dataesr/scientific-paragraphs-categorization huggingface dataset:
| Data analyses We assessed species richness each year and compared it with a twofactor (year and treatment) ANOVA in a repeated-measures design with five replicates. Before ANOVA, we performed the Cochran and Shapiro-Wilk tests to check for homoscedasticity and normality respectively. We log-transformed heteroscedastic variables. The post-hoc mean separation was based on Fisher's protected least significant difference (LSD) test  (Steel & Torrie, 1980). We adopted the same procedure to compare the summed cover of plants belonging to each biological life-form, the specialists of Mediterranean temporary ponds, the generalists of wet habitats and the opportunistic terrestrial species, and bare soil. The differences in vegetation inter-annual dynamics and treatments were tested for significance with PERMANOVA  (Anderson, 2001)  with the treatments and the years as variables.
After hybridization, microarrays were washed using the NimbleGen Wash Buffer Kit (Roche) and scanned with a NimbleGen MS200 scanner (Roche). The DEVA 1. 2. 1 software was used to extract and segment the scanned images (Roche) and to perform the normalization of the fluorescence intensities using Robust Microarray Averaging (RMA). Normalized data was used to determine differential expression between conditions and assayed molecule using the TM4 Microarray Software Suite  (Saeed et al. , 2003 ; Supplementary Table  S1  contains the normalized intensity with error, one transcript per line, used for the analysis presented in this work). Clustering was performed using K-means clustering (KMC). Enrichment analysis was performed using the R library ReactomePA  (Yu and He, 2016)  which implements gene set enrichment analysis based on the REACTOME (https://reactome.
Molecular Studies These studies were carried out using the program GOLD  (Verdonk et al. , 2003)  version 5. 2 and the geometries found in the available crystal structures of eNOS/Heme/BH4/L-Arg (PDB code 4D1O, 1. 82 Å  (Li et al. , 2014) ) and iNOS/Heme/BH4/L-Arg (PDB code 1NSI, 2. 55 Å  (Li et al. , 1999) ) enzyme complexes. Ligand geometries were minimized using the AM1 Hamiltonian as implemented in the program Gaussian 09  (Frisch et al. , 2009)  and used as MOL2 files. Each ligand was docked in 25 independent genetic algorithm (GA) runs, and for each of these a maximum number of 100000 GA operations were performed on a single population of 50 individuals. Operator weights for crossover, mutation and migration in the entry box were used as default parameters (95, 95, and 10, respectively), as well as the hydrogen bonding (4.
Molecular Dynamics Simulation Studies 1) Ligand minimization. The ligand geometries of the highest score solution obtained by docking were minimized using a restricted Hartree-Fock (RHF) method and a 6-31G(d) basis set, as implemented in the ab initio program Gaussian 09  (Frisch et al. , 2009). Partial charges were derived by quantum mechanical calculations using Gaussian 09, as implemented in the R. E. D. Server (version 3. 0),  (Frisch et al. , 2009; Vanquelef et al. , 2011) , according to the RESP  (Dupradeau et al. , 2010)  model. Ligands were manually docked into the active site as it was obtained by docking. The missing bonded and nonbonded parameters were assigned, by analogy or through interpolation, from those already present in the AMBER  (Case et al. , 2005)  database (GAFF)  (Wang et al.
, 2004; Wang et al. , 2006). 2) Generation and minimization of the complexes. Simulations of the NOS/Heme/BH4/L complexes (L CC, CA) were carried out using the enzyme geometries in PDB codes 4D1O and 1NSI obtained by docking. These studies were also carried out with the natural substrate (Arg) as a control. For the eNOS enzyme, the coordinates of the unsolved residues 106-119 were modeled using the web-based ModLoop server  (Fiser and Sali, 2003). Computation of the protonation state of titratable groups at pH 7. 0 was carried out using the H ++ Web server  (Gordon et al. , 2005). Addition of hydrogen and molecular mechanics parameters from the ff14SB  (Maier et al. , 2015)  and GAFF force fields, respectively, were assigned to the protein and the ligands using the LEaP module of AMBER Tools 15  (Wang et al.
A positional restraint force of 50 kcal mol -1 Å -2 was applied to the whole protein during the stages a-b and to a carbons during the stages c-d, respectively. For the iNOS complex, the system was minimized in only four stages (b-e). 3) Simulations. MD simulations were performed using the pmemd. cuda_SPFP  (Goetz et al. , 2012; Le Grand et al. , 2013; Salomon-Ferrer et al. , 2013)  module from the AMBER 14 suite of programs. Periodic boundary conditions were applied, and electrostatic interactions were treated using the smooth particle mesh Ewald method (PME)  (Darden et al. , 1993)  with a grid spacing of 1 Å. The cutoff distance for the non-bonded interactions was 9 Å. The SHAKE algorithm  (Ryckaert et al. , 1977)  was applied to all bonds containing hydrogen using a tolerance of 10 -5 Å and an integration step of 2.
