dataesr/scientific-paragraphs-categorization huggingface dataset:
25, 26 sing a label-free quantitative proteomics pipeline, we quantified 3551 proteins in a comparative analysis of control vs DTX mouse TIF (Supplemental Dataset 1B). A total of 1812 TIF proteins were significantly different (P <. 05) between control and DTX mice (Figure  1B ). DTX caused a significant reduction in 1433 (41%) of TIF proteins (Figure  1B , Supplemental Dataset 1B and 2A) whereas n = 379 (11%) were up-regulated (Figure  1B ,C, Supplemental Dataset 2B). Two functional markers of peritubular myoid cells  26  were present in TIF but not decreased in DTX mice (CNN1, MYH11, Supplemental Dataset 1B). This first in-depth proteomic analysis of mouse TIF reveals that the seminiferous epithelium is a major determinant of the TIF proteome.
| Germ cell proteins are present in mouse TIF We next investigated TIF proteins that were decreased by DTX (Supplemental Dataset 2A). These included several well-known sperm-specific proteins such as ODF1, AKAP3, and AKAP4  41, 42  that are thought to be restricted inside the blood-testis barrier. If these proteins were present because of non-specific leakage into TIF during fluid collection, we reasoned that most high-abundance spermatid-specific proteins should also be present. However, of the proteins whose mRNA is highly and specifically expressed in mouse spermatids but not detected in Sertoli cells (Supplemental Dataset 4A) or those with the highest expression in round spermatids (Supplemental Dataset 4B)  40  only a subset was identified in TIF (Supplemental Dataset 4A,B) and only 39% of spermatid-specific transcripts had protein products identified in TIF (Supplemental Dataset 4A).
Furthermore, leakage should favor the release of germ cell proteins into TIF in DTXtreated mice, where there is degeneration of the seminiferous epithelium, marked germ cell apoptosis, and disruption of tight junctions between Sertoli cells. However, we saw the opposite; sperm-specific proteins ODF1, AKAP3, and AKAP4 were detected in high abundance in normal mouse TIF but were reduced ~90% after DTX-induced seminiferous epithelial disruption (Supplemental Dataset 2A). These observations indicate some sperm-specific proteins are deposited into TIF in the normal mouse testis. To identify TIF proteins of germ cell origin, we investigated their testicular mRNA expression in an RNASeq dataset of germ cell ablation in mice. 39 Proteins likely to originate from germ cells were identified using previously established criteria (a >70% reduction in mRNA expression in whole testis after the administration of the germ cell-specific toxicant, busulfan).
39 Of the 1433 proteins that were decreased in TIF after DTX treatment (Supplemental Dataset 2A), 498 were potential of germ cell origin (Supplemental Dataset 3A). These proteins showed a broad molecular weight range, from 5 to 541 kDa. We further defined the cellular origin of these 498 proteins using mRNA data from purified mouse Sertoli and germ cells. 40 Most were very highly expressed in germ cells, but also detectable in Sertoli cells. However, 141 TIF proteins were specifically expressed, or very highly enriched, in pachytene spermatocytes and round spermatids but were very low to undetectable in Sertoli cells and spermatogonia (Supplemental Dataset 3B). Thus, 141 proteins were highly enriched in germ cells that reside inside the blood-testis barrier (ie, adluminal germ cells) and were significantly decreased in TIF by DTX ablation, suggesting that these are adluminal germ cell-specific proteins contributed to TIF via Sertoli cells.
We next investigated which of these proteins were testis-specific by assessing expression in a range of other adult male mouse tissues including brain, liver, and lung. 40 Of the 141 spermatocyte and spermatid proteins, 95 had mRNA expression restricted to the testis (Supplemental Dataset 3B) and their abundance in TIF varied over a large range (Supplemental Dataset 3B). Therefore, at least 141 proteins are likely contributed to TIF solely by adluminal germ cells via Sertoli cells, and 95 of these proteins are only expressed in these cells and nowhere else in the adult mouse body. Because sperm proteins often undergo translational delay during spermiogenesis  43  it is likely that many of the TIF proteins with high mRNA expression in round spermatids (Supplemental Dataset 3B) are translated during | 7 of 18 O'DONNELL Et aL the elongation phase of spermatids and are located in mature sperm.
