EMBO/soda-nel huggingface dataset:
HUWE1 interacts with PCNA to alleviate replication stress (2016)
Figure 7

Figure 7. HUWE1 is a novel ubiquitin ligase for H2AX. A, B. The impact of HUWE1 on H2AX phosphorylation and ubiquitination, under normal conditions (A) or upon induction of replication stress (2mM HU for 18h, or 2h after exposure to 40J/m2 UV). Control or HUWE1-knockout HeLa cells were analyzed. C. In vitro ubiquitination assay showing that recombinant HUWE1C-ter can mono-ubiquitinate H2AX. D. Wild-type, but not the PIP-mutant can correct the defective H2AX ubiquitination in HUWE1-knockout 293T cells. E. Chromatin immunoprecipitation showing that H2AX binding to the common fragile site FRA3B is reduced in HUWE1-knockout 8988T cells. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 4 experiments -/+ SD. P-value is 4.4x10-6. F. Western blots showing that several H2AX ubiquitin ligases participate in H2AX ubiquitination following replication stress. HUWE1, RNF168, and BMI1 were knocked-down in HeLa cells. The efficiency of the knockdown is shown in Fig EV4. Cells were treated with 600nM aphidicolin for 24h, 2mM HU for 24h, or analyzed 2h after exposure to 40J/m2 UV. G. Chromatin immunoprecipitation from HeLa cells, showing that knockdown of H2AX ubiquitin ligases can reduce H2AX binding to FRA3B. Cells were treated with 600nM aphidicolin for 24h. Binding was quantified relative to input material. Shown is the average of 3 experiments -/+ SD. The p-values shown indicate the statistical significance relative to siControl (0.0016, 0.0005, and 0.0009 respectively). H. Chromatin fractionation experiments showing that HUWE1-knockout HeLa cells, treated with 2mM HU for 24h, fail to efficiently recruit BRCA1 and BRCA2 proteins to DNA. In contrast, 53BP1 chromatin association is not induced by HU treatment, and not affected by HUWE1 knockout, and thus can serve as loading control. Shown are blots of whole cell extract (WCE) samples, representing input material, and of chromatin pellet samples.
α-Actinin-1 promotes activity of the L-type Ca2+ Channel Cav1.2 (2019)
Figure 1

Fig. 1 | α-Actinin-1 forms electrostatic and hydrophobic contacts with CaV1.2.

a, Linear model of α-actinin domains (CH, calponin homology domains; SR, spectrin repeats; EF, EF hands). Bars and numbers on top of the model depict the segments used in this work.

b, Ensemble of 10 lowest energy NMR-derived structures of α-actinin-1_EF34 (cyan) bound to IQ peptide (red). Structural statistics are given in Table 1.

c, Energy minimized average structure of α-actinin-1_EF34 (cyan) bound to IQ (red), revealing intermolecular salt bridges between K1647 of IQ and E847/E851 of α-actinin-1. The K1647 side chain amino nitrogen atom is 2.8 Å and 2.5 Å away from the side chain carbonyl oxygen atoms of E847 and E851, respectively.

d, Intermolecular hydrophobic contacts between I1654 of IQ (red) and F833 of α-actinin-1 (cyan). The I1654 side chain methyl carbon atom is 2.9 Å away from the closest aromatic ring atom of F833. Side-chain atoms are colored yellow.

e, Lack of direct contacts between F1658 of IQ (red) and α-actinin-1 (cyan). The aromatic side chain of F1658 is primarily solvent exposed and does not directly contact α-actinin-1. The aromatic ring of F1658 is closest to the aromatic ring of Y859 and β-methylene carbon of Q856 of α-actinin-1, which are 5.2 Å and 4.3 Å apart, respectively.

f, FP titrations show binding of WT α-actinin-1_EF34 to IQ peptides WT (black) and K1647E (red), of α-actinin-1_EF34 mutant E847K/E851K to IQ peptides WT (green) and K1647E (purple), of full-length α-actinin-1 to IQ WT (o), and lack of IQ binding to α-actinin-1_CH1-CH2 (+) and α-actinin-1_EF12 (x; see Table 2 for binding parameters and standard errors).

