Journal: mBio

Article Title: Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth

doi: 10.1128/mbio.03563-21

Figure Lengend Snippet: Generation of hsa-miR-24-3p is dependent on CR3 and TLR4. (a) The level of hsa-miR-24-3p 1 h after infection was higher in MEV Ca than in MEV. Blocking of CR3 or TLR4 on blood monocytes had a minor effect on hsa-miR-24-3p content in vesicles. However, when both CR3 and TLR4 were blocked, hsa-miR-24-3p was absent from vesicles. Data represent mean values ± SD, P = 0.0025, unpaired two-tailed t test, n = 3 different donors. EVs were isolated from 10 6 human monocytes, and hsa-miR-24-3p levels were determined using qPCR. (b) Mmu-miR-24-3p content was significantly higher in REV Ca (vesicles from opsonized C. albicans -induced RAW 264.7 cells) than in REV (vesicles from untreated RAW 264.7 cells). The level of mmu-miR-24-3p was not elevated in CR3- or TLR4 -silenced RAW 264.7 cells. Data represent mean values ± SD, P = 0.0245 and P = 0.0166, unpaired two-tailed t test, n = 3 or 4 different experiments. (c) Candida -treated CD11b- and TLR4 -silenced RAW 264.7 cells generated the same amount of REV as nonsilenced cells. EVs isolated from the same numbers of cells were counted by DLSM. (d) CD11b and TLR4 colocalized on blood monocytes upon incubation with soluble β-glucan (sβG) and mannan but not on untreated cells, as observed by CLSM. Green, CD11b; red, TLR4. Bars, 10 μm. Data are representative of n = 3 independent experiments. (e) Colocalization of CD11b and TLR4 in the presence of soluble β-glucan (sβG) and mannan on monocytes, as confirmed by PLA. Red, CD11b–TLR4 complexes; blue, DNA. Bars, 10 μm. Data are representative of n = 3 experiments. (f) EVs isolated from sβG and mannan-treated human monocytes for 1 h (MEV sβG+mannan ) contained significantly more hsa-miR-24-3p than MEV. Data represent mean values ± SD, P = 0.0025, unpaired two-tailed t test, n = 3 different donors. REV Ca but not REV Ca(TLR4 and CD11b silenced) induced significant growth (g) and hyphal filamentation (h) in C. albicans . EVs were isolated from opsonized C. albicans -infected or control RAW 264.7 cells (REV and REV Ca , respectively). EVs were also isolated from opsonized C. albicans - infected or control TLR4- and CD11b-silenced RAW 264.7 cells [REV (TLR4 and CD11b silenced) and REV Ca(TLR4 and CD11b silenced) , respectively]. Isolated EVs were counted by DLSM, and C. albicans was incubated with identical amounts of vesicles obtained from the indicated treatments.

Article Snippet: CD14 was stained with Alexa Fluor 488 anti-human CD14 antibody (no. 367130; BioLegend) (1:100), CD9 was stained with Alexa Fluor 647 anti-human CD9 antibody (no. MCA469A647T; Bio-Rad) (1:1,000), TLR4 was stained with mouse anti-TLR4 antibody (no. NBP1-51697; R&D Systems) (3 μg/ml) and Alexa Fluor 647 goat anti-mouse IgG (H+L) secondary antibody (no. A-21235; Thermo Fisher) (1:500), and CD11b was stained with rabbit anti-CD11b antibody (no. 133357; Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody.

Techniques: Infection, Blocking Assay, Two Tailed Test, Isolation, Generated, Incubation

Journal: mBio

Article Title: Candida albicans Induces Cross-Kingdom miRNA Trafficking in Human Monocytes To Promote Fungal Growth

doi: 10.1128/mbio.03563-21

Figure Lengend Snippet: hsa-miRNA-24-3p acts across species boundaries (cross-kingdom) as it regulates C. albicans gene expression. C. albicans induces the release of miRNA-containing vesicles in human monocytes upon binding of mannan and soluble β glucan to TLR4 and CR3, respectively, followed by receptor colocalization. Subsequently, EVs are released from multivesicular bodies transporting miRNAs. EVs attach via CR1 on the vesicle to opsonized C. albicans . hsa-miRNA-24-3p but not hsa-miRNA-21-5p inhibits sol1 translation in C. albicans , leading to enhanced growth and filamentation of the fungus. Graphic created with BioRender.com.

Article Snippet: CD14 was stained with Alexa Fluor 488 anti-human CD14 antibody (no. 367130; BioLegend) (1:100), CD9 was stained with Alexa Fluor 647 anti-human CD9 antibody (no. MCA469A647T; Bio-Rad) (1:1,000), TLR4 was stained with mouse anti-TLR4 antibody (no. NBP1-51697; R&D Systems) (3 μg/ml) and Alexa Fluor 647 goat anti-mouse IgG (H+L) secondary antibody (no. A-21235; Thermo Fisher) (1:500), and CD11b was stained with rabbit anti-CD11b antibody (no. 133357; Abcam) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) secondary antibody.

Techniques: Expressing, Binding Assay

Journal: Ecology and evolution

Article Title: Expression of TLR2/4 in the sperm-storing oviduct of the Chinese soft-shelled turtle Pelodiscus sinensis during hibernation season.

doi: 10.1002/ece3.1726

Figure Lengend Snippet: Figure 6. The Expression of TLR4 protein in November was Similar in the Magnum, Isthmus, Uterus, and Vagina. Cross-sections of the magnum, isthmus, uterus, and vagina are shown at low magnification in A, E, I, and M, respectively; B, F, J, and n refer to the amplification of the epithelium in the magnum, isthmus, uterus, and vagina, respectively; the secretory glands in magnum, isthmus, uterus, and vagina are described in C, G, K, and o; respectively; and D, H, I, and P represent the negative controls for the magnum, isthmus, uterus, and vagina, respectively. No immunoreaction products were observed. All sections were counterstained with hematoxylin. Positive staining was observed on the ciliated cell superior surface and cilium surface (white thin arrow head), secretory cell superior surface (white thin arrow), secretory cell lateral membrane (white fat arrow head), secretory cell basal membrane (white fat arrow), secretory gland vesicles membrane (black thin arrow), longitudinal muscle (black fat arrow), circular muscle (black fat arrow head), blood vessel endothelium (black thin arrow head). Scale bars: 100 lm (E and M), 50 lm (A, D, H, I, L, and P), and 10 lm (B, C, F, G, J, K, N, and O).

Article Snippet: The sections were incubated at 4°C for 15 h with 200 lg/mL of a rabbit polyclonal antibody to human, mouse, and rat TLR4 (Boster, China).

Techniques: Expressing, Staining, Membrane

Journal: Ecology and evolution

Article Title: Expression of TLR2/4 in the sperm-storing oviduct of the Chinese soft-shelled turtle Pelodiscus sinensis during hibernation season.

doi: 10.1002/ece3.1726

Figure Lengend Snippet: Figure 7. The Location of TLR4 protein was Similar in the Magnum, Isthmus, Uterus, and Vagina. Cross-sections of the magnum, isthmus, uterus, and vagina are depicted in the pictures (A, E, I, and M) at low magnification: B, F, J, and n refer to the amplification for the epithelium in the magnum, isthmus, uterus, and vagina, respectively; the secretory glands in the magnum, isthmus, uterus, and vagina are described C, G, K, and O, respectively; and D, H, I, and p show the negative controls for the magnum, isthmus, uterus, and vagina, respectively. All sections were counterstained with hematoxylin. No immunoreaction products were observed. Positive staining was observed on the ciliated cell superior surface and cilium surface (white thin arrow head), secretory cell superior surface (white thin arrow), secretory cell lateral membrane (white fat arrow head), secretory cell basal membrane (white fat arrow), secretory gland vesicles membrane (black thin arrow), longitudinal muscle (black fat arrow), circular muscle (black fat arrow head), and blood vessel endothelium (black thin arrow head). Scale bars: 50 lm (A, D, E, H, I, L, M, and P), 20 lm (B, F, J, and N), and 10 lm (C, G, K, and O).

Article Snippet: The sections were incubated at 4°C for 15 h with 200 lg/mL of a rabbit polyclonal antibody to human, mouse, and rat TLR4 (Boster, China).

Techniques: Staining, Membrane

Journal: Ecology and evolution

Article Title: Expression of TLR2/4 in the sperm-storing oviduct of the Chinese soft-shelled turtle Pelodiscus sinensis during hibernation season.

doi: 10.1002/ece3.1726

Figure Lengend Snippet: Figure 8. The IOD of TLR2/4 Positive Reaction Parts in Oviduct during Hibernation. (A) Image shows changes in numbers of TLR2-positive reaction parts in four different parts of oviduct in two different months. (B) The IOD of TLR4- positive reaction parts in oviduct during hibernation. Data are presented as mean SE of five turtles per group. Significant differences are identified as *, P < 0.05; **, P < 0.01.

Article Snippet: The sections were incubated at 4°C for 15 h with 200 lg/mL of a rabbit polyclonal antibody to human, mouse, and rat TLR4 (Boster, China).

Techniques:

Journal: Molecular medicine reports

Article Title: Effect of S100A8 and S100A9 on expressions of cytokine and skin barrier protein in human keratinocytes.

doi: 10.3892/mmr.2019.10454

Figure Lengend Snippet: Figure 6. HaCaT cells were treated with (A) S100A8 or (B) S100A9 (10 µg/ml) for 24 h and TLR4 expression was increased as assessed by western blotting. Data are expressed as the mean ± standard deviation. **P<0.01 vs. the control. S100, calcium binding protein; TLR4, toll‑like receptor 4.

Article Snippet: Tlr4 inhibitor cli-095 (Tlr4i), protein kinase δ (PKcδ) inhibitor (rottlerin), p38 mitogen-activated Correspondence to: dr in Sik Kim, department of Biomedical laboratory Science, School of Medicine, eulji university, 77 Gyeryoung-ro 771 beon-gil, Jung-Gu, daejeon 34824, republic of Korea e-mail: orientree@eulji.ac.kr Key words: atopic dermatitis, S100 calcium binding protein a8, S100 calcium binding protein a9, keratinocyte, skin barrier protein protein kinase (MaPK) inhibitor (SB202190), MeK inhibitor (Pd98059) and nuclear factor-κB (nF-κB) inhibitor (BaY-11-7085) were obtained from calbiochem (Merck KGaa). antibodies against p38 MaPK (cat. no. 9212), phospho-p38 MaPK (cat. no. 9211), phospho-extracellular signal regulated kinase 1/2 (erK1/2; cat. no. 9101), rabbit igG-HrP (cat. no. 7074), and mouse igG-HrP (cat. no. 7076) were acquired from cell Signaling Technology, inc. anti-erK2 (cat. no. sc-154) and anti-Tlr4 (cat. no. sc-10741) antibodies were obtained from Santa cruz Biotechnology, inc. Production of recombinant S100A8 and S100A9 proteins. in our previous report, the cdna of human S100a8 and S100a9 was cloned into peT28 expression vector (Merck KGaa) (10). recombinant S100a8 and S100a9 expression was induced with 1 mM isopropyl β-d-thiogalactoside in E. coli Bl21 (de3; Merck KGaa).

Techniques: Expressing, Western Blot, Standard Deviation, Control, Binding Assay

Journal: Journal of neuropathology and experimental neurology

Article Title: Broad expression of Toll-like receptors in the human central nervous system.

doi: 10.1093/jnen/61.11.1013

Figure Lengend Snippet: Fig. 3. Immunocytochemical localization of TLR3 and TLR4 in cultures of glia cells. Primary cultures of glial cells were obtained from white matter samples of control donors. Microglial cells were double stained for TLR3 or TLR4 (green) and CD68 (red) (A, B). Both TLRs were found exclusively localized in distinct vesicular structures inside microglia rather than on the surface of the cells. Astrocytes were stained for GFAP (C), TLR3 (D), and TLR4 (E). The subcellular localization of TLR3 and TLR4 in astrocytes was distinctly different from microglia in that TLR3 and TLR4 were found exclusively on the cell surface. Magnification: A–E, 3400.

Article Snippet: Following heat treatment, the sections were incubated overnight with anti-human TLR3 or anti-TLR4 antibodies (Santa Cruz Biotechnologies) diluted in 0.1% BSA in PBS plus 1% pooled human serum.

Techniques: Control, Staining

Journal: Journal of neuropathology and experimental neurology

Article Title: Broad expression of Toll-like receptors in the human central nervous system.

doi: 10.1093/jnen/61.11.1013

Figure Lengend Snippet: Fig. 4. Elevated expression of TLR3 and TLR4 in multiple sclerosis lesions. Expression of TL3 and TLR4 in healthy control white matter and sections representing MS lesions. Low-power photomicrographs (3100) of white matter from MS stained for either TLR3 (C, E, G) or TLR4 (D, F, H). Numbers of TLR-expression cells are strongly increased in MS lesions, with relatively high expression of TLR3 and TLR4 observed particularly in perivascular areas (*). TLR3 (A) and TLR4 (B) were almost absent from control brains. Inserts show TLR-positive cells at high magnification (3400).

Article Snippet: Following heat treatment, the sections were incubated overnight with anti-human TLR3 or anti-TLR4 antibodies (Santa Cruz Biotechnologies) diluted in 0.1% BSA in PBS plus 1% pooled human serum.

Techniques: Expressing, Control, Staining