Bacterial strains and tradition situations
P. anaerobius pressure VPI 4330 (catalogue quantity 27337) and E. coli pressure MG1655 (catalogue quantity 700926) have been bought from the American Sort Tradition Assortment (ATCC). P. anaerobius was cultured in Wilkins–Chalgren anaerobe broth (Thermo Fisher Scientific, CM0643) at 37 °C underneath anaerobic situations (80% N2, 10% H2 and 10% CO2). E. coli pressure MG1655 was cultured in a single day at 37 °C in Luria–Bertani medium with shaking (220 rpm min−1).
Mouse fashions
The Animal Experimentation Ethics Committee of the Chinese language College of Hong Kong authorized the entire animal experiments performed on this examine. Mice have been saved in particular pathogen-free services, following a cycle of 12 h gentle and 12 h darkish. Meals and water have been offered advert libitum. No statistical strategies have been used to pre-determine pattern dimension however our pattern sizes are just like these in earlier experiences7. The mice have been randomly assigned to completely different teams and no animals have been excluded from the analyses. Information assortment and evaluation weren’t performed in a blinded method.
For the Apcmin/+ mice mannequin, male C57BL/6 Apcmin/+ mice (six weeks outdated) have been administered an antibiotic cocktail of their consuming water (0.2 g l−1 ampicillin, neomycin and metronidazole, and 0.1 g l−1 vancomycin) for 2 weeks to deplete their intestine microbiota. One week after the antibiotic remedy, the mice have been orally administrated with 1 × 108 c.f.u. P. anaerobius, E. coli MG1655 or PBS each day for seven weeks.
For the AOM-induced CRC mouse mannequin, male typical C57BL/6 wild-type mice have been subjected to intraperitoneal injections of AOM (10 mg kg−1) as soon as per week for six weeks, adopted by the identical remedy because the Apcmin/+ mice mannequin for twenty-four weeks. For integrin α2β1 blockade, RGDS peptide (200 μg per mouse) was given to 2 further teams of mice (one group handled with PBS and the opposite handled with P. anaerobius) 3 times per week by intraperitoneal injection for twenty-four weeks.
To review the tumour-promoting impact of P. anaerobius in an AOM and DSS-induced CRC mannequin, male typical C57BL/6 wild-type mice have been injected with AOM (10 mg kg−1) on day 0. After 7 d, the mice have been supplied with consuming water containing 1% DSS (MPBio) for 7 d, adopted by consuming water for 14 d after which uncovered to 2 extra remedy cycles with 1% (wt/vol) DSS. On days 14, 35 and 56, we administered P. anaerobius (1 × 108 c.f.u.), E. coli (1 × 108 c.f.u.) or PBS to the mice each day for 2 weeks after suspending DSS remedy.
To review the impact of P. anaerobius on anti-PD1 remedy utilizing an AOM and DSS-induced CRC mannequin, male typical C57BL/6 wild-type mice have been injected with AOM (10 mg kg−1) on days 0 and 19. The animals got 1% DSS solubilized in water 3 instances for five d every time, beginning on days 7, 19 and 38, adopted by common water for 7 d. After consuming an antibiotic cocktail for 2 weeks, the mice got P. anaerobius (1 × 108 c.f.u.) or PBS orally each day till the tip of the experiment. Monoclonal anti-mPD1 (100 μg) or an isotype management monoclonal antibody was administered by intraperitoneal injection twice per week for a complete of three weeks after starting P. anaerobius remedy for 10 d.
For the allograft experiments, MC38 cells (5 × 105) have been subcutaneously injected into the precise flank of male C57BL/6 mice. After 6 d, P. anaerobius(1 × 107 c.f.u. per 50 μl), an equal quantity of PBS and RGDS peptide (200 μg per 50 μl) have been administered by multipoint intratumoral injection each 3 d till the tip of the experiments. The mice have been intraperitoneally injected with isotype management or anti-mouse PD1 (BioXcell) twice per week (100 μg per injection) after the primary bacterial remedy. For the MDSC depletion, mice have been handled with anti-mouse Ly6G or management rat IgG (250 μg per injection; BioXcell) by way of intraperitoneal injections each 48 h for 14 d after the primary bacterial remedy. To validate the impact of recombinant lytC_22 protein in vivo, lytC_22 (25 µg protein in 50 µl PBS) was given by multipoint intratumoral injection each 3 d as soon as tumours had turn out to be measurable. For Slamf4 receptor blockade, the mice have been intravenously injected, by way of the tail vein, 3 times per week (complete of six doses) with anti-CD244 (eBioscience) or isotype antibody (each at a dose of fifty µg antibody in 100 µl PBS) beginning after the primary lytC_22 remedy.
