dataesr/scientific-paragraphs-categorization huggingface dataset:
At the time of writing, SLC19A1 is the only cGAMP conduit for which a direct interaction is demonstrated and the exact mechanisms used by other conduits are still under investigation. Although SLC19A1's function has been observed in human monocytic cell lines (THP1 and U937), only a minimal role in cGAMP uptake has been shown in primary human monocytes  (12). This suggests SLC19A1 plays a non-dominant role in monocytic cGAMP import and highlights the caution and need for primary cell data to delineate the cell types in which these channels transport cGAMP in vivo. Other cyclic dinucleotides in addition to 2'3'-cGAMP are imported into cells by SLC19A1, both synthetic (2'3'-RR CDA, 2'3'-cG S A S MP, 2'3'-CDA, 2'3'-CDA S ) and bacterially derived CDNs (3'3'-cGAMP). In contrast, some bacterially derived CDNs were imported poorly or not at all (e.
g. 3'3'-cGAMP) produced by bacteria as secondary messengers have also shown resistance to ENPP1 mediated hydrolysis  (27). This is the strategy adopted by companies developing CDN based immunotherapeutics, which are currently in clinical trials. Notably, the CDNs 2'3'CDA S (also known as ADU-S100) and MK-1454 are being tested for activity against advanced/ metastatic solid tumors and lymphomas (trial IDs -NCT03172936 and NCT03010176)  (12, 121). As these CDNs show different specificities for the cGAMP conduits, knowing which cell types express the different cGAMP conduits could allow for cell targeted CDN treatments based on CDN channel expression profiles  (12). This is of particular importance for the development of these CDNbased therapies as CDN mediated STING activation can inhibit the proliferation of T cells required for effective tumor rejection.
0. For data collection, the crystals were cryoprotected by quick immersion into various cryoprotectant solutions  [36]  before flash freezing in liquid nitrogen. The data sets of several complexes were collected at synchrotron facility SOLEIL (beamlines PROXIMA-1 and PROXIMA-2, Gif-sur-Yvette, France) using both standard rotation and helical scan method  [37]. About 130 samples were tested and the best crystal diffracted to 2. 19 Å. X-ray diffraction data were collected from single crystals at 100 K on the Pilatus detector on beamline PROXIMA-1. The crystal belongs to the space group P4 1 2 1 2 with unit cell parameters of a ¼ b ¼ 52. 3 Å and c ¼ 265. 2 Å. Structure deposited into the PDB Data Bank: 7ANQ.
The cells were washed with ice-cold PBS, collected, and lysed in RIPA buffer (50 mM TriseHCl pH 7. 5, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 1% Nonidet P-40, 0. 5% sodium deoxycholate, 0. 1% SDS, and PIC). The resulting total cell lysates and media were analysed by Western blotting, imaged using ChemiDoc MP imaging system (Bio-Rad) and the protein bands quantified by Image LabÔ Software (Bio-Rad) as previously described  [40]. The following antibodies were used in this study: anti-V5-epitope (1:5,000; Sigma), monoclonal antibody to detect V5-tagged proteins (PCSK9 and LDLR), anti-Flag (1:2,000; Sigma); anti-HA (1:4,000; Sigma), anti-hLDLR (1:1,000; R&D system), anti-hPCSK9 (1:2,000  [14] ), anti-b-actin (1:3000; Sigma), anti-CAP1 (1:2,000; Santa Cruz), anti-Caveolin-1 (1:1,000; abcam), anti-Clathrin Heavy Chain (1:1,000; Invitrogen); anti-goat-conjugated Alexa-488 (1:500; Molecular probes, Invitrogen), and anti-rabbit (1:10,000; Amersham), anti-goat (1:10,000; Sigma) and anti-mouse (1:10,000; VWR)-conjugated to horseradish peroxidase HRP.
