Journal: International Journal of Molecular Sciences

Article Title: Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches

doi: 10.3390/ijms23010412

Figure Lengend Snippet: Identification of cleavage sites of SpeBm in peptide substrates derived from the E. coli proteome by PICS analysis. A cumulative number of 1975 unique cleavage events were determined in trypsin- ( A , B ) and GluC-derived ( C , D ) peptide libraries employing stable isotope labeling and HPLC-MS analysis. Heatmaps depict the fold-change in occurrence of specific amino acids at certain positions in the substrate in relation to their natural abundance using either ( A ) trypsin or ( C ) GluC as library protease. Sequence logos ( B , D ) demonstrate the difference in percent of amino acids at specific positions in prime and non-prime sites as obtained by the IceLogo algorithm. Data are based on ( C ) a tryptic or ( D ) a GluC-derived peptide library. The color code represents the physico-chemical properties of amino acids: hydrophobic residues (blue), positively charged residues (red), negatively charged residues (green), hydrophilic residues (black), and others (violet). Amino acids that were not found in any of the determined peptide substrates at a specific position are depicted in gray.

Article Snippet: PICS experiments were carried out with the commercially available SpeBm enzyme (FABulous; Genovis, Lund, Sweden).

Techniques: Derivative Assay, Quantitative Proteomics, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches

doi: 10.3390/ijms23010412

Figure Lengend Snippet: Proteolysis of IgG subclasses by SpeBm. Total ion chromatograms of human polyclonal IgG digested by SpeBm under ( A ) reducing or ( B ) physiological conditions. Main cleavage products are annotated: fragment crystallizable (Fc) and fragment antigen-binding (Fab), as well as their derivatives. Numbers denote the corresponding subclasses from which these subunits originate. Single peaks may be annotated by multiple numbers, as proteolytic products from different subclasses may co-elute. The asterisk (*) denotes a chromatographic peak corresponding to an incompletely alkylated IgG1 subunit cleaved at KTHT|CPPC. § Putative chromatographic peak of the Fd subunit and the light chain (LC). §§ Putative chromatographic peak of Fab 2 and Fab 2 -comprising C H 2 domains. The exact nature of these species could not be determined due to the high amino acid sequence variability of their variable portions and was derived from the identified cleavage sites. ( C ) SpeBm cleavage sites are mapped to a schematic of an IgG1 antibody for all four subclasses in a combined manner. Whether the cleavage also occurs under physiological conditions is specified in the respective small box with a red cross. ( D ) Likelihood of cleavage sites calculated for the hinge region (upper sequence) and the joint region (lower sequence) of IgG1. High green values suggest likely cleavage, whereas low red values suggest unlikely cleavage. Values were calculated based on amino acid occurrences relative to natural abundance of combined tryptic and GluC PICS data, considering P3 to P3′. Black vertical lines show experimentally observed cleavages.

Article Snippet: PICS experiments were carried out with the commercially available SpeBm enzyme (FABulous; Genovis, Lund, Sweden).

Techniques: Binding Assay, Sequencing, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: In vitro citrullination of bacterially produced chemokines leads to their partial degradation. ( a ) Colloidal Coomassie stained SDS-PAGE analysis of the MCP-1/CCL2 products of in vitro citrullination. In vitro citrullination of commercially available recombinant MCP-1/CCL2 and self-made bacterially produced MCP-1/CCL2 performed with recombinant human PAD2 and PAD4 or rabbit PAD2, respectively. Colloidal Coomassie stained MCP-1 bands are shown while recombinant humanPAD2/4 or rabbit PAD2 (Sigma Aldrich/Merck, Waltham, MA, USA) were added to reactions into the quantities below the Colloidal Coomassie sensitivity limits and cannot be visualized. ( b ) Immunoblot analysis of bacterially produced MCP-1/CCL2 upon an in vitro citrullination reaction. Self-made full-length bacterially produced MCP-1/CCL2 was citrullinated in vitro with rabbit PAD2 and resolved on SDS-PAGE, stained with Ponceau S. detection of total MCP-1/CCL2 and modified citrullines with Senshu’s antibody that recognizes that modified citrullines were made according to Senshu’s protocol . Results shown are a representative of three or more repetitive experiments.

Article Snippet: A monoclonal mouse antibody specific for human MCP-1/CCL2 (BioLegend, San Diego, CA, USA) was used at a dilution of 1:1000.