The PCA plot reveals a clear separation between the control and DTX-treated mice. Heat map data are log 2 protein ratio in DTX mice relative to control We next investigated whether the appearance of a sperm protein in TIF was related to its location in spermatids (Supplemental Datasets 4B-D). Proteins highly expressed in round spermatids and detected in TIF (Supplemental Dataset 4B) were significantly associated with sperm flagellar and cytoplasmic functions (Supplemental Dataset 4D), whereas those not detected in TIF were significantly enriched in nuclear/nucleosome functions and mitochondria (Supplemental Datasets 4B,C). Of note, the highly abundant spermatid nuclear proteins PRM1 and TNP1 were not detected in TIF, yet sperm flagellar proteins such as ODF1 and 2, AKAP3 and 4, were identified (Supplemental Dataset 4B).
These data suggest that sperm proteins are differentially deposited into TIF based on their cellular location. The sperm-specific protein LDHC has recently been shown to promote peripheral immune tolerance in mice, via interacting with immune cells outside of the seminiferous tubules. 23 In contrast, another sperm protein, zonadhesin (ZAN), was non-tolerogenic and thus assumed to remain within the seminiferous tubules. 23 In support, we found LDHC was the fifth most abundant protein in mouse TIF (Supplemental Dataset 1A) but ZAN was not detected. LDHC showed a highly significant reduction in TIF after DTX treatment (P = 5. 06 × 10 -8 ), confirming that it is contributed to TIF by the seminiferous tubules in mice. 23 hus, mouse TIF contains hundreds of germ cell proteins, many of which are predominantly or only expressed in developing sperm that reside "inside" the blood-testis barrier.
| Identification of germ cell proteins in human TIF There is no data on the proteome of human TIF, and whether sperm-specific proteins are also deposited into TIF by the seminiferous tubules in men is not known. Accordingly, we characterized the human TIF proteome from men with normal testis function. TIF was taken from three men undergoing surgical retrieval of sperm due to the obstruction of the distal reproductive tract (obstructive azoospermia, OA). These men had normal testis function based on a variety of clinical parameters (see Methods). It is important to note that the method of TIF collection in humans was somewhat different from that in mice (see Methods); however, both methods ensured that the seminiferous tubules remained intact. A total of 4720 proteins was identified in TIF from these men (Figure  2 , Supplemental Dataset 5).
To identify proteins in human TIF that could arise specifically from germ cells, we utilized two different approaches. First, we looked for TIF proteins that are highly enriched in human spermatocytes and spermatids. We used an independent dataset of n = 1079 human testis-enriched proteins (www. prote inatl as. org/human prote ome/testis 51), whose mRNA expression is enriched >5-fold in human testis compared to all other tissues as assessed by RNASeq. This revealed 84 proteins in TIF whose expression is deemed to be enriched in human testis (Supplemental Dataset 6). The cellular localization of these human testis-enriched TIF proteins was then further assessed using immunohistochemistry data in human testis sections (www. prote inatl as. org). We found 30 human TIF proteins that are highly enriched or specifically expressed in adluminal germ cells of the human testis (Supplemental Dataset 7A).
This list included proteins involved in sperm motility (AKAP4, GAPDHS, LDHC, PGK2, and ROPN1L) and fertilization (ACR, ADAM2, CLGN). Twenty-three of these proteins had orthologs that were present in mouse TIF and were decreased by DTX (Supplemental Dataset 7A). In the second approach, we analyzed whether the 95 sperm-specific proteins in mouse TIF (Supplemental Dataset 3B) have orthologs in human TIF and whether there is information supporting their enrichment or specificity in adluminal germ cells in men. Of these, 28 proteins had orthologs detectable in human TIF, and 21 have been shown to be specific to, or highly enriched in, human testis based on multiple human RNASeq-based datasets (Supplemental Dataset 7B). Using the above data (Supplemental Dataset 7A,B), and relevant published datasets, we identified 33 TIF proteins that are likely to originate from adluminal germ cells in both mice and humans (Table  1 , Figures  1A  and 3 , Supplemental Dataset 7C).
