Journal: Regenerative Therapy

Article Title: Orexin-A increases the differentiation of human olfactory sensory neurons through orexin receptor type 1

doi: 10.1016/j.reth.2024.10.014

Figure Lengend Snippet: Immunofluorescence of orexin receptors and dose-response analyses of orexin-A. (a) Immunofluorescence images of OSNs stained with OX1R and OX2R (green). (b) Schematic showing the proposed mechanism of orexin-mediated differentiation of OSNs and dose-response analyses of suvorexant. Western blot densitometric analysis of (c) ASCL1, (d) βIII Tubulin, and (e) OMP, respectively. (p > 0.05, compared to controls, n = 5).

Article Snippet: The orexin receptor type 1 (OX1R) antagonist, SB-674042 (HY-10898, MedChem Express, NJ, USA), and orexin receptor type 2 (OX2R) antagonist, TCS-OX2-29 (HY-100452, MedChem Express) were diluted in the dimethyl sulfoxide, DMSO with 1 μg/mL and 10 μg/mL, respectively.

Techniques: Immunofluorescence, Staining, Western Blot

Journal: Regenerative Therapy

Article Title: Orexin-A increases the differentiation of human olfactory sensory neurons through orexin receptor type 1

doi: 10.1016/j.reth.2024.10.014

Figure Lengend Snippet: The effect of OX1R antagonists (SB674042) and OX2R antagonists (TCS-OX2-29) on the differentiation of OSN. The expression of various markers was assessed through western blots (a and g) and subsequently analyzed densitometrically for ADCY3, Golf, ASCL1, βIII tubulin, and OMP (b-f and h-l). (Asterisks indicate p < 0.05, compared to controls, n = 5–6).

Article Snippet: The orexin receptor type 1 (OX1R) antagonist, SB-674042 (HY-10898, MedChem Express, NJ, USA), and orexin receptor type 2 (OX2R) antagonist, TCS-OX2-29 (HY-100452, MedChem Express) were diluted in the dimethyl sulfoxide, DMSO with 1 μg/mL and 10 μg/mL, respectively.

Techniques: Expressing, Western Blot

Journal: Endocrinology

Article Title: Inhibition of Lysyl Oxidase by Cortisol Regeneration in Human Amnion: Implications for Rupture of Fetal Membranes.

doi: 10.1210/en.2016-1406

Figure Lengend Snippet: Figure 1. Changes of 11-HSD1, cortisol and LOX in human amnion tissue at parturition and reciprocal regulation of 11- HSD1 and LOX by cortisol in human amnion tissue explants. (A and B) Comparison of 11-HSD1 protein, cortisol (A) and LOX activity (B) between amnion tissues collected at term after elective Cesarean section (term nonlabor) and after vaginal delivery with spontaneous rupture of the membranes (term labor). n 7 for 11-HSD1, n 6 for LOX and n 10 for cortisol. (C) Effects of cortisol (1 M) on 11-HSD1 and LOX protein abundance in cultured human amnion tissue explants. n 3. The top panels of A and C are the representative immunoblots. Data are the mean SEM. Statistical analysis was performed using unpaired (A, B) and paired (C) Student’s t test. *P .05, **P .01 vs. nonlabor or control.

Article Snippet: Following blocking, antibodies against 11 -HSD1 (Origene, Rockville, MD), LOX (Abcam, Cambridge, UK), cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA) and vimentin (Santa Cruz) at 1:100 dilutions were applied.

Techniques: Comparison, Activity Assay, Quantitative Proteomics, Cell Culture, Western Blot, Control

Journal: Endocrinology

Article Title: Inhibition of Lysyl Oxidase by Cortisol Regeneration in Human Amnion: Implications for Rupture of Fetal Membranes.

doi: 10.1210/en.2016-1406

Figure Lengend Snippet: Figure 2. Distribution of LOX and 11-HSD1 in human fetal membranes and the effects of cortisol in human amnion epithelial and fibroblast cells. (A-C) Immunofluorescence staining of 11-HSD1 (A, orange) and LOX (B, green) in human fetal membranes. Yellow color after merge represents the colocalization of 11-HSD1 and LOX (C). Nuclei were counterstained blue with DAPI. Scale bar, 10 m. (D) Immunofluorescent staining of LOX and LOX activity in cultured amnion epithelial and fibroblast cells. The top panel is the double staining of LOX (green) and vimentin (red) in amnion fibroblasts, and LOX (green) and cytokeratin (red) in amnion epithelial cells. Nuclei were counterstained blue with DAPI. Arrows indicate amnion epithelial cells stained positive for LOX. Scale bar, 10 m. The bottom panel illustrates the effect of cortisol (1 M) on LOX activity in amnion fibroblasts and epithelial cells. n 3. E, The reciprocal effects of cortisol (1 M) on 11-HSD1 and LOX protein abundance in human amnion epithelial and fibroblast cells. (F-H) Immunofluorescent double staining of 11-HSD1 (F, orange) and LOX (G, green) in human amnion fibroblasts. Nuclei were counterstained blue with DAPI. Yellow color after merge represents the colocalization of 11- HSD1 and LOX (H). Scale bar, 10 m. (I and J) Concentration-dependent induction of 11-HSD1 (I, n 5) and reduction of LOX (J, n 5 for mRNA, n 7 for protein) by cortisol (0.01–1 M) in human amnion fibroblasts. The top panels of E, I and J are the representative immunoblots. ae: amnion epithelial cells; af: amnion fibroblasts; ct: chorion trophoblasts. Data are the mean SEM. Statistical analysis was performed with paired Student’s t test (E, K) or one-way ANOVA test (I and J). *P .05, **P .01, ***P .001 vs. control or 0 M.

Article Snippet: Following blocking, antibodies against 11 -HSD1 (Origene, Rockville, MD), LOX (Abcam, Cambridge, UK), cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA) and vimentin (Santa Cruz) at 1:100 dilutions were applied.

Techniques: Immunofluorescence, Staining, Activity Assay, Cell Culture, Double Staining, Quantitative Proteomics, Concentration Assay, Western Blot, Control

Journal: Endocrinology

Article Title: Inhibition of Lysyl Oxidase by Cortisol Regeneration in Human Amnion: Implications for Rupture of Fetal Membranes.

doi: 10.1210/en.2016-1406

Figure Lengend Snippet: Figure 3. Involvement of 11-HSD1 in the down-regulation of LOX by cortisone in human amnion fibroblasts. (A) Attenuation of cortisone (5 M)-induced reduction in LOX mRNA and protein by 11-HSD1 inhibitor CBX (1 M). n 3. (B) Attenuation of cortisone (5 M)-induced reduction in LOX mRNA and LOX protein by 11-HSD1 inhibitor 10j (1 M). n 3 for mRNA, n 4 for protein. (C) Attenuation of cortisone (5 M)-induced reduction in LOX mRNA, protein and activity by siRNA (50 nM)-mediated knock-down of 11-HSD1. n 5. The top panels of A-C are the representative immunoblots. Data are the mean SEM. Statistical analysis was performed with one-way ANOVA test. *P .05, ***P .001 vs. control or negative scrambled siRNA control; #P .05, ##P .01, ###P .001 vs. cortisone alone.

Article Snippet: Following blocking, antibodies against 11 -HSD1 (Origene, Rockville, MD), LOX (Abcam, Cambridge, UK), cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA) and vimentin (Santa Cruz) at 1:100 dilutions were applied.

Techniques: Activity Assay, Knockdown, Western Blot, Control

Journal: Endocrinology

Article Title: Inhibition of Lysyl Oxidase by Cortisol Regeneration in Human Amnion: Implications for Rupture of Fetal Membranes.

doi: 10.1210/en.2016-1406

Figure Lengend Snippet: Figure 6. Involvement of GR and nGRE in the inhibition of LOX promoter activity by cortisol and the working model illustrating LOX inhibition in human amnion fibroblasts. (A) Inhibition of LOX promoter (-850bp 126bp, -450bp 126bp) activities by cortisol (1 M). n 4. (B) Blockade of cortisol (1 M)-induced inhibition of LOX promoter (-850bp 126bp) activity by RU486 (1 M, n 4) or siRNA (50 nM, n 5)-mediated knock-down of GR. (C) Enhanced enrichment of GR in LOX promoter spanning the putative nGRE upon cortisol (1 M) stimulation of human amnion fibroblasts as measured with the ChIP assay. n 4. TSS: transcription start site, Fwd and Rev indicate the aligning position of the forward primer (Fwd) and reverse primer (Rev). (D) Abolition of cortisol (1 M)-induced inhibition of LOX promoter (-850bp 126bp) activity by introduction of nucleotide mutations (nucleotides in red) into the putative nGRE (-260 to -252bp) but not by nucleotide mutations in the adjacent CRE (-332 to -325bp) (nucleotides in red). n 3. Data are the mean SEM. Statistical analysis was performed with paired Student’s t test (A, C and D) or one-way ANOVA test (B). *P .05, **P .01 vs. control or negative scrambled siRNA control; #P .05 vs. control or cortisol alone. (E) A working model illustrating the participation of the feed-forward regeneration of cortisol by 11-HSD1 in the structural remodeling in human amnion. Toward the end of gestation, accumulation of cortisol regenerated by 11-HSD1 in the amnion not only induces 11-HSD1 expression, but also down-regulates LOX expression via GR-mediated inhibition of LOX expression in amnion fibroblasts. At the same time, accumulation of cortisol also up-regulates PGE2 production via induction of cPLA2 and COX-2 expression. Increased PGE2 production, in turn, inhibits LOX expression via EP2/EP4 receptors in amnion fibroblasts. Cortisol and PGE2 may act in a coordinated fashion to cause dramatic down-regulation of LOX expression toward the end of gestation resulting in the reduction of collagen cross-linking and weakened tensile strength of the amnion. Reduction in collagen cross-linking may also predispose it to degradation by MMP, which further reduces the tensile strength of the amnion. All of these effects may be implicated in ROM during normal parturition in humans. Preterm activation of this feed-forward mechanism may result in PPROM and preterm birth. Blue arrows indicate stimulation and red lines indicate inhibition. E, cortisone; F, cortisol.

Article Snippet: Following blocking, antibodies against 11 -HSD1 (Origene, Rockville, MD), LOX (Abcam, Cambridge, UK), cytokeratin (Santa Cruz Biotechnology, Santa Cruz, CA) and vimentin (Santa Cruz) at 1:100 dilutions were applied.

Techniques: Inhibition, Activity Assay, Knockdown, Control, Expressing, Activation Assay

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes

doi: 10.1016/j.jcmgh.2023.03.011

Figure Lengend Snippet: Functional role of association of HBV with lipoproteins. ( A ) HepG2-NTCP cells were infected with high pure luciferase expressing recombinant HBV in presence or absence of fetal calf serum ( left ) or in presence of serum from ApoE knockout or control mice ( right ). On day 7 cell culture supernatants were analyzed for luminescence intensities. ( B ) Differentiated HepaRG-cells were incubated with an LDL-receptor blocking antibody (1/500) either before or before and during HBV infection or 4 days after infection. On day 12 after HBV infection HBeAg was determined in the cell supernatant by enzyme-linked immunosorbent assay, and cccDNA was quantified in cell lysates by quantitative real-time polymerase chain reaction. ( C–E ) Isolated liver macrophages were incubated for 1 hour with VP 594 in medium containing 10% human serum and washed. In ( C ) 5 μg/mL chylomicron NBD-cholesterol was added for 2 hours; in ( D ) 2 μg/mL HDL 488 was added for 1 hour before fixation. ( E ) Cells were fixed and stained for ApoA1. Merged pictures show VP 594 in red , NBD-cholesterol, HDL 488 , and ApoA1 staining in green , and nuclear staining with DAPI in blue . Scale bars indicate 10 µm. ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: Other reagents were BODIPY 493/503 , Alexa Fluor 594 carboxylic acid, succinimidyl ester, NBD-cholesterol (Invitrogen, Germany), 32 P (Hartmann Analytic, Germany), purified ApoA1, HDL (Acris Antibodies, Germany), filipin, Fluorescein-5-Isothiocyanat (FITC), PFA (Sigma-Aldrich, Germany), and DAPI (Roche, Germany).

Techniques: Functional Assay, Infection, Luciferase, Expressing, Recombinant, Knock-Out, Control, Cell Culture, Incubation, Blocking Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Isolation, Staining

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Hepatitis B Virus Targets Lipid Transport Pathways to Infect Hepatocytes

doi: 10.1016/j.jcmgh.2023.03.011

Figure Lengend Snippet: Recycling of HBV along intracellular transport pathways of free cholesterol. ( A–C ) Monocyte derived macrophages were loaded for 16 hours with cholesterol using medium containing 50 μg/ml acLDL, incubated with VP 594 containing medium for 1 hour, and further incubated for 4 hours with acLDL containing medium without VP 594 . After fixation cells were stained with filipin to detect free cholesterol. ( A ) Total cellular (total) and perinuclear (pn) areas of a representative cell are indicated by dashed lines . Boxed area is exemplarily shown enlarged below the main panels separately for each channel. Merged pictures show VP 594 in red and filipin staining in green . ( B and C ) 5 μmol/L U18666A was added to the culture medium as indicated. ( B ) Representative vision fields of several macrophages. ( C ) Relative fluorescence intensity of filipin and Alexa 594 in the cell periphery were calculated by subtracting pn from total signals and multiplying these with the corresponding area to quantify the content of free cholesterol and VP 594 . The diagram gives the ratio of pn to peripheral cholesterol (filipin, dots ) and VP 594 ( triangles ) in analyzed cells. ( D and E ) THP macrophages were incubated with HBV in serum-free medium for 3 hours, washed, and further cultured for 16 hours with 0.2% bovine serum albumin (BSA) containing medium only or medium supplemented with 100 or 200 μg/ml HDL as indicated. ( D ) Supernatants of macrophages were collected and analyzed for HBsAg or ( E ) HBV-DNA content. ( F ) THP macrophages were incubated with HBV in 0.2% BSA containing medium for 3 hours, washed, and further cultured for 16 hours with 0.2% BSA containing medium only or supplemented with 25 μg/mL ApoA1. ( G ) THP cells were incubated with HBV and 1 μg/mL [3H]-cholesterol-chylomicrons in serum-free medium (w/o) or medium containing 10% human serum. Cells incubated with neither HBV nor [3H]-cholesterol-chylomicrons were used as negative control (–). In cell lysates and supernatant, HBV-DNA was quantified by real-time polymerase chain reaction, and [3H]-cholesterol content was determined by liquid scintillation counting. Mean values ± standard deviation of 4 independent experiments are given. Scale bars are 10 μm ( A ) and 100 μm ( B ).

Article Snippet: Other reagents were BODIPY 493/503 , Alexa Fluor 594 carboxylic acid, succinimidyl ester, NBD-cholesterol (Invitrogen, Germany), 32 P (Hartmann Analytic, Germany), purified ApoA1, HDL (Acris Antibodies, Germany), filipin, Fluorescein-5-Isothiocyanat (FITC), PFA (Sigma-Aldrich, Germany), and DAPI (Roche, Germany).

Techniques: Derivative Assay, Incubation, Staining, Fluorescence, Cell Culture, Negative Control, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Molecular & cellular proteomics : MCP

Article Title: Cross-Linking Mass Spectrometry Uncovers Interactions Between High-Density Lipoproteins and the SARS-CoV-2 Spike Glycoprotein.

doi: 10.1016/j.mcpro.2023.100600

Figure Lengend Snippet: FIG. 1. XL-MS upon plasma derived from patients with COVID-19. A, plasma isolated from patients in intensive care with confirmed COVID-19 (n = 6) was pooled and crosslinked using DSSO. The subsequent interprotein crosslinks identified by XL-MS are represented as an interaction network, 125 crosslinks across 68 proteins. Crosslinks had a minimum of two associated crosslink spectral matches (CSMs) and an XlinkX score of >40. The amino acid sequence of ApoD has been expanded, highlighting different regions of protein interaction. Apolipoproteins are in orange, serum amyloid proteins (SAA) are in red, and complement factors are in yellow. B, HDL interactions in healthy controls were determined following the same XL-MS workflow and the resulting inter-protein crosslinks identified are visualised as an interaction network. XL- MS data visualisation was conducted in xiview.org. All protein abbreviations are gene names as determined by Uniprot.

Article Snippet: Lipoprotein Immuno-Depletion HDL was immuno-isolated from healthy volunteer plasma (n = 8) using human HDL-specific IgY affinity columns according to the manufacturer’s instructions (Genway Biotech).

Techniques: Structural Proteomics, Clinical Proteomics, Derivative Assay, Isolation, Sequencing

Journal: Molecular & cellular proteomics : MCP

Article Title: Cross-Linking Mass Spectrometry Uncovers Interactions Between High-Density Lipoproteins and the SARS-CoV-2 Spike Glycoprotein.

doi: 10.1016/j.mcpro.2023.100600

Figure Lengend Snippet: FIG. 3. XL-MS upon isolated HDL and SARS-CoV-2 spike protein. A, a schematic representation of the XL-MS workflow conducted upon mixtures of ultracentrifuge-isolated HDL and spike protein. B, inter-protein crosslinks identified within isolated HDL supplemented with spike protein are represented as an interaction network, 404 crosslinks across 60 proteins. Crosslinks had a minimum of two associated crosslink spectral matches (CSMs) and an XlinkX score of >10. C, protein-protein interactions identified by XL-MS between ApoD and spike are separately visualized. Spike structural domains are coloured; NTD: blue, RBD: red and S2: green. XL-MS data visualization was conducted in xiview.org. All protein abbreviations are gene names as determined by Uniprot.

Article Snippet: Lipoprotein Immuno-Depletion HDL was immuno-isolated from healthy volunteer plasma (n = 8) using human HDL-specific IgY affinity columns according to the manufacturer’s instructions (Genway Biotech).

Techniques: Structural Proteomics, Isolation, Protein-Protein interactions