P. anaerobius quantification in fecal and mucosal samples from naive and AOM and DSS-induced CRC mice
Six-week-old male typical C57BL/6 wild-type mice have been divided into two teams. One group was injected with AOM (10 mg kg−1) on days 0 and 19, and 1% DSS solubilized in water was given to the animals 3 times for five d every time, beginning on days 7, 19 and 38, adopted by common water for 7 d. The mice within the different group have been fed in parallel underneath regular situations. After tumour formation had occurred, the mice have been orally gavaged with P. anaerobius (1 × 108 c.f.u.) each day for 2 weeks. The mice have been sacrificed at particular time factors (days 0–5 and seven) following P. anaerobius gavage to acquire stool samples and colon tissue. Stool DNA was extracted from collected samples utilizing a Fast-DNA fecal/soil microbe miniprep equipment (Zymo Analysis). Amplification and detection of P. anaerobius DNA within the stool samples have been carried out utilizing a Common SYBR Inexperienced Grasp response on the QuantStudio 7 flex system (Thermo Fisher Scientific). Fluorescence in situ hybridization was employed to evaluate the abundance of P. anaerobius in colon tissue samples.
Cell tradition
The colon most cancers cell strains HCT116, Caco-2 and MC38 have been obtained from the ATCC. The traditional colon epithelial cell line NCM460 was obtained from INCELL. The cells have been cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U penicillin and 100 µg ml−1 streptomycin. The cells have been cultured at 37 °C in a cell tradition incubator with 5% CO2. The bacterial co-culture assay was carried out in response to beforehand reported protocols7. For the inhibitor-treatment experiments, cells have been pretreated with 30 μM NF-κB inhibitor JSH-23 (MCE) or 100 μM integrin α2β1 inhibitor RGDS peptide (MCE) for twenty-four h after which handled with P. anaerobius.
Conditional tradition medium preparation
Most cancers cells or clean medium have been incubated with P. anaerobius for 4 h underneath anaerobic situations. The medium containing micro organism was then changed with DMEM medium supplemented with 10% FBS, 1% penicillin–streptomycin and 200 mg ml−1 gentamycin for two h, which was used to kill all of the extracellular micro organism. The medium containing the killed micro organism was changed with full DMEM medium. After culturing for twenty-four h, the tradition medium was collected, necrotic cell particles was eliminated by centrifugation at 2,000g for 10 min and conditioned supernatant was obtained by filtration by means of a 0.45 μm membrane filter (Millipore).
CXCL1 detection
CXCL1 protein in most cancers cell tradition medium and serum was detected utilizing ELISA assay kits (Abcam) in response to the producer’s directions. To find out the extent of CXCL1 in tumour tissue, complete protein was remoted from tumour tissue utilizing Cytobuster (Novagen) and complete 50 μg protein was used to detect CXCL1 utilizing an ELISA equipment (Abcam).
Integrin α2β1 and CXCL1 knockdown
Cells have been transfected with siRNA focusing on integrin α2β1, CXCL1 or CXCL3, or non-targeting RNA utilizing Lipofectamine 2000 (Thermo Fisher Scientific) in response to the producer’s directions. Integrin α2β1 have been transiently silenced utilizing a pool of two completely different siRNAs—that ism siRNA to ITGA2 (Ambion, S7537) and ITGB1 (GenePharma). CXCL1 and CXCL3 have been silenced by designed siRNA (Ambion). A silencer destructive transcription management was launched into every experiment. Following 6 h of transfection, the medium was changed with full DMEM medium supplemented with serum and antibiotic. CXCL1, CXCL3 and integrin α2β1 knockdown was validated by ELISA and western blotting.
