dnase i  (Roche)


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    Structured Review

    Roche dnase i
    Real-time PCR quantitation of DNase I-resistant genomes after incubation with nuclear extract or cytoplasmic extract. A total of 10 10 particles of purified AAV2/2-hF.IX vector were incubated for 30 min at 37°C with 50 μg of nuclear extract, cytoplasmic extract, or the same volume of buffer alone, before digesting with <t>DNase</t> I for a further hour at 37°C (final volume, 100 μl). (A) DNase I-resistant VG in 5 μl were quantified by real-time PCR. Error bars show the standard error of the mean of four samples per group. (B) Twenty microliters of the remaining sample was analyzed by Western blotting with the anti-VP1,2,3 antibody to detect the integrity of the capsid proteins following incubation with nuclear or cytoplasmic extract. Lanes 1 and 2, AAV particles incubated with buffer only; lanes 3 to 6, AAV particles incubated with cytoplasmic extract; lanes 7 to 10, AAV particles incubated with nuclear extract.
    Dnase I, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Rapid Uncoating of Vector Genomes Is the Key to Efficient Liver Transduction with Pseudotyped Adeno-Associated Virus Vectors"

    Article Title: Rapid Uncoating of Vector Genomes Is the Key to Efficient Liver Transduction with Pseudotyped Adeno-Associated Virus Vectors

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.6.3110-3122.2004

    Real-time PCR quantitation of DNase I-resistant genomes after incubation with nuclear extract or cytoplasmic extract. A total of 10 10 particles of purified AAV2/2-hF.IX vector were incubated for 30 min at 37°C with 50 μg of nuclear extract, cytoplasmic extract, or the same volume of buffer alone, before digesting with DNase I for a further hour at 37°C (final volume, 100 μl). (A) DNase I-resistant VG in 5 μl were quantified by real-time PCR. Error bars show the standard error of the mean of four samples per group. (B) Twenty microliters of the remaining sample was analyzed by Western blotting with the anti-VP1,2,3 antibody to detect the integrity of the capsid proteins following incubation with nuclear or cytoplasmic extract. Lanes 1 and 2, AAV particles incubated with buffer only; lanes 3 to 6, AAV particles incubated with cytoplasmic extract; lanes 7 to 10, AAV particles incubated with nuclear extract.
    Figure Legend Snippet: Real-time PCR quantitation of DNase I-resistant genomes after incubation with nuclear extract or cytoplasmic extract. A total of 10 10 particles of purified AAV2/2-hF.IX vector were incubated for 30 min at 37°C with 50 μg of nuclear extract, cytoplasmic extract, or the same volume of buffer alone, before digesting with DNase I for a further hour at 37°C (final volume, 100 μl). (A) DNase I-resistant VG in 5 μl were quantified by real-time PCR. Error bars show the standard error of the mean of four samples per group. (B) Twenty microliters of the remaining sample was analyzed by Western blotting with the anti-VP1,2,3 antibody to detect the integrity of the capsid proteins following incubation with nuclear or cytoplasmic extract. Lanes 1 and 2, AAV particles incubated with buffer only; lanes 3 to 6, AAV particles incubated with cytoplasmic extract; lanes 7 to 10, AAV particles incubated with nuclear extract.

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitation Assay, Incubation, Purification, Plasmid Preparation, Western Blot

    Immunoprecipitation of intact AAV2/2 particles from purified liver nuclei. Liver nuclei purified at different time points after intraportal injection of 5 × 10 11 VG of AAV2/2-hF.IX16 into C57BL/6 mice were incubated with DNase I for 1 h at 37°C. (A and B) Intact AAV capsids were immunoprecipitated from solubilized, DNase I-treated nuclei using the A20 antibody (A) or heparin-Sepharose beads (B). Immunoprecipitated capsids were boiled for 5 min in alkaline buffer, and the released VG were separated on a 1% alkaline agarose gel, blotted, and probed with a sequence-specific probe. (C) Control immunoprecipitations were performed with the anti-VP1,2,3 antibody, which recognizes dissociated, but not intact, capsid proteins. Positive and negative controls were performed for immunoprecipitations with all antibodies. For the positive control, purified nuclei were spiked with 10 9 VG of AAV2/2-hF.IX.16 prior to solubilization (lane 6). For the negative control, solubilized nuclei were spiked with 10 10 VG of AAV-hF.IX16 DNA extracted from purified AAV2/2-hF.IX16 particles (lane 5). Lanes 1 to 4 show copy number standards (purified AAV2/2-hF.IX particles boiled in alkaline buffer prior to loading). Lanes 7 to 15 represent individual mice. (D) Ten microliters of supernatant from solubilized nuclei (from a total volume of 1 ml for each mouse) removed prior to immunoprecipitation was also boiled in alkaline buffer and loaded on a gel.
    Figure Legend Snippet: Immunoprecipitation of intact AAV2/2 particles from purified liver nuclei. Liver nuclei purified at different time points after intraportal injection of 5 × 10 11 VG of AAV2/2-hF.IX16 into C57BL/6 mice were incubated with DNase I for 1 h at 37°C. (A and B) Intact AAV capsids were immunoprecipitated from solubilized, DNase I-treated nuclei using the A20 antibody (A) or heparin-Sepharose beads (B). Immunoprecipitated capsids were boiled for 5 min in alkaline buffer, and the released VG were separated on a 1% alkaline agarose gel, blotted, and probed with a sequence-specific probe. (C) Control immunoprecipitations were performed with the anti-VP1,2,3 antibody, which recognizes dissociated, but not intact, capsid proteins. Positive and negative controls were performed for immunoprecipitations with all antibodies. For the positive control, purified nuclei were spiked with 10 9 VG of AAV2/2-hF.IX.16 prior to solubilization (lane 6). For the negative control, solubilized nuclei were spiked with 10 10 VG of AAV-hF.IX16 DNA extracted from purified AAV2/2-hF.IX16 particles (lane 5). Lanes 1 to 4 show copy number standards (purified AAV2/2-hF.IX particles boiled in alkaline buffer prior to loading). Lanes 7 to 15 represent individual mice. (D) Ten microliters of supernatant from solubilized nuclei (from a total volume of 1 ml for each mouse) removed prior to immunoprecipitation was also boiled in alkaline buffer and loaded on a gel.

    Techniques Used: Immunoprecipitation, Purification, Injection, Mouse Assay, Incubation, Agarose Gel Electrophoresis, Sequencing, Positive Control, Negative Control

    Southern blot analysis of rAAV VG extracted from purified liver nuclei without preincubation with DNase I (A), or after incubation with DNase I for 5 h at 37°C (B). Vector forms in DNA samples extracted 3 weeks after injection of pseudotyped AAV-hF.IX16 vectors are shown. Forty micrograms of undigested total DNA (A) or the equivalent volume of DNase I-treated sample (B) was separated on a 1% agarose gel, blotted, and probed with a vector sequence-specific probe. Lanes 1 and 2, 1- and 0.1-VG/DGE standards, respectively (40 μg of DNA extracted from naïve mice was spiked with a 6.4-kb plasmid containing the AAVhF.IX16 sequence and then digested with Sac I prior to loading). A total of 10 7 VG of AAV-hF.IX16 extracted from purified vector stock was denatured by boiling for 5 min in the presence of formamide and loaded in lane 3 as a size marker for ss AAV-hF.IX genomes. Lanes 5 to 15 represent individual mice. Arrowheads indicate the different molecular forms of the AAV-hF.IX16 genome. ds indicates either relaxed circular, supercoiled circular, or linear ds forms. c, concatemers.
    Figure Legend Snippet: Southern blot analysis of rAAV VG extracted from purified liver nuclei without preincubation with DNase I (A), or after incubation with DNase I for 5 h at 37°C (B). Vector forms in DNA samples extracted 3 weeks after injection of pseudotyped AAV-hF.IX16 vectors are shown. Forty micrograms of undigested total DNA (A) or the equivalent volume of DNase I-treated sample (B) was separated on a 1% agarose gel, blotted, and probed with a vector sequence-specific probe. Lanes 1 and 2, 1- and 0.1-VG/DGE standards, respectively (40 μg of DNA extracted from naïve mice was spiked with a 6.4-kb plasmid containing the AAVhF.IX16 sequence and then digested with Sac I prior to loading). A total of 10 7 VG of AAV-hF.IX16 extracted from purified vector stock was denatured by boiling for 5 min in the presence of formamide and loaded in lane 3 as a size marker for ss AAV-hF.IX genomes. Lanes 5 to 15 represent individual mice. Arrowheads indicate the different molecular forms of the AAV-hF.IX16 genome. ds indicates either relaxed circular, supercoiled circular, or linear ds forms. c, concatemers.

    Techniques Used: Southern Blot, Purification, Incubation, Plasmid Preparation, Injection, Agarose Gel Electrophoresis, Sequencing, Mouse Assay, Marker

    Real-time PCR quantitation of the proportions of DNase I-resistant AAV genomes localized within the nucleus over time. Numbers of AAV genomes were quantified in total DNA extracted from purified liver nuclei prepared at different time points after intraportal injection of 10 11  VG of pseudotyped AAV-hF.IX16 vectors into C57BL/6 mice. Solid black bars indicate the total number of nuclear-localized AAV genomes at each time point (expressed as copy numbers per DGE), and open bars indicate the relative number of DNase I-resistant AAV genomes. The ratio of DNase I-resistant to total nuclear-localized VG at each time point is expressed as a percentage above each graph.  n  = 4 mice per vector per time point. Error bars show standard errors of the means.
    Figure Legend Snippet: Real-time PCR quantitation of the proportions of DNase I-resistant AAV genomes localized within the nucleus over time. Numbers of AAV genomes were quantified in total DNA extracted from purified liver nuclei prepared at different time points after intraportal injection of 10 11 VG of pseudotyped AAV-hF.IX16 vectors into C57BL/6 mice. Solid black bars indicate the total number of nuclear-localized AAV genomes at each time point (expressed as copy numbers per DGE), and open bars indicate the relative number of DNase I-resistant AAV genomes. The ratio of DNase I-resistant to total nuclear-localized VG at each time point is expressed as a percentage above each graph. n = 4 mice per vector per time point. Error bars show standard errors of the means.

    Techniques Used: Real-time Polymerase Chain Reaction, Quantitation Assay, Purification, Injection, Mouse Assay, Plasmid Preparation

    2) Product Images from "Synergistic Action of GA-Binding Protein and Glucocorticoid Receptor in Transcription from the Mouse Mammary Tumor Virus Promoter"

    Article Title: Synergistic Action of GA-Binding Protein and Glucocorticoid Receptor in Transcription from the Mouse Mammary Tumor Virus Promoter

    Journal: Journal of Virology

    doi:

    B-cell factors binding in vitro to the HRE of MMTV. DNase I footprinting analysis with nuclear extracts of the M12 B-cell line shows two protected sites, fp1 and fp2 (arrowheads), not seen with nuclear extracts of the fibroblastic Ltk − cell line. A DNA fragment comprising the sequences from the Sty I restriction site at positions −303 to +133, where a synthetic Bam HI linker was inserted, was 5′ end labeled at the Sty ]).
    Figure Legend Snippet: B-cell factors binding in vitro to the HRE of MMTV. DNase I footprinting analysis with nuclear extracts of the M12 B-cell line shows two protected sites, fp1 and fp2 (arrowheads), not seen with nuclear extracts of the fibroblastic Ltk − cell line. A DNA fragment comprising the sequences from the Sty I restriction site at positions −303 to +133, where a synthetic Bam HI linker was inserted, was 5′ end labeled at the Sty ]).

    Techniques Used: Binding Assay, In Vitro, Footprinting, Labeling

    3) Product Images from "Dead or alive: Deoxyribonuclease I sensitive bacteria and implications for the sinus microbiome"

    Article Title: Dead or alive: Deoxyribonuclease I sensitive bacteria and implications for the sinus microbiome

    Journal: American Journal of Rhinology & Allergy

    doi: 10.2500/ajra.2016.30.4278

    Diagram of experimental design. 1st: Sample collection. 2nd: Deoxyribonuclease I (DNase) treatment. 3rd: DNA isolation. 4th: Downstream applications. 5th: Comparative analysis.
    Figure Legend Snippet: Diagram of experimental design. 1st: Sample collection. 2nd: Deoxyribonuclease I (DNase) treatment. 3rd: DNA isolation. 4th: Downstream applications. 5th: Comparative analysis.

    Techniques Used: DNA Extraction

    4) Product Images from "An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus"

    Article Title: An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00726-15

    Immunofluorescence microscopy of GAPDH in fixed biofilms. Untreated and proteinase K- or DNase I-treated biofilms were fixed and probed with a primary anti-GAPDH antibody and a secondary anti-rabbit antibody conjugated to Alexa Fluor 488 (green), and cell nuclei were strained with DAPI (blue). Size bars, 10 μm.
    Figure Legend Snippet: Immunofluorescence microscopy of GAPDH in fixed biofilms. Untreated and proteinase K- or DNase I-treated biofilms were fixed and probed with a primary anti-GAPDH antibody and a secondary anti-rabbit antibody conjugated to Alexa Fluor 488 (green), and cell nuclei were strained with DAPI (blue). Size bars, 10 μm.

    Techniques Used: Immunofluorescence, Microscopy

    Quantification of eDNA by qPCR. (A) Quantification of eDNA in untreated and proteinase K- or DNase I-treated biofilms by qPCR. eDNA was quantified in biofilm medium supernatant (pH approximately 4.5 to 5) following resuspension after remaining cells were removed by filtration. (B) Quantification of eDNA release upon suspension of biofilms in PBS at pH 5 or 7.5. (C) Quantification of eDNA release of untreated and DNase I- or proteinase K-treated biofilms upon suspension in PBS at pH 7.5 with or without washing. The amount of eDNA measured was normalized to that in unwashed, untreated cells. For panels B and C, eDNA was quantified after biofilms were resuspended in PBS and cells were removed by centrifugation and filtration. For all experiments, primers for the gene for gyrase A ( gyrA ) were used and dilutions of genomic  S. aureus  DNA were included to calculate absolute DNA concentrations. Average values and standard deviations of at least three independent experiments with three biological and three technical replicates are shown. Significant differences were calculated with Student's  t  test. Not significant (ns),  P >  0.05; *,  P  ≤ 0.05; ***,  P  ≤ 0.001.
    Figure Legend Snippet: Quantification of eDNA by qPCR. (A) Quantification of eDNA in untreated and proteinase K- or DNase I-treated biofilms by qPCR. eDNA was quantified in biofilm medium supernatant (pH approximately 4.5 to 5) following resuspension after remaining cells were removed by filtration. (B) Quantification of eDNA release upon suspension of biofilms in PBS at pH 5 or 7.5. (C) Quantification of eDNA release of untreated and DNase I- or proteinase K-treated biofilms upon suspension in PBS at pH 7.5 with or without washing. The amount of eDNA measured was normalized to that in unwashed, untreated cells. For panels B and C, eDNA was quantified after biofilms were resuspended in PBS and cells were removed by centrifugation and filtration. For all experiments, primers for the gene for gyrase A ( gyrA ) were used and dilutions of genomic S. aureus DNA were included to calculate absolute DNA concentrations. Average values and standard deviations of at least three independent experiments with three biological and three technical replicates are shown. Significant differences were calculated with Student's t test. Not significant (ns), P > 0.05; *, P ≤ 0.05; ***, P ≤ 0.001.

    Techniques Used: Real-time Polymerase Chain Reaction, Filtration, Centrifugation

    Quantitative assays of biofilms undergoing different treatments at different time points. Proteinase K, DNase I, or water was added to biofilms at inoculation (bars 0), after 7 h of incubation (bars 7), or after 23 h of incubation (bars 23). The OD 600 s of the washed resuspended biofilm, the two combined PBS washes, and the growth medium were measured after 24 h of incubation.
    Figure Legend Snippet: Quantitative assays of biofilms undergoing different treatments at different time points. Proteinase K, DNase I, or water was added to biofilms at inoculation (bars 0), after 7 h of incubation (bars 7), or after 23 h of incubation (bars 23). The OD 600 s of the washed resuspended biofilm, the two combined PBS washes, and the growth medium were measured after 24 h of incubation.