Binding Free Energies Calculations The binding free energy for CC, CA, and Arg was calculated by the MM/PBSA  (Miller et al. , 2012)  approach implemented in Amber Tools 1. 5. ante-MMPBSA. py module was used to create topology files for the complex, enzyme and ligands and binding free energies were calculated with the MMPBSA. py module. A single trajectory approach was used to calculate binding free energies considering only the last 100 ns (401 snapshots) of the 150 ns MD trajectories (NOS/Heme/BH4/L) (L CC, CA, Arg). The Poisson-Boltzmann (PB) and Generalized Born (GB) implicit solvation models were employed. The latter model provided relative free energy values more in agreement with the experimental results.
In silico Molecular Analysis To get an insight on the molecular basis of the potentiating effect of CC on the NOS activity, the interaction in atomic detail of CC on the active site of iNOS and eNOS enzymes was first studied by molecular docking using the program GOLD and the enzyme coordinates found in the crystal structures of eNOS and iNOS in complex with the natural substrate and cofactors, i. e. , L-arginine, 5,6,7,8-tetrahydrobiopterin (BH4) and heme group. In this study CA was also included, to get a better understanding of the reasons at molecular level behind the opposite observed effects, thus, while CC induced high NO production and vasodilation, NO production for CA was not identified. The proposed binding modes were further analyzed by MD simulation studies to assess the stability and therefore the reliability of the postulated binding.
5 mm interelectrode distance) from the rostral craniotomy (see Supplementary material). Oxygen was withdrawn from the anesthetic gas mixture 50 min after ischemia induction to create an episode of anoxia, which produced anoxic/ischemic depolarization (recurrent SD: rSD, or simultaneous depolarization: SiD). The condition of anoxia was resolved by re-oxygenation 5 min later, followed by an additional 25 min of data acquisition. Intrinsic optical signal (IOS) imaging at green light illumination was performed as described previously, and detailed in the Supplement to this paper. 26, 27 asurement of extra-synaptic glutamate concentration. Extra-synaptic glutamate concentrations were acquired using oxidase enzyme-based microelectrode biosensors (tip diameter: 30-40 mm; see Supplementary material for details) with constant potential amperometry.
Biosensors were connected to a dedicated 3electrode potentiostat (Quadstat) equipped with an eDAQ data acquisition system (eDAQ Pty Ltd. , Colorado Springs, CO, USA) and a dedicated eDAQ Chart program. Histology. Animals were transcardially perfused with physiological saline followed by 4% paraformaldehyde (PFA). The brains were removed and post-fixed in 4% PFA for 24 hours. Twenty-mm brain slices were cut with a freezing microtome after cryoprotection with 30% sucrose in PBS. Cleaved caspase-3 labeling was co-localized with the neuron marker NeuN or the astrocyte marker glial fibrillary acidic protein (GFAP). A detailed protocol for immunolabeling and image processing is given in the Supplement to this paper. Nissl staining to visualize necrosis was carried out on 20 mm thick brain slices mounted on Superfrostþ slides in 0.
3% polyvinyl-alcohol solution and dried at room temperature. Slides were incubated with 1% cresyl violet (containing 0. 08% acetic acid) for 20 min at 50 C, rinsed with distilled water, dehydrated, and mounted with Depex V R mounting medium. The slides were examined under a Nikon Eclipse 80i brightfield microscope at 4Â and 20Â magnification, and images were captured with a camera (QImaging MicroPublisher) operated via ImagePro Plus software.
Statistical analysis Data analysis was conducted offline and was assisted by the inbuilt tools of dedicated software (AcqKnowledge 4. 2 for MP 150, Biopac Systems, Inc. , USA and eDAQ Chart, eDAQ Pty Ltd. , Colorado Springs, CO, USA). Blinding data analysis was intended by assigning codes to files and recordings, which do not reveal the experimental condition (i. e. , date of the experiment). The exact steps of analysis are given in the Supplementary material. Image processing was performed after background subtraction and thresholding in Fiji detailed in the Supplementary material. Quantitative data are given as mean AE standard deviation (stdev). Statistical analysis was conducted with the software SPSS (IBM SPSS Statistics for Windows, Version 22. 0, IBM Corp. ). Data sets were evaluated first with a Shapiro-Wilk test of normality.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grants from the  National Research, Development and Innovation Office of Hungary  (No. PD128821  to AM,  K134377  to EF,  K120358  to FB,  K135425  to IAK,  FK132638  to AEF); the  Ministry of Human Capacities of Hungary  (.