g, FP titrations showing binding of wild type α-actinin-1_EF34 to IQ peptides WT (black) and I1654A (purple) and of ACTN1_EF34 mutant F833 to IQ WT (red; see Table 2 for binding parameters and standard errors).
α-Actinin-1 promotes activity of the L-type Ca2+ Channel Cav1.2 (2019)
Figure 2

Fig. 2 | ApoCaM forms electrostatic and hydrophobic contacts with CaV1.2.

a, Ensemble of 10 lowest energy NMR-derived structures of apo CaM/IQ complex. Structural statistics are given in Table 3.

b, Energy minimized average structure of apoCaM (cyan) bound to IQ (red), showing an intermolecular salt bridge between IQ K1662 and apoCaM E88. The K1662 side chain amino nitrogen atom is 2.7 Å away from the side chain carbonyl oxygen of E88.

c, Intermolecular hydrophobic contacts between IQ I1654 (red) and apoCaM F90 (cyan). Side chain atoms colored yellow. The I1654 side chain methyl carbon atom is 2.2 Å away from the closest aromatic ring atom of F90.

d, Intermolecular hydrophobic contacts between F1658 of IQ and F90/M110 in apoCaM. The aromatic side chain atoms of F1658 are 2.3Å and 2.6 Å away from the closest side chain atom of F90 and M110, respectively.

e, FP titrations showing binding of apoCaM to IQ peptides WT (black) and K1662E (red) and of apoCaM mutant E88K to IQ WT (green) and IQ peptide K1662E (purple, "charge inversion"; see Table 4 for binding parameters and standard errors).

f, FP titrations showing binding of apo-CaM to IQ peptides WT (black), I1654A (red), and F1658A (green) and of apoCaM mutant F90A to IQ WT (blue; see Table 4 for binding parameters and standard errors).
α-Actinin-1 promotes activity of the L-type Ca2+ Channel Cav1.2 (2019)
Figure 3

Fig. 3 | CaV1.2 mutations that affect α-Actinin-1 drastically decrease channel open probability.

HEK293 cells were transfected with α11.2, α2δ-1, and β2A and cultured for 22-24h before cell attached patch recording in 110 mM Ba2+.

a, Single CaV1.2 channel recordings of CaV1.2 WT and K1647A. Holding potential (HP) was

-80 mV and test potential (TP) 0 mV. Shown are 10 consecutive sweeps from representative experiments (see Fig. EV 4 for more sweeps).

b, Mean assemble averages for all experiments with CaV1.2 WT and K1647A, which are based on a total of 2744 and 868 sweeps, respectively (see Table 5).

c-g, Dot plots and means + SEM for availability (i.e., likelihood that a sweep had at least one event; c), NPo (d), Po (e), unitary current amplitude i (e), and the mean of the current of a single channel at any point in time calculated from the ensemble averages of each experiment (g) (**p<0.01, ***p<0.001 compared to WT; one-way ANOVA with Bonferroni post-hoc test (c) or Welch ANOVA with Tamhane T2 test (d-g); n = 7 to 35 see Table 5 for more details).
α-Actinin-1 promotes activity of the L-type Ca2+ Channel Cav1.2 (2019)
Figure 4

Fig. 4 | Ectopic α-actinin-1 expression increases CaV1.2 open probability.

HEK293 cells were transfected with α11.2, α2δ-1, and β2A plus, if indicated, α-actinin-1 before cell attached patch recording.

a, Representative single CaV1.2 channel recordings of WT CaV1.2 alone or with WT or E847K/E851K mutant α-actinin-1.

b, Mean assemble averages for all experiments for each combination.

c-g, Dot plots and means + SEM for availability (c), NPo (d), Po (e), unitary current amplitude i (f), and the mean of the current of a single channel calculated from the ensemble averages of each experiment (g) (*p<0.05, **p<0.01, ****p<0.001; unpaired t-test; n = 13 to 35; see Table 6 for more details).
α-Actinin-1 promotes activity of the L-type Ca2+ Channel Cav1.2 (2019)
Figure 5

Fig. 5 | α-Actinin-1 E847K/E851K partially rescues open probability for CaV1.2 K1647E.