Techniques: In Vitro, Produced, Staining, SDS Page, Recombinant, Western Blot, Modification

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Mass-spectrometry verification of successful in vitro citrullination of MCP-1/CCL2. ( a ) Amino acid sequence of MCP-1/CCL2 (mature form of the protein spans residues 24–99). The first row shows 23 amino acid-long signaling peptide and the second row–mature protein of 76 residues. All arginine residues are highlighted with circles. The boxed areas show the peptide that contains the citrullinated arginine residues that were detected. ( b ) Annotated tandem mass spectrometry (MS/MS) fragmentation spectrum for the citrullinated MCP1/CCL2 peptide, showing the citrullinated arginine residue at position 3 of the studied peptide (R45). The MS/MS fragmentation data were annotated using Expert System (Max Planck Institute of Biochemistry). The precursor ion was observed with a mass error of 1 part per million, and the error for the fragment ions was 0.02 daltons.

Article Snippet: A monoclonal mouse antibody specific for human MCP-1/CCL2 (BioLegend, San Diego, CA, USA) was used at a dilution of 1:1000.

Techniques: Mass Spectrometry, In Vitro, Sequencing, Tandem Mass Spectroscopy, Residue

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Detection of citrullinated recombinant human MCP-1/CCL2. Standard curves of citrullinated recombinant human MCP-1/CCL2 were set up using enzyme-linked immunosorbent assay in duplicate ( a ) standard curve of citrullinated E. coli produced recombinant human MCP-1/CCL2 chemokine (R&D Systems), ( b ) standard curve for citrullinated MCP-1/CCL2 chemokine produced by and purified from human HEK 293T cells. Absorbance at 450 nm is shown. Coefficients of determination R 2 are indicated in red on the relevant plots. Results shown are a representative of three or more repetitive experiments.

Article Snippet: A monoclonal mouse antibody specific for human MCP-1/CCL2 (BioLegend, San Diego, CA, USA) was used at a dilution of 1:1000.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Produced, Purification

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Site-directed mutagenesis of potential glycosylation site at asparagine-14 in mammalian cell-produced MCP-1 significantly destabilizes recombinant human MCP-1/CCL2. ( a ) Amino acid sequence of MCP-1/CCL2 with indicated positions of predicted N-glycosylation and citrullination site confirmed by a mass-spectrometry. ( b ) Immunoblot analysis of bacterially versus mammalian cell-produced MCP-1/CCL2 upon an in vitro citrullination reaction. Bacterially produced wild-type MCP-1/CCL2 or HEK293T cell-produced wild-type and N14Q mutant version of chemokine were incubated with EDTA-free proteinase inhibitor cocktail supplemented cellular lysates prepared from the control (vehicle-transfected) or indicated hPAD enzyme-transfected HEK293T cells. Citrullination reactions that were performed directly within cellular lysates essentially as published before . Recombinant chemokine was concentrated with immunoprecipitation and resolved on 10–18% polyacrylamide gradient SDS-PAGE. Results shown are a representative of three experiments.

Article Snippet: A monoclonal mouse antibody specific for human MCP-1/CCL2 (BioLegend, San Diego, CA, USA) was used at a dilution of 1:1000.

Techniques: Mutagenesis, Produced, Recombinant, Sequencing, Mass Spectrometry, Western Blot, In Vitro, Incubation, Transfection, Immunoprecipitation, SDS Page

Journal: bioRxiv

Article Title: MAFB surrogates the glucocorticoid receptor ability to induce tolerogenesis in dendritic cells

doi: 10.1101/2021.07.27.453975

Figure Lengend Snippet: (A) Schematic representation of the experimental approach, comparing dendritic cell (DC) with tolerogenic dendritic cell (tolDC) differentiation. (B) DC and tolDC were cocultured with CD8+ cells for 5 days. The final CFSE signal of CD8+ cells is shown (left panel). CD8+ with only CD3/CD28 T-activator beads (C+) or alone (C-) are also shown. In the right panel, the average proliferation of the quadruplicate is shown (mean ± standard error of the mean (SEM)). (C) IL-10, TNFα, IL-12p70 and IL1-β production of DC and tolDC, after 5 days of differentiation and 24 h of LPS (10 ng/μL) and IFNg (20 ng/μL) stimuli. P-values of paired t-tests are shown. (D) Box-plots of CD80, CD83, CD86 and HLA-DR surface expression (Median Fluorescence Intensity) in DCs and tolDCs in steady-state or stimulated with LPS (10 ng/μL) and IFNg (20 ng/μL) (ns p > 0.05, ** p < 0.01, *** p ≤ 0.001). (E) Gene expression heatmap of differentially expressed genes comparing tolDCs with DCs and also displaying the gene expression values of the precursor cell type (MO) (logFC > 0.5, FDR < 0.05). Scaled fluorescence values of expression arrays are shown, ranging from -2 (lower gene expression, green) to +2 (higher gene expression, orange). (F) Gene ontology (GO) over-representation of GO Biological Process categories. Fold change of tolDC induced genes over background and -log10(FDR) of Fisher’s exact tests are shown. (G) Discriminant regulon expression analysis (DoRothEA) of tolDC compared with DC. Only transcription factors with FDR < 0.05 are shown. NES and logFC of transcription factor expression are depicted. (H) T-distributed stochastic neighbor embedding (t-SNE) plot of the aggregated and batch-corrected gene expression data from our study (MO, DC and tolDC) and two additional public datasets (GSE40484 (moMAC, moDC, cDC2, CM (Classical MOs) and NCM (Non-Classical MOs) and GSE99056 (M-MAC (M2 Macrophages) and GM-MAC (M1 Macrophages)). The 4 different groups obtained using k-means clustering are represented with grey ellipses of multivariate t-distributions.

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were performed, following the manufacturer’s instructions: Human IL-10, Human IL-12p70, and Human TNFα from BioLegend, and Human IL-1β from ThermoFisher.

Techniques: Expressing, Fluorescence, Gene Expression

Journal: bioRxiv

Article Title: MAFB surrogates the glucocorticoid receptor ability to induce tolerogenesis in dendritic cells

doi: 10.1101/2021.07.27.453975

Figure Lengend Snippet: (A) Volcano plot comparing tolDCs treated with control siRNA (siCTL) and MAFB siRNA (siMAFB). Dashed lines indicate significance thresholds (FDR < 0.05, absolute logFC > 0.5). tolDC-induced and tolDC-repressed genes are shown in blue and orange, respectively. (B) Gene set enrichment analysis (GSEA) of tolDCs (siCTL) vs. tolDCs (siMAFB), using tolDC-induced and tolDC-repressed gene sets. The running enrichment score is represented and the normalized enrichment score (NES) is shown above (FDR < 0.01). (C) DNA methylation heatmap of previously obtained differentially methylated CpGs (C1-CpGs and C2-CpGs) in tolDCs (siCTL) and tolDCs (siMAFB). Scaled β-values are shown (lower DNA methylation levels in blue and higher methylation levels in red). On the right side, violin plots of Cluster 1 (C1) and Cluster 2 (C2) depict β-values (ns p > 0.05, *** p ≤ 0.001). (D) Methylated CpG set enrichment analysis (mCSEA) of tolDCs (siCTL) vs. tolDCs (siMAFB), using MAFB-only CpGs, GR/MAFB CpGs and GR-only CpGs as CpG-sets (depending on the overlap of CpGs with GR or MAFB peaks). The running enrichment score is represented and the normalized enrichment score (NES) and FDR are shown above. (E) Box-plots of median fluorescence intensity (MFI) of CD14, CD16, CD163 and CD1a flow cytometry data from DCs (siCTL), tolDCs (siCTL) and tolDCs (siMAFB) (n = 7) (ns p > 0.05, * p < 0.05, ** p ≤ 0.01). (F) Box-plots of supernatant concentration from DCs (siCTL), tolDCs (siCTL) and tolDCs (siMAFB) (n = 7) of IL-10 in steady-state and stimulated conditions (LPS 10 ng/μL and IFNγ 20 ng/μL) and IL-12p70 and TNFα under stimulated conditions (pg/mL). TNFα and IL-12p70 in steady state were not detected. (ns p > 0.05, * p < 0.05, ** p ≤ 0.01) (G) DC (siCTL), tolDC (siCTL) and tolDC (siMAFB) were cocultured with CD8+ cells for 5 days (n = 4). The final CFSE signal of CD8+ cells is shown (left panel). CD8+ with only CD3/CD28 T-activator beads (C+) or alone (C-) are also shown. On the right panel, the average proliferation of the quadruplicate is shown (mean ± standard error of the mean (SEM)) (** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were performed, following the manufacturer’s instructions: Human IL-10, Human IL-12p70, and Human TNFα from BioLegend, and Human IL-1β from ThermoFisher.

Techniques: Control, DNA Methylation Assay, Methylation, Fluorescence, Flow Cytometry, Concentration Assay

Journal: Journal of dairy science

Article Title: Efficacy of prolonged ingestion of Lactobacillus acidophilus L-92 in adult patients with atopic dermatitis.

doi: 10.3168/jds.2015-10605

Figure Lengend Snippet: Figure 2. Changes in IL-12 (p70). Values are presented as aver- age ± SE. Wilcoxon signed rank test was used. L-92 = Lactobacillus acidophilus L-92. *P < 0.05.

Article Snippet: The IL-12 (p70) and TGF-β were measured by ELISA using a human IL-12 (p70) ELISA kit (BD Biosciences, San Jose, CA) and LEGEND Free Active TGF-β1 kit (BioLegend, San Diego, CA), respectively.

Techniques: