dataesr/scientific-paragraphs-categorization huggingface dataset:
9. 3. 2. A possible link between geographic (in Km) and genetic distances (Fst) was assessed using the Mantel test. Second, we used the principal components analysis (PCA) implemented in Genetix  [46]. Because we sampled several miracidia per patient, these miracidia are related, which could influence the genetic structure. In order to assess a possible bias of our sampling strategy, we performed PCA by randomly sampling two miracidia per patient. Third, we used the Bayesian clustering approach implemented in the Structure software to determine the uppermost level of genetic structure  [47]. We tested the number of clusters from K = 1 to K = 12, by computing three runs for each cluster which is made up of 10 6 iterations after a "burn-in" period of 250,000 iterations with other parameters set by default and an admixture model.
The mean logarithm probability for each cluster (K) was taken for the three runs with the corrsieve package in R. The ∆K-values were then computed in R to determine the probable cluster number from the total clusters (∆K) tested according to Evanno et al. [48] , from which we identified K = 2 as the most probable genetic clusters. Lastly, an additional 10 runs were computed for K = 2 using 10 6 iterations and setting the same parameters as earlier described. The mean probability for a miracidium to belong to each cluster over the 10 runs was taken as Q-values, and we used Clumpp version 1. 1. 2 according to Francis R. M. [49] , and Distruct version 1. 1 according to Rosenberg N. A  [50].
Supplementary Materials: The following supporting information can be downloaded at: https: //www. mdpi. com/article/10. 3390/pathogens11040425/s1, Table  S1. Accession numbers for Supple-mentary Figure  S1 ; Table  S2. Microsatellite database; Figure  S1. Maximum likelihood phylogenetic tree built with 21 (2 S. haematobium NigeriaHap1&2, and 19 S. bovis NigeriaHap3-21) haplotypes from the present study and haplotypes from Cameroon, Benin, Senegal, Cote d'lvoire, Kenya and Tanzania from Genbank database. See supplementary Table  S1  for AN database; Figure  S2. The sequence chromatograms show the pure and mixed signal in the nuclear ITS2 marker. The double sequence chromatogram (heterozygous) showing bi-parental inheritance of the nuclear DNA; Figure  S3. Population genetic structure graph assessed by principal component analysis using 2 miracidia by patient.
Funding: This research was funded by Campus France, France, and Tertiary Education Trust Fund (TETfund), Nigeria and was supported by the program HySWARM (ANR-18-CE35-0001) from the French Research National Agency respectively awarded to O. Rey and J. Boissier. Amos Onykwere is funded by Campus France. This study is set within the framework of the "Laboratoires d'Excellence (LABEX)" TULIP (ANR-10-LABX-41).
Methods Patients. Primary lung tumor samples were obtained from NSCLC patients operated at Institut Mutualiste Montsouris, Hotel Dieu, and Cochin hospitals (Paris, France). A retrospective cohort of 338 NSCLC patients operated between the years 2001 to 2005 was enrolled in this study. Pathologic staging of lung cancer was determined according to the new TNM staging classification  50. Histological subtypes were determined according to the classification of the WHO  51. Among the retrospective cohort of patients, the patients treated with neoadjuvant chemotherapy and radiotherapy were excluded. The time between the surgery and the last follow-up or death is considered as the observation time for this cohort. The data on long-term outcomes were obtained after interaction from municipality registers or the family of the patient.
Briefly, tissue sections were deparaffinized, rehydrated and treated with the antigen retrieval buffer. The antigen retrieval was performed using TRS buffer (Dako, France) at 97 °C for 30 min in water bath, in case of the CD3/FoxP3 and CD62L/FoxP3 double staining. The sections were incubated in the 3% H 2 O 2 and ready-to-use protein bloc (Dako, France) solution for 30 min before the addition of the appropriate primary and secondary antibodies. The antibodies and reagents are listed in Supplementary Table  2. The enzymatic activity was performed using the AEC (3-amino-9-ethylcarbazole) kit and APS (Vector blue alkaline phosphatase) substrate kits. Images were acquired using Nanozoomer (Hamamatsu) with NDPview software. The same pretreatments were used for the 5 plex-immunofluorescence staining.
