Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: PLY-EVs induce dendritic cell maturation and inflammatory cytokine release upon internalization (A) Confocal microscopy images showing the internalization of CFSE-labelled PLY (0.1) and naive EVs (green) by THP-1-monocyte-derived DCs at 24 h post-treatment. Scale bars, 25 μm. (B) Flow cytometry histograms ( N = 3) to quantify the DC uptake of CFSE-labeled PLY(0.5)EVs and naive EVs. (C) Dose-dependent uptake of PLY (0.1, 0.5) EVs by DCs. (D) Phase-contrast microscopy images of immature day 5 DCs coincubated with PLY (0.1, 0.5) EVs and naive EVs for 24 h. Arrows indicate matured DCs (magnified in inset). Scale bars, 50 μm. Images are representative of three independent experiments. (E–G) Flow cytometry histograms ( N = 3) to quantify the expression levels of (E) CD80, (F) CD86, and (G) CD83 on THP-1-monocyte-derived DCs treated with PLY(0.5) and naive EVs. (H and I) Flow cytometry histograms ( N = 2) showing the expression levels of DC maturation marker CD83 at 96 h post-incubation of primary human monocytes with (H) PLY(0.5) and naive EVs and (I) naive EVs pre-treated with recombinant PLY protein (naive EVs+rPLY). (J and K) Cytokine ELISA showing the levels of secreted TNF-α from (J) DCs treated with PLY (0.1) EVs or naive EVs alone ( N = 3) for 24 h and (K) DCs pre-treated with PLY (0.1,0.5) or naive EVs for 24 h followed by subsequent infection with S. pneumoniae , T4R strain ( N = 2). Recombinant PLY (0.5 μg/mL) was used as positive control. All data are represented as mean ± SEM. ∗ p < 0.05, ∗∗ p < 0.005, and ∗∗∗ p < 0.001 by one-way ANOVA with Tukey’s multiple comparisons test. n.s., not significant. See also Figures S6–S9 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Confocal Microscopy, Derivative Assay, Flow Cytometry, Labeling, Microscopy, Expressing, Marker, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Infection, Positive Control

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet: Adoptive transfer of EVs from infected mice drives inflammation and pathology in a PLY-dependent manner (A) C57BL/6 mice were intranasally administered with 4 × 10 6 CFU of serotype 4 strain, T4 or the isogenic PLY mutant strain, T4Δply. At day 4 post-infection, EVs isolated from BALF were labeled and administered to healthy recipient mice at 35 μg/mice. The EV retention in murine respiratory tract was imaged by IVIS imaging and immune infiltration into lungs, and cytokine levels in BALF was measured. (B) Bacterial load in murine BALF ( N = 5 mice/group) upon infection with T4 and T4Δply strains was measured by CFU dilution assay. ∗∗ in (B) indicates p < 0.01 by Mann-Whitney test. (C) Quantification of relative total EV protein content from mice ( N = 3 mice/group) infected with T4 and T4Δply strains by BCA protein assay. PBS-treated mice served as control. ∗ and ∗∗ in (C) indicates p < 0.05 and p < 0.005, respectively, by unpaired t test. (D) IVIS imaging of mice intranasally administered with Nile-red-labeled EVs isolated from mice infected with T4 (EVs-T4) or T4Δply (EVs-T4Δply). EVs from PBS-treated mice (naive EVs) served as control. ROI intensity values indicate the total flux (photons/sec) recorded from the given region showing higher intensity of EVs from T4-infected mice in the respiratory tract. The color scale (photons/sec/cm 2 ) indicates the relative intensities of individual signals. (E and F) Flow cytometry analysis of inflammatory macrophages (F4/80 + ) and neutrophils (Ly6G + ) in BALF of mice ( N = 6 mice/group) administered with EVs from infected or untreated mice at 18 h. (G) TNF-α levels in the BALF of mice ( N = 5 mice/group) treated with EVs isolated from infected or untreated mice were measured post-sacrifice at 18 h by ELISA. ∗∗ and ∗∗∗ in (G) indicates p < 0.01 and p < 0.001, respectively, by unpaired t test. (H) Hematoxylin and eosin (H&E) staining of mouse lungs ( N = 6 mice/group) at 18 h post-administration of EVs from infected or PBS-treated mice. Mice treated with EVs from T4-infected mice showed tissue microlesions (MLEs) and immune cell infiltration in the alveolar interstitium indicative of PLY-induced tissue damage (magnified in the inset). BR, bronchiole; MLE, microlesions. Scale bars, 200 μm. Blind histopathological scoring was performed based on presence or absence of cellularity in alveolar interstitium and lesions. A score of “0” was given when no lesions were found, and a score of “1” was given to tissue showing increasing cellularity and lesions. Mouse BALF flow cytometry and histology data are representative of three independent experiments. All data are represented as mean ± SEM. See also Figure S12 .