To analyze total LDLR and CAP1 expression, cells were treated with the same protocol but were permeabilized with 0. 2% TritonX-100 in PBS for 5 min prior blocking. To test LDLR functionality, cells were incubated for 2 h at 37 C with 5 mg/ml DiI-LDL (1,1 0dioctadecyl-3,3,3 0 ,3 0 -tetramethyl-indocarbocyanine perchlorate, Cedarlane/Kalen Biomedical) in SFM media before fixation. Coverslips were mounted on a glass slide with ProLong Gold antifade reagent with DAPI (Thermo Fisher). Samples were visualized using Plan-Neofluar 40Â/0. 60 or Plan-Apochromat 63Â1. 4 oil immersion objectives of LSM-710 confocal laser-scanning microscope (Carl Zeiss) with sequential excitation and capture image acquisition with a digital camera. Images were processed with ZEN software. Image analysis to quantify the fluorescence intensities was accomplished using VolocityÒ6.
0. Dil-LDL uptake assay was achieved as previously described  [40]. 2. 8. PCSK9 and MHC-I 2. 8. 1. Database search for the motif RXE A python code was written to analyze all non-redundant protein sequences from NCBI's BLAST database (with entries from GenPept, Swissprot, PIR, PDF, PDB, and RefSeq; downloaded June 14, 2021, at: https://ftp. ncbi. nlm. nih. gov/blast/db/). Briefly, all human nonredundant sequences with one or more occurrences of the motif RXE (X ¼ any residue) were filtered to a tab separated values file (. tsv) corresponding to the following fields: the fasta header, the protein sequence, the list of motifs that were found and, the total count of motifs for the sequence. A multi-fasta file was generated using all listed motifs. Next, using WebLogo3. 7  [42]. 2. 8. 2. Molecular modeling of PCSK9/HLA-I and CHRD/CAP1 complexes PCSK9 (Pdb ID: 2P4E) and HLA-I (Pdb ID: 6TRN) structures were docked using GRAMM-X web server (http://vakser.
compbio. ku. edu/ resources/gramm/grammx/  [43] , respectively, as receptor and ligand. All available chains in PDB were used and the residues R68 and E70 of HLA-I, comprised in the motif RXE, were assumed as potential interface residues. The interface residues of the docking model were subjected to energy minimization using GROMOS96 43B1  [44]  implementation of Swiss-PdbViewer (100 steepest descent steps, 1 conjugate gradient step, 100 steepest descent steps). The quality of the final model was evaluated using PROCHECK  [45]. The CHRD/CAP1 complex was modeled using AlphaFold2 multimer interactive python notebook at https://colab. research. google. com/github/sokrypton/ ColabFold/blob/main/AlphaFold2. ipynb using default parameters.
40 (Supplementary Figure  5B ). To explain the higher affinity of P1. 40 for the CHRD alone versus fulllength PCSK9, the 3D structure of the CHRD/P1. 40 (PDB code 7ANQ) was superimposed on that of PCSK9 (PDB code 2P4E) with a PyMOL software (https://pymol. org/2/). This overlay highlights that a large part of the CHRD surface involved in the P1. 40 interaction is hidden in the crystal structure of PCSK9. Complex formation would thus require opening of the PCSK9 "closed conformation" to expose to solvent the surface involving P1. 40 interactions. Such movement should be possible as the two domains of PCSK9, the catalytic domain and CHRD, are connected through a 19-residue linker (Lys 421 -Asn 439 ) essential for LDLR degradation  [9]. To validate this hypothesis, size exclusion chromatography followed by Small Angle X-ray Scattering (SAXS) were performed with PCSK9 alone or in complex with P1.