HEK293 cells were transfected with α11.2 with the K1647E mutation, α2δ-1, and β2A plus α-actinin-1 WT or E847K/E851K before cell attached patch recording.

a, Representative single CaV1.2 channel recordings of CaV1.2 K1647E with WT or E847K/E851K α-actinin-1.

b, Mean assemble averages for all experiments for both combinations.

c-g, Dot plots and means + SEM for availability (c), NPo (d), Po (e), unitary current amplitude i (f), and mean of the current of a single channel calculated from the ensemble averages of each experiment (g) (**p<0.01; unpaired t-test; n = 16 to 30; see Suppl. Fig. 8 and Table 6 for more details).
α-Actinin-1 promotes activity of the L-type Ca2+ Channel Cav1.2 (2019)
Figure 6

Fig. 6 | Modulation of CaV1.2 surface expression via its interaction with α-actinin-1.

a, HEK293 cells were transfected with WT α11.2, α2δ-1, and β2A + WT α-actinin-1 and cultured for 22-24h before surface biotinylation. Shown are representative immunoblots of NeutrAvidin pull-down samples (from lysate containing 600 μg protein) and total lysate samples containing 20 μg protein. α11.2 was detected with an antibody against its HA tag (top), which is present in all constructs used throughout this work. Pull-down and lysate samples are from the same blot but different exposures because signals from lysate samples were much stronger than from pull-down samples. Immunoblotting for vinculin (middle) and tubulin (bottom) indicated that comparable amounts of these intracellular control proteins were present in lysate samples. Their absence in pull-down samples as seen on the same blots showed that these prominent intracellular proteins did not undergo biotinylation as control for membrane integrity during surface biotinylation. Bar graph shows means + SEM of the pull-down immunosignals in mutants relative to surface labelling of control α11.2 samples lacking α-actinin-1 co-expression (mean set to 100%; see Methods; **p<0.01 two-tailed t-test; n = 7).

b, HEK293 cells were transfected with WT or K1647E mutant α11.2, α2δ-1, and β2A, and WT or E847K/E851K mutant α-actinin-1, or CFP alone as negative control and cultured for 22-24h before surface biotinylation. Shown are representative immunoblots of pull-down and lysate samples as in (a). Bar graph shows means + SEM of the pull-down immunosignals in mutants relative to surface labelling in the WT/WT control (mean set to 100%; see Methods). **p<0.01, ***p < 0.001; one-way ANOVA with Tukey post hoc test; n = 4-5).
α-Actinin-1 promotes activity of the L-type Ca2+ Channel Cav1.2 (2019)
Figure 7

Fig. 7 | CaV1.2 mutations that affect α-actinin-1 impair gating charge movement and its coupling to channel opening.

HEK293 cells were transfected with α11.2, α2δ-1, and β2A before whole cell patch recording in 20 mM Ba2+.

a, Representative current traces of the first 2 ms obtained from recordings upon depolarizations from a holding potential of -80 mV to the indicated potentials (the voltage protocol is schematized in the upper left corner).

b, Representative current traces upon step depolarizations to the reversal potential (Erev) for 20 ms to obtain movement of the ON-gating charges (Qon), and subsequent to -50 mV for 10 ms to obtain tail currents (Itail). Insets: magnifications of exemplary Qon for CaV1.2 WT, K1647A, Y1649A, I1654A, F1658A and K1662E.

c, Plots of Itail (in this panel corrected for variations in cell capacitance) versus total detectable charge transfer for Qon. Slopes of regression curves are strongly reduced for CaV1.2 K1647A, Y1649A and I1654A versus WT (see Table 7 for more details).

d, Dot plots and means + SEM of Qon (in this panel corrected for variations in cell capacitance; *p<0.05; **p<0.01; one-way ANOVA with Bonferroni post-hoc test; n = 13-18; see Table 7 for more details).

e, The reduction in slope of the regression curve of combined population data for K1647A, Y1649A and I1654A versus WT indicates reduced coupling of Itail with Qon when α-actinin binding to the IQ motif is diminished.
Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway (2022)
Figure 1

Figure 1. The intestinal microenvironment in hypoganglionosis negatively influences the phenotype of enteric neural crest-derived cells

Comparison of the phenotype of ENCCs treated with the intestinal extract from hypoganglionosis rats at weeks 2, 4 and 6 after BAC treatment. The intestinal extracts from the sham-treated guts were used as controls.

(A) Morphology of the ENCC-derived neurospheres on days 2 and 4. Scale bars represent 200 µm.

(B-C) The proliferation of ENCC-derived neurospheres is represented by neurosphere diameter (B) and change in diameter on day 4 compared with day 2 (C).

(D) ENCC viability assessed by CCK-8 assay.

(E) ENCCs number analyzed by nuclear staining with DAPI.

(F) Migration of ENCCs evaluated by transwell plate assay and stain with crystal violet, scale bar, 200 µm.

(G) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry.

Data information: All data are represented as means ± SEM (n=6 biological replicates per group). * p<0.05, ** p<0.01, and *** p<0.001, defined by one-way ANOVA or Kruskal-Wallis test (>2 groups) followed by multiple comparisons with Bonferroni post hoc test. BAC, benzalkonium chloride; ENCCs, enteric neural crest-derived cells.
Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway (2022)
Figure 2

Figure 2. Microbial dysbiosis and inflammation in hypoganglionosis

Comparison of fecal microbial species and inflammation in the hypoganglionosis and sham rats. For hypoganglionosis, fecal samples from the narrow segments (NS, BAC-treated, hypoganglionic) and expanded segments (ES, proximal to narrow segments, with abnormal ganglion) colon are analyzed, respectively.

(A-D) Cluster analysis at the (A) phylum and (C) genus levels. A column accumulation diagram of the top 10 most abundant (B) phyla and (D) genera.

(E-F) Alpha-diversity indices demonstrate species abundance (E: OTUs, ACE and Chao1) and diversity (F: Shannon and Simpson).

(G) The functional distribution of intestinal microbiota using principal component analysis.

(H-I) Inter-group diversity analysis and metabolic mass spectrometry to compare the differences in (H) intestinal microbiota and (I) SCFAs metabolites between the hypoganglionosis and sham rats. The species indicated in red are linked to SCFAs production.

(J) Representative HE staining and quantification to assess the intestinal mucosal injury. Scale bar, 100 µm.

(K) Representative IHC stain and quantification of CRP and TNF-α levels. Scale bar, 100 µm.

(L) Representative immunofluorescence stain and quantification of β-catenin levels. Nuclei were stained blue with DAPI. Scale bar, 200 µm.

Data information: Data (in A-I) are represented as means ± SEM (n=3 biological replicates per group). Data (in J-L) are represented as means ± SEM (n=6 rats per group) and presented relative to values from sham rats. * p<0.05, ** p<0.01, and *** p<0.001, defined by two-tailed Student's t-test (2 groups) or one-way ANOVA and Kruskal-Wallis test (>2 groups) followed by multiple comparisons with Bonferroni post hoc test. CRP, C-reactive protein; ES, expanded segments; NS, narrow segments; PC, principal component; SCFAs, short-chain fatty acids. TNF-α, tumor necrosis factor α.
Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway (2022)
Figure 3

Figure 3. Fecal microbiota transplantation enhances the ENCC phenotype and promotes cell therapy for hypoganglionosis

Comparison of ENCC-derived neurospheres treated with the intestinal extract from hypoganglionosis rats, hypoganglionosis treated with Fecal microbiota transplantation (FMT) and sham rat.

(A-B) The diameter of ENCC-derived neurospheres on day 2 (A). ENCC viability assessed by CCK-8 assay (B).

(C-D) Migration of ENCCs using transwell plate assay and stain with crystal violet (C), scale bar, 200 µm. (D) Quantification of C.

(E-F) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry (E). (F) Quantification of E.

(G) Graphical protocol and weight change in the hypoganglionosis: untreated and treated with ENCCs only, or ENCCs+FMT.

(H) Laparoscopic analysis indicating that the same segments were treated with BAC in all the groups.

(I) Delay in distal colonic transit time.

