dataesr/scientific-paragraphs-categorization huggingface dataset:
These complex host-microbiota interactions are still the subject of intense investigations and are very likely specific for each organ as suggested by the important disparities between each microbiome and mucosal surface (skin, gut, oral, vaginal). According to the antimicrobial peptide database (http://aps. unms. edu), the squamous epithelial cells (keratinocytes) lining the lower part of the gynecologic tract secrete a dozen of innate (antimicrobial) peptides  29, 30. By laser capture microdissection, 44 independent frozen human tissue specimens (11 normal squamous epithelia, 10 LSIL, 10 HSIL, and 13 SCC) were sampled. In order to avoid bias related to HPV status, all (pre)neoplastic lesions displayed diffuse (basal or full-thickness) p16 INK4a immunoreactivity in keeping with carcinogenic HPV infection.
The other analyzed peptides were down-regulated at a lower extent. HPV impairs TNFα/LPS-induced innate peptide expression through E7-dependent NEMO degradation and subsequent suppression of NF-κB activation. Proinflammatory factors such as TNFα, IL-1β and LPS have been previously shown to induce the expression of elafin as well as several members of the defensin family  [31] [32] [33]. Given these results and the identification of putative NF-κB/p65 binding sites within the promoters of DEFB2-4, DEFA5-6 and PI3 (elafin) genes [estimations made using the Eukaryotic promoter database 34 ], we first evaluated the requirement of NF-κB signaling pathway activation in TNFα/LPSinduced innate peptide expression. As expected, knockdown of p65 with siRNA or indirectly via blockade of the degradation of IκBα (BAY 11-7082) resulted in a significant decrease of defensin/ elafin mRNA levels in normal/uninfected keratinocytes (Fig.
ns: not significant (p > 0. 1). Source data are provided as a Source Data file. each individual viral oncoprotein on c-myc expression, its mRNA level was investigated in HPV16 E6 or E7-transduced keratinocytes. Compared to control cells, a significant increase in c-myc transcripts was only detected in HPV16 E7-positive cells (Fig. 4c ) and the requirement of β-catenin in this up-regulation was highlighted (Fig. 4d ). Confirming these data, an increased nuclear c-myc immunoreactivity was clearly observed in HPV18-positive organotypic raft cultures, E7-transduced keratinocytes as well as in HPVpositive (pre)neoplastic lesions compared to respective controls (Fig. 4e, f, i ). Exclusively detected at the cell membrane in uninfected cells/tissues, an intense cytoplasmic/nuclear β-catenin immunoreactivity was reported in case of HPV infection/E7 transduction (Fig.
P values were determined using two-sided unpaired t-tests a, b, one-way ANOVA followed by Dunnett's multiple comparison post-hoc test c, i, Fisher's exact test h and one-way ANOVA followed by Bonferroni post-hoc test k. ns: not significant (p > 0. 1). The Western blot and co-IP analyses were independently performed three times and twice, respectively. One representative experiment is shown. Source data are provided as a Source Data file. directly or needs some latency, elafin and S100A7 were added to lactic acid bacteria and several time points were investigated. Importantly, for all Lactobacillus species, a significant increased percentage of surviving colonies was only detected after 3 or 6 h of incubation (Fig. 6a ). As commonly practiced for both eliminating variables and focusing on the compounds of interest, no nutrient (serum) was added during the incubation (starvation assay), explaining why the number of surviving colonies was decreasing over the time.
One representative experiment is shown. Source data are provided as a Source Data file. group clustering and clearly illustrated by the β-diversity plot (Fig. 7e ), the microbial profiles detected in HSIL-positive K14-HPV16 mice were significantly distinct to those from hyperplasia/ LSIL-positive (p = 0. 002) or control mice (p = 0. 0005). In addition, the homogeneity in the cervico-vaginal microflora tended to be different in mice diagnosed with a precancerous lesion compared to the control group, as indicated by HOMOVA testing (HSIL versus control, p = 0. 09; hyperplasia/LSIL versus control, p = 0. 08). The distribution of the twelve main bacterial orders and their relative abundance in the four groups of mice are shown in Fig. 7f. Accounting for over 90% of the total microbial community in the vast majority of samples (44/48, 91.
