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: The Science of the Total Environment

Article Title: Sampling methods and assays applied in SARS-CoV-2 exposure assessment

doi: 10.1016/j.scitotenv.2021.145903

Figure Lengend Snippet: Data obtained from the chosen articles.

Article Snippet: , 43. Aerosol and surface contamination of SARS-CoV-2 observed in quarantine and isolation care , USA , No , Surface and air samples from COVID-19 patient rooms , Air sampling: Sartorius Airport MD8 air sampler operating at 50 Lpm for 15 min. Surface samples: sterile swabs , Viral RNA Extractions: using a Qiagen DSP Virus Spin Kit. RT-qPCR: using Invitrogen Superscript III Platinum One-Step Quantitative RT-qPCR System. Primers and probe used target the E gene of SARS-CoV-2. , • We detected viral contamination among all samples. , ( ) .

Techniques: Sampling, Lysis, RNA Extraction, Environmental Monitoring, Virus, Multiplex Assay, Northern Blot, Marker, RNA Detection, Isolation, Membrane, Control, Environmental Sampling, Amplification, Transmission Assay, Aerosol, Diagnostic Assay, Infection, Sterility, Real-time Polymerase Chain Reaction, Nested PCR, Reverse Transcription, Extraction, Purification, Digital PCR, Preserving, Quantitative RT-PCR, cDNA Synthesis, Magnetic Beads, Incubation, Modification, One Step RT-PCR, Cell Culture, Sequencing

Journal: The FASEB Journal

Article Title: Protease-activated receptor 2 activates airway apical membrane chloride permeability and increases ciliary beating

doi: 10.1096/fj.201700114RRR

Figure Lengend Snippet: Expression of PAR-2 in airway epithelial cells. A) PAR-2 expression in primary airway epithelial cell lines and primary cells from 3 patients was assayed by reverse transcription-PCR. Oral squamous epithelium cDNA was used as a control for PAR-4. b.p., base pair; Pt, patient; rt, reverse transcription. B) PAR-2 expression was confirmed by Western blot in primary sinonasal ALI culture samples (6 separate patients, unrelated to patients in A. C) Immunofluorescence using a PAR-2 antibody in submerged BEAS-2B cells revealed plasma membrane–localized staining. GLUT1 was a control for membrane localization. D, E) Immunofluorescence of PAR-2 in primary dissociated sinonasal ciliated cells showing colocalization with NKCC1 (D) and Na+K+ ATPase (E). A distinct gap was noted (arrows) between the base of the cilia (labeled with β-tubulin IV), corresponding to the apical cell body membrane and the start of basolateral NKCC1/Na+K+ ATPase immunofluorescence. PAR-2 rabbit pAb was used with mouse mAb anti-NKCC1 (top). PAR-2 mouse mAb was used with rabbit mAb anti-Na+K+ATPase (bottom). Scale bars, 10 µm. F) Representative scatter plots of PAR-2 intensity and either NKCC or Na+K+ ATPase. For all dissociated cells imaged (n = 15 with NKCC1 and n = 17 with Na+K+ ATPase), Pearson’s correlation coefficient (Pearson’s R) and Mander’s overlap coefficient (Mander’s R) for basolateral marker and PAR-2 were both ≥0.95.

Article Snippet: Human PAR-2 and PAR-4 samples were amplified by PCR using an iCycler (Bio-Rad, Hercules, CA, USA), RedTaq DNA Polymerase (MilliporeSigma), and the following primers: 5′-GCTGGTCACCATCCCTTTGT-3′ and 5′-TCTGCTTTACAGTGCGGACA-3′ (hPAR-2) and 5′-CCTGGTTTATCTCTACCGGCG-3′ and 5′-CTCTCGTCGTAGACGCTGGG-3′ (hPAR-4) from Integrated DNA Technologies (Coralville, IA, USA). cDNA was prepared from cultured cells using Trizol (Thermo Fisher Scientific) with rDNAse I (Ambion, Austin, TX, USA) and an iScript cDNA Synthesis Kit (Bio-Rad).

Techniques: Expressing, Reverse Transcription, Control, Western Blot, Immunofluorescence, Clinical Proteomics, Membrane, Staining, Labeling, Marker

Journal: Molecular Genetics & Genomic Medicine

Article Title: Clinical application of single‐molecule optical mapping to a multigeneration FSHD1 pedigree

doi: 10.1002/mgg3.565

Figure Lengend Snippet: Molecular diagnosis of FSHD1. (a) Southern blot hybridization of selected family members with FSHD1. Membrane‐bound EcoRI/HindIII (E/H), EcoRI/HindIII/BlnI (E/H/B), and HindIII (H) digested genomic DNA from affected and normal family members were hybridized to p13E‐11 (4q35 and 10q26), 4qA, and 4qB labeled probes. 4qA (*) and 4qB (+) fragments. (b) FISH analysis of family member 28‐III (FSHD1). Green signals represent D4Z4 repeat numbers. (c) SMOM analysis of FSHD1 family members. BssSI maps (vertical green bars) of 4qA (purple) and 4qB (orange) alleles for family members 28‐III and 58‐IV (FSHD1) and 59‐IV (normal). The position and number ( n ) of D4Z4 repeats for each allele are indicated by horizontal purple and orange bars

Article Snippet: The WBCs were resuspended in cell suspension buffer, embedded into agarose plugs (CHEF Genomic DNA Plug Kit, Bio‐Rad).