ScRNA-seq
Tumours remoted from AOM and DSS-induced mice have been minced and digested in RPMI1640 medium containing 1 mg ml−1 collagenase kind IV (Roche) and 0.5 mg ml−1 DNase Ι for 20 min at 37 °C with mild rocking. The cell suspension was filtered by means of a 70 μm cell strainer. Single cells have been stained with BV421-labelled anti-CD45.2 (BioLegend) and 7AAD viability staining resolution (BioLegend) for 30 min at 4 °C, washed and resuspended in PBS (2% FBS) buffer for stream cytometric sorting utilizing a FACS Aria II cell sorter (BD Biosciences). Sorted CD45+7AAD− cells have been diluted with trypan blue and counted utilizing a haemocytometer. Tumours from three completely different mice have been pooled per pattern. Single-cell capturing for scRNA-seq and library preparation have been accomplished by the SBS core laboratories in CUHK. Briefly, a single-cell suspension of 500–1,000 cells µl−1 in FACS buffer was added to a real-time PCR grasp combine to pattern as much as 8,000 cells; these have been then mixed with Single Cell 3′ gel beads and dispense oil onto a single cell 3′ chip (10x Genomics). RNA transcripts from particular person cells have been uniquely barcoded and reverse transcribed in droplets. Complementary DNA molecules have been pre-amplified and pooled, adopted by library building. All libraries have been quantified utilizing a Qubit fluorometer and real-time PCR was carried out on a LightCycler 96 system (Roche Life Science). The scale profiles of pre-amplified cDNA and sequencing libraries have been examined utilizing the Agilent excessive sensitivity D5000 and D1000 ScreenTape techniques, respectively. All single-cell libraries have been sequenced on a NextSeq 500 system (Illumina) utilizing a NextSeq 500 excessive output v2 equipment (Illumina).
Evaluation of scRNA-seq information
Uncooked information (uncooked reads) of FASTQ information have been assembled from the Uncooked BCL information utilizing Illumina’s bcl2fastq converter. Pre-processing of the sequencing outcomes have been performed utilizing the 10x Genomics Cell Ranger pipeline (v6.0.0) with default settings and evaluation was carried out in R. The dataset was filtered to exclude low-quality cells utilizing the next standards: (1) <5% or >95% distinctive molecular identifier or gene depend, or (2) proportion of mitochondrial genes > 20%. All the downstream analyses have been based mostly on the clear information with top quality. All downstream single-cell analyses have been then carried out utilizing Cell Ranger and Seurat33,34, until talked about in any other case. Briefly, for every gene and every cell barcode (filtered by Cell Ranger), distinctive molecule identifiers have been counted to assemble digital expression matrices. Secondary filtration carried out utilizing Seurat, the place a gene was deemed to be expressed if it confirmed expression in additional than three cells. Every cell was mandated to have a minimal of 200 expressed genes. Cells expressing fewer than 200 genes or multi-cells have been excluded from futher evaluation. Cell Ranger depend takes FASTQ information and performs alignment, filtering, barcode counting and distinctive molecular identifier counting. It makes use of the Chromium mobile barcodes to generate characteristic barcode matrices by Cell Ranger depend or Cell Ranger aggr and reruns the dimensionality discount, clustering and gene expression algorithms utilizing Cell Ranger default parameter settings.
Blocking of Slamf4 on MDSCs
For blocking of lytC_22–Slamf4 interplay, main MDSCs have been pre-incubated with purified monoclonal anti-mouse Slamf4 (Invitrogen, eBio244F4) at 20 μg ml−1 for 1 h on ice earlier than the addition of lytC_22 protein.
Human chemokine array
Chemokines have been quantified utilizing a human chemokine array equipment (Abcam, ab169812) in response to the producer’s directions. Briefly, array membranes have been blocked in blocking buffer at room temperature for 1 h after which incubated in a single day at 4 °C with 1.5 ml of conditioned tradition medium from Caco-2 cells pre-incubated with or with out P. anaerobius. The subsequent day, the membranes have been washed and incubated in a single day with 1 ml main biotin-conjugated antibody combination at 4 °C. After washing, the membranes have been incubated with 2 ml horseradish peroxidase-conjugated streptavidin at room temperature for two h. The membrane spots have been uncovered to ECL plus western blotting detection reagents. Optimistic controls on every membrane have been used to normalize spot intensities throughout completely different teams.