Tissue sections were successively stained with the appropriate primary antibody (CD3, CD20, CD62L, FoxP3, DAPI) and HRP-coupled secondary antibodies. The sections were then washed three times for 5 min and then incubated with the fluorophore tyramide reagent that covalently binds the tissue. Finally, a stripping step was performed by heating slides for 10 min at 90 °C before proceeding to another staining cycle. The specificity of each antibody was tested by using isotype control antibodies. The effectiveness of the stripping step was checked for each multiplex staining. Nucleus staining was performed by using ProLong® Gold with DAPI (Molecular Probes). IF slides were scanned with an Axio Observer microscope (Zeiss) and analyzed by Zen (Zeiss) and Halo (Excilone) software. Cell quantification.
The area of interest was manually drawn on the scanned images of tumor tissue sections. The surface area of this region of interest was also determined using the Calopix software (Tribvn, France). CD3 + total T cells and CD3 + FoxP3 + T cells were quantified in the whole tumor section using the same software and expressed as a number of cells/mm 2 (number of total cells divided by the surface area in mm 2 ). The region of TLS was determined manually by referring to the double staining DC-Lamp/CD3 to determine the TLS area. The DC-Lamp/ CD3 staining was performed on the serial tumor sections from the same set of patients. The surface area of the TLS was also determined by the Calopix software. The density of CD3 + FoxP3 + cells in TLS was determined with an automatic counting using Calopix.
Data were analyzed using Flow Jo 9. 7. 6 (Tree Star Inc, Ashland, OR) and Spice 5. 3. 5 (developed by Mario Roederer, Vaccine Research Center, NIAID, NIH) software programs. The gating strategies are detailed in Supplementary Fig. 1. Cell sorting. For the gene expression study, Tregs and Tconv subsets were sorted from 20 tumors and non-tumoral specimens by the in house designed protocol  21. The combination of Easysep TM untouched human CD4 + T cell kit (stem cell technologies, France) and flow cytometry cell sorting with the cocktail of antibodies (anti-CD2, CD4, CD8, CD25, CD127, and CD62L) was used to achieve the high purity of the cell subsets. Four populations of cells, namely TLS Tregs (defined as CD2 + CD4 + CD8 -CD25 hi CD127 -CD62L + ), non-TLS Tregs (defined as CD2 + CD4 + CD8 -CD25 hi CD127 -CD62L -), TLS CD4 + Tconv (Tconv defined as CD2 + CD4 + CD8 -CD25 -CD127 + CD62L + ) and non-TLS CD4 + conventional T cells (Tconv defined as CD2 + CD4 + CD8 -CD25 -CD127 + CD62L -) were then sorted from fresh tumor and non-tumoral tissue specimens (n = 20).
The medium was filtered and maintained in sterile conditions. RNA extraction, reverse transcription, and gene expression analysis. Total mRNA from the sorted cells was extracted with the RNeasy micro kit (Qiagen, France) according to manufacturer's instructions, and RNA quantity and quality were determined using the 2100 Bioanalyzer (Agilent Technologies, France). The mRNA was reverse transcribed to cDNA using a superscript VILO kit (Life Technologies, France). The samples below 1 ng of mRNA were amplified by 9 cycles. When the quantity of mRNA was more than 1 ng of mRNA, it was amplified by 7 cycles of PCR using Taqman PreAmp 2x and MTE primers (NanoString technologies, Seatle, USA). Two specific probes (capture and reporter) for each gene of interest were applied. The customized reporter probe and capture probe code-set of selected 125 genes, including five housekeeping controls (β-actin, GAPDH, EEF1G, OAZ1, and RPL19) and cell lineage controls (CD3, CD4, CD8, CD19, CD138, and EpCAM) were used for the hybridization according to the manufacturer's instructions (Nanostring Technologies) (Supplementary Data 1).
Water was used as a negative control to check the background noise. The hybridized samples were recovered using the NanoString Prep-station and the mRNA molecules counted with the digital nCounter. The number of counts represented the gene expressed. The high expression level of gene means the readout in terms of a high number of counts of that particular gene. The positive and negative controls and one patient's RNA sample as an internal control were used to check the technical consistency between each strip of experiments. Raw data are available in Supplementary data 2. Immunosuppression assay. Tregs and CD4 + Tconv cells were purified from the lung tumor and blood specimens (3 × 10 3 /well). CD4 + Tconv cells were labeled with CTV (life technologies, France, working concentration: 10 µM), and then cultured with anti-CD3/anti-CD28 beads (1 bead/10 cells, Invitrogen) and without any exogenous cytokine in 96 well round bottom plates in complete RPMI 1640 with 10% AB + heat-inactivated human serum for 72 h at 37 °C.
