Clostridioides difficile infection (CDI). While bile salt hydrolases (BSHs) shape the BA pool by deconjugating conjugated BAs, the basis for their substrate selectivity and impact on C. difficile remain elusive. Here we survey the diversity of BSHs in the gut commensals Lactobacillaceae, which are commonly used as probiotics, and other members of the human gut microbiome. We structurally pinpoint a loop that predicts BSH preferences for either glycine or taurine substrates. BSHs with varying specificities were shown to restrict C. difficile spore germination and growth in vitro and colonization in pre-clinical in vivo models of CDI. Furthermore, BSHs reshape the pool of microbial conjugated bile acids (MCBAs) in the murine gut, and these MCBAs can further restrict C. difficile virulence in vitro. The recognition of conjugated BAs by BSHs defines the resulting BA pool, including the expansive MCBAs. This work provides insights into the structural basis of BSH mechanisms that shape the BA landscape and promote colonization resistance against C. difficile.Bile acids (BAs) mediate the crosstalk between human and microbial cells and influence diseases including  Clostridioides difficile infection in the murine gut.Bile salt hydrolases encoded by the gut microbiome shape the bile acid pool, including microbial conjugated bile acids, which impact  Host-encoded BA receptors recognize BAs as signalling molecules to regulate host immunity3, metabolism4 and circadian rhythms6. BAs also shape the microbiota, where specific taxa exhibiting resistance to bile stress harbour mechanisms to survive BA exposure8. The influence of BAs on the microbiota is reciprocated by the microbial transformation of BAs to establish the chemical complexity of the intestinal BA pool9. The resulting mosaicism is a hallmark of host health and is dependent on the collective activity of microbiome-encoded BA-altering enzymes10.Bile acids (BAs) are host-synthesized and microbial-derived metabolites that support intestinal health and homoeostasis by providing a scaffold for host-microbiome crosstalk and adaptation11. Primary conjugated BAs synthesized in the liver, such as the tauro- and glycoconjugates of cholic acid , are deconjugated by BSHs to generate cholic acid (CA) 11.Bile salt hydrolases (BSHs) comprise a diverse family of microbial enzymes that catalyse critical BA transformation12, cancer13, inflammatory bowel disease3 and colonization resistance against pathogens including Clostridioides difficile18. C. difficile infection (CDI) is an important public health problem that is difficult to treat with conventional antibiotics19. Even after a course of vancomycin or fidaxomicin, there remains a high recurrence rate (~30%)21, making a faecal microbiota transplant (FMT) the last line of treatment22. Although FMTs can treat recurrent CDI (rCDI), the long-term consequences remain unclear, making a targeted therapeutic approach appealing for safety and efficacy reasons. Susceptibility to CDI is predominantly driven by antibiotic-induced perturbations to the intestinal microbiota resulting in a loss of colonization resistance24. Strong evidence suggests that microbial BA metabolism is an important mechanism of colonization resistance against C. difficile25, as antibiotic usage depletes BSHs, increasing conjugated BAs and decreasing secondary BAs17.Dysregulation of BA biosynthesis and BSH activity has been directly implicated in obesityC. difficile pathogenic life cycle: TCA acts as a germinant for C. difficile spores26, whereas chenodeoxycholic acid (CDCA) can inhibit germination and growth27. Most studies have focused on the production of deconjugated secondary BAs as inhibitors of C. difficile pathogenesis, while relatively few have examined the contributions of BSH activity alone28. The therapeutic potential of BSHs has been proposed, but gaps in our knowledge of their pleiotropic effects on intestinal biology and their exact mechanism of action have hampered their development30. Additionally, the recent discovery of microbial conjugated bile acids (MCBAs) has considerably increased the complexity of the BA pool and altered the paradigm of BSH-BA biology33, warranting new investigations into BSH–substrate relationships  were grouped into 84 clusters and a representative sequence from each cluster was used to construct a phylogenetic tree. Nearly all BSH clusters were identified from vertebrate-adapted species and ~40% were human-associated, underscoring their role in host-microbiota symbiosis  enzymes are all present within a single major clade, while the LsalBSH  stands alone in a distantly related branch  and two taurine-preferring  , as demonstrated using its promoter (PtcdA) fused to an mCherry reporter  ml−1 to measure ex vivo spore germination and growth, respectively. The addition of the BSH cocktail significantly inhibits C. difficile spore germination in the small intestinal contents of all mice and suppresses growth at 8 and 24 h , we hypothesized that a set of MCBAs similar to those seen in Fig. 33, and initial BA deconjugation was quantified using liquid chromatography-ion mobility spectrometry-mass spectrometry (LC–IMS–MS)  Fig. . LingBSHMS) Fig.  and 9. AMS) Fig. . Ala andMS) Fig. . NotablyLactobacillus gasseri cells. Wild-type, ΔbshA, ΔbshB and ΔbshAB L. gasseri were incubated with the AA-CDCA pool, and the resultant supernatants were examined to detect deconjugation. We note that the ΔbshAB double mutant displays basal deconjugation potentially arising from non-specific hydrolases or cell sequestration of supernatant BAs , with the BSH reference set as the query and the Lactobacillaceae database as the BLAST database. Search results were filtered using custom code to identify sequences with >30% identity and at least 100 amino acids in length for downstream analysis.Briefly, the whole-genome sequence data for lactobacilli were downloaded from NCBI in July 2021, resulting in a total of 3,712 nucleotide files. The data were subsequently built into a local BLAST Lactobacillaceae database. Updated BSH (15 sequences) and penicillin V acylase (PVA) (11 sequences) reference sets were manually created on the basis of previously characterized BSH and PVA proteins49. A Stockholm multiple sequence alignment file generated using the alignment was then used to run the Hidden Markov Model using HMMER (v3.3.2)50 on the results of the BLASTx search to identify likely BSH proteins. Custom code was employed to remove results with E values <1 × 10−99 as false positives and to determine whether the amino acid sequence was likely a BSH or a PVA on the basis of its E value.The BSH and PVA reference sets were separately aligned using MUSCLE aligner (v3.8.2)52. Custom code was employed to extract the consensus sequence from each cluster for the construction of a phylogenetic tree. CD-HIT cluster data were hand-curated to confirm the amino acid sequence of the representative BSH proteins from each of the 84 clusters and to remove redundant sequences. Geneious Primer (v2022.1) and CLC Genomics Workbench (v12) were used to visualize protein sequence alignments, construct phylogenetic trees and add metadata.To facilitate the downstream phylogenetic analysis, CD-HIT (v4.8.1) clustering was used to cluster similar BSH sequences that shared at least 95% identitydl-dithiothreitol , trichloroacetic acid (Sigma Aldrich), ninhydrin (Sigma Aldrich), sodium citrate tribasic dihydrate (Sigma Aldrich), glycerol (Thermo Fisher) and crystal screens MCSG1-4 (Anatrace).The following chemicals were used in this study: sodium taurocholate , sodium glycocholate (Sigma Aldrich), sodium taurochenodeoxycholate (Sigma Aldrich), taurine (Sigma Aldrich), glycine (Sigma Aldrich), sodium acetate trihydrate (Thermo Fisher), sodium phosphate dibasic dihydrate (Sigma Aldrich), sodium phosphate monobasic dihydrate (Sigma Aldrich), Escherichia coli Rosetta (DE3) pLysS were cultured at 37 °C, shaking overnight in LB broth supplemented with 30 μg ml−1 kanamycin and 20 μg ml-1 chloramphenicol (Cam) or in Terrific Broth (TB) supplemented with the same antibiotics for BSH overexpression. Lactobacillaceae species were cultured statically in MRS media at 37 °C in a Coy anerobic chamber (5% H2/10% CO2/85% N2) and L. gasseri ATCC 33323 bsh mutants were previously described8. C. difficileR20291 was statically cultured anaerobically using BHIS broth supplemented with 100 mg l−1l-cysteine. BHIS agar supplemented with 0.1% TCA (TBHIS) was used to start cultures from spore stocks. C. difficile enumeration from intestinal contents or stool was performed on CCFA  or TCCFA (containing 0.1% TCA). All agar plates were made using 1.5% agar. C. difficile growth curves were carried out in BHIS broth in clear flat-bottom plates containing 200 μl of media per well. Growths were performed in a Tecan plate reader within the anaerobic chamber at 37 °C for 24 h.BSH-expressing Selecting BSHs for characterization was informed by the Lactobacillaceae BSH phylogenetic tree in Fig. E. faecalis BSH were codon-optimized and synthesized. The genes for Bifidobacterium longum, Bacteroides thetaiotaomicron, Faecalibacterium sp., Clostridiales sp. and Bacteroidales sp. BSHs were codon-optimized, synthesized and subcloned into the pLic-His Cterm vector (ampicillin resistant) by BioBasic. PCR was performed using Phusion Flash High-Fidelity PCR master mix (Thermo Fisher) and products were purified using the QIAquick PCR Purification kit (Qiagen). Amplicons were cloned into the pETite C-His vector (Lucigen), purified using the Monarch Plasmid Miniprep kit (NEB) and sequenced. Overexpression E. coli cultures were grown at 37 °C with shaking in 1 l of TB with kanamycin and Cam until an OD600 ≈ 0.6 was reached. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added to cultures to induce expression and cells were grown at 30 °C with shaking for 16–20 h. Cells were collected by centrifugation and stored at −80 °C.All BSH sequences cloned in this study were amplified from genomic DNA or were codon-optimized in synthesized genes (Integrated DNA Technologies (IDT)) using custom oligonucleotide primers (IDT) or were synthesized by BioBasic, as reported in Supplementary Tables E. coli BL21-G (DE3) cells and purified as described in the ‘Protein expression and purification for crystallography and mutant proteins’ section.All BSH mutants were created with site-directed mutagenesis using Pfusion High-Fidelity DNA Polymerase (New England Biolabs). Primers were synthesized by IDT , DNAse (Sigma), pH 8.0) and lysed by sonication. Cell debris were pelleted by centrifugation at 25,000 × g for 30 min at 4 °C. Lysates were run over a gravity column containing 4 ml of fresh HisPur cobalt resin (Thermo Fisher) that was equilibrated in wash buffer . Bound BSHs were washed on the column at a rate of ~1 ml min−1 with 20 ml of wash buffer, eluted with 10 ml of elution buffer (wash buffer plus 150 mM imidazole and 10 mM dl-dithiothreitol) and flash frozen in liquid N2 to prevent oxidation. BSHs were quantified using the Qubit Protein Assay kit (Invitrogen) and protein purity was assessed using 10%, 14% or 4–20% SDS–PAGE gels (Thermo Fisher)  and cultured under kanamycin (25 µg ml−1) and Cam (34 µg ml−1) or ampicillin (100 µg ml−1) selection. A single colony was selected the next day and grown overnight in 100 ml TB media  at 37 °C with shaking at 210 r.p.m. with kanamycin (25 µg ml−1) and Cam (34 µg ml−1) or ampicillin (100 µg ml−1). The next day, 50 ml of the overnight culture was added to 1.5 l TB media with the appropriate antibiotics and ~40 µl of Antifoam 204. The culture was then incubated at 37 °C with shaking at 210 r.p.m. for expression. Expression was induced when optical density (OD)600 reached 0.6–0.8 with 100 µM IPTG, and the culture was incubated overnight at 18 °C.Expression plasmids were transformed into BL21-G competent Clostridiales sp. BSH was expressed using autoinduction instead of IPTG induction53. A single colony or 20 µl of glycerol stock was added to 10 ml MDAG-135 media (9.4 ml double distilled (ddH2) O; 20 µl 1 M MgSO4; 2 µl 1,000x trace metals ; 70 µl 50% (w/v) glucose; 40 µl 25% (w/v) aspartate; 200 µl 50x M ; 200 µl 17 amino acids ; 80 µl 25 mg ml−1 methionine; and 100 µg ml−1 ampicillin). The culture was grown overnight at 37 °C with shaking at 210 r.p.m. The next day, the overnight culture was added to 1 l ZYM-5052 media ; and 20 ml 50x M) with ampicillin and ~40 µl of Antifoam 204. The culture was then incubated at 37 °C with shaking at 210 r.p.m. for expression. Expression was autoinduced via incubation at 37 °C; when the OD600 reached 1–2, the culture was then incubated for approximately 24 h at 18 °C until optical density reached 13–15.g for 20 min at 4 °C. The cell pellet was resuspended in lysis buffer , lysozyme, 50 µg ml−1 DNAse and 10 mM 2-mercaptoethanol). Cells were then lysed by sonication on ice using a Thermo Fisher Sonic dismembrator model 500 with 0.5 s pulses for 1.5 min three times at 40%, 50% and 60% intensity. The cell suspension was pelleted by centrifugation in a Beckman Coulter J2-HC centrifuge at 17,000 × g for 50 min at 4 °C. The supernatant was syringe-filtered using a 0.22 µm filter.The cells were then pelleted in a Sorvall Instruments RC-3B centrifuge at 4,500 × −1. The final protein concentration was determined using an ND-1000 spectrophotometer. Dithiothreitol was added to the concentrated fractions at a final concentration of 10 mM, and samples were snap-frozen in liquid nitrogen and stored at −80 °C.The filtrate was flowed over a HisTrap HP 5 ml column (Cytiva) using the Aktaexpress FPLC (Amersham Bioscience) and washed with BSH buffer A. The bound protein was eluted in a stepwise fashion with 100% BSH buffer B . Fractions containing the protein of interest were combined and flowed over a HiLoad 16/60 Superdex 200 gel filtration column (GE Life Sciences). Samples were eluted in S200 sizing buffer . Fractions containing the protein of interest were analysed for purity using SDS–PAGE, and those with >95% purity were combined and concentrated using 30 kDa cut-off molecular weight centrifuge concentrators (EMD Millipore) to ~10 mg ml8). Briefly, the assay reacted 10–25 nM BSH with 9 mM conventional BAs for 5 min, or 100 nM BSH with 4.5 mM MCBAs/penV, for 1 h in 50 μl volumes. Reactions were carried out in 0.1 mM sodium phosphate, 10 mM dithiothreitol (pH 6.0) and stopped with 50 μl of trichloroacetic acid. To determine the quantity of amino acid or 6-APA released, the colorimetric ninhydrin reaction was carried out. Of the quenched BSH reaction, 25 μl was added to 475 μl of ninhydrin buffer , 0.25% ninhydrin reagent) and boiled for 14 min. A standard curve of the respective conjugated amino acid or 6-APA was prepared for each assay. Absorbance was measured at 570 nm in clear flat-bottom plates in a Tecan Infinite F200 Pro plate reader. Specific activity is reported as μmol amino acid released per second per μmol BSH.Activity assays were performed as previously described for the BSHs surveyed in Fig. Lactobacillus BSHs) or by performing a single pH screen (one replicate) as described below using both TCA and GCA for all 5 pH values tested. The substrate with faster turnover was then used to complete the full pH screen in triplicate.The optimal pH of each BSH enzyme tested in Fig. To determine the optimal pH, the activity of each BSH was tested with the preferred substrate at pH 5.0, 5.5, 6.0, 6.5 and 7.0. pH screen reactions were prepared to a final volume of 50 µl consisting of 35 µl buffer (50 mM sodium acetate (for pH 5.0 and 5.5) or 50 mM sodium phosphate ), each with 10 mM dithiothreitol, 5 µl preferred substrate  and 10 µl enzyme . Substrate and buffer were plated and allowed to incubate at 37 °C for 5 min before 10 μl enzyme was added to initiate the reaction. Reactions were carried out at 37 °C and quenched with 50 µl of 15% (w/v) trichloroacetic acid at five time points ranging from 2 min to 3 h based on enzyme activity.g for 2 min. Quenched reaction (10 µl) was added to 190 µl ninhydrin reaction (62.5 ml of 1% ninhydrin in 0.5 M sodium citrate (pH 5.5), 150 ml glycerol, 25 ml 0.5 M sodium citrate (pH 5.5)) in a 96-well PCR plate. A standard curve of glycine or taurine was also created for analysis using the ninhydrin reaction. Using a thermocycler, the mixtures were heated to 90 °C for 14 min and then cooled at 25 °C for 15 min. Ninhydrin mixture (150 µl) was then added to a 96-well flat-black plate (Grenier 655906) and the absorbance was measured at 570 nm using a Clariostar Plus plate reader (BMG Labtech). The standard curve was used to quantitate the amount of free amino acid liberated by the reaction. Reaction curves over time were fit using linear regression and the reaction rate was divided by protein concentration to yield specific activity. Specific activities are the mean ± s.d of three biological replicates. Optimal pH was determined by selecting the pH at which turnover of the substrate was the fastest  PEG 3350. Crystals formed in a 2:1 protein (11.57 mg ml−1) to crystallant ratio. LgasBSHa with taurine: crystallant composed of 0.2 M potassium sulfate, 20% (w/v) PEG 3350. Crystals formed in a 1:2 protein (9.5 mg ml−1) to crystallant ratio. LgasBSHa with taurine and CDCA: a condition containing 18% (w/v) PEG 3350, 0.2 M ammonium chloride, pH 6.3, in a hanging drop tray was streak seeded with crystals grown using the sitting drop method in 20% (w/v) PEG 3350, 0.2 M ammonium chloride (pH 6.3). Crystals were grown in a 1:2 protein (9.55 mg ml−1) to crystallant ratio. The resultant crystals were soaked in 1:1 ratio of crystallant:100 mM taurochenodeoxycholic acid for 24 h before looping. LgasBSHb: crystallant composed of 0.2 M magnesium chloride, 0.1 M sodium cacodyolate:HCl, pH 6.5, 20% (w/v) PEG 1000. Crystals formed in a 1:2 protein (10.75 mg ml−1) to crystallant ratio. LjohBSHc: crystallant composed of 0.1 M sodium citrate:HCl, pH 5.6, 10% (w/v) PEG 4000, 10% (v/v) isopropanol. Crystals formed in a 2:1 protein (10.34 mg ml−1) to crystallant ratio. LingBSH: crystallant composed of 0.2 M calcium acetate hydrate, 0.1 M Tris:HCl, pH 7.0, 20% (w/v) PEG 3000. Crystals formed in a 1:2 protein (13.8 mg ml−1) to crystallant ratio. LreuBSH: crystallant composed of 0.1 M Bis-Tris Propane:HCl, pH 7.0, 1.5 M ammonium sulfate. Crystals formed in a 2:1 protein (13.3 mg ml−1) to crystallant ratio.Crystals were generated at 20 °C using sitting drop vapour diffusion for all specimens obtained except those of LgasBSHa grown with taurine and CDCA, which employed hanging drop vapour diffusion. Sitting drop trays were set up using the Douglas Instruments Oryx4 instrument and Hampton Research 3-well midi crystallization plates (Swissci). Hanging drops were manually plated using EasyXtal 15-well tool trays (Qiagen). Specific crystallization conditions follow. LaciBSHa: crystallant composed of 0.15 M 54 and Phenix (v1.17.1-3660) was employed to perform molecular replacement with the model PDB 2HEZ. Output models from Phaser were improved with Autobuild55 and structures were refined using phenix.refine55 with iterative cycles of manual adjustment using Coot (v0.9.4.1)56. Final coordinates were deposited into the RCSB Protein Data Bank under the codes 7SVE, 7SVF, 7SVG, 7SVH, 7SVI, 7SVJ and 7SVK.Crystals were cryo-protected in the conditions described above with 20% glycerol before looping and flash cooling in liquid nitrogen. Diffraction data were collected at 100 K at either APS 23ID-D , APS 23ID-B  or ALS 5.0.2 (LreuBSH). Data were processed and scaled with XDS (v2.3.2)Glycine was modelled into the active site of the unliganded structure of LgasBSHb by superimposing this enzyme’s active site with that of the structure of LgasBSHa in complex with both taurine and CDCA. Glycine was then manually positioned over the backbone of taurine using PyMOL (v2.3.2) to approximate the binding mode of glycine.57. Enzyme (0.125 mg ml−1) in CD buffer  was placed into a 1 mm cuvette. Scan spectra from 185 to 260 nm were acquired at 20 °C using the Chirascan Plus instrument (Applied Photolysis). A background spectrum of buffer alone was acquired and subtracted out to correct for background signal. Spectra were smoothed before plotting. Calculation of protein secondary structure was performed using the DichroWeb server (http://dichroweb.cryst.bbk.ac.uk/html/process.shtml)58. Data were assessed using the CONTIN-LL method59, with reference set SMP180  using Protein-Protein BLAST (BLASTP v2.5.0+)67. Candidate sequences with ≥25% identity to any representative BSH enzyme were then assessed for the presence of 5 conserved residues: C2, R18, D21, N175 and R228 (relative to PDB: 2BJF). Sequences that both met the identity threshold and contained all 5 conserved residues were accepted as BSH enzymes (Supplementary Table 68. Accepted sequences were processed with the Enzyme Function Initiative–Enzyme Similarity Tool (https://efi.igb.illinois.edu/efi-est/) to create a sequence similarity network, represented using an E value of 1 × 10−100 (ref. 69). Each node represents sequences bearing ≥95% identity.The Integrated Gene Catalog contains metagenomic sequencing data from 249 human samples examined in the Metagenomics of the Human Intestinal Tract (MetaHit) project along with 1,018 collections of extant human gut metagenomics data to form an