40 to detect large conformational shifts in solution (Supplementary Table  3 ; Supplementary Figure  6 ). We demonstrated by size exclusion chromatography that in solution PCSK9 is monomeric and that the PCSK9/P1. 40 complex remains stable and does not dissociate (Supplementary Figure  6A ). The information deduced from the average experimental SAXS curves (Supplementary Figure  6B-D ) allowed us to estimate a molecular weight of PCSK9 alone (70. 6 kDa) and of the PCSK9/P1. 40 complex (88. 3 kDa), indicating that a single protein P1. 40 (14. 4 kDa) binds to PCSK9 (72. 5 kDa) in solution (Supplementary Figure  6E ) and that the PCSK9/ P1. 40 complex is more extended than PCSK9 alone (Supplementary Figure  6F ). To understand the conformational changes induced by P1. 40 binding to PCSK9, atomic models in "closed" and "open" conformations were generated using the MODELLER software  [47]  from the 3D structure of PCSK9 (PDB code 2P4E) and the 3D structure of PCSK9 and CHRD-P1.
40 (PDB code 2P4E þ 7ANQ). These latter models in "open" and "closed" conformations were then used to generate atomic models under the constraints of average experimental SAXS curves using the DADIMODO software  [48]. Our data show that the formation of the PCSK9/P1. 40 complex implies a significant movement of the CHRD domain relative to the catalytic domain that leads to a transition from a "closed" to an "open" conformation of PCSK9 (the best DADIMODO models in Figure  1C ). This rationalizes the inhibitory effect of P1. 40 on the PCSK9 activity on the LDLR, as P1. 40 would primarily bind the CHRD in the "open" PCSK9 conformation and interact more effectively with the CHRD alone. Indeed, the presence of two PCSK9 conformations that bind the LDLR was previously predicted from the first reported crystal structure of PCSK9  [9].
3 mg/ml of WT-PCSK9. Cell extracts and media were analyzed by Western blot. LDLR protein levels were normalized to b-actin and set to 100 for EV. Representative blots of two independent experiments are shown. Data represent means AE SEM. P values for LDLR (**, P < 0. 001) were obtained by two-way ANOVA coupled to Tukey's multiple comparisons tests. Ns: non-significant. overexpression (Figure  6D ). Furthermore, upon CAP1 overexpression and unlike WT-PCSK9 or PCSK9-3RA, the prodomain mutant PCSK9-DEE/A is inhibitable by P1. 40. While PCSK9-D33-58 is more active, the combined PCSK9-D33-58/3RA is no longer sensitive to CAP1, but is inhibitable by P1. 40, like PCSK9-D33-58 (Figure  6D ). Altogether, these results highlight the role of key N-terminal acidic residues in modulating the CAP1 stimulation of the activity of PCSK9.
ds. czbiohub. org/), while based on tissue RNA-sequencing, CAP1 mRNA levels (https://www. ncbi. nlm. nih. gov/geo/ query/acc. cgi?acc¼GSE205008) are 1. 5-fold higher in extracts of mouse liver  [63]. It was proposed that the primarily cytosolic CAP1 could somehow associate with the extracellular surface of hepatocytes by an unspecified membrane-flipping mechanism and hence interacts with extracellular PCSK9 at the cell surface  [30]. In this study, we showed for the first time that CAP1 is well secreted from hepatic cell lines (Figures  2A  and 3A ). Indeed, CAP1 was reported to circulate in the plasma of metastatic cancer  [64]  patients and in mouse models of breast cancer  [65]. We next tested whether the secretion of CAP1 from the cytosol occurs through an unconventional secretory pathway  [66]  that, like for Annexin-A2, might implicate caveoli  [67].
ACKNOWLEDGEMENTS This work was supported in part by a  CIHR Foundation  grant (NGS: #  148363 ), a  Canada Research Chairs in Precursor Proteolysis  (NGS: #  950-231335 ) and a  Leducq Foundation  grant (NGS: #  13 CVD 03 ). Sepideh Mikaeeli  was supported by an  FRQS  fellowship. We acknowledge SOLEIL for provision of synchrotron radiation facilities, and we would like to thank the staff of beamlines SWING, PROXIMA-1, and  PROXIMA-2  for assistance in setting up our experiments. The authors would also like to acknowledge the expert secretarial help of  Habiba Oueslati  in the preparation of this manuscript.