    Techniques Used: Incubation

    SDS-PAGE of matrix proteins released from biofilm cells and corresponding cell lysates. (A) Untreated and DNase I-treated biofilms were resuspended and incubated in PBS at pH 5 or 7.5, and cells were removed by centrifugation and filtration. Proteins in the resulting supernatant were concentrated by TCA precipitation and separated by SDS-PAGE. (B) Pelleted cells from biofilms resuspended in PBS at pHs 5 and 7.5 were lysed with zirconium beads, and whole-cell lysates were separated by SDS-PAGE. Molecular size markers are shown on the left.
    Figure Legend Snippet: SDS-PAGE of matrix proteins released from biofilm cells and corresponding cell lysates. (A) Untreated and DNase I-treated biofilms were resuspended and incubated in PBS at pH 5 or 7.5, and cells were removed by centrifugation and filtration. Proteins in the resulting supernatant were concentrated by TCA precipitation and separated by SDS-PAGE. (B) Pelleted cells from biofilms resuspended in PBS at pHs 5 and 7.5 were lysed with zirconium beads, and whole-cell lysates were separated by SDS-PAGE. Molecular size markers are shown on the left.

    Techniques Used: SDS Page, Incubation, Centrifugation, Filtration, TCA Precipitation

    Quantification of clump sizes from phase-contrast microscopy images of untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of DNA (DNA from S. aureus [SA] or salmon sperm [Sal] at 24 μg ml −1 ) after DNase I and proteinase K treatment of biofilms. Clumps of 1 to 4, 5 to 10, 11 to 20, or > 20 cells were identified. Data represent average values of three experiments with standard deviations of quantifications of 20 randomly picked microscopic fields per experiment. Significantly more clumps of > 20 cells were detected in untreated biofilms than in proteinase K- or DNase I-treated biofilms, as well as in DNase I-treated samples with added DNA (Sal or SA) versus only DNase I-treated biofilms. Significant differences were calculated with Student's t test. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001.
    Figure Legend Snippet: Quantification of clump sizes from phase-contrast microscopy images of untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of DNA (DNA from S. aureus [SA] or salmon sperm [Sal] at 24 μg ml −1 ) after DNase I and proteinase K treatment of biofilms. Clumps of 1 to 4, 5 to 10, 11 to 20, or > 20 cells were identified. Data represent average values of three experiments with standard deviations of quantifications of 20 randomly picked microscopic fields per experiment. Significantly more clumps of > 20 cells were detected in untreated biofilms than in proteinase K- or DNase I-treated biofilms, as well as in DNase I-treated samples with added DNA (Sal or SA) versus only DNase I-treated biofilms. Significant differences were calculated with Student's t test. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001.

    Techniques Used: Microscopy

    Microscopic images of clumping of resuspended cells from untreated biofilm and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of exogenous DNA to DNase I- and proteinase K-treated biofilms. (A) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase. (B) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from biofilms treated with DNase I or proteinase K, with subsequent addition of exogenous DNA from S. aureus (SA) or salmon sperm (Sal) at 24 μg ml −1 .
    Figure Legend Snippet: Microscopic images of clumping of resuspended cells from untreated biofilm and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of exogenous DNA to DNase I- and proteinase K-treated biofilms. (A) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase. (B) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from biofilms treated with DNase I or proteinase K, with subsequent addition of exogenous DNA from S. aureus (SA) or salmon sperm (Sal) at 24 μg ml −1 .

    Techniques Used: Microscopy

    5) Product Images from "PU.1 binds to a distal regulatory element that is necessary for B-cell specific expression of the class II transactivator"

    Article Title: PU.1 binds to a distal regulatory element that is necessary for B-cell specific expression of the class II transactivator

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1000079

    B cell and plasma cell specific DNase I hypersensitive sites are located in conserved regions upstream of CIITA promoter I ). The screen shot from that analysis is shown with an annotated schematic.
    Figure Legend Snippet: B cell and plasma cell specific DNase I hypersensitive sites are located in conserved regions upstream of CIITA promoter I ). The screen shot from that analysis is shown with an annotated schematic.

    Techniques Used:

    6) Product Images from "DNase Pretreatment of Master Mix Reagents Improves the Validity of Universal 16S rRNA Gene PCR Results"

    Article Title: DNase Pretreatment of Master Mix Reagents Improves the Validity of Universal 16S rRNA Gene PCR Results

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.4.1763-1765.2003

    Influence of DNase I dosages on the sensitivity of PCR assays. Rate of positive PCR results at the detection limit are given depending on DNase I dosages for assays I (A), II (B), and III (C) (for details, see text). The threshold dosage of DNase I that was required for the complete elimination of false-positive PCR results is underlined. Amounts of DNase I increasing stepwise reduced the sensitivity of the PCRs.
    Figure Legend Snippet: Influence of DNase I dosages on the sensitivity of PCR assays. Rate of positive PCR results at the detection limit are given depending on DNase I dosages for assays I (A), II (B), and III (C) (for details, see text). The threshold dosage of DNase I that was required for the complete elimination of false-positive PCR results is underlined. Amounts of DNase I increasing stepwise reduced the sensitivity of the PCRs.

    Techniques Used: Polymerase Chain Reaction

    DNase I dosages required to improve universal PCR results. Percentages of PCR runs delivering valid (white bars), false-positive (grey bars), and false-negative (black bars) results depending on DNase I dosages are shown for assays I (A), II (B), and III (C) (for details, see text). Complete elimination of false-positive results and significant increases in valid results were achieved with highly varying amounts of DNase I (0.1 through 70 IU). The asterisks mark the significantly high proportion of valid PCR results obtained with DNase pretreatment compared to the results from PCR runs without DNase pretreatment.
    Figure Legend Snippet: DNase I dosages required to improve universal PCR results. Percentages of PCR runs delivering valid (white bars), false-positive (grey bars), and false-negative (black bars) results depending on DNase I dosages are shown for assays I (A), II (B), and III (C) (for details, see text). Complete elimination of false-positive results and significant increases in valid results were achieved with highly varying amounts of DNase I (0.1 through 70 IU). The asterisks mark the significantly high proportion of valid PCR results obtained with DNase pretreatment compared to the results from PCR runs without DNase pretreatment.

    Techniques Used: Polymerase Chain Reaction

    7) Product Images from "DNase I improves corneal epithelial and nerve regeneration in diabetic mice. DNase I improves corneal epithelial and nerve regeneration in diabetic mice"

    Article Title: DNase I improves corneal epithelial and nerve regeneration in diabetic mice. DNase I improves corneal epithelial and nerve regeneration in diabetic mice

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.15112

    DNase I restored the resolution of corneal inflammation. A, Immunofluorescence staining was performed with the macrophage marker anti‐F4/80 (green fluorescence) and the M2 macrophage marker anti‐CD206 (red fluorescence) 48 h after removal of the corneal epithelium. B, mRNA expression levels of iNOS, CD86, TNF‐α, MCP‐1, IL‐12, IL‐10, arginase‐1 and CD206 (48 h after epithelial scrape) were analyzed by RT‐qPCR from the control and diabetic mouse corneas (n = 6). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I restored the resolution of corneal inflammation. A, Immunofluorescence staining was performed with the macrophage marker anti‐F4/80 (green fluorescence) and the M2 macrophage marker anti‐CD206 (red fluorescence) 48 h after removal of the corneal epithelium. B, mRNA expression levels of iNOS, CD86, TNF‐α, MCP‐1, IL‐12, IL‐10, arginase‐1 and CD206 (48 h after epithelial scrape) were analyzed by RT‐qPCR from the control and diabetic mouse corneas (n = 6). Data were given as the mean ± SD; * P

    Techniques Used: Immunofluorescence, Staining, Marker, Fluorescence, Expressing, Quantitative RT-PCR

    DNase I restored the resolution of corneal inflammation. A, Expression of H3Cit and Ly6G was examined with immunofluorescence staining 48 h after corneal epithelial removal in the control, diabetic and DNase I‐treated diabetic mice. B‐D, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of H3Cit, H3 and Ly6G (B), accompanied by the quantified results of Western blot experiments (C‐D; n = 6). E‐F, Corneas harvested 48 h after injury were homogenized and examined for levels of myeloperoxidase (MPO) activity (E) and neutrophil elastase (NE) expression (F) with enzyme‐linked immunosorbent assay (ELISA; n = 5). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I restored the resolution of corneal inflammation. A, Expression of H3Cit and Ly6G was examined with immunofluorescence staining 48 h after corneal epithelial removal in the control, diabetic and DNase I‐treated diabetic mice. B‐D, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of H3Cit, H3 and Ly6G (B), accompanied by the quantified results of Western blot experiments (C‐D; n = 6). E‐F, Corneas harvested 48 h after injury were homogenized and examined for levels of myeloperoxidase (MPO) activity (E) and neutrophil elastase (NE) expression (F) with enzyme‐linked immunosorbent assay (ELISA; n = 5). Data were given as the mean ± SD; * P

    Techniques Used: Expressing, Immunofluorescence, Staining, Mouse Assay, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

    Anti‐NETs treatment promoted the regeneration of corneal epithelium in diabetic mice. A, Diabetic mice were topically treated with 1 mg/mL DNase I (5 μL/eye, six times per day) after the removal of the corneal epithelium. Meanwhile, healthy and diabetic control mice were topically treated with PBS. The residual epithelial defect was examined at 0, 24 and 48 h after the removal of the corneal epithelium with fluorescein staining. B, The histogram of the residual epithelial defect was presented as the percentage of the original wound area (n = 5). C, Corneas harvested 48 h after injury were homogenized and examined for levels of eDNA with spectrophotometer (n = 4). D‐E, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of PAD4 (n = 6). Data were given as the mean ± SD; ** P
    Figure Legend Snippet: Anti‐NETs treatment promoted the regeneration of corneal epithelium in diabetic mice. A, Diabetic mice were topically treated with 1 mg/mL DNase I (5 μL/eye, six times per day) after the removal of the corneal epithelium. Meanwhile, healthy and diabetic control mice were topically treated with PBS. The residual epithelial defect was examined at 0, 24 and 48 h after the removal of the corneal epithelium with fluorescein staining. B, The histogram of the residual epithelial defect was presented as the percentage of the original wound area (n = 5). C, Corneas harvested 48 h after injury were homogenized and examined for levels of eDNA with spectrophotometer (n = 4). D‐E, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of PAD4 (n = 6). Data were given as the mean ± SD; ** P

    Techniques Used: Mouse Assay, Staining, Spectrophotometry, Western Blot

    Effects of DNase I on the regeneration of diabetic corneal nerves and the restoration of mechanical sensation. A‐C, Twenty‐one days after injury, the renewing corneas were harvested and the regenerated corneal nerve fibres were examined with corneal whole‐mount staining, and the immunofluorescence intensity of central (B) and peripheral (C) nerve fibres at 21 d after injury was calculated with ImageJ software (n = 5). D, A Cochet‐Bonnet esthesiometer was used to test the mechanical sensitivity of the cornea in healthy, diabetic and DNase I‐treated diabetic mice at 3, 7, 14 and 21 d after the corneal epithelial removal (n = 5). Data were given as the mean ± SD; * P
    Figure Legend Snippet: Effects of DNase I on the regeneration of diabetic corneal nerves and the restoration of mechanical sensation. A‐C, Twenty‐one days after injury, the renewing corneas were harvested and the regenerated corneal nerve fibres were examined with corneal whole‐mount staining, and the immunofluorescence intensity of central (B) and peripheral (C) nerve fibres at 21 d after injury was calculated with ImageJ software (n = 5). D, A Cochet‐Bonnet esthesiometer was used to test the mechanical sensitivity of the cornea in healthy, diabetic and DNase I‐treated diabetic mice at 3, 7, 14 and 21 d after the corneal epithelial removal (n = 5). Data were given as the mean ± SD; * P

    Techniques Used: Staining, Immunofluorescence, Software, Mouse Assay

    DNase I reactivated epithelial regeneration‐related signaling pathways. A, Immunofluorescence staining and (B) Western blot were used to examine the activation levels of epithelial regeneration‐related signaling pathways, including pAkt, IGF‐1R and Sirt1, in the regenerated corneal epithelium 48 h after injury. C‐E, The histogram showed the quantified results of the Western blot (n = 6). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I reactivated epithelial regeneration‐related signaling pathways. A, Immunofluorescence staining and (B) Western blot were used to examine the activation levels of epithelial regeneration‐related signaling pathways, including pAkt, IGF‐1R and Sirt1, in the regenerated corneal epithelium 48 h after injury. C‐E, The histogram showed the quantified results of the Western blot (n = 6). Data were given as the mean ± SD; * P

    Techniques Used: Immunofluorescence, Staining, Western Blot, Activation Assay

    DNase I inhibited the increased ROS accumulation and NADPH oxidase 2/4 expression. A, Generation of reactive oxygen species (ROS) and expression of NADPH oxidase 2/4 were examined with immunofluorescence staining in healthy, diabetic and 5‐day DNase I‐treated diabetic corneal epithelia. B, Quantification of fluorescence intensity of ROS by ImageJ software (n = 3). C‐E, Corneas harvested from healthy and diabetic (with or without 5‐day DNase I treatment) mice were evaluated with Western blot to examine the protein levels of NADPH oxidase 2/4, and the quantified data of the Western blot results were shown (D, E; n = 6). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I inhibited the increased ROS accumulation and NADPH oxidase 2/4 expression. A, Generation of reactive oxygen species (ROS) and expression of NADPH oxidase 2/4 were examined with immunofluorescence staining in healthy, diabetic and 5‐day DNase I‐treated diabetic corneal epithelia. B, Quantification of fluorescence intensity of ROS by ImageJ software (n = 3). C‐E, Corneas harvested from healthy and diabetic (with or without 5‐day DNase I treatment) mice were evaluated with Western blot to examine the protein levels of NADPH oxidase 2/4, and the quantified data of the Western blot results were shown (D, E; n = 6). Data were given as the mean ± SD; * P

    Techniques Used: Expressing, Immunofluorescence, Staining, Fluorescence, Software, Mouse Assay, Western Blot

    8) Product Images from "Insights Into the Role of Extracellular DNA and Extracellular Proteins in Biofilm Formation of Vibrio parahaemolyticus"

    Article Title: Insights Into the Role of Extracellular DNA and Extracellular Proteins in Biofilm Formation of Vibrio parahaemolyticus

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.00813

    Representative CLSM images of  V. parahaemolyticus  biofilms treated with DNase I, proteinase K, and their combination at 12, 24, and 48 h of cultivation. The scale bar represents 50 μm. Pictures are representative of at least three individual scans from three independent experiments.
    Figure Legend Snippet: Representative CLSM images of V. parahaemolyticus biofilms treated with DNase I, proteinase K, and their combination at 12, 24, and 48 h of cultivation. The scale bar represents 50 μm. Pictures are representative of at least three individual scans from three independent experiments.

    Techniques Used: Confocal Laser Scanning Microscopy

    Raman spectrum and intensity changes of mature biofilms of  V. parahaemolyticus  after treatment with DNase I, proteinase K, and their combination.  (A)  Raman spectrum and  (B)  intensity changes. Error bars show standard deviations of three independent experiments with five measurements each, and different letters represent significant differences among treatments ( P
    Figure Legend Snippet: Raman spectrum and intensity changes of mature biofilms of V. parahaemolyticus after treatment with DNase I, proteinase K, and their combination. (A) Raman spectrum and (B) intensity changes. Error bars show standard deviations of three independent experiments with five measurements each, and different letters represent significant differences among treatments ( P

    Techniques Used:

    Structural characteristics changes of  V. parahaemolyticus  biofilms treated with DNase I, proteinase K, and their combination at 12, 24, and 48 h of cultivation.  (A)  Biovolume,  (B)  mean thickness,  (C)  biofilm roughness, and  (D)  porosity. Error bars show standard deviations of three independent experiments, and the different letters represent significant differences among treatments ( P
    Figure Legend Snippet: Structural characteristics changes of V. parahaemolyticus biofilms treated with DNase I, proteinase K, and their combination at 12, 24, and 48 h of cultivation. (A) Biovolume, (B) mean thickness, (C) biofilm roughness, and (D) porosity. Error bars show standard deviations of three independent experiments, and the different letters represent significant differences among treatments ( P

    Techniques Used:

    Effect of eDNA and extracellular proteins on biofilm formation of  V. parahaemolyticus . To confirm whether the eDNA and extracellular proteins serve as structural components in biofilms of  V. parahaemolyticus , DNase I, proteinase K, and their combination were added to the biofilms incubated at different times (2, 12, 24, 36, and 48 h). Error bars show standard deviations of three independent experiments, and the different letters represent significant differences among treatments ( P
    Figure Legend Snippet: Effect of eDNA and extracellular proteins on biofilm formation of V. parahaemolyticus . To confirm whether the eDNA and extracellular proteins serve as structural components in biofilms of V. parahaemolyticus , DNase I, proteinase K, and their combination were added to the biofilms incubated at different times (2, 12, 24, 36, and 48 h). Error bars show standard deviations of three independent experiments, and the different letters represent significant differences among treatments ( P

    Techniques Used: Incubation

    9) Product Images from "Interbacterial Macromolecular Transfer by the Campylobacter fetus subsp. venerealis Type IV Secretion System ▿"

    Article Title: Interbacterial Macromolecular Transfer by the Campylobacter fetus subsp. venerealis Type IV Secretion System ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00798-10

    Transcriptional organization of C. fetus subsp. venerealis PAI region harboring the fic genes. (A) Gene organization of the region extending from transposase orfA to the putative nickase gene downstream of the fic genes was analyzed in strain ATCC 19438 by ORF-spanning amplification of cDNA produced with reverse transcriptase PCR (upper panel). Genomic DNA served as positive controls for each primer combination (lower panel). Numbers above the lanes indicate the ORFs common to the amplified cDNA according to the gene designations shown in panel B. Negative controls lacked reverse transcriptase (upper panel) or template (lower panel). (B) The 3′ region of the genomic island of C. fetus subsp. venerealis ) (below) and putative functional assignments are shown (above). fic genes are shaded gray. (C) Transcription map depicts two distinct mRNA fragments encoding ORFs 24 to 30 and ORFs 33 to 40, respectively (solid lines). ORFs 31 and 32 of unknown function are not transcribed with the other genes nor each other. Transcripts initiated within units 33 to 40 extend into the putative relaxase gene cpp17 in reverse orientation (dotted line). The separately amplified cDNA regions shown in panel A are indicated schematically (above).
    Figure Legend Snippet: Transcriptional organization of C. fetus subsp. venerealis PAI region harboring the fic genes. (A) Gene organization of the region extending from transposase orfA to the putative nickase gene downstream of the fic genes was analyzed in strain ATCC 19438 by ORF-spanning amplification of cDNA produced with reverse transcriptase PCR (upper panel). Genomic DNA served as positive controls for each primer combination (lower panel). Numbers above the lanes indicate the ORFs common to the amplified cDNA according to the gene designations shown in panel B. Negative controls lacked reverse transcriptase (upper panel) or template (lower panel). (B) The 3′ region of the genomic island of C. fetus subsp. venerealis ) (below) and putative functional assignments are shown (above). fic genes are shaded gray. (C) Transcription map depicts two distinct mRNA fragments encoding ORFs 24 to 30 and ORFs 33 to 40, respectively (solid lines). ORFs 31 and 32 of unknown function are not transcribed with the other genes nor each other. Transcripts initiated within units 33 to 40 extend into the putative relaxase gene cpp17 in reverse orientation (dotted line). The separately amplified cDNA regions shown in panel A are indicated schematically (above).

    Techniques Used: Amplification, Produced, Polymerase Chain Reaction, Functional Assay

    10) Product Images from "Analysis of Jmjd6 Cellular Localization and Testing for Its Involvement in Histone Demethylation"

    Article Title: Analysis of Jmjd6 Cellular Localization and Testing for Its Involvement in Histone Demethylation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013769

    For nuclear localization of Jmjd6 an intact ribonuclear matrix is needed. Wildtype MEFs were treated with Trition X-100, RNase A, DNase I or a combination of Trition X-100 with RNase A or DNase I before fixation and staining. Time intervals and concentration of reagents used in the experiments are indicated on the right side. Untreated cells were used as controls. Horizontal rows correspond from left to right to a phase contrast view and immunofluorescence imaging of Hoechst DNA stain (blue), H3K36tri (red) and Jmjd6 (green). Shown is one representative result of at least three experiments performed.
    Figure Legend Snippet: For nuclear localization of Jmjd6 an intact ribonuclear matrix is needed. Wildtype MEFs were treated with Trition X-100, RNase A, DNase I or a combination of Trition X-100 with RNase A or DNase I before fixation and staining. Time intervals and concentration of reagents used in the experiments are indicated on the right side. Untreated cells were used as controls. Horizontal rows correspond from left to right to a phase contrast view and immunofluorescence imaging of Hoechst DNA stain (blue), H3K36tri (red) and Jmjd6 (green). Shown is one representative result of at least three experiments performed.

    Techniques Used: Staining, Concentration Assay, Immunofluorescence, Imaging

    11) Product Images from "CRX directs photoreceptor differentiation by accelerating chromatin remodeling at specific target sites"

    Article Title: CRX directs photoreceptor differentiation by accelerating chromatin remodeling at specific target sites

    Journal: Epigenetics & Chromatin

    doi: 10.1186/s13072-018-0212-2

    CRX activates a subset of Group C distal enhancers over development. a Plots display read density of DNase I experiments centered on CRX-binding sites of Dependent and Independent Group C sites. b Analysis of RNA-seq of nearest gene to each peak, displayed as boxplot of normalized RPKM values at P2 and P21 in WT and P21 in Crx − / −. (Wilcoxon rank sum test, paired; * p
    Figure Legend Snippet: CRX activates a subset of Group C distal enhancers over development. a Plots display read density of DNase I experiments centered on CRX-binding sites of Dependent and Independent Group C sites. b Analysis of RNA-seq of nearest gene to each peak, displayed as boxplot of normalized RPKM values at P2 and P21 in WT and P21 in Crx − / −. (Wilcoxon rank sum test, paired; * p

    Techniques Used: Binding Assay, RNA Sequencing Assay

    CRX binds a subset of active regulatory sites in the rod photoreceptor.  a  Browser track displays ATAC-seq, DNase I, and CRX ChIP-seq read depth. (Scale bar 5 kb)  b  Venn diagram depicting number of CRX ChIP-seq-defined binding sites that overlap with regulatory sites defined by ATAC-seq. ( c ,  d ) Meta-gene plots of all genes expressed in P21  WT  and  Crx − / − retinas, ordered by [log2] fold-change (as detailed on left). Black dots represent the center of ATAC regulatory site relative to TSS of  c  all ATAC peaks and of  d  only the subset bound by CRX. Histograms on X and Y axes display density and distribution of the data points
    Figure Legend Snippet: CRX binds a subset of active regulatory sites in the rod photoreceptor. a Browser track displays ATAC-seq, DNase I, and CRX ChIP-seq read depth. (Scale bar 5 kb) b Venn diagram depicting number of CRX ChIP-seq-defined binding sites that overlap with regulatory sites defined by ATAC-seq. ( c , d ) Meta-gene plots of all genes expressed in P21 WT and Crx − / − retinas, ordered by [log2] fold-change (as detailed on left). Black dots represent the center of ATAC regulatory site relative to TSS of c all ATAC peaks and of d only the subset bound by CRX. Histograms on X and Y axes display density and distribution of the data points

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay

    CRX is only required for activity and remodeling of a subset of Group A local regulatory sites. a Plots display read density of DNase I experiments centered on CRX-binding site of Dependent and Independent Group A sites. b Analysis of RNA-seq of nearest gene to each peak, displayed as boxplot of normalized RPKM values at P2 and P21 in WT and P21 in Crx − / −. (Wilcoxon rank sum test, paired; * p
    Figure Legend Snippet: CRX is only required for activity and remodeling of a subset of Group A local regulatory sites. a Plots display read density of DNase I experiments centered on CRX-binding site of Dependent and Independent Group A sites. b Analysis of RNA-seq of nearest gene to each peak, displayed as boxplot of normalized RPKM values at P2 and P21 in WT and P21 in Crx − / −. (Wilcoxon rank sum test, paired; * p

    Techniques Used: Activity Assay, Binding Assay, RNA Sequencing Assay

    12) Product Images from "Redundant and Distinct Roles of Secreted Protein Eap and Cell Wall-Anchored Protein SasG in Biofilm Formation and Pathogenicity of Staphylococcus aureus"

    Article Title: Redundant and Distinct Roles of Secreted Protein Eap and Cell Wall-Anchored Protein SasG in Biofilm Formation and Pathogenicity of Staphylococcus aureus

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00894-18

    SasG is a DNA binding protein. (A) DNase I sensitivities of the biofilms formed by the indicated strains. Relative biofilm biomasses are shown (nontreated biofilms are defined as 100%). Original data are shown in Fig. S1 in the supplemental material. −, absence; +, presence. (B) The DNA binding capacity of SasG was analyzed by a gel shift assay. Purified SasG (0.1, 0.5, and 1 μM) was mixed with lambda DNA (λDNA) prior to agarose gel electrophoresis. (C) Degradation of λDNA by DNase I was analyzed in the presence and absence of purified SasG. After treatment, DNase I and SasG were degraded by proteinase K, and the residual DNA was analyzed by agarose gel electrophoresis. Band intensities were measured using an LAS-4000 image analyzer, and the relative intensities are shown in the graph.
    Figure Legend Snippet: SasG is a DNA binding protein. (A) DNase I sensitivities of the biofilms formed by the indicated strains. Relative biofilm biomasses are shown (nontreated biofilms are defined as 100%). Original data are shown in Fig. S1 in the supplemental material. −, absence; +, presence. (B) The DNA binding capacity of SasG was analyzed by a gel shift assay. Purified SasG (0.1, 0.5, and 1 μM) was mixed with lambda DNA (λDNA) prior to agarose gel electrophoresis. (C) Degradation of λDNA by DNase I was analyzed in the presence and absence of purified SasG. After treatment, DNase I and SasG were degraded by proteinase K, and the residual DNA was analyzed by agarose gel electrophoresis. Band intensities were measured using an LAS-4000 image analyzer, and the relative intensities are shown in the graph.

    Techniques Used: Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Lambda DNA Preparation, Agarose Gel Electrophoresis

    13) Product Images from "DNase I improves corneal epithelial and nerve regeneration in diabetic mice. DNase I improves corneal epithelial and nerve regeneration in diabetic mice"

    Article Title: DNase I improves corneal epithelial and nerve regeneration in diabetic mice. DNase I improves corneal epithelial and nerve regeneration in diabetic mice

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.15112

    DNase I restored the resolution of corneal inflammation. A, Immunofluorescence staining was performed with the macrophage marker anti‐F4/80 (green fluorescence) and the M2 macrophage marker anti‐CD206 (red fluorescence) 48 h after removal of the corneal epithelium. B, mRNA expression levels of iNOS, CD86, TNF‐α, MCP‐1, IL‐12, IL‐10, arginase‐1 and CD206 (48 h after epithelial scrape) were analyzed by RT‐qPCR from the control and diabetic mouse corneas (n = 6). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I restored the resolution of corneal inflammation. A, Immunofluorescence staining was performed with the macrophage marker anti‐F4/80 (green fluorescence) and the M2 macrophage marker anti‐CD206 (red fluorescence) 48 h after removal of the corneal epithelium. B, mRNA expression levels of iNOS, CD86, TNF‐α, MCP‐1, IL‐12, IL‐10, arginase‐1 and CD206 (48 h after epithelial scrape) were analyzed by RT‐qPCR from the control and diabetic mouse corneas (n = 6). Data were given as the mean ± SD; * P

    Techniques Used: Immunofluorescence, Staining, Marker, Fluorescence, Expressing, Quantitative RT-PCR

    DNase I restored the resolution of corneal inflammation. A, Expression of H3Cit and Ly6G was examined with immunofluorescence staining 48 h after corneal epithelial removal in the control, diabetic and DNase I‐treated diabetic mice. B‐D, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of H3Cit, H3 and Ly6G (B), accompanied by the quantified results of Western blot experiments (C‐D; n = 6). E‐F, Corneas harvested 48 h after injury were homogenized and examined for levels of myeloperoxidase (MPO) activity (E) and neutrophil elastase (NE) expression (F) with enzyme‐linked immunosorbent assay (ELISA; n = 5). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I restored the resolution of corneal inflammation. A, Expression of H3Cit and Ly6G was examined with immunofluorescence staining 48 h after corneal epithelial removal in the control, diabetic and DNase I‐treated diabetic mice. B‐D, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of H3Cit, H3 and Ly6G (B), accompanied by the quantified results of Western blot experiments (C‐D; n = 6). E‐F, Corneas harvested 48 h after injury were homogenized and examined for levels of myeloperoxidase (MPO) activity (E) and neutrophil elastase (NE) expression (F) with enzyme‐linked immunosorbent assay (ELISA; n = 5). Data were given as the mean ± SD; * P

    Techniques Used: Expressing, Immunofluorescence, Staining, Mouse Assay, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

    Anti‐NETs treatment promoted the regeneration of corneal epithelium in diabetic mice. A, Diabetic mice were topically treated with 1 mg/mL DNase I (5 μL/eye, six times per day) after the removal of the corneal epithelium. Meanwhile, healthy and diabetic control mice were topically treated with PBS. The residual epithelial defect was examined at 0, 24 and 48 h after the removal of the corneal epithelium with fluorescein staining. B, The histogram of the residual epithelial defect was presented as the percentage of the original wound area (n = 5). C, Corneas harvested 48 h after injury were homogenized and examined for levels of eDNA with spectrophotometer (n = 4). D‐E, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of PAD4 (n = 6). Data were given as the mean ± SD; ** P
    Figure Legend Snippet: Anti‐NETs treatment promoted the regeneration of corneal epithelium in diabetic mice. A, Diabetic mice were topically treated with 1 mg/mL DNase I (5 μL/eye, six times per day) after the removal of the corneal epithelium. Meanwhile, healthy and diabetic control mice were topically treated with PBS. The residual epithelial defect was examined at 0, 24 and 48 h after the removal of the corneal epithelium with fluorescein staining. B, The histogram of the residual epithelial defect was presented as the percentage of the original wound area (n = 5). C, Corneas harvested 48 h after injury were homogenized and examined for levels of eDNA with spectrophotometer (n = 4). D‐E, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of PAD4 (n = 6). Data were given as the mean ± SD; ** P

    Techniques Used: Mouse Assay, Staining, Spectrophotometry, Western Blot

    Effects of DNase I on the regeneration of diabetic corneal nerves and the restoration of mechanical sensation. A‐C, Twenty‐one days after injury, the renewing corneas were harvested and the regenerated corneal nerve fibres were examined with corneal whole‐mount staining, and the immunofluorescence intensity of central (B) and peripheral (C) nerve fibres at 21 d after injury was calculated with ImageJ software (n = 5). D, A Cochet‐Bonnet esthesiometer was used to test the mechanical sensitivity of the cornea in healthy, diabetic and DNase I‐treated diabetic mice at 3, 7, 14 and 21 d after the corneal epithelial removal (n = 5). Data were given as the mean ± SD; * P
    Figure Legend Snippet: Effects of DNase I on the regeneration of diabetic corneal nerves and the restoration of mechanical sensation. A‐C, Twenty‐one days after injury, the renewing corneas were harvested and the regenerated corneal nerve fibres were examined with corneal whole‐mount staining, and the immunofluorescence intensity of central (B) and peripheral (C) nerve fibres at 21 d after injury was calculated with ImageJ software (n = 5). D, A Cochet‐Bonnet esthesiometer was used to test the mechanical sensitivity of the cornea in healthy, diabetic and DNase I‐treated diabetic mice at 3, 7, 14 and 21 d after the corneal epithelial removal (n = 5). Data were given as the mean ± SD; * P

    Techniques Used: Staining, Immunofluorescence, Software, Mouse Assay

    DNase I reactivated epithelial regeneration‐related signaling pathways. A, Immunofluorescence staining and (B) Western blot were used to examine the activation levels of epithelial regeneration‐related signaling pathways, including pAkt, IGF‐1R and Sirt1, in the regenerated corneal epithelium 48 h after injury. C‐E, The histogram showed the quantified results of the Western blot (n = 6). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I reactivated epithelial regeneration‐related signaling pathways. A, Immunofluorescence staining and (B) Western blot were used to examine the activation levels of epithelial regeneration‐related signaling pathways, including pAkt, IGF‐1R and Sirt1, in the regenerated corneal epithelium 48 h after injury. C‐E, The histogram showed the quantified results of the Western blot (n = 6). Data were given as the mean ± SD; * P

    Techniques Used: Immunofluorescence, Staining, Western Blot, Activation Assay

    DNase I inhibited the increased ROS accumulation and NADPH oxidase 2/4 expression. A, Generation of reactive oxygen species (ROS) and expression of NADPH oxidase 2/4 were examined with immunofluorescence staining in healthy, diabetic and 5‐day DNase I‐treated diabetic corneal epithelia. B, Quantification of fluorescence intensity of ROS by ImageJ software (n = 3). C‐E, Corneas harvested from healthy and diabetic (with or without 5‐day DNase I treatment) mice were evaluated with Western blot to examine the protein levels of NADPH oxidase 2/4, and the quantified data of the Western blot results were shown (D, E; n = 6). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I inhibited the increased ROS accumulation and NADPH oxidase 2/4 expression. A, Generation of reactive oxygen species (ROS) and expression of NADPH oxidase 2/4 were examined with immunofluorescence staining in healthy, diabetic and 5‐day DNase I‐treated diabetic corneal epithelia. B, Quantification of fluorescence intensity of ROS by ImageJ software (n = 3). C‐E, Corneas harvested from healthy and diabetic (with or without 5‐day DNase I treatment) mice were evaluated with Western blot to examine the protein levels of NADPH oxidase 2/4, and the quantified data of the Western blot results were shown (D, E; n = 6). Data were given as the mean ± SD; * P

    Techniques Used: Expressing, Immunofluorescence, Staining, Fluorescence, Software, Mouse Assay, Western Blot

    14) Product Images from "The Composition and Structure of Biofilms Developed by Propionibacterium acnes Isolated from Cardiac Pacemaker Devices"

    Article Title: The Composition and Structure of Biofilms Developed by Propionibacterium acnes Isolated from Cardiac Pacemaker Devices

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00182

    Three-dimensional live/dead imaging of P. acnes biofilms. (A–D) Three-dimensional images of a P. acnes JK12.2 biofilm cultured for 3 days were obtained by CLSM. Living and dead cells in the biofilm were stained by SYTO9 (green) and propidium iodide (red), respectively. Biofilm structures of JK12.2 (A) , JK17.1 (B) , JK12.2 cultured with DNase I (C) , and JK17.1 cultured with DNase I (D) are shown.
    Figure Legend Snippet: Three-dimensional live/dead imaging of P. acnes biofilms. (A–D) Three-dimensional images of a P. acnes JK12.2 biofilm cultured for 3 days were obtained by CLSM. Living and dead cells in the biofilm were stained by SYTO9 (green) and propidium iodide (red), respectively. Biofilm structures of JK12.2 (A) , JK17.1 (B) , JK12.2 cultured with DNase I (C) , and JK17.1 cultured with DNase I (D) are shown.

    Techniques Used: Imaging, Cell Culture, Confocal Laser Scanning Microscopy, Staining

    Biofilm formation by Propionibacterium acnes isolates and biofilm inhibition by enzymes. (A) Biofilm formation by P. acnes isolates cultured in GAMG broth, using 96-well plates and quantified by measuring ABS 595 . The dotted line represents the arbitrarily set threshold value (ABS 595 = 0.5). (B) Enzymatic inhibition of biofilm formation using DNase I (blue), proteinase K (red), and dispersin B (green). No-enzyme controls (white) were set to 100 and relative values are shown. Means and standard deviations represent two independent experiments performed in triplicate. ∗ p
    Figure Legend Snippet: Biofilm formation by Propionibacterium acnes isolates and biofilm inhibition by enzymes. (A) Biofilm formation by P. acnes isolates cultured in GAMG broth, using 96-well plates and quantified by measuring ABS 595 . The dotted line represents the arbitrarily set threshold value (ABS 595 = 0.5). (B) Enzymatic inhibition of biofilm formation using DNase I (blue), proteinase K (red), and dispersin B (green). No-enzyme controls (white) were set to 100 and relative values are shown. Means and standard deviations represent two independent experiments performed in triplicate. ∗ p

    Techniques Used: Inhibition, Cell Culture

    15) Product Images from "Genome-wide mapping of transcriptional enhancer candidates using DNA and chromatin features in maize"

    Article Title: Genome-wide mapping of transcriptional enhancer candidates using DNA and chromatin features in maize

    Journal: Genome Biology

    doi: 10.1186/s13059-017-1273-4

    Heatmaps of chromatin, DNA and transcript features at enhancer candidates. DNase I hypersensitivity, H3K9ac enrichment, mCG, mCHG and mCHH levels, presence of TEs and transcript levels at and around (±1 kb) DHSs in enhancer candidates. DHSs were scaled to equal size. The colour scales are in RPM for DNase I hypersensitivity, H3K9ac enrichment and transcript levels, and in methylation frequency (0–1) for DNA methylation. For TE sequences, red and white show the presence or absence of TEs, respectively. DHSs were clustered based on H3K9ac enrichment using a k-means (k = 4) clustering algorithm. The categories identified were numbered from 1 to 4 from the top to the bottom. All the DHSs were oriented based on H3K9ac enrichment intensity values 300 bp away from the DHS boundaries; the side with higher H3K9ac enrichment was defined as 3' end
    Figure Legend Snippet: Heatmaps of chromatin, DNA and transcript features at enhancer candidates. DNase I hypersensitivity, H3K9ac enrichment, mCG, mCHG and mCHH levels, presence of TEs and transcript levels at and around (±1 kb) DHSs in enhancer candidates. DHSs were scaled to equal size. The colour scales are in RPM for DNase I hypersensitivity, H3K9ac enrichment and transcript levels, and in methylation frequency (0–1) for DNA methylation. For TE sequences, red and white show the presence or absence of TEs, respectively. DHSs were clustered based on H3K9ac enrichment using a k-means (k = 4) clustering algorithm. The categories identified were numbered from 1 to 4 from the top to the bottom. All the DHSs were oriented based on H3K9ac enrichment intensity values 300 bp away from the DHS boundaries; the side with higher H3K9ac enrichment was defined as 3' end

    Techniques Used: Methylation, DNA Methylation Assay

    Examples of candidate rankings. From the top : identified candidate region with its ID ( V V2-IST, H husk candidate) and coordinates, DNase I hypersensitivity and H3K9ac enrichment signal intensities in V2-IST and husk tissues. In these examples, the DNase I hypersensitivity and H3K9ac enrichment signal differences do not positively correlate to each other as assumed
    Figure Legend Snippet: Examples of candidate rankings. From the top : identified candidate region with its ID ( V V2-IST, H husk candidate) and coordinates, DNase I hypersensitivity and H3K9ac enrichment signal intensities in V2-IST and husk tissues. In these examples, the DNase I hypersensitivity and H3K9ac enrichment signal differences do not positively correlate to each other as assumed

    Techniques Used:

    Average profiles of the enhancer candidates in ( a ) V2-IST and ( b ) husk. Average signal intensities of DNase I hypersensitivity, H3K9ac enrichment in RPM and DNA methylation levels in methylation frequency at DHSs and their 1-kb flanking regions. DHSs were scaled to equal size. Prior to calculation of the average, all the DHSs were oriented based on H3K9ac enrichment intensity values 300 bp away from the DHS boundaries; the sides with higher H3K9ac enrichment were defined as 3' end. The profiles show a clear preferential enrichment of H3K9ac 3’ of the DHSs and high levels of DNA methylation (CG and CHG context) around the DHSs and H3K9ac-enriched regions. The level of mCHH is low throughout the regions with a slight increase at the 5’ side of DHSs
    Figure Legend Snippet: Average profiles of the enhancer candidates in ( a ) V2-IST and ( b ) husk. Average signal intensities of DNase I hypersensitivity, H3K9ac enrichment in RPM and DNA methylation levels in methylation frequency at DHSs and their 1-kb flanking regions. DHSs were scaled to equal size. Prior to calculation of the average, all the DHSs were oriented based on H3K9ac enrichment intensity values 300 bp away from the DHS boundaries; the sides with higher H3K9ac enrichment were defined as 3' end. The profiles show a clear preferential enrichment of H3K9ac 3’ of the DHSs and high levels of DNA methylation (CG and CHG context) around the DHSs and H3K9ac-enriched regions. The level of mCHH is low throughout the regions with a slight increase at the 5’ side of DHSs

    Techniques Used: DNA Methylation Assay, Methylation

    Example of data on ( a ) DICE and ( b ) b1 repeat enhancer. From the top : AGPv4 annotation and candidate annotation from our prediction ( V V2-IST, H husk candidate), DNase I hypersensitivity and H3K9ac enrichment signal (all replicates pooled) and peak position (indicated as blue and green bars , respectively) in V2-IST and in husk tissue, mCG, mCHG and mCHH levels and unique mappability in percentage. The numbers under gene names indicate relative gene expression levels (V2-IST/husk). Although the b1 locus is on chromosome 2, in the current version of the AGPv4 assembly, the b1 gene is located in contig 44 (B, on the right of the grey vertical line ). The dark blue bars in the gene annotation tracks indicate previously annotated known enhancers and putative cis- regulatory elements. The vertical red boxes indicate enhancer candidates identified in this study. Peaks at those tracks might not be present in each replicate, affecting enhancer candidate prediction
    Figure Legend Snippet: Example of data on ( a ) DICE and ( b ) b1 repeat enhancer. From the top : AGPv4 annotation and candidate annotation from our prediction ( V V2-IST, H husk candidate), DNase I hypersensitivity and H3K9ac enrichment signal (all replicates pooled) and peak position (indicated as blue and green bars , respectively) in V2-IST and in husk tissue, mCG, mCHG and mCHH levels and unique mappability in percentage. The numbers under gene names indicate relative gene expression levels (V2-IST/husk). Although the b1 locus is on chromosome 2, in the current version of the AGPv4 assembly, the b1 gene is located in contig 44 (B, on the right of the grey vertical line ). The dark blue bars in the gene annotation tracks indicate previously annotated known enhancers and putative cis- regulatory elements. The vertical red boxes indicate enhancer candidates identified in this study. Peaks at those tracks might not be present in each replicate, affecting enhancer candidate prediction

    Techniques Used: Expressing

    Average DNase I hypersensitivity and H3K9ac enrichment at genic regions. Average signal (in RPM) for DNase I hypersensitivity in ( a ) V2-IST and ( b ) husk, and for H3K9ac enrichment in ( c ) V2-IST and ( d ) husk at genes and their 1-kb flanking regions. Genes were binned based on their expression levels, from no expression ( light colour ) to high expression ( dark colour ): the lowest expression level bin contains all genes with an expression lower than 1 RPKM. The thresholds (in RPKM) are at 1.94, 4.17, 8.58, 16.64 and 36.28 for V2-IST and 1.88, 4.00, 8.34, 15.83 and 32.99 for husk tissue
    Figure Legend Snippet: Average DNase I hypersensitivity and H3K9ac enrichment at genic regions. Average signal (in RPM) for DNase I hypersensitivity in ( a ) V2-IST and ( b ) husk, and for H3K9ac enrichment in ( c ) V2-IST and ( d ) husk at genes and their 1-kb flanking regions. Genes were binned based on their expression levels, from no expression ( light colour ) to high expression ( dark colour ): the lowest expression level bin contains all genes with an expression lower than 1 RPKM. The thresholds (in RPKM) are at 1.94, 4.17, 8.58, 16.64 and 36.28 for V2-IST and 1.88, 4.00, 8.34, 15.83 and 32.99 for husk tissue

    Techniques Used: Expressing

    16) Product Images from "The Composition and Structure of Biofilms Developed by Propionibacterium acnes Isolated from Cardiac Pacemaker Devices"

    Article Title: The Composition and Structure of Biofilms Developed by Propionibacterium acnes Isolated from Cardiac Pacemaker Devices

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00182

    Three-dimensional live/dead imaging of P. acnes biofilms. (A–D) Three-dimensional images of a P. acnes JK12.2 biofilm cultured for 3 days were obtained by CLSM. Living and dead cells in the biofilm were stained by SYTO9 (green) and propidium iodide (red), respectively. Biofilm structures of JK12.2 (A) , JK17.1 (B) , JK12.2 cultured with DNase I (C) , and JK17.1 cultured with DNase I (D) are shown.
    Figure Legend Snippet: Three-dimensional live/dead imaging of P. acnes biofilms. (A–D) Three-dimensional images of a P. acnes JK12.2 biofilm cultured for 3 days were obtained by CLSM. Living and dead cells in the biofilm were stained by SYTO9 (green) and propidium iodide (red), respectively. Biofilm structures of JK12.2 (A) , JK17.1 (B) , JK12.2 cultured with DNase I (C) , and JK17.1 cultured with DNase I (D) are shown.

    Techniques Used: Imaging, Cell Culture, Confocal Laser Scanning Microscopy, Staining

    Biofilm formation by Propionibacterium acnes isolates and biofilm inhibition by enzymes. (A) Biofilm formation by P. acnes isolates cultured in GAMG broth, using 96-well plates and quantified by measuring ABS 595 . The dotted line represents the arbitrarily set threshold value (ABS 595 = 0.5). (B) Enzymatic inhibition of biofilm formation using DNase I (blue), proteinase K (red), and dispersin B (green). No-enzyme controls (white) were set to 100 and relative values are shown. Means and standard deviations represent two independent experiments performed in triplicate. ∗ p
    Figure Legend Snippet: Biofilm formation by Propionibacterium acnes isolates and biofilm inhibition by enzymes. (A) Biofilm formation by P. acnes isolates cultured in GAMG broth, using 96-well plates and quantified by measuring ABS 595 . The dotted line represents the arbitrarily set threshold value (ABS 595 = 0.5). (B) Enzymatic inhibition of biofilm formation using DNase I (blue), proteinase K (red), and dispersin B (green). No-enzyme controls (white) were set to 100 and relative values are shown. Means and standard deviations represent two independent experiments performed in triplicate. ∗ p

    Techniques Used: Inhibition, Cell Culture

    17) Product Images from "Detection of a Fourth Orbivirus Non-Structural Protein"

    Article Title: Detection of a Fourth Orbivirus Non-Structural Protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025697

    DNase I competition assay. Lane DL: dsDNA ladder labelled in base pairs. Lane 1: dsDNA ladder pre-incubated with GIV NS4 followed by DNase I. GIV NS4 protected the ladder against DNase cleavage. Lane 2: dsDNA ladder pre-incubated with BTV-8 NS4, followed by DNase I, showing that BTV NS4 protected against DNase cleavage. Lane 3: Ladder incubated with DNase I as positive control of digestion. Lane 4 : dsDNA ladder pre-incubated with VP9 of BAV followed by DNAse I. VP9 of BAV did not prevent DNAse I from degrading dsDNA. Lane 5: dsDNA ladder incubated with VP9 of BAV. VP9 of BAV did not affect the integrity of dsDNA.
    Figure Legend Snippet: DNase I competition assay. Lane DL: dsDNA ladder labelled in base pairs. Lane 1: dsDNA ladder pre-incubated with GIV NS4 followed by DNase I. GIV NS4 protected the ladder against DNase cleavage. Lane 2: dsDNA ladder pre-incubated with BTV-8 NS4, followed by DNase I, showing that BTV NS4 protected against DNase cleavage. Lane 3: Ladder incubated with DNase I as positive control of digestion. Lane 4 : dsDNA ladder pre-incubated with VP9 of BAV followed by DNAse I. VP9 of BAV did not prevent DNAse I from degrading dsDNA. Lane 5: dsDNA ladder incubated with VP9 of BAV. VP9 of BAV did not affect the integrity of dsDNA.

