dataesr/scientific-paragraphs-categorization huggingface dataset:
Side-Chain Refinement in Ligand Binding Sites As starting positions, we used the coordinates of the calcium and zinc ions, ATP and caffeine molecules, as previously determined by electron microscopy  (Georges et al. , 2016). For ryanodine, we used a Ryd model provided by Ngo and the coordinates from their MD study of ligand placement  (Ngo et al. , 2017). The models were created using YASARA. For each model, the most likely protonation state was calculated at pH 7. 0. The program YASARA was used for atom typing and hydrogen atom assignment, followed by virtual titration. Sidechains of residues forming ligand binding sites and ligands were then refined using a YASARA macro within the VINALS method (VINA with Local Search) for local ligand docking plus minimization steps. The simulation cell was enlarged by 10 Åin this step.
Normal Mode Analyses Due to the large size of the RyR1 system, we carried out Normal Modes Analysis (NMA) using the Non-Linear Rigid Block (NOLB by  Hoffmann and Grudinin (2017) ) software, which uses a coarse-grained block approach  (Durand et al. , 1994). In addition to efficiently analyzing large assemblies, NOLB decomposes the block motions into instantaneous rotational and translational components, whose nonlinear extrapolation allows the computation of periodic trajectories for visualization that preserve structural integrity better than standard linear approaches. The 100 lowest frequency vibrational modes were calculated for each of the superimposed RyR1 conformations using an all-atom elastic network model with an interaction cutoff distance of 10 Å. Ligand atoms, when present, were included in the analyses.
Starting Point for the Present Study The starting point for this system was the results of a preliminary coarse-grained study of the densely packed association complex between the RecA nucleoprotein filament (RecA*) and DNApolymerase IV (DinB), which had been obtained by interactive simulations using the BioSpring software also used for the RyR1 modelling above (Section 2. 1. 4), and which represents each component of a complex system by a variable network of harmonic springs, allowing interactive dynamic testing of possible component orientations and deformations  (Molza et al. , 2014; Tashjian et al. , 2019). In this case the components were the nucleoprotein filament (RecA and DNA) resulting from our earlier strand exchange simulations  (Yang et al. , 2015)  and the crystal structure of DinB bound to its cognate DNA (PDB code 4IRC) (Figure  1B ).
Modelling the RyR1 Ryanodine Receptor Channel and Its Dynamics Ryanodine Receptors (RyRs) regulate cytosolic calcium concentration, which is critical in numerous signaling pathways. Mutations in these receptors in muscle cells can lead to severe skeletal muscle and heart disease. RyRs belong to the six-transmembrane helix ion channel superfamily  (Lanner et al. , 2010)  and are localized in the endoplasmic and sarcoplasmic reticulum. Here, RyR1 provides a case study to demonstrate general aspects of the pipeline that lead from deposited cryo-EM datasets to complete models that can be used for molecular modelling studies on mechanistic and structural aspects. We were particularly interested in 1) addressing issues related to the channel's large size and 2) benefitting from the availability of maps for multiple functional/conformational sub-states, which provide a good test for the usability of the final models and the level of detail and accuracy that can be expected from them.
Secondary Structures and Propensity to Embed into the Membrane PPSIPRED predicted five putative alpha-helices within the big disordered region (see Supplementary Figures  S3A, B ). The TOPCONS and CCTOP server predictions for the transmembrane region predicted multiple transmembrane parts. Interestingly, most tools predicted the same auxiliary transmembrane helix in the region of residues 4320 to 4336 as shown in Supplementary Figure  S3C. To evaluate these results, we attempted to model some parts of this loop using a de novo approach with the PEP-FOLD3 package  (Lamiable et al. , 2016).
Model-Building Procedure Despite many available maps for RyR, complete models of the protein are not yet available. This lack of completed models hampers many efforts to obtain insight into the structuredynamics-function relationship. In addition, RyR serves here as a use case to show how information from the pool of deposited cryo-EM datasets can be used to complete these models. Given that the number of relatively high-resolution datasets is continuously increasing, we hope that the model building procedures developed here will provide a general reference protocol that can be further adapted and optimized for exploring mechanistic and structural aspects of other systems under investigation. Figure  2  provides a global schematic overview of the entire modelling procedure. In the following sections, the individual steps for the reconstruction of the receptors are described in detail.
