scfa elisa kit  (Thermo Fisher)

Journal: Gut Microbes

Article Title: Alterations in mucosa branched N -glycans lead to dysbiosis and downregulation of ILC3: a key driver of intestinal inflammation

doi: 10.1080/19490976.2025.2461210

Figure Lengend Snippet: Reduction of branched N -glycans in mice promotes intestinal permeability and gut dysbiosis. (a and b) levels of β1,6-branching N -glycans at steady state in epithelial cells (CD45- cells) from Mgat5 −/− mice and Mgat5 WT controls. (a) L-PHA lectin was used to detect branched N -glycans and the median fluorescence intensity (MFI) was determined by flow cytometry. MFI was normalized for the average of Mgat5 −/− mice MFI; in the representative histogram, dark gray, light gray and orange depicts unstained control, Mgat5 WT and Mgat5 -/- , respectively. (b) Lectin histochemistry staining with L-PHA in mouse colonic samples. Scale bar = 50 μm. (c and d) levels of high-mannose N -glycans at steady state in epithelial cells (CD45 − cells) from Mgat5 -/- mice and Mgat5 WT controls. (c) GNA lectin was used to detect mannose N -glycans and the median fluorescence intensity (MFI) was determined by flow cytometry. MFI was normalized for the average of Mgat5 −/− mice MFI; in the representative histogram, dark gray, light gray and orange depicts unstained control, Mgat5 WT and Mgat5 -/- , respectively. (d) Lectin histochemistry staining with GNA in mouse colonic samples. Scale bar = 50 μm. (e) Linear discriminant analysis (LDA) of the gut microbiota composition based on 16S rRNA sequencing in the fecal samples from Mgat5 -/- and Mgat5 WT mice at steady state. (f) Principal component analysis (PCoA) of gut microbiota composition generated on Jaccard based on 16S rRNA sequencing of fecal samples from Mgat5 -/- and Mgat5 WT mice at steady state. (g) Concentration of short-chain fatty acids (SCFAs) measured by ELISA in the colon of Mgat5 -/- and Mgat5 WT mice at steady state. (h-k) the mRNA expression levels at steady state of genes encoding SFCAs receptors (h) Gpr43, (i) Gpr109a, and SFCAs transporters (j) Smct1 and (k) Mct1 in the colonic tissue from Mgat5 -/- and Mgat5 WT mice measured by RT-qPCR. Expression of target gene mRNA was calculated based on housekeeping gene ( Gapdh ). mRNA expression levels were normalized for the average of mRNA levels of Mgat5 −/− mice. (l) Intestinal permeability measured by FITC-labeled dextran in Mgat5 −/− and Mgat5 WT mice at steady state. (m-p) the mRNA expression levels at steady state of genes encoding for (m) claudin-1, (n) claudin-2, (o) claudin-3, and (p) claudin-4 in the colonic tissue from Mgat5 -/- mice and Mgat5 WT controls measured by RT-qPCR. Expression of target gene mRNA was calculated based on housekeeping gene ( Gapdh ). mRNA expression levels were normalized for the average of mRNA levels of Mgat5 −/− mice. (a and c) n = 14–19 per group. (f) n = 6–7 per group. (g) n = 7 per group. (h-k) n = 9–12 per group. (l) n = 7–8 per group (m-p) n = 9–16 per group. Each datapoint represents an individual animal. Data is represented as mean ± SD. * p < 0.05; ** p < 0.01; **** p < 0.0001 using an unpaired two-tailed Student’s t -test or Mann-Whitney test.

Article Snippet: The quantitative determination of SCFAs in colonic tissue was determined using the Mouse Short-Chain Fatty Acids (SCFAs) ELISA kit (Amsbio), according to the manufacturer’s instructions.