(J-K) Comparison of neuronal markers. (J) Immunofluorescence stain and number of PGP9.5+ cells. Nuclei were stained blue with DAPI; Triangles indicate the PGP9.5+ cell. Scale bars represent 200 µm. (K) Representative immunofluorescence images of Tuj1 and GFP localization at the injection and adjacent sides. Asterisks and triangles indicate the Tuj1+/GFP+ cells and Tuj1+/GFP- cells, respectively. Nuclei were stained blue with DAPI. Scale bar, 200 µm.

(L-M) Number of Tuj1+, Tuj1+/GFP+ cells (L) and Tuj1+/GFP- cells(M).

Data information: Data (in A-M) are represented as means ± SEM (n=6 biological replicates per group) and presented relative to values of the hypoganglionosis group. * p<0.05, ** p<0.01, and *** p<0.001, defined by two-tailed Student's t-test (2 groups) or one-way ANOVA and Kruskal-Wallis test (>2 groups) followed by multiple comparisons with Bonferroni post hoc test. BAC, benzalkonium chloride; ENCCs, enteric neural crest-derived cells; FMT, fecal microbiota transplantation; GFP, green fluorescent protein; PGP9.5, protein gene product; Tuj1, neuron-specific class III beta-tubulin.
Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway (2022)
Figure 4

Figure 4. FMT improves microbial dysbiosis, which coincides with increased short-chain fatty acid production and reduced IL-17 levels

Comparison of fecal microbial species, short-chain fatty acids and inflammation between hypoganglionosis: untreated (Hypoganglionosis) or treated with broad-range antibiotics (, Antibiotic), broad-range antibiotics followed by FMT (FMT) and sham groups.

(A-D) Cluster analysis at the (A) phylum and (C) genus. Column accumulation diagrams of the top abundant microbiota at the (B) phylum and (D) genus levels.

(E-F) Sparse curve (E) shows the relative abundance, and (F) Principal component analysis (PCA) demonstrates the functional distribution of microbiota.

(G) SCFAs subsets in the feces and intestinal extracts, analyzed by targeted mass spectrometry.

(H) IL-10, IL-17, and TNF-α levels in serum and intestinal extracts, assessed by ELISA.

(I) Correlation analysis of acetate, propionate or butyrate with IL-17 in the intestinal extracts. (J-K) Environmental association analysis shows Bacteroides and Clostridium (K: circled in red) correlated significantly with SCFAs at the species level.

Data information: Data are represented as means ± SEM (n=6 rats/samples per group). * p<0.05, ** p<0.01, and *** p<0.001, # Indicates significant difference compared with the sham group, defined by one-way ANOVA or Kruskal-Wallis test (>2 groups) followed by multiple comparisons with Bonferroni post hoc test. FMT, fecal microbiota transplantation; IL, interleukin; PCA, principal component analysis; RDA, redundancy analysis; SCFAs, short-chain fatty acids.
Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway (2022)
Figure 5

Figure 5. SCFAs and IL-17 differentially affect the ENCC phenotype

Phenotype analysis of ENCCs treated with SCFAs.

(A) ENCC viability assessed by CCK-8 assay. Data are normalized to the NC group.

(B-C) Migration of ENCCs evaluated using transwell plate assay and stain with crystal violet (B); Scale bars represent 200 µm. (C) Quantitative analysis of B.

(D-F) Analysis of different IL-17 concentrations on ENCC viability (D), and 50 ng/mL IL-17 on ENCC migration (E, F); scale bars represent 200 µm.

(G-H) ENCC apoptosis determined by AnnexinV-FITC/PI stain and flow cytometry (G). (H). Quantification of G.

Data information: Data are represented as means ± SEM (n=6 biological replicates per group). * p<0.05, ** p<0.01 and *** p<0.001, # Indicates significant difference compared with NC group, defined by two-tailed Student's t-test (2 groups) or one-way ANOVA and Kruskal-Wallis test (>2 groups) followed by multiple comparisons with Bonferroni post hoc test. CCK-8, cell counting kit-8; ENCCs, enteric neural crest-derived cells; IL, interleukin; NC, normal control; OD, optical density; PI, propidium iodide; SCFAs, short-chain fatty acids.
Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway (2022)
Figure 6

Figure 6. SCFAs synergize with ENCCs in inducing neurogenesis in hypoganglionic gut

(A) Graphical protocol of ENCC transplantation and SCFA enema in the hypoganglionosis rat model.