    Techniques Used: Competitive Binding Assay, Incubation, Positive Control

    18) Product Images from "Discovery of functional noncoding elements by digital analysis of chromatin structure"

    Article Title: Discovery of functional noncoding elements by digital analysis of chromatin structure

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0407387101

    Modeling genome-scale discovery of HSs with DACS. Shown are results of an in silico simulation of DACS indicating the number of HSs ( y axis) that would be identified at a fixed 90% PPV threshold as a function of the number of tags ( x axis) mapped for a given input tag-population enrichment for HSs (colored curves; range 2–20%). DACS was simulated against a model genome in which 50,000 model DNase I HSs were distributed against the complete human genome sequence. As expected, the number of HSs predicted with > 90% accuracy grows rapidly and then levels off. Larger numbers of tags will eventually enable identification of most HSs in the population (not shown).
    Figure Legend Snippet: Modeling genome-scale discovery of HSs with DACS. Shown are results of an in silico simulation of DACS indicating the number of HSs ( y axis) that would be identified at a fixed 90% PPV threshold as a function of the number of tags ( x axis) mapped for a given input tag-population enrichment for HSs (colored curves; range 2–20%). DACS was simulated against a model genome in which 50,000 model DNase I HSs were distributed against the complete human genome sequence. As expected, the number of HSs predicted with > 90% accuracy grows rapidly and then levels off. Larger numbers of tags will eventually enable identification of most HSs in the population (not shown).

    Techniques Used: In Silico, Sequencing

    19) Product Images from "Isolation, purification and in vitro differentiation of cytotrophoblast cells from human term placenta"

    Article Title: Isolation, purification and in vitro differentiation of cytotrophoblast cells from human term placenta

    Journal: Reproductive Biology and Endocrinology : RB & E

    doi: 10.1186/s12958-015-0070-8

    Comparison of yield and viability of purified cytotrophoblast cells among a variety of enzymatic degradation protocols. Yield ( a ) and viability ( b ) of purified cytotrophoblast cells using different enzymatic digestion protocols were assessed. Protocol 1: digestion three times in 0.25 % trypsin for 30 min each [ 10 ]; Protocol 2: digestion two times in 0.25 % trypsin for 10 min each [ 11 ]; Protocol 3: digestion in 0.125 % trypsin and 0.2 mg/ml DNase I for 45 min [ 14 ]; Protocol 4: digestion three times in 0.125 % trypsin and 0.2 mg/ml DNase I for 30 min each [ 15 ]; Protocol 5: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 15 min each; Protocol 6: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 20 min each; Protocol 7: digestion two times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 30 min each. Data are presented as mean ± SD of six independent experiments
    Figure Legend Snippet: Comparison of yield and viability of purified cytotrophoblast cells among a variety of enzymatic degradation protocols. Yield ( a ) and viability ( b ) of purified cytotrophoblast cells using different enzymatic digestion protocols were assessed. Protocol 1: digestion three times in 0.25 % trypsin for 30 min each [ 10 ]; Protocol 2: digestion two times in 0.25 % trypsin for 10 min each [ 11 ]; Protocol 3: digestion in 0.125 % trypsin and 0.2 mg/ml DNase I for 45 min [ 14 ]; Protocol 4: digestion three times in 0.125 % trypsin and 0.2 mg/ml DNase I for 30 min each [ 15 ]; Protocol 5: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 15 min each; Protocol 6: digestion three times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 20 min each; Protocol 7: digestion two times in 1 mg/ml Dispase II, 0.5 mg/ml collagenase I and 0.1 mg/ml DNase I for 30 min each. Data are presented as mean ± SD of six independent experiments

    Techniques Used: Purification

    20) Product Images from "Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation"

    Article Title: Defective NET clearance contributes to sustained FXII activation in COVID-19-associated pulmonary thrombo-inflammation

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2021.103382

    Delayed NET clearance due to defective DNase activity in COVID-19. (a) DNase activity measured in plasma samples from COVID-19 patients (n=43) or healthy donors (n=39) using the DNase Alert QC System. Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test. (b) DNase activity measured in plasma samples from COVID-19 patients (n=43) or healthy donors (n=39) using the SRED assay. Representative pictures are shown for COVID-19 (orange box) and healthy donors (grey box). Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test. (c) NET degradation capacity of COVID-19 plasma. DNA fluorescence was quantified from COVID-19 plasma (n=36) and healthy donor plasma (n=24). Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test. Representative immunofluorescence images of in vitro generated NETs from healthy neutrophils are shown upon incubation with healthy donor or COVID-19 plasma. (d) Activation of purified FXII by NETs was measured by conversion of the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroaniline (S-2302) at an absorption of λ=405 nm. S-2302 was added either in the presence or absence of the FXIIa inhibitor rHA-infestin-4 (INH) or DNase I (Pulmozyme). Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test (n=6 replicates per time point from two independent experiments). Arbitrary units (AU), Scale bar: 25 µm (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
    Figure Legend Snippet: Delayed NET clearance due to defective DNase activity in COVID-19. (a) DNase activity measured in plasma samples from COVID-19 patients (n=43) or healthy donors (n=39) using the DNase Alert QC System. Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test. (b) DNase activity measured in plasma samples from COVID-19 patients (n=43) or healthy donors (n=39) using the SRED assay. Representative pictures are shown for COVID-19 (orange box) and healthy donors (grey box). Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test. (c) NET degradation capacity of COVID-19 plasma. DNA fluorescence was quantified from COVID-19 plasma (n=36) and healthy donor plasma (n=24). Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test. Representative immunofluorescence images of in vitro generated NETs from healthy neutrophils are shown upon incubation with healthy donor or COVID-19 plasma. (d) Activation of purified FXII by NETs was measured by conversion of the chromogenic substrate H-D-Pro-Phe-Arg-p-nitroaniline (S-2302) at an absorption of λ=405 nm. S-2302 was added either in the presence or absence of the FXIIa inhibitor rHA-infestin-4 (INH) or DNase I (Pulmozyme). Data represent mean ± s.e.m., p-value, two-tailed unpaired Student's t-test (n=6 replicates per time point from two independent experiments). Arbitrary units (AU), Scale bar: 25 µm (c). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

    Techniques Used: Activity Assay, Two Tailed Test, Fluorescence, Immunofluorescence, In Vitro, Generated, Incubation, Activation Assay, Purification

    21) Product Images from "Transcriptional regulation of BRCA1 expression by a metabolic switch"

    Article Title: Transcriptional regulation of BRCA1 expression by a metabolic switch

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.1941

    TSA mimics estrogen induced activation of BRCA1 by increasing p300 dependent histone acetylation at the BRCA1 promoter. ( a ) Time course of TFF1 , NBR2 , and BRCA1 expression in MCF-7 cells treated 0–24 h with either E2, E2 + cycloheximide (10 µg ml −1 ), TSA (500 ng ml −1 ), or TSA + cycloheximide as indicated. Error bars represent the s.e.m. for N=2 independent biological replicates. ( b ) DNase I hypersensitivity profile of the BRCA1 promoter and an ( HBB ) locus control from MCF-7 cells treated with either estrogen or TSA. The error bars represent the s.e.m. for N=3 biological replicates. ( c ) Acetylated histone H3, acetylated histone H4, HDAC1, BRCA1, p130, and CtBP ChIP profiles at the BRCA1 promoter in control or MCF-7 cells treated 1 h with 500 ng ml −1 TSA. Error bars represent the s.e.m. for N=2 biological replicates. ( d ) Upper panel: TSA stimulated expression of BRCA1 nascent and mature RNA levels in either control or p300 depleted MCF-7. Error bars represent the s.e.m. for N=2 biological replicates. Lower panel: ChIP enrichment for H3 and H4 histone acetylation at the BRCA1 locus in control versus p300 depleted MCF-7 cells with or without TSA stimulation. Means from N=2 independent biological replicates are shown.
    Figure Legend Snippet: TSA mimics estrogen induced activation of BRCA1 by increasing p300 dependent histone acetylation at the BRCA1 promoter. ( a ) Time course of TFF1 , NBR2 , and BRCA1 expression in MCF-7 cells treated 0–24 h with either E2, E2 + cycloheximide (10 µg ml −1 ), TSA (500 ng ml −1 ), or TSA + cycloheximide as indicated. Error bars represent the s.e.m. for N=2 independent biological replicates. ( b ) DNase I hypersensitivity profile of the BRCA1 promoter and an ( HBB ) locus control from MCF-7 cells treated with either estrogen or TSA. The error bars represent the s.e.m. for N=3 biological replicates. ( c ) Acetylated histone H3, acetylated histone H4, HDAC1, BRCA1, p130, and CtBP ChIP profiles at the BRCA1 promoter in control or MCF-7 cells treated 1 h with 500 ng ml −1 TSA. Error bars represent the s.e.m. for N=2 biological replicates. ( d ) Upper panel: TSA stimulated expression of BRCA1 nascent and mature RNA levels in either control or p300 depleted MCF-7. Error bars represent the s.e.m. for N=2 biological replicates. Lower panel: ChIP enrichment for H3 and H4 histone acetylation at the BRCA1 locus in control versus p300 depleted MCF-7 cells with or without TSA stimulation. Means from N=2 independent biological replicates are shown.

    Techniques Used: Activation Assay, Expressing, Chromatin Immunoprecipitation

    22) Product Images from "Expanded Role for the Nitrogen Assimilation Control Protein in the Response of Klebsiella pneumoniae to Nitrogen Stress ▿"

    Article Title: Expanded Role for the Nitrogen Assimilation Control Protein in the Response of Klebsiella pneumoniae to Nitrogen Stress ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00931-09

    Template strand footprints of NAC bound to  oppA  or  dppA  promoter fragments. (A) End-labeled DNA fragments were mixed with buffer only or increasing concentrations of NAC prior to treatment with DNase I. A chemical A+G ladder of each fragment was
    Figure Legend Snippet: Template strand footprints of NAC bound to oppA or dppA promoter fragments. (A) End-labeled DNA fragments were mixed with buffer only or increasing concentrations of NAC prior to treatment with DNase I. A chemical A+G ladder of each fragment was

    Techniques Used: Labeling

    23) Product Images from "Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass"

    Article Title: Targeting of cell-free DNA by DNase I diminishes endothelial dysfunction and inflammation in a rat model of cardiopulmonary bypass

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-55863-8

    Effects of DNase I on endothelial activation. ( a ) Aortas were collected from rats that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) at the end of surgery. Relative expression levels of intercellular adhesion molecule-1 ( ICAM-1) , vascular cell adhesion molecule-1 ( VCAM-1) , inducible NO synthase ( iNOS) , IL-6 , TNF-α , and IL-10 in aortic tissue were analyzed by quantitative real-time PCR. 18 S rRNA was used to normalize the data. Two-time DNase I administration (before CPB and before reperfusion) significantly reduced the aortic expression of ICAM-1 and VCAM-1 . * P
    Figure Legend Snippet: Effects of DNase I on endothelial activation. ( a ) Aortas were collected from rats that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) at the end of surgery. Relative expression levels of intercellular adhesion molecule-1 ( ICAM-1) , vascular cell adhesion molecule-1 ( VCAM-1) , inducible NO synthase ( iNOS) , IL-6 , TNF-α , and IL-10 in aortic tissue were analyzed by quantitative real-time PCR. 18 S rRNA was used to normalize the data. Two-time DNase I administration (before CPB and before reperfusion) significantly reduced the aortic expression of ICAM-1 and VCAM-1 . * P

    Techniques Used: Activation Assay, Expressing, Real-time Polymerase Chain Reaction

    Effects of DNase I on vascular function. Rat aortas were removed at the end of surgical procedure and immediately used for ex vivo functional analyses. ( a ) Aortic rings (4 mm width) were pre-constricted with 0.1 µM Phenylephrin (PE) and endothelial-dependent vasorelaxation was achieved by the addition of different concentrations of Acethylcholin (ACh, range 0 nM-10 µM). Pre-constriction was defined as 100%. Improved vasorelaxation was found in aortic vessels of rats treated with two doses of DNase I . * P
    Figure Legend Snippet: Effects of DNase I on vascular function. Rat aortas were removed at the end of surgical procedure and immediately used for ex vivo functional analyses. ( a ) Aortic rings (4 mm width) were pre-constricted with 0.1 µM Phenylephrin (PE) and endothelial-dependent vasorelaxation was achieved by the addition of different concentrations of Acethylcholin (ACh, range 0 nM-10 µM). Pre-constriction was defined as 100%. Improved vasorelaxation was found in aortic vessels of rats treated with two doses of DNase I . * P

    Techniques Used: Ex Vivo, Functional Assay

    Assessment of DNase I effects on plasma cytokine levels. Rats were subjected to cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) as described in the Methods section. Rats received DNase I treatment before CPB (1 × DNase , n = 7) or a second DNase I dose before reperfusion (2 × DNase , n = 8). Animals without DNase I treatment served as control (n = 7). Plasma levels of IL-6, TNF-α, IFN-ɣ and IL-10 were quantified using Procartaplex Multiplex Assays before CPB (T1), before (T2) and after (T3) reperfusion. Rats that received two doses of DNase I showed significant reduction of IL-6 at the end of reperfusion (T3).* P
    Figure Legend Snippet: Assessment of DNase I effects on plasma cytokine levels. Rats were subjected to cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) as described in the Methods section. Rats received DNase I treatment before CPB (1 × DNase , n = 7) or a second DNase I dose before reperfusion (2 × DNase , n = 8). Animals without DNase I treatment served as control (n = 7). Plasma levels of IL-6, TNF-α, IFN-ɣ and IL-10 were quantified using Procartaplex Multiplex Assays before CPB (T1), before (T2) and after (T3) reperfusion. Rats that received two doses of DNase I showed significant reduction of IL-6 at the end of reperfusion (T3).* P

    Techniques Used: Multiplex Assay

    Impact of DNase I on leukocyte extravasation. Rats with or without DNase I treatment that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) were sacrificed and lung tissue was collected at the end of surgery. ( a ) Immunofluorescence detection of CD45 in lung tissue of control rats (Ctrl) and those treated with DNase I (1 × DNase I , 2 × DNase I ). Three animals per group were examined. Representative images are depicted. Scale bar: 50 µm. ( b ) Quantification of CD45 fluorescence expressed as mean fluorescence intensity. Ten random fields were examined from each specimen at 400 × magnification. DNase I treatment significantly reduced the number of CD45-positive cells.* P
    Figure Legend Snippet: Impact of DNase I on leukocyte extravasation. Rats with or without DNase I treatment that underwent cardiopulmonary bypass (CPB) with deep hypothermic cardiac arrest (DHCA) were sacrificed and lung tissue was collected at the end of surgery. ( a ) Immunofluorescence detection of CD45 in lung tissue of control rats (Ctrl) and those treated with DNase I (1 × DNase I , 2 × DNase I ). Three animals per group were examined. Representative images are depicted. Scale bar: 50 µm. ( b ) Quantification of CD45 fluorescence expressed as mean fluorescence intensity. Ten random fields were examined from each specimen at 400 × magnification. DNase I treatment significantly reduced the number of CD45-positive cells.* P

    Techniques Used: Immunofluorescence, Fluorescence

    Evaluation of plasma cell-free DNA (cfDNA) levels and DNase activity. Rats underwent cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA) as described in the Methods section. Plasma samples were collected from control rats without DNase I therapy (n = 7), rats receiving DNase I before CPB (1 × DNase , n = 7) and those receiving a second DNase I dose before reperfusion (2 × DNase , n = 8) at following times: before CPB (T1), before reperfusion (T2) and at the end of reperfusion (T3). ( a ) Plasma cfDNA levels were quantified by PicoGreen staining and were found to be significantly decreased upon DNase I administration. ( b ) Additionally, relative plasma DNase activity was determined and significantly increased in rats that received DNase I . ** P
    Figure Legend Snippet: Evaluation of plasma cell-free DNA (cfDNA) levels and DNase activity. Rats underwent cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA) as described in the Methods section. Plasma samples were collected from control rats without DNase I therapy (n = 7), rats receiving DNase I before CPB (1 × DNase , n = 7) and those receiving a second DNase I dose before reperfusion (2 × DNase , n = 8) at following times: before CPB (T1), before reperfusion (T2) and at the end of reperfusion (T3). ( a ) Plasma cfDNA levels were quantified by PicoGreen staining and were found to be significantly decreased upon DNase I administration. ( b ) Additionally, relative plasma DNase activity was determined and significantly increased in rats that received DNase I . ** P

    Techniques Used: Activity Assay, Staining

    Visual summary of DNase I -mediated vasoprotection during CPB. Systemic application of DNase I during CPB efficiently degraded cfDNA/NETs released by activated neutrophils and prevented reperfusion-induced cellular damage. DNase I improved endothelial function, reduced endothelial activation and leukocyte extravasation.
    Figure Legend Snippet: Visual summary of DNase I -mediated vasoprotection during CPB. Systemic application of DNase I during CPB efficiently degraded cfDNA/NETs released by activated neutrophils and prevented reperfusion-induced cellular damage. DNase I improved endothelial function, reduced endothelial activation and leukocyte extravasation.

