dataesr/scientific-paragraphs-categorization huggingface dataset:
Indirect immunofluorescence using confocal microscopy Intracellular parasites were fixed with 4% paraformaldehyde prepared in PBS for 30 minutes at room temperature, and were washed three times with PBS before staining with DAPI. Samples were observed with a Zeiss confocal microscope and the obtained images were processed using Image J software. When antibodies were utilized, the samples were permeabilized with 0. 2% Triton X-100 prepared in PBS with 100 mM of glycine to block free aldehyde groups, incubations were done for 30 min at room temperature all along the experiment. After blocking with 10% of FBS in PBS containing 0. 1% Triton X-100, monoclonal or polyclonal antibodies were added at 1:500 dilutions and incubated at 37°C for 30 min. After three washes, Alexa 488 nm or 560 nm secondary antibodies were added to the same buffer with DAPI and blue Evans, and after incubation at 37°C for 30 min, and three washes with PBS containing 0.
The experiments were done in quadruplicate and DMSO was used as the negative control. Chloroquine, a wellknown anti-plasmodial drug used at 0. 005-150 µM served as the positive control. Plates were incubated for 48 hours at 37°C under the same conditions. Thin smears were created by spreading 6 µL of parasitized RBCs (pRBCs) on a glass slide, which was fixed and stained using the RAL 555 kit before using the AxioCam MRc (color) CCD Rev3 camera of ZEISS microscope at X63 to observe and photograph the RBC smears and process the data using ImageJ software.
Flow cytometry analysis Both untreated and drug-treated infected RBCs (2. 5 µL) were collected from each well and were incubated at 37°C for 60 minutes in a Hoechst 33258-Thiazole Orange mixture at 0. 001 mg/mL and 0. 005 mg/mL final concentrations, respectively  (Grimberg et al. , 2008). Samples were analyzed using BD FACS Canto flow cytometer, whereby FlowJO was used for data analysis and statistical analyses were conducted using GraphPad Prism.
Image-based miniaturized assay For the assay, HFF cells were first infected with a pre-culture of T. gondii expressing both TgSORT-GFP and ROP1-mCherry, as shown in Figure  2  depicting assay workflow. The infected cells were subsequently harvested onto a compound containing 384-well plates and were incubated for 24 hours, after which the samples were fixed and stained with nuclear label-DAPI. Next, confocal images were acquired using an automated microscope and wellbased analysis was performed using Columbus software (Supplementary Table  1 ; Figure  3 ). To establish the assay, the number of HFF cells, the multiplicity of infection (MOI), and the DMSO impact have to be determined to ensure a homogenous T. gondii-infected HFF layer in each well at read-out (Supplementary Figure  2 ). We have chosen pyrimethamine (Pyr) for this purpose, as this well-known drug is frequently used in toxoplasmosis treatment  (Hill and Dubey, 2002; Halonen and Weiss, 2013; Dunay et al.
Small molecule screen for identifying known compounds that inhibit T. gondii intracellular growth As a part of the present study, a pre-screen optimization was also performed by filling two 384-well plates with two Pyr doses (0. 6 µM and 10 µM) in a checkerboard style (Figure  4A ). For the DMSO negative control, 20 parasites were noted on average per HFF cell, declining to about 2 parasites in cells containing 10 µM Pyr. Next, the first 384-well plate from the Tocris library in Supplementary Table  2  was analyzed under the conditions described above. For this purpose, we generated a data processing script utilizing the signal emitted by the ROP1-mCherry and correlating it with the mislocalisation of the rhoptry signal. We first tested 300 compounds of Tocris library and the initial results pertaining to the wells corresponding to a compound yielding positive results were visually validated to concur with the findings related to the rhoptry marker mislocalisation.
Thus, the vector expressing the TgSORT-GFP was transfected (C) and one positive clone (green signal), which replicates into eight intracellular tachyzoites (D) for the expression of this receptor in the post-Golgi and endosome-like compartment (ELC) is shown. Bar scale = 3 µm. screening of the 900 remaining small molecules distributed in three additional 384-well plates, as shown in Figure  5. During the analysis script creation, the dual GFP and mCherry labelling approaches were tested but it appears that ROP1-mCherry signal was sufficient to determine the effect of drugs on the parasite morphology inside the parasitophorous vacuole. Indeed, the TgSORT-GFP fluorescence was concentrated and created puncta, whereas the ROP1-mCherry created larger structured area in which the vacuole disruption can be captured in a more visible manner.
Acknowledgments We would like to thank  Dr. Vern Carruthers  ( University of Michigan, USA ) for providing us with the transgenic T. gondii RH (clone 2F1) strain. Also thanks to  Gaspard Deloison ,  Viriginie Pottiez ,  Thomas Mouvaux ,  Florence Leroux  and  Omar Ndao  for their technical assistance during high content screening and validation of positive compounds.
Funding Financial support for this work was provided by the  European Community  ( ERC-STG INTRACELLTB Grant n°260901 ), the  Agence Nationale de la Recherche  ( ANR-10-EQPX-04-01 ,  ANR-11-LABX-0024 ), the  Feder  ( 12001407  ( D-AL) Equipex Imaginex BioMed ) and the  Re ́gion Nord Pas de Calais  (convention n°1 2000080), the  Agence Nationale de la Recherche  grants  N°A NR-14-CE14-0002-01  and  N °ANR-19-CE44-0006  to ST.
Post-infection pulmonary cell recruitment We focused on major myeloid immune cell types using the macrophage markers F4/80 and CD11c (Figure  1 ). As previously described, the first cells recruited to the lung following infection with IAV PR8 on D0 were granulocytes (Ly6G + ). The number of granulocytes rapidly and significantly increased in the infected group during the first day post-infection (p>0. 05), peaking on D2, and then rapidly decreased on D3 and D4 back to basal levels (data not shown). CD11c + cells were then recruited, consisting mostly of monocytes, which peaked on D3. The number of pulmonary macrophages (F4/80 + ) increased slowly between days 1 and 2 (p<0. 05), reaching a peak at D4 (four times higher by D4). Over the same period, myeloid cells remained at basal levels in the control animals.
Acknowledgments The authors gratefully acknowledge the  Direction Geńeŕale de l'Armement (DGA)  (grant  NBC-4-B-4116 ) and the  Service de Santed es Armeés (SSA)  of the  French Ministry of Armed Forces  for their sustained financial support. We are grateful to  Cleḿence Rougeaux  ( IRBA ) and  Marie Mura  ( IRBA ) for their invaluable help in data management.
We report here a novel HLA-DPB1 allele, now named HLA-DPB1*1463:01N that carries one nucleotide substitution in exon 3 when compared with the HLA-DPB1*02:01:02:04 allele, identified in a volunteer bone marrow donor. The HLA typing was performed using Next Generation Sequencing (AllType NGS, One Lambda, Canoga Park, CA) on the Ion S5 system platform (ThermoFisher Scientific, Waltham, MA),  1   1 ). This nucleotide change was confirmed using other NGS reagents provided by GenDX NGSgo-MX6-1 (Utrecht, Netherlands) run on the Illumina MiSeq system (San Diego, CA) and analyzed with the NGSEngine software (GenDX, version 2. 26). We were very confident in the phasing as the sample displayed a mean read length of 306 base pairs over all the loci, the mismatched T base was attributed 771 times to the new HLA-DPB1*1463:01N allele and can be only attributed to this allele because it was possible to discriminate from the associated HLA-DPB1*03:01:01:01 allele by virtue of four variant positions each distant by less than 100 base pairs.
HLA typing by Luminex reverse sequence-specific oligonucleotide (SSO) was performed (One Lambda Labtype XR, Canoga Park, CA). 3 ith this assay (lot 004, catalog RSSO2P_010_04), the most likely HLA-typing of the donor was DPB1*02: EFWDV, 03:EBKTD (most likely allele DPB1*02:01, 03:01, respectively) without any bead modification. Indeed the IPD-IMGT/HLA Database 3. 51. 0 release describe few other HLA-DPB1 alleles displaying a TGA sequence in codon 128, also identified as null alleles, the manufacturer did not include probes targeting this codon. As a null allele, the HLA-DPB1*1463:01N is clinically significant in both organ and allogeneic hematopoietic cell transplantation. The coding nucleotide sequence of the new allele has been submitted to the GenBank database (Accession No. OQ378319) and to the IPD-IMGT/HLA database (Submission No.
from exons 2 to 5. The reads were analyzed using the Type-Stream Visual Software version 3. 0 (One Lambda). This donor was found to have a new DPB1 allele and was consequently typed A*02:01, 02:06; B*51:01, 51:01; C*14:02, 15:02; DRB1*04:07, 11:01; DRB3*02:02; DRB4*01:03; DQA1*03:03, 05:05; DQB1*03:01, 03:01; DPA1*01:03, 01:03; DPB1*1463:01N, 03:01. Using the IPD-IMGT/HLA database, 2 nucleotide sequence alignment with HLA-DPB1 alleles shows that this new allele has one nucleotide change from HLA-DPB1*02:01:02:04 in codon 128 in exon 3 where C ! T, resulting in a coding change (CGA ! TGA, Arginine ! STOP, Figure.