Moreover, studies have shown that it is possible to identify patients at risk of polymetastatic progression from patients who will have oligometastatic progression using molecular biomarkers  (95). Thus, one could avoid proposing RT to patients at risk of polymetastatic progression. In agreement with this concept, a study of colorectal cancer liver metastases suggested the use of a molecular classification to identify patients at risk of progression after metastasectomy  (96). This could be a relevant tool to select patients with oligometastatic tumors eligible for curative treatment, i. e. who could benefit from the combination of RT and ICIs. Finally, an ongoing study (NCT04574635) collects blood samples to assess if the DNA of HPV that causes cervical cancer can be detected in patients with CC that is new (primary), recurrent, or metastatic and are undergoing treatment with surgery, RT, CT, and/or ICI.
Active compounds and proteins Active compounds were synthesized as described in detail in the Supplementary Material 1 -Chemistry. The structure of the compounds is shown in Fig. 1. The N-terminal, 306 amino acids long, region of HSV capsid scaffolding protein (UniProt accession number: P10210) containing the serine protease domain was amplified based on a codon-optimized synthetic gene using PCR and oligonucleotides containing NcoI and KpnI restriction sites. The gene was ligated into a plasmid coding for the sequences of His 6 tag followed by a cleavage site of tobacco etch virus protease (TEV) at the N-terminal end of the VP24 protease. The expression of the protein was carried out in E. coli BL21 lysY (New England Biolabs) in the LB medium  (Amrane et al. , 2014). Initially, the culture was grown at 37 • C until OD 600 ~0.
F-actin was stained during 1-h incubation with AlexaFluor 647-conjugated phalloidin (Invitrogen) diluted at 1:50. For counterstaining of nuclear DNA. Slides were incubated with 0. 1 μg/ml 4 ′ ,6 ′diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 20 min. Preps were then mounted on glass slides using ProLong Diamond Antifade Mountant (Thermo Scientific). Confocal images were acquired under a ZEISS LSM 710 (release version 8. 1) confocal microscope using ZEN 2012 SP1 (black edition, version 8. 1. 0. 484) software. Stack acquisition parameters: frame size 2048 × 2048 pixels, pixel size 0. 13 μm. Fluorescent images were acquired using EVOS Cell Imaging System (Thermo Scientific). ImageJ FIJI version was used for image processing. For the imaging of infection inhibition by protease inhibitors, the cells were stained on 16 × 16mm glass slides with the field of view as wide as possible to see the outlines of the cells.
Acknowledgments This study was supported by the  National Science Centre, Poland  grants No. 2017/27/N/NZ6/02328  to MP and  2014/13/B/ST5/04510  to MN. RG is grateful to the  Wroclaw Centre of Biotechnology , program The  Leading National Research Centre (KNOW)  for years 2014-2018 for financial support. RG would like to thank  Michał Jewgiński  for scientific support,  Magda Wysocka  for synthesizing a peptide for HPLC analysis, and  Wictoria Łuczak  for her technical help during the realization of the project. RG and MS are thankful to  Wrocław University of Science and Technology  for its support ( Statute Funds 8211104160 ).
Materials and Methods A 15-member technical working group (TWG) was formed to develop a BRM to support the application of laboratory biological risk management and improve laboratory sustainability by providing an evidence base for biosafety measures. The TWG conducted a gap analysis for a selected list of priority pathogens on procedures related to diagnostic testing and associated research for those pathogens, including but not limited to sample processing, testing, animal models, tissue processing, necropsy, culture, storage, waste disposal, and decontamination. The TWG screened databases, websites, publications, reviews, articles, and reference libraries for relevant data to achieve this. The main research domains used to perform the literature searches were the ABSA International database, Belgian Biosafety Server, Centers for Disease Control and Prevention (CDC) reports, WHO reports, PubMed, and internet searches for terms related to biosafety matters, including, for example, inactivation, decontamination, laboratory-acquired infections, laboratory releases, and modes of transmission.