0 (Becton Dickinson). Statistics and reproducibility. For the flow cytometry data, Wilcoxon-Mann-Whitney U test was used to compare the density of cells in the different tumors. For gene expression study and volcano plot demonstrations, the 'nSolver' (Nanostring Technologies) and R (CRAN) software were used. The raw data were normalized with an average count of the five housekeeping genes using the "nSolver" software. The Student T and ANOVA tests were used to compare the gene expression profile of the different groups. To avoid the inclusion of the false positive results, we computed the P-values with the false discovery rate (FDR) method. The data were represented as volcano plots. The overall survival (OS) curves were estimated by the Kaplan-Meier method, and differences between groups of patients were calculated using log-rank test.
Acknowledgements The authors thank  Mr. Sylvain Leveugle  for technical support in statistical analysis, and  Dr. Hélène Froher-Ting ,  Dr. Estelle Devevre , and  Dr. Christophe Klein  (platform "Center of cellular imaging and cytometry",  Cordeliers Research Center, Paris, France)  for their excellent technical support and assistance in cell sorting, and cell quantification from digital slides, respectively. We also thank  Dr. David Gentien  and  Mr. Bertrand Albaud  ( Genomic platform, Department of Translational Research of Curie Institute, Paris, France ) for the technical support in the gene expression profiling. We thank  Marie Laviron  and  Alexandre Boissonnas  for both technical assistance and helpful scientific discussions. Finally, we thank  Ms. P. Bonjour  and  Ms. Nathalie Jupiter  for their help in tissue section.
Data availability All data relevant to the study are included in the article or uploaded as supplementary data: supplementary data 1 (list of genes and accession number), supplementary data 2 (Figs. 4  and 5a-c ), supplementary data 3 (Figs. 8  and 9 ), supplementary data 4 (Fig. 2a ), supplementary data 5 (Fig. 3a ), supplementary data 6 (Fig. 3b ), supplementary data 7 (Fig. 3c ), supplementary data 8 (Fig. 5d ), supplementary data 9 (Fig. 6b ).
Viral strains The viral strains of human SARS-CoV-2 VOCs were obtained from a positive nasopharyngeal PCR sample. The viruses have been treated in biosafety level-3 laboratory (BSL-3). The SARS-CoV-2 primo-culture stocks used as B (EPI_ISL_4537783), Alpha ( E P I _ I S L _ 4 5 3 6 4 5 4 a n d E P I _ I S L _ 4 5 3 6 9 9 6 ) , B e t a (EPI_ISL_4537125 and EPI_ISL_4537284), Gamma (EPI_ISL_4536760), Delta (EPI_ISL_4536228) and Omicron (EPI_ISL_13017139) were produced in Vero E6 cells. The infected Vero E6 cells were incubated at 37°C in a humidified atmosphere with 5% of CO2. The supernatants were purified and concentrated in Amicon ® Ultra-15 diafiltration devices (Merck Milipore Ltd). Then, supernatants were quantified for viral RNA levels by RT-qPCR (Altona ® , Roche) and viral infectivity by lysis plaque assay titration  (13)  (see below).
Statistics Comparison between paired data set was done with paired Wilcoxon signed-rank test or Friedman test for comparison of more than two groups. Comparison of unpaired data sets was done with Mann-Whitney test or Kruskal-Wallis test for comparison of more than two groups followed by Dunn posttest. Analyses were done in Graphpad Prism v9. 3. 1 (GraphPad Software, LLC. ). A p-value <0. 05 was considered significant.
The time step used is 0. 001 days. The descriptions and values of all parameters used in the model are summarized in Table  1. Note that the values used in simulations were selected from the ranges extracted from various studies. Furthermore, to investigate the effects of factors on the transmission dynamic, particularly at equilibrium, we used steady-state equations by solving ODEs numerically. In the case of no treatment, ϕ and z are set to zero; in the case of no incoming prevalence, u and y are set to zero. A change in R 0 resulted from varying δ. All the figures and calculations were generated using MATLAB software (version R2020b; The MathWorks, Inc).
Acknowledgements We would like to thank  Max Maurin  for introducing us to the Klebsiella pneumoniae problem and for fruitful discussions at an early stage of this work. This project was financially supported by the  Royal Golden Jubilee PhD Programme  (Grant No. PHD/0079/2560 ). This work was performed during the visit of  Suttikiat Changruenngam  to the  Theory group of the Laue-Langevin Institute, France.