P values were determined using one-way ANOVA followed by Dunnett's multiple comparison post-hoc test (c, e). ns: not significant (p > 0. 1). Source data are provided as a Source Data file. their antimicrobial activity. For example, defensin(-like) peptides (e. g. , HβD2, HD-5, and S100A7) have a well-documented chemotactic capacity for lymphocytes, monocytes and dendritic cells and their reduced secretion would greatly help the virus to escape the host immunity  [54] [55] [56]. Further promoting a tolerogenic environment, the lack of HβDs would also disrupt the activation and maturation of antigen-presenting cells  57. Moreover, some defensins (most notably HD-5) have been demonstrated to block HPV infection via various mechanisms  58, 59. Therefore, the low HD-5 expression detected in infected epithelial cells could be particularly beneficial for the virus during the productive replication stage.
g. , ASC-US, ASC-H); lack of data related to vaginal bacterial flora; immunosuppressive Fig. 6  Host defense peptides are metabolized and used as amino acid source by Lactobacillus species. a Elafin and S100A7 were incubated with Lactobacillus species for 45 min to 9 h in the absence of nutrients (starvation assay) and the percentage of surviving colonies was determined. Results (each time point) represent the means ± SEM of six (L. iners: CTRL, elafin and S100A7), eight (L. crispatus/L. jensenii: elafin and S100A7) or eleven (L. crispatus/L. jensenii: CTRL) independent experiments. b The hydrolysis of host defense peptides (elafin and S100A7) by the dominant Lactobacillus species constituting the vaginal microbiome was evaluated at different time points (0 h, 3 h and 6 h) by mass spectrometry.
ns: not significant (p > 0. 1). Source data are provided as a Source Data file. conditions. In total, six thousand one hundred seventeen women [mean age: 48 ± 13 years (range, 21-84)] were selected. Thirty-two patients (32/6,117, 0. 52%) were excluded for missing data (incomplete viral-cytological information) during the follow-up period. Mean number of visits was 3 (range, 2-13). All Pap smear samples were analyzed by experienced cytopathologists according to the Bethesda system for reporting cervical cytology. HPV testing from residual liquid-based cytology specimens was performed using the Abbott RealTime High-Risk HPV assay (Abbott, Wiesbaden, Germany) which enables simultaneous detection of all WHO/IARC-classified carcinogenic (high-risk) HPV types (HPV16, 18, 31, 33, 35,  39, 45, 51, 52, 56, 58, 59, 66, and 68).
Overall, the STROBE (STrengthening the Reporting of OBservational studies in Epidemiology) guidelines has been followed as accurately as possible. The data collection from personal health records was approved by the Ethics Committee of the University Hospital of Liege (#2021-117). Human tissue samples, classification and laser capture microdissection. A total of 44 frozen and 127 paraffin-embedded cervical/vulvar specimens were retrieved from the Tissue Biobank of the University Hospital Center of Liege (Belgium). All patients underwent a surgical procedure between 2011 and 2015. An informed consent was obtained from all participants and the protocol was approved by the local ethics committee. These tissue samples included 31 normal squamous tissues (ectocervix/TZ), 65 dysplastic lesions (30 LSIL and 35 HSIL) and 75 primary invasive squamous cell carcinomas (SCC) (33 cervical and 42 vulvar cancers).
The Quant-iT PicoGreen dsDNA Kit (Invitrogen) and the KAPA Library Quantification Kit for Illumina platforms (KapaBiosystems, Wilmington, MA, USA) were used to normalize the DNA libraries and to determine the pooled library concentration, respectively. Pair-end sequencing (2 × 300 cycles) was done on the Illumina MiSeq platform. As previously described  79, 80  , both the alignment and taxonomical assignment (from phylum to species) of post-sequencing data were performed using the MOTHUR software package (v141. 1), based on the SILVA database (v1. 38) of full-length 16S rDNA sequences. Potentially chimeric sequences were detected using the VSEARCH algorithm. For each sample, a subsampling dataset containing 10,000 representative cleaned reads was retained and used to generate OTUs (cut off: 0.
Bacterial suspensions were then plated on Columbia Blood agar plates for 24-48 h to determine CFU. High-resolution images for each condition were taken. Colony detection and counts were finally performed using ImageJ software (NIH, Bethesda, MD, USA) and verified by manual counting. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The different innate peptides were incubated with or without DTT (2 mM, pH 7. 4) for 30 min at 37 °C and then alkylated with 20 mM iodoacetamide for 30 min at 25 °C. The peptides were deposited on MALDI plate with α-cyano-4-hydroxy cinnamic acid (HCCA) saturated solution for co-crystallization (equal volume). The spectra were acquired with a rapifleX TOF/TOF instrument (Bruker, Bremen, Germany) in linear positive mode. Flow cytometry. Cells were scraped and washed twice with PBS before staining reaction (30 min at 4 °C).