Tumor-associated Tregs upregulate surface CD74 To evaluate whether Tregs present in the tumor microenvironment exhibit specific molecular features that distinguish them from Tregs in other tissues, we used an in silico approach. We analyzed a single cell RNA sequencing (scRNAseq) dataset of CD4+ T conventional (Tconv) and Treg cells from non-small cell lung cancer (NSCLC) patients. Among the genes with a significantly higher differential expression in tumor-Tregs (tumTregs) compared to tumor-Tconvs, we observed several ones associated to activated tumTregs like CD25, HLA-DR, CTLA-4 and CCR8, and also CD74, whose biological function in Tregs has not been previously described (Fig. 1A ). We extended this analysis to blood-derived Tregs and Tconvs from the same scRNAseq dataset (Fig. 1B  and S1A ) and observed that tumTregs overexpressed CD74 transcripts as compared to blood Tregs and to blood and tumor Tconvs.
Fig. S2B  shows the compilation of CD74 KO efficacy data for all Treg donors used. We first assessed the effect of CD74 on Treg survival and proliferation. Similar in vitro cell expansion was observed for cultured WT and CD74KO Tregs along a 20-day observation period (Fig. 2C ). Upon 7 days of culture, no significant difference was observed in the protein expression of GITR, CCR8, ICOS, OX40, CTLA-4, CD25, TIGIT, 4-1BB and Foxp3, while PD1 was mildly upregulated (Fig. 2D-E, S2C-E ). As expected, expression of HLA-DR was statistically significantly reduced at the surface of CD74KO Tregs compared to their WT counterparts, consistent with CD74 being an MHC-II chaperone (13) (Fig. 2D -F, S2C, E). Finally, CD74 WT and KO Tregs exhibited a similar capacity to inhibit Tconv proliferation across the range of evaluated Treg:Tconv ratios (Fig.
Then, DAPI-stained WT or CD74KO Tregs are loaded in the chip and let 2 hours to enter the microchannel / micropillar area. Images are recorded in a 37°C-5%CO2 chamber with a frequency of one image every 3 minutes for 16 hours, using an epifluorescence Nikon TiE microscope equipped with a cooled CCD camera (HQ2, Photometrics), using a 10X (NA=0. 3) dry objective. Image analysis is performed using home-made custom macros in Fiji software. Bulk-RNA sequencing of primary Tregs 1. 10 6 WT Tregs and 1. 10 6 CD74KO Tregs are washed in PBS 14 days after electroporation. Three biological replicates of each condition are used for RNA sequencing. Total RNA is isolated using RNeasy kit following the manufacturer's protocol (NORGEn -Cat#51800). After RNA quantification and quality assessment, retrotranscription is done to obtained cDNA with ultralow input kit.
cDNA is amplified by PCR and cDNA library is sequenced with Illumina HiSeq 2500. All the 6 samples are processed at the same time and on the same pipeline for sequencing. Reads are trimmed with TrimGalore using standard settings to remove adaptaters, sequenced poly-A tails and low-quality input. The trimmed reads are mapped on the human genome with the reference hg38 using STAR (version 2. 7. 1. a). Gene expression is quantified using featureCounts from the software package Subread. Downstream analysis is performed using R (version 4. 1. 1) in which raw counts are loaded and then normalized using R package DESeq2 (version 1. 34. 0). Statistical analysis to identify the differential expression genes is performed using DESeq2. Genes are considered significantly differentially expressed according to Benjamini-Hochberg adjusted pvalue.
Violin plots and volcanoplots are created using ggplot2 (version 3. 3. 6). Heatmaps are computed with R package Pheatmap (version 1. 0. 12). Enrichment pathways analyses are performed using enrichr function from EnrichR (version 3. 0) and gseGO function from clusterProfiler (version 4. 2. 2). Enriched pathways for the two conditions are identified using the GeneOntolgy database (cellular component, molecular function, and biological process pathways).
