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: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Schematic representation of mechanistic strategies of barcoding. (A–C) Barcodes can be introduced to a template using adaptors through direct ligation (A) , using RT- or PCR primers at the reverse transcription or PCR amplification step (B) , and using hybridizing molecular inversion probes (C) . (D) Schematic representation of the difference between “barcodes” and “sample indexes”. Barcodes aim to correct sequencing errors. For example, a misreading nucleotide, guanosine (G) can be corrected in final consensus sequences for a pool of Sample 1 (top panel). Sample indexes are used to multiplex different sequencing amplicons generated from different pools of samples (Sample 1, 2, and 3) (bottom panel). Panel (A) is modified based on in and panel (C) is modified based on in .

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Ligation, Reverse Transcription, Amplification, Sequencing, Multiplex Assay, Generated, Modification

Journal: Frontiers in Molecular Biosciences

Article Title: A systematic review of the barcoding strategy that contributes to COVID-19 diagnostics at a population level

doi: 10.3389/fmolb.2023.1141534

Figure Lengend Snippet: Systematic comparison of barcoding strategies used in the category of molecular barcodes.

Article Snippet: Primer-associated approach , Sequence-based barcodes , SQK-RBK004: transposase carrying barcodes to the site of the cleavage , - , - , Whole genome , Oxford Nanopore Rapid Barcoding kit (SQK-RBK004) , SARS-CoV-2 patient samples (nasopharyngeal swab) , Oxford Nanopore , Guppy version 3.6.0; ARTIC Network bioinformatics protocol , Multiplex samples , Propose a method to sequence the whole genome of SARS-CoV-2 in a rapid and cost-efficient manner , .

Techniques: Comparison, Software, Sequencing, Multiplex Assay, CRISPR, Plasmid Preparation, Microarray, Binding Assay, Amplification, Extraction, Ligation, DNA Sequencing, Multiplexing, Generated, Reverse Transcription, Staining, Flow Cytometry, High Throughput Screening Assay, Inhibition, Blocking Assay, Conjugation Assay, RNA Sequencing Assay, Transmission Assay, Incubation, Diagnostic Assay, Next-Generation Sequencing, Infection

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: Investigative Ophthalmology & Visual Science

Article Title: T Cell–Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration

doi: 10.1167/iovs.16-20789

Figure Lengend Snippet: The frequency of GM-CSF+ Th17 cells is increased in DED mice. Flow cytometry analysis showing GM-CSF+IL-17+CD4+ T cells in the lymph nodes of normal ( white bar ) and DED ( gray bar ) mice (gated on CD4+; n = 6 mice per group; data from one experiment of two is shown).

Article Snippet: Using a flow cytometer (LSR II; BD Biosciences), 100,000 events were collected and analyzed using flow cytometry software (Summit v4.3; Dako Colorado, Inc., Fort Collins, CO, USA).

Techniques: Flow Cytometry

Journal: Investigative Ophthalmology & Visual Science

Article Title: T Cell–Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration

doi: 10.1167/iovs.16-20789

Figure Lengend Snippet: Blocking T cell–derived GM-CSF reduces CD11b+ cell maturation, proliferation, and migration in vitro. We isolated CD4+ T cells from the draining lymph nodes of DED mice and stimulated the cells with anti-CD3 antibody overnight. Culture supernatant was then incubated with either anti–GM-CSF or IgG control antibody and then added to a CD11b+ cell culture derived from naïve mice. Flow cytometry analysis shows MHC-II expressing ( A ) CD11b+ cells and ( B ) Ki67+ cells 24 hours after coculture ( gray line : isotype control). In a transwell migration assay, GM-CSF neutralized or control supernatant was cultured with naïve CD11b+ cells ( upper well ). One hour later, CD11b+ cells that migrated to the lower well were enumerated. (Each group run in triplicate, data from one experiment of two is shown). CON, IgG isotype control; α-GM-CSF, anti–GM-CSF.

Article Snippet: Using a flow cytometer (LSR II; BD Biosciences), 100,000 events were collected and analyzed using flow cytometry software (Summit v4.3; Dako Colorado, Inc., Fort Collins, CO, USA).

Techniques: Blocking Assay, Derivative Assay, Migration, In Vitro, Isolation, Incubation, Cell Culture, Flow Cytometry, Expressing, Transwell Migration Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: T Cell–Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration

doi: 10.1167/iovs.16-20789

Figure Lengend Snippet: Topical neutralization of GM-CSF in a murine model of DED decreases CD11b+ cell maturation and migration to the ocular surface. We induced DED for 7 days and mice were treated topically 3 times daily with anti–GM-CSF or isotype control. Flow cytometry was used to determine frequencies and expression of MHC-II by ( A ) corneal CD11b+ cells and ( B ) frequencies of conjunctival CD11b+ cells. Gray line : isotype. n = 5 mice per group; data from one of two experiments is shown. CON, IgG isotype control; α-GM-CSF, anti–GM-CSF.

Article Snippet: Using a flow cytometer (LSR II; BD Biosciences), 100,000 events were collected and analyzed using flow cytometry software (Summit v4.3; Dako Colorado, Inc., Fort Collins, CO, USA).

Techniques: Neutralization, Migration, Flow Cytometry, Expressing

Journal: Investigative Ophthalmology & Visual Science

Article Title: T Cell–Derived Granulocyte-Macrophage Colony-Stimulating Factor Contributes to Dry Eye Disease Pathogenesis by Promoting CD11b+ Myeloid Cell Maturation and Migration

doi: 10.1167/iovs.16-20789

Figure Lengend Snippet: Topical treatment with anti–GM-CSF reduces pathogenic Th17 cells and improves clinical signs of DED. We induced DED for 7 days and mice were treated topically 3 times daily with anti–GM-CSF or isotype control. ( A ) Flow cytometry was used to determine frequencies of IL-17+ and CD4+ Th17 cells in the draining lymph nodes of treated versus control mice. ( B ) Clinical severity of DED after treatment with IgG isotype control ( gray ) versus anti–GM-CSF ( white ) was evaluated using CFS and the NEI scoring rubric (based on a scale of 0–15). Anti–GM-CSF significantly decreases corneal epitheliopathy compared to IgG control ( n = 10 eyes per group; data from one of two experiments is shown). CON, IgG isotype control; α-GM-CSF, anti–GM-CSF.

Article Snippet: Using a flow cytometer (LSR II; BD Biosciences), 100,000 events were collected and analyzed using flow cytometry software (Summit v4.3; Dako Colorado, Inc., Fort Collins, CO, USA).

Techniques: Flow Cytometry

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