overall database with 9,878,647 unique translated bacterial protein sequencesC. difficile spores were prepared as previously described70. Briefly, C. difficile was grown at 37 °C anaerobically for 1 week in Clospore media71. Spores were collected by centrifugation, washed with water, heat treated for 20 min at 65 °C and stored at 4 °C. Spores were plated on BHIS and TBHIS agar to make sure no viable cells were present.72. Conventional BAs were dissolved in water and MCBAs were dissolved in methanol. For the inhibition of germination experiments, the germinant TCA was used across conditions at 2 mM. CDCA was used at 0.75 mM as a positive control for germination inhibition and all other BAs were tested at the same concentration (0.75 mM). Germination reactions were carried out anaerobically in 100 μl volumes in PBS for 30 min. Reactions were subsequently serially diluted and plated on TBHIS and BHIS agar. Percent germination for each BA was calculated as (100 × (c.f.u.s on BHIS / c.f.u.s on TBHIS)) and was normalized to germination with TCA alone. For the germination reactions with MCBAs, all BAs were used at 1.9 mM and percent germination was calculated and normalized as stated above.Spore germination experiments were modified from refs. 73. Overnight C. difficile cultures (~108 c.f.u.s ml−1) were inoculated at 1% into BHIS containing a range of BA concentrations. Cultures were anaerobically incubated for 24 h at 37 °C. Following incubation, cultures were serially diluted in PBS and plated on BHIS agar to determine whether the concentration of BA tested inhibited growth relative to the starting inoculum.BA tolerance measured by MIC was adapted from the bile tolerance assayC. difficile cultures were captured and washed 3 times with PBS before being back diluted to a final OD600 = 0.1 into PBS containing BAs at 0.25× the MIC for 30 min. Bacteria were exposed to 150 μM SDS as positive controls for detergent-induced membrane damage and PI staining. Following ΒΑ exposure, bacteria were stained for 30 min at 37 °C with 20 μg ml−1 PI using slight modifications to a previous method74. Stained bacteria were diluted 1:10 in PBS and PI fluorescence was measured from flat-bottom clear plates  in 100 μl volumes in a Tecan Infinite F200 Pro plate reader and background fluorescence was subtracted.Exponentially growing C. difficileR20291 containing either pDSW1728-PtcdA-mCherry or the empty vector pDSW1728 was grown anaerobically on BHIS agar + 3 μg ml−1 thiamphenicol75. A single colony was inoculated into a 5 ml culture of BHIS broth + 3 μg ml−1 thiamphenicol and grown overnight at 37 °C. The overnight culture was diluted 1:100 into a fresh 3 ml culture of BHI broth + 3 μg ml−1 thiamphenicol with individual BAs and grown at 37 °C for 24 h. Culture (500 μl) was mixed with 120 μl of a 5X fixation cocktail: 100 μl 16% paraformaldehyde and 20 μl 1 M NaPO4 buffer (pH 7.4). The sample was incubated anaerobically at room temperature for 30 min, then incubated in the dark for 30 min on ice outside of the anaerobic chamber. Fixed cells were washed 3 times with PBS, resuspended in 30 μl of PBS and left in the dark at 4 °C overnight to allow for chromophore maturation. mCherry fluorescence was measured at excitation/emission wavelengths of 550/610 nm using a Tecan Infinite F200 Pro plate reader. Absorbance at 600 nm was measured on the same plate reader. Data are displayed as the ratio of fluorescence to absorbance. Baseline fluorescence from the empty vector was subtracted from all measurements.All consenting patients undergoing FMT for rCDI at the University of North Carolina from January to December 2017 in a prospective registry were enrolled for faecal collection. rCDI was defined as a patient having at least the third episode of CDI. There were no exclusion criteria for participation in the registry specifically, although participants were by definition undergoing FMT under the care of a physician who judged the benefits to outweigh the risks. Patient stool samples from 2 weeks before FMT (pre-FMT) were collected. The study was approved by the UNC Insitutional Review Board (no. 16-2283). Informed written consent was obtained from recipients. Stool samples were collected and de-identified by the research team.2 asphyxiation. This protocol is approved by NC State’s Institutional Animal Care and Use Committee (IACUC).Male and female C57BL/6J mice (5 weeks old) were purchased from Jackson Labs (Bar Harbor) for use in infection experiments. The food, bedding and water were autoclaved, and all cage changes were performed in a laminar flow hood. The mice were subjected to a 12 h light and 12 h dark cycle, and were housed at an average temperature of 70 °F and 35% humidity. Animal experiments were conducted in the Laboratory Animal Facilities located on the NCSU CVM campus. The animal facilities are managed by full-time animal care staff coordinated by the Laboratory Animal Resources (LAR) division at NCSU. The NCSU CVM is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Trained animal handlers in the facility fed and assessed the status of animals several times per day. Those assessed as moribund were humanely euthanized by COn = 16 total) were given 0.5 mg ml−1 cefoperazone in their drinking water for 5 d to make them susceptible to CDI76. Subsequently, the mice were then given normal water (Gibco) for 2 d, after which groups of mice (n = 3–4) were killed at day 0. Small intestinal and caecal contents from each mouse were collected and samples were flash frozen in liquid N2.Male C57BL/6J mice , the glycine-preferring BSH cocktail G-BSH  or the combined broad acting G&T-BSH cocktail for 30 min at 37 °C. Mouse contents were treated with PBS or the G&T-BSH cocktail. All BSHs had a final concentration of 0.1 μM each. Immediately afterwards, exponentially growing C. difficile was inoculated into samples to a final concentration of 105 c.f.u.s ml−1. C. difficile was cultured anaerobically at 37 °C and enumerated on TCCFA at 8 and 24 h.To measure BSH-dependent inhibition of C. difficile spores (106 ml−1) were then inoculated in the small intestinal contents for 30 min and samples were promptly diluted and enumerated on CCFCA and TCCFA. Percent germination was calculated as (100% × (c.f.u.s on CCFA / c.f.u.s on TCCFA)).To measure BSH-dependent inhibition of germination in mouse small intestinal contents, 1:3 diluted samples were treated with PBS or the G&T-BSH cocktail for 8 h at 37 °C. −1 cefoperazone in their drinking water for 5 d to make them susceptible to CDI76. Subsequently, the mice were given normal water (Gibco) for 2 d. Mice were orally gavaged with the T-BSH cocktail containing 10 μg each of LgasBSHa, LjohBSHc and LsalBSH prepared by exchanging the BSHs from their storage buffer into PBS. PBS or the BSH cocktail was administered once on day −1, twice on days 0 and 1, and once on day 2 before necropsy. Infected mice were challenged with 105C. difficile spores at day 0, weighed daily and monitored for clinical signs of distress . At day 2, all mice  were humanely killed and necropsy was performed. Small intestinal and caecal contents were collected for enumeration of vegetative C. difficile c.f.u.s using TCCFA. Contents were also immediately frozen and stored at −80 °C for later metabolomic analysis.Male C57BL/6J mice (6–8 weeks old) housed at 3 mice per cage were given 0.5 mg ml78. Synthesis of the conjugated bile acids was adapted from a previously published method79. Bile acid  was dissolved in anhydrous tetrahydrofuran  and cooled to 0 °C with stirring. Ethyl chloroformate  was added, followed by triethylamine  and the reaction was stirred for 1.5 h at 0 °C. After conversion of starting material by thin-layer chromatography (TLC), a cold solution of amino acid  and either NaHCO3 or NaOH  in H2O  was added in one portion. Then, the reaction was stirred for 2 h, allowing it to gradually warm to r.t. After this time, tetrahydrofuran was removed in vacuo, then 2 M HCl was added to acidify the reaction mixture to pH < 2, producing a white precipitate. The mixture was extracted in ethyl acetate (3 × 20 ml), then the combined organic layers were washed with brine (50 ml), dried over Na2SO4 and concentrated in vacuo. The crude residue was purified over silica gel using methanol and dichloromethane with 1% acetic acid.All MCBAs were purified according to literature precedent and characterization data are consistent with reported dataOrganic solutions were concentrated under reduced pressure on a Büchi rotary evaporator using a water bath. Chromatographic purification of products was accomplished by flash chromatography on Silicycle F60 silica gel. All reactions were carried out in well-ventilated fume hoods. TLC was performed on Silicycle 250 μm silica gel plates. Visualization of the developed chromatogram was performed by irradiation with 254 nm UV light or treatment with a solution of ceric ammonium molybdate stain, followed by heating. Yields refer to purified compounds unless otherwise noted.1H and 13C NMR spectra are displayed in Supplementary Fig. 1H and 13C, respectively) instrument using Bruker TopSpin (v3.6.0) and are internally referenced to residual protiosolvent signals of CD3OD at δ 3.31 and 49.00 and (CD3)2SO at 2.50 and 39.52. Data for 1H NMR were analysed using MNova (v14.2.0) and are reported as follows: chemical shift (δ ppm), integration, multiplicity  and coupling constant (Hz). Data for 13C NMR are reported in terms of chemical shift and no special nomenclature is used for equivalent carbons.33.Glu-CDCA, Ile-CA, Leu-CA, Phe-CA, Trp-CDCA and Tyr-CA were purified according to literature precedent and characterization data are consistent with reported data3 was used as the inorganic base. Product was purified using 3–10% CH3OH in CH2Cl2 with 1% acetic acid to obtain a 92% yield as a white amorphous solid. 1H NMR  δ 4.40–4.29 , 3.96 , 3.80 , 3.41–3.36 , 2.33–2.21 , 2.20–2.12 , 2.00–1.93 , 1.92–1.77 , 1.77–1.70 , 1.69–1.63 , 1.63–1.50 , 1.47–1.40 , 1.38 , 1.36–1.24 , 1.11 , 1.03 , 0.98 , 0.91 , 0.71 . 13C NMR  δ 176.46, 74.01, 72.83, 69.02, 49.85, 48.04, 47.46, 43.14, 42.93, 40.97, 40.40, 36.83, 36.47, 35.86, 35.82, 33.85, 33.09, 31.13, 29.52, 28.65, 27.81, 24.21, 23.18, 17.80, 17.76, 13.01. HRMS (ESI) exact mass calculated for (M + H)+ (C27H46NO6) requires m/z 480.3320, found 480.3320 with a difference of 0.00 ppm.Ala-CA: NaHCO3OH in CH2Cl2 with 1% acetic acid to obtain a 41% yield as a white amorphous solid. 1H NMR  δ 8.52 , 8.24 , 7.20 , 4.58 , 3.79 , 3.38 , 3.24 , 3.05 , 2.34–2.21 , 2.20–2.08 , 2.03–1.93 , 1.93–1.81 , 1.78–1.69 , 1.69–1.57 , 1.57–1.40 , 1.40–1.22 , 1.21–1.06 , 1.02–0.96 , 0.95 , 0.93 , 0.68 . 