dataesr/scientific-paragraphs-categorization huggingface dataset:
5. One of the strengths of our study is the extended follow-up duration, giving both chance to observe a high number of events and time to observe immune restoration. To our knowledge, this study is the first to analyse morbidity on cART with such an extended follow-up duration. In addition, by recruiting patients in 47 medical centres in France with a standardised data collection carried out by qualified personnel, this study gave us a good representation of the population of patients who started cART in France in the late 1990s. Moreover, all severe events in the cohort were monitored, which gave us completeness in the collection of events. These events were validated by an endpoint review committee constituted by experienced clinicians (see events validation committee in appendix), which reinforces the validity of the diagnoses.
Acknowledgements We would like to thank all participating IMD experts without whom this survey would not have been possible: Austria,  Sigrid Heuberger ;  Belgium ,  Valeska Laisnez  and  Carole Schirvel; Bulgaria ,  Teodora Georgieva ; Croatia,  Sanja Kurečić Filipović ; Cyprus,  Maria Koliou ;  Czech Republic ,  Pavla Křížová; Denmark ,  Palle Valentiner-Branth ; Estonia,  Natalia Kerbo ; Finland,  Markku Kuusi ;  France, Isabelle Parent du Chatelet; Germany, Wiebke Hellenbrand ; Greece,  Georgina Tzanakaki  and  Theano Georgakopoulou ; Hungary,  Katalin Krisztalovics ; Iceland,  Thorolfur Gudnason ; Ireland,  Suzanne Cotter ; Italy,  Fortunato D'Ancona; Latvia ,  Irina Lucenko; Liechtenstein ,  Sabine Erne ; Lithuania, Egle Pauzaite; Luxembourg, Pierre Weicherding; Malta,  Jackie Maistre Melillo; Netherlands ,  Aura Timen ; Norway,  Hans Blystad ; Poland,  Iwona Paradowska-Stankiewicz ; Portugal,  Ana Leça ; Romania, Lavinia Cipriana Zota; Slovakia,  Maria Avdičová ; Slovenia,  Alenka Kraigher; Spain ,  Rosa Cano; Sweden ,  Tiia Lepp ; Switzerland,  Elisabetta Peduzzi ;  United Kingdom ,  Shamez Ladhani.
EEG signals were acquired at 200Hz using band pass filter (50Hz) and were analyzed using a combination of commercially available software (p-Clamp 9. 2; Origin Microcal) and a routine (Matlab 2013b; Matworks Inc. ) optimized in our laboratory. Sham animals (IN PBS) displayed no EEG changes. Acute SE was characterized by generalized EEG convulsions (on/off episodes lasting >2 minute in the acute phase) developing to synchronized continuous generalized activity 45 to 60 minutes after. Spike activity was defined as symmetrical EEG signals with amplitude of at least 2-3 fold the baseline. In the chronic phase SE was characterized by generalized EEG activity lasting at least 10-15 sec. Video review was performed to rule out EEG artifacts (scratching, chewing or sleep). For each mouse we determined the time spent in spike activity and SE during seizure progression.
EEG data were obtained as X/Y (msec/ V) Excel or European Data Format (. edf) files. We applied a Short Time Fourier Transform to the EEG files calculating signal energy as a function of frequency and time. Power spectra were estimated as the square of the Fourier transform for each period analyzed. STFTðo; tÞ ¼ Z 1 À1 eegðtÞ cðt À tÞe Àiot dt ð1Þ In particular, ψ(t) = Bartlett function; ω = frequency in hertz; τ = translation time form segment to segment; t = time in seconds; eeg (t) = EEG signal. A qualitative measure for EEG frequency bands (δ, θ, α, β, γ) was performed. The latter measurement is defined as: Z o i max o i min Iðo; tÞ do i ¼ d; y; a; b; g ð2Þ Where (o i min ,o i max ) are the frequency limits for the band I and I(ω,t) is the power spectrum as a function of frequency and time t.
