ll37  (Santa Cruz Biotechnology)

Journal: Science Advances

Article Title: Bacteriocin-transport–inspired oral peptide-probiotic delivery ameliorates IBD complications via autophagy and gut homeostasis

doi: 10.1126/sciadv.adz9069

Figure Lengend Snippet: ( A ) Bioinspired design of BTB biocomposites. ( B ) Protective mechanisms of orally delivered probiotics and LL37 in the GI tract via microbial mimicry. ( C ) Following oral administration, BTB-Alg undergoes sequential intestinal adhesion, payload release, tissue penetration, and lesion-specific targeting. ( D ) BTB-Alg alleviates AC, resolves fibrosis, and suppresses C. difficile infection (CDI). ( E ) Mechanism of BTB-Alg–mediated intestinal niche reprogramming. ROS, reactive oxygen species; MDS, molecular dynamics simulation; SCFA, short-chain fatty acid; BP, biological process; CC, cellular component; MF, molecular function.

Article Snippet: Then, CCD-18Co cells were stimulated with TGF-β (10 ng ml −1 ) and treated with PBS, Met (MedChemExpress, HY-B0627) (1.00 mM), LL37 (1.11 μM), LL37 (1.11 μM) + CC (MCE, HY-13418A) (20 μM), Lac/LL@ALg (C LL37 = 1.11 μM), and Lac/LL@ALg (C LL37 = 1.11 μM) + CC for 24 hours, respectively.

Techniques: Probiotics, Infection

Journal: Science Advances

Article Title: Bacteriocin-transport–inspired oral peptide-probiotic delivery ameliorates IBD complications via autophagy and gut homeostasis

doi: 10.1126/sciadv.adz9069

Figure Lengend Snippet: ( A to D ) Antifibrosis efficacy of LL37 by quantitative polymerase chain reaction (qPCR) analysis of fibrotic markers (α-SMA, Col1α1, and FN) in TGF-β–stimulated CCD-18Co cells treated with LL37-1 (1.11 μM) or LL37-2 (11.1 μM). ( E ) Western blot assays of Col1α1 and FN protein expression. AU, arbitrary units; NC, negative control. ( F to H ) Cytotoxicity assessment of LL37 in (F) HT29 cells, (G) unstimulated CCD-18Co cells, and (H) TGF-β–pretreated (24 hours) CCD-18Co cells. ( I and J ) Cellular uptake of Cy5.5-labeled LL37 (20 μg ml −1 ) in unstimulated versus TGF-β–stimulated intestinal fibroblasts (I), with fluorescence intensity quantification (J). Scale bars, 100 μm. ( K ) Chromatogram profiles of LL37 detected by high-performance liquid chromatography (HPLC) following different treatments. ( L ) Fabrication schematic of BTB-Alg. ( M ) Zeta potential of Lac, LL37, and Alg. ( N ) Growth kinetics of Lac with LL37 coculture. ( O ) Fluorescence imaging of LL37 loading optimization [37.5, 75, 150, 300, 600, 1000, 1500, and 2500 μg of LL37 per 1 × 10 9 colony-forming units (CFU) of Lac]. ( P ) Fluorescence intensity of different BTB models. ( Q ) Zeta potential stabilization at saturation (Lac/LL 6 versus Lac/LL 7 ). n.s., not significant. ( R ) HPLC quantification of unbound LL37 in supernatants. ( S ) Alg coating optimization using Cy5.5-labeled Alg (0.032, 0.063, 0.125, 0.25, 0.5, 1, or 2 mg per 1 × 10 9 CFU of Lac). ( T ) Fluorescence intensity of Alg deposition. ( U and V ) Protective efficacy of Alg against pepsin degradation. HPLC chromatograms (U) and residual ratio of LL37 quantification (V). In (B) to (D), (F) to (H), (J), (M), (N), (P), (Q), (T), and (V), data are shown as mean ± SD ( n = 3 biological replicates). Statistical analysis was performed via a one-way analysis of variance (ANOVA) or an unpaired Student’s two-sided t test. Experiments in (E), (I), (K), (O), (R), (S), and (U) repeated three times with consistent results.