Enzymatic routes to xylan deconstruction employ Carbohydrate Active enZymes (CAZymes), which are classified into protein domain families by the CAZy database; www. cazy. org  [14]. Glycoside hydrolases (GH) like endo-β-1,4-xylanases (GH5, GH10, GH11, GH30, GH98), xylobiohydrolases (GH30), exo-xylanases (GH8), β-xylosidases (GH3, GH39, GH43, GH52), α-xylosidases (GH31), α-l-arabinofuranosidases (GH43, GH51, GH62), α-glucuronidases (GH43, GH67, GH115) and α-lgalactosidases (GH95) cleave glycosidic bonds in the xylan backbone or of substituted sugars like Araf, MeGlcpA and galactopyranoside  [3, 15]. Carbohydrate esterases (CE) like acetyl xylan esterases (AcXE, CE1-7, CE16  [16] [17] [18] [19] ) and feruloyl esterases (FAE, distantly related to CE1  [20] [21] [22] [23] [24] ) in turn release the ester-bound substituents acetic acid and ferulic acid.
BD-FAE comprises parts of two Pfam domains, namely Abhydrolase_3 and Peptidase_S9 (PF07859 and PF00326, respectively). More precisely the N-terminal sequence matches the first half of PF07859 family model, while the C-terminal sequence matches the second half of PF00326 family model. Unlike other described FAEs, BD-FAE does not display the remote homology to CE1 family, and likely belongs to a novel broad esterase family to be created in dedicated databases (e. g. , ESTHER  [48] ). Sequence similarity search against the non-redundant NCBI database  [49]  revealed that BD-FAE homologs mostly belong to the Bacteroidetes phylum and to the α/β-hydrolase superfamily. Given this taxonomic specificity, a sequence similarity search was conducted against the 1,283 Bacteroidetes genomes integrated in PULDB (www.
Crystal structures and substrate binding of BD-FAE and its truncated form The crystal structure of BD-FAE was solved (PDB: 6TKX) to predict structural determinants that likely drive AcXE and FAE activities of the enzyme. The BD-FAE structure belonged to space group P4 3 2 1 2 and contained one molecule in an asymmetric unit. The final model of BD-FAE was refined to 2. 06 Å resolution and it contained the residues from Gln2 to Glu292 (Additional file 3: Table  S5 ). Clear electron density permitted unambiguous modeling of all residues except Met1, Leu293 and Glu294. The His-tag was not visible and the N-terminal tail showed weaker electron density than other residues of the protein, most likely due to its flexible protruding nature. Overall, the BD-FAE crystal structure adopted a typical α/β-hydrolase fold (Fig.
7C ). A comparison with other solved CE crystal structures can be found in the Additional file 3: Figure  S7  with GH43-CE of Bacteroides eggerthii (PDB: 6MLY) being the closest hit sharing only 55% sequence identity. The solved BD-FAE structure revealed higher-order oligomers within the corresponding crystal (Fig. 6B-D ). The N-terminal tail (Gln2-Pro7) protruded out of the core protein and packed against an adjacent symmetry molecule in the crystal (Fig. 6A, B ). Such crystal packing did not form a closed-ended dimer but an unusual fourfold spiral shaped polymer, in which the active sites pointed to the center of the spiral (Fig. 6C, D ). This openended oligomerization could be described as fibril formation  [57]. The interaction area of sequential molecules within the polymer was determined using PISA server  [58]  and revealed 1055 Å 2 , which was typical for strong intermolecular interactions.
To investigate the role of the first seven N-terminal residues in oligomerization, the crystal structure of a truncated form (ΔMet1-Pro7, PDB: 6XYC) was solved. The final model of the truncated form was refined to 1. 85 Å resolution and contained residues from Met8 to Lys292 (Additional file 3: Table  S5 ). Surprisingly, also in the crystal of the truncated form, higher-order oligomers were observed indicating that N-terminal deletion was not sufficient for disrupting this assembly. Substrate binding in BD-FAE was investigated by crystal soaking followed by co-crystallization and docking studies. The first approach was used to obtain a complex structure with a bound XOS ligand (degree of polymerization (DP) 1-6), however, no electron density for any ligand was detected and no changes in loop orientation or oligomerization were found.