After washing/centrifugation, cells were fixed with 1% paraformaldehyde. Flow cytometry analyses were performed with a FACSCanto II flow cytometer and data (mean fluorescence intensity and positive cell percentages) were acquired using FACSDiva software (version 8. 0. 1, BD Biosciences, San Jose, CA, USA). Results were analyzed with FlowJo (version 10. 5. 3, TreeStar, Ashland, OR, USA). The following antibodies were used: anti-CD14 Phycoerythrin (PE) (1/ 33, clone TUK4, Dako, Glostrup, Denmark), anti-TLR4 PE (1/33, FAB6248P, R&D systems), anti-TNFR1 PE (1/33, clone W15099A, Biolegend, San Diego, CA, USA) and anti-TNFR2 PE (1/33, clone 3G7A02, Biolegend). The gating strategy is detailed in Supplementary Fig. 14. To determine the incorporation of selected peptides (or their hydrolyzed products) within the cytoplasm of several bacterial species, free amine groups (lysine residues and N-terminus) were first conjugated with Atto 488 dye according to manufacturer's recommendations (Atto 488 protein labeling kit, Sigma Aldrich).
Other operational conditions as well as data-dependent acquisition settings were previously described  85. Datafiles were opened using MassHunter Qualitative Analysis software (version 7. 0, Agilent Technologies) and individual extracted ion chromatograms were generated at the MS level for each m/z value of interest. To precisely verify whether the innate peptides highly secreted by the vaginal/ cervical mucosa can be used by the predominant Lactobacillus species as amino acid source, two elafin fragments (H-AQEPVK*GPVSTK*PGSCPII-OH and H-PGIK*K*CCEGSCGMACFVPQ-OH), each containing two 13 C 6  15  N 2 -labeled lysines, were synthesized (Pepscan, Lelystad, The Netherlands). Five µg/ml of each peptide were incubated with 6 × 10 8 CFU/ml for 6 h at 37 °C in 10 mM SPB. Following two steps of washing, bacterial cells were suspended in 20 µl of Tris-HCl (50 mM, pH 8) containing urea (8 M) and then sonicated for 15 min using a Bioruptor Plus (Diagenode, Seraing, Belgium).
A 2 m/z isolation window was used, the maximum injection time was 50 ms and a normalized collision energy of 28 was utilized for the activation of ions. Data analysis/protein identification was performed using SEQUEST HT search engine and Proteome Discoverer 2. 1 software (Thermo Scientific). Databases were downloaded from Uniprot for each bacterial strain of interest (L. jensenii: 3763 sequences; L. crispatus: 5120 sequences; L. iners: 1872 sequences). The parameters for the search were as follows: maximum 2 miss-cleavages and cysteine carbamidomethylation as fixed modification and deamidation (asparagine, glutamine), oxidation (methionine) and label 13 C 6  15  N 2 of Lysine as variable modifications. The tolerances of precursor and fragment mass were 5 ppm and 0. 02 Da, respectively. Percolator node was used to select peptides with a Target FDR set at maximum 1%.
In order to determine the stability of a given protein, monolayer cultures were pretreated with 100 µM cycloheximide (Sigma Aldrich). Where indicated, DNA methyltransferases were inhibited with 5 µM 5-aza-deoxycytidine (Sigma Aldrich). To analyze the CK1dependent phosphorylation of β-catenin (ser45), cells were treated with 50 nM calyculin A (Sigma Aldrich) for 30 min before protein extraction. Immunohistochemistry, immunofluorescence and in situ hybridization. Immunohistochemical/fluorescence analyses were performed using standard protocols previously described  86, 87  and included the following primary antibodies: anti-SLPI (1/400, NBP1-76803, Novus Biologicals, Centennial, CO, USA), anti-S100A7 (1/500, HPA006997, Atlas Antibodies, Bromma, Sweden), anti-elafin (1/ 150, clone FL-117, Santa Cruz Biotechnology, Dallas, TX, USA), anti-LL37/CAMP (1/1000, HPA029874, Atlas Antibodies), anti-HβD1 (1/50, PA5-51286, Thermo Fisher Scientific), anti-HβD2 (1/100, ab63982, Abcam, Cambridge, MA, USA), anti-HβD3 (1/300, NB200-117, Novus Biologicals), anti-HβD4 (1/100, clone L13-10-D1, Abcam), anti-HD-5 (1/200, HPA015775, Atlas Antibodies), anti-HD-6 (1/ 50, NBP1-84281, Novus Biologicals), anti-p65 (1/500, clone D14E12, Cell Signaling Technology, Danvers, MA, USA), anti-E-cadherin (ready to use, clone 36, Ventana Medical Systems, Tucson, AZ, USA), anti-β-catenin (1/100, clone E247, Abcam), anti-c-Myc (1/100, ab32072, Abcam), anti-p16  INK4a  (1/300, clone JC8; Santa Cruz Biotechnology) and anti-ki67 (ready to use, clone 30-9; Ventana Medical Systems).
