dataesr/scientific-paragraphs-categorization huggingface dataset:
Treatment with ITZ Results in Relocalization of OSBP to the Golgi Complex After having established that OSBP and, possibly, ORP4 are novel targets of ITZ, we wanted to study how ITZ targets these proteins. Because of available tools, we focused on OSBP for the remainder of this study. In line with published data  (Burgett et al. , 2011) , OSW-1 caused a massive recruitment of overexpressed GFP-OSBP to the Golgi apparatus (as marked by staining endogenous PI4KIIIb) (Figure  4A ). A similar relocalization was observed for endogenous OSBP (Figure  4B ). ITZ and posaconazole, but not the azoles that lacked antiviral activity, redistributed OSBP in a manner that is similar to OSW-1. Live-cell imaging was performed to study the dynamics of GFP-OSBP relocalization by the compounds. Before addition of the compounds, GFP-OSBP primarily localized in the cytosol with a Golgi pattern faintly visible.
ITZ Binds Directly to OSBP The inhibitory effect of ITZ on OSBP function in a minimal in vitro system implied that ITZ directly inhibits OSBP. To biochemically define the binding in more detail, we measured binding of ITZ to GFP-OSBP using microscale thermophoresis (MST). Each molecule or complex distributes differently in a temperature field, depending on size, charge, and the hydration shell. Binding of ITZ to OSBP will affect the hydration shell and thereby its thermophoretic behavior. ITZ altered the thermophoretic profiles of purified GFP-OSBP (Figures  S7B  and S7C ) in a dose-dependent manner, indicating direct binding. Normalization and fitting of data from three independent measurements demonstrated that ITZ binds to OSBP with a K D of 430 nM (Figure  5M ). The monophasic shape of the binding curve indicates that there is likely only a single binding site for ITZ on OSBP, although our data cannot rule out that there are two sites with nearly identical K D 's.
OSBP Localizes to ROs in a PI4P-Dependent Manner To test whether OSBP plays a role in formation and/or maintenance of the ROs, we examined its localization in infected cells. In uninfected cells, OSBP is mainly distributed throughout the cytosol with some OSBP localized to the Golgi apparatus (Figure  6A ), where it colocalized with PI4KIIIb and the trans-Golgi network marker TGN46 (data not shown). In infected cells, OSBP localization was markedly changed, i. e. , the Golgi pattern was lost and OSBP appeared in dispersed structures throughout (G-J) The effect of ITZ on binding of an N-terminal OSBP fragment (amino acids 76-408; PH-FFAT) to ER-like (G and H) and Golgi-like (I and J) liposomes was examined by liposomal float-up assays as outlined in (G) and (I). Liposomal fractions were analyzed for binding of proteins by SDS-PAGE (H and J).
(K and L) The effect of ITZ and control compounds on DHE (K) or PI4P (L) transfer by trypsinized OSBP was studied as with full-length OSBP (A-F). (M) The interaction of ITZ with GFP-tagged OSBP was investigated using MST. Data from three separate measurements were normalized and plotted, and a sigmoidal dose-response curve was fitted. Shown are mean values ± SEM. Statistical significance for the drug-treated conditions was calculated compared to the ''no drug'' control. See also Figure  S7. the cytoplasm where it colocalized with viral protein 3A as a marker for ROs (Manders' coefficient for overlap of 3A with OSBP: 0. 36) (Figures  6A  and 6B ). To examine whether OSBP localized to ROs in a PI4P-dependent manner, cells were infected with CVB3 and replication was allowed to progress uninhibited for 4.
Live-Cell Imaging For live-cell imaging experiments, HeLa R19 cells were transfected with pEGFP-hOSBP; treated with ITZ, OSW-1, or solvent control (DMSO); and imaged using a Nikon A1R confocal laser scanning microscope. Images were processed and quantified using the Nikon NIS-Elements software. For additional details, see Supplemental Experimental Procedures.
(K and L) The effect of ITZ and control compounds on DHE (K) or PI4P (L) transfer by trypsinized OSBP was studied as with full-length OSBP (A-F). (M) The interaction of ITZ with GFP-tagged OSBP was investigated using MST. Data from three separate measurements were normalized and plotted, and a sigmoidal dose-response curve was fitted. Shown are mean values ± SEM. Statistical significance for the drug-treated conditions was calculated compared to the ''no drug'' control. See also FigureS7.