ACKNOWLEDGMENTS This work was supported by grants from the  "Cancéropole Ile de France ," the " Intergroupe Francophone du Myelome " and from the  "Fondation Française pour la Recherche contre le Myélome et les Gammapathies monoclonales. "A. Abecassis was supported by a fellowship from the  "Agence Nationale pour la Recherche Scientifique , CIFRE" in partnership with Servier. We are also grateful to Car-olina  Martinez-Cingolani , to the animal facility, and to the cytometry platform of the  Institut de Recherche Saint Louis  for technical help.
Sequence Analyses We directly sequenced positive-PCR products in both directions using appropriate internal primer sets (Supplementary Table  3 ). We conducted DNA sequencing by capillary electrophoresis using the BigDye R Terminator chemistry (Applied Biosystems) on an ABI PRISM 3130 automated DNA sequencer. We visually inspected the obtained chromatograms for quality control and for detecting the presence of ambiguous (double peak) positions. Sequences obtained in this study were deposited in GenBank under accession numbers OK285278-OK285280 (Cryptosporidium spp. ), OK318919-OK318938 and OL456212 (Giardia duodenalis), OK285223-OK285250 (Blastocystis sp. ), OK533569-OK533571 and OL458611 (Enterocytozoon bieneusi), OK493778 (Balantioides coli), and OK493782 (Troglodytella abrassarti).
Molecular Characterisation of Intestinal Protist Species in NHP and Humans We summarised in Table  4  the distribution of intestinal protist species and genotypes (confirmed by Sanger sequencing) by zoological institution and primate genera obtained in the present study. The in-depth molecular features of the obtained protist isolates including sub-genotyping data, presence of single nucleotide polymorphisms, and GenBank accession numbers of representative sequences are shown in Supplementary Tables  6, 7  (NHP) and Supplementary Table  8  (humans). We identified two Cryptosporidium species including C. hominis (66. 7%, 4/6; present in members of the genera Callithrix, Homo, and Pan), and C. parvum (33. 3%, 2/6; present in NHP of the genera Macaca and Saguinus). All Cryptosporidiumpositive samples were obtained in the MZA and the SZ in Spain (Table  4 ).
Molecular Evidence of Zoonotic Transmission We considered evidence of zoonotic transmission the finding of the same protist species and genetic variant circulating simultaneously among NHP and their handlers in the same zoological institution and during the same sampling period. Under these premises, we detected C. hominis in two zookeepers and a captive chimpanzee (Pan troglodytes) investigated during the first sampling campaign at the MZA (Table  4 ). Lack of genotyping data at the gp60 locus precluded us to determine the gp60 family sub-genotype involved in these three infections. We obtained strong evidence of zoonotic transmission for Blastocystis sp. At the BZ, we identified ST3 allele 34 in a gorilla (Gorilla gorilla) and a zookeeper (Table  4 ; Figure  3 ). At Faunia, we confirmed ST4 allele 42 in four lemurs (Lemur catta) and a zookeeper during the first sampling campaign, and ST2 allele 12 in a lemur (L.
Available epidemiological data seem to indicate that E. bieneusi is a relatively common finding in African captive NHP including resident chimpanzees in sanctuaries from Cameroon and Kenya  (89)  and recently captured olive baboons in the latter country  (44). Reported infection rates ranged from 1-12% and involved genotypes A, D, EbpA, KB-1 to KB-6, PigEBITS5, Peru7, and Peru11. Much higher infection rates and genetic diversity frequencies have been documented in NHP collections from zoological gardens in Asian countries, particularly in China. In that country, E. bieneusi infection rates varied from 11-46%  (46, (90) (91) (92) (93). All these studies included sample panels over 150 animals. The highest infection rates were detected in golden snub-nosed monkeys (46%)  (92) , and longtailed macaques (31%)  (93).
Furthermore, ST3 allele 34 was identified in a gorilla and a zookeeper at BZ, and ST4 allele 42 in lemurs and a zookeeper at Faunia. Because of lack of intra-subtype molecular data within ST2, ST3, and ST4 of NHP origin, the directionality of these zoonotic events remains to be fully elucidated. Of note, zoonotic transmission of Blastocystis ST1 and ST8 between captive NHP and their zookeepers has been previously documented in zoological gardens in Spain and UK, respectively  (12, 13). Similarly, C. hominis zoonotic transmission was highly suspected between a chimpanzee and two zookeepers at MZA, although lack of genotyping data at the gp60 locus precluded us to ascertain the extent and directionality of this event. In conclusion, this molecular-based survey revealed that a high proportion of the captive NHP (41%) and their zookeepers (30%) investigated were infected/colonised by intestinal protist species of potential or uncertain pathogenicity.