Regarding p65, the percentage (<10% versus > 10%) of cells exhibiting a nuclear immunoractivity was determined. Bisulfite genomic sequencing. Freshly extracted genomic DNA (500 ng) was processed using Bisulfite Conversion kit (Active Motif, La Hulpe, Belgium) according to supplier's recommendations. Conversed CDH1 (E-cadherin) promoter was amplified using the specific primers listed in Supplementary Table  1. These latter were designed with the Methprimer software (https://www. urogene. org/methprimer/). PCR products were cloned into pJET1. 2 vector using CloneJET PCR cloning kit (Thermo Fisher Scientific) and then ligation products were transformed into competent JM109 bacteria using heat shock. Resistant colonies were isolated and recombinant plasmids were purified (NucleoSpin Plasmid kit, Macherey Nagel).
Finally, at least 6 independent plasmids for each condition were sequenced using Sanger method. RT-qPCR. Total RNA was extracted and purified using NucleoSpin RNA isolation kit (Macherey Nagel) according to manufacturer's instructions. The ReliaPrep RNA Miniprep system (Promega) was used for both human and mouse microdissected samples. One microgram was reverse transcribed using RevertAid Reverse Transcriptase and oligo dT primers (Thermo Scientific). Quantitative PCR was performed using the FastStart Universal SYBR Green Master mix (Roche, Basel, Switzerland). Both human and mouse primer sequences used for PCR analysis are listed in Supplementary Table  1. The public database PrimerBank was used for the retrieval of most of these sequences  89. The experiments were performed in triplicate and threshold cycle numbers (Ct) were determined using the ABI-Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA).
The protein bands were detected using an enhanced chemiluminescence system (Westar ECL-Sun, Cyanagen, Bologna, Italy) and finally quantified by densitometry using ImageJ software. Actin, HSC-70, or NBS-1 was used for the normalization. For uncropped blots, see the Source Data file. Gaussia princeps luciferase complementation assay (GPCA). The coding (or mutated/truncated) sequences of HPV E7 from several alpha and beta genotypes (alpha: HPV16, 18, 33, and 39; beta: HPV8, 38, and 49) were transferred into Gatewaycompatible GPCA destination vector pSPICA-N2. The open reading frames (ORFs) encoding proteins involved in IKK or β-catenin destruction complex were obtained from the human ORFeome v7. 1 and 8. 1 (Dana-Farber Cancer Institute, Boston, MA, USA) and were transferred into pSPICA-N1 plasmid.
After washing and reversal of crosslinking, immunoprecipitated DNA was purified (phenol/chloroform extraction) and subjected to qPCR using PI3/elafin and HβD2 promoter NF-kB binding site specific primers. Sequences targeting a more distal region (-2 kb) on both promoters were used as negative control (Supplementary Table  1 ). Statistical analysis. Statistical analysis was performed using the GraphPad Prism 8 software (San Diego, CA, USA). For normally distributed variables, two group comparisons were performed using unpaired t-tests. One-way or two-way ANOVA was applied to determine the statistical significance of differences between more than two groups/conditions. Dunnett's or Bonferroni post-hoc test was used for multiple comparisons. In the case of discrete (staining scores for assessing innate peptide expression) or non-normally distributed (bacterial population abundances) variables, a Mann-Whitney or Kruskal-Wallis test was performed according to the number of groups.
The comparison of phenotypic/immunohistochemical variables (0-5%, 6-33%, 34-66%, and >67% of cells displaying a nuclear c-myc/p65 immunoreactivity or membrane versus cytoplasmic/nuclear β-catenin localization) between independent groups was performed using a Fisher's exact test or a χ 2 test according to the number of variables. For the different microbial population ecological indices (α-diversity, richness, evenness), the differences between defined groups were evaluated using a non-parametric Kruskal-Wallis test followed by a two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli. Using the MOTHUR software, sample clustering and beta-dispersion were assessed in a Bray-Curtis dissimilarity matrix with AMOVA (analysis of molecular variance) and HOMOVA (homogeneity of molecular variance) tests, respectively.