ACKNOWLEDGMENTS We thank  Stijn Delmotte ,  Katrien Geerts ,  Caroline Collard ,  Gerrit Koen , and  Katja Wolthers  for assistance in acquisition of part of the antiviral data;  Patrick Celie  and  Alex Fisch  ( Netherlands Cancer Institute, Amsterdam ) for help with the MST measurements; and the  Center for Cell Imaging  ( Faculty of Veterinary Medicine, Utrecht University ) for support with microscopy experiments. This work was supported by grants from the  ''Convenant K. U. Leuven-Radboud University Nijmegen '' framework (L. v. d. L. , J. N. , and F. J. M. v. K. ), the  European Union   FP7 Marie Curie Initial Training Network ''EUVIRNA' ' (grant agreement number  264286 ) (F. J. M. v. K. ) and  FP7 Large Scale Collaborative Project ''SIL-VER' ' (grant agreement number  260644 ) (F. J.
Search terms A scoping study was conducted in January 2015 using Web of Knowledge (WoK), CAB Abstracts and Scopus to identify a comprehensive search strategy (Table  2 ). This resulted in 6376, 8346 and 8926 hits for the abovementioned databases, respectively. The search iteration was compared against a test library, which contained all references from Hertel and Leuschner (Hertel and Leuschner 2010) and Finér et al. (Finér et al. 2011 ) (Table  S2 ). In the final iteration of the search, 18 of 20 studies were included and one study  (Castellanos et al. 1991 ) that did not include fine root dynamic estimates, was missing. The search terms within each category of 'Subject/ Population', 'Outcomes', 'Geographical focus' and 'Exposures' will be combined with the Boolean operator 'AND'. The use of Boolean operators and truncations will be modified according to the requirements of the bibliographic databases and recorded in an appendix of the full review.
Critical appraisal After completion of full text screening, the remaining studies will be assessed for robustness of their study design. The appraisal will be based on sampling period, number of replicates and sampling depth. Each study will be placed into one of three categories: below acceptable quality, acceptable quality and high quality (Table  3 ). Two researchers will first critically appraise a subset of 20% of the included studies and calculate a kappa score. Upon agreement, one researcher will carry out the complete assessment. Where studies have missing or abbreviated data sets, the reviewers will request the additional data from the authors. Any studies that are rejected during the critical appraisal process will be listed in an appendix to the review, and the reasons for their exclusion will be included.
Data synthesis and presentation This review will use a quantitative synthesis to describe how land-use change from tropical forest, degraded forest and/or agriculture affects fine root dynamics. A meta-analysis will be conducted on the subgroups, i. e. root production, root mortality and root decomposition, to show the effects of land-use change on the different components of fine root dynamics. We expect that the effects of land-use change from forest to shifting cultivation on fine root dynamics in Africa will be impossible to assess because of the data limitation on the different types of shifting cultivation practices used, which are very region specific. The meta analysis will use R software (R Core Team 2014). Different weighted effect sizes will be computed to test for differences in fine root production, fine root mortality and fine root decomposition between land uses, including tests for heterogeneity and temporal changes of the effect sizes.
For some proteins, wild-type sequence (TB Database, www. tbdb. org) has been conserved, deleted or mutated. The first fusion, RpfB-RpfD-Ag85B-TB10. 4-ESAT-6, was constituted by the residues 30 to 284 of RpfB fused to 54-147 fragment of mutated RpfD (E61K, T84A and Q113A), followed by the mature Ag85B polypeptide , and the full-length TB10. 4 and ESAT-6 proteins. The second fusion, SF-Rv2029-Rv2626-Rv1733-Rv0111, was constituted by Rv2029 deleted of the last C-terminal 25 residues and mutated (D265N), followed by the full-length Rv2626 protein, the mature Rv1733 polypeptide (63-210) and Rv0111 deleted of all except one transmembrane domain (393-685). The SF signal peptide present at the N-terminus of the F protein of measles virus (Hallé strain, described in Genbank n°X05597-1) was positioned inframe, 5' to the Mtb fusion.