Techniques: Permeability, Fluorescence, Flow Cytometry, Control, Staining, Sequencing, Generated, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Labeling, Two Tailed Test, MANN-WHITNEY

Journal: Gut Microbes

Article Title: Alterations in mucosa branched N -glycans lead to dysbiosis and downregulation of ILC3: a key driver of intestinal inflammation

doi: 10.1080/19490976.2025.2461210

Figure Lengend Snippet: Glycan supplementation has a protective effect against colitis development and restores the levels of SCFAs. (a) Schematic representation of 800 mg/Kg/day N -acetylglucosamine (GlcNAc) supplementation of Mgat5 −/− mice for 8 weeks followed by 2% DSS treatment for 7 days. (b) Disease activity score (DAI) of Mgat5 WT , Mgat5 -/- , and GlcNAc-supplemented Mgat5 -/- mice upon DSS-induced colitis. (c and d) Histological analysis of hematoxylin & eosin staining and qualitative scores of intestinal immune infiltration of colon sections from Mgat5 WT , Mgat5 -/- , and GlcNAc-supplemented Mgat5 -/- mice upon DSS-induced colitis. There is no statistical significance between Mgat5 -/- and GlcNAc-supplemented Mgat5 − / - mice. Zoomed images highlight immune infiltrate. Scale bar = 50 μm. (e) The mRNA expression levels at steady state of Cldn4 in Mgat5 WT , Mgat5 -/- , and GlcNAc-supplemented Mgat5 -/- mice at steady state. Expression of target gene mRRNA was calculated based on housekeeping gene ( Gapdh ). mRNA expression levels were normalized for the average of mRNA levels of Mgat5 -/- mice. (f) Quantification of SCFAs measured by ELISA in the colon of Mgat5 WT , Mgat5 -/- , and GlcNAc-supplemented Mgat5 -/- mice at steady state. (g) The mRNA expression levels at steady state of SCFAs transporter Smct1 in Mgat5 WT , Mgat5 -/- , and GlcNAc-supplemented Mgat5 -/- mice at steady state. Expression of target gene mRNA was calculated based on housekeeping gene ( Gapdh ). mRNA expression levels were normalized for the average of mRNA levels of Mgat5 -/- mice. (h) Frequency of ILC3 in CD45 + population in Mgat5 WT , Mgat5 -/- , and GlcNAc-supplemented Mgat5 -/- mice upon DSS-induced colitis. (i) Frequency of IL22–producing ILC3 and (j) mean fluorescence intensity (MFI) of intracellular IL-22 in ILC3, in Mgat5 WT , Mgat5 -/- , and GlcNAc-supplemented Mgat5 -/- mice upon DSS-induced colitis. (b) n = 5 per group. (d) n = 3–4 per group. (e) n = 11–14 per group; (f) n = 7–9 per group. (g) n = 11–12 per group. (h-j) n = 4–7 per group. Each datapoint represents an individual animal. Data is represented as mean ± SD. * p < 0.05; ** p < 0.01; using an unpaired two-tailed Student’s t-test or Mann–Whitney test.

Article Snippet: The quantitative determination of SCFAs in colonic tissue was determined using the Mouse Short-Chain Fatty Acids (SCFAs) ELISA kit (Amsbio), according to the manufacturer’s instructions.

Techniques: Activity Assay, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Fluorescence, Two Tailed Test, MANN-WHITNEY

Journal: Gut Microbes

Article Title: Alterations in mucosa branched N -glycans lead to dysbiosis and downregulation of ILC3: a key driver of intestinal inflammation

doi: 10.1080/19490976.2025.2461210

Figure Lengend Snippet: Changes in mucosa branched N -glycosylation impacts gut microbial balance, leading to an intestinal inflammatory response. In a healthy gut glycocalyx, epithelial branched N -glycans sustain commensal gut microbiota composition, contributing to the integrity of the epithelial barrier. ILC3 subsets, including LTi-like ILC3 and CCR6 − NCR − ILC3, promote gut homeostasis through IL-22 release. Short-chain fatty acids (SCFAs) produced by commensal microbiota regulate mucosal homeostasis through activation of Gpr43/Gp109a signaling pathways or entering cells through Mct1 and Smct1 transport. Intestinal epithelial fucosylation (via Fut2 expression) and microbiota recognition (through Nod2 expression) also support the symbiotic relationship between microbes and the intestinal immune system in homeostasis. During health to intestinal inflammation transition, there is an altered host glycome profile, characterized by a deficiency in the mucosa expression of complex branched N -glycans and exposure of mannose-enriched glycans, perturbing mucosal integrity. This imbalanced glycoenvironment leads to a dysbiotic gut microbiota and deficient SCFAs release. Signaling and transport of SCFAs is also impaired. Concomitantly, this mucosa glycan switch hampers a protective immune response, through decreased frequency of ILC3 subsets, reduction in homeostatic cytokines (IL-22 and IL-10) and downregulation of Nod2 and Fut2 expression in the intestinal mucosa. An ILC3-ILC1 plasticity occurs imposed by the mucosa glycosylation alteration, leading to a pathogenic ILC1 proinflammatory phenotype, with increased TNFα and IL-6 production and expression of inhibitory IL-22 binding protein, which has the ability to block IL-22. The frequency of dendritic cells (DCs) is also increased upon deficiency in Mgat5 -mediated branched N -glycans. Taken together, this results in a pro-inflammatory environment in the gut and a consequent shift from health to inflammation. Prophylactic supplementation with GlcNAc, a key metabolite in the hexosamine biosynthetic pathway, restores the expression of the protective ILC3-IL-22 module, suppressing health to intestinal inflammation transition. This ultimately highlights the beneficial impact of mucosa glycosylation remodeling through nutritional intervention in order to promote intestinal homeostasis.