All 33 proteins were significantly reduced in mouse TIF by DTX-induced seminiferous epithelial disruption and had immunohistochemical and/or mRNA expression data to support their enrichment in adluminal germ cells in both species (Table  1 , Figure  3 , Supplemental Dataset 7C). These conserved TIF proteins include flagellar components (AKAP3 and 4), acrosomal proteins (ACR, ACRBP, F I G U R E 2 The human TIF proteome. iBAQ analysis and dynamic range estimation of n = 4720 proteins identified in human TIF. iBAQ expression values for the proteins quantified are plotted with the log10 iBAQ intensity on the y axis and proteins are ranked by iBAQ intensity on the x axis. Proteins that have been identified as cancer-testis antigens (CTAs) are highlighted in orange (see Supplemental Dataset 8) SPACA3), and sperm-specific enzymes (LDHAL6B, LDHC, GAPDHS) (Figure  3 ).
| Sperm-specific TIF proteins can be detected in human blood plasma Because proteins in TIF can enter the circulation,  52  we next investigated whether these advanced germ cell-specific human TIF proteins could be detected in human plasma by performing in silico analysis of the Plasma Proteome Database. 53, 54 e found that 22 human sperm-specific TIF proteins are detected in human plasma; ACR, ACTL7B, ADAM2, AKAP3, AKAP4, CABS1, CCT6B, CLGN, DDX20, GAPDHS, HSPA4L, LDHAL6B, LDHC, ODF2, PFN3, PGK2, PRSS50, PSMA8, SHCBP1L, TRIM36, TTC21A, YBX2 (Supplemental Dataset 7A,C). These proteins are also testis and germ cell-specific in mice (Supplemental Dataset 7C). These findings contradict the widely held assumption that sperm-specific proteins do not have access to the circulation and suggest that sperm proteins released into TIF can enter the circulation.
| Cancer-testis antigens are present in mouse and human TIF We noted several well-known cancer-testis antigens (CTAs) were detected in both mouse and human TIF. These are proteins whose normal expression is restricted to advanced germ cells/sperm but are aberrantly expressed in cancer. 7, 8, 10, 15 e investigated human CTAs in TIF utilizing published O'DONNELL Et aL datasets,  8, 55, 56  including a recent study identifying germ cellspecific CTAs in humans. 56 Human TIF contained 82 proteins that are classified as CTAs (Figure  2 , Supplemental Dataset 8). Of these, 43 are detected in human plasma according to the Plasma Proteome Database (Supplemental Dataset 8), challenging the assumption that germ cell-specific CTAs do not appear in the circulation. Furthermore, 41 of the 82 human TIF CTAs have orthologs detectable in mouse TIF (Supplemental Dataset 8).
Importantly, LDHC levels were significantly reduced in men with nonobstructive azoospermia (Figure  4 , P =. 03, n = 8/group). The phenotypes of these men were broadly classified as Sertoli cell-only (SCO) based on testicular biopsy (Supplemental Dataset 10A,B), and when their testes were biopsied for the purpose of sperm retrieval, no sperm were retrieved. However, the variation in the levels of LDHC levels, ranging from very low to similar to those with obstructive azoospermia, suggests there could be varying levels of spermatid development in the seminiferous tubules of these individuals (Figure  4 ). These results are the first to demonstrate that the levels of a sperm-specific protein and CTA in human TIF vary according to the status of spermatogenesis.
