and 5′-C m5 CTC-3′, were all detected in strain 13377 but not in other strains of our panel (Figs  1  and 3 ). The case of the 5′-C m5 G-3′ motif is addressed below, in the section dedicated to CpG methyltransferase. blast analyses using the REBASE database retrieved a cognate MTase gene for the other two motifs (Table  3  and Fig. 3 ). Interestingly, BS-seq data showed that the 5′-C m5 CTC-3′ was methylated on the cytosine, while its reverse complement, 5′-GA m6 GG-3′, was shown by SMRT analysis to be methylated on the adenine and was further classified as a Type II motif (see above).
The dcm MTase responsible for 5′-C m5 CTC-3′ methylation in 13377 is J7894_00205 (Fig. 3 ) and had 92 % identity with M. bovis M. MboH1ORF688P, a Type II cytosine-5 DNA methyltransferase, predicted to recognise the CCTC motif. Interestingly, the non-mycoplasma MTase that had the most significant homology with 13377 MTase J7894_00205 (63 % identity) was Carnobacterium alterfunditum M. Cal5972II, annotated as a m5C MTase. Yet, PacBio data suggested that this MTase is responsible for both the GA m6 GG and T m6 CCTCY methylations. In their REBASE comments, the authors suggested that methylation of this second motif indeed involved m5C on the complementary strand but that this modified base was not being called correctly (see REBASE PacBio data for M. Cal5972II). By analogy, our data strongly suggest that a single MTase, MTase J7894_00205, is responsible for the methylation of the two asymmetric motifs 5′-C m5 CTC-3′ and 5′-G m6 AGG-3 (Fig.
3 ), was identified by blast analysis against REBASE dcm databases (Table  S1 ). This coding sequence displayed 40 % identity with the MSssI and MmpeI CpG MTases found in two other Mollicutes, Spiroplasma spp. strain MQ1 and Mycoplasma penetrans, respectively  [48, 49]. Spiroplasma MSssI was the first prokaryotic MTase described that specifically and exclusively methylates the 5′-CG-3′ sequence  [48]. In this Mollicute, as in M. agalactiae, these CpG MTases methylate both strands, while their eukaryote counterparts preferentially generate hemimethylated DNA  [52]. This is the first description of a ruminant pathogenic mycoplasma strain harbouring an active CpG MTase. Comparative genome analysis revealed that all M. agalactiae genomes contained CpG MTase homologs (Fig. 3 ). A common feature of these coding sequences is that they all displayed a poly(G) region having between six and 11 'G' located in their middle (Fig.
Role of M. agalactiae RM systems in HGT In prokaryotes, RM systems are known to provide a defence against foreign incoming DNA. Mycoplasmas are notoriously difficult to manipulate genetically, and this may in part be attributed to RM systems that prevent the entry of unprotected DNA. For instance, the 5632 strain can be efficiently transformed by specific mycoplasma vectors carrying the selective gentamicin or tetracycline resistance marker (pMT85, pminiO/T)  [35]. Replacing these with the puromycin resistance gene (pac) designed by Algire and Lartigue (2009)  [55]  was successful in generating transformants using PG2 but not 5632. A close examination of the pac gene sequence revealed the presence of an Hsd-5632 recognition motif (5′-A m6 YC(N)5KTR-3′) at the beginning of the gene (position 15/600, GenBank accession no.