Techniques: Biomarker Discovery, Southern Blot, Hybridization, Membrane, Labeling

Journal: Developmental Cell

Article Title: The imprinted Igf2 - Igf2r axis is critical for matching placental microvasculature expansion to fetal growth

doi: 10.1016/j.devcel.2021.12.005

Figure Lengend Snippet:

Article Snippet: Total RNA was extracted from E19 male Lz using RNeasy Midi Kits (Qiagen – 75144) and quantity and quality were verified using RNA 6000 Nano Kit (Agilent – 5067-1511) and an Agilent 2100 Bioanalyzer.

Techniques: Plasmid Preparation, Recombinant, Blocking Assay, BIA-KA, Western Blot, Stripping Membranes, Reverse Transcription, SYBR Green Assay, Red Blood Cell Lysis, Staining, Enzyme-linked Immunosorbent Assay, In Situ, TUNEL Assay, Imaging, Flow Cytometry, Transfection, Gene Expression, Expressing, Microarray, Isolation, Software

Journal: Cell Death & Disease

Article Title: The oncogene Etv5 promotes MET in somatic reprogramming and orchestrates epiblast/primitive endoderm specification during mESCs differentiation

doi: 10.1038/s41419-018-0335-1

Figure Lengend Snippet: a Comparison of Etv5 expression levels between mESC lines and somatic cell lines. The relative expression was based on the microarray data from BioGPS database. b The interactions between pluripotency relevant regulators and Etv5 . ChIP-seq and ChIP-chip data with Etv5 as target were extracted from ESCAPE database and used for drawing these interactions. c Growth curve of J1 mESCs stably infected with shCtrl and Etv5 shRNA (shEtv5-7). d RT-qPCR analysis of Etv5 and Tet2 in mESCs stably infected with shCtrl, Etv5 shRNA (shEtv5-7), and shEtv5-7 plus lentiviral Etv5 . Data are shown as mean ± SD ( n = 3). * P < 0.05, *** P < 0.001. Two-way ANOVA with Sidak’s multiple comparisons test was used for c . One-way ANOVA with Dunnett’s multiple comparisons test for d . e Western blotting of TET2 in mESCs stably infected with shCtrl, shEtv5-7, and shEtv5-7 + Etv5 . GAPDH was used as internal control. The relative quantification is also shown. f Dot blot of global 5hmC in mESCs stably infected with shCtrl, shEtv5-7, and shEtv5-7 plus lentiviral Etv5 . The blotting result of serially diluted genomic DNA (100-3.125 ng) was shown (left panel). The same membrane stained with methylene blue as DNA loading control was also presented (right panel)

Article Snippet: For transgenes integration detection, genomic DNA of mouse iPSCs was extracted using TIANamp Genomic DNA Kit (TIANGEN).

Techniques: Comparison, Expressing, Microarray, ChIP-sequencing, ChIP-chip, Stable Transfection, Infection, shRNA, Quantitative RT-PCR, Western Blot, Control, Quantitative Proteomics, Dot Blot, Membrane, Staining

Journal: bioRxiv

Article Title: Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial metabolic reprogramming

doi: 10.1101/2023.01.19.524841

Figure Lengend Snippet: ( A ) mtROS production from HBEC3-KT cells after treatment with pathogen associated molecular patterns. ( B ) mtROS production from HBEC3-KT cells after treatment with the indicated ODNs. mtROS production after treatment with ODN from mouse lung epithelial cell lines ( C ) and primary human ( D ) and primary mouse ( E ) lung epithelial cells. ( F ) Representative fluorescence images primary tracheal epithelial cells harvested from mt-roGFP mice treated with PBS or ODN. Images shown as gradient of color intensity from the reduced (blue) form to the oxidized (green) form of roGFP. Scale bar, 50 μm. ( G ) Ratio of the fluorescence intensity of the oxidized:reduced roGFP from F , quantified at 488 nm and 405 nm, respectively. ( H ) Oxygen consumption following the indicated treatment by Seahorse XFe96 Flux Analyzer, shown as mean ± SEM. ( I ) Mitochondrial membrane potential Δ Ψm measurement in HBEC3-KT cells after ODN treatment. * p<0.001 vs. PBS by one-way ANOVA using Holm-Sidak method, except A which use Tukey method due to failed normality testing; † p<0.001 vs PBS by two-way Student’s t test. ODN, oligodeoxynucleotide; ISD, immune stimulating DNA; mTEC, primary mouse tracheal epithelial cells; NHBE, primary normal human bronchial epithelial cells; GFP, green fluorescent protein; OCR, oxygen consumption rate; TMRM, tetramethylrhodamine.