Article Snippet: The quantitative determination of SCFAs in colonic tissue was determined using the Mouse Short-Chain Fatty Acids (SCFAs) ELISA kit (Amsbio), according to the manufacturer’s instructions.

Techniques: Produced, Activation Assay, Expressing, Binding Assay, Blocking Assay

Journal: International Journal of Molecular Sciences

Article Title: Oral Microbiome and CPT1A Function in Fatty Acid Metabolism in Oral Cancer

doi: 10.3390/ijms252010890

Figure Lengend Snippet: Impact of CPT1A siRNA treatment on cell viability, CPT1A depletion, immunohistochemical and biomarker analysis in oral cancer. ( A ) Representative images of immunohistochemistry of CPT1A protein detected from oral tissues. Stained tissues are shown at 100× magnification. Scale bar represents 100 μm. ( B ) IHC staining of control and OC tissues; the staining is visualized using a yellow color (Opal 480 yellow with scale bar: 200 μm) and DAPI in OC, oral cancer. ( C ) Quantified intensity of CPT1A in OC. Error bars indicate the mean ± SEM for three independent experiments. ( D ) Quantified CD4+T-helper cell counts. ( E ) Oxidative Stress Responsive 1 (OXRS1). ( F ) Human plasma interleukin-6 levels (IL6). ( G ) Tumor necrosis factor-alpha (TNFα). ( H ) Short-chain fatty acid (SCFA) concentrations in oral saliva. OC: oral cancer. ( I ) Western blot analysis showing the depletion of CPT1A by siRNA treatment in normal and oral cancer cells. The data are representative of at least three independent experiments. ( J ) HGF-1, YD-10B, CAL27 and SCC1 cells were reverse-transfected with either siCTL or siCPT1A; after 48 h, cell viability of HGF-1, YD-10B, CAL27 and SCC1 cells was analyzed using MTT assay. ( K ) Spearman correlation heatmap for four specific microbiomes with cytokines and enzyme in oral cancer. (The color chart range has been set from −1.00 to 1.00.) Statistical significance * p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant.

Article Snippet: Total human SCFAs in each saliva sample was measured using an SCFA ELISA Kit (MBS7269061; MyBioSource, San Diego, CA, USA), which has a sensitivity of 0.92 pg/mL.

Techniques: Immunohistochemical staining, Biomarker Discovery, Immunohistochemistry, Staining, Control, Clinical Proteomics, Western Blot, Transfection, MTT Assay

Journal: International Journal of Molecular Sciences

Article Title: Oral Microbiome and CPT1A Function in Fatty Acid Metabolism in Oral Cancer

doi: 10.3390/ijms252010890

Figure Lengend Snippet: Exploring the impact of oral microbiota on carnitine O-palmitoyltransferase 1A (CPT1A) function in fatty acid metabolism and its potential immunomodulatory effects in oral cancer. The oral microbiome may modulate the upregulation of CPT1A, Oxidative Stress Responsive Kinase 1 (OXSR1), Interleukin-6 (IL-6), and Tumor Necrosis Factor-alpha (TNF-α) in oral cancer patients, while concurrently influencing the downregulation of short-chain fatty acids. Red: increased, blue: decreased.

Article Snippet: Total human SCFAs in each saliva sample was measured using an SCFA ELISA Kit (MBS7269061; MyBioSource, San Diego, CA, USA), which has a sensitivity of 0.92 pg/mL.

Techniques: