dataesr/scientific-paragraphs-categorization huggingface dataset:
statistics For arthritis studies, serial measurements of clinical variables were analyzed by the area under the receiver operating characteristic curve for each mouse as a summary measure, then as raw data for inter-group comparisons (ANOVA). Differences in arthritis onset were analyzed by ANOVA. Posttest data were compared by data distribution (Student-Newman-Keuls). For Listeria infection studies, evaluation of survival involved the Kaplan-Meier method (log-rank test), and bacterial burden data were analyzed by one-way ANOVA with Newman-Keuls posttest. p < 0. 05 was considered statistically significant.
postinfection, but the other mice did not experience body weight loss (data not shown) and were euthanized during early infection (day 28) or established infection (day 56). Blood was collected before (day -10) and during infection (day 28) to evaluate levels of anti-TNF-α antibodies. As expected, ELISA revealed a sustained serum production of anti-TNF-α antibodies in mice vaccinated with TNFKi (Figures  S3A, B  in Supplementary Material). At euthanasia, weights of lungs, spleens, and livers were evaluated as indicators of inflammation. During early infection, all TNF -/-mouse organs showed a marked increase in weight as compared with the other groups, with the exception of lung and spleen of etanercept-treated mice (Figures  4A-C ). At day 56, established infection, organ weights were similar between all infected mice (Figures  4D-F ).
In the lungs of TNFKi, KLH, and PBS groups, granulomas were more discrete, with the typical   granuloma structure at day 56 (Figures  6A-H ). Etanercepttreated mice showed granulomas on day 28, but these were more developed and less structured by day 56 (Figures  6C, G ). With Archimed software quantification, during early infection, the lung granuloma surface area was increased but not significantly in TNF -/-and etanercept-treated mice (Figure  6I ). At day 56, the granuloma surface area of TNFKi-and KLH-vaccinated mice appeared decreased, contrary to etanercept treatment (*p < 0. 05 vs etanercept group, ANOVA) (Figure  6J ). Granuloma numbers were similar between mouse groups at both phases (data not shown). During both early and established infection, all infected mice showed less free alveolar space as compared with naïve mice (Figures  6K, L ), which was most prominent with etanercept treatment on day 56 (Figure  6L ).
fr/products/tnf-kinoid/, NCT01911234). The TNFKi vaccine used in the present study consisted of mouse TNF-α linked to the KLH carrier protein. TNFKi induced the production of anti-TNF-α polyclonal antibodies and alleviated arthritis in two experimental To assess to what extent this novel anti-TNF strategy would affect immunity against infectious agents, we used two experimental models of infection, with L monocytogenes and M. tuberculosis. In both cases, we compared the effect of TNFKi to that of complete lack of TNF-α (TNF -/-mice) and a current anti-TNF-α treatment (etanercept). Etanercept is considered the safest anti-TNF inhibitor in terms of the possible risk of serious infection (notably tuberculosis reactivation). It is a pharmacological reference for anti-TNF-α blockade in animal models of infection  (25).
bovis BCG, effectively protecting these highly susceptible mice  (42). There are some limitations to this work that warrant further studies. The exact mechanisms by which the two different anti-TNF strategies affect the immune response to infectious agents need to be further dissected. Another issue is the extrapolation of the results obtained in the animal model to humans, which is not the aim of the present study. Although we used validated models of arthritis (CIA) and infection, these models only partially mimic human disease. Moreover, differences between the human and murine immune system both in response to vaccination and infection may lead to deep discrepancies that warrant caution. No safety issues or increased risk of infection were reported when anti-TNF vaccination was administered in humans (Crohn's disease: NCT00808262; RA: NCT01040715).
Introduction Dominant optic atrophy (DOA) (Online Mendelian Inheritance in Man database #165500) is characterized by moderate to severe loss of visual acuity. The insidious onset of the disease in early childhood is caused by the selective loss of retinal ganglion cells (RGCs) and degeneration of the optic nerve. The majority of patients with genetically confirmed DOA (about 60-80%) harbour mutations in the OPA1 gene coding for a mitochondrial GTPase. This dynamin GTPase mediates the fusion of mitochondrial inner membranes and is consequently a key moderator of the organization of the mitochondrial network. OPA1 is also involved in other functions including maintenance of the mitochondrial membrane potential  [1] , the integrity of the mitochondrial genome  [2]  and the control of apoptosis through its regulatory role in the organization of cristae and the subsequent trapping of cytochrome c  [3].
Mitochondrial morphology Fibroblasts were seeded either on Lab-tek glass coverslips (Thermo Fisher Scientific) at least one day before imaging, or on micropatterned coverslips (Cytoo â ) (Cytoo SA, Grenoble, France) at least 4 hrs before image acquisitions. Mitochondrial morphology was assessed by staining cells with 10 nM Mitotracker Red (Life Technologies, Carlsbad, CA, USA) for 15 min. at 37°C. Wide-field cellular fluorescence images were acquired with an inverted Leica microscope (Leica Microsystems, Wetzlan, Germany) equipped with a Roper CoolSnap HQ2 camera (Roper Scientific, Munich, Germany). Images were collected using a 9 100/1. 5 oil objective. Data were acquired and analysed using Metamorph software â 7. 7 (Universal Imaging Corporation, Downingtown, PA, USA). An average of 21 image planes was acquired along the z-axis with an increment of 0.
2 lm between each step. Measurements were performed at room temperature (20°C). During acquisition and image data analysis, only well-spread triangular cells were selected to minimize biases arising from the variation of cell size and shape  [19]. An average of 100 cell acquisitions from micropatterned slides was used. The images were deconvolved with Huygens Essential Software (Scientific Volume Imaging, Hilversum, Netherlands). All the selected images were iteratively deconvolved with a maximum iteration scored 50 and a quality threshold of 0. 05. Bitplane's Imaris 7. 1. 1 â software (Bitplane, Zurich, Switzerland) was used for 3D processing and morphometric analysis.
Fluorescence microscopy and quantitative analysis of mitophagy For mitophagy experiments, cells were loaded with Lyso-ID (Enzo Life Technologies, Farmingdale, NY, USA) to mark lysosomal structures (green) and MitoTracker Red FM (Life Technologies) to visualize mitochondria (red) for 30 min. The degree of colocalization of mitochondria with lysosomes was measured via live cell imaging microscopy at 37°C in an atmosphere of 5% CO 2 , using an average of 100 cell acquisitions from micropatterned slides. Bitplane's Imaris 7. 1. 1 â software was used for analysis. Spot detection software and surface tools were used for the 3D detection of lysosome and autophagosome structures (see Movie S1). Contacts between these structures and mitochondria, considered as markers of cell degradation, were determined using the colocalization tool provided with Bitlane's Imaris 7.
Anti-mouse and anti-rabbit peroxidase-linked secondary antibodies (GE Healthcare, Life Sciences) were used at 1/ 10,000 and 1/20,000 respectively, and signals were detected using a chemiluminescence system (Super signal â West Femto, Thermo Scientific, #34095) (Thermo Fisher Scientific). The images acquired were analysed quantitatively using Image Studio 2. 1 software (Li-Cor, Lincoln, NE, USA).
The thermal cycler conditions consisted of 95°C for 3 min. , followed by cycles of 95°C for 15 sec. and 59°C for 40 sec. The analyses were performed in triplicates. To quantify mtDNA content through live microscopy, nucleoids were stained by diluting stock PicoGreen â solution (Molecular Probes, Eugene, OR, USA) at 2 ll/ml directly into the cell culture medium for 15 min. Co-staining was performed with a mitochondrion-selective dye, tetramethylrhodamine methyl ester perchlorate â (TMRM) at 50 nM for 15 min. Image treatment was performed using Imaris spot detection software (Bitplane) to visualize and quantify mtDNA/nucleoid labelling. An average of 50 cell acquisitions from micropatterned slides was used.
(A-C) The results, represented as LC3II conversion (LC3I/ total LC3I+II) and P62, show ratios of protein band densities to the densities of the respective tubulin alpha bands (TUBA) (See Fig. S4 ). To modulate and test efficiency in these cell lines, control and OPA1 mutated fibroblasts were incubated in presence of either DMSO (A), a mitochondrial uncoupler CCCP (B), or the autophagy inhibitor chloroquine (C). Data obtained from five independent experiments were analysed. P1-P7: patients; CTL: controls. Fig. 2  Morphology of mitochondrial network and bioenergetics in control and OPA1 mutated fibroblasts. Cell volumes and shapes were standardized using micropatterned coverslips. Mitochondrial lengths were assessed and colour-coded (e. g. the red colour represents mitochondria >20 lm). (A) Fibroblasts were incubated in 2/3 Dulbecco's modified Eagle medium and 1/3 Amniomax for 48 hrs, and then loaded with MitoTracker Red as described in Materials and methods.
Representatives of 5-10 similar images are shown for each cell line. Scale bar = 10 lm. (B) Bar graphs show the distribution of the mitochondrial population in three different categories on the basis of mitochondrial morphology: 'filamentous', mitochondria >20 lm; 'intermediate', mitochondria 5-20 lm; and 'fragmented', mitochondria <5 lm. (C) Bar graphs show mitochondrial volume quantified as described in Materials and methods. Data were obtained from three independent experiments. (D) Mitochondrial oxidative phosphorylation was analysed on control (CTL) and OPA1 mutated fibroblasts. Oxygen consumption was measured under basal conditions (Basal respiration graph), in the presence of oligomycin (4 lg/ml) (oligomycin-insensitive respiration graph), and in the presence of FCCP (4 mM). The respiratory control ratio (RCR graph) was calculated as the ratio of the FCCP-uncoupled to the oligomycin-insensitive respiration.
Respiration coupled to ATP production (RCRp graph) was calculated as the ratio of the difference between the rates of basal and oligomycin-insensitive respiration to the uncoupled FCCP respiration. Respiratory reserve capacity was calculated as the ratio of basal respiration to the FCCP-uncoupled respiration (R/FCCP graph). Data obtained from six independent experiments were analysed. P1-P7: patients; CTL: controls. accumulation of P62 in fibroblasts from patients with mono-or biallelic OPA1 mutations, in conformity with various reports on impaired autophagy  [36, 37]. Indeed, P62 is degraded by the autophagic pathway to prevent the formation of inclusion bodies. Thus, if the pathway functions normally, then there should be no accumulation of P62  [36]. We then assessed the consequences of the induction of autophagy using the mitochondrial uncoupler CCCP which collapses the mitochondrial membrane potential to boost the elimination of damaged mitochondria by autophagy.
Acknowledgements We are grateful to  Kanaya Malkani  for critical reading and comments on the manuscript. This work was supported by grants from the following patients' foundations: ' Ouvrir les Yeux', 'Association Contre les Maladies Mitochondriales', 'Retina France', 'Union Nationale des Aveugles et D eficients Visuels', 'Fondation VISIO' and 'Kjer France '. We acknowledge support from the  INSERM, CNRS ,  Universit e d'Angers, CHU d'Angers, la R egion Pays de Loire , and Angers Loire M etropole. We thank the  SCIAM 'Service Commun d'Imageries et d'Analyses Microscopiques, Universit e d'Angers'  for transmission electron microscopy acquisitions. We thank  Dr. Majida CHARIF  for her help in determining the pathogenicity score of OPA1 variants.
Detection and characterization of microsporidian ncRNAs Conventional approaches used to annotate coding DNA sequences (CDS) consider the first AUG codon in an open reading frame as the transcriptional initiation site (TIS). However, this is often inaccurate and can cause the erroneous inclusion of intergenic regions and/or other small overlapping unpredicted genes in the 5' end of the CDS. To mitigate this and help identify ncRNA that could potentially be found in these overlapping segments, we first revisited the latest annotations from Encephalitozoon cuniculi (accession codes AL391737. 2, AL590442. 2-AL590451. 2), Encephalitozoon intestinalis (accession codes CP001942. 1-CP001952. 1), Encephalitozoon hellem (accession codes CP002713. 1-CP002724. 1) and Encephalitozoon romaleae (accession codes CP003518.
20 BLASTN results were manually explored and flanking regions analysed to identify putative transcriptional regulation signals. Multiple sequence alignments were performed with Clustal Omega  24  to validate sequence conservation between the four Encephalitozoon genomes. The presence of ncRNAs in species from the genus Nosema was first investigated by screening the Nosema genomes (N. ceranae (accession codes JPQZ01000001-JPQZ01000536), N. apis (accession codes ANPH01000001-ANPH01001133) and N. bombycis (accession codes ACJZ01000001-ACJZ01003558)) for the Encephalitozoon ncRNA genes identified as described above using BLASTN searches (word size: 7 nts, Match/Mismatch Scores (1, À1), Gap Costs (Existence: 0 Extension: 2) and low-complexity filter disabled). Next, because accurate annotation and TIS prediction in Nosema genomes has been exclusively carried out for N.
ceranae,  25  only the intergenic regions from this species were extracted and used as queries against the N. apis and N. bombycis genomes for genus-specific BLASTN searches (same parameters), with putative hits investigated for the presence of transcriptional regulation signals as described above. Canonical secondary structures were predicted using locARNA  26  and refined manually. In silico functional annotations of identified ncRNAs were conducted using the RFAM 12. 0 database. 27 ncRNAs lacking predicted functions after this step were annotated according to the expertize available in our group as follows. The C/D box snoRNA genes were annotated based on their conserved C (RUGAUGA) and D (CUGA) sequence motifs located at the 5' and 3 0 ends, respectively, and based on a short terminal stem located 2 nt upstream and of the C and D boxes, respectively.
cuniculi snoRNA and rRNA sequences. rRNA alignments were built using multalin,  28  (http:// multalin. toulouse. inra. fr) for human, yeast and Encephalitozoon genomes. Positions modified in S. cerevisiae and human rRNA were obtained from the Human snoRNA  29  and the S. cerevisiae snoRNA 30 database, respectively. rRNA targets of Encephalitozoon box C/D and H/ACA snoRNA were then predicted by searching in rRNAs for putative base pairings to the expected regions in snoRNA (upstream the D or D' box for C/D box snoRNA and in the pocket of the stem loop for H/ACA box snoRNA). E. cuniculi candidate targets were reported in the alignment. Positions labeled as modified in human and/or yeast and predicted as modified in E. cuniculi were kept as true positive modifications. SnoRNA actors of these modifications were assigned the same identifier than in human/yeast when possible.
Identification and validation of non-coding RNA genes in Encephalitozoon and Nosema spp Using a synteny-driven "all-versus-all" BLASTN approach, a total of 10 new putative ncRNAs were predicted in the genomes of the four closely related Encephalitozoon species E. cuniculi, E. intestinalis, E. romaleae and E. hellem (Supplementary Table  S1 ), raising the total to 18 with the eight ncRNAs (RNase P, MRP, SRP, U3 C/D snoRNA, U2, U4, U5 and U6 snRNAs) previously described in the RFAM 12. 0 database  27  for at least one of these genomes. Similarity and synteny-based ortholog searches in three AT-rich Nosema genomes (N. ceranae, N. apis and N. bombycis) retrieved only four out of the ten Encephalitozoon newly predicted ncRNAs, suggesting that the primary sequences of ncRNAs are overall poorly conserved throughout Microsporidia.
However, using only intergenic regions of N. ceranae to screen N. apis and N. bombycis ones, we were able to identify seven additional ncRNAs. Thus, with the RNase P, U2, U3 and U6 ncRNA genes available in the RFAM 12. 0 database, Nosema species would possess 15 ncRNA genes that are orthologous to Encephalitozoon ones (Supplementary Table  S1 ). All 18 E. cuniculi ncRNAs were then validated by 5 0 and 3 0 RACE-PCR (Supplementary Fig. S1 ) and Sanger sequencing of the full-length products. This approach allowed to precisely define their transcriptional initiation and termination sites (Fig. 1 ). The lengths of ncRNA primary sequences were found to be relatively well conserved between the four Encephalitozoon species and the three Nosema species (Supplementary Fig. S2 ) but otherwise to be generally shorter when compared with their respective yeast and human orthologs (Supplementary Table  S1 ).
Sense and antisense primers for each AMP (Table  1 ) were generated using Primer Designer software (Scientific and Educational Software, Inc. ) as previously reported  (Santoro et al. , 2013). The relative mRNA expression levels were quantified using SYBR® Green (Qiagen, Valencia, CA, USA) and ABI (Applied Biosystems Inc. , Foster City, CA, USA) quantitative RT-PCR methodology. All samples were tested in triplicate 25 μL reactions in an ABI 7500 Real Time PCR System (Applied Biosystems Inc. , Foster City, CA, USA). PCR amplifications were carried out as followed: 50 °C for 2 min; 95 °C for 10 min; 40 cycles of 95 °C for 15 s; 60 °C for 60 s. Amplifications were followed by dissociation (melting) curves to ensure specificity of the primers. The results were analyzed using the comparative C T (cycle threshold) method, and the relative mRNA expression of each AMP was compared using the ΔΔC T method (2 -[ΔCT experimental sample -ΔCT control sample] ) where ΔC T is the difference between the target gene and the normalizer gene expression.
For RRMS patients, the revised 2005 McDonald Criteria  [13]  were used to diagnose MS. Demographic and clinical characteristics, including gender, age, neurological symptoms and signs, and CSF white blood cell (WBC) count were retrieved from our CSF biobank database as previously described  [14]. Sera were collected from healthy controls (HC) and RRMS patients under different disease-modifying treatments. Analysis of IL-27 in the sera and CSF IL-27 was measured using an enzyme immune-assay (Human IL-27 ELISA, Kit: Duo Set (R&D) Ref:DY2526).