A third mechanism could be via Sertoli cell-mediated release of germ cell proteins in extracellular vesicles at the mid-spermatogenic stages (stages VII-VIII in mice and stages IV-V in humans; these stages occur before residual body release), where there is significant stage-specific apoptosis of pachytene spermatocytes and round spermatids  80  (Figure  5 ). MHCII+ macrophages preferentially accumulate around these tubules,  65  and could access antigens deposited by Sertoli cells. The mouse and human testis-specific proteins in TIF, HDGFL1, and LYAR, are detected in round spermatids during these stages but are absent from mature sperm (Figure  3 ), suggesting that they are not deposited via residual bodies or the tubuli recti. Of interest, we observed that very few of the germ cell-derived proteins in TIF had secreted annotations, but several mice and human TIF germ cell and sperm-specific proteins have been detected in exosomes (Supplemental Dataset 7B).
Our data also suggest that CTAs could show sex-specific responses, another crucial consideration in the design of immunotherapies for different cancers. There are currently more than 39 cancer trials using CTAs registered in the Clinical Trials database (http://www. clini caltr ials. gov). While initial data suggest that CTAs are useful for stratifying patient outcomes for various tumor types, there are still issues with the induction of a sufficient immune response. 15 ur findings strongly suggest that all CTAs should not be presumed to be neoantigens and that the measurement of a particular CTA in serum, levels of antibodies, and a comprehensive analysis of tissue expression,  56  in a variety of men and women is necessary prior to determining its potential clinical utility. CTAs are also being pursued as cancer biomarkers, as the protein (and antibodies to the protein) are assumed to be absent from circulation in healthy individuals.
ACKNOWLEDGMENTS LBS and PJO received a BBSRC Project Grant Award (BB/ J015105/1) and DR received a Society for Endocrinology, UK (SFE) Early Career Grant. LBS received a Medical Research Council Program Grant Award (MR/N002970/1). LO'D and PS were supported by NHMRC Project Grant #10099002 and by the Victorian State Government Operational Infrastructure Support Program. LO'D, PS, AP, DF, RS, and TD received research support from the Deutsche Forschungsgemeinschaft (DFG) as part of an International Research Training Group between Justus Liebig University (Giessen, Germany) and Monash University (Clayton, Australia) on the "Molecular pathogenesis of male reproductive disorders. " The authors thank Laura Milne and Michael Curley (MRC Center for.
Gene symbol (mouse, human) Protein name (human) Relative abundance (mouse) Adluminal germ cell proteins in TIF conserved between mice and humans a Relative abundance (human) 9230110C19Rik, CFAP300/ Cilia-and flagella-associated protein 300, +++ ++ C11orf70 Uncharacterized protein C11orf70 Acr, ACR Acrosin +++ ++ Acrbp, ACRBP Acrosin-binding protein ++++ ++ Actl7b, ACTL7B Actin-like protein 7B ++++ ++ Adad1, ADAD1 Adenosine deaminase domain-containing protein 1 +++ ++ Akap3, AKAP3 A-kinase anchor protein 3 ++++ ++ Akap4, AKAP4 A-kinase anchor protein 4 ++++ ++ Cabs1, CABS1 Calcium-binding protein, spermatid associated 1 ++++ ++ Cct6b, CCT6B T-complex protein 1 subunit zeta-2 +++ ++ Clgn, CLGN Calmegin +++ ++ Ddx20, DDX20 DEAD-box helicase 20 +++ ++ Gapdhs, GAPDHS Glyceraldehyde-3-phosphate dehydrogenase, ++++ ++ spermatogenic Hdgfl1, HDGFL1 Hepatoma-derived growth factor-like 1 ++++ ++ Hspa4l, HSPA4L Heat shock protein family A (Hsp70) member 4 like ++++ +++ Klhl10, KLHL10 Kelch-like family member 10 +++ ++ Ldhal6b, LDHAL6B Lactate dehydrogenase A-like 6B ++++ ++ Ldhc, LDHC Lactate dehydrogenase C +++++ +++ Lrrc46, LRRC46 Leucine-rich repeat-containing protein 46 ++++ ++ Lyar, LYAR Ly1 antibody reactive ++++ +++ Odf2, ODF2 Outer dense fiber of sperm tails 2 +++ ++ Pdcl2, PDCL2 Phosducin-like 2 ++++ ++ Pfn3, PFN3 Profilin-3 +++ ++ Pgk2, PGK2 Phosphoglycerate kinase 2 ++++ ++ Piwil1, PIWIL1 Piwi-like protein 1 ++++ ++ Psma8, PSMA8 Proteasome subunit alpha type-7-like ++++ +++ Ropn1, ROPN1/ROPN1B Rhophilin associated tail protein 1B ++++ ++ Ropn1l, ROPN1L Rhophilin associated tail protein 1 like ++++ ++ Shcbp1l, SHCBP1L SHC-binding and spindle associated 1 like ++++ +++ Spaca3, SPACA3 Sperm acrosome associated 3 ++++ ++ Spata33, SPATA33 Spermatogenesis-associated protein 33 +++ ++ Tktl2, TKTL2 Transketolase-like protein 2 ++++ +++ Trim36, TRIM36 E3 ubiquitin-protein ligase TRIM36 +++ ++ Ybx2, YBX2 Y-box-binding protein 2 ++++ +++ a See Supplemental Dataset 7C for further details.
Chromatin immunoprecipitation (ChIP) and binding site analysis The detailed procedure is described in Additional file 1. The ChIP-Seq performed are: RUNX1 in REH cells (n = 2), Nalm6 (n = 2), BCP-ALL patients not expressing ETV6-RUNX1 (n = 3), H3K4me1, H3K4me3, H3K27ac, and CBFA2T3 in REH (n = 1) and Nalm6 (n = 1). Antibodies used are listed in Table  S2. All sequencing data are available at NCBI's GEO (https ://www. ncbi. nlm. nih. gov/ geo/query ) through #GSE109377 for RUNX1 in Nalm6 and patients  [26]  and #GSE117684 for the other data.
Results CBFA2T3 transcript level is upregulated in blasts from BCP-ALL patients and is correlated with RUNX1 expression. Among BCP-ALL subtypes, RUNX1 and CBFA2T3 transcripts are upregulated in ETV6-RUNX1 BCP-ALL blasts (Fig. 1a , Additional file 2: Fig. S1A ). This result, obtained from the analysis of the RNA-Seq database of St. Jude Children's Research Hospital Pediatric Cancer Data Portal cohort  [31] , is coherent with microarray data from  previous cohorts  [19] [20] [21]. Moreover, RUNX1 transcript level positively and significantly correlates with CBFA2T3 transcript level in ETV6-RUNX1 BCP-ALL samples compared to other childhood BCP-ALL (Fig. 1b ). Nevertheless, this correlation between RUNX1 and CBFA2T3 expression is retrieved under enforced expression or depletion of RUNX1 or CBFA2T3 in two BCP-ALL cell lines, the REH cells (that express ETV6-RUNX1 transcript and protein) and Nalm6 cells (that do not express ETV6-RUNX1) (Fig.
Acknowledgements We are grateful to all patients and patients' families from  Rennes University Hospital, CHRU Rennes, France. We thank our colleagues from the team ECLA from Brest and  GEO  from  Rennes  for technical assistance and scientific advices. We thank the  Human and Environmental Genomics of Rennes  facilities from  BIOSIT SFR UMS CNRS 3480 INSERM 018. We thank  David F. Callen  and  Andrew Turner  from the  University of Adelaide, Australia  for providing CBFA2T3-myc truncated plasmids.