The primer pairs were either commercially available (Quantitec primers, Qiagen) or designed using Primer 3 input software, and their specificity and efficacy was experimentally validated. Cq for individual genes were determined using the second Derivative Max tool of the LightCycler480 software. The relative RNA expression was calculated using Gapdh as a reference gene. Indeed there was no statistical variation in the level of expression of Gapdh across the experimental groups. Relative expression between KA-treated and control groups were calculated using the ΔΔCq method  [16]. For genes undetectable under control conditions, the fold change (FC) increase was determined by attributing a Cq value of 35 corresponding to the maximal value in our experimental conditions. Thus, under these conditions FC values were underestimated.
Microgliosis was observed as early as 24 hours after IN KA induced SE. Our results show that both microglial M1 and M2 genes are dysregulated following IN KA induced SE. M1 corresponds to a pro-inflammatory status, correlating to or facilitating neuronal damage. In contrast, the anti-inflammatory M2 phenotype may be beneficial following brain pathological events  [25]. Although, we cannot rule out that some of these genes may also be expressed by activated astrocytes, and the number of genes analyzed is limited, our results suggest complex microglia activation following IN KA SE. Our data are consistent with earlier studies performed using 20-40 mg/kg IP KA  [6, 14]. Remarkably, inflammatory signs were present 15 days after SE onset. Although significant, the inflammatory changes developing after IN KA induced SE were less prominent as compared to the IP protocols, suggesting that the IN KA dose can be still modified to induce a more severe phenotype.
Habitat type and the multispecies characteristics did not show any significant effects, but showed marginal interaction. The multispecies sites show higher infection rates, compared to monospecific sites, for caves compared to buildings and forest capture sites (Fig. 2 ), reinforcing the important role of multispecies bat environments on PV infection. Diet was also associated with viral infection (Table  2 ), with higher infection among insectivorous bat species, whereas, age and sex did not show any significant relationships. Generalized Linear Mixed Model (GLMM) with species, locality, and province as random factors were tested separately and did not improve the fit, but models with family, species and locality failed to converge due to numerical issues in model estimation. We conducted a Bayesian analysis on the PV sequences generated from positive Malagasy bats together with PV GenBank sequences from Madagascar and elsewhere in the world.
Methods Fieldwork and sampling. This study used samples collected in the context of a long-term project to document the land vertebrates of Madagascar based on voucher specimens and for a variety of studies  26. From February 2012 to March 2013, bats were captured in the six different provinces of Madagascar using harp traps, hand nets, and mist nets. Some Pteropus fruit bats were purchased in markets. Individual bats were identified to species using external and cranio-dental characters and comparison to museum specimens. For each animal, different parameters, including age, sex, and reproductive condition  45  were recorded and this information deposited in DRYAD (http://dx. doi. org/10. 5061/dryad. 06g12). Bat tissue samples were collected in the field and immediately stored in liquid nitrogen, then transferred to -80 °C storage upon arrival in the laboratory.
The geographic ranges of the captured bat species were variable, with some having broad distributions nearly across the complete island and others distinctly more restricted. Several species, especially insectivorous bats, occur sympatrically in the same cave systems and in the same forest blocks, or synanthropically in human-built structures. Information on the species, province and specific collection locality, sampling season, geographic coordinates, elevation, habitat type, and the number of bat species found at each site and the associated species composition are presented in Tables  S1  and S2. Mean climatic conditions of the sampling sites were extracted from the WorldClim database (http://www. worldclim. org/). We used the resolution proposed by WorldClim as 30 arc seconds (∼1 km).
An open-source GIS software, QGIS  46  , was used to generate the map for visualizing Madagascar bioclimatic regions proposed by Cornet  47. Ethics statement. Animals used in this study were manipulated in strict accordance with the guidelines for the handling of wild mammals  48. All protocols strictly followed the terms of research permits and regulations and were approved by licencing authorities: Direction du Système des Aires Protégées et Direction Générale de l'Environnement et des Forêts and Madagascar National Parks under different research permits (n°194/12/MEF/SG/ DGF/DCB. SAP/SCB, 067/12/MEF/SG/DGF/DCB. SAP/SCBSE, and 032/12/MEF/SG/DGF/DCB. SAP/SCBSE). Animals were captured, manipulated, and dispatched with thoracic compression following procedures accepted by the scientific community for the handling of wild mammals  48.
Bioinformatics analysis. The sequences were first compared to the published sequences from the Paramyxoviridae and published UMRVs in GenBank (National Center for Biotechnology Information, Bethesda, Maryland, USA) online (www. ncbi. nih. gov) using BLASTn and BLASTx. The sequence quality of individual reads was assessed, and all sequences were processed using the Geneious Pro software package v7. 1. 8  53. DNA sequences obtained from at least three independent bacterial clones were aligned to correct for most sequencing or PCR introduced errors. M13 Primer sequences were trimmed from the finalized sequences. The resulting partial sequences (~490 bp) of the L-gene polymerase gene were then aligned with Translation Alignment using the default ClustalW cost matrix in Geneious Pro software package.
PVs sequences from bats reported in a previous study by Wilkinson et al. 17  were used for phylogenetic analysis (GenBank number to KF928225 to KF928256 and to K928261 to KF928265). PVs and bat Cytochrome b (Cyt-b) sequences used for the present analyses were deposited in GenBank under the reference numbers in respectively Table  S3  and Table  S4. Information concerning amplified PV sequences is given in Table  S3. In order to classify the detected new paramyxoviruses, viral family-level phylogenetic analyses were performed. A total of 209 partial L-gene paramyxovirus sequences collected in Genbank were used. Sequences were trimmed to remove any free end gaps or were entirely removed if the obtained alignment did not provide at least 462 bp of non-gap overlap. Internal gaps were permitted.
The tree was performed in 5,000,000 iterations in MrBayes with the GTR+ G+ I evolutionary model and a 10% burn-in rooted with an Aquaparamyxovirus sequence (GenBank number EF646380). Genbank accession numbers used for each virus genera are indicated in Table  S6. A best-fit nucleotide substitution model of the alignment was determined using jModelTest  54  with the Corrected Akaike Information Criterion (AICc)  55  , and the most appropriate one for URMVs from Malagasy bats was GTR+ G. Phylogenetic trees were constructed using MrBayes v3. 2  56  employing a Bayesian Markov Chain Monte Carlo (MCMC) method, rooted with a Mumps virus sequence (GenBank number AY309060). A minimum of two independent runs were made, with four chains in each run, for a total of 50 000 000, sampling every 5000 generations.
The first 5000 trees burn-in were discarded. The obtained effective sample size values (ESS) for each parameter were all superior to 200. Trees obtained after the convergence point were summarized and visualized by FigTree 1. 4. 2 (http://treebioedacuk/software/figtree). Available full-length Cyt-b gene sequences corresponding to each bat species that were positive for UMRV infection were downloaded from GenBank. When Cyt-b sequences were not available for a given bat species, PCR using primers targeting the Cyt-b gene were performed  57  to generate ~1140 bp sequences. All bat Cyt-b sequences were aligned, the GTR+ I+ G model was also the most appropriate substitution model, and the phylogenetic relationship among bat species were analyzed using RAxML 7. 2. 8 Geneious plugin 53 using 1000 generations.
Two subsets of operational taxonomic units (OTUs) were defined using Mothur  58  , and based on two genetic distance cutoffs (90. 0% and 98. 0%), generated 24 and 39 representative sequences, respectively. To study the history of co-evolution of UMRVs with respect to their associated bat hosts, we performed event-based co-phylogenetic reconciliation, using the tool CoRe-PA  59. CoRe-PA is an event-based maximum parsimony method, which attempts to construct the most parsimonious co-evolutionary history of hosts and associated parasites. A cost is assigned to each type of co-evolutionary event (co-speciation, host-switching, duplication, and sorting) and then, the parasite phylogeny is mapped onto the host phylogeny, while trying to minimize the total costs of all occurring events. In contrast to many other co-evolutionary software packages, CoRe-PA does not require an a priori assignment of a cost scheme.
We quantified the degree of congruence between bat and UMRVs topologies, and the potential individual associations leading to a potential co-phylogenetic structure using a global-fit method, ParaFit  60. The latter program tests the independence of host and symbiont genetic or patristic distances, and specifically herein, tests the hypothesis of evolution independence between bats and UMRVs, i. e. , one partner randomly evolving with respect to the other. Statistical analyses were done using the R software package  49.
Acknowledgements For kindly providing research permits for our work on Madagascar, we are grateful to  Département de Biologie Animale, Université d' Antananarivo ;  Direction du Système des Aires Protégées et Direction Générale de l'Environnement et des Forêts ; and  Madagascar National Parks. We are indebted to the  Institut Pasteur de Madagascar  for their help in sample preservation and exportation and  Pr. Xavier N. de Lamballerie  at  Aix Marseille University, IRD , and  Dr. David A. Wilkinson  from  CRVOI  for their technical assistance. This work was financially supported by  CRVOI  and from the  European Regional Development Funds FEDER POCT   ParamyxOI  project ( N°33857 ). The postdoctoral fellowships of B. Ramasindrazana were funded by  RUN-Emerge ,  European Commission   FP7 Regpot Capacity program , the  Fonds de Coopération régionale, Prefecture de La Réunion , and  The Field Museum of Natural History, Chicago , through the  Dr.
1. 3 (FlowJo, Ashland, OR). The gating strategies are shown in figures and all analyses exclude dead cells based on 7-actinomycin D (Sigma Aldrich) staining. Statistical analysis. Significant differences between mean values were determined using unpaired two-tailed Student's test or Mann-Whitney depending on normality using GraphPad Prism software version 7. 6. 5. P-value under 0. 05 was considered statistically significant.
ACKNOWLEDGMENTS The authors are grateful to their colleagues at  Genethon in the groups "Bioexperimentation", "Animal Facility" , and " Flow cytometry " for help with experiments. The authors are also grateful to  CJ Buchholz  for criticial review of the manuscript. Financial support for this study was obtained from  PERSIST  ( EC FP7  large-scale integrating project GA number  222878 ), from  AFM/Telethon , from " Fondation pour la Recherche Médicale " and from the  French Ministry of Research via Ecole Doctorale   B2T ,  University Paris Diderot.
2C ). Quantitative analysis of EhPC4 and lamin B colocalization using the WCIF ImageJ software, suggested that both proteins colocalized in great extent as indicates the Pearson's correlation coefficient (0. 79) and Mander's overlap coefficient (0. 63) (Fig. 2D ). To study EhPC4 cellular functions, we first performed knock-down assays to inhibit its expression using the pEh-hyg-TetR-O-EhPC4 tetracycline inducible vector. However, the inhibition of EhPC4 was not maintained over the days even at high tetracycline concentrations. In addition, viability of trophozoites was compromised (data not shown). Therefore, we decided to overexpress the EhPC4 protein in trophozoites and investigated the biological effects through diverse cellular approaches. A Myc-tagged EhPC4 protein was produced in trophozoites via transfection of the pKT-3M-EhPC4 plasmid.
Methods Cell cultures. E. histolytica trophozoites (HM1:IMSS strain) were axenically grown at 37 °C in Diamond's TYI-S-33 medium and harvested during exponential growth phase for experiments. In silico analysis. PC4 orthologues were identified by BLAST in NCBI database using the EhPC4 amino acid sequence (UNIPROT: C4M1H2) as query. Functional domains were predicted using MyHits server. Multiple alignments were performed using the ClustalW program. The phylogenetic tree was constructed by the Neighbor-Joining method using the MEGA software (version 5. 0). EhPC4 tertiary structure was predicted using the Phyre program and the construction of structural alignment was performed using VMD viewer. Molecular modeling of CTD-EhPC4 dimer in complex with a 19-mer oligonucleotide (oligo-dT (18) G) was generated by homology using the MOE program.
DNA was counterstained with 2. 5 μ g/ml DAPI (Invitrogen) for 1 min. For coimmunolocalization studies staining was carried out with anti-EhPC4 (1:100) and anti-lamin B1 (Abcam ab16048; 1:100) antibodies, followed by incubation with anti-rabbit Alexa 647 (Invitrogen; 1:200) and anti-mouse Alexa 488 (Invitrogen; 1:200), respectively. Light optical sections were obtained through a Nikon inverted microscope attached to a laser confocal scanning system (TCS SP2, Leica Microsystems) and analyzed using the Confocal Assistant software Image J. A three-laser beam (350, 488 and 568 nm) was used for excitation of the fluorophores, using a 25 Plan-NEOFLUAR oil immersion lens (0. 8 numerical aperture). Immunofluorescence staining experiments were done by triplicate and multiple serial sections were examined.
Cell proliferation, cell viability and cell size determinations. 1. 5 × 10 5 trophozoites were inoculated into culture tubes with TYI-S-33 medium and grown at 37 °C during 24, 48, 72 and 96 h. At each time, cell number, cell diameter and viability were measured using the TC20 TM Automated Cell Counting System (BIO-RAD) and the TC10 trypan blue dye method as described by the manufacturer. Data were expressed as the mean ± standard deviation (SD) of three independent experiments. DNA replication assays. DNA replication experiments were performed using the DNA Replication Assay kit (Upstate Biotechnology) according to manufacturer's recommendations. Trophozoites incorporating the fluorescent BrDU in DNA were examined through a Nikon inverted microscope attached to a laser confocal scanning system (TCS SP2, Leica Microsystems) and analyzed with Image J software.
Fluorescence intensity of 250 randomly selected cells was measured with the Leica confocal software. Three experiments were performed and data were expressed as the mean fluorescence ± SD. Determination of nuclei number. Trophozoites were synchronized by serum starvation for 12 h as described 3 ; 1. 5 × 10 5 cells were inoculated into culture tubes with TYI-S-33 complete medium and grown at 37 °C for 24, 48, 72 and 96 h. Then, trophozoites were fixed with 4% paraformaldehyde on sterile coverslips, nuclei were stained with DAPI (Invitrogen) and cells were analyzed through a Nikon inverted microscope attached to a laser confocal scanning system (TCS SP2, Leica Microsystems). The number of nuclei per cell at 0, 24, 48, 72 and 96 h post-synchronization was determined from 250 randomly selected trophozoites.
Determination of nuclear DNA content. DNA content was analyzed by FACS in paraformaldehyde-fixed trophozoites after staining with propidium iodide (PI) using standard procedures. Flow cytometry was performed using the FACScalibur (Becton Dickinson) and data from 10,000 cells were recorded for each experiment and analyzed using CellQuest software (Becton Dickinson). Real-time microscopy. Trophozoites were synchronized as described  3  , plated on POC-R2 Chambers (LaCon, Germany) with TYI-S-33 medium supplemented with adult bovine serum (10%) for 24 h at 37 °C and visualized under a Axiovert 200 M fluorescence microscope (Zeiss Germany) (40× objective). The time-lapse images were captured with 2 s intervals, analyzed and further processed using the Axiovision v4. 8 software (Zeiss, Germany).