(B) Delay in distal colonic transit time.

(C-D) Representative immunofluorescent images (C) and (D) number of PGP9.5+ cells. Nuclei were stained blue with DAPI; Triangles indicate the PGP9.5+ cell. Scale bars represent 200 µm.

(E) Representative immunofluorescent images of Tuj1 and GFP localization at the injection and adjacent sides. Asterisks and triangles indicate the Tuj1+/GFP+ cells and Tuj1+/GFP- cells, respectively. Scale bar, 200 µm.

(F-G) Number of Tuj1+ and Tuj1+/GFP+ cells (F) and Tuj1+/GFP- cells(G).

Data information: Data are represented as means ± SEM (n=6 biological replicates per group) presented relative to values of the sham group. * p<0.05, ** p<0.01, and *** p<0.001. # Indicates significant difference compared with the sham group, defined by two-tailed Student's t-test (2 groups) or one-way ANOVA and Kruskal-Wallis test (>2 groups) followed by multiple comparisons with Bonferroni post hoc test. FMT, fecal microbiota transplantation; ENCCs, enteric neural crest-derived cells; GFP, green fluorescent protein; PGP9.5, protein gene product; SCFAs, Short-chain fatty acids; Tuj1, neuron-specific class III beta-tubulin.
Fecal microbiota transplantation enhances cell therapy in a rat model of hypoganglionosis by SCFA-induced MEK1/2 signaling pathway (2022)
Figure 7

Figure 7. SCFAs modify neurogenesis via the MEK1/2 signaling pathway

Volcano diagram shows differentially expressed genes (DEGs) in ENCCs after SCFAs and IL-17A intervention, with up-regulated and down-regulated genes shown in yellow and blue, respectively (|log fold change|≥0.5 and adjusted p<0.05).

(B) Venn diagram shows 24 potential downstream pathways of SCFAs in ENCCs shared between acetate, propionate, and butyrate (left). Gene ontology/Kyoto Encyclopedia of Genes and Genomes enrichment analyses are detailed in the histogram, MAPK, PI3K-Akt signaling pathways (indicated in red), as well as the cytokine signaling pathways (indicated in blue) are significantly enriched.

(C) Venn diagram shows 19 shared downstream DEGs of acetate, propionate, and butyrate by Gene Set Enrichment Analysis, details in the histogram suggest that SCFAs are associated with cytokine signal transduction (indicated in blue).

(D) Interaction networks of the potential target genes of acetate, propionate, and butyrate from the Comparative Toxicogenomic Database and BindingDB databases present MAPK (circled in purple) and cytokine (circled in red) signaling pathways.

(E) ENCC viability assessed by CCK-8 assay after intervention with MEK1/2 inhibitor (U0126, 50 μM), p38 inhibitor (SB203580, 1 μM), JNK inhibitor (SP600125, 50 μM) and MER5 inhibitor (BIX 02189, 10 μM).

(F-H) Diameter analyses of the ENCC-derived neurospheres (F), migration of ENCCs evaluated using transwell plate assay and stain with crystal violet on day 2 (G, H) show that MEK1/2 inhibitor U0126 suppressed SCFA-induced ENCC proliferation and migration. Scale bars, 200 µm.

Data information: Data are represented as means ± SEM (n=6 biological replicates per group). * p<0.05, ** p<0.01 and *** p<0.001, # Indicates significant difference compared with NC group, defined by one-way ANOVA or Kruskal-Wallis test (>2 groups) followed by multiple comparisons with Bonferroni post hoc test. AceP, treated with acetate; ProP, treated with propionate; ButP, treated with butyrate; AceNC, control to acetate; ProNC, control to propionate; ButNC, control to butyrate; CCK-8, cell counting kit-8; DEGs, differentially expressed genes; ENCCs, enteric neural crest-derived cells; IL, interleukin; NC, normal control; NES, normal enrichment score; SCFAs, short-chain fatty acids.