Acknowledgements We thank the  Biobank of the University of Liege , the  Laboratory of Clinical Microbiology  as well as the  GIGA-Immunohistochemistry , in vitro imaging, genomics and viral vectors facilities ( University of Liege ) for their assistance. We are also grateful to  Dr Stephanie Gofflot ,  Dr Wouter Coppieters ,  Dr Emmanuel Di Valentin ,  Manon Deckers ,  Latifa Karim ,  Cecile Meex ,  Raphael Thonon ,  Kamilia El Kandoussi , and  Nancy Rosiere  for their technical assistance. We sincerely thank  Prof Bernard Joris  and  Dr Ana Amoroso (Laboratory of Bacterial Physiology and Genetics, University of Liege) ,  Prof Christine Jacobs-Wagner  ( Microbial Sciences Institute, Yale University ) as well as  Dr Olivier Peulen  ( Metastasis Research Laboratory, University of Liege ) for helpful discussions.
Data availability Raw amplicon sequencing libraries have been deposited in the NCBI Sequence Read Archive (SRA) repository with the accession code: PRJNA670165. Both the alignment and taxonomical assignment (from phylum to species) of post-sequencing data were performed, based on the SILVA database (v1. 38). The proteomics data have been deposited at the ProteomeXchange Consortium via the PRIDE partner repository with the following dataset identifier: PXD022113. Source data for all Figures are provided with this paper. Source data are provided with this paper.
For each stage a single section is presented in phase, Gal-3, and CellMask (CM) channels, as well as an inset (yellow frame) containing a composite view of the Gal-3 (green) and CM (magenta) channels. Cell death (if it occurred) is marked with a red 'x' in the corresponding invasion stage. The distribution of cell death timing as a function of the invasion stage is presented for each scenario. ND stands for no cell death during invasion. Scale bars are 10 µm or 5 µm in insets. Source data are provided as a Source Data file. 1 )  11, [14] [15] [16] 30, 32. While LDH release was not detected at 6 h post infection using a MOI of 0. 1 (used in our live cell imaging pipeline), it was detected using a MOI of 1 at this time point. This difference is likely due to lower sensitivity of the LDH release assay in detecting cell damage compared to its detection via Gal-3 at the single cell level.
We therefore studied invasion using serial block face-scanning electron microscopy (SBF-SEM), an emerging "volume" EM technique capable of imaging entire C. albicans filaments invading multiple host cells within a single 3D dataset at nano-scale resolution  56, 57  (see Fig. 4 ). As invasion into Caco-2 occurs via a single non-damaging invasion scenario, we targeted this cell line for SBF-SEM. Caco-2 were infected by WT C. albicans for 6 h, followed by EM sample preparation, which resulted in excellent sample contrast for both C. albicans internal structures like the Spitzenkörper and for membranes and organelles of the host  58  (see Materials and Methods). Host cells presented clear microvilli at their apical surface, indicating monolayer polarization. Datasets were acquired in three different sessions, with a resolution of 10 nm in the acquisition plane and an axial resolution of 100 nm.
Overall, 11 volumes were acquired containing 11 invasion sites in which the entire or nearly the entire invading hypha and the sequence of invaded host cells were captured, and an additional 30 invasion sites in which only a partial segment of an invading hypha was captured. A representative dataset containing a C. albicans filament invading three host cells in succession is presented in detail (see Fig. 4A , Supplementary Movie 10). A quantification of the features found in the 11 volumes containing complete or nearly complete invasion sites can be found in Supplementary Table  2. Invading filaments are enveloped by host membranes in a tunnel structure progressively remodelled with each invaded cell. In all invasion events examined, invading hyphae were fully enveloped by host membranes on all sides (except at contact points with glycogen stores (see below)) so that hyphae were always found within a continuous tunnel structure that could extend through successively invaded host cells.
This opening was found towards the posterior side (away from the hypha tip) of the inflation in all three cases (see Fig. 4D , Supplementary Movie 13, Supplementary Table  2 ). In the dataset presented here, a portion of the tunnel inflation was found outside of the host cell, though it was directly linked through a narrow passage to the intracellular inflation. The contact points between tunnel inflations and host glycogen stores may represent points of nutrient uptake from the host by C. albicans (see Discussion). Membrane layer accumulation around invading hyphae corresponding to the invasion sequence is a hallmark of invasion into Caco-2. Systematic quantification of the organization of the layers surrounding C. albicans hyphae in relation to the invasion sequence showed that in every invasion site analysed by SBF-SEM (n = 11), the same pattern was reproduced (see Supplementary Table  2 ): a thin membrane and an exclusion layer in cell 1, a thick membrane in cell 2 and two thick membranes in cell 3.