Article Snippet: Then, CCD-18Co cells were stimulated with TGF-β (10 ng ml −1 ) and treated with PBS, Met (MedChemExpress, HY-B0627) (1.00 mM), LL37 (1.11 μM), LL37 (1.11 μM) + CC (MCE, HY-13418A) (20 μM), Lac/LL@ALg (C LL37 = 1.11 μM), and Lac/LL@ALg (C LL37 = 1.11 μM) + CC for 24 hours, respectively.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Negative Control, Labeling, Fluorescence, High Performance Liquid Chromatography, Zeta Potential Analyzer, Imaging

Journal: Science Advances

Article Title: Bacteriocin-transport–inspired oral peptide-probiotic delivery ameliorates IBD complications via autophagy and gut homeostasis

doi: 10.1126/sciadv.adz9069

Figure Lengend Snippet: ( A ) Surface charge evolution during the decoration. ( B and C ) Particle size (B) and transmission electron microscopy (TEM) images (C) showing structural transitions. Scale bars, 2 and 1 μm. ( D ) Triple-channel confocal imaging confirming core-shell architecture: FITC-labeled Lac [green; excitation/emission (Ex/Em): 490/520 nm], Cy3-labeled LL37 (red; Ex/Em: 550/570 nm), and Cy5-labeled Alg (purple; Ex/Em: 646/664 nm). Scale bars, 100 and 50 μm. ( E ) XRD patterns demonstrating LL37 crystalline structure attenuation after Alg encapsulation. ( F ) Growth curves of Lac and Lac/LL@Alg at 37°C recorded at 2-hour interval for 24 hours. ( G ) Schematic illustration of the physiological responsiveness behavior of Lac/LL@Alg. ( H ) Fluorescence images of Lac/LL@Alg after incubation with SGF supplemented with pepsin for 2 hours and SIF supplemented with pancreatin for 2, 4, 6, and 8 hours. FITC-labeled Lac (green), Cy3-labeled LL37 (red), and Cy5-labeled Alg (purple), respectively. Scale bar, 50 μm. ( I and J ) Survival of Lac and Lac/LL@Alg measured on the basis of plate counts after incubation with SGF supplemented with pepsin and SIF supplemented with pancreatin at 0 point (I) and indicated times (J). ( K ) Cumulative release profiles of LL37 from Lac/LL or Lac/LL@Alg after incubation with SGF and SIF. In (A), (B), (F), and (I) to (K), data are shown as mean ± SD ( n = 3 biological replicates). Experiments in (C) to (E) and (H) repeated three times with consistent results.

Article Snippet: Then, CCD-18Co cells were stimulated with TGF-β (10 ng ml −1 ) and treated with PBS, Met (MedChemExpress, HY-B0627) (1.00 mM), LL37 (1.11 μM), LL37 (1.11 μM) + CC (MCE, HY-13418A) (20 μM), Lac/LL@ALg (C LL37 = 1.11 μM), and Lac/LL@ALg (C LL37 = 1.11 μM) + CC for 24 hours, respectively.

Techniques: Transmission Assay, Electron Microscopy, Imaging, Labeling, Encapsulation, Fluorescence, Incubation

Journal: Science Advances

Article Title: Bacteriocin-transport–inspired oral peptide-probiotic delivery ameliorates IBD complications via autophagy and gut homeostasis

doi: 10.1126/sciadv.adz9069

Figure Lengend Snippet: ( A ) The workflow of in vivo tissue distribution experiments. p.o., per os. ( B to D ) IVIS imaging of the GI tract (top) and major organs (bottom) after oral administration of (B) FITC-labeled native Lac, (C) Cy5.5-labeled free LL37, and (D) triple-labeled Lac/LL@Alg at specified time points ( n = 3). ( E ) The quantification of total colonic fluorescence intensity for Lac and LL37 over time. ( F ) Time-dependent fluorescence changes of Lac, LL37, and Alg components in Lac/LL@Alg at 2, 4, 6, and 8 hours postadministration. ( G and H ) Colonic mucosal retention analysis of (G) representative images and (H) quantification of total intensity of different groups after 8 hours postadministration ( n = 3). ( I to K ) Representative images (I), penetration ratios (J), and a schematic diagram (K) demonstrate the effective diffusivity of Lac, LL37, and Lac/LL@Alg in mucus. ( L ) Effective penetration of Lac/LL@Alg in intestinal tissues derived from patients with CD. Data in (E), (F), (H), and (J) presented as mean ± SD ( n = 3 biological replicates). Significance determined by unpaired two-tailed Student’s t test. Experiments in (B) to (D), (G), (I), and (L) repeated three times with consistent results.

Article Snippet: Then, CCD-18Co cells were stimulated with TGF-β (10 ng ml −1 ) and treated with PBS, Met (MedChemExpress, HY-B0627) (1.00 mM), LL37 (1.11 μM), LL37 (1.11 μM) + CC (MCE, HY-13418A) (20 μM), Lac/LL@ALg (C LL37 = 1.11 μM), and Lac/LL@ALg (C LL37 = 1.11 μM) + CC for 24 hours, respectively.

Techniques: In Vivo, Imaging, Labeling, Fluorescence, Derivative Assay, Two Tailed Test

Journal: Science Advances

Article Title: Bacteriocin-transport–inspired oral peptide-probiotic delivery ameliorates IBD complications via autophagy and gut homeostasis

doi: 10.1126/sciadv.adz9069

Figure Lengend Snippet: ( A ) Schematic representation of DSS-induced AC in mice and therapeutic design. ( B ) Daily body weight changes throughout the study. ( C ) DAI changes during the whole study. ( D to F ) Colon length (D), weight length index (E), and morphology (F) across different groups. ( G ) Hematoxylin and eosin (H&E) and Alcian blue-periodic acid schiff (AB-PAS) staining of intestines from each group. Scale bars, 500 μm. ( H ) H&E-based histopathological scoring of colonic tissues posttreatment. ( I ) Spleen weight index in each group. ( J to M ) Measurement of TNF-α (J), IL-10 (K), MPO (L), and MDA (M) in different groups. prot, protein. ( N ) Analysis of Sobs index at the genus level. ( O ) Principal components analysis elucidated the similarity of gut microbial communities at the genus level. PC1, principal component 1. ( P and Q ) The composition of microbial communities was characterized at both phylum (P) and genus (Q) levels from the control, AC model, and Lac/LL@Alg. Bars represent relative abundance. ( R ) Relative abundance of Lac in feces from AC model and Lac/LL@Alg groups. ( S ) Linear discriminant analysis Effect Size (LEfSe) was implemented to identify differentially enriched taxa in gut microbiota among control, AC model, and Lac/LL@Alg-treated groups. Data in (B) to (M) are derived from n = 6 biological independent samples. In (B) to (E) and (H) to (M), data are shown as mean ± SD, with statistical analysis performed via one-way ANOVA. P values denote the statistical significance between AC model and LL37, Lac, and Lac/LL@Alg. In (N) to (S), data are shown as mean ± SD ( n = 4 biological independent samples), with statistical analysis analyzed via one-way ANOVA or a Student’s two-sided t test.

Article Snippet: Then, CCD-18Co cells were stimulated with TGF-β (10 ng ml −1 ) and treated with PBS, Met (MedChemExpress, HY-B0627) (1.00 mM), LL37 (1.11 μM), LL37 (1.11 μM) + CC (MCE, HY-13418A) (20 μM), Lac/LL@ALg (C LL37 = 1.11 μM), and Lac/LL@ALg (C LL37 = 1.11 μM) + CC for 24 hours, respectively.

Techniques: Staining, Control, Derivative Assay

Journal: Science Advances

Article Title: Bacteriocin-transport–inspired oral peptide-probiotic delivery ameliorates IBD complications via autophagy and gut homeostasis

doi: 10.1126/sciadv.adz9069

Figure Lengend Snippet: ( A ) Schematic representation of DSS and C. difficile –induced colitis in mice and therapeutic design. ( B ) TcdB expression in feces from control, CD, and saline (DSS + CD)–treated groups on day 8 ( n = 3). ( C ) Survival curves of mice with different treatments. ( D ) Representative colon morphology images from different groups ( n = 5). ( E to I ) Body weight changes (E), colon lengths (F), and proinflammatory markers [IL-6 (G), IL-17 (H), and IL-1β (I) mRNA levels] between control and CD-treated groups. ( J to N ) Body weight changes (J), colon lengths (K), and proinflammatory markers [IL-6 (L), IL-17 (M), and IL-1β (N) mRNA levels] between CD and saline-treated groups. ( O to S ) Body weight changes (O), colon lengths (P), and proinflammatory markers [IL-6 (Q), IL-17 (R), and IL-1β (S) mRNA levels] from different treatment groups. ( T ) Western blot analysis of ZO-1 expression in different treatment groups ( n = 3). ( U ) H&E staining of intestinal tissues from each group ( n = 3). Scale bars, 500 μm. ( V ) Western blot analysis of NF-κB pathway in different treatment groups ( n = 3). p–NF-κB, phosphorylated NF-κB. ( W ) Immunofluorescence staining of macrophages with distinct phenotypes ( n = 3). CD206 staining (green): M2 macrophage; CD86 staining (red): M1 macrophage. Scale bars, 200 μm. In (G) to (I) and (L) to (N), data are shown as mean ± SD ( n = 4 biological replicates), with statistical analysis conducted via an unpaired Student’s two-sided t test. In (Q) to (S), data are shown as mean ± SD ( n = 4 biological replicates), with statistical analysis conducted via one-way ANOVA. P values denote the statistical significance among the saline, VAN, LL37, Lac, and Lac/LL@Alg groups.

Article Snippet: Then, CCD-18Co cells were stimulated with TGF-β (10 ng ml −1 ) and treated with PBS, Met (MedChemExpress, HY-B0627) (1.00 mM), LL37 (1.11 μM), LL37 (1.11 μM) + CC (MCE, HY-13418A) (20 μM), Lac/LL@ALg (C LL37 = 1.11 μM), and Lac/LL@ALg (C LL37 = 1.11 μM) + CC for 24 hours, respectively.

Techniques: Expressing, Control, Saline, Western Blot, Staining, Immunofluorescence

Journal: Science Advances

Article Title: Bacteriocin-transport–inspired oral peptide-probiotic delivery ameliorates IBD complications via autophagy and gut homeostasis

doi: 10.1126/sciadv.adz9069

Figure Lengend Snippet: ( A ) Experimental design of DSS-induced IF model and the timeline of therapeutic intervention. ( B ) Body weight changes during whole treatment. ( C and D ) Colon morphology (C) and length quantification (D) in different groups. ( E ) Histopathological analysis via H&E, Masson’s trichrome, and Van Gieson (VG) staining. Scale bars, 500 μm. ( F and G ) Quantitative histological scoring of inflammation (F) and fibrosis severity (G). ( H and I ) Immunofluorescence imaging of Col I (H) and α-SMA (I). Scale bars, 200 μm. ( J to L ) qPCR analysis of fibrotic markers α-SMA (J), Col1α1 (K), and FN (L) in CCD-18Co cells (C LL37 = 1.11 μM). ( M ) EMT modulation with vimentin (green) and E-cadherin (red) costaining. Scale bars, 100 μm. In (B), (D), (F), and (G), data are shown as mean ± SD ( n = 6 biological replicates). In (J) to (L), data are shown as mean ± SD ( n = 3 biological replicates). Statistical analysis was studied via one-way ANOVA, with P values denoting the statistical significance among the IF model, LL37, Lac, and Lac/LL@Alg groups. The images in (E), (H), (I), and (M) are representative of five biological replicates.

Article Snippet: Then, CCD-18Co cells were stimulated with TGF-β (10 ng ml −1 ) and treated with PBS, Met (MedChemExpress, HY-B0627) (1.00 mM), LL37 (1.11 μM), LL37 (1.11 μM) + CC (MCE, HY-13418A) (20 μM), Lac/LL@ALg (C LL37 = 1.11 μM), and Lac/LL@ALg (C LL37 = 1.11 μM) + CC for 24 hours, respectively.

Techniques: Staining, Immunofluorescence, Imaging

Journal: Science Advances

Article Title: Bacteriocin-transport–inspired oral peptide-probiotic delivery ameliorates IBD complications via autophagy and gut homeostasis

doi: 10.1126/sciadv.adz9069

Figure Lengend Snippet: ( A ) Workflow for antifibrotic mechanism investigation. ( B ) AMPK activation levels in intestinal stricture tissues versus normal tissues from patients with CD. ( C ) Western blot analysis of AMPK activation and fibrotic marker gene expression after treatment with LL37 or Lac/LL@Alg (C LL37 = 20 μg ml −1 ). ( D and E ) Grayscale analysis of Western blot bands for phospho-AMPK (pAMPK)/AMPK (D) and FN (E). ( F to H ) qPCR level of α-SMA (F), Col1α1 (G), and FN (H) in CCD-18Co cells after different treatments. ( I ) Expression levels of autophagy-related genes between patients with active-stage CD and healthy controls in the GSE75214 dataset. ( J ) Dysregulation of autophagy-related genes in structured intestinal tissues compared to nonstructured tissues from patients with CD ( n = 9). ( K and L ) Autophagic flux dynamics were quantified through LC3B-II/I ratio modulation, p62 accumulation (K), and phospho-mTOR signaling activity (L). ( M ) GSEA of G protein signaling pathways and apelin signaling pathway activation in colon tissues after Lac/LL@Alg treatment. ( N ) Dynamic snapshot of the interaction between LL37 and apelin receptor (APJ). ( O ) Root mean square fluctuation (RMSF) analysis of the LL37-APJ and apelin-36–APJ complexes. ( P ) Average binding free energies of the simulated LL37-APJ or apelin-APJ complexes. In (B), (C), (K), (L), and (M) to (P), experiments were repeated three times with consistent results. In (D) to (H), data are shown as mean ± SD ( n = 3 biological replicates). Statistical analysis was studied via one-way ANOVA comparing phosphate-buffered saline (PBS), Met, LL37, LL37 + CC, Lac/LL@Alg, and Lac/LL@Alg + CC.

Article Snippet: Then, CCD-18Co cells were stimulated with TGF-β (10 ng ml −1 ) and treated with PBS, Met (MedChemExpress, HY-B0627) (1.00 mM), LL37 (1.11 μM), LL37 (1.11 μM) + CC (MCE, HY-13418A) (20 μM), Lac/LL@ALg (C LL37 = 1.11 μM), and Lac/LL@ALg (C LL37 = 1.11 μM) + CC for 24 hours, respectively.

Techniques: Activation Assay, Western Blot, Marker, Gene Expression, Expressing, Activity Assay, Protein-Protein interactions, Binding Assay, Saline