Isolation of MDSCs and MDSC migration assay
We collected MDSCs from the splenocytes of tumour-bearing mice utilizing an MDSC isolation equipment. To watch MDSC migration, 1 × 105freshly remoted mouse MDSCs have been added to transwell inserts with DMEM containing 10% FBS, DMEM from tumour cells contaminated with micro organism or DMEM containing completely different concentrations of conditioned medium from P. anaerobius have been put within the backside chamber of the transwell. Migrated MDSCs have been counted underneath microscopy. To dam the CXCR2 receptor, freshly remoted MDSCs have been pre-incubated with 10 μM SB265610 (CXCR2 antagonist) for 1 h at 37 °C after which seeded in transwell inserts. For the drug remedy assay, MDSCs have been cultured in RPMI 1640 medium containing 10% FBS, 10 ng ml−1 IL-6 and 10 ng ml−1 GM-CSF to maintain its exercise.
Isolation of colonic lamina propria cells and stream cytometry
Colonic lamina propria cells have been remoted from mice as described beforehand. Briefly, colon tissue was minced and incubated in a digestion buffer consisting of PBS, 1% BSA, 1 mg ml−1 collagenase kind IV and 0.5 mg ml−1 DNase Ι for 30 min at 37 °C. The cell suspensions have been filtered by means of a 70 μm strainer after which centrifuged at 750g for 10 min. For floor marker staining, lamina propria cells have been stained with fluorescent dye-conjugated antibodies in cell staining buffer for 20 min. For intracellular staining, the cells have been stimulated for 4 h in RPMI full medium with 30 ng ml−1 phorbol 12-myristate 13-acetate (Abcam) and 1 μg ml−1 ml ionomycin (STEMCELL Applied sciences) within the presence of two.5 μg ml−1 monomycin (BioLegend). The cells have been then stained with cell floor markers. Samples for stream cytometry evaluation have been fastened with cell fixation buffer (BioLegend), permeabilized with FOXP3 Perm buffer (eBioscience) in response to the producer’s suggestions and stained with intracellular antibody. Optimistic cells have been acquired and quantified utilizing the FlowJo software program. Samples for single-cell sequencing have been sorted in collaboration with the LKSIHS core facility.
Analysis of MDSC immunosuppressive exercise
T cells have been collected from the splenocytes of non-tumour-bearing mice utilizing a T cell isolation equipment (STEMCELL Applied sciences), adopted by CFSE staining. An EasySep mouse MDSC (CD11b+Gr-1+) isolation equipment (STEMCELL Applied sciences, 19867) was used to gather MDSCs from the spleen of tumour-bearing mice on days 17–19 after subcutaneous inoculation with 5 × 105 cells MC38 cells. CFSE-labelled T cells (1 × 105 cells) and 1 × 105 cells, the half (5 × 104) of MDSCs have been seeded in a 96-well plate. T cell proliferation was induced with Dynabeads mouse T-activator CD3/CD28 (Thermo Fisher Scientific, 11452D) for 3 d, adopted by stream cytometry evaluation utilizing the FlowJo software program to judge the CFSE dilution price and the share of practical cells within the T cell subpopulations.
Immunofluorescence staining
Antigen retrieval was carried out by incubation of the slides in Tris–HCI buffer at 100 °C for 20 min, adopted by two washes with PBST (PBS containing 0.1% Tween 20). The slides have been incubated with 1% BSA for 1 h to dam non-specific binding of immunoglobulin after which in a single day incubation with main antibody to CD11b (dilution, 1:200; Abcam, ab133357) and Gr-1 (dilution, 1:50; BioLegend, 108448) at 4 °C. Following two washes with PBST, the specimens have been incubated with Alexa Fluor 488 secondary antibody (dilution, 1:500; Jackson Immuno, 111-545-003 (X2)). The accuracy of automated measurements was confirmed by means of impartial analysis by two pathologists. Cells stained with the indicated antibodies have been counted at ×63 magnification in at the very least ten fields per part.