Funding This work was funded by grants from  Ligue Régionale  contre le cancer (comité  22 ,  29 ,  35 ,  56 ,  79 ,  41 ) (MBT, LD, VG),  Région Bretagne  (LD, MBT), The  Société Française d'Hématologie (LD) ,  French Research Ministry (HJ) , the société française de lutte contre les cancers et les leucémies de l'enfant et de l'adolescent and the  Fédération Enfants et Santé (MBT) ,  The Société Française de Biochimie et Biologie Moléculaire (HJ) ,  The association Gaetan Salaun (BS, MBT) ,  The association Halte au Cancer (MBT)  and the  People Programme  ( Marie Curie Actions ) of the European Union's  Seventh Framework Programme  ( FP7/2007-2013)  under  REA  grant agreement n°291851 (MBT).
After initially hypothesizing a positive relationship between use of renin-angiotensin-aldosterone system inhibitors and risk of coronavirus disease 2019 (COVID-19), more recent evidence suggests negative associations. We examined whether COVID-19 risk differs according to antihypertensive drug class in patients treated by ACE (angiotensin-converting enzyme) inhibitors and angiotensin receptor blockers (ARBs) compared with calcium channel blockers (CCBs). Three exclusive cohorts of prevalent ACE inhibitors, ARB and CCB users, aged 18 to 80 years, from the French National Health Insurance databases were followed from February 15, 2020 to June 7, 2020. We excluded patients with a history of diabetes, known cardiovascular disease, chronic renal failure, or chronic respiratory disease during the previous 5 years, to only consider patients treated for uncomplicated hypertension and to limit indication bias.
5 ] [8] [9] [10] [11] [12] [13]  The most recent meta-analysis showed that ACE inhibitors use may not increase the susceptibility to severe acute respiratory coronavirus 2 infection, disease severity, and mortality. 14 ost studies were carried on patients who were hospitalized, and not on the overall hypertensive population, with a risk of collider bias. 6 e used the SNDS (Système National des Données de Santé) database to study a large population of patients treated for hypertension. While ensuring a sufficient number of events to provide good statistical power, we wanted to consider patients treated for hypertension only, with no known severe cardiometabolic diseases, to limit indication bias. We examined whether the COVID-19 risk differs according to the pharmacological class of antihypertensive drugs in patients treated by ACE inhibitors or ARBs compared with CCBs.
Setting and Data Sources We conducted a retrospective cohort study comparing 3 classes of antihypertensive drugs using data from the SNDS, which covers the entire French population, that is, 67 million inhabitants. Since 2006, an anonymous unique individual identifier links information from two main data sources: DCIR (Données de Consommation Inter-Régimes, national health insurance claims database) and PMSI (Programme de Médicalisation des Systèmes d'Information, national hospital and discharge database). The DCIR includes individual information on outpatient medical care, laboratory tests, and reimbursed drugs coded according to the Anatomic Therapeutic Chemical classification. The health expenditure of patients with serious and costly long-term diseases, such as cancer, or diabetes, is fully reimbursed, and their diagnosis is recorded according to the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision.
The DCIR also collects a few patient data. 15, 16 he PMSI includes dates of admission and discharge to all public and private hospitals in France, discharge diagnoses coded according to International Statistical Classification of Diseases and Related Health Problems, Tenth Revision and type of medical or surgical procedures coded according to the French common classification of medical procedures (Classification Commune des Actes Médicaux). Disease and expenditure mapping is a tool developed from the DCIR and PMSI databases by using 58 medical algorithms identifying diseases. 17 [20].
Quintiles of the social deprivation index were used as a measure of socioeconomic status based on the area's median household income, percentage of high school graduates in the population aged 15 years and older, percentage of manual workers in the active population, and unemployment in individual's city of residence. Smoking was defined by the presence of specific hospitalization diagnoses, nicotine replacement therapy, or annual lump sum payment for smoking cessation. Patients who did not take another class of antihypertensive drug (eg, betablockers, diuretics) were classified as patients on monotherapy. History of known comorbidities was assessed using both the disease and expenditure mapping medical algorithms and additional data from the DCIR and PMSI databases. Hospitalization for comorbidities was identified in the 5 years before the last antihypertensive drug dispensing; drugs other than the 3 classes of antihypertensive drugs considered were identified in the 12 months before the last antihypertensive drug dispensing.