dataesr/scientific-paragraphs-categorization huggingface dataset:
pertussis (alleles  1, 4, 7, 8, 9, 42, 43, 44, and 47). Figure  S1  presents the polymorphisms of the 47 different cyaA alleles. Focusing on the nine alleles from B. pertussis and considering the 603 isolates for which data are publicly available for this locus in the database, we found that 90. 0% of the isolates (i. e. , 543/603) had allele 4, as in the Tohama reference strain; 7. 6% (i. e. , 41/603) had allele 7, as in the 18323 reference strain; 1. 5% (i. e. , 9/603) had allele 9; 0. 9% (i. e. , 6/603) had allele 42; and the remaining alleles  (8, 43, 44, and 47)  were each present in a single isolate (1/603). Four of these nine alleles are synonymous and five encode proteins with amino-acid substitutions. Non-synonymous alleles are nevertheless infrequent, because such alleles were found in only 2.
9% (18/603) of isolates, most of which were collected between 1960 and 1995. Data for only five isolates of B. parapertussis were publicly available from the Bordetella MLST database, including the human reference strain Bpp12822 and the ovine reference strain Bpp5. Only three different alleles were identified for B. parapertussis: two in human isolates (alleles 3 and 5) and one in an ovine isolate (allele 37). We extended this analysis to recently obtained human B. parapertussis isolates, which were also all found to contain allele 3, illustrating the high degree of conservation of the cyaA gene in B. parapertussis isolates (data not shown). We found that two polymorphic sites resulting in amino-acid substitutions were specific to allele 37 of the ovine Bpp5 (M37V and S560A) strain, one was specific to allele 5 (T1139S), and two others were specific to human isolates (i.
Discussion The AC-Hly protein is encoded by the cyaA gene (BP0760 in B. pertussis, BPP0321 in B. parapertussis). Unlike other virulence genes encoding vaccine antigen proteins displaying allelic variation (such as the ptxA and prn genes), cyaA is stable, with few polymorphisms detected in the two species. Most B. pertussis isolates carried the same cyaA allele as the Tohama reference strain (i. e. , allele 4 in the PubMLST database) and all human B. parapertussis isolates carried the same allele as the 12,822 reference strain (i. e. , allele 3). This finding is consistent with the results of the SNP study by Bart et al. [22] , which reported only two different SNPs within cyaA, relative to the Tohama strain, in only seven isolates (corresponding to alleles 7 and 4) from a set of more than 300 isolates collected from around the world in different vaccination eras.
We loaded 0. 5 µg of peptides onto a C4 trap column (Acclaim™ PepMap™ 300, 5 µm, 300 Å, 300 µm i. d. × 5 mm-Thermo Fisher Scientific, Waltham, MA, USA) and separated them on a custom-built 20 cm C4 column (5 µm particles, 300 Å pore size, ReproSil-Pur 300 C4, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany) with a one-step gradient from 2. 0% to 90% solvent B (80% ACN/0. 1% FA) over 43 min, at a flow rate of 300 nL/min over 78 min. Mass spectrometry (MS) data were acquired with Xcalibur software in survey scan (300-1700 m/z) mode and were analyzed in the Orbitrap mass analyzer at a resolution of 60,000. Five ETD fragmentations were then analyzed in the linear ion trap. The AGC targets for MS and MS/MS scans were set to 1E6 and 5E3, respectively. The isolation width was set at 2. 0 m/z and the activation time was set at 150 ms.
Selected ions were dynamically excluded for 20 s. Raw data were analyzed with MaxQuant software version 1. 5. 1. 2 [49]  and the Andromeda search engine  [50]  against the Bordetella pertussis and Bordetella parapertussis UniProt databases, containing 3258 proteins and 4161 proteins, respectively. The digestion mode was set to trypsin and a maximum of two missed cleavages were allowed. N-terminal acetylation and methionine oxidation were allowed as variable modifications and cysteine carbamidomethylation as a fixed modification. For the PTM analysis, additional N-acetylated lysines (myristoyl-4H, myristoleylation, myristoylation, palmitoleylation, palmitoylation) were considered as variable modifications. The minimum peptide length was fixed at five amino acids and the required false discovery rate was set at 1% for PSMs and proteins.