The third fusion, SR-Rv0569-Rv1813-Rv3407-Rv3478-Rv1807-TMR, was constituted by the full-length Rv0569 protein, the mature Rv1813 polypeptide (35-143) followed by the full-length Rv3407, Rv3478 and Rv1807 proteins. The SR signal peptide present at the N-terminus of the glycoprotein precursor of rabies virus ERA strain (Genbank n°M38452) was positioned in-frame, 5' to the Mtb fusion while the TMR membrane-anchoring peptide derived from the rabies glycoprotein (Genbank n°AY009097) was added in frame, 3' to the Mtb fusion. Synthetic genes coding for the different Mtb fusions were synthesized by Geneart (Regensburg, Germany). The sequences were optimized for human codon usage, a Kozak sequence (ACC) was added before the ATG starting codon and a 3'TTTTTNT transcriptional terminator sequence was added immediately after the stop codon.
The Multiphasic MVATG18377 Induces Broad IFNγ Response in mice Although IFNγ is not recognized per se as being a correlate of protection in TB infection, it is believed to be an important contributor to control of mycobacterial replication  [31]. ELISpot analysis was performed one week after mice immunization to investigate the specific IFNγ response induced by the MVATG18377. Immune response specific of each expressed Mtb antigen was evaluated in three different inbred mouse strains, BALB/c (H-2 d ), C57BL/6 (H-2 b ) and C3H/HeN (H-2 k ). Concerning large antigens (Rv2029, Rv2626, Rv1733, Rv0111, RpfB-RpfD, Ag85B, Rv3478 and Rv1807) for which sequence is covered by several pools of peptides, only results obtained with the pool leading to the highest response are shown. Data of all individual pools of peptides are shown in S2 Fig.
06% and 0. 04%, respectively) as well as a high proportion of TNFα + CD8 + T cells (1. 23%). TB10. 4-specific IFNγ + cell subsets were unexpected as no IFNγ response was detected by ELI-Spot assay (Fig  2B ). As in BALB/c mice, in C57BL/6 mice no triple cytokine CD8 + T cell population was detected. The distribution of cell subsets showed that single cytokine producers constitute the main population (e. g. up to 96% in case of TB10. 4). In conclusion, these results reinforce the data obtained by ELISpot assay. In fact the seven antigens those giving a positive response in IFNγ ELISpot assays (Fig 2B ) were also shown to be positive for IFNγ staining in ICS.
Mtb Antigen-Specific CD8 T Cells Induced by MVATG18377 Are Cytolytic In Vivo We investigated whether the CD8 + T cells elicited following MVATG18377 vaccination display cytotoxicity in vivo. In vivo CTL assays were performed in BALB/c mice immunized twice with vaccine candidate using target cells pulsed with pool of antigen-specific peptides. We first analyzed the kinetics of the CTL response by adoptively transferring target cells at different times after the last immunization (3, 5 or 7 days after the last immunization). The broadest CTL killing response in terms of number of antigens was observed 7 days after the last immunization (data not shown). This time point was selected in the next experiments. Mice immunized with either the MVATG18377 or the control vector generated a strong CTL killing response to the MVA peptide-pulsed targets (80-90%, data not shown).
Additional data collected in phase 2, led to enumerate four 'career paths' as representative elements to understand trajectory or state (illustrative quotes). • A natural pathway, which takes form in time as a continuity. A priori it concerns to the older members of the profession who refer to the 'generational' dimension where entry into the profession appears to have been recorded in a conflict or a rupture but as a kind of 'evidence', in an environment when entry was easier than nowadays (training development, growth, recognition and need for external expertise). This 'natural' dimension is based on a high education and training level, a network and credibility in the field or intervention. It seems that the independent trainers-consultants appear less in a system of 'hard proof' but more in a legitimacy built 'along the way' than resulting from a struggle.
Ultrasonographic examination Ultrasonography were performed by the same operators using an Esaote Piemedical MyLab30 ultrasound device equipped with a linear LA332 probe and a volumetric BL433 probe (all materials from Hospimedi France, Valdampierre, France). Ultrasonography of individual teats was performed at 5 MHz immediately after milking by placing the probes along the teat skin, taking great care not to crush or flatten the teats. Data were acquired on the four quarters of each cow one week before challenge to select teats without morphological defects. Teats displaying cistern inflammation before challenge were excluded from the experiment. Ultrasonography measurements were carried out on the challenged and contralateral quarters just before challenge and at 16, 48, 96 and 168 hpi.
Cells were surface stained with anti-CD4 Ab, fixed, permeabilized, and stained with anti-IL-17A and anti-IFN-γ (Bio-Rad, AbD Serotec) Abs using the Cytofix/Cytoperm kit (BD Pharmingen). Data were acquired on a FACScalibur cytometer using Cellquest software (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR, USA). Gates were set according to appropriate isotype/control staining.
Statistical analysis of data Statistical analyses were performed with the Prism software (GraphPad, version 5. 0). A probability level of < 0. 05 was considered significant. Comparisons of paired samples were done with the Friedman's test followed by Bonferroni post-test comparison when appropriate. Correlation analyses were done with the Spearman's rank test. A principal component analysis (PCA) was performed with the ADE4 software  [31]  to summarize the variance observed in each of the following variable: decrease in teat cistern volume, IFN-γ in milk 14 hpi, IgG1 titer D45, IgG2 titer D45, IgM D45, IL-17A in milk 48 hpi, Peak SCC, Skin test D45 24h, Temperature 24 hpi, WBA IFN-γ D45, WBA IL-17A D45. A permutation test was used to estimate the correct number of components to retain (10 000 permutations of measurements in each variable)  [32].
Statistical analysis All the datasets were checked for missing and doublecrossed (more than one boxes selected in an item, either by mistake or because the answer was between 2 categories) values. The double-crossed values were corrected where possible, by choosing the valid selection or selecting the more frequent of the two options selected. The total sample analyses included all subjects from all communities. Due to minor discrepancies between the translated versions of the questionnaire, i. e. missing response categories in certain items, minor adaptations in the response categories were made when necessary. The Mann-Whitney U test for the ordinal variables and Pearson's Chi-square test for the binary variables were used to detect differences in behaviours and determinants between the two socio-economic groups.
Acknowledgements We acknowledge the significant contribution of the  EPHE Operational Board  to the data acquisition and all related processes. We would like to thank the involved school personnel of all countries for permitting and assisting the data collection. In addition, we would like to thank all parents/caretakers for their 358 participation to the study. The  EPHE  Project is co-funded by the  DG SanCO. Additional funding is contributed by  Ferrero ,  Mars and Orangina-Schweppes  together with Danone, whereas these partners are not involved to the design, analysis nor interpretation of the current study.
Experimental and Theoretical Methods 2. 1. DFT Calculations. Density Functional Theory (DFT) calculations have been performed with the CRYSTAL09 code  [34, 35]. DFT calculations have been performed using the B3LYP hybrid functional, corresponding to Becke's threeparameter exchange functional  [36] , and the Lee-Yang-Parr gradient-corrected correlation functional  [37]. All electron basis sets were used for C, O, and H. To test the effect of different basis sets we calculated the INS spectrum of toluene using the 3-21G+ * * , 6-31G+(d, p), 6-31G ++(2d, 2p), 6-311G++(2d, 2p), 6-311G++(3d, 3p), DZVP, and TZVP. The first three basis sets did not reproduce adequately the mode frequencies and intensities. The other entire basis sets showed the same good agreement on mode frequencies in the range 0-1300 cm -1 , but the relative intensity of the modes is best represented by the TZVP calculations.
INS. Inelastic neutron scattering experiment was performed on the TOSCA spectrometer at ISIS spallation source  [40]. The INS spectrum was measured with a high statistic by integrating for 1500 A of incoming proton current (proton accelerator was working at about 150 A/hour). Signals from detectors both in forward and in backward directions have been extracted and combined using Mantid software  [41] , without any sign of degradation of the resolution. The measured sample weighted 7. 7 grams and was inserted in a thin aluminium envelope and successively placed into inwire sealed Al cells. All the manipulations were performed inside a glove-box to prevent contamination by moisture. The sample was prepared so that it fits the beam dimension of 40 mm × 40 mm. Finally, the sample was inserted in a duplex CCR cryostat allowing measurements to be run at 20 K.
The calculated inelastic neutron scattering spectra have been obtained from the output of vibrational analysis using the aClimax software  [42]. For coronene, the contribution of second overtones has been included, while any effect of phonon tails has been considered. A spectrum convolution of Δ/ ≈ 2% has been chosen. For carbon clusters, contributions up to 10th overtone and isotropic phonon tails have been included and a broader Δ/ ≈ 4% spectral convolution has been included. The experimental spectrum of coronene, used as a reference, was downloaded from TOSCA INS database  [28, 43].
Predicting function-related amino acids in proteins with unknown function or unknown allosteric binding sites in drug-targeted proteins is a task of paramount importance in molecular biomedicine. In this paper we introduce a simple, light and computationally inexpensive structure-based method to identify catalytic sites in enzymes. Our method, termed cutoff lensing, is a general procedure consisting in letting the cutoff used to build an elastic network model increase to large values. A validation of our method against a large database of annotated enzymes shows that optimal values of the cutoff exist such that three different structure-based indicators allow one to recover a maximum of the known catalytic sites. Interestingly, we find that the larger the structures the greater the predictive power afforded by our method.
In turn, the latter should combine different indicators, related to the physical-chemical properties of amino acid environments and to patterns of chemical and topological connectivity. In this paper we focus on the prediction of catalytic sites in enzymes based on an original ENM-based strategy. Atomistic approaches devised to identify residues involved in catalysis in enzymes are not new  55. More recently, approaches specifically relying on sophisticated electrostatic calculations have been introduced  56, 57. Conversely, coarse-grained models have been relatively less exploited to solve this specific problem  29, 58, 59. Yet, ENM-based tools are light (they can be applied to large databases of structures) and can be readily extended to perform all-residue searches in many structures. Moreover, CG topology-based methods have the advantage to strip the structure of most chemical details so as to bring to the surface purely topological features.
Following previous studies, we fix k 2 = 5 kcal/mol/Å  2 63. Constructing structural hotspot indicators. The basic idea of our method rests on the evidence reported by several studies that hotspot/functional sites in proteins are generally found in stiff/rigid regions  59, [69] [70] [71]. Analogously, it has been shown that functional residues tend to move independently from the rest of the structure, involving high-frequency localized vibrations (the stiffer the bonds, the higher the frequency)  62, 63, 72, 73. Structural rigidity can be gauged by many indicators, that assess the different flavours associated with it. The simplest and more intuitive method, albeit unsuitable for automated screening of large structure databases, would be to measure fluctuations directly via MD simulations, such as in ref.
The CSA is a major resource in the field of structural biology, and provides up-to-date catalytic residue annotation for enzymes in the Protein Data Bank based on experimental structural data. The results of our ensemble analysis are reported in Fig. 4. For each indicator, we have calculated the fraction of catalytic sites that are found within a prescribed distance Δ n (in units of residues) along the sequence from a peak. For example, the curves at Δ n = 0 indicate the fraction of catalytic sites that coincide with a peak for a given indicator. A number of interesting observations can be made by inspecting Fig. 4. The reduced connectivity c increases its predictive power at increasing values of the cutoff. However, this is a trivial consequence of the fact the number of peaks also increases as the systems become more and more connected (top right panel).
Discussion, Conclusions and Perspectives In this paper we have investigated the ability of different structure-related indicators to pinpoint the location of known catalytic sites in a large number of enzyme structures in the framework of the elastic network model. More precisely, we defined reduced peak patterns of (i) local connectivity, (ii) closeness centrality and (iii) structural stiffness, where the peaks retained along the protein sequence are assumed to flag potentially interesting sites. Our method is general and computationally inexpensive (see supplementary material for a benchmark test). Our analysis shows that all three considered indicators display a considerable predictive power (up to 50% of the catalytic sites recovered within a distance of two amino acids along the sequence), when the computed peak structures are compared with the location of annotated catalytic sites in a large database of enzymes (the Catalytic Site Atlas  75  ).
An analysis performed over the whole CSA database shows that the fraction of structures where the combined algorithm returns a prediction is 0. 61 for Δ n = 1 and 0. 68 for Δ n = 2 (see also Supplementary Material). In order to elucidate the meaning of the site scores S i and global score S Δn , it proves useful to concentrate on a specific enzyme. In Fig. 7 , we consider the classic case of HIV-1 protease. Let us first concentrate on the profile of the combined score (9). Two facts immediately apparent: (i) the catalytic sites appear to be all captured but (ii) there are a number of orphan peaks. The global scores for this enzyme are S Δn=1 = 0. 27 and S Δn=2 = 0. 29. Thus, despite the algorithm flags correctly all the catalytic sites, it does so with some degree of over-prediction (incidentally, we observe that the orphan peaks shown in the combined score profile in Fig.