    Techniques Used: Activation Assay

    24) Product Images from "Effects of pH on the Properties of Membrane Vesicles Including Glucosyltransferase in Streptococcus mutans"

    Article Title: Effects of pH on the Properties of Membrane Vesicles Including Glucosyltransferase in Streptococcus mutans

    Journal: Microorganisms

    doi: 10.3390/microorganisms9112308

    Role of eDNA for effects of initial pH condition on the MVs-dependent biofilm formation. Biofilm formation of S. mutans UA159 gtfBC - was quantitatively assessed in TSB with 0.25% raffinose with 0.125 mg/ ml of MVs from S. mutans UA159 in HC, LA, AA and NO, with or without DNase I and iDNase I. The data indicate the mean ± SD of three independent experiments. The asterisks indicate a significant difference between the groups (*: p
    Figure Legend Snippet: Role of eDNA for effects of initial pH condition on the MVs-dependent biofilm formation. Biofilm formation of S. mutans UA159 gtfBC - was quantitatively assessed in TSB with 0.25% raffinose with 0.125 mg/ ml of MVs from S. mutans UA159 in HC, LA, AA and NO, with or without DNase I and iDNase I. The data indicate the mean ± SD of three independent experiments. The asterisks indicate a significant difference between the groups (*: p

    Techniques Used:

    25) Product Images from "Staphylococcus aureus utilizes environmental RNA as a building material in specific polysaccharide-dependent biofilms"

    Article Title: Staphylococcus aureus utilizes environmental RNA as a building material in specific polysaccharide-dependent biofilms

    Journal: NPJ Biofilms and Microbiomes

    doi: 10.1038/s41522-022-00278-z

    RNA from the surrounding milieu affects biofilm formation. a Nucleic acids were isolated from BHI medium via ethanol precipitation, treated with DNase I and RNase A or left untreated (Control), and detected on an agarose gel. A white arrowhead denotes RNA. b The presence of RNA identified by RNA-seq was confirmed by reverse transcriptase-polymerase chain reaction using RNA purified from the BHI medium as a template and primers specific for the fragments identified through RNA-seq (Supplementary Table 1 ). + added, − not added. c The effects of the RNA purified from BHI medium (BHI–RNA) on the formation of MR10 biofilms in Roswell Park Memorial Institute medium supplemented with 1% glucose. The data are presented as the mean and standard deviation (error bar) of relative biofilm biomass from triplicate experiments. The biofilm biomass in the absence of BHI–RNA was defined as 100%. ** P
    Figure Legend Snippet: RNA from the surrounding milieu affects biofilm formation. a Nucleic acids were isolated from BHI medium via ethanol precipitation, treated with DNase I and RNase A or left untreated (Control), and detected on an agarose gel. A white arrowhead denotes RNA. b The presence of RNA identified by RNA-seq was confirmed by reverse transcriptase-polymerase chain reaction using RNA purified from the BHI medium as a template and primers specific for the fragments identified through RNA-seq (Supplementary Table 1 ). + added, − not added. c The effects of the RNA purified from BHI medium (BHI–RNA) on the formation of MR10 biofilms in Roswell Park Memorial Institute medium supplemented with 1% glucose. The data are presented as the mean and standard deviation (error bar) of relative biofilm biomass from triplicate experiments. The biofilm biomass in the absence of BHI–RNA was defined as 100%. ** P

    Techniques Used: Isolation, Ethanol Precipitation, Agarose Gel Electrophoresis, RNA Sequencing Assay, Polymerase Chain Reaction, Purification, Standard Deviation

    26) Product Images from "Genome-wide mapping of transcriptional enhancer candidates using DNA and chromatin features in maize"

    Article Title: Genome-wide mapping of transcriptional enhancer candidates using DNA and chromatin features in maize

    Journal: Genome Biology

    doi: 10.1186/s13059-017-1273-4

    Heatmaps of chromatin, DNA and transcript features at enhancer candidates. DNase I hypersensitivity, H3K9ac enrichment, mCG, mCHG and mCHH levels, presence of TEs and transcript levels at and around (±1 kb) DHSs in enhancer candidates. DHSs were scaled to equal size. The colour scales are in RPM for DNase I hypersensitivity, H3K9ac enrichment and transcript levels, and in methylation frequency (0–1) for DNA methylation. For TE sequences, red and white show the presence or absence of TEs, respectively. DHSs were clustered based on H3K9ac enrichment using a k-means (k = 4) clustering algorithm. The categories identified were numbered from 1 to 4 from the top to the bottom. All the DHSs were oriented based on H3K9ac enrichment intensity values 300 bp away from the DHS boundaries; the side with higher H3K9ac enrichment was defined as 3' end
    Figure Legend Snippet: Heatmaps of chromatin, DNA and transcript features at enhancer candidates. DNase I hypersensitivity, H3K9ac enrichment, mCG, mCHG and mCHH levels, presence of TEs and transcript levels at and around (±1 kb) DHSs in enhancer candidates. DHSs were scaled to equal size. The colour scales are in RPM for DNase I hypersensitivity, H3K9ac enrichment and transcript levels, and in methylation frequency (0–1) for DNA methylation. For TE sequences, red and white show the presence or absence of TEs, respectively. DHSs were clustered based on H3K9ac enrichment using a k-means (k = 4) clustering algorithm. The categories identified were numbered from 1 to 4 from the top to the bottom. All the DHSs were oriented based on H3K9ac enrichment intensity values 300 bp away from the DHS boundaries; the side with higher H3K9ac enrichment was defined as 3' end

    Techniques Used: Methylation, DNA Methylation Assay

    Examples of candidate rankings. From the top : identified candidate region with its ID ( V V2-IST, H husk candidate) and coordinates, DNase I hypersensitivity and H3K9ac enrichment signal intensities in V2-IST and husk tissues. In these examples, the DNase I hypersensitivity and H3K9ac enrichment signal differences do not positively correlate to each other as assumed
    Figure Legend Snippet: Examples of candidate rankings. From the top : identified candidate region with its ID ( V V2-IST, H husk candidate) and coordinates, DNase I hypersensitivity and H3K9ac enrichment signal intensities in V2-IST and husk tissues. In these examples, the DNase I hypersensitivity and H3K9ac enrichment signal differences do not positively correlate to each other as assumed

    Techniques Used:

    Average profiles of the enhancer candidates in ( a ) V2-IST and ( b ) husk. Average signal intensities of DNase I hypersensitivity, H3K9ac enrichment in RPM and DNA methylation levels in methylation frequency at DHSs and their 1-kb flanking regions. DHSs were scaled to equal size. Prior to calculation of the average, all the DHSs were oriented based on H3K9ac enrichment intensity values 300 bp away from the DHS boundaries; the sides with higher H3K9ac enrichment were defined as 3' end. The profiles show a clear preferential enrichment of H3K9ac 3’ of the DHSs and high levels of DNA methylation (CG and CHG context) around the DHSs and H3K9ac-enriched regions. The level of mCHH is low throughout the regions with a slight increase at the 5’ side of DHSs
    Figure Legend Snippet: Average profiles of the enhancer candidates in ( a ) V2-IST and ( b ) husk. Average signal intensities of DNase I hypersensitivity, H3K9ac enrichment in RPM and DNA methylation levels in methylation frequency at DHSs and their 1-kb flanking regions. DHSs were scaled to equal size. Prior to calculation of the average, all the DHSs were oriented based on H3K9ac enrichment intensity values 300 bp away from the DHS boundaries; the sides with higher H3K9ac enrichment were defined as 3' end. The profiles show a clear preferential enrichment of H3K9ac 3’ of the DHSs and high levels of DNA methylation (CG and CHG context) around the DHSs and H3K9ac-enriched regions. The level of mCHH is low throughout the regions with a slight increase at the 5’ side of DHSs

    Techniques Used: DNA Methylation Assay, Methylation

    Example of data on ( a ) DICE and ( b ) b1 repeat enhancer. From the top : AGPv4 annotation and candidate annotation from our prediction ( V V2-IST, H husk candidate), DNase I hypersensitivity and H3K9ac enrichment signal (all replicates pooled) and peak position (indicated as blue and green bars , respectively) in V2-IST and in husk tissue, mCG, mCHG and mCHH levels and unique mappability in percentage. The numbers under gene names indicate relative gene expression levels (V2-IST/husk). Although the b1 locus is on chromosome 2, in the current version of the AGPv4 assembly, the b1 gene is located in contig 44 (B, on the right of the grey vertical line ). The dark blue bars in the gene annotation tracks indicate previously annotated known enhancers and putative cis- regulatory elements. The vertical red boxes indicate enhancer candidates identified in this study. Peaks at those tracks might not be present in each replicate, affecting enhancer candidate prediction
    Figure Legend Snippet: Example of data on ( a ) DICE and ( b ) b1 repeat enhancer. From the top : AGPv4 annotation and candidate annotation from our prediction ( V V2-IST, H husk candidate), DNase I hypersensitivity and H3K9ac enrichment signal (all replicates pooled) and peak position (indicated as blue and green bars , respectively) in V2-IST and in husk tissue, mCG, mCHG and mCHH levels and unique mappability in percentage. The numbers under gene names indicate relative gene expression levels (V2-IST/husk). Although the b1 locus is on chromosome 2, in the current version of the AGPv4 assembly, the b1 gene is located in contig 44 (B, on the right of the grey vertical line ). The dark blue bars in the gene annotation tracks indicate previously annotated known enhancers and putative cis- regulatory elements. The vertical red boxes indicate enhancer candidates identified in this study. Peaks at those tracks might not be present in each replicate, affecting enhancer candidate prediction

    Techniques Used: Expressing

    Average DNase I hypersensitivity and H3K9ac enrichment at genic regions. Average signal (in RPM) for DNase I hypersensitivity in ( a ) V2-IST and ( b ) husk, and for H3K9ac enrichment in ( c ) V2-IST and ( d ) husk at genes and their 1-kb flanking regions. Genes were binned based on their expression levels, from no expression ( light colour ) to high expression ( dark colour ): the lowest expression level bin contains all genes with an expression lower than 1 RPKM. The thresholds (in RPKM) are at 1.94, 4.17, 8.58, 16.64 and 36.28 for V2-IST and 1.88, 4.00, 8.34, 15.83 and 32.99 for husk tissue
    Figure Legend Snippet: Average DNase I hypersensitivity and H3K9ac enrichment at genic regions. Average signal (in RPM) for DNase I hypersensitivity in ( a ) V2-IST and ( b ) husk, and for H3K9ac enrichment in ( c ) V2-IST and ( d ) husk at genes and their 1-kb flanking regions. Genes were binned based on their expression levels, from no expression ( light colour ) to high expression ( dark colour ): the lowest expression level bin contains all genes with an expression lower than 1 RPKM. The thresholds (in RPKM) are at 1.94, 4.17, 8.58, 16.64 and 36.28 for V2-IST and 1.88, 4.00, 8.34, 15.83 and 32.99 for husk tissue

    Techniques Used: Expressing

    27) Product Images from "Neutrophil Extracellular Traps Augmented Alveolar Macrophage Pyroptosis via AIM2 Inflammasome Activation in LPS-Induced ALI/ARDS"

    Article Title: Neutrophil Extracellular Traps Augmented Alveolar Macrophage Pyroptosis via AIM2 Inflammasome Activation in LPS-Induced ALI/ARDS

    Journal: Journal of Inflammation Research

    doi: 10.2147/JIR.S321513

    NET-targeting agents and inhibition of alveolar macrophage pyroptosis provide similar protection against ARDS. ( A ) Administration of Ac-YVAD, DNase I or BB-Cl-amidine significantly reduced lung damage in LPS-challenged mice. Representative H E-stained sections showing a significant improvement in the lungs of Ac-YVAD-, DNase I- or BB-Cl-amidine-treated mice. ① Sham-treated mice, ② LPS-treated mice, ③ DNase I plus LPS-treated mice, ④ BB-Cl-amidine plus LPS-treated mice, and ⑤ Ac-YVAD plus LPS-treated mice (200X). Administration of Ac-YVAD, DNase I or BB-Cl-amidine improved ( B ) the PaO2 of LPS-challenged mice and reduced ( C ) the weight/dry ratio of lung tissue and ( D ) the protein level in the BALF of LPS-challenged mice. BALF and serum ( E and H ) IL-1β, ( F and I ) KC, and ( G and J ) TNF-ɑ levels were all notably decreased in the DNase I, BB-Cl-amidine or Ac-YVAD plus LPS groups compared with the LPS-challenged group (n=5 mice). (*p
    Figure Legend Snippet: NET-targeting agents and inhibition of alveolar macrophage pyroptosis provide similar protection against ARDS. ( A ) Administration of Ac-YVAD, DNase I or BB-Cl-amidine significantly reduced lung damage in LPS-challenged mice. Representative H E-stained sections showing a significant improvement in the lungs of Ac-YVAD-, DNase I- or BB-Cl-amidine-treated mice. ① Sham-treated mice, ② LPS-treated mice, ③ DNase I plus LPS-treated mice, ④ BB-Cl-amidine plus LPS-treated mice, and ⑤ Ac-YVAD plus LPS-treated mice (200X). Administration of Ac-YVAD, DNase I or BB-Cl-amidine improved ( B ) the PaO2 of LPS-challenged mice and reduced ( C ) the weight/dry ratio of lung tissue and ( D ) the protein level in the BALF of LPS-challenged mice. BALF and serum ( E and H ) IL-1β, ( F and I ) KC, and ( G and J ) TNF-ɑ levels were all notably decreased in the DNase I, BB-Cl-amidine or Ac-YVAD plus LPS groups compared with the LPS-challenged group (n=5 mice). (*p

    Techniques Used: Inhibition, Mouse Assay, Staining

    NET-targeting agents alleviates alveolar macrophage pyroptosis in ARDS mice. Pulmonary instillation of LPS caused alveolar macrophage pyroptosis in mice, and alveolar macrophage pyroptosis was alleviated in the Ac-YVAD, DNase I or BB-Cl-amidine plus LPS group, as evidenced by cytometry analysis of active caspase-1- and PI-positive alveolar macrophages( A and C )and analysis of active caspase-1- and TUNEL-positive alveolar macrophages( B and E ) in random fields by confocal microscopy. ( D ) The proportion of alveolar macrophages in BALF was increased in the Ac-YVAD plus LPS group compared to the LPS alone group or the DNase I and BB-Cl-amidine plus LPS group (n=4 mice). (*p
    Figure Legend Snippet: NET-targeting agents alleviates alveolar macrophage pyroptosis in ARDS mice. Pulmonary instillation of LPS caused alveolar macrophage pyroptosis in mice, and alveolar macrophage pyroptosis was alleviated in the Ac-YVAD, DNase I or BB-Cl-amidine plus LPS group, as evidenced by cytometry analysis of active caspase-1- and PI-positive alveolar macrophages( A and C )and analysis of active caspase-1- and TUNEL-positive alveolar macrophages( B and E ) in random fields by confocal microscopy. ( D ) The proportion of alveolar macrophages in BALF was increased in the Ac-YVAD plus LPS group compared to the LPS alone group or the DNase I and BB-Cl-amidine plus LPS group (n=4 mice). (*p