7A, C ). The amino acid residues that bound Fa-Araf in BD-FAE were similar to those binding the AEBS moiety (Additional file 3: Figure  S6 ), and were in a long α-helical loop after the β5 strand. A stabilizing disulfide bond between Cys186 and Cys242 prevented extensive movement of that loop. The phenol of ferulic acid was sandwiched between Pro196 and Val240 by π-CH stacking and van der Waals interactions and the furanose ring of Araf interacted with Phe127 via CH-π stacking (Fig. 7A ). Overall, similarities in binding small substrates were observed to the characterized fungal FAE of Anaeromyces mucronatus (AmCE1/Fae1A, PDB: 5CXX  [59, 60] , Fig. 7 ). The hydroxyl group of ferulic acid bound in BD-FAE to Glu197 in a bidentate way (Fig. 7A ). In AmCE1, the corresponding residue was Asp190 and overall hydrogen bonding of ferulic acid with surrounding amino acid residues was stronger than in BD-FAE (Fig.
Together with the repeated occurrence of BD-PH_PUL30-like clusters in the metagenomic dataset  [27] , this supports the likelihood that BD-FAE and its homologs have an important function in microbial xylan degradation (Fig. 2 ). To ensure a nonbiased characterization, however, the initial screening of BD-FAE was performed on a broad substrate library covering 10 simple pNP-glycosides and pNP-Ac as well as 17 complex natural, polymeric substrates (Additional file 2: Table  S2 ). Following detection of acetyl esterase activity on pNP-Ac in the initial screening and a rather low k cat value of 0. 89/s, catalytic activity of BD-FAE was further studied on other substrates with increasing complexity. Of the tested pNP-esters, pNP-Ac and pNP-Fa BD-FAE released 0. 22 nmol pNP/ μg and 0. 22 mmol pNP/ μg after 2 h, respectively, suggesting a preference for the feruloylated substrate (Additional file 2: Figure  S3 , Fig.
5B ). On X2Ac5 positional specificity of BD-FAE towards 1-O-Ac was observed (Fig. 4 ), consistent with the degradation of X2Ac5 into X2Ac4 (Fig. 3 ). This linkage, however, does not occur in natural xylans  [6, 8, 9]  but might be the most accessible or reactive acetyl group in this synthetic substrate. We also showed that BD-FAE was capable of releasing 37% of total acetic acid from AcGX within 19 h (Fig. 5B ) and 13% of total acetic acid from X2Ac4 after 4 h, in which the acetyl groups are linked 2-O and/or 3-O to a Xylp. These results point out that catalytic activity and positional specificity can differ on synthetic and natural substrates  [24, 68]. BD-FAE did not act on AcGGM containing 2-O and/or Fig. 7  Comparison of substrate binding in BD-FAE (beige) and FAE from Anaeromyces mucronatus (AmCE1, PDB: 5CXX, green).
The role of the N-terminal tail in oligomerization In crystals of BD-FAE, the protruding N-terminal tail packed as a β-strand against a small β-sheet on the surface of another molecule, leading to an unusual fourfold spiral shaped polymer (Fig. 6 ). Surprisingly, the crystal structure of an N-terminal truncated form of BD-FAE (∆Met1-Pro7) adopted a similar spiral shaped polymer. The interaction surface area of sequential molecules in the truncated form, however, was determined to be 764 Å 2 , which is 28% smaller compared to BD-FAE (1055 Å 2 ). N-terminal residues were previously shown to participate in protein packing; for example the N-terminal β-domains of two adjacent BiFae1A monomers, an FAE from Bacteroides intestinalis, lead to dimerization and subsequent tetramerization (PDB: 5VOL  [73] ).
Candidate selection Annotation of protein domains in the previously published metagenomic dataset  [27]  relied on HMMER searches  [78]  using Pfam  [47]  and CAZy library  [14]  with recommended thresholds. PUL prediction was performed similarly to the PULDB (www. cazy. org/ PULDB/  [31] ), with a relaxed procedure that only require susD presence to start, and not necessarily susC, to cope with the fragmentary aspect of metagenomic dataset. PULs containing more than five genes encoding CAZy family members related to xylan degradation (GH10, GH11, GH43, GH51, GH115 and CE1) as well as PUFs were further investigated. To verify the quality of the selected PUL (BD-PH PUL30, Fig. 1 ) each protein was analyzed for its sequence length (> 250 bp) and homologs (BLASTp  [49] ), the present of a signal peptide of Gram-negative bacteria (SignalP5.