Full-Length Models of the Receptor (RyR1) at the Atomic Level To model missing parts of the protein, several software packages were tested. For the specific use case of the RyR protein, given the challenges presented by its size, the best results were obtained by de novo modelling using Rosetta (https://www. rosettacommons. org/) to complete the models. The process is shown schematically in Supplementary Figure  S4. To optimize the geometry of these initial models for further computations after de novo modelling, we applied some model refinement routines using minimization steps within the Yasara software (Yasara Structure 17. 12. 24; http://www. yasara. org/) to remove possible clashes, knots, and some bond errors. We then refined the models for all atoms using the MDFF method (https:// www.
In practice, the low-frequency (or low-energy) modes are of the most interest, as they have been extensively correlated with directions of conformational changes and dynamics in biological macromolecules (e. g. ,  Tama and Sanejouand, 2001; Rueda et al. , 2007). Here we used NMA to better understand the dynamics of our models of the ryanodine receptor in its different conformational states and how these movements differ from one state to another. For such large molecules, the analyses were facilitated through the use of the NOLB software  (Hoffmann and Grudinin, 2017). We performed NMA on the full-length RyR-1 models and the restricted channel regions after removing the modelled loop regions (structure "no-loops" in Table  3 ). These calculations were carried out on the five conformational states of RyR-1 (Table  2 ): the closed or closed-like states apo and primed, the intermediate state, and the open states activated and locked.
FUNDING We acknowledge the  Servier group  for funding part of this project. HPC resources from  GENCI  were obtained via the grant numbers  A0070701714 ,  A0080707438  and  A0100707438. This work was supported by the  French "Initiative d'Excellence" program  (Grants " DYNAMO ",  ANR-11-LABX-0011 , and " CACSICE ",  ANR-11-EQPX-0008 ), the  Chu Family Foundation  to MP, and VG-C research was supported in part by a grant to the  Northeastern University Skills for Capacity and Inclusion Program  by the  Howard Hughes Medical Institute  through the  Science Education Program. MB thanks " Sesame Ile-de-France " for cofunding the display wall used for data analysis.
Experimental design Experiments were performed in March and April 2021 in the stables where the horses lived, 26 locations (private homes or private stables) from Southern Finland. The two tests were performed in a place familiar to the horses and where they are used to be taken for training (empty outdoor paddock/riding arena or indoor riding arena). In total 76 horses participated in the novel surface test and 71 in the novel object test due to practical reasons. The materials were set up in the test area prior to the horses' arrival. Both tests were recorded using cameras fixed on tripods for later analysis of the behaviours using the Behavioural Observation Research Interactive Software (BORIS)  (Friard and Gamba, 2016). Novel surface tests: The horses were led once by the familiar handler and once by an unfamiliar handler to walk on a novel surface.
Statistical Analysis Data are provided in Online Resources  (Liehrmann, 2022). All analyses were carried out using the statistical software R, version 3. 6. 3 (R Core Team, 2021) and figures were created using the 'ggplot2' package  (Wickham, 2016). We checked for potential collinearity with bivariate analyses between our variables of interests (number of regular handlers, number of owner changes, relationship length, age and sex) using Wilcoxon, Spearman and χ 2 independence tests. Correlations appeared between the relationship length and the age of the horse (as a quantitative variable, r s = 0. 54, P < 0. 0001, N = 76). For this reason, if the relationship length appeared to significantly affect the response variable, the models were run again on a smaller portion of the dataset (11-22 years old) with no correlation between the age and the relationship length (r s = 0.
There was no significant difference in the reluctance levels between the two surfaces, confirming that there was no habituation (Estimate ± SE = -0. 65 ± 0. 42, Z = -1. 57, P = 0. 116). The success of stepping on the surface (1: success/0: fail) was investigated using Generalized Linear Mixed Models (GLMM) from the package 'glmmTMB'  (Brooks et al. , 2017). The best suited distribution was selected using the 'DHARMa' package  (Hartig, 2021)  to be a complementary log-log binomial family. Again, we controlled for potential habituation between the first and second surface and there was no significant difference in a horse's success at stepping on surface 1 or surface 2 (Estimate ± SE = 0. 44 ± 0. 29, Z = 1. 51, P = 0. 129). The latency to touch the novel object was investigated using survival analyses from the package 'coxme'  (Therneau, 2020).