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Adoptive Transfer Assay, Infection, Mutagenesis, Isolation, Labeling, Imaging, Dilution Assay, MANN-WHITNEY, Bicinchoninic Acid Protein Assay, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining

Journal: iScience

Article Title: Bacterial pore-forming toxin pneumolysin drives pathogenicity through host extracellular vesicles released during infection

doi: 10.1016/j.isci.2024.110589

Figure Lengend Snippet:

Article Snippet: Mouse TNF-α DuoSet ELISA kit , R & D Systems , DY410-05.

Techniques: Virus, Mutagenesis, Isolation, Recombinant, Modification, Saline, Labeling, Staining, Electron Microscopy, Lysis, Western Blot, Buffer Exchange, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, Clone Assay, Software, Membrane

Journal: STAR Protocols

Article Title: Efficient generation of locus-specific human CAR-T cells with CRISPR/cCas12a

doi: 10.1016/j.xpro.2022.101321

Figure Lengend Snippet:

Article Snippet: DNA Clean & Concentrator-25 (Capped) , Zymo Research , Cat# D4033.

Techniques: Recombinant, Electroporation, Modification, SYBR Green Assay, Transfection, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, DNA Extraction, Software, Real-time Polymerase Chain Reaction, Protein Purification, Plasmid Preparation, Synthesized, Flow Cytometry, Spectrophotometry

Journal: bioRxiv

Article Title: Cell binding, uptake and infection of influenza A virus using recombinant antibody-based receptors

doi: 10.1101/2024.07.29.605726

Figure Lengend Snippet: (A) Representative fluorescent microscopy images of fixed cells stained with fluorescein-labeled SNA or MAL I. Relative fluorescence level of (B) SNA- and (C) MAL I, stained live cells quantified by flow cytometry. MFI intensities were normalized relative to HEK WT Sia expression (B, C). Background (unstained) subtracted MFI values were analyzed using PRISM software; MFI = Mean fluorescent intensity. *, Error bars show mean ± standard mean error. Statistics were calculated using ANOVA. P<0.05; ****P<0.0001; n=6. (D) Unmodified (WT), and Slc35A1 KO A549 cells transduced with an Slc35A1 encoding lentivirus (LV- Slc35A1 ) were fixed and stained with biotinylated SNA or MAL II (magenta) and DAPI (blue). The Alexa Fluor 647-Streptavidin signal intensities were assessed via microscopy. Images are representative of n=3 independent replicates. Scale bar represents 50µm. (E, F) Cells from (D) were incubated with biotinylated SNA or MAL II and the APC-streptavidin in live cells analyzed via flow cytometry. Following subtraction of the median APC signal of corresponding background samples, the median APC signal was normalized to that of WT cells. Data are means +/- standard error of mean/standard deviation from n=3 independent experiments. Statistical significance was inferred by two-tailed one sample t test with a theoretical mean of 100. ***P<0.001, **P< 0.01.

Article Snippet: Protein concentration was determined by Qubit (Invitrogen) and stored at -80°C in aliquots. pMslc expressing the human Slc35A1 gene was a gift from Dr. Christopher Buck (Addgene plasmid #32095).

Techniques: Microscopy, Staining, Labeling, Fluorescence, Flow Cytometry, Expressing, Software, Transduction, Incubation, Standard Deviation, Two Tailed Test

Journal: bioRxiv

Article Title: Cell binding, uptake and infection of influenza A virus using recombinant antibody-based receptors

doi: 10.1101/2024.07.29.605726

Figure Lengend Snippet: (A) Representative fluorescent microscopic images of fixed cells incubated with HA-hFc probe and detected with fluorescein-labelled anti-human Fc. (B) Relative fluorescent intensities of live HA-hFc-stained cells quantified by flow cytometry. Fluorescent intensities were normalized relative to fTfR-5J8 expressing cells. (C) The fTfR and fTfR-5J8 expression was analyzed in A549 Slc35A1 KO cells transduced with lentiviruses encoding fTfR (LV-fTfR) or fTfR-5J8 (LV-fTfR-5J8) via qPCR. Primers targeting GAPDH, the extracellular domain of fTfR and 5J8 were utilized. Data are 40-Ct values from technical triplicates from n=1 experiment. A Ct of 40 was assigned to samples where no amplification was detected. (D) Cells from (C) and A549 Slc35A1 KO cells stably expressing SLC35A1 (LV- Slc35A1 ) were inoculated with A/Netherlands/602/09 at a MOI 25 for 1.5hr on ice. Cells were incubated with an anti-HA antibody and the signal from bound virus quantified via flow cytometry. A representative histogram from n=3 independent experiments is shown. (E) Quantification of the percentage of IAV positive cells from (D). The gating strategy was established using the mock-infected sample. Data are means ± standard deviation and statistical significance was inferred by one-way ANOVA with Sidak’s multiple comparisons test. ****P<0.0001.

Article Snippet: Protein concentration was determined by Qubit (Invitrogen) and stored at -80°C in aliquots. pMslc expressing the human Slc35A1 gene was a gift from Dr. Christopher Buck (Addgene plasmid #32095).

Techniques: Incubation, Staining, Flow Cytometry, Expressing, Transduction, Amplification, Stable Transfection, Virus, Infection, Standard Deviation

Journal: bioRxiv

Article Title: Cell binding, uptake and infection of influenza A virus using recombinant antibody-based receptors

doi: 10.1101/2024.07.29.605726

Figure Lengend Snippet: (A) Representative fluorescence microscopy images of fixed Ca’09 infected cells. Receptors (fTfR-5J8 and full-length fTfR) were transiently expressed on HEK Slc35A1 KO for 24hrs before viral infection (MOI = 0.2) for 8hrs. Cells were fixed and stained with anti-NP antibody (green) and DAPI (blue). (B and C) Infection data for Ca’09 or PR8 infected cells respectively. Four different fields of view were imaged for each condition and % infection was determined. Data was normalized to HEK WT infected cells. (D) Infection curve for the Neth/09 -Renilla at MOI = 3 in A549 LV-fTfR, LV-fTfR-5J8 and LV- Slc35A1 cells. (E) AUC plot from D, where AUC values of LV-fTfR-5J8 and LV-fTfR are shown relative to LV- Slc35A1. ND = No infection detected, NS = not significant, AUC = Area under the curve, MOI = Multiplicity of infection. All experiments were performed in three independent replicates (n=3). Error bars show mean ± standard mean error. Statistics were calculated using ANOVA. *P<0.05; **P<0.01, ****P<0.0001.

Article Snippet: Protein concentration was determined by Qubit (Invitrogen) and stored at -80°C in aliquots. pMslc expressing the human Slc35A1 gene was a gift from Dr. Christopher Buck (Addgene plasmid #32095).

Techniques: Fluorescence, Microscopy, Infection, Staining

Journal: bioRxiv

Article Title: Cell binding, uptake and infection of influenza A virus using recombinant antibody-based receptors

doi: 10.1101/2024.07.29.605726

Figure Lengend Snippet: A549 Slc35A1 KO cells stably expressing SLC35A1 (LV- Slc35A1 ) or fTfR-5J8(LV-5J8-TfR) were pre-treated with Dynasore (40-20µM), Pitstop 2 (15-10µM), NH 4 Cl (25mM) or DMSO (0.15%) for 30 minutes at 37°C and then infected with Neth/09- Renilla at a MOI 3 in their presence. (A) Cell viability at 24 hours post-inhibitor treatment relative to the DMSO control. (B) Neth/09 -Renilla infection curves. Dashed line indicates luminescence from the mock-infected sample. (C) AUC values from (B) were normalized to the DMSO-treated sample within each cell line. (A-C) Data are means ± standard deviation from n=3 independent experiments. Statistical significance in (C) was determined by two-tailed one sample t-test with a theoretical mean of 100. *P<0.05, ns = not significant.

Article Snippet: Protein concentration was determined by Qubit (Invitrogen) and stored at -80°C in aliquots. pMslc expressing the human Slc35A1 gene was a gift from Dr. Christopher Buck (Addgene plasmid #32095).

Techniques: Stable Transfection, Expressing, Infection, Control, Standard Deviation, Two Tailed Test

Journal: Stem Cell Research & Therapy

Article Title: Identification of CD141 + vasculogenic precursor cells from human bone marrow and their endothelial engagement in the arteriogenesis by co-transplantation with mesenchymal stem cells

doi: 10.1186/s13287-024-03994-9

Figure Lengend Snippet: CD141 is a critical marker that distinguishes BM-VPCs from BM-MSCs. A List of markers with an expression difference of > 50% between BM-VPCs and BM-MSCs screened among 376 surface markers. B Differential expression of CD141 and CD282 in 15–20 donors assessed using flow cytometry analysis (*** p < 0.001, Student's t-test, two-tailed). C–F BM-MNCs were sorted using magnetic bead-conjugated CD141 antibody. The selected MNCs were plated in EGMPL. C Colonies that appeared from CD141 + sorted BM-MNCs on days 5, 7, and 9. Scale bar = 500 μm. D Marker expression profile of ex vivo-cultured cells from CD141 + sorted colonies at passages 1 to 4 analyzed using flow cytometry. E, F CD141 + sorted BM-MNCs were cultured in EGMPL at passage 0; from passage 1 to 3 and then, the cells were divided and cultured in EGMPL or StemMACS. E Morphology of replated CD141 + sorted cells in EGMPL and StemMACS at passages 1 to 3, and their tube-forming ability in MEMα + 0.2%hPL at passage 2. Scale bar = 500 μm. F Marker expression profile of CD141 + sorted cells cultured in StemMACS

Article Snippet: StemMACS is an abbreviation for StemMACS MSC expansion medium XF, human (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Marker, Expressing, Quantitative Proteomics, Flow Cytometry, Two Tailed Test, Ex Vivo, Cell Culture