Statistical analysis All continuous variables were reported as mean + -S. D. and categorical variables as number (percentage). Release of MP and cytokines over all sampling time points after PCI was quantified by area under the curve (AUC) + -S. E. M. Total AUC for each group or levels at specific time points between and within groups were compared using Student's t test continuous variable associations, including Gensini score and MP levels were assessed with linear regression. All tests were two-tailed with acceptable type 1 error set at P<0. 05. Statistical analyses were performed in Graph Pad Prism Version 6. 0 (Graph Pad Software, LA Jolla, CA) and SPSS for Macintosh version 23 (IBM, Armonk, NY).
Background: This study aims to assess the role that Pleistocene refugia, rivers and local habitat conditions may have played in the evolutionary diversification of three central African duiker species (Cephalophus dorsalis, C. callipygus and Philantomba monticola). Genetic data from geo-referenced feces were collected from a wide range of sites across Central Africa. Historical patterns of population genetic structure were assessed using a ~650 bp fragment of the mitochondrial control region and contemporary patterns of genetic differentiation were evaluated using 12 polymorphic microsatellite loci. Results: Mitochondrial analyses revealed that populations of C. callipygus and P. monticola in the Gulf of Guinea refugium are distinct from other populations in west central Africa. All three species exhibit signatures of past population expansion across much of the study area consistent with a history of postglacial expansion.
Mitochondrial DNA sequencing and microsatellite genotyping A fragment of the mitochondrial control region of approximately 650 base pairs (bp) in length was amplified and sequenced following previously optimized conditions  [62]. Mitochondrial sequences from the three most commonly sampled species (C. dorsalis, C. callipygus and P. monticola) were collapsed into representative haplotypes using the program COLLAPSE v. 1. 2 (available from http://collapse. sharewarejunction. com/). An intraspecific Minimum Spanning Network (MSN) was created with the program NETWORK v. 4. 6. 1. 0 [63]  in order to visualize relationships between haplotypes. Fecal samples from these three species were also genotyped at 12 polymorphic microsatellites assembled into three multiplex reactions of four loci each, following previously optimized conditions  [61].
A quantitative PCR assay  [55]  based on a 96 bp of the vertebrate p53 tumor suppressor gene  [64]  was used to assess nuclear DNA quantity and to determine the number of PCR replicates required to obtain a reliable genotype  [65]. In order to minimize the risk of allelic dropout, duiker fecal samples with concentrations of 25-49. 9 pg/μl of nuclear DNA or higher were genotyped a minimum of four times and those of 50 pg/μl of DNA or more were typed a minimum of three times  [55]. Samples with concentrations <25 pg/ μl were not genotyped because of the risk of allelic dropout. The program ARLEQUIN v. 3. 5. 1. 3 [66]  was used to calculate expected heterozygosity, and deviations from Hardy Weinberg Equilibrium (HWE) and Linkage Equilibrium (LE) within each population. The significance of HWE and LE tests was assessed using an extension of Fisher's exact probability test with contingency tables  [67]  and a test analogous to Fishers's exact test extended to a triangular contingency table of arbitrary size, respectively  [68].
Species identification Fecal samples were identified to species level using an established mitochondrial control region diagnostic  [62]. We also carried out a phylogenetic analysis to assess whether the highly divergent mitochondrial sequences obtained from samples collected in the Nigerian highlands were from a new species of dwarf duiker P. walteri described by Colyn and colleagues  [71]. To test this hypothesis, a 402 bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified from fecal samples using published primers  [71]. These sequences were then aligned with COI sequences from P. maxwelli (GenBank HQ644099-644100, HM144021, HM 144027), P. monticola (GenBank GQ144522-144545, HM144015, HM144016, HM144020, HM144022, HM144024, HM144026), P. walteri (GenBank HM144017-144019) and an outgroup species C.
callipygus using the program MEGA7  [72]. A neighbor-joining tree was then inferred using the Tamura-Nei distance  [73]  and a bootstrap consensus trees was constructed from 1000 replicates. Although the majority of fecal samples could be identified on the basis of their mitochondrial DNA, fecal samples from NGO could not be sequenced due to their poor quality and were instead identified on the basis of their multi-locus microsatellite genotypes using a cluster-based analysis implemented in the Bayesian program STRUC-TURE v. 2. 3. 3  [74]. To do this, multi-locus microsatellite genotypes were first obtained from reference samples of all six duiker species known to occur in sympatry in the study area (P. monticola, C. callipygus, C. dorsalis, C. silvicultor, C. leucogaster and C. nigrifons).