83% G + C content. Genome annotation was done by using CLC genomics workbench 10. 0. 1. The genome contains 11 genes and has a genome sequence and genome organization similar to the reference BRSV strain ATCC51908 (GenBank accession number NC_038272). The complete genome sequence of the Swedish BRSV strain (Sweden_HPIG-SLU-620-Lövsta_2016) was submitted to the GenBank database under accession number MG947594.
Design and recovery of an infectious recombinant BRSV-mCherry from cDNA An expression cassette was designed for the expression of the antigenomic RNA of the field strain isolate Sweden_ HPIG-SLU-620-Lövsta_2016 with an additional transcription unit coding for the red fluorescent mCherry protein (Genbank accession numbers ON110491). The additional mCherry gene was placed between P and M genes with gene start and gene end sequences from the N gene upstream and downstream the mCherry gene. A T7 RNA polymerase promoter was placed upstream the Leader region such that antigenomic RNA would be transcribed with three supplementary G residues at the 5' end. The hepatitis delta virus (HDV) antigenome ribozyme sequence was fused to the last nucleotide of the trailer sequence to achieve correct cleavage at the 3' end (Figure  1A ).
We therefore  tested CPM antiviral activity against BRSV on BT cells infected with rBRSV-mCherry at MOI = 0. 02. Two hours post-infection, the medium was changed for medium containing serial dilutions of CPM (with an initial concentration of 10 µM in DMSO and ½ serial dilutions). Quantification of viral replication was performed by fluorescence measurement on live cells at 7 dpi using the Tecan spectrofluorometer. Values of nontreated infected and noninfected BT cells were used for standardization and normalization. The half maximal effective concentration (EC 50 ) was determined by fitting the data to the dose-response curve implemented in GraphPad version 8 software. Cell survival was quantified using the CellTiter-Glo Luminescent cell viability assay (Promega). As previously determined on human cells [17], CPM was not toxic for BT cells up to 10 µM concentration (not shown).
The slight difference between EC 50 measured by either mCherry or F fluorescence (76nM versus 49 nM) could be due to the treatments of cells for F immunofluorescence compared to the epifluorescence of mCherry on live cells. But these results correlate with those previously obtained for HRSV in similar experimental conditions, with an EC 50 close to 126 nM  [17]. Altogether, these data show that CPM displays similar antiviral activity against HRSV and BRSV and validate the use of rBRSV-mCherry for screening antivirals.
Funding This work was carried out with the financial support of the  French Agence Nationale de la Recherche , specific program  ANR PRC DecRisp   ANR-19-CE11-0017  and  ANR  EpiLungCell  ANR-17-CE20-0018. The BRSV isolate was collected through funding from the  European Union   Horizon 2020 Program  for research, technological development, and demonstration under the Grant Agreement n633184 (Project name SAPHIR) and sequenced through the project which was funded by the  Swedish Research Council (Formas, Sweden) , grant no. 2016 -00463.
Study design We used data from the BIRDY cohort (Bacterial Infections and antibiotic Resistant Diseases among Young children in LMICs), a prospective, multicentric, community-based motherand-child cohort. The study took place across urban and rural sites in Madagascar (pilot phase: 2012 to 2014 and complete phase: 2014 to 2018), Senegal (2014 to 2018), and Cambodia (2014 to 2018). The primary objective of this cohort study was to estimate the incidence of antibiotic-resistant bacterial infections in children. The formal analysis plan for the BIRDY cohort was established in 2012  [14]. There were no changes in the protocol between the pilot phase and complete phase that may bias results of the present study. No additional formal prospective analysis plan specific to this study on inappropriate antibiotic prescribing was made.
In Senegal, the study was conducted in an urban district of Guediawaye (population: 20,529) and in the rural community of Sokone (population: 14,500). To recruit study participants, a first phase of exhaustive identification of all pregnant women was carried out in the study areas. Women were enrolled, after having given written informed consent, during their third trimester of pregnancy and followed until delivery to include their newborns at birth. Inclusion criteria were the following for both the mother and her newborn: routine residence in the study area with no plan to move away during the follow-up period, and no opposition to the research being conducted, nor to the collection of epidemiological data and biological samples. Data regarding sociodemographic, medical, and obstetric characteristics of mothers were collected, as well as data regarding the delivery and health characteristics of newborns.
In case of a health event, the child was referred to an outpatient consultation or directly to hospital if the child's condition was serious. Passive surveillance consisted of asking mothers to contact the investigation team whenever the child showed signs of infection in order to schedule an outpatient consultation. Thus, children were able to benefit from outpatient consultations whenever they were sick, whether identified by their guardians or by study investigators. Clinical decisions regarding antibiotic prescribing were not monitored or imposed, but were rather left to the attending physicians to decide according to local protocols. Importantly, epidemiological data collected during these consultations included children's diagnoses, symptoms, and treatments received, including details of any antibiotics prescribed.
Study population and outcomes For the present study, the population considered was all children from the BIRDY cohort who had at least 1 outpatient consultation during their follow-up. Only systemic antibiotics were taken into account (oral, intramuscular, and intravenous). We did not consider local antibiotics (dermal and ophthalmic) because their impact on antibiotic resistance is probably different from systemic antibiotics. For each consultation, diagnoses were categorized by the study team using data collected during children consultation. First, we ensured that all diagnoses made by the physician not of infectious origin were concordant with data collected on the signs and symptoms presented by the child. Then, all infectious diagnoses were determined using a syndromic algorithm based on the World Health Organization (WHO) Integrated Management of Childhood Illness (IMCI) guidelines.
Acknowledgments We are grateful to all the mothers and their newborns for their participation. We thank all physicians, laboratory staff, field interviewers, and community workers for their involvement in this project. We thank all collaborators of the  BIRDY program :  Rasoanaivo Fanjalalaina Vololonirina ,  Andrianonimiadana Lova Maminirina ,  Rabearitiana Lydos ,  Rasoloson Dimitri ,  Ratsima Hariniaina Elisoa ,  Volahasina Antsa Tanjona ,  Volahasina Antso Fenitra ,  Randriamamonjiarison Aina Nirina ,  Andriatahina Todisoa ,  Marguerite Diatta ,  Joseph Faye ,  Armya Youssouf Abdou ,  Sophie Goyet ,  Veronique Ngo ,  Frederique Randrianirina ,  Abdoulaye Seck ,  Elisoa Hariniaina-Ratsima ,  Michael Padget , and  Benoit Garin. WeAU : PerPLOSstyle ; recognition also thank the  Department of International Affairs  and the  Centre de Recherche Translationnelle-Coordination Clinique of Institut Pasteur  for their support in the management of the study.
Methods The SRLF appointed its Reference and Evaluation Committee to organize a consensus conference to better define the indications and conditions of use for these new antibiotics. The members of the committee defined six generic questions (Table  1 ), and then Patient, Intervention, Control, Outcome (PICO) questions were submitted to experts (Additional file 1)  [15]. One expert was appointed for each generic question asked. A group of intensive care fellows and members of the committee carried out the bibliographic research in PubMed (contributors are listed in Additional file 1). Keywords were defined based on PICO questions. Grading of Recommendations Assessment, Development and Evaluation (GRADE) tables of published data were drawn up  [16]. The level of evidence was assessed according to the type of study for each of the quoted references and then reassessed (increased or decreased) according to the quality of the study's methodology.
The experts presented their analyses and the specific scientific data for the question they were responsible for, and they answered the questions and comments of the panel and the public. After the public meeting, the panel met privately to draft the text answering the questions. Recommendations were formulated according to GRADE methodology. The proposed recommendations were presented and discussed individually. The aim was not necessarily to obtain a convergent opinion of the panel members for all of the proposals but to uncover points of agreement and points of disagreement or indecision. Each recommendation was then assessed by each panel member and scored individually from 1 (totally disagree) to 9 (strongly agree). The panel score was defined using a GRADE grid  [17]. To achieve a strong recommendation, at least 70% of the participants had to agree.