Quantification of the P. anaerobius relative abundance within the TCGA colon adenocarcinoma cohort
We downloaded the unaligned RNA-seq information, in FASTQ format, of colon adenocarcinoma from the TCGA database37. The pathseq38 pipeline was used to map the unaligned RNA-seq of TCGA colon adenocarcinoma samples to gut-related micro organism after eradicating human reads and low-quality reads. The pre-built host genome was obtained from the GATK Useful resource Bundle FTP server in /bundle/pathseq/. The microbe references used right here included 1,520 cultivated bacterial genomes39 and colon cancer-related micro organism that have been recognized by means of intensive and statistically rigorous validation. The normalized rating generated by pathseq was used because the relative transcriptome abundance for every species. We calculated the imply expression degree of marker genes because the signature rating for every immune cell kind. For MSDC cells, the markers CD33, ITGAM and ARG1 have been utilized, whereas effector T cells have been characterised utilizing CD44, CD8A and IFN-γ.
Fluorescence in situ hybridization
A P. anaerobius Alexa Fluor 488-conjugated particular probe (5′-ATATCTACGATGCCGTAAATATA-3′) was labelled with Spectrum-Inexperienced (Thermo Fisher Scientific). Recent colon tissues have been embedded in paraffin and lower into 5 μm sections. The colon tissue sections have been then subjected to hybridization utilizing hybridization buffer (0.9 M NaCl, 20 mM Tris–HCl pH 7.3 and 0.01% SDS) combined with oligonucleotide probe (1:50). After incubation for 48 h at 37 °C in a darkish humid chamber, every slide was rinsed with sterile double-distilled water for five min, air-dried in the dead of night and eventually mounted utilizing ProLong gold antifade mountant with 4,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific).
Prediction of lytC_22–Slamf4 binding websites and building of lytC_22 mutant protein
The protein constructions of lytC_22 (UniProt identifier: A0A379CGB7) and CD244 (UniProt identifier: P02679) have been retrieved from the UniProt database (https://www.uniprot.org/). The HDOCK software program was employed to foretell the protein–protein binding conformation, with choice based mostly on the scoring operate and identification of probably the most destructive vitality conformation. Molecular dynamics simulations have been carried out utilizing Gromacs 2020, making use of the AMBER99SB-ILDN pressure subject for proteins and using the TIP3P dominant water mannequin. To neutralize system cost, sodium or chloride ions have been launched in response to the docking outcomes. The system was then subjected to a 100 ns molecular dynamics simulation underneath the isothermal-isobaric ensemble.
All through the molecular dynamics simulation, key properties of curiosity similar to system vitality, the basis imply sq. deviation of protein construction fluctuations and root imply sq. fluctuation evaluation of amino acid residues have been monitored over time to analyze adjustments in system-related traits. Following optimization of the binding conformation, the Pymol 2.1 software program was utilized to visualise and map the protein interplay websites.
To acquire the mutant protein that’s unable to bind with Slamf4, after figuring out which key residues on lytC_22 bind to Slamf4, alanine residues have been launched to exchange these binding residues. LytC_22 mutant was then purified as described earlier.
Silver staining and protein identification
The 50–100 kDa fraction from PA-CM was separated by 10% SDS–PAGE. Silver staining was carried out following the directions offered by the producer (Thermo Fisher Scientific). After staining, particular bands situated between 50 and 100 kDa have been excised into 1.5 ml plastic tubes for in-gel digestion and mass spectrometry evaluation by Shanghai Biotree Biotech.
Expression, purification and in vitro exercise of lytC_22
LytC_22 proteins have been produced from E. coli BL21 (DE3) containing plasmids pGEX-4T1 for lytC_22. The cells have been induced by incubation with 0.5 mM isopropyl-thio-β-d-galactopyranoside (Sangon Biotech) for 16 h at 25 °C. Subsequent, the bacterial pellets have been collected and resuspended in PBS containing 1 mg ml−1 lysosome. After three freeze–thaw cycles, the samples have been centrifuged at 4 °C and 20,000gfor 15 min and the supernatant containing the GST–lytC_22 protein was collected. GST–lytC_22 was additional purified utilizing a GST-tag protein purification equipment (Beyotime). The GST tag was eliminated by means of incubation with 80 U thrombin protease (Cytiva) for 16 h at 25 °C, adopted by incubated with glutathione Sepharose resins for 1 h at 4 °C. The thrombin was then eliminated by passing by means of a HiTrap Benzamidine FF column (Cytiva). Fractions containing the goal protein have been concentrated and additional purified by centrifugation at 4,000g and 4 °C for 1 h utilizing 50 kDa MWCO Amicon Extremely-4 centrifugal filters. To detect the impact of lytC_22 on MDSCs, 10 or 20 nM purified lytC_22 was added into RPMI 1640 medium when culturing main MDSCs. After remedy for twenty-four h, the MDSCs have been collected for additional experiments.
RNA extraction and quantitative PCR
Whole RNA was extracted from cells and flash-frozen colon tissues utilizing TRIzol reagent (Thermo Fisher Scientific). Complementary DNA was synthesized from 1 μg of complete RNA utilizing a PrimeScript RT–PCR equipment (Takara). Quantitative real-time PCR was carried out utilizing a LightCycler 480 real-time PCR system (Roche Utilized Sciences). The goal mRNA degree was normalized to that of β-actin and calculated utilizing the two−∆∆Ct methodology.
GST pulldown assay
Major MDSCs have been lysed utilizing IP lysis buffer (1% NP-40, 150 mM NaCl, 5 mM MgCl2 and 25 mM HEPES pH 7.5) for 1 h and complete protein from MDSCs was acquired by centrifugation at 20,000g and 4 °C for 15 min. For the GST pulldown assay, GST–lytC_22 and MDSC cell lysate or recombinant Slamf4 protein (ABclonal, RP00167) have been incubated in a single day at 4 °C with anti-GST (dilution, 1:200; Cell Signaling Know-how, 2624S) or anti-Slamf4 (dilution, 1:100; Cell Signaling Know-how, 54560S). Protein A/G beads have been added to the samples and incubated at 4 °C for two h. The beads have been then washed 5 instances with IP lysis buffer and boiled in SDS–PAGE gel loading buffer. The eluted proteins have been separated by SDS–PAGE and analysed by western blotting.
Western blots
Whole protein was separated by 10% SDS–PAGE after which transferred onto polyvinylidene difluoride membranes for two h. After blocking with 5% non-fat milk for 1 h at room temperature, the blots have been incubated with main antibodies in a single day at 4 °C after which washed. Subsequent, the blots have been incubated with secondary antibodies for 1 h at room temperature. The protein bands have been uncovered to ECL plus western blotting detection reagents. The next main antibodies have been used at a dilution of 1:1,000: anti-integrin α2 (Abcam, ab133557; dilution,), anti-integrin β1 (Abcam, ab179471), anti-β-actin (Cell Signaling Know-how, 4970S), anti-phospho-NF-κB p65 (Cell Signaling Know-how, 3033S), anti-NF-κB p65 (Cell Signaling Know-how, 8242S), anti-lamin A/C (Cell Signaling Know-how, 4777) and anti-Slamf4 (Cell Signaling Know-how, 54560). Anti-GST tag (Cell Signaling Know-how, 2625) was used at a dilution of 1:5,000. Quantification was carried out utilizing Picture Lab 6.1 (Bio-Rad).
MST assay
Analyses of direct binding of between Slamf4 and lytC_22 have been carried out utilizing a Monolith NT.115 instrument (NanoTemper Applied sciences). Recombinant Slamf4 was fluorescently labelled upfront. To measure binding between Slamf4 and GST–lytC_22, gradient concentrations of GST–lytC_22 (11, 5.5, 2.75, 1.375, 0.6875 and 0.34375 μg μl−1) have been incubated with 0.2 μg μl−1 pre-labelled recombinant Slamf4 at a ratio of 1:1 (vol/vol) for 30 min at room temperature. For measurement, the incubated combination was loaded into capillaries (NanoTemper Applied sciences) and loaded onto a Monolith NT.115 instrument. A cap scan of all of the capillaries was carried out and the MST experiment was performed utilizing auto LED/excitation energy and medium MST energy. Information have been analysed utilizing the NT Analyses 1.5.41 software program.
Statistical analyses
A Mann–Whitney U-test or Pupil’s t-test was used to analyse the variations between two pattern teams. A one-way ANOVA was used to analyse the variations between a number of teams. Information are offered because the imply ± s.e.m. Pilot experiments have been used to estimate the pattern dimension. Statistical evaluation was carried out utilizing the GraphPad Prism model 9 software program (GraphPad Software program). Comparisons with P < 0.05 have been thought-about to be statistically important.
Reporting abstract
Additional data on analysis design is obtainable within the Nature Portfolio Reporting Abstract linked to this text.