Pleistocene refugia hypothesis All mitochondrial DNA analyses only considered sites where three or more individuals had been sampled. In cases where sampling was relatively limited, sequences from the following nearby sites (< 100 km) were pooled: (1) TAK, NIG and BAM in the Gulf of Guinea refugium; (2) MTA and MM within the Equatorial Guinea and northern Gabon refugium; (3) ND and CO within the Parc National de Nouabelé Ndoki. A Spatial Analysis of Molecular Variance (SAMOVA) of each species was carried out using the program SAMOVA v. 1. 0 [78]  where the number of groups (K) was varied from 2 to 5. The program ARLEQUIN was also used to conduct a series of Analyses of Molecular Variance (AMOVA) in order to test the role that putative refugia have played in shaping regional genetic structure.
A Mann-Whitney-Wilcoxon test was used to test the hypothesis that molecular diversity was higher in candidate refugial sites than in intervening sites. Evidence for past demographic expansion within regional groups identified by SAMOVA analyses was assessed using Fu's Fs and Tajima's D, as implemented in the program ARLEQUIN. Patterns of nuclear genetic structure were investigated using STRUCTURE. The total number of steps in the Markov Chain Monte Carlo (MCMC) and burn-in were set respectively to one million and 100,000 generations with 10 separate simulations for each value of K. The admixture model was used and allele frequencies among populations were assumed to be correlated. Clustering of K populations within species was used to assess whether population differentiation was structured by putative refugia.
Riverine barrier hypothesis An AMOVA analysis of mitochondrial DNA sequence datasets was also carried out to test the effects of the Ogooué and Sanaga rivers on genetic structure of all three species. To do this, sites immediately flanking the north and south banks of each river were placed into two separate groups. To test the combined effects of both Sanaga and Ogooué River bodies, the entire dataset was divided into three groups made up of: (a) sites to the north of the Sanaga; (b) between both rivers and (c) to the south of the Ogooué. In the case of C. callipygus, we could not test the effect of the Sanaga River barrier due to insufficient sample sizes. STRUCTURE analyses of nuclear genetic data were also carried out to examine whether genetic variation was partitioned by either river.
The modeled species distributions were then converted into binary maps where genetic variation was only projected onto areas where the species were predicted to be present. The second step then maps environmentallyassociated intraspecific variation across these inferred species distributions using the GDM framework  [82]. The environmental variables included in both MAXENT and GDM modeling procedures were similar to those used previously  [44]. These were comprised of a set of climate variables from the WorldClim database  [84]  at 30 arcsec resolution (~1 km), representing annual means, ranges, and seasonality in temperature and precipitation averaged over a 50-year period from 1950 to 2000 and a set of satellite remotely sensed variables comprising: a measure of vegetation biomass or surface moisture from the QuickScat archive for the year 2001  [85] ; elevation from the Shuttle Radar Topography Mission (SRTM); percent tree cover, computed from the Vegetation Continuous Field (VCF) for the year 2001.
These environmental variables and their database sources are listed in Additional file 1: Table  S2. In addition to these environmental variables a set of predictor variables were generated to model the effect of Pleistocene refugia and rivers. A GIS layer was first generated for two refugia; one north of the Sanaga River in the Gulf of Guinea and another south of the Sanaga River encompassing southern Cameroon, Equatorial Guinea, and northern Gabon  [12]. To create the GIS layer, the approximate center points of the refugia were coded as 0 and 1 respectively, and then an inverse distance weighted spatial interpolation in ArcGIS 9. 3 was to compute a raster layer in which the influence of a refugium is highest closest to its centroid, but diminishes further away from it. This layer thus represented divergence between populations in the two refugia, with subsequent dispersal away from each refugium in all directions and at the same speed, allowing for admixture where dispersing individuals would have met.