This process was selected because 19 horses did not approach the object at all and therefore had no timing recorded. The survival analysis allows these individuals to be considered in the analyses. The reluctance when approached with the novel object (3-level ordinal variables, see Table  1 ) were investigated using Cumulative Linked Models (CLM) from the package 'ordinal'  (Christensen, 2019). To avoid over-parameterisation of the models due to the sample size of the datasets, for each of the four response variablesthe reluctance to the novel surface (ordinal: None <Mild <Strong), the success to step on the surface (binomial 0. 1), the reluctance to be touched with the novel object (ordinal: None <Mild <Refused) and the latency to touch the novel object (sec) -six small models were always run assessing the effect of the handlers' familiarity in interaction with one of the six other independent variables (age, sex, relationship length, number of owner changes, number of regular handlers, breed and previous habituation to novelty) plus one model with the effect of the handlers' familiarity alone.
The most appropriate version of each of the 28 models was selected using a model comparison with two tailed ANOVA  (Rouder et al. , 2016)  comparing the null model against the original model (variable of interest in interactions with the handler familiarity) and simpler models (without the interaction and then without the variable of interest). For each of this selection process, the selected models are presented in Tables  2  and 3. When necessary, a post hoc test based on Tukey's methods was performed with the function emmeans () from the package 'emmeans'  (Lenth, 2021). Significance was evaluated at P < 0. 05, with tendencies recognised at P < 0. 10.
2a ). The relationship length between the familiar handler and the horse tended to affect the reluctance towards the novel surfaces regardless of the handler's familiarity (estimate ±SE = -0. 27 ±0. 14. Z= -1. 93. P=0. 054). The probability for the horses to approach the novel surfaces without reluctant behaviour (reluctance level = None) increased with the relationship length between the horse and the familiar handler while the probability for the horses to display mild reluctant behaviours and strong reluctant behaviours decreased with the relationship length. We can summarise by saying that after six years of relationship horses had significantly more chances to approach the novel surfaces without showing reluctant behaviours (Fig. 2b ). The results were similar when the models were run with the smaller subsets of the dataset without collinearity with the horses' age: Novel object (estimate ± SE = -0.
We found that the handler's familiarity mattered for older horses. Horses older than 18 years old (n = 15) refused more often to step on the novel surfaces when they were led by an unfamiliar handler compared to when they were led by the familiar handler. One could argue that older horses may also have the longest relationship with the familiar person. In our dataset, horses older than 18 years had relationship lengths with their owners ranging from 1 to 15 years and the relationship length variable did not affect the horses' success at stepping on the surfaces. Geriatric horses often suffer from corneal degeneration and loss of eyesight  (Berryhill et al. , 2017) , and it has been shown that older horses may feel more anxiety towards novelty than younger horses  (Lee et al. , 2021). Therefore, older horses may perceive someone familiar as a secure base, feeling safer to walk over an unknown material when led by a familiar person.
Phospholipases are esterases involved in lipid catabolism. In pathogenic micro-organisms (bacteria, fungi, parasites) they often play a critical role in virulence and pathogenicity. A few phospholipases (PL) have been characterised so far at the gene and protein level in unicellular parasites including African trypanosomes (AT). They could play a role in different processes such as host-pathogen interaction, antigenic variation, intermediary metabolism. By mining the genome database of AT we found putative new phospholipase candidate genes and here we provided biochemical evidence that one of these has lipolytic activity. This protein has a unique non-canonical glycosome targeting signal responsible for its dual localisation in the cytosol and the peroxisomes-related organelles named glycosomes.
Results In silico identification and sequence analysis of a new putative lipase in Trypanosoma brucei. Previous studies strongly suggested the existence of more than one phospholipase in African trypanosomes playing important role(s) in host-pathogen interaction  10, 11. Our goal was to identify such new phospholipase(s) in T. brucei. We rationalised our research by combining an in silico analysis with a literature analysis, including global approaches described below. We found a gene, Tb927. 8. 6390 (TriTrypDB accession number), with a predicted α/β hydrolase/phospholipase domain  20  and annotated as TbLysoPLA (standing for Trypanosoma brucei Lyso-phospholipase A) based on sequences homology. According to the literature  21, 22  this gene encodes for a protein located in the glycosomes which are peroxisome-like organelles  23  and also is possibly excreted/secreted  24.
As mentioned above, Tb927. 8. 6390 has been detected in glycosomes in several mass-spectrometry analysis  21, 22  and this localisation could be due to the tripeptide "SKS" at the extreme C-terminus. This sequence does not correspond strictly to a canonical PTS1 and has not been observed as a PTS1 in other kinetoplastid species. Since the "SKS" is unusual, we searched in the TriTryp genomic database for other T. brucei encoded proteins that possess the same motif. The table presented in Fig. 1B  shows that two other proteins contain this C-terminal tripeptide. The transmembrane adaptator Erv26 is found in COPII vesicles  28  and CIF1 is located at the cell tip  29.
8. 6390 is therefore a lipase. A further thorough investigation of its enzymatic specificities in vitro and in vivo is required to fully understand the catalytic properties of this protein. Nevertheless, given actual annotations we have chosen to keep the actual name TbLysoPLA. TbLysoPLA is constitutively expressed. The purified recombinant TbLysoPLA described before was used to immunize rabbits in order to raise a serum directed against it (see "Materials and methods" section). This immune serum recognises a single 30 kDa protein corresponding to the size predicted in Tritryp database (Fig. 3A, left ). This signal is detected in both bloodstream and procyclic cultivated stages indicating that this enzyme is constitutively expressed. As observed in the figure only one protein could be detected by the polyclonal serum, and we could not detect any signal either in the pre-immune serum or cell-lines depleted of TbLysoPLA showing the specificity of our antibody.
Immunofluorescence assay. Parasites grown in culture were collected by centrifugation, washed and fixed in paraformaldehyde as described elsewhere  37. Slides were incubated with primary antibodies followed by Alexa Fluor 488-conjugated goat anti-mouse secondary antibody or Alexa Fluor 594-conjugated goat anti-rabbit secondary antibody (diluted 1:400) (Invitrogen). The nuclei were stained with DAPI (10 µg/mL) and cells were observed using a Zeiss Axio imager Z1 microscope; images were captured using Metamorph software (Molecular Devices). Images were processed using ImageJ software. RNA interference and gene knock-out. The inhibition by RNAi of the expression of the TbLysoPLA gene in the 427 BSF was performed by expression of stem-loop « sens/antisens » RNA molecules of the targeted sequences introduced into the pLew100 as previously described  31.
Funding Experiment costs were supported by  Université de Bordeaux  (https:// www. u-borde aux. fr),  CNRS  (https:// www. cnrs. fr) and the  Agence Nationale de la Recherche  through the grants GLYCONOV (grant number  ANR-15-CE-15-0025-01 ) and ADIPOTRYP (grant number  ANR19-CE15-0004-01 ). This work was also funded by the  Laboratoire d'Excellence (LabEx)  " French Parasitology Alliance For Health Care " ( ANR-11-LABX-0024-PAR-AFRAP , https:// labex-paraf rap. fr). This work was also partially supported by the  European Research Council  (FatTryp, ref. 771714) and by  Fundacao para a Ciencia e Tecnologia  ( CEECIND/03322/2018 ) awarded to LMF. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
Candidatus Riesia pediculicola housekeeping gene analysis Primer design In order to investigate the genotypic profile of the endosymbiotic bacteria, four standard PCR systems were designed targeting three Candidatus Riesia pediculicola housekeeping genes: ftsZ, groEL and two regions of rpoB. Four genomes of Candidatus Riesia pediculicola belonging to clade A and B lice deposited in the Gen-Bank database (accession numbers CP012841, CP012843, CP012845, CP001085)  [17, 18]  were aligned using Muscle in MEGA7 software  [24]  and screened for conserved and discriminative genes. Based on the variability in the available Riesia genomes, three housekeeping genes were selected as candidates for primer design. In order to find a suitable and conserved region for primers, two sequence fragments (approx.
100 bp) separated by a minimum of 500 bp for each gene were submitted to Primer3 software v. 0. 4. 0 (http:// prime r3. ut. ee/). The melting temperature of each primer was tested using the free online software programme Oligo Analyser 3. 1 (https:// eu. idtdna. com/ calc/ analy zer)  [25]. Designed primers (Table  1 ) were then tested in silico, using the NCBI BLAST nucleotide sequence similarity tool (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi).
