Journal: Langmuir : the ACS journal of surfaces and colloids

Article Title: Determining Molecular-Level Interactions of Carboxyl-Functionalized Nanodiamonds with Bacterial Membrane Models as the Basis for Antimicrobial Activity.

doi: 10.1021/acs.langmuir.4c05173

Figure Lengend Snippet: Figure 1. Schematic illustration for (A) E. coli (Gram-negative) and (B) S. aureus (Gram-positive) bacteria along with their respective membrane structures (insets). The lipid membrane regions mimicked in this study are indicated, and the molecular structures of the lipids in the composition of (A) E. coli lipid extract and (B) S. aureus lipid mixture are shown. The numbers 1 and 2 refer to the lipid chains of PE and PG, which can have different sizes and be unsaturated or not.

Article Snippet: Escherichia coli (E. coli) total lipid extract (strain B; ATCC 11303; Avanti Polar Lipids, 100500P) consists of 57.5% phosphatidylethanolamines (PE), 15.1% phosphatidylglycerols (PG), 9.8% cardiolipins (CL), and 17.6% unidentified components.

Techniques: Bacteria, Membrane

Journal: Langmuir : the ACS journal of surfaces and colloids

Article Title: Determining Molecular-Level Interactions of Carboxyl-Functionalized Nanodiamonds with Bacterial Membrane Models as the Basis for Antimicrobial Activity.

doi: 10.1021/acs.langmuir.4c05173

Figure Lengend Snippet: Figure 2. Surface pressure (mN/m) versus area (102 cm2/mL lipid extract) isotherms and surface compression modulus (Cs −1) for (A) E. coli and (B) E. coli:NDCOOHs (1:1, 1:2, and 1:4, v/v) and for (C) S. aureus and (D) S. aureus:NDCOOHs monolayers (1:1, 1:2, and 1:4, v/v).

Article Snippet: Escherichia coli (E. coli) total lipid extract (strain B; ATCC 11303; Avanti Polar Lipids, 100500P) consists of 57.5% phosphatidylethanolamines (PE), 15.1% phosphatidylglycerols (PG), 9.8% cardiolipins (CL), and 17.6% unidentified components.

Techniques:

Journal: Langmuir : the ACS journal of surfaces and colloids

Article Title: Determining Molecular-Level Interactions of Carboxyl-Functionalized Nanodiamonds with Bacterial Membrane Models as the Basis for Antimicrobial Activity.

doi: 10.1021/acs.langmuir.4c05173

Figure Lengend Snippet: Figure 3. PM-IRRAS spectra for (A) E. coli and E. coli:NDCOOHs (1:1) and (B) S. aureus and S. aureus:NDCOOHs (1:2) monolayers on the PBS subphase at 30 mN/m. The left panels highlight bands related to polar head groups, while the right panels refer to those associated with alkyl chain groups.

Article Snippet: Escherichia coli (E. coli) total lipid extract (strain B; ATCC 11303; Avanti Polar Lipids, 100500P) consists of 57.5% phosphatidylethanolamines (PE), 15.1% phosphatidylglycerols (PG), 9.8% cardiolipins (CL), and 17.6% unidentified components.

Techniques:

Journal: Langmuir : the ACS journal of surfaces and colloids

Article Title: Determining Molecular-Level Interactions of Carboxyl-Functionalized Nanodiamonds with Bacterial Membrane Models as the Basis for Antimicrobial Activity.

doi: 10.1021/acs.langmuir.4c05173

Figure Lengend Snippet: Figure 4. Schematic representation of the binding mechanism of NDCOOHs in (A) E. coli extract and (B) S. aureus lipid mixture monolayers. Green arrows indicate attractive electrostatic interactions, red arrows show repulsive electrostatic interactions, and blue dashed arrows illustrate secondary interactions, such as van der Waals and hydrogen bonding.

Article Snippet: Escherichia coli (E. coli) total lipid extract (strain B; ATCC 11303; Avanti Polar Lipids, 100500P) consists of 57.5% phosphatidylethanolamines (PE), 15.1% phosphatidylglycerols (PG), 9.8% cardiolipins (CL), and 17.6% unidentified components.

Techniques: Binding Assay

Journal: Langmuir : the ACS journal of surfaces and colloids

Article Title: Determining Molecular-Level Interactions of Carboxyl-Functionalized Nanodiamonds with Bacterial Membrane Models as the Basis for Antimicrobial Activity.

doi: 10.1021/acs.langmuir.4c05173

Figure Lengend Snippet: Figure 5. Comparative evolution of relative area (A/A0) versus time (100 min) for (A) E. coli and E. coli:NDCOOHs (1:1) and for (B) S. aureus and S. aureus:NDCOOHs (1:2) recorded at constant pressure of 30 mN/m. A0 is the extrapolated area of the isotherms at 30 mN/m down to zero pressure.

Article Snippet: Escherichia coli (E. coli) total lipid extract (strain B; ATCC 11303; Avanti Polar Lipids, 100500P) consists of 57.5% phosphatidylethanolamines (PE), 15.1% phosphatidylglycerols (PG), 9.8% cardiolipins (CL), and 17.6% unidentified components.

Techniques:

Journal: Cellular and Molecular Life Sciences

Article Title: Polysialic acid and Siglec-E orchestrate negative feedback regulation of microglia activation

doi: 10.1007/s00018-020-03601-z

Figure Lengend Snippet: Validation of polySia expression and polysialylated proteins in BV2 microglia. a Immunofluorescence staining of polySia co-localized with the Golgi marker giantin. Cell shape is highlighted by co-staining with the microglia/macrophage marker CD11b. Nuclei were counterstained with DAPI (blue). Scale bar, 20 µm. b Immunoprecipitation (IP) of polysialylated proteins from lysate of 10 7 BV2 cells using polySia-specific mAb 735-conjugated magnetic beads followed by Western blot (WB) detection with polySia-specific antibody (left), or by joint incubation with NRP2- and ESL-1-specific antibodies (right). Where indicated, IP fractions were treated with endosialidase (endo +), to remove polysialic acid. Protein bands were assigned according to the apparent molecular weights of NRP2 and ESL-1 as previously detected in primary and stem cell-derived murine microglia or in mouse brain tissue [ ; see text for details]. c Compared to untreated controls (ctrl), incubation of BV2 cultures with 1-µg/ml LPS for 24 h leads to the loss of polySia signals in almost all cells. Nuclei were counterstained with DAPI (blue). Scale bar, 50 µm. d Loss of polySia-positive cells after LPS treatment, as indicated. Individual values and means of 5 evaluated frames per culture condition, with a minimum of 20 cells each are plotted and significant difference by two-tailed t-test is indicated (*** p < 0.001). e PolySia with DP > 4 but not trisialic acid (DP3) attenuates the LPS-induced production of NO. Nitrite levels in the supernatant of BV2 cells cultured for 24 h in the presence or absence of 30-nM trisialic acid (DP3), 500-ng/ml poly—Sia (approximately 30 nM, see text for details), and/or 1-µg/ml LPS, as indicated. Individual values and means from 3 independent treatments per group are plotted. One-way ANOVA indicated significant differences ( p < 0.0001) and results from Tukey’s post hoc test are shown for comparisons between the LPS-treated groups (* p < 0.05)

Article Snippet: Lipopolysaccharide (LPS) extracted from E. coli serotype O127:B8, 4-chloro- m -cresol (4-C m C), TAK-242, 1,1′‐diheptyl‐4,4′‐bipyridinium dibromide (DHBP), genistein, and biotinyl tyramide were from Merck, Darmstadt, Germany.

Techniques: Biomarker Discovery, Expressing, Immunofluorescence, Staining, Marker, Immunoprecipitation, Magnetic Beads, Western Blot, Incubation, Derivative Assay, Two Tailed Test, Cell Culture

Journal: Cellular and Molecular Life Sciences

Article Title: Polysialic acid and Siglec-E orchestrate negative feedback regulation of microglia activation

doi: 10.1007/s00018-020-03601-z

Figure Lengend Snippet: PolySia staining patterns in BV2 cells treated for 10 or 20 min with 1-µl/ml DMSO ( a ), 50-µM RyR agonist 4-C m C ( b ), 1-µg/ml LPS ( c ), or 1-µg/ml LPS together with 10-µM RyR antagonist DHBP ( d ), and quantitative assessment ( e ), as indicated. Per well, a minimum of 10 cells in 3 randomly selected frames with at least 3 cells each were evaluated and percentages of cells with detectable polySia signals at the cell surface (upper graph) or co-localized with the Golgi marker giantin (lower graph) were calculated. Individual values and means of 3 wells per condition are plotted. For each data set, one-way ANOVA indicated significant differences ( p < 0.0001) and results from Tukey’s post hoc test are shown for comparisons against the DMSO control and for selected group comparisons (** p < 0.01, *** p < 0.001, **** p < 0.0001). 4-C m C and DHBP were added as 1-µl/ml stock solution in DMSO. Giantin and CD11b were co-stained to visualize the Golgi compartment and the cells’ shape, respectively. Nuclei were counterstained with DAPI (blue). Scale bar, 20 µm. See text for a description of treatment effects

Article Snippet: Lipopolysaccharide (LPS) extracted from E. coli serotype O127:B8, 4-chloro- m -cresol (4-C m C), TAK-242, 1,1′‐diheptyl‐4,4′‐bipyridinium dibromide (DHBP), genistein, and biotinyl tyramide were from Merck, Darmstadt, Germany.

Techniques: Staining, Marker, Control

Journal: Cellular and Molecular Life Sciences

Article Title: Polysialic acid and Siglec-E orchestrate negative feedback regulation of microglia activation

doi: 10.1007/s00018-020-03601-z

Figure Lengend Snippet: Detection of polysialylated proteins released by LPS-induced BV2 microglia. a Elution profile of cell culture supernatants collected from 2.5 × 10 7 BV2 cells treated with 10-µg/ml LPS for 24 h and applied to immunoaffinity chromatography with polySia-specific antibody. The increase of conductivity (brown line) denotes the onset of elution with 2-M NaCl. Detection at 214 nm and 280 nm (red and blue line), indicative for the presence of sialic acid and protein, respectively, resulted in peaks during washing (fractions 18–20) and during elution (fractions 22–24). b Western blot detection of polysialylated protein in the cell culture supernatant prior to immunopurification (left panel) and in the pooled fractions 22–24, but not in fractions 18–20 and 26–28 of the chromatogram shown in a (right panel). Specificity of polySia detection in the supernatant was controlled by enzymatic removal of polySia with endosialidase (+ endo). c Elution profile of cell culture supernatants as in a , but collected from 3 × 10 7 BV2 cells during the first 4 h or, after changing the medium, between 4 and 24 h after the onset of LPS treatment, respectively. The left panel shows an elution profile without sample (blank)

Article Snippet: Lipopolysaccharide (LPS) extracted from E. coli serotype O127:B8, 4-chloro- m -cresol (4-C m C), TAK-242, 1,1′‐diheptyl‐4,4′‐bipyridinium dibromide (DHBP), genistein, and biotinyl tyramide were from Merck, Darmstadt, Germany.

Techniques: Cell Culture, Chromatography, Western Blot, Immu-Puri

Journal: Cellular and Molecular Life Sciences

Article Title: Polysialic acid and Siglec-E orchestrate negative feedback regulation of microglia activation

doi: 10.1007/s00018-020-03601-z

Figure Lengend Snippet: LPS-induced changes of Siglec-E in BV2 microglia. a Analysis by quantitative real-time RT-PCR reveals a strong increase of Siglec-E mRNA in cells treated with 1-µg/ml LPS for 24 h. Individual values and means from 3 independent treatments per group are plotted. ** p < 0.01, unpaired t test. b Immunofluorescence detection of Siglec-E (red) and polySia (green). Nuclear counterstain with DAPI (blue). Staining patterns before (control, ctrl) and after treatment with 1-µg/ml LPS for 24 h. Lower panels show 3D reconstructions at higher magnification. c Colocalization of Siglec-E (red) and polySia (green) with the endosomal marker EEA1 (cyan). Staining patterns before (control, ctrl) and after treatment with 1-µg/ml LPS for 10 min. Lower panels show 3D reconstructions of single and merged channels with nuclear counterstain (DAPI, blue) at higher magnification. d – h Detection of Siglec-E (red) and polySia (green) as in b . d Staining patterns after 20 min of LPS treatment in the presence of solvent (1-µl/ml DMSO, left) or 200-µM genistein (right). e , f Staining patterns after incubation for 20 h without or with LPS followed by 10 min with 1-µl/ml DMSO ( e ), 200-µM genistein, or 1-µM TAK-242 ( f ), as indicated. Genistein and TAK-242 were added as 1-µl/ml stock solution in DMSO. g Incubation of LPS-treated cells with DMSO and TAK-242 as in e and f , but this time the cell culture medium was changed to apply DMSO and TAK-242. h Staining patterns after incubation with polySia (10 µg/ml) for 1 min (left) or 20 min (right). The strong Siglec-E signals under control conditions without LPS ( b , c , e , h ) and after genistein treatment ( d , f ) are overexposed to enable a visualization of the weak signals in LPS-treated cells with the same camera setting. Scale bars, 50 µm in b and c (upper panels) and in d – h ; 10 µm in b and c, lower panels. i Quantitative assessment of reduced Siglec-E cell surface staining under the conditions shown in d – h . Based on the densitometric evaluation of signal intensities of 108 cells treated for 10 min with DMSO in the absence of LPS, intensities below 50% of the mean Siglec-E signal intensity under these conditions were considered “reduced”. Per well, a minimum of 20 cells in 3 randomly selected frames with at least 5 cells each were evaluated and the percentage of cells with reduced Siglec-E immunoreactivity was calculated. Individual values and means of 3 wells per condition are plotted. One-way ANOVA indicated significant differences ( p < 0.0001) and results from Tukey’s post hoc test are shown for selected comparisons (**** p < 0.0001)

Article Snippet: Lipopolysaccharide (LPS) extracted from E. coli serotype O127:B8, 4-chloro- m -cresol (4-C m C), TAK-242, 1,1′‐diheptyl‐4,4′‐bipyridinium dibromide (DHBP), genistein, and biotinyl tyramide were from Merck, Darmstadt, Germany.

Techniques: Quantitative RT-PCR, Immunofluorescence, Staining, Control, Marker, Solvent, Incubation, Cell Culture

Journal: Cellular and Molecular Life Sciences

Article Title: Polysialic acid and Siglec-E orchestrate negative feedback regulation of microglia activation

doi: 10.1007/s00018-020-03601-z

Figure Lengend Snippet: Loss of Siglec-E abrogates responsiveness to polySia and enhances LPS-induced activation. a Compared to wildtype BV2 cells ( Siglece +/+ ), the immunoreactivity of Siglec-E (red), but not polySia (green) is abolished by CRISPR/spCas9-mediated knockout of Siglece ( Siglece −/− , clone D19). Nuclear counterstain with DAPI (blue). Scale bar, 50 µm. b Comparable to the effect of preincubation with 60-µM minocycline for 2 h, application of polySia (5 µg/ml) inhibits the LPS-induced NO production of Siglece +/+ but not Siglece −/− BV2 microglia (clone D19). In addition, the LPS-induced NO production of Siglece −/− microglia was significantly higher. Where indicated (LPS +), 1-µg/ml LPS was applied for 24 h. c , d During 24 h of LPS treatment, Siglec-E-negative cells also showed a significantly more pronounced increase of TNF and IL-6 mRNA levels. In b – d , individual values and means from n = 3 independent treatments per group are plotted. Mixed two-way ANOVA indicated significant differences and results from Holms–Sidak post hoc test are shown for selected group comparisons (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001)

Article Snippet: Lipopolysaccharide (LPS) extracted from E. coli serotype O127:B8, 4-chloro- m -cresol (4-C m C), TAK-242, 1,1′‐diheptyl‐4,4′‐bipyridinium dibromide (DHBP), genistein, and biotinyl tyramide were from Merck, Darmstadt, Germany.

Techniques: Activation Assay, CRISPR, Knock-Out