Acknowledgements The authors would like to acknowledge  Catherine Juillard-Mochon  for technical assistance. Funding P. H. L. received support from the  Swiss National Foundation  (# 310030-153164 ), D. M. receives support by the  Swiss National Science Foundation  ( 310030_173010 ), and C. P. holds stipendiary professorships of the  Swiss National Science Foundation  (No. PP00P3_157426 ). P. H. L. , D. M. and C. P. received supports from the  Swiss Multiple Sclerosis Society.
Day 84 sera were tested using a 2-fold dilution series starting at 1:5 and through to 1:640, with percentage binding inhibition calculated for each volunteer using their matched day 0 serum sample as the baseline control. Example binding-inhibition curves are shown (Supplemental Figure  7A ), and 50% binding-inhibition titers were interpolated from these data (Figure  6A ). One sample in group 2A showed a weak 50% binding-inhibition titer of 1:16. All samples from groups 2B and 2C showed binding inhibition with median 50% titers of 1:137 (range 1:14-1:248) and 1:168 (range 1:52-1:352), respectively. To further assess the quality of the vaccineinduced antibody response, these titers were used to calculate the concentration of anti-PvDBP_RII polyclonal IgG that gives 50% binding inhibition in each individual (Figure  6B ).
Methods Detailed methods are provided in supplemental methods. ChAd63 and MVA PvDBP_RII vaccines. The design, production, and preclinical testing of the viral vector vaccines have been reported previously  (27). Briefly, both recombinant viruses express the same 984-bp coding sequence of PvDBP_RII from the SalI strain of P. vivax, aa D194-T521 (GenBank Accession DQ156512). ChAd63 PvDBP_RII was manufactured under current Good Manufacturing Practice (cGMP) conditions by the Clinical Biomanufacturing Facility (CBF), University of Oxford, UK, and MVA PvDBP_RII was manufactured under cGMP conditions by IDT Biologika GmbH, Germany, both as previously described  (37). Study design and approvals. The VAC051 study was a phase Ia open-label, dose-escalation, first-inhuman, nonrandomized trial of the viral vectored vaccines ChAd63 PvDBP_RII and MVA PvDBP_RII given in a prime-boost regimen with an 8-week interval.
The study was conducted at the Centre for Clinical Vaccinology and Tropical Medicine (CCVTM), University of Oxford, Oxford, UK. The study received ethical approval from the Oxfordshire Research Ethics Committee A in Oxford, UK (REC reference 13/ SC/0001). The study was also reviewed and approved by the UK Medicines and Healthcare products Regulatory Agency (MHRA, reference 21584/0312/001-0001). Volunteers signed written consent forms and consent was verified before each vaccination. The trial was registered on Clinicaltrials. gov (NCT01816113) and was conducted according to the principles of the current revision of the Declaration of Helsinki 2008 and in full conformity with the ICH guidelines for Good Clinical Practice (GCP). The primary endpoint of the study was to assess the safety of ChAd63 PvDBP_RII and MVA PvDBP_RII, with a secondary endpoint to assess immunogenicity.
Acknowledgments The authors are grateful for the assistance of  Julie Furze ,  Fay Nugent ,  Susanne Hodgson ,  Sharon Sheehan ,  Alexandra Spencer ,  Sumi Biswas ,  Alexander Douglas ,  Mary Smith ,  Adrienne Cook ,  Raquel Lopez-Ramon , insight. jci. orghttps://doi. org/10. 1172/jci. insight. 93683.
ACKNOWLEDGMENTS We acknowledge the support from  Cécile Laurier-Nadalié  for helpful discussion on statistical analyses and  Damon Getman  from  Hologic, Inc. for support in analyzing discrepant results using Alt1-and Alt2-TMA assays and resistance testing based on the nested RT-PCR sequencing assay. This evaluation was supported by a research grant from  Hologic, Inc. (USA). The study funders had no role in data collection or interpretation.
Study population-Alexishafen, Papua New Guinea Women attending prenatal care at Alexishafen Health Centre, Madang Province, Papua New Guinea (PNG) between August 2005 and September 2007 were recruited into a longitudinal study of malaria and pregnancy following voluntary informed consent  [41]. Peripheral blood (5ml) was obtained from women at enrolment, delivery and any additional antenatal clinic visits. At delivery a placental biopsy and 10ml of cord blood were collected. Clinical and demographic data were obtained at enrolment and delivery visits. Presence of microscopic parasitaemia was determined from blood smears by two independent microscopists. A subset of 204 participants with available delivery and cord samples were included in the present study. The study was approved by The Medical Research Advisory Council of Papua New Guinea (MRAC 05/05) and the Human Research Ethics Committee of Melbourne Health, Australia (06/06).