hominis. Molecular-based studies constitute a powerful tool for the monitoring of intestinal protist species in captive NHP and their zookeepers, assisting in the identification of clinical cases and sources of infection, and assessing potential transmission risk to (or from) other resident animals and visitors. under project PI16CIII/00024. DG-B was recipient of a Sara Borrell Postdoctoral Fellowship (CD19CIII/00011) funded by the Spanish Ministry of Science, Innovation and Universities. AD was recipient of a PFIS contract (FI20CIII/00002) funded by the Spanish Ministry of Science and Innovation and Universities.
Acknowledgements The work of the authors is supported by Grants from  Agence Nationale pour la Recherche   CE17-0014 ,  ERA-Net Narcomics ,  Fondation pour l'Aide à la Recherche sur la Sclérose en Plaques (ARSEP)-French MS society, and Biomarkers in autoimmune EncephaliTis and Paraneoplastic neurological SYndromes Recherche Hospitalo-Universitaire  ( BETPSY RHU) RHU-18-0012  the  Inspire Geroscience  project (to RSL), and from the  Swiss National Science Foundation   310030B_201271 & 310030_185321 , and  European Research Council (ERC)   865026  (to DM).
Discussion This analysis showed that a sufficient quantity of DNA was extracted from tumor excision blocks embedded in paraffin originating from an MT-ND4 LHON patient. DNA from a housekeeping gene was amplified successfully by PCR from tumor slides, indicating that the DNA quality was adequate for PCR amplification. Lenadogene nolparvovec DNA was not detected in glioblastoma tumor samples from the patient 3 years post-administration of lenadogene nolparvovec, and no PCR inhibition was detected. These results, along with the data accumulated in the five clinical trials conducted with lenadogene nolparvovec to date, demonstrate the long-term safety of lenadogene nolparvovec gene therapy as regards the potential for causing tumoral transformation. Data from a study in cynomolgus monkeys have shown that lenadogene nolparvovec DNA penetrates the central nervous system (CNS) visual pathways (optic chiasm, optic tract and contralateral optic nerve) following intravitreal administration  [12, 14].
Funding The Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, USA, processed and performed brain tissue/tumor analyses with no funding from the sponsor of the study. Charles River Laboratories, funded by GenSight Biologics, performed sample analyses. All authors contributed to interpretation of the data and writing of the manuscript without payment as part of a scientific collaboration. NJN is supported in part by an ophthalmology department core grant from the NIH/NEI (P30 EY006360). PYWM is supported by a Clinician Scientist Fellowship Award (G1002570) from the Medical Research Council (UK), and receives funding from.
Peptidylarginine deiminases: structure, function and tissue expression Peptidylarginine deiminases or protein arginine deiminases (PADs), first reported in 1977, belong to the cysteine hydrolases enzyme family (EC 3. 5. 3. 15). PADs drive arginine deimination via a nucleophilic attack of the active site cysteine on the arginine's guanidinium group, leading to the formation of an intermediate acyl-enzyme which is then hydrolyzed to citrulline  [13]. There are five known isotypes of PADs: PAD1, PAD2, PAD3, PAD4 and PAD6, with PAD6 being catalytically inactive, that are distributed in a myriad of human cell types showing a highly tissuespecific expression pattern and consequently perform various functions. All the human PAD isozymes have a length ranging from 663 to 694 amino acids, a molecular weight around 74 kDa and share 70-95% homology within their sequences  [13] , UniProtKB database].
Diagnostic and therapeutic potential of citrullination Recent years brought significant progress into the understanding of the role of the citrullination process in immune disorders. The growing body of evidence highlighting the importance of citrullination, and the identification of autoantibodies directed to the citrullinated peptides or proteins (ACPAs) has significantly broadened our understanding, and clinical characterization, of many immune disorders. The number of the ongoing clinical studies targeting PADs and the citrullination process is, however, currently limited to a few. The most advanced studies concern rheumatoid arthritis, and include diagnostic tests (Table  5 ) as well as interventional trials (Table  6 ). The non-interventional studies aim at the identification and quantification of the prevalence of anti-citrullinated proteins antibodies directed against different citrullinated antigens, including collagen, fibrinogen, filaggrin or vimentin, to evaluate their diagnostic and prognostic value in rheumatoid arthritis progression (Table  5 , NCT01078597, NCT03265236, NCT03224377).