    Techniques Used: Mouse Assay, Cytometry, TUNEL Assay, Confocal Microscopy

    Alveolar macrophage pyroptosis inhibition reduces NET content in ARDS mice. Pulmonary instillation of LPS caused NET formation in mice, and NET levels were decreased in the Ac-YVAD, DNase I or BB-Cl-amidine plus LPS groups, as evidenced by immunofluorescence staining of lung sections for ( A ) Cit-H3 and NE, ( B and C ) Western blotting of lung tissues for cit-H3 and ( D ) measurement of the MPO-DNA complex level in BALF (n=5 mice). (*p
    Figure Legend Snippet: Alveolar macrophage pyroptosis inhibition reduces NET content in ARDS mice. Pulmonary instillation of LPS caused NET formation in mice, and NET levels were decreased in the Ac-YVAD, DNase I or BB-Cl-amidine plus LPS groups, as evidenced by immunofluorescence staining of lung sections for ( A ) Cit-H3 and NE, ( B and C ) Western blotting of lung tissues for cit-H3 and ( D ) measurement of the MPO-DNA complex level in BALF (n=5 mice). (*p

    Techniques Used: Inhibition, Mouse Assay, Immunofluorescence, Staining, Western Blot

    28) Product Images from "CopR, a global regulator of transcription to maintain copper homeostasis in Pyrococcus furiosus"

    Article Title: CopR, a global regulator of transcription to maintain copper homeostasis in Pyrococcus furiosus

    Journal: bioRxiv

    doi: 10.1101/2020.08.14.251413

    Putative model of allosteric CopR regulation in P. furiosus. A , Domain architecture of CopR (monomer shown schematically in red) highlighting the DNA-binding HTH domain and the metal-sensing TRASH domain. B , Binding of copper presumably triggers a conformational switch from a closed state to a complex, which is opened between dimer 1 and 4. In combination with the rigid connection between dimers 2 and 3 it is possible that this conformational switch could facilitate TBP/TFB binding (+ square) and/or TBP-induced bending to the corresponding promoter regions (+ turned square). C , CopR-regulated promoter regions include CopR-binding sites (orange/red) and the archaeal-specific promoter elements BRE (recruits TFB, purple) and the TATA box (bound by TBP, light-green). Transcription start sites (TSS) are indicated by vertical lines. Transcription is either repressed (3 lines) or stimulated (arrows) under copper-shock conditions (lower panel, light-blue). The octameric CopR assembly in open conformation allows bending of critical promoter regions and facilitates binding of TBP/TFB depending on the distance of the divergent TSS: While CopR prevents binding of general transcription factors to the copR promoter, TBP/TFB can bind to the second promoter ( copA ). Simultaneous binding of CopR and two sets of GTFs stimulates transcription in both directions for pf0722/pf0271 and pf0738/pf0738.1n. D , Major groove bendability of selected promoters revealed increased rigidity between dimers 2 and 3. Major groove bendability of promoter sequences was estimated based on trinucleotide scales derived from DNase-I cutting frequencies ( 80 , 81 ). More negative values are the result of less cutting by DNase-I and indicate that the DNA is not bend towards the major groove and is therefore less flexible. Trinucleotides were extracted from CopR-target promoters (n=9) and all available promoters defined in P. furiosus previously ( 41 ) from −150 bp to +50 from the TSS. Each line represents the smoothed conditional mean with confidence intervals (0.95) displayed as shaded areas. Boxes represent area between dimers 2 and 3 (grey), BRE (purple) and TATA box (light-green). E , Summary of the TSS-distance dependent stimulation of divergent CopR-regulated transcripts.
    Figure Legend Snippet: Putative model of allosteric CopR regulation in P. furiosus. A , Domain architecture of CopR (monomer shown schematically in red) highlighting the DNA-binding HTH domain and the metal-sensing TRASH domain. B , Binding of copper presumably triggers a conformational switch from a closed state to a complex, which is opened between dimer 1 and 4. In combination with the rigid connection between dimers 2 and 3 it is possible that this conformational switch could facilitate TBP/TFB binding (+ square) and/or TBP-induced bending to the corresponding promoter regions (+ turned square). C , CopR-regulated promoter regions include CopR-binding sites (orange/red) and the archaeal-specific promoter elements BRE (recruits TFB, purple) and the TATA box (bound by TBP, light-green). Transcription start sites (TSS) are indicated by vertical lines. Transcription is either repressed (3 lines) or stimulated (arrows) under copper-shock conditions (lower panel, light-blue). The octameric CopR assembly in open conformation allows bending of critical promoter regions and facilitates binding of TBP/TFB depending on the distance of the divergent TSS: While CopR prevents binding of general transcription factors to the copR promoter, TBP/TFB can bind to the second promoter ( copA ). Simultaneous binding of CopR and two sets of GTFs stimulates transcription in both directions for pf0722/pf0271 and pf0738/pf0738.1n. D , Major groove bendability of selected promoters revealed increased rigidity between dimers 2 and 3. Major groove bendability of promoter sequences was estimated based on trinucleotide scales derived from DNase-I cutting frequencies ( 80 , 81 ). More negative values are the result of less cutting by DNase-I and indicate that the DNA is not bend towards the major groove and is therefore less flexible. Trinucleotides were extracted from CopR-target promoters (n=9) and all available promoters defined in P. furiosus previously ( 41 ) from −150 bp to +50 from the TSS. Each line represents the smoothed conditional mean with confidence intervals (0.95) displayed as shaded areas. Boxes represent area between dimers 2 and 3 (grey), BRE (purple) and TATA box (light-green). E , Summary of the TSS-distance dependent stimulation of divergent CopR-regulated transcripts.

    Techniques Used: Binding Assay, Derivative Assay

    Mechanistic and structural characterisation of CopR. A , Influence of CopR on in vitro transcription. 2 nM of the gdh and B , the copR/copA templates were transcribed in the presence of increasing concentrations of CopR (0.3, 0.6, 1.2, 2.3 μM). C , DNase I footprint on the copR/copA template in the presence of CopR, TBP/TFB and all three components. TBP/TFB-protected regions (purple lines), CopR-protected regions (red asterisks) and hypersensitive sites (black triangles) are highlighted. D , Summary of the protected regions determined in the in vitro DNAse I footprinting assay. Promoter elements, transcript boundaries and the semi-palindromic CopR recognition motif are highlighted. E , Representative 2D class averages of CopRΔTRASH in the absence and presence of a copR/copA DNA template reveal an octameric assembly formed by a tetramer of dimers in both states. F , Putative model of CopR bound to DNA. The octameric cartoon structure (PDB: 1I1G) represents LrpA from P. furiosus, that forms a similar structure ( 61 ).
    Figure Legend Snippet: Mechanistic and structural characterisation of CopR. A , Influence of CopR on in vitro transcription. 2 nM of the gdh and B , the copR/copA templates were transcribed in the presence of increasing concentrations of CopR (0.3, 0.6, 1.2, 2.3 μM). C , DNase I footprint on the copR/copA template in the presence of CopR, TBP/TFB and all three components. TBP/TFB-protected regions (purple lines), CopR-protected regions (red asterisks) and hypersensitive sites (black triangles) are highlighted. D , Summary of the protected regions determined in the in vitro DNAse I footprinting assay. Promoter elements, transcript boundaries and the semi-palindromic CopR recognition motif are highlighted. E , Representative 2D class averages of CopRΔTRASH in the absence and presence of a copR/copA DNA template reveal an octameric assembly formed by a tetramer of dimers in both states. F , Putative model of CopR bound to DNA. The octameric cartoon structure (PDB: 1I1G) represents LrpA from P. furiosus, that forms a similar structure ( 61 ).

    Techniques Used: In Vitro, Footprinting

    29) Product Images from "Neutrophil extracellular traps participate in the development of cancer-associated thrombosis in patients with gastric cancer"

    Article Title: Neutrophil extracellular traps participate in the development of cancer-associated thrombosis in patients with gastric cancer

    Journal: World Journal of Gastroenterology

    doi: 10.3748/wjg.v28.i26.3132

    NETs promote platelet adhesion and prothrombotic state.  A: Isolated platelets were incubated on glass slides which were coated with 1% dBSA, NETs (μg DNA/mL), or NETs pretreated with DNase I, APC, and sivelestat alone or together, followed by the F-actin components of platelets with 594-phalloidin staining. Magnification 63×; scale bars: 10 μm. Red-platelets; B: The percentage of area coverage of platelet adhesion was defined as the rate of red area in the total area and analyzed with ImageJ software; C: Isolated platelets were cocultured with different concentrations of NETs for 30 min with or without DNase I, APC and sivelestat treatment alone or together, and plasma fibrin formation was tested using turbidity measurements and monitored OD at 405 nm; D: TAT complex level of activated platelets was analyzed by ELISA. All values are the mean ± SD.  a P
    Figure Legend Snippet: NETs promote platelet adhesion and prothrombotic state. A: Isolated platelets were incubated on glass slides which were coated with 1% dBSA, NETs (μg DNA/mL), or NETs pretreated with DNase I, APC, and sivelestat alone or together, followed by the F-actin components of platelets with 594-phalloidin staining. Magnification 63×; scale bars: 10 μm. Red-platelets; B: The percentage of area coverage of platelet adhesion was defined as the rate of red area in the total area and analyzed with ImageJ software; C: Isolated platelets were cocultured with different concentrations of NETs for 30 min with or without DNase I, APC and sivelestat treatment alone or together, and plasma fibrin formation was tested using turbidity measurements and monitored OD at 405 nm; D: TAT complex level of activated platelets was analyzed by ELISA. All values are the mean ± SD. a P

    Techniques Used: Isolation, Incubation, Staining, Software, Enzyme-linked Immunosorbent Assay

    Tumor-bearing mice show a greater ability to form thrombi by inferior vena cava flow restriction. A and B: The values for weight and length of thrombi present in control, tumor-bearing mice, DNase I infused tumor-bearing mice at 6 h after surgery. Each group, n = 9; C and D: Values for weight and length of thrombi present in mice at 48 h after surgery. Each group, n = 5; E and F: Confocal imaging of thrombi derived from control mice and tumor-bearing mice with Ly6G and citH3 staining. Magnification 10×; scale bars: 200 μm. Red-Ly6G, Green-citH3, and Blue-DAPI; G and H: Magnified (40×) part of thrombi derived from control mice and tumor-bearing mice. Scale bars: 50 μm. Red-Ly6G, Green-citH3, and Blue-DAPI; I and J: Fibrin formation levels in the plasma of control, tumor-bearing mice, or DNase-I-infused tumor-bearing mice were detected by turbidity measurement at 405 nm, and TAT complex levels were detected by ELISA. Each group, n = 8. All values are the mean ± SD. a P
    Figure Legend Snippet: Tumor-bearing mice show a greater ability to form thrombi by inferior vena cava flow restriction. A and B: The values for weight and length of thrombi present in control, tumor-bearing mice, DNase I infused tumor-bearing mice at 6 h after surgery. Each group, n = 9; C and D: Values for weight and length of thrombi present in mice at 48 h after surgery. Each group, n = 5; E and F: Confocal imaging of thrombi derived from control mice and tumor-bearing mice with Ly6G and citH3 staining. Magnification 10×; scale bars: 200 μm. Red-Ly6G, Green-citH3, and Blue-DAPI; G and H: Magnified (40×) part of thrombi derived from control mice and tumor-bearing mice. Scale bars: 50 μm. Red-Ly6G, Green-citH3, and Blue-DAPI; I and J: Fibrin formation levels in the plasma of control, tumor-bearing mice, or DNase-I-infused tumor-bearing mice were detected by turbidity measurement at 405 nm, and TAT complex levels were detected by ELISA. Each group, n = 8. All values are the mean ± SD. a P

    Techniques Used: Mouse Assay, Imaging, Derivative Assay, Staining, Enzyme-linked Immunosorbent Assay

    NETs contribute to hypercoagulation of platelets.  A: PS exposure and P-selectin expression were measured when isolated platelets were cocultured with BETs (μg, DNA/mL) or in the presence of DNase I, activated protein C, and sivelestat alone or together by confocal microscopy. Magnification 63×; scale bars: 10 μm. Red-platelets, Green-Lactadherin, and Blue-P-selectin; B and C: PS exposure and P-selectin expression are indicated as MFI. MFI was defined as the ratio of total fluorescence intensity to the area; D and E: The rates of PS-positive platelets and P-selectin-positive platelets were detected by flow cytometry. All values are the mean ± SD.  c P
    Figure Legend Snippet: NETs contribute to hypercoagulation of platelets. A: PS exposure and P-selectin expression were measured when isolated platelets were cocultured with BETs (μg, DNA/mL) or in the presence of DNase I, activated protein C, and sivelestat alone or together by confocal microscopy. Magnification 63×; scale bars: 10 μm. Red-platelets, Green-Lactadherin, and Blue-P-selectin; B and C: PS exposure and P-selectin expression are indicated as MFI. MFI was defined as the ratio of total fluorescence intensity to the area; D and E: The rates of PS-positive platelets and P-selectin-positive platelets were detected by flow cytometry. All values are the mean ± SD. c P

    Techniques Used: Expressing, Isolation, Confocal Microscopy, Fluorescence, Flow Cytometry

    NETs drive hypercoagulation of ECs.  A: ECs were cocultured with NETs (μg DNA/mL) or PBS in the presence of DNase I, APC, or sivelestat alone or together for 4 h and analyzed by confocal microscopy. The intercellular junctions of ECs were stained with VE–cadherin and phalloidin. Magnification 63×; scale bars: 10 μm. Red-phalloidin, Green-VE, and Blue-DAPI; B: EC activation was stained with CD31 and TF. Magnification 63×; scale bars: 10 μm. Red-TF, Green-CD31, and Blue-DAPI; C and D: VE–cadherin expression and TF expression on ECs were detected by confocal microscopy and analyzed with ImageJ software (expression indicated as MFI). MFI was defined as the ratio of total fluorescence intensity to the area; E and F: EC monolayers were stimulated with various concentrations of NETs for 4 h, followed by determination of fibrin formation by turbidity measurement at 405 nm, and the TAT complex level was detected by ELISA. All values are the mean ± SD.  a P
    Figure Legend Snippet: NETs drive hypercoagulation of ECs. A: ECs were cocultured with NETs (μg DNA/mL) or PBS in the presence of DNase I, APC, or sivelestat alone or together for 4 h and analyzed by confocal microscopy. The intercellular junctions of ECs were stained with VE–cadherin and phalloidin. Magnification 63×; scale bars: 10 μm. Red-phalloidin, Green-VE, and Blue-DAPI; B: EC activation was stained with CD31 and TF. Magnification 63×; scale bars: 10 μm. Red-TF, Green-CD31, and Blue-DAPI; C and D: VE–cadherin expression and TF expression on ECs were detected by confocal microscopy and analyzed with ImageJ software (expression indicated as MFI). MFI was defined as the ratio of total fluorescence intensity to the area; E and F: EC monolayers were stimulated with various concentrations of NETs for 4 h, followed by determination of fibrin formation by turbidity measurement at 405 nm, and the TAT complex level was detected by ELISA. All values are the mean ± SD. a P

    Techniques Used: Confocal Microscopy, Staining, Activation Assay, Expressing, Software, Fluorescence, Enzyme-linked Immunosorbent Assay

    30) Product Images from "An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus"

    Article Title: An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00726-15

    Immunofluorescence microscopy of GAPDH in fixed biofilms. Untreated and proteinase K- or DNase I-treated biofilms were fixed and probed with a primary anti-GAPDH antibody and a secondary anti-rabbit antibody conjugated to Alexa Fluor 488 (green), and cell nuclei were strained with DAPI (blue). Size bars, 10 μm.
    Figure Legend Snippet: Immunofluorescence microscopy of GAPDH in fixed biofilms. Untreated and proteinase K- or DNase I-treated biofilms were fixed and probed with a primary anti-GAPDH antibody and a secondary anti-rabbit antibody conjugated to Alexa Fluor 488 (green), and cell nuclei were strained with DAPI (blue). Size bars, 10 μm.