ACKNOWNLEDGEMENTS We thank  Lea Guyonnet ,  Anna Chipont ,  Annick Viguier , and  Coralie Guerin  from the  Curiecoretech Cytometry  platform at  Institut Curie ; and  Virginie Reynal ,  Benoit Albaud ,  Laura Baudrin , and  Patricia Legoix  at the  Curiecoretech Next Generation Sequencing platform at Institut Curie. We acknowledge all the U932 members for the helpful discussion,  Yago A. Arribas  and  Blandine Baudon  for the technical support; as well as  Egle-Tx  employees,  Mercedes Tkach  and  Irena Chacon  for experiment advice. In addition, we thank the  AVIESAN -IFB Bioinformatic school  for the training to analyze Bulk RNA-seq data,  Adrien Pain  for the tutorial. This work has received the support of the  LabEx DCBIOL  ( ANR-10-IDEX-0001-02 PSL ;  ANR-11-LABX-0043 );  INCa-DGOS-Inserm_12554 , and  Center of Clinical Investigation  ( CIC IGR-Curie 1428 ) and Egle-therapeutics.
1: CD74 is overexpressed in Tregs from different tumor types compared to Tconvs and CD8+ cells. A: Single-cell RNA sequencing (scRNAseq) of CD4+ T cells from blood of 5 non-small cell lung cancer (NSCLC) patients (from Tosello, Richer et al. , under review). Volcano plot (P-value versus fold change) of the gene expression profile of Tregs and Tconvs. Genes highlighted in blue are overexpressed by peripheral Tregs and highlighted in green by peripheral Tconvs. B: Bulk RNA sequencing (bulk-RNAseq) of CD4+ T cells from tumor (5 colorectal carcinoma (CRC) patients from De Simone et al. dataset, and 2 luminal-breast cancer (TNBC) patients from Nunez, Tosello et al. dataset. Violin plot of CD74 normalized expression in Tregs versus Tconvs from tumor samples of CRC patients (left) and TNBC patients (right).
An integration assay performed on naked 601 DNA also showed that MN was a preferential substrate and that integration sites detected on MN were specific to this structure. All these data fully confirmed that functional intasomes and MN nucleocomplexes could be assembled using differently tagged partners, allowing their use in alphaLISA and integration assays. The PFV intasome was assembled as described in Materials and Methods section and subsequently purified by size exclusion chromatography (A). Protein and DNA elution was monitored by 280 nm and 254 nm absorbance respectively. MN was assembled by salt dialysis protocol using 5' biotinylated 601 Widom sequence and human octamers. The structure of the nucleosome was checked by native 5 % polyacrylamide gel (B). The functionality of the intasome complex was checked by in vitro concerted integration assay performed on the assembled MN using 0-710 nM of intasome and 100ng of MN for 30mn.
The AlphaLISA interaction signal (AU) was monitored using increasing concentrations of each partners and data are reported in (B) as a representative experiment. Comparison of the interaction signal obtained with both partners (1 nM), each partner or beads alone is reported in (C) as mean of three independent experiments ±SD. Competition experiment performed with increasing concentration of untagged intasome is reported in (D) as mean of three independent experiments ±SD. Taken together, these data indicate that the functional intasome/MN complex can be monitored using the alphaLISA approach, leading to a robust interaction signal even when using low amounts of each partner. This allowed us to further use this system for better characterization of the complex and validated the feasibility of drug screening assays.
Based on these data, both selected drugs were assayed for HIV-1 infection in primary PBMCs. Infection assays performed with the clinical B subtype HIV-1 strain in PBMCs treated with doxorubicin showed a strong inhibition of replication by the drug, leading to an IC50 of 2-20 nM (Figure  9A ). The CA3 compound was also found to decrease viral replication with an IC50 of 1-2 µM (Figure  9B ). qPCR quantification of the total DNA at 20 h postinfection showed no effect of the Doxotubicin, indicating that the drug does not affect the reverse transcription or the entry step (Figure  9C ). In contrast, integrated DNA was found to be decreased in doxorubicin-treated cells, while two-LTR circle amounts were increased (Figure  9C ). These data confirmed that doxorubicin inhibited the postnuclear integration steps.