Article Snippet: Normal human bronchial epithelial (NHBE) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit (ATCC, Manassas, VA).

Techniques: Fluorescence, Membrane

Journal: bioRxiv

Article Title: Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial metabolic reprogramming

doi: 10.1101/2023.01.19.524841

Figure Lengend Snippet: ( A ) RPPA heatmap from HBEC3-KT cells treated with PBS or ODN. ( B ) Immunoblot of AMPK and ACC proteins after ODN treatment. ( C ) Immunoblot for phospho-AMPKα1 following treatment with the indicated mitochondrial permeability modulators in HBEC3-KT cells. ( D ) Phospho-AMPK immunofluorescence in mouse lungs after treatment with ODN. Scale bar, 50 μm. ( E ) Quantification of fluorescence in D . ( F ) mtROS production in primary Prkaa1 fl/fl ;Prkka2 fl/fl mouse tracheal epithelial cells infected with empty or Cre + adenovirus, then treated with PBS or ODN. ( G ) Acetyl-CoA levels in ODN-treated HBEC3-KT cells. ( H ) Fatty acid oxidation after ODN treatment. ( I ) mtROS production following treatment with ODN and/or β-oxidation inhibitor etomoxir. ( J ) Oxygen consumption following the indicated treatments, shown as mean ± SEM. ( K ) HBEC3-KT cell complex II activity following treatment with the indicated agents.ODN-induced mtROS production in cells with knockdowns of gene CPT1A ( L ) and the genes for electron shuttles GPD2 ( M ) or ETFDH ( N ). ( O ) Ratio of reduced:oxidized CoQ in mitochondria isolated from HBEC3-KT cells treated with PBS or ODN. ( P ) Schematic model of mtROS formation induced by ODN via metabolic reprogramming. * p <0.01 vs 0 min; † p <0.001 vs. (syngeneic) PBS treated; ǂ p < 0.02 vs (syngeneic) PBS treated. RPPA, reverse phase protein array; AMPK, AMP-activating protein kinase; ACC, acetyl-CoA carboxylase; AdV, adenovirus; OCR, oxygen consumption rate; Scr, scrambled shRNA control; CPT1A, carnitine palmitoyltransferase 1A; GPD2, glycerol-3-phosphate dehydrogenase 2; ETFDH, electron transfer flavoprotein-ubiquinone dehydrogenase.

Article Snippet: Normal human bronchial epithelial (NHBE) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit (ATCC, Manassas, VA).

Techniques: Western Blot, Permeability, Immunofluorescence, Fluorescence, Infection, Activity Assay, Isolation, Protein Array, shRNA, Control

Journal: bioRxiv

Article Title: Antimicrobial mitochondrial reactive oxygen species induction by lung epithelial metabolic reprogramming

doi: 10.1101/2023.01.19.524841

Figure Lengend Snippet: ( A ) Representative fluorescence images primary tracheal epithelial cells harvested from mt-roGFP mice, pre-treated (or not) with TTFA and FCCP, then treated with PBS or ODN. Images shown as gradient of color intensity from the reduced (blue) form to the oxidized (green) form of roGFP. Scale bar, 50 μm. ( B ) Ratio of the fluorescence intensity of the oxidized:reduced roGFP from A , quantified at 488 nm and 405 nm, respectively. ( C ) Bacterial burden of HBEC3-KT cells treated with the indicated ligands with or without TTFA-FCCP treatment. ( D ) Survival of wild type mice challenged with P. aeruginosa one day after nebulized treatment with PBS or Pam2 and ODN with or without TTFA-FCCP (n=15 mice/group). ( E ) Survival of Tlr9 -/- mice challenged with P. aeruginosa one day after nebulized treatment with PBS or Pam2 and ODN with or without TTFA-FCCP (n=15 mice/group). ( F ) Bacterial burden of HBEC3-KT cells treated with Pam2 and erastin or ODN. ( G ) Mouse survival of P. aeruginosa challenge given one day after nebulized treatment with the indicated agents (n=15 mice/group). ( H ) Mouse lung bacterial burden immediately after P. aeruginosa challenge following treatment with the indicated agents (n=4 mice/group). * p <0.02 vs PBS, † p< 0.05 vs ODN, ‡ p < 0.05 vs same ligand without TTFA-FCCP, ¶ P <0.0001 vs. PBS.

Article Snippet: Normal human bronchial epithelial (NHBE) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and cultured in airway epithelial cell basal medium supplemented with bronchial epithelial cell growth kit (ATCC, Manassas, VA).

Techniques: Fluorescence