Phylogenetic analysis In total, 28 lice specimens harbored common sequences for the cytb and the endosymbionte genes, but only 21 specimens had good quality sequences and were chosen for the phylogenetic analysis. The obtained cytb sequences were combined and compared with the worldwide cytb data previously reported and deposited in the GenBank database  [8, 26]. Alignments were performed using MEGA7. 0. 26 software, and a maximum-likelihood (ML) tree was constructed using the Kimura2-parameter model under 1000 bootstrap replicates  [27]. Candidatus Riesia pediculicola sequences were combined with data reported previously by Boyd et al. [16, 17]  and were analysed as described for the cytb gene. Specifically, we used an Orthologous Average Nucleotide Identity Tool (OAT)  [28]  to define the overall similarities between the published Candidatus Riesia pediculicola genomes reported previously by Boyd et al.
13 objective for the placement of the stimulating electrode and a 40x/0. 80 water-immersion objective for localizing cells for whole-cell recordings. The series resistance was not compensated. Recordings were sampled at 10 kHz, using the Patchmaster v2x32 program (HEKA Elektronik). For paired-pulse protocols, electrical stimulations were performed with a bipolar electrode (Phymep, Paris, France) placed in the layer V of the somatosensory S2 cortex. Electrical stimulations were monophasic at constant current (ISO-Flex stimulator, AMPI, Jerusalem, Israel). Currents were adjusted to evoke 50-200pA EPSCs. Repetitive control stimuli were applied at 0. 1Hz. For each ISI, 20 successive EPSCs were individually measured and then averaged. Variation of input and series resistances above 20% led to the rejection of the experiment.
Electrophysiology analysis For each ISI, 20 successive EPSCs were individually measured and then averaged. Variation of input and series resistances above 20% led to the rejection of the experiment. Off-line analysis was performed with Fitmaster (Heka Elektronik). Statistical analysis was performed with Prism 5. 02 software (San Diego, CA, USA). All results are expressed as mean ± SEM. Statistical significance was assessed in non-parametric Mann Whitney, one-sample t-tests using the indicated significance threshold (p).
Acknowledgements We thank  Sebastien Carnicella ,  Sandrine Humbert ,  Alain Marty , members of the  Saudou, Humbert, and Venance labs  for comments;  Vicky Brandt  for critical editing;  Béatrice Blot ,  Aurélie Genoux ,  Nagham Badreddine ,  Claire Seris  and  Camille Brodier  for technical help;  T. Ryan  for the gift of pHluorin plasmid;  C. Hoogenraad  for the gift of sh-KIF1A plasmid;  Y. Saoudi  for help with image acquisitions and the  Photonic Imaging Center of Grenoble Institut Neuroscience (PIC-GIN)  which is part of the  ISdV core facility  and is certified by the  IBiSA label. We acknowledge the contribution of  Gisèle Froment ,  Didier Nègre  and  Caroline Costa  and the AniRA lentivectors production facility from the  CELPHEDIA Infrastructure and SFR Biosciences  ( UAR 3444/CNRS ,  US8/Inserm ,  ENS de Lyon, UCBL ).
This work was supported by grants from the  European Research Council (ERC)  under the European Union's  Horizon 2020 research and innovation program  AdG grant agreement no. 834317 ,  Fueling Tranport  (F. S. );  Agence Nationale de la Recherche :  ANR-15-IDEX-02  NeuroCoG (F. S. ) in the framework of the " Investissements d'avenir" program ;  ANR-18-CE16-0009-01  AXYON (F. S. );  Fondation pour la Recherche Médicale, FRM ,  DEI20151234418  (F. S. ) and  AGEMED program  from  INSERM  (F. S. ). The Saudou laboratory is part of the  Grenoble Center of Excellence in Neurodegeneration (GREEN). C. S. was supported by a Postdoctoral fellowship from:  FRM  ( SPF20140129405 ) and  EMBO LTF  ( ALTF 693-2015 ). H. V. was supported by a PhD fellowship from  Association Huntington France  and by a  FRM fellowship  ( FDT201904008035 ).