Mitochondrial DNA sequencing and microsatellite genotyping Owing to the high amount of variation in the control region dataset, sequences were collapsed into composite haplotypes prior to construction of haplotype networks. This collapse step left 16 composite haplotypes for C. dorsalis (sequences differing at ≤17 nucleotide positions), 27 for C. callipygus (sequences differing at ≤27 nucleotide positions), and 25 for P. monticola (sequences differing ≤31 nucleotide positions). Within C. dorsalis, most haplotypes were widespread across the study area except for haplotypes K-P which were restricted to single sites (Additional file 2: Figure  S2a  and Additional file 1: Table  S3 ). Similarly, C. callipygus did not reveal any obvious pattern of genetic structure, although haplotypes P, Q, S, T, V, X, Y, Z and Φ were also unique to specific sites (Additional file 2: Figure  S2b  and Additional file 1: Table  S4 ).
Lastly, within P. monticola, most of the NIG samples formed a cluster (haplotypes J, N, and R) that was highly differentiated from other sequences in the dataset (Additional file 2: Figure  S2c  and Additional file 1: Table  S5 ). Phylogenetic analysis of mitochondrial COI sequences showed that these samples are from P. walteri, the new duiker species described by  [71]  (Additional file 2: Figure  S3 ). The average pairwise Tamura-Nei distances (Additional file 3) were lowest between candidate P. walteri samples from NIG and diagnosed P. walteri samples (average 0. 012 base substitutions/site) and highest when compared to P. monticola (average 0. 111 base substitutions/site). These sequences were removed from any further mitochondrial DNA analyses. Other haplotypes within this species were also widespread with the exception of P, Q, S, U, W, X, and Y which were restricted to individual sites.
With respect to the microsatellite data, we detected very few instances of deviations from HWE (Additional file 1: Tables  S7-9 ) or LE (Additional file 1: Table  S10 ) after correcting for table-wise error. GIMLET analyses revealed that the minimum number of typed microsatellite loci needed to identify first-order relatives was five for C. dorsalis, six for C. callipygus, and four for P. monticola (Additional file 1: Table  S11 ). KINGROUP analyses found 11 out of 65 (C. dorsalis), 77 out of 310 (C. callipygus), and 82 out 332 (P. monticola) samples were first-order relatives of other individuals in the dataset. These individuals were subsequently removed from all further analyses.
Pleistocene refugia hypothesis There was no evidence for any obvious mitochondrial genetic structure within C. dorsalis (Table  2 , Additional file 1: Table  S12 ), although SAMOVA uncovered a significant among group variance component (F CT = 16. 31%; P = 0. 014) for three clusters comprising a riverine site in Cameroon (NKO), Gabon (OLO S ) and the rest of the dataset. SAMOVA analysis of C. callipygus identified a significant among group variance component (F CT = 18. 25%; P = 0. 007) comprising samples from the Gulf of Guinea refugium (NIG, TAK), central D. R. C. (SA) and all other sites. Partitioning the dataset into two groups comprising only the Gulf of Guinea refugium and all remaining sites also revealed a marginally significant among group variance component (Table  2 , Additional file 1: Table  S13 ).
Similarly, SAMOVA analysis of P. monticola identified a high among group variance component (F CT = 36. 831%; P = 0. 010) for three clusters comprising Gulf of Guinea refugium (NIG, TAK, BAM), Sanaga River mouth (EDEA) and elsewhere across the study region. Once more, partitioning the dataset into the Gulf of Guinea refugium versus all other sites revealed an among group component that was only marginally significant (Table  2 , Additional file 1: Table  S14 ). There was no evidence of significant genetic structure when samples were clustered by additional candidate refugia except in the case of P. monticola when a significant among group variance component was detected (F CT = 6. 41%, P = 0. 019) when sites were grouped according to the Sanaga River basin (Table  2 , Additional file 1: Table  S14 ).
86, P = 0. 036) and P. monticola (Fu's Fs = -24. 41, P < 0. 001). STRUCTURE analyses of C. dorsalis indicated that the best estimate of K = 2 although there was no obvious geographic pattern in the dataset (Additional file 2: Fig. S4a-b ). For C. callipygus, there was no obvious geographic pattern and the best K could not be reliably determined (Additional file 2: Fig. S5a-b ). However, the raw mean estimated likelihood probability of data at K = 1 was not the highest, indicating that a model of population subdivision was more plausible than one single panmictic population. For P. monticola, the best K = 2 with NGO constituting one population and all other sites falling into the other (Additional file 2: Fig. S6a-b ). Lastly, GDM analyses revealed no effect of a Pleistocene refugium in any of the three species datasets (Table  3 ).