Phylogeny A BLASTp  [49]  search against the NCBI non-redundant database was performed using BD-FAE as a query with a 10E-10 e-value threshold and resulted in 153 hits out of which only seven belonged to other phyla than the Bacteroidetes. Subsequently, a BLASTp search against all proteins encoded by the 1283 genomes integrated in the PULDB (www. cazy. org/ PULDB/  [31] , accessed on 14. 06. 2020) was performed. The first 200 hits were retrieved and further analyzed (Additional file 1: Table  S1 ). Genomic contexts of the homologs were manually inspected using the genome browsers of the PULDB. Glycan structure targeted by PULs was determined based on known CAZymes specificities. To visualize the phylogenetic relationship and the genomic context of these 200 homologs of BD-FAE, a phylogenetic analysis was performed at ngphylogeny.
1 M MES at pH 6. 0. A 4 μL drop, including 2 μL of protein (12 mg/mL) and 2 μL of crystallization solution, was allowed to equilibrate against 500 μl of crystallization solution per well. Both fulllength and truncated forms were crystallized under the same conditions. Thin needle like crystals were obtained within a week. They were cryoprotected with 30% ethylene glycol. Crystals were mounted in nylon loops and plunged into liquid nitrogen prior to data collection. Data collection for BD-FAE (PDB 6TKX) was carried out at Diamond Light Source on beamline i04 and for truncated form (PDB: 6XYC) at ESRF on beamline ID23-1. The data sets were processed and scaled with XDS  [89]. The structure was solved using the automated molecular replacement and model building software phenix. mr_rosetta [90].
As templates the 250 closest structural homologs of the Protein Data Bank (PDB) obtained with a HHPred multiple sequence alignment (MPI Bioinformatics Toolkit  [91] ) were used. A clear molecular replacement solution was found with lowest Rfree of 0. 357 which was refined with phenix. refine  [90]  and manual editing in Coot  [92]. The structure of truncated BD-FAE was solved using the crystal structure of BD-FAE as template. In an attempt to obtain a complex structure with a bound XOS ligand, the BD-FAE crystals were soaked with 30 mM xylobiose, 30 mM xylotriose or 10 mM xylopentose. Binding of XOS was also studied by docking d-xylose, xylobiose, xylotetraose and xylohexaose into BD-FAE (PDB: 6TKX) with AutoDock Vina (ADV1. 1. 2, http:// www. vina. scrip ps. edu/  [93] ). The receptor molecules used in were the monomeric form of BD-FAE and two BD-FAE molecules packed in a way they packed in the crystal.
Data acquisition In this study, we examined the mortality data from 22 countries participating in the international consortium (Supplementary Figure  S1  and Table  S1 , available as Supplementary data at IJE online) that have collected and provided data until the end of either June (n ¼ 5) or August 2020 (n ¼ 17), depending on data availability. Information was collected for total and sex-specific all-cause mortality (for 2015-2020), as well as total and sex-specific COVID-19 deaths (for 2020). Anonymous data were collected from national vital-statistics databases, either publicly available or with restricted access, from each participating country to the latest available data point of 2020 (Supplementary Table  S1 , available as Supplementary data at IJE online). Countries reported all-cause mortality and COVID-19 deaths by week [either International Organization for Standardization (ISO) week, starting on Monday; or epidemiological (Epi) week, starting on Sunday; or another national counting week system, depending on the country].
COVID-19-death reporting also differed between countries, as shown in Supplementary Table  S1 , available as Supplementary data at IJE online. Some countries (n ¼ 11) reported as COVID-19 deaths any deaths among positive cases irrespective of where COVID-19 was listed on the death certificate. Thus, COVID-19 was either listed among the chain of causes leading to death or as a contributing condition on the death certificate [cause of death (COD) or contributing condition]. 20 Other countries (n ¼ 11) reported as COVID-19 deaths only the deaths for which COVID-19 was listed among the chain of causes leading to death (COD); 20 of these latter countries, France reported only hospital and nursing-home COVID-19 deaths. The national primary data sources used in this study and endorsed by the national partners might differ from publicly available repositories/databases, primarily due to the retrospective addition of cases and deaths declared with some delay.
Statistical analysis Excess mortality for 2020 was calculated by comparing weekly or monthly 2020 mortality (observed deaths) against a baseline mortality obtained from 2015-2019 data for the same week or month using two different methods. The choice of 5 years for the baseline mortality estimation was based on widely adopted practices from well-established surveillance consortia  3, 23, 24  as well as on other published studies. 2  In the first method, the baseline was computed as the average mortality rate of the previous 5 years. 8 [26]  Method 1: Observed 2020 vs 2015-2019 average mortality rates For each country (n ¼ 22) and year, the weekly or monthly observed number of deaths was divided by the country's population at the beginning of the particular year to obtain weekly and monthly mortality rates.