Acknowledgements The authors thank the members of the bibliography group for their commitment and work:  Pierrick Bauduin  (médecine intensive réanimation,  CHU, Caen, France ),  Sylvain Le Pape  (médecine intensive réanimation,  CHU, Poitiers, France ),  Lionet Tchatat-Wangueu ,  Hélène Messet-Charrière ,  Misylias Bouaoud  (médecine intensive réanimation,  CHU, Tours, France ) and  Dima Siblani  (médecine intensive réanimation, CH, Mulhouse, France).
| Biological analysis All the PLWHA enrolled in this study gave their written informed consent to having their medical records stored in the Nadis electronic medical record system, or local databases, which was designed for medical follow-up of PLWHA, including the use of their biological data for research studies (www. dataids. org Fedialis Medica, Marly Le Roi, France, CNIL number: 1171457 24 May 2006). DTG and RPV plasma concentrations were measured locally in each centre using a validated liquid chromatography-tandem mass spectrometry method. Blood collection time and time of the last administration were recorded according to the nurse's recording and the statement of people living with HIV/AIDS, respectively. All pharmacology laboratory contributing to TDM data in this study is registered to the same external quality control programme.
Among covariates, only body weight was included in the modelling. Smoking status of PLWHA was not available and none of the comedications influencing the model (i. e. rifampicin as other inducers, atazanavir and darunavir boosted ritonavir) was given to PLWHA. For RPV, individual parameters were estimated using the model developed by Néant and colleagues,  18  with the same method. We used the saemix package in R  19  to estimate individual parameters by fixing the population parameters for each drug separately to obtain the conditional distributions of the individual parameters for each subject at each occasion. R was then used to compute the trough concentrations as well as their confidence interval, which can be obtained from the standard deviation of the conditional distributions on each parameter.
Sequence and phylogenetic analysis PCR products were purified using NucleoFast 96 PCR ultrafiltration plates (Macherey Nagel EURL, Hoerdt, France) according to the manufacturer's instructions. The amplicons were sequenced using a Big Dye Terminator Cycle Sequencing Kit (Perkin Elmer Applied Biosystems, Foster City, CA, USA) with an ABI automated sequencer (Applied Biosystems, Foster City, CA, USA). The obtained electropherograms were assembled and edited using ChromasPro software (ChromasPro 1. 7, Technelysium Pty Ltd. , Tewantin, Australia). DNA sequences were compared with the records available in GenBank using the Basic Local Alignment Search Tool (Blast version 2. 0). The data of obtained sequences were submitted to GenBank (OK044081-84). A flaB gene-based phylogenetic tree was inferred with MEGA7 using the maximum likelihood and Kimura 2-parameter method with 1000 bootstrap replicates  (Kumar et al.
Results In this study, blood samples from 536 rodents trapped in four provinces of Turkey (Fig. 1  According to the qPCR screening results targeting the Borrelia 16S rRNA region, 11 out of 536 blood samples were determined to be positive. Amplification by conventional PCR targeting the Borrelia species flaB gene confirmed positivity in nine out of 11 qPCR positive samples. When compared with Borrelia species sequences deposited in Gen-Bank (accession numbers KT452930, KT932823, JF951386), it was observed that nine sequence results of the FlaB gene were between 99. 7% and 100% identical with B. miyamotoi sequences. The features of the sequence data which were obtained from GenBank and compared with this study are presented in detail in the phylogenetic tree. Phylogenetic analysis with a high bootstrap value showed that all positive samples belonged to B.
Cells were then incubated with 2. 5 mM Mito-SOX Red (MSR) (Invitrogen, Thermo Fisher Scientific) in staining media for 10 minutes at 37°C, 5% CO 2 , followed by LIVE/DEAD Aqua staining for 10 minutes at 4°C. Macrophages were washed and subsequently fixed with 1% formalin in flow staining medium, and data were acquired on a BDX20 (BD Biosciences). Live nondebris singlets were analyzed for fluorescence in the MSR channel to quantify mROS.
Samples were acquired on a 4-laser BD Fortessa Flow Cytometer, and the analysis was performed using FlowJo (Treestar) software. Alveolar macrophages (CD11c + CD11b -), neutrophils (CD11b + C-D11c -Gr-1 hi ), monocytes (CD11b + CD11c -Gr-1 med ), and recruited macrophages (CD11b + CD11c -Gr-1 lo ) were defined as previously reported by our group  (50).