13C NMR  δ 176.35, 175.72, 132.60, 118.14, 72.84, 69.06, 57.31, 54.33, 51.56, 43.67, 43.15, 41.06, 40.74, 40.46, 36.96, 36.54, 36.21, 35.91, 34.06, 34.00, 33.10, 31.35, 29.27, 24.63, 23.39, 21.78, 18.92, 12.18. HRMS (ESI) exact mass calculated for (M + H)+ (C30H48N3O5) requires m/z 530.3589, found 530.3591 with a difference of 0.38 ppm.His-CDCA: NaOH was used as the inorganic base. Product was purified using 3–10% CH3 was used as the inorganic base. Product was purified using 6–12% CH3OH in CH2Cl2 with 1% acetic acid to obtain a 74% yield as an off-white amorphous solid. 1H NMR  δ 4.33 , 3.95 , 3.88–3.75 , 3.41–3.36 , 2.40–2.33 , 2.31–2.16 , 2.03–1.98 , 1.95–1.71 , 1.69–1.63 , 1.63–1.51 , 1.47–1.35 , 1.34–1.26 , 1.11 , 1.04 , 0.98 , 0.91 , 0.71 . 13C NMR  δ 176.47, 74.03, 72.88, 69.04, 63.77, 49.85, 48.04, 47.50, 43.20, 42.99, 41.02, 40.46, 37.00, 36.49, 35.90, 35.86, 34.17, 33.04, 31.18, 29.58, 28.72, 27.88, 24.24, 23.17, 17.80, 13.01. HRMS (ESI) exact mass calculated for (M + H)+ (C27H46NO7) requires m/z 496.3269, found 496.3267 with a difference of 0.40 ppm.Ser-CA: NaHCO3OH in CH2Cl2 with 1% acetic acid to obtain 49% yield as an off-white amorphous solid. 1H NMR  δ 7.58 , 7.34 , 7.14–7.07 , 7.02 , 4.75 , 3.93 , 3.81 , 3.42–3.35 , 3.17 , 2.34–2.20 , 2.14–2.06 , 2.03–1.93 , 1.86–1.76 , 1.76–1.65 , 1.64–1.50 , 1.49–1.41 , 1.41–1.29 , 1.28–1.15 , 1.13–1.05 , 1.05–1.00 , 0.98 , 0.92 , 0.67 . 13C NMR  δ 176.70, 175.49, 137.97, 128.83, 124.30, 122.35, 119.78, 119.21, 112.27, 111.07, 74.04, 72.84, 69.08, 54.62, 47.94, 47.40, 43.10, 42.90, 40.91, 40.40, 36.71, 36.43, 35.84, 35.80, 33.79, 32.96, 31.12, 29.47, 28.54, 28.45, 27.80, 24.19, 23.14, 17.70, 12.96. HRMS (ESI) exact mass calculated for (M + H)+ (C35H51N2O6) requires m/z 595.3742, found 595.3737 with a difference of 0.84 ppm.Trp-CA: NaOH was used as the inorganic base. Product was purified using 6–12% CH1H NMR  δ 7.30 – 7.16 , 4.64 , 3.58 , 3.48 , 3.22 , 2.94 , 2.19 , 2.11–2.00 , 1.94 , 1.81 , 1.77–1.66 , 1.66–1.57 , 1.50–1.32 , 1.27–1.12 , 1.10–1.00 , 0.93 , 0.69 . 13C NMR  δ 176.63, 138.67, 130.26, 129.37, 127.70, 77.16, 74.34, 71.83, 57.19, 56.46, 49.21, 44.75, 41.38, 41.13, 39.50, 38.44, 36.76, 36.70, 36.48, 34.92, 33.85, 33.21, 30.67, 29.58, 28.19, 26.16, 21.98, 19.01, 12.63. HRMS (ESI) exact mass calculated for (M + H)+ (C33H50NO6) requires m/z 556.3633, found 556.3636 with a difference of 0.54 ppm.Phe-βMCA: product isolated in 91% yield as a white solid. 1H NMR  δ 7.03 , 6.68 , 4.53 , 3.58 , 3.53–3.42 , 3.12 , 2.88–2.80 , 2.25–2.17 , 2.10–2.00 , 1.97–1.90 , 1.88–1.79 , 1.78–1.67 , 1.66–1.57 , 1.51–1.34 , 1.27–1.14 , 1.09 , 1.08–1.01 , 0.93 , 0.70 . 13C NMR  δ 157.06, 131.33, 129.71, 116.05, 77.18, 74.36, 71.84, 57.20, 56.48, 49.23, 44.77, 41.39, 41.15, 39.51, 37.97, 36.77, 36.76, 36.48, 34.93, 34.07, 33.29, 30.68, 29.60, 28.21, 26.15, 21.99, 19.02, 12.63. HRMS (ESI) exact mass calculated for (M + H)+ (C33H50NO7) requires m/z 572.3582, found 572.3585 with a difference of 0.52 ppm.Tyr-βMCA: product isolated in 56% yield as a white solid. −1) AA-CA, AA-CDCA, AA-DCA or AA-βMCA for 1 h in 50 μl volumes in triplicate. Samples (10 μl) were taken at 5 min, 30 min and 1 h, immediately quenched by diluting them into 90 μl methanol and stored at −80 °C. Reactions containing no BSH were used as a negative control. Percent deconjugation was calculated at each time point as follows:BSH activity assay with the pooled AA-BAs was carried out using similar reaction conditions as described above in the BSH activity assays with purified BAs. BSH (100 nM) was reacted with  and 10 μl of the extracted sample were added into the wells of a 96-well filter plate  fixed on top of a deep-well plate (Waters QuanRecovery) and extracted with 100 μl methanol by shaking at 600 r.p.m. for 20 min with a plate mixer (Thermo Fisher). Elution of the methanol extracts was performed using a positive-pressure manifold (Waters) into the lower receiving deep-well plate, which was then detached from the upper filter plate. After adding 50 μl MS-grade water to the extracts and shaking briefly , samples from the microbial conjugated AA-BA pooled activity assays were injected (5 μl) without dilution or extraction.For metabolomic analysis of treated pre-FMT samples and murine intestinal contents, thawed ex vivo samples were homogenized using a genie disrupter (Scientific Industries) for 15 min after vortex mixing for 30 min. Samples were then centrifuged at 7,000 × For the quantification of the bile acids, an internal standard mixture of 16 deuterated bile acids (containing 6 unconjugated and 10 conjugated bile acids) was created by combining equal volumes of one vial of the unconjugated  and one vial of the conjugated  bile acids to make a 50 μM solution in 1.0 ml 1:1 methanol:water. This stock was further diluted and added in equal volumes (10 μl) to each sample, blank, calibration point and quality control at a final concentration of 10 μM. Calibration curves were prepared by making a mixture of 16 unlabelled bile acids (containing 6 unconjugated and 10 conjugated bile acids) by combining equal volumes of a vial of the unconjugated  and a vial of the conjugated  bile acids to make a 400 μM solution in 1:1 methanol:water. Serial dilutions were prepared to create a 9-point calibration curve ranging from 500 nM to 400 μM and using the concentrations 500 nM, 1 μM, 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, 200 μM and 400 μM.−1 and 15% B with isocratic elution for 2 min, followed by a stepped gradient going to 80% B over the next 7.7 min . The flow rate and mobile phase composition were then increased to 0.8 ml min−1 and 85% B for an additional 0.8 min, followed by re-equilibration at initial conditions for 2 min, resulting in a total run time of 12.5 min. Samples were analysed at negative ion mode  and data were collected from 50–1,700 m/z with an IMS drift potential of 17.2 V cm−1, frame rate of 0.09 frames per second, IMS transient rate of 16 IMS transients per frame, maximum IMS drift time of 60 ms and TOF transient rate of 600 transients per IMS transient. LC–IMS–MS spectra were acquired using MassHunter Acquisition Software and raw files (.d) were uploaded to Skyline-daily (v22.2) for peak picking and molecular annotation using a library built from the synthesized bile acid mixtures and standard mixtures. Peak areas per compound per sample were exported to Microsoft Excel and utilized for statistical analysis in GraphPad Prism. All LC–IMS–MS data are publicly available on Panorama under the Panorama dashboard of the Baker Lab-NCSU within the ‘0821 BSH Assessment’, ‘1121 Mouse Germination and Growth Ex-Vivo’ and the ‘1121 Theriot FMT Ex-Vivo Growth’ projects.LC–IMS–MS analyses were performed using an Agilent 1290 Infinity UPLC system coupled with an Agilent 6560 IM-QTOF MS instrument. Chromatographic separation was achieved using a Restek Raptor C18 column  heated to a temperature of 60 °C. Mobile phase A was composed of 5 mM ammonium acetate, while mobile phase B was 1:1 methanol:acetonitrile. The LC initially started at 0.5 ml min80. Briefly, individual samples were subjected to methanol extraction and then split into aliquots for analysis by ultrahigh-performance liquid chromatography-mass spectrometry. The global biochemical profiling analysis comprised four unique arms consisting of reverse-phase chromatography positive-ionization methods optimized for hydrophilic compounds (LC–MS Pos Polar) and hydrophobic compounds (LC–MS Pos Lipid), and reverse-phase chromatography performed under negative-ionization conditions (LC–MS Neg) as well as a hydrophilic interaction liquid chromatography (HILIC) method coupled to negative ionization (LC–MS Polar). All the methods alternated between full-scan MS and data-dependent MSn scans. The scan ranges differed slightly between methods but generally covered 70–1,000 m/z.Metabolomic analyses of untreated pre-FMT samples were performed by Metabolon in the same manner as described in our previous studym/z), preferred adducts, in-source fragments as well as associated MS spectra, and were curated by visual inspection for quality control using software developed at Metabolon. Identification of known chemical entities was based on comparisons to metabolomic library entries of purified standards81.Metabolites were identified by automated comparison of the ion features in the experimental samples to a reference library of chemical standard entries that included retention time, molecular weight  with Tukey’s multiple comparisons test. Comparisons were made separately between the wild-type and mutant versions of LgasBSHa and LgasBSHb. Inhibition of C. difficile spore germination, membrane integrity and PtcdA-mCherry reporter assays were analysed from n = 3 experiments with a one-tailed Welch’s t-test. Statistical comparisons were only made between conditions containing related BAs . C. difficile growth and bile acid metabolomics in pre-FMT samples were analysed from n = 3 experiments using a one-way ANOVA with Sidak’s correction for multiple comparisons. In the cases where c.f.u. or bile acid metabolomic data were generated from intestinal contents that were split and treated with PBS or a BSH cocktail, one-tailed ratio paired t-tests were used to analyse findings. In vivo c.f.u.s were analysed with a Mann-Whitney test. Bile acid metabolomics from in vivo samples were analysed using a Kruskal-Wallis with Dunn’s test for multiple comparisons. All graphed bars represent mean ± s.d. Asterisks indicate significant differences . See Reporting Summary and figure legends for more details.All statistical analysis was performed in GraphPad Prism 8 or 9. No statistical methods were used to pre-determine sample sizes, but our sample sizes are similar to those reported in previous publications. All data met the assumptions of the statistical tests used and data distribution was assumed to be normal in logFurther information on research design is available in the Supplementary InformationSupplementary Figs. 1–10.Reporting SummaryPeer Review FileSupplementary Tables 1–6Table 1: Lactobacillaceae BSH cluster and phylogeny information. Table 2: Purified BSH sequences and information. Table 3: Mutagenesis primers. Table 4: IGC sequences and selectivity loop. Table 5: CD analysis. Table 6: X-ray crystallography statistics.Supplementary Data 1P values from all statistics.Supplementary Data 27SVE PDB validation report.Supplementary Data 37SVF PDB validation report.Supplementary Data 47SVG PDB validation report.Supplementary Data 57SVH PDB validation report.Supplementary Data 67SVI PDB validation report.Supplementary Data 77SVJ PDB validation report.Supplementary Data 87SVK PDB validation report.