Acknowledgements We thank  Universidad Autonoma de la Ciudad de Mexico  ( UACM ) for support. This work was also supported by  CONACYT  (59481 grant) and  Mexico-France program  ( SEP-CONACYT-ANUIES; ECOS NORD  grant  M08-S02 ). Olga Hernández de la Cruz  was a scholarship recipient from  ECOS-NORD Mexico-France  grant  M08-S02  ( 2008-2012 ). We thank  Tomoyoshi Nozaki  for provide us the pKT-3M plasmid, to the Genopole platform from  Pasteur Institute Paris  for DNA microarray analysis, and to  Dr. Rosaura Hernández  for provide us the anti-Lamin B1 antibodies.
Mass spectrometry Mass spectrometry (MS) measurements were performed with an electrospray Q/TOF mass spectrometer (Q/TOF Premier, Waters) equipped with the Nanomate device (Advion). The HD_A_384 chip (5 μm I. D. nozzle chip, flow-rate range 100-500 nL/min) was calibrated before use. The Q/TOF instrument was operated in the RF quadrupole mode and data were acquired in the 400-3990 m/z range. Collision energy was set to 6 eV and argon was used as collision gas. Mass spectrometry of the intact proteins was performed after 5 min denaturation in 50% acetonitrile and 1% formic acid. Acquisition and data processing were performed with Mass Lynx 4. 1 software. Deconvolution of multiply charged ions was performed by applying the MaxEnt1 algorithm. The average protein masses are annotated in the spectra, and the estimated mass accuracy is ± 2 Da.
Acknowledgments The authors are grateful to  Dr. J. S. Lomas  and  Dr. M. Kirkpatrick  for constructive discussion, to  Prof. E Gautier  from the  Etablissement Français du Sang (EFS) , and to  Prof. A. Bénachi  and  Dr. A. G. Cordier  from the  Antoine Béclère Hospital (Clamart)  for the placentas. We also thank  X. Baudin  and  R. Leborgne  for their technical assistance in confocal and transmission electron microscopies and the  ImagoSeine Facility of the Institute Jacques Monod, Paris, France.
This trial was, however, halted due to safety concerns related to the adjuvant. In order to improve the immunogenicity of Pfs25-based vaccines, several methods are being investigated. Pfs25 fused to a detoxified form of Pseudomonas aeruginosa exoprotein A (EPA), forming a nanoparticle with hydrodynamic radius ranging in size from 5 to 25 nm  10  , has been shown to increase antibody responses versus unconjugated Pfs25 between 75-and 100-fold  11. A batch of Pfs25-EPA has been manufactured according to Good Manufacturing Practice (GMP) and has entered Phase Ia/b clinical trials (ClinicalTrials. gov Identifier: NCT01434381 and NCT01867463). Similarly, Pfs25 has been fused to the outer-membrane protein complex (OMPC) of Neisseria meningitidis serogroup B. This Pfs25-OMPC induced a substantial increase in anti-Pfs25 antibodies in mice compared to a similar dose of Pfs25 alone, as well as demonstrating a response sustained for over 18 months in rhesus monkeys  12.
Other studies have demonstrated that fusion of an antigen to IMX313 has a number of beneficial adjuvant effects. Immunization of mice with the Mycobacterium tuberculosis antigen 85A fused to IMX313 in both DNA vaccines and viral vectors showed consistent increases in CD4 + and CD8 + T cell responses. This same fusion induced higher IFN-γ responses in rhesus macaques and improved the quantity of the immune response in both mice and monkeys without changing the quality  16. A viral vector vaccine encoding 85A-IMX313 has now entered Phase I clinical trial in healthy UK adults (NCT01879163). In this study, we investigated the potential of using the IMX313 multimerization strategy to improve the immunogenicity and transmission-blocking efficacy of vaccines targeting Pfs25. We have fused Pfs25 to IMX313 and expressed it from the leading clinical viral vectors, chimpanzee adenovirus serotype 63 (ChAd63) and modified vaccinia virus Ankara (MVA)  17.