Discussion The present study is one of the first to simultaneously assess the roles of Pleistocene refugia, riverine barriers, isolation by distance and environmental heterogeneity in shaping the diversification of central African forest duiker species. While analyses of mitochondrial structure suggest that the Gulf of Guinea may have constituted a historical refugium for at least two of three of the species studied here, subsequent population expansion during the warmer inter-glacial periods has erased any signature of population structure in nuclear microsatellite markers. Unlike several vertebrate species that have been studied to date  [17, 19, 45] , the effect of riverine barriers appears negligible in rainforest duikers. GDM analyses of nuclear microsatellite data confirm that neither historical forest refugia nor rivers play an   S2  for explanation of environmental variables important role and that population structure is instead driven by environmental heterogeneity.
Field observations in a naturally fragmented landscape in central Gabon provide some support to this hypothesis  [103]  and suggest that although duikers are generally tied to forest habitats, they are able to cross intervening savanna areas to gain access to forest fragments or even inhabit thicket habitats  [56]. Several previous studies have also suggested that river basins  [26]  or gallery forests flanking smaller rivers and streams  [27, 104]  may have constituted fluvial refugia during the dry phases of the Pleistocene. AMOVA analysis of mitochondrial datasets does provide limited support for this hypothesis in showing that P. monticola populations within the Sanaga River basin are genetically differentiated from the surrounding area and that all three taxa exhibit a signature of population expansion.
STRUCTURE analyses also indicate that P. monticola populations in the NGO area of southern Central African Republic are differentiated from the rest of the study region, possibly due to a riverine refugium within the Sangha River basin  [17]. However, more detailed sampling is needed to evaluate this hypothesis more fully. It is also important to note that the mitochondrial break between the Gulf of Guinea refugium and the rest of the study area was not observed in the nuclear microsatellite dataset and that refugia added little or nothing to the microsatellite-based GDM model. This discrepancy is probably due to the fact that mitochondrial data is more likely to reflect historical genetic structure than microsatellites, which in turn are better able to capture contemporary drivers of genetic structure.
Ecological differences between species Although mitochondrial data indicate that the Gulf of Guinea refugium has played a role in structuring C. callipygus and P. monticola populations, this pattern was not evident in C. dorsalis. This effect could be simply due to limited number of samples obtained for C. dorsalis within the Gulf of Guinea refugium or due its wider geographic distribution  [56] , larger home range  [58] , and broad dietary flexibility  [109]  that may have made it less sensitive to past forest refugial dynamics. Nevertheless, all three species exhibit signatures of past population expansion, indicating a common demographic history. Future work should include sampling from candidate refugia in West Africa  [12]  in order to better assess the impact of Pleistocene history on C.
Acknowledgements The authors would like to thank the following institutions for permission to carry out scientific research: the  Centre National de Recherche Scientifique et Technologique (CENAREST) of Gabon , the  Conseil National des Parcs Nationaux (CNPN)  and its successor the  Agence National des Parcs Nationaux (ANPN) of Gabon , the  Ministère de l'Enseignement Supérieur, de la Recherche et de l'Innovation Technologique of Gabon , the  Ministère de la Recherche Scientifique et de l'Innovation of Cameroon, Ministère des Forêts et de la Faune of Cameroon  and the  Instituto Nacional de Desarrollo Forestal y Manejo del Sistema de Áreas Protegidas of Equatorial Guinea , the  Department of Fauna and Protected Areas (DFAP) of the Republic of the Congo, the Ministère des Eaux et Forêts, Chasse, Pêche et Environnement of C.
Duration of ischemia was based on our and previous data demonstrating that 3 h ischemia is needed to result in significant skeletal muscle injury, which is still more severe after 6 h ischemia  (Belkin et al. , 1988; Thaveau et al. , 2007). Chronological sequences about the cellular and molecular events occurring during skeletal muscle IR has been recently reported  (Paradis et al. , 2016). Interestingly, as compared to the heart which is still beating, such a long duration might be explained by the noncontracting, resting skeletal muscle generally examined. Like in the heart however  (Ma et al. , 2016) , a no reflow occurrence might increase with long ischemia time and we therefore choose a 3 h ischemia period for our experimental protocol. At the end of reperfusion, lactate, which is considered as a good marker of anaerobic state, was determined on total blood obtained from the tail before and after ischemia (lactate Pro device, LT710, Arkray R , KGK, Kyoto, Japan).