Introduction: Polysaccharides are important components of Panax notoginseng that contribute to its immunomodulatory ability. This study aimed to isolate polysaccharides from notoginseng and investigate the structural feature and potential immunomodulatory activity.Methods: The polysaccharide was isolated from notoginseng by anion exchange and gel permeation chromatography. Its preliminary structure was characterized by Fourier transform infrared (FT-IR) spectroscopy, gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. The immunoregulatory function was further investigated in cyclophosphamide induced immunosuppressive mice, murine splenocytes and macrophages.Results: A novel homogeneous polysaccharide (PNPB1) was isolated from notoginseng with the molecular weight of 9.3 × 105 Da. Monosaccharide composition analysis indicated that PNPB1 consisted of Glc (88.2%), Gal (9.0%), Ara (2.4%) and trace GlcA, with the major backbone of (1→4)-linked α-Glcp, (1→6)-linked β-Glcp, and -linked β-Glcp. The polysaccharide was found to significantly enhance murine body weight, improve their thymus and spleen indices and increase the white blood cells (WBC). PNPB1 significantly enhanced splenic lymphocyte proliferation, NO and cytokine  production, as well as the phagocytosis and TLR2 expression of peritoneal macrophages, indicating potent immunoenhancement effect.Discussion: These findings provide a theoretical basis for elucidating the structure and immune activity of notoginseng polysaccharides. Panax notoginseng (Burk.) F.H. Chen, known as Sanqi or Sanchi in Chinese, is a precious medicinal plant cultivated in south-western China. The root and rhizome of P. notoginseng have been traditionally used as a tonic and haemostatic in East Asian countries for centuries. Notoginseng possesses various bioactive ingredients such as saponins, flavonoids and polysaccharides, exhibiting numerous pharmacological effects including haemostatic, antioxidant, hypolipidaemic, hepatoprotective, anti-tumour and immunomodulatory activities (P. notoginseng polysaccharides (PNP), was ignored previously and has been attracting more attention due to its immunomodulating activities. Notoginseng saponins, most of which are dammarane-type triterpene saponins, have been intensively studied in recent decades, revealing their favorable roles in the improvement of nervous, cardiovascular, endocrinic and immune functions  spectroscopy, gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Consequently, the immunomodulatory activities were evaluated using immunosuppressive mice, murine splenocytes and peritoneal macrophages. This work will provide chemical and bioactive information for the structure elucidation and potential application of notoginseng polysaccharides.P. notoginseng was obtained from Panlongyunhai Pharmaceutical Co. Ltd . DEAE Bestarose FF was purchased from Bestchrom . Sephadex G-200 was purchased from Solarbio . Standard monosaccharides, trifluoroacetis acid (TFA), methyl iodide were supplied by Adama-beta Co. . Pyridine, dimethyl sulphoxide (DMSO), acetic anhydride and sodium hydride were purchased from General-Reagent Co. . Methanol (HPLC grade) was from Tedia . 1-pheny-3-methyl-5-pyrazolone (PMP) was from Macklin Biochemical Co., Ltd. . Cyclophosphamide (CTX) was provided by Ammonium acetate was from Anaqua Chemicals . All other chemicals and solvents were of analytical grade.g for 5 min, then the supernatant was combined and concentrated by rotary evaporator at 60°C under reduced pressure. The concentrated extract was precipitated by adding 4 volumes of ethanol and placed at 4°C for overnight. The resulting precipitate was dissolved in appropriate volumes of distilled water to remove protein by Sevag reagent, and then lyophilized to obtain crude polysaccharide (The crude polysaccharide was prepared following the ethanol subsiding method . Grindedccharide .The crude polysaccharide (500 mg) was redissolved in distilled water and passed through DEAE Bestarose FF column (50 × 600 mm) and eluted successively with distilled water, 0.1, 0.2, 0.3 and 0.5 M NaCl solutions at a flow rate of 2 mL/min . The fouPWXL column . The sample solution  was injected and eluted with distilled water at 30°C with a flow rate of 1.0 mL/min (The homogeneity and molecular weight of PNPB1 were determined by high-performance gel permeation chromatography (HPGPC) using a Thermo Ultimate 3,000 system  equipped with an Alltech 2000 ES evaporative light scattering detector (ELSD) and a TSK gel G40000 mL/min . The mol18 column  and UV detection at 245 nm according to a previously described method  indicated complete methylation (PNPB1 was methylated according to a previously described method . The methylation . The met−1.PNPB1 (2.0 mg) was dissolved in 1 mL of distilled water, and detected the UV-Vis absorption spectra in the wavelength rang of 190–700 nm. PNPB1 (2.0 mg) was mixed with about 200 mg of KBr and pressed into a pellet for FT-IR determination in the frequency range of 4,000–400 cm2O (1.0 mL) followed by H/D exchange reaction for three times. The 1H, 13C, HMBC and HSQC spectra of PNPB1 were recorded by Agilent DD2 600 MHz NMR spectrometer  with chemical shifts given in ppm.The dried PNPB1 of 40 mg were dissolved in DThe polysaccharide powder was coated with conductive gold, and then examined using a JSM-6510 SEM system  under different magnifications.Twenty-four male BALB/c mice  purchased from Liaoning Changsheng Biotechnology Co., Ltd.  were housed at a constant temperature (21°C ± 2°C) with a 12 h light-dark cycle with free access to food and water. The experimental protocols were approved by the Ethics Committee of Hubei University of Chinese Medicine. After 1 week acclimatization, the mice were randomly divided into 4 groups with 6 for each. The low and high dose treatment groups were given PNPB1 by intraperitoneal injection with the dosage of 5 and 20 mg/kg/d respectively for 14 days, whereas the mice of control and model group were treated with normal saline. The model and treatment groups were subjected to immunosuppression by intraperitoneal injection of CTX (80 mg/kg/d) at day 10, 11, 12 and 13. The weight of each animal was recorded daily throughout the experiment. Twenty-four hours after the last drug administration, the animals were subjected to carbon clearance test and then sacrificed for harvesting immune organs.Whole blood samples (100 μL) were collected from the retro-orbital plexus of mice 12 h after the last treatment and the WBC and RBC count of peripheral blood were analyzed using Mindray Vet2800 blood analyzer .The phagocytosis of macrophages in liver and spleen was assessed based on the scavenging efficiency of carbon particles in blood . At the Mice were weighed and killed by cervical dislocation after carbon clearance test, and thymus and spleen were excised from the mice and weighted immediately. The thymus and spleen indexes were calculated according to the following formula:6 cells/mL) were seeded into the 96-well plate containing indicating concentrations of PNPB1  or ConA (5 μg/mL) or only RPMI-1640 medium in triplicate and then incubated for 2 days at 37°C in humidified 5% CO2. Cell proliferation was measured using a CCK-8 assay kit according to the manufacturer’s instructions. The ODs were measured using a Tecan Sunrise microplate reader  at 450 nm. The proliferation of splenocytes was defined with stimulation index (SI) showed as follow: SI = sample absorbance/control absorbance.BALB/c mice at 8–10 weeks of age were used for obtaining splenocytes and peritoneal macrophages. Splenocytes were prepared as described by 6 cells/mL and incubated with PNPB1 at different concentrations  or ConA (5 μg/mL) or RPMI-1640 medium alone. After 48-h stimulation, NO content in cell supernatant was determined by Griess reaction and the concentrations of cytokines  were measured by ELISA kits  according to the manufacturer’s instructions.Splenocytes (150 μL) were seeded into a 96-well plate at a density of 2.5 × 106 cells/mL in 96-well plate with or without PNPB1  or LPS (1 μg/mL) and cultured for 24 h. Supernatants were removed and 100 μL of 0.1% (w/v) neutral red solution was added and incubated for 30 min. Subsequently, the cells were washed 3 times with PBS to remove excess dye and lysed in 200 μL of 50% ethanol containing 1% glacial acetic acid. The absorbance was read at 550 nm on a microplate reader.Phagocytosis of peritoneal macrophages was measured by neutral red uptake assay as previously described . PeritonIn-Cell Western (ICW) is an immunofluorescence approach utilizing near infrared fluorophore-conjugated antibodies to measure individual proteins in fixed cells, which was performed to detect TLR2/4 protein level in present study using Odyssey Infrared Imaging System (LI-COR Biosciences). The In-Cell Western Kits provide detection reagents for cell-based ICW assays, which includes blocking buffer, IRDye 800CW secondary antibody for detection of a specific protein in 800 nm channel, and two fluorescent cell stains used in 700 nm channel to normalize variations in cell number. Peritoneal macrophages were collected and cultured in DMEM (containing 10% FBS) with or without PNPB1  or FSL (1 μg/mL) and then cultured for 24 h. After removing the medium, cells were fixed by adding 25 μL of 4% formaldehyde and incubating for 20 min, and then washed by 0.1% Triton X-100. Cells were blocked by LI-COR blocking buffer for at least 2 h at room temperature followed by incubation with primary antibodies  and celltag 700 stand internal reference  solutions diluted in blocking buffer, respectively, at 4 °C overnight. After washing with 0.1% (v/v) Tween-20, cells were incubated with IRdye 800CW Goat anti-rabbit secondary antibody at 1:5,000 in blocking buffer for 2 h at room temperature, and then washed with 0.1% Tween-20 again. The plate was scanned at 700 and 800 nm channels at the same time to obtain fluorescent images and output data.p < 0.05 was considered statistically significant.Statistical analysis was performed by GraphPad Prism 6 . Statistical significance between two groups was analyzed by Student’s t-test and multigroup comparisons were compared using one-way analysis of variance (ANOVA). Data was presented as the mean value ±SD, and P. notoginseng by hot water and ethanol precipitation and the yield was determined as 12.48% after removing protein with Sevag method. As shown in 5 Da.The crude polysaccharides were isolated from −1 was attributed to the stretching vibration of O-H, and the band at 2,927.65 cm−1 was the stretching vibration of C-H. The absorption peaks at 1,639.83 and 1,413.30 cm−1 were assigned to the stretching vibration of carboxylic carbonyl group and C-O, respectively, indicating the presence of uronic acid in PNPB1 -β-Glcp- -β-Galp--α-Glcp--β-Galp--β-Glcp-, δ4.55/70.14 (DH1/GC6), δ5.30/76.77 (EH1/EC4), δ5.15/76.40 (AH1/FC4), δ4.99/66.44 (BH1/AC5), δ4.55/70.33 (CH1/DC6), δ4.55/76.40 (DH1/FC4), δ4.45/69.29 (FH1/CC6), δ4.55/77.62 (CH1/GC4), δ99.70/3.71 (BC1/GH4) and δ4.55/76.77 (DC1/EH4) were presented as the linkages of residue E O-1 with residue D C-6, residue D O-1 with residue G C-6, residue E O-1 with residue E C-4, residue A O-1 with residue F C-4, residue B O-1 with residue A C-5, residue C O-1 with residue D C-6, residue D O-1 with residue F C-4, residue F O-1 with residue C C-6, residue C O-1 with residue G C-4, residue G O-4 with residue B C-1 and residue E O-4 with residue D C-1. Based on the results of monosaccharide composition, methylation analysis and NMR analysis, the possible repetitive structure unit of PNPB1 was deduced and depicted in The sample was morphologically characterized by SEM as a laminar structure with smooth surface under low magnification (×100), while the microstructure up to 3,000 times magnification revealed the arrays of irregular dotted pits in the surface .9/L, while those in low and high PNPB1 group markedly increased to 1.1 × 109/L and 1.9 × 109/L, respectively (p < 0.05). In addition, the peripheral blood cell levels of mice were unaffected by CTX or PNPB1 treatment and each group displayed no statistical difference.Being an anti-mitotic and apoptosis inducing agent, CTX has strong immunosuppressive effect and is often used for the preparation of immunosuppressive model, which causes body weight loss and bone marrow suppression in animals. Previous studies have found some notoginseng polysaccharides, such as NPPN, a neutral polysaccharide (232.50 kDa), played a bone marrow protective role through increasing peripheral blood counts, relieving cell cycle arrest, and decreasing the apoptosis rate of bone marrow cells in myelosuppression mice induced by CTX . By contp < 0.05) than those of model group and showed a dose-dependent manner. These results suggested that PNPB1 could increase macrophage phagocytosis to a normal level in immunosuppressed mice, which was consistent with the results of p < 0.05) in a dose-dependent manner, which were better than the corresponding immune effects reported for other notoginseng polysaccharides . The productions of IFN-γ and IL-2 were also increased remarkably with PNPB1 concentrations up to 50 μg/mL (p < 0.05), especially the secretions of IFN-γ, which were elevated to 57 and 68 times those in the control group after treatment with PNPB1 at 50 and 200 μg/mL, respectively (p < 0.01). However, PNPB1 showed little impact on the IL-4 production in splenocytes over 5 μg/mL, but strongly elevated the concentration of IL-10 even at the lowest concentration (5 μg/mL), indicating that PNPB1 could enhance both Th1 and Th2 cytokines production with varying degrees in vitro, and has the potential to alter Th1/Th2 immune balance.Besides the stimulation on the proliferation of immune cells, promoting the cytokine secretion is another important way for polysaccharides to exert immune function. Cytokines are small molecular proteins or polypeptides mainly secreted by activated immune cells, which are vital for lymphocytes to mediate immune response. Naive CD4h2 cells . Liu et h2 cells . CurrentNO, a cytotoxic substance secreted by activated immune cells to kill pathogens and tumor cells, is also an important mediator of intercellular information transmission that can enhance the nonspecific immune function through complex interaction with various cytokines . As showToll like receptor 2/4 (TLR2/4) are considered to play important roles in the innate immune response and involved in the immediate immune response induced by polysaccharides, which can be directly induced by stimulus and mediate the secretion of NO, TNF, IFNs, ILs and other substances from immunocytes. The receptor affinity of polysaccharides is closely related to their structure, for instance, glucose and mannose fractions in polysaccharides are prone to be recognized by TLR2, whereas carboxylic or acetyl groups are essential in TLR4 binding. The above mentioned polysaccharide Pnp can bind to TLR2, while PNPS-0.3 might primarily bind to TLR4 due to the abundant carboxylic group in GalA fractions . The proin vivo and in vitro. PNPB1 possesses a high molecular weight up to 930 kDa with a complex structure involving three kinds of monosaccharides and seven types of linkages. The present work demonstrated that PNPB1 could significantly enhance body weight, improve thymus and spleen indices and increase the WBC in immunosuppressive mice. The polysaccharide might mediate immunopotentiation through the enhancement of splenocyte proliferation, secretion of TNF-α, IFN-γ, IL-2, IL-10 as well as phagocytosis of macrophages, and involve the protein regulation of TLR2, which could be used as a potential immunological response modifier.A novel water-soluble polysaccharide PNPB1 was isolated from notoginseng. Its chemical structure was characterised by FT-IR, GC–MS, 1D and 2D NMR spectroscopic techniques and the immunomodulatory activities were studied


Human epidermal growth factor receptor 2 (HER2) positive breast cancer is an aggressive subtype, accounting for around 20% of all breast cancers. The development of HER2-targeted therapy has substantially improved patient outcomes. Nevertheless, the increasing rate of side effects and resistance to targeted drugs limit their efficacy in clinical practice. In this study, we designed and synthesized a new immunotoxin, 4D5Fv-PE25, which targets HER2-positive breast cancer, and evaluated its effectiveness in vitro and in vivo.Escherichia coli (E. coli.) using the fermentor method and refined via hydrophobicity, ion exchange, and filtration chromatography, achieving a 56.06% recovery rate. Additionally, the semi-manufactured product with 96% purity was prepared into freeze-dried powder by the lyophilized process. Flow cytometry was used to detect the expression of HER2 in SK-BR-3, BT-474, MDA-MB-231, and MDA-MB-468 breast cancer cell lines. The 3--2,5-diphenyl tetrazolium bromide (MTT) method was used for cytotoxicity assay, and the half-maximal inhibitory concentration (IC50) of 4D5Fv-PE25 lyophilized products to HER2-positive cell line SK-BR-3 was 12.53 ng/mL. The 4D5Fv-PE25 was injected into xenograft tumor mice via the tail vein on the 1st, 4th, and 8th day, it indicated that the growth of tumor volume was effectively inhibited for 24 days, although the 4D5Fv-PE25 was metabolized within 60 min by measuring the release of 3 H-Thymidine radiation.The 4D5Fv-PE25 was expressed in high-density we succeeded in producing the 4D5Fv-PE25 freeze-dried powder using the prokaryotic expression method, and it could be employed as a potential drug for treating HER2-positive breast cancer. Breast cancer is the most prevalent neoplasm and a significant contributor to cancer-related deaths in women . Based oIn the first-line category of drugs approved for treating HER2-positive breast cancer, trastuzumab is the gold standard. Small molecule drugs such as Lapatinib (a protein tyrosine kinase inhibitor for HER2-positive breast cancer) and Antibody-drug conjugates (ADCs) like trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd) belong to the second-line drugs –7. The APseudomonas exotoxin A), approved by the Food and Drug Administration (FDA) [Immunotoxin is a heterogenous molecule that exerts specific targeted killing effects, consisting of tumor-specific recognition antibodies and toxin molecules. Compared to conventional drugs, it is better suited due to its strong effect and diminished incidence of drug resistance , 13. Immon (FDA) . As suchIn this research, a novel immunotoxin, 4D5Fv-PE25, was developed for HER2-positive breast cancer management. We evaluated the pharmaceutical properties of 4D5Fv-PE25 via cell viability assays and mice experiments. The findings demonstrated the capacity of 4D5Fv-PE25 to recognize and inhibit HER2 targets effectively in vivo and in vivo, making it a highly suitable candidate drug for the management of HER2-positive breast cancer.4S)3) and a truncated form of Pseudomonas exotoxin A (PE25)  Fig. . The varE. coli. BL21 strain used in this study was engineered to overexpress the target gene through a constructed expression plasmid named 4D5Fv-PE25. Bacterial growth was monitored periodically during fermentation, and the process was terminated upon reaching the stable phase , failed to completely elute 4D5Fv-PE25 using chromatography buffer A or deionized water. Significant amounts of 4D5Fv-PE25 remained on the gel and could only be eluted using sodium hydroxide  through Sephadex G25 chromatography, and the products were collected for concentration optimization. After the configuration, filtration sterilization, filling, and freeze-drying processes, the lyophilized powder of the 4D5Fv-PE25 with a loose texture was obtained Fig. B.Breast cancer cell lines were analyzed for HER2 expression using flow cytometry. SK-BR-3 and BT-474 were used as the positive control due to their high HER2 expressions. SK-BR-3 expressed significantly higher levels of HER2 than BT-474. The triple-negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468, were used as negative controls. MDA-MB-468 had no HER2 expression while HER2 expression in MDA-MB-231 was observed at low levels   through the tail vein on the 1st, 4th, and 8th day when the tumor volume of mice reached 200 mmHER2-positive breast cancer has the highest incidence rate in the world, accounting for 7.26% of all new malignant tumors . Early-sPseudomonas aeruginosa cytotoxin. The 4D5Fv-PE25 was produced using a highly controllable, low-cost, high-yield, and time-saving prokaryotic expression system [E. coli. as the expression strain effectively reduced the toxicity of PE25 to the host, while increasing expression efficiency [Immunotoxins are targeted drugs obtained via heterologous expression by fusing a targeted vector with a gene fragment of a toxin protein through gene recombination technology. Immunotoxins can be recognized by specific receptors and internalized through receptor-mediated endocytosis. This enables the toxin to be enriched and activated in the target cells, leading to an increase in therapeutic efficiency and a reduction in effects on non-specific cells . This tan system , 34. Usificiency . After hficiency , making ficiency . Therapeficiency . The molficiency –43. Thro50 of 4D5Fv-PE25 against HER2-positive breast cancer cell line SK-BR-3 was found to be 11.29 ng/mL, significantly lower than the IC50 value of 3.00 µg/mL observed for HER2-negative breast cancer cell line MDA-MB-468. This result confirmed that 4D5Fv-PE25 effectively recognizes and inhibits the growth of HER2-positive breast cancer cells. Furthermore, normal cells expressing low levels of HER2 should remain unaffected by this toxin [50 of the 4D5Fv-PE25 can still reach 12.53 ng/mL with an 89% activity recovery rate. In comparison to monoclonal antibodies and other small-molecule drugs, 4D5Fv-PE25 demonstrates significant anticancer activity [Subsequently, we assessed the potential of using 4D5Fv-PE25 as a treatment for HER2-positive breast cancer. To determine the appropriate dosage for in vivo and in vitro assays, we conducted a cell survival assay. The ICis toxin , indicatactivity . Accordiactivity , 48. Theactivity , 50. Thiactivity . Of courIn summary, we designed and manufactured a lyophilized powder immunotoxin 4D5Fv-PE25 with stable purity above 96% for HER2-positive breast cancer. The preparation process of 4D5Fv-PE25 has the advantages of good controllability, high preparation efficiency, and low-quality risk, which is conducive to industrialization. Our in vivo and in vitro studies confirmed the significant targeted recognition and inhibition of 4D5Fv-PE25 to HER2-positive breast cancer cell lines and solid tumors. Mouse experiments further showed that it had a long-term inhibitory effect on tumors despite the rapid loss of 4D5Fv-PE25 activity in plasma, providing perspective for future mechanistic exploration of 4D5Fv-PE25. Overall, our findings suggest that 4D5Fv-PE25 is a promising candidate drug for targeted therapy in HER2-positive breast cancer, given its high development value.4S, VH, and Linker, Furin cleavage site, PE25, KDEL sequence were synthesized by Songon Biotech Company. The target gene sequence was digested by the EcoR I and Spe I (Takara Bio) restriction endonucleases, and then, T4 ligase (Takara Bio) was used to connect it to a new plasmid. The 4D5Fv-PE25 sequence was inserted into the pET-28a(+) vectorusing the Xho I/Xba I (Takara Bio) restriction enzyme sites. The recombinant plasmid was transformed into BL21 (DE3) E. coli.The plasmids containing the VL, G+) and cultured at overnight 37℃. A single clone was selected and inoculated in 120 mL liquid LB medium (Kan+), cultured at 37℃ for 6 h. Then the culture medium was transferred into a 6,000 mL LB medium (Kan+) and continued to culture for 10 h.The 4D5Fv-PE25 strain (BL21 (DE3)) was inoculated onto a solid Luria-Bertani (LB) medium plate containing 50 µg/mL kanamycin  cultured in the fermentation tank (NEW BRUNSWICK BF-5000), and added 15 mL of trace element mother liquor . It was incubated for 155 min at 37℃, 220 rpm, and 100% dissolved oxygen, then added 2,400 mL glucose supplement (glucose 0.5 g/mL) and 1,500 mL LB supplement , continued to incubate for 30 min. Afterward, Isopropyl-beta-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM to induce the 4D5Fv-PE25 expression, and ammonia was used for adjusting pH to 6.90–7.20 during the fermentation process. The absorbance (OD600) of the fermentation broth was measured by random sampling. The fermentation broth was collected after 280 min of induced expression, and the pelleted bacteria were collected using a continuous flow centrifuge at 7,227 g, 10℃.The seed liquid was inoculated into 50 L M9 medium The pelleted bacteria were washed with Tris-EDTA (TE) buffer ). Then use the cell crusher to crush the bacteria and collect the bacteria pellet. The pellet was washed successively with lysis bufferI, lysis bufferII and 2 M guanidine-HCl buffer , pH 8.0) at the ratio of 10 mL/mg, the clean 4D5Fv-PE25 inclusion bodies were obtained finally. The denature buffer  was added into 4D5Fv-PE25 inclusion bodies at a ratio of 12 mL/mg for denaturation, then centrifuged at 16,260 g for 20 min to collect the supernatant. The denatured solutions were dropwise added into refolding buffer , 1 mM EDTA, 0.5 M Urea, 0.5 M Arginine, pH 8.5), standing for 12 h at 10℃. Pre-cooled ammonium sulfate solid was added to 30% saturation and stored overnight at 4℃. The 4D5Fv-PE25 crude products containing ammonium sulfate were obtained by centrifugation at 16,260 g, 4℃ for 30 min.Hydrophobic chromatography was implemented using the protein purification system from GE Healthcare (ÄKTA pure 150 Convenience) with columns consisting of strong hydrophobic gel Capto Phenyl (HS) and weak hydrophobic gel Capto Butyl .Chromatography buffer A  with 30% saturation ammonium sulfate was used to equilibrate the gel column (GE XK 26/40) with 5 column volumes, then the crude products were loaded at a flow rate of 2.0 mm/min. The effects of gradient elution  of 30% saturation ammonium sulfate) and eluent containing ethanol ) were compared. The Phenyl/Butyl columns were eluted with water, followed by 0.5 M NaOH. Finally, the eluates were analyzed to assess the purification efficiency of the hydrophobic chromatography by 12% polyacrylamide gel electrophoresis (SDS-PAGE).The chromatographic column GE XK 16/40 and GE XK 26/40 were separately filled with Capto S ImpAct  gel and Capto Q  gel. 5 column volumes of chromatography buffer A and chromatography buffer B  were used respectively for regeneration. The hydrophobic chromatographic eluent was separately sampled on Capto S ImpAct and Capto Q gel for purification. Capto S ImpAct was sampled at 6.0 mm/min and 15.0 mm/min, the column was equilibrated with chromatography buffer A after loading, and the flow-through liquid was collected to detect the purity. The 4D5Fv-PE25 on Capto Q was eluted with chromatography buffer C , and elution peaks were collected to detect protein purity by SDS-PAGE.GE XK 26/100 column was filled with Sephadex G25  gel and equilibrated with 5-column volumes of phosphate buffer saline (PBS). The elution peaks were collected after loading the sample. The high-resolution gel filtration column Superdex 75 10/300 GL  was used to detect purity with a flow rate of 6.5 mm/min, and the wavelength for UV detection was 280 nm.The 4D5Fv-PE25 was prepared according to the dosage of 10mL products  human albumin, 4% (w/w) mannitol) in the 50 mL vial. The semi-products were filtered by the 0.22 µM filter membrane, then filled and lyophilized by a freeze-drying machine (Christ Epsilon 60D Lscplus).2. The cells were detached using trypsin when they reached the logarithmic growth phase to prepare cell suspensions for future use.The human breast cancer cell lines MDA-MB-231 (ATCC HTB-26), MDA-MB-468 (ATCC HTB-132), BT-474 (ATCC HTB-20), and SK-BR-3 (ATCC HTB-30) were cultured in DMEM medium with 10% fetal serum, at 37℃ and 5% COBreast cancer cells were blocked with 5% rabbit serum  in PBS for 20 min, then added 2.5 µg mIgG  or an anti-HER2 antibody . FITC (fluorescein isothiocyanate) goat anti-mouse IgG  was added and incubated for 30 min, followed by a washing step with IF buffer. Finally, t the sample was fixed with 4% paraformaldehyde (PFA) and measured using flow cytometry.4, 3.0 × 103, 1.0 × 103, 3.0 × 102, 1.0 × 102, 3.0 × 101, 1.0 × 101, 3.0, 1.0, 3.0 × 10− 1, 1.0 × 10− 1 ng/mL, and determined the cell viability according to Juliette Sauveur’ method after 72 h culture [The 4D5Fv-PE25, both before and after lyophilization, were prepared as a 1 mg/mL solution. Four different types of cell lines  were seeded at a density of 8,000 cells per well in 96-well plates. A co-culture experiment was conducted by seeding SK-BR-3 and MDA-MB-231 cell lines, each with a density of 4,000 cells, into the same well. The 4D5Fv-PE25 solution was added with the final concentration of 1.0 × 10 culture .The solution of 4D5Fv-PE25 utilized in the experiments involving mice was a freeze-dried product redissolved in water for injection (WFI). The initial concentration was 1 mg/mL, and the sample was diluted to the appropriate concentration. The control group was injected with the lyophilization protective agent.3, the 4D5Fv-PE25 (5 mg/kg) was injected via the tail vein on the 1st, 4th, and 8th day, and the tumor volume was monitored for 24 days after the final injection. Tumor dimensions of mice were measured using calipers, and their volumes were calculated using the formula: Volume = 4/3 × π × length × width×depth/8 [BT-474 xenograft mice  with high expression of humanized HER2 were used as animal models. Upon reaching a tumor volume of 200 mm×depth/8 .4, 3.0 × 103, 1.0 × 103, 3.0 × 102, 1.0 × 102, 3.0 × 101, 1.0 × 101, 3.0, 1.0, 3.0 × 10− 1, 1.0 × 10− 1 ng/mL and plasma sample were diluted 10 times. The samples were added to 96-well plates containing 1.5 × 104 SK-BR-3 cells (200 µL) per well and cultured at 37℃ for 2 days. Added 10 µL 50 µCi/mL 3 H-Thymidine to each well, and recorded the number of ray counts per minute (CPM) [C57BL/6 mice were injected with 5 mg/kg 4D5Fv-PE25, and 25 µL blood samples were collected at 5, 15, 30, 60, 120, and 300 min after injection. The blood samples were collected in EDTA coated centrifuge tube and plasma was collected after centrifuging at 2,000 g for 3 min. The 4D5Fv-PE25 samples were prepared at the concentration of 1.0 × 10te (CPM) , 53. TheData were analyzed by GraphPad Prism 5.0 software. The data in the curve were presented as means (Ā) ± standard deviation (SD), two way ANOVA was performed to determine the difference between the data. and differences were considered statistically significant when p < 0.05. The software Image J version 1.48 was used to roughly calculate the purity of protein by gray analysis.The recovery was calculated by the following formula.aft represents the protein concentration after treatment, Vaft represents the sample volume after treatment, Cbef represents the protein concentration before treatment, Vbef represents the sample volume before treatment. The concentration of protein was measured by the Bradford assay [Crd assay .The purity of the sample was calculated by the formula:tar represents the peak area of the target protein and Amis represents the peak area of miscellaneous protein.A


Angiogenesis plays important roles in physiological and pathologic conditions, but the mechanisms underlying this complex process often remain to be elucidated. In recent years, liquid–liquid phase separation (LLPS) has emerged as a new concept to explain many cellular functions and diseases. However, whether LLPS is involved in angiogenesis has not been studied until now. Here, we investigated the potential role of LLPS in angiogenesis and endothelial function.We found 1,6-hexanediol , an inhibitor of LLPS, but not 2,5-hexanediol  dramatically decreases neovascularization of Matrigel plug and angiogenesis response of murine corneal in vivo. Moreover, 1,6-HD but not 2,5-HD inhibits microvessel outgrowth of aortic ring and endothelial network formation. The endothelial function of migration, proliferation, and cell growth is suppressed by 1,6-HD. Global transcriptional analysis by RNA-sequencing reveals that 1,6-HD specifically blocks cell cycle and downregulates cell cycle-related genes including cyclin A1. Further experimental data show that 1,6-HD treatment greatly reduces the expression of cyclin A1 but with minimal effect on cyclin D1, cyclin E1, CDK2, and CDK4. The inhibitory effect of 1,6-HD on cyclin A1 is mainly through transcriptional regulation because proteasome inhibitors fail to rescue its expression. Furthermore, overexpression of cyclin A1 in HUVECs largely rescues the dysregulated tube formation upon 1,6-HD treatment.CCNA1, implying LLPS as a possible novel player to modulate angiogenesis, and thus, it might represent an interesting therapeutic target to be investigated in clinic angiogenesis-related diseases in future.Our data reveal a critical role of LLPS inhibitor 1,6-HD in angiogenesis and endothelial function, which specifically affects endothelial G1/S transition through transcriptional suppression of The online version contains supplementary material available at 10.1186/s12915-023-01580-8. Angiogenesis, a process of blood vessel formation from pre-existing vascular structures, has a pivotal impact on human health and the pathological process of many diseases, such as female menstrual cycle, tissue growth, wound healing, cancer, ischemia, inflammatory diseases, and blinding eye diseases . The proLiquid–liquid phase separation (LLPS) is a reversible and dynamic process by formation of weak and multivalent interactions between biomolecules, which attracts scientist’s extensive attention, and is believed as a new concept to understand the mechanisms of some complex diseases recently. It was used to explain the problem with the different non-membrane-bound compartments coexisting in cytoplasm at the very beginning . The nonTo assess the potential role of LLPS in angiogenesis, we employed 1,6-hexanediol , which is considered as an inhibitor of LLPS and a powerful tool to study the physical function of membrane-less compartments. 1,6-HD, an aliphatic alcohol, specifically disrupts multivalent hydrophobic interactions between protein-RNA or protein–protein that are favorable to the formation of LLPS , 19, 20.To access whether liquid–liquid phase separation might play a role in pathological angiogenesis, we used 1,6-hexanediol, an inhibitor of LLPS, which mixed into Matrigel to carry out in vivo pathological angiogenesis assay. Matrigel mixed without or with 1,6-HD was injected into C57BL/6 mice subcutaneously. The liquid Matrigel solidifies to a form of plug when injected into mice at the body temperature. This is a widely used an in vivo assay to evaluate angiogenesis. Five days later after injection, the Matrigel plugs were isolated. H&E staining and immunohistochemistry were then performed to determine the formation of neovasculature. Interestingly, the data showed that Matrigel plugs containing 1,6-HD robustly inhibits blood vessel formation, while the plugs from control group showed a proper vessels imbuement from bright field images, H&E, CD31, and VE-cadherin immunohistochemistry (IHC) staining , since 1,6-HD has been reported to affect cell viability in HeLa cells  but mainTo further examine the effect of 1,6-HD on endothelial function, we checked the cell migration, cell growth and proliferation in HUVECs in the treatment with 1,6-HD. HUVECs were placed in a 6-well plate before a pipette tip to scratch and then cultured with low fetal bovine serum condition. Twelve and twenty-four hours later, compared to control cells, cells treated by 1,6-HD showed an impaired scratch healing ability with a dose-dependent manner Fig. A. This rBecause we found that 1,6-HD but not 2,5-HD controls microvessel outgrowth and network formation, we then checked the effects of 2,5-HD on the proliferation of HUVECs to further verify it. Three representative doses were chosen to use of 1,6-HD and 2,5-HD respectively. As expected, 2,5-HD failed to impair the proliferation of HUVECs, while 1,6-HD also showed an inhibitory effect on the proliferation of HUVECs Fig. D, which CCNA1 is the greater changed gene  inhibitors JQ1 and I-BET to treat HUVECs. The expression of CCNA1 is significantly downregulated . It worth to notice that the expression of G1/S transition-related cell cycle genes, such as CDK2, CDK4, cyclin D1, and cyclin E1, has no big change in the present of 1,6-HD  regulated by 1,6-HD in endothelial cell also need to be further investigated.Cyclin A1 was firstly cloned in murine testis which binds to both CDK1 and CDK2  and then7In summary, we showed that suppression of LLPS impairs angiogenesis by cyclin A1 medicated-EC function. LLPS might play a critical role in angiogenesis. To our knowledge, this is the first time to use 1,6-HD to study the potential function of LLPS in angiogenesis. Although the underlying molecular mechanism regarding how LLPS inhibitor specifically regulates the expression of cyclin A1in EC is unaddressed, it is important to realize that the new concept LLPS is very likely play critical role in angiogenesis. LLPS may be a novel therapeutic target for the treatment of angiogenesis-related diseases.In this study, our data demonstrated that LLPS inhibitor 1,6-hexanediol dramatically decreases neovascularization by specific suppression of cyclin A1-mediated endothelial function, implying that the new concept LLPS is very likely play critical role in angiogenesis. LLPS could be a novel promising therapeutic target for the treatment of angiogenesis-related diseases.2 inhalation or cervical dislocation.C57BL/6 male mice 6 to 10-week-old were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd, and housed in the animal facility of Nantong University. The mice were kept on a 12 h light/12 h dark cycle at 22–23 °C. Mice were euthanized by COM199 medium was purchased from Hyclone (SH30253.01). ECM was purchased from Sciencell (1001). Fetal bovine serum was purchased from Gibco (10099141). DMSO was purchased from sigma (D2650). Trizol Reagent was purchased from TAKARA (9108). Protein marker (P0076) and SDS-PAGE (P0690) were purchased from Beyotime. PVDF membrane was purchased from BBI (c62-393–0100). BSA kit (C102301-0002) and DEPC water (B501005-0500) were purchased from Sangon. The Matrigel matrix was purchased from CORNING (356234). CCK-8 kit was purchased from Vazyme (A311-01/02). Flow cytometry kit was purchased from Beyotime (C1062S). Collagenase I was purchased from Maokangbio (MX1001-1000MG). MG132 and bortezomib were purchased from AbMole (M1902 and M1686). 1,6-HD and 2,5-HD were purchased from Sigma-Aldrich (240117 and H11904).Male mice (8 weeks old) were sacrificed by cervical dislocation. Thoracic aorta was then isolated and transferred into a dish with sterilized 1 × PBS buffer. After removing the fibroadipose tissue, arteries were sectioned into 1-mm-long cross-sections and placed on the collagen-coated wells with corresponding concentration of 1,6-HD or 2,5-HD. Micrographs were taken after incubation at 37 °C for 2 or 3 days.2 inhalation 5 days later. The Matrigel plugs were retrieved and fixed with 4% PFA for further analysis. Hematoxylin and eosin (H&E) staining, CD31 , and VE-cadherin  immunohistochemistry were carried out.Male mice (7–8 weeks old) were anesthetized with oxygen and 2–3% isoflurane. A mixture containing of Matrigel  with corresponding concentration of 1,6-HD, 2,5-HD, or vehicle was subcutaneously injected into the dorsal surface of mice under anesthesia. Two Matrigel plugs were implanted in each mouse. One was for 1,6-HD or 2,5-HD treatment group; another was for control group. Mice were sacrificed by COMale mice (8–9 weeks old) were anesthetized with an intraperitoneal injection of a combination of ketamine 100 mg/kg and xylazine 10 mg/kg. The eye is further anesthetized with few drop of 0.5% proparacaine. Then, an incision into the cornea approximately 1 mm from the limbus was made. The incision should be 1–1.5 mm in length. A pellet with 1,6-HD or 2,5-HD was implanted into the micropocket in one eye. A pellet without 1,6-HD or 2,5-HD was implanted into the micropocket in another eye. Pellets  were prepared ahead. Seven days after pellet implantation, observations of the implants were made by a slip lamp microscope on anaesthetized mice with isoflurane. Immunofluorescence of CD31  and FITC-lectin  was further performed.Umbilical cord was obtained from a healthy donor. Human umbilical vein endothelial cells were then isolated by digestion with 0.1% collagenase I at 37 °C for 15 min in incubator. The solution was collected and centrifuged at 1000 × g for 5 min. The pellets were resuspended in M199 medium. Then cells were cultured in M199 full medium. The study on the protocol of HUVECs isolation was in compliance with the Declaration of Helsinki.−ΔΔCt method and normalized to the internal reference gene 18S.Total RNA was extracted with Trizol reagent . The quality and concentration were tested by the Nanodrop. Then, approximately 1 μg of RNA was reverse transcribed with HiScript II-RT SuperMix for qPCR (+ gDNAwiper) . Quantitative PCR was carried out in triplicate with AceQ Universal SYBR qPCR Master Mix  on a StepOne Plus Real-Time PCR System . The 18S served as the housekeeping control. Relative mRNA expression levels were calculated using the 2The primers used are listed as follows: Human cyclinA1: Forward: GAGGTCCCGATGCTTGTCAG, Reverse: GTTAGCAGCCCTAGCACTGTC. Human cyclinD1: Forward: CAATGACCCCGCACGATTTC, Reverse: CATGGAGGGCGGATTGGAA. Human cyclinE1: Forward: AAGGAGCGGGACACCATGA, Reverse: ACGGTCACGTTTGCCTTCC. Human CDK2: Forward: GTACCTCCCCTGGATGAAGAT, Reverse: CGAAATCCGCTTGTTAGGGTC. Human CDK4: Forward: TCAGCACAGTTCGTGAGGTG, Reverse: GTCCATCAGCCGGACAACAT. Mouse cyclinA1: Forward: TGATGCTTGTCAAATGCTCAGC, Reverse: AGGTCCTCCTGTACTGCTCAT. Mouse CD31: Forward: CTGCCAGTCCGAAAATGGAAC, Reverse: CTTCATCCACCGGGGCTATC. Mouse VE-cadherin: Forward: CACTGCTTTGGGAGCCTTC, Reverse: GGGGCAGCGATTCATTTTTCT. 18S: Forward: GAACGAGCTGGCATGCTA, Reverse: CACGTGAGCCAGTCAGTGTA.All the cells were lysed in radioimmunoprecipitation (RIPA) buffer supplemented with 1% proteinase inhibitors  on ice for 30 min. Protein were extracted by centrifuged for 15 min at 4 °C and quantitatively analyzed using a BCA kit . An equal amount of protein from each sample was separated by 10% SDS PAGE and then transferred to the PVDF membrane . The membranes were incubated overnight at 4 °C with indicated primary antibodies and following incubated with the corresponding secondary antibodies. Anti-cyclinA (sc-271682), anti-cyclin D (sc-8396), anti-cyclin E (sc-377100), anti-CDK2 (sc-6248), and anti-CDK4 (sc-23896) antibodies were purchased from Santa Cruz; anti-GAPDH (AF1186) were purchased from Beyotime Biotechnology. The anti-mouse IgG secondary antibodies were purchased from Sino biological (SSA007), and anti-rabbit IgG secondary antibodies were purchased from Bioworld Technology (BS13278).HUVECs  were treated by 1 mg/ml and 3.3 mg/ml 1,6-HD for 16 h. RNA sequencing was conducted with the help of Beijing Genomic Institution (BGI). Briefly, mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads. After mRNA fragment, cDNA synthesis, end repair, and add A and adaptor ligation, cDNA fragments were amplified and PCR product were purified with Ampure XP Beads (AGENCOURT). Then, library was validated on the Agilent Technologies 2100 bioanalyzer. After circularization, the single strand circle DNA (ssCir DNA) were formatted as the final library. Then, the library was amplified with phi29 to make DNA nanoball (DNB), which were load into patterned nanoarray, and 150 pair end bases reads were generated in the way of combinatorial Probe-anchor Synthesis (cPAS). After quality control, clean and high-quality data obtained and stored in FASTQ format. The clean reads were mapped to the reference genome using HISAT2 (v2.0.4) Bowtie2 (v2.2.5) was applied to align the clean reads to the reference coding gene set, and then expression level of gene was calculated by RSEM (v1.2.12). The most downregulated genes were analyzed by online gene set enrichment analysis (GSEA) program.3 cells per well for 24 h, and then the medium was replaced by 200 μl complete medium with indicated concentration of 1,6-HD or 2,5-HD. After 24 h, 10 μl of CCK-8 solution was added into each well and incubated at 37 °C for 2 h. Finally, cell optical density was determined at 450 nm through a microplate reader (Thermo Fisher Scientific). In order to detect the ability of cell migration, wound healing assay was used. Approximately 4 × 105 cells/well were seeded in six-well plates. After the cells reached 70% confluency, wounds were created using a 10-μl pipette tip. Cells were treated with corresponding concentration of 1,6-HD. Then, the cells were incubated for at 37° C, 5% CO2. Images were taken 0, 24, 36, and 48 h later. For network formation assay, approximately 4 × 105 cells/well were seeded in six-well plates with ECM overnight, and then starved using DMEM for 12 h. After starvation, replacing DMEM with ECM including different concentrations of 1,6-HD or 2,5-HD. A 96-well plate was coated with 50 μl Matrigel (Corning) and kept at 37 °C for 30 min. A total of 2 × 104 HUVECs suspended in 100 μl conditioned medium were applied to the precoated 96-well plate. Micrographs were taken after incubation at 37 °C for another 6 h.Cell proliferation was assessed by CCK-8 kit  following the instructions of manufacturer. Briefly, HUVECs were seeded into 96-well plates at a density of 2 × 105 HUVECs/well were seeded in six-well plates and then treated with different concentrations of 1,6-HD for 36 h, and using trypsin (without EDTA) to collect cells. The cells were fixed with 70% ethanol overnight at 4 °C in a refrigerator. Then, the cells were washed gently with PBS twice and incubated for 15–20 min with PI in dark. The samples were analyzed using a BD flow cytometer (BD Biosciences).Approximately 3 × 10t test was used to determine the differences between two groups, and one-way ANOVA was used for multiple-group comparison. P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.All the data were analyzed using Prism. The unpaired two-side Additional file 1: Figure S1-S9. Fig. S1 2,5-hexanediol has no effect on blood vessel formation in Matrigel plugs. (A) Matrigel mixed with/without 2,5-HD (20 mg/ml) was injected into C57BL/6 mice subcutaneously. The gross morphology of Matrigel plugs was shown after five days’ injection. (B-D) Matrigel plugs were fixed by PFA. After section, the hematoxylin and eosin (H&E) staining, CD31 and VE-cadherin immunohistochemistry of Matrigel plugs was performed and shown. Scale bar: 50 μm. Fig. S2 Morphology of endothelial cell after 1,6-hexanediol treatment. HUVECs were treated with indicated concentrations of 1,6-HD. Images were taken on day 1, day 2 and day 3, respectively. Scale bar: 100 μm. Fig. S3 Effect of 1,6-hexanediol on endothelial cell viability. Cell viability was monitored by propidium iodide (PI) which can stain the dead cells with red. Scale bar: 100 μm. Fig. S4 1,6-HD has no effect on other G1/S transition-related genes. (A-D) HUVECs were treated by 1,6-HD at indicated concentrations for 16 h. Total RNA was extracted. The mRNA expressions of CCND1, CCNE1, CDK2 and CDK4 were determined by qRT-PCR. Fig. S5 1,6-HD but no 2,5-HD affects Cyclin A1 expression. (A) Matrigel plug assay was performed. The mRNA expression of CCNA1 was examined in plugs with or without 1,6-HD by qRT-PCR. (B) HUVECs were treated by 2,5-HD at different time points. Protein were extracted. The expression of Cyclin A1 was analyzed by western blot. Mean viability was shown and standard error of the mean presented the standard deviations of triplicate samples. ***** p < 0.00001. Fig. S6 1,6-HD cannot affect the distribution of Cyclin A1 in HUVECs. (A) HUVECs were treated with 1,6-HD for 2 h. Immunofluorescence of Cyclin A1 was performed. Confocal images were shown. (B) Magnification of the areas indicated in A. Scale bar: 10 μm. Fig. S7 Inhibiting super-enhancer downregulates the expression of CCNA1. (A) The published ChIP sequencing data were extracted and analyzed the H3K27ac levels in HUVECs around CCNA1. (B) HUVECs were treated with or without JQ1 and I-BET. The expression of CCNA1 was determined by qRT-PCR. Mean viability was shown and standard error of the mean presented the standard deviations of triplicate samples. *** p < 0.001, **** p < 0.0001. Fig. S8 1,6-HD but not 2,5-HD disrupts BRD4 accumulation in HUVECs. (A) HUVECs were treated by 1,6-HD and 2,5-HD with indicated time. Cells were fixed and immunofluorescent staining of BRD4 were carried out. Images were obtained by confocal microscope. (B) Magnification of the areas indicated in A. Scale bar: 10 μm. Fig. S9 1,6-HD fails to further reduced CCNA1 after reduction of BRD4 protein. (A) HUVECs were treated with dBET6 for 6 h. The expression of BRD4 was analyzed by western blot.  HUVECs were treated with 3 μM dBET6, then 10 mg/ml 1,6-HD were further treated. The expression of CCNA1 was examined by qRT-PCR and Western blot. Mean viability was shown and standard error of the mean presented the standard deviations of triplicate samples. ** p < 0.01.