    Techniques Used: Immunofluorescence, Microscopy

    Quantification of eDNA by qPCR. (A) Quantification of eDNA in untreated and proteinase K- or DNase I-treated biofilms by qPCR. eDNA was quantified in biofilm medium supernatant (pH approximately 4.5 to 5) following resuspension after remaining cells were removed by filtration. (B) Quantification of eDNA release upon suspension of biofilms in PBS at pH 5 or 7.5. (C) Quantification of eDNA release of untreated and DNase I- or proteinase K-treated biofilms upon suspension in PBS at pH 7.5 with or without washing. The amount of eDNA measured was normalized to that in unwashed, untreated cells. For panels B and C, eDNA was quantified after biofilms were resuspended in PBS and cells were removed by centrifugation and filtration. For all experiments, primers for the gene for gyrase A ( gyrA ) were used and dilutions of genomic  S. aureus  DNA were included to calculate absolute DNA concentrations. Average values and standard deviations of at least three independent experiments with three biological and three technical replicates are shown. Significant differences were calculated with Student's  t  test. Not significant (ns),  P >  0.05; *,  P  ≤ 0.05; ***,  P  ≤ 0.001.
    Figure Legend Snippet: Quantification of eDNA by qPCR. (A) Quantification of eDNA in untreated and proteinase K- or DNase I-treated biofilms by qPCR. eDNA was quantified in biofilm medium supernatant (pH approximately 4.5 to 5) following resuspension after remaining cells were removed by filtration. (B) Quantification of eDNA release upon suspension of biofilms in PBS at pH 5 or 7.5. (C) Quantification of eDNA release of untreated and DNase I- or proteinase K-treated biofilms upon suspension in PBS at pH 7.5 with or without washing. The amount of eDNA measured was normalized to that in unwashed, untreated cells. For panels B and C, eDNA was quantified after biofilms were resuspended in PBS and cells were removed by centrifugation and filtration. For all experiments, primers for the gene for gyrase A ( gyrA ) were used and dilutions of genomic S. aureus DNA were included to calculate absolute DNA concentrations. Average values and standard deviations of at least three independent experiments with three biological and three technical replicates are shown. Significant differences were calculated with Student's t test. Not significant (ns), P > 0.05; *, P ≤ 0.05; ***, P ≤ 0.001.

    Techniques Used: Real-time Polymerase Chain Reaction, Filtration, Centrifugation

    Quantitative assays of biofilms undergoing different treatments at different time points. Proteinase K, DNase I, or water was added to biofilms at inoculation (bars 0), after 7 h of incubation (bars 7), or after 23 h of incubation (bars 23). The OD 600 s of the washed resuspended biofilm, the two combined PBS washes, and the growth medium were measured after 24 h of incubation.
    Figure Legend Snippet: Quantitative assays of biofilms undergoing different treatments at different time points. Proteinase K, DNase I, or water was added to biofilms at inoculation (bars 0), after 7 h of incubation (bars 7), or after 23 h of incubation (bars 23). The OD 600 s of the washed resuspended biofilm, the two combined PBS washes, and the growth medium were measured after 24 h of incubation.

    Techniques Used: Incubation

    SDS-PAGE of matrix proteins released from biofilm cells and corresponding cell lysates. (A) Untreated and DNase I-treated biofilms were resuspended and incubated in PBS at pH 5 or 7.5, and cells were removed by centrifugation and filtration. Proteins in the resulting supernatant were concentrated by TCA precipitation and separated by SDS-PAGE. (B) Pelleted cells from biofilms resuspended in PBS at pHs 5 and 7.5 were lysed with zirconium beads, and whole-cell lysates were separated by SDS-PAGE. Molecular size markers are shown on the left.
    Figure Legend Snippet: SDS-PAGE of matrix proteins released from biofilm cells and corresponding cell lysates. (A) Untreated and DNase I-treated biofilms were resuspended and incubated in PBS at pH 5 or 7.5, and cells were removed by centrifugation and filtration. Proteins in the resulting supernatant were concentrated by TCA precipitation and separated by SDS-PAGE. (B) Pelleted cells from biofilms resuspended in PBS at pHs 5 and 7.5 were lysed with zirconium beads, and whole-cell lysates were separated by SDS-PAGE. Molecular size markers are shown on the left.

    Techniques Used: SDS Page, Incubation, Centrifugation, Filtration, TCA Precipitation

    Quantification of clump sizes from phase-contrast microscopy images of untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of DNA (DNA from S. aureus [SA] or salmon sperm [Sal] at 24 μg ml −1 ) after DNase I and proteinase K treatment of biofilms. Clumps of 1 to 4, 5 to 10, 11 to 20, or > 20 cells were identified. Data represent average values of three experiments with standard deviations of quantifications of 20 randomly picked microscopic fields per experiment. Significantly more clumps of > 20 cells were detected in untreated biofilms than in proteinase K- or DNase I-treated biofilms, as well as in DNase I-treated samples with added DNA (Sal or SA) versus only DNase I-treated biofilms. Significant differences were calculated with Student's t test. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001.
    Figure Legend Snippet: Quantification of clump sizes from phase-contrast microscopy images of untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of DNA (DNA from S. aureus [SA] or salmon sperm [Sal] at 24 μg ml −1 ) after DNase I and proteinase K treatment of biofilms. Clumps of 1 to 4, 5 to 10, 11 to 20, or > 20 cells were identified. Data represent average values of three experiments with standard deviations of quantifications of 20 randomly picked microscopic fields per experiment. Significantly more clumps of > 20 cells were detected in untreated biofilms than in proteinase K- or DNase I-treated biofilms, as well as in DNase I-treated samples with added DNA (Sal or SA) versus only DNase I-treated biofilms. Significant differences were calculated with Student's t test. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001.

    Techniques Used: Microscopy

    Microscopic images of clumping of resuspended cells from untreated biofilm and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of exogenous DNA to DNase I- and proteinase K-treated biofilms. (A) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase. (B) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from biofilms treated with DNase I or proteinase K, with subsequent addition of exogenous DNA from S. aureus (SA) or salmon sperm (Sal) at 24 μg ml −1 .
    Figure Legend Snippet: Microscopic images of clumping of resuspended cells from untreated biofilm and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of exogenous DNA to DNase I- and proteinase K-treated biofilms. (A) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase. (B) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from biofilms treated with DNase I or proteinase K, with subsequent addition of exogenous DNA from S. aureus (SA) or salmon sperm (Sal) at 24 μg ml −1 .

    Techniques Used: Microscopy

    31) Product Images from "Purification and characterization of transcription factor IIIA from Acanthamoeba castellanii"

    Article Title: Purification and characterization of transcription factor IIIA from Acanthamoeba castellanii

    Journal: Nucleic Acids Research

    doi:

    The DNase I footprint of Ac TFIIIA is similar to that of Xenopus TFIIIA. Promoter–DNA affinity fractions indicated above each gel were used for footprinting. The relative DNA binding activity determined by EMSA is graphically represented above each lane. The gel on the left shows DNase I cleavage of the template strand, and the gel on the right shows cleavage of the RNA-like strand. The positions of the A-box, the intermediate element (IE) and the C-box are shown to the left of each gel. Footprinted regions are identified by brackets. Nucleotide positions +44 and +95 are indicated on the template strand, and nucleotide positions +47 and +97 are indicated on the RNA-like strand. The arrow identifies a hypersensitive cleavage site at +64 on the RNA-like strand.
    Figure Legend Snippet: The DNase I footprint of Ac TFIIIA is similar to that of Xenopus TFIIIA. Promoter–DNA affinity fractions indicated above each gel were used for footprinting. The relative DNA binding activity determined by EMSA is graphically represented above each lane. The gel on the left shows DNase I cleavage of the template strand, and the gel on the right shows cleavage of the RNA-like strand. The positions of the A-box, the intermediate element (IE) and the C-box are shown to the left of each gel. Footprinted regions are identified by brackets. Nucleotide positions +44 and +95 are indicated on the template strand, and nucleotide positions +47 and +97 are indicated on the RNA-like strand. The arrow identifies a hypersensitive cleavage site at +64 on the RNA-like strand.

    Techniques Used: Footprinting, Binding Assay, Activity Assay

    32) Product Images from "DNase I improves corneal epithelial and nerve regeneration in diabetic mice. DNase I improves corneal epithelial and nerve regeneration in diabetic mice"

    Article Title: DNase I improves corneal epithelial and nerve regeneration in diabetic mice. DNase I improves corneal epithelial and nerve regeneration in diabetic mice

    Journal: Journal of Cellular and Molecular Medicine

    doi: 10.1111/jcmm.15112

    DNase I restored the resolution of corneal inflammation. A, Immunofluorescence staining was performed with the macrophage marker anti‐F4/80 (green fluorescence) and the M2 macrophage marker anti‐CD206 (red fluorescence) 48 h after removal of the corneal epithelium. B, mRNA expression levels of iNOS, CD86, TNF‐α, MCP‐1, IL‐12, IL‐10, arginase‐1 and CD206 (48 h after epithelial scrape) were analyzed by RT‐qPCR from the control and diabetic mouse corneas (n = 6). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I restored the resolution of corneal inflammation. A, Immunofluorescence staining was performed with the macrophage marker anti‐F4/80 (green fluorescence) and the M2 macrophage marker anti‐CD206 (red fluorescence) 48 h after removal of the corneal epithelium. B, mRNA expression levels of iNOS, CD86, TNF‐α, MCP‐1, IL‐12, IL‐10, arginase‐1 and CD206 (48 h after epithelial scrape) were analyzed by RT‐qPCR from the control and diabetic mouse corneas (n = 6). Data were given as the mean ± SD; * P

    Techniques Used: Immunofluorescence, Staining, Marker, Fluorescence, Expressing, Quantitative RT-PCR

    DNase I restored the resolution of corneal inflammation. A, Expression of H3Cit and Ly6G was examined with immunofluorescence staining 48 h after corneal epithelial removal in the control, diabetic and DNase I‐treated diabetic mice. B‐D, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of H3Cit, H3 and Ly6G (B), accompanied by the quantified results of Western blot experiments (C‐D; n = 6). E‐F, Corneas harvested 48 h after injury were homogenized and examined for levels of myeloperoxidase (MPO) activity (E) and neutrophil elastase (NE) expression (F) with enzyme‐linked immunosorbent assay (ELISA; n = 5). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I restored the resolution of corneal inflammation. A, Expression of H3Cit and Ly6G was examined with immunofluorescence staining 48 h after corneal epithelial removal in the control, diabetic and DNase I‐treated diabetic mice. B‐D, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of H3Cit, H3 and Ly6G (B), accompanied by the quantified results of Western blot experiments (C‐D; n = 6). E‐F, Corneas harvested 48 h after injury were homogenized and examined for levels of myeloperoxidase (MPO) activity (E) and neutrophil elastase (NE) expression (F) with enzyme‐linked immunosorbent assay (ELISA; n = 5). Data were given as the mean ± SD; * P

    Techniques Used: Expressing, Immunofluorescence, Staining, Mouse Assay, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay

    Anti‐NETs treatment promoted the regeneration of corneal epithelium in diabetic mice. A, Diabetic mice were topically treated with 1 mg/mL DNase I (5 μL/eye, six times per day) after the removal of the corneal epithelium. Meanwhile, healthy and diabetic control mice were topically treated with PBS. The residual epithelial defect was examined at 0, 24 and 48 h after the removal of the corneal epithelium with fluorescein staining. B, The histogram of the residual epithelial defect was presented as the percentage of the original wound area (n = 5). C, Corneas harvested 48 h after injury were homogenized and examined for levels of eDNA with spectrophotometer (n = 4). D‐E, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of PAD4 (n = 6). Data were given as the mean ± SD; ** P
    Figure Legend Snippet: Anti‐NETs treatment promoted the regeneration of corneal epithelium in diabetic mice. A, Diabetic mice were topically treated with 1 mg/mL DNase I (5 μL/eye, six times per day) after the removal of the corneal epithelium. Meanwhile, healthy and diabetic control mice were topically treated with PBS. The residual epithelial defect was examined at 0, 24 and 48 h after the removal of the corneal epithelium with fluorescein staining. B, The histogram of the residual epithelial defect was presented as the percentage of the original wound area (n = 5). C, Corneas harvested 48 h after injury were homogenized and examined for levels of eDNA with spectrophotometer (n = 4). D‐E, Corneas harvested 48 h after injury were evaluated with Western blot to examine the protein contents of PAD4 (n = 6). Data were given as the mean ± SD; ** P

    Techniques Used: Mouse Assay, Staining, Spectrophotometry, Western Blot

    Effects of DNase I on the regeneration of diabetic corneal nerves and the restoration of mechanical sensation. A‐C, Twenty‐one days after injury, the renewing corneas were harvested and the regenerated corneal nerve fibres were examined with corneal whole‐mount staining, and the immunofluorescence intensity of central (B) and peripheral (C) nerve fibres at 21 d after injury was calculated with ImageJ software (n = 5). D, A Cochet‐Bonnet esthesiometer was used to test the mechanical sensitivity of the cornea in healthy, diabetic and DNase I‐treated diabetic mice at 3, 7, 14 and 21 d after the corneal epithelial removal (n = 5). Data were given as the mean ± SD; * P
    Figure Legend Snippet: Effects of DNase I on the regeneration of diabetic corneal nerves and the restoration of mechanical sensation. A‐C, Twenty‐one days after injury, the renewing corneas were harvested and the regenerated corneal nerve fibres were examined with corneal whole‐mount staining, and the immunofluorescence intensity of central (B) and peripheral (C) nerve fibres at 21 d after injury was calculated with ImageJ software (n = 5). D, A Cochet‐Bonnet esthesiometer was used to test the mechanical sensitivity of the cornea in healthy, diabetic and DNase I‐treated diabetic mice at 3, 7, 14 and 21 d after the corneal epithelial removal (n = 5). Data were given as the mean ± SD; * P

    Techniques Used: Staining, Immunofluorescence, Software, Mouse Assay

    DNase I reactivated epithelial regeneration‐related signaling pathways. A, Immunofluorescence staining and (B) Western blot were used to examine the activation levels of epithelial regeneration‐related signaling pathways, including pAkt, IGF‐1R and Sirt1, in the regenerated corneal epithelium 48 h after injury. C‐E, The histogram showed the quantified results of the Western blot (n = 6). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I reactivated epithelial regeneration‐related signaling pathways. A, Immunofluorescence staining and (B) Western blot were used to examine the activation levels of epithelial regeneration‐related signaling pathways, including pAkt, IGF‐1R and Sirt1, in the regenerated corneal epithelium 48 h after injury. C‐E, The histogram showed the quantified results of the Western blot (n = 6). Data were given as the mean ± SD; * P

    Techniques Used: Immunofluorescence, Staining, Western Blot, Activation Assay

    DNase I inhibited the increased ROS accumulation and NADPH oxidase 2/4 expression. A, Generation of reactive oxygen species (ROS) and expression of NADPH oxidase 2/4 were examined with immunofluorescence staining in healthy, diabetic and 5‐day DNase I‐treated diabetic corneal epithelia. B, Quantification of fluorescence intensity of ROS by ImageJ software (n = 3). C‐E, Corneas harvested from healthy and diabetic (with or without 5‐day DNase I treatment) mice were evaluated with Western blot to examine the protein levels of NADPH oxidase 2/4, and the quantified data of the Western blot results were shown (D, E; n = 6). Data were given as the mean ± SD; * P
    Figure Legend Snippet: DNase I inhibited the increased ROS accumulation and NADPH oxidase 2/4 expression. A, Generation of reactive oxygen species (ROS) and expression of NADPH oxidase 2/4 were examined with immunofluorescence staining in healthy, diabetic and 5‐day DNase I‐treated diabetic corneal epithelia. B, Quantification of fluorescence intensity of ROS by ImageJ software (n = 3). C‐E, Corneas harvested from healthy and diabetic (with or without 5‐day DNase I treatment) mice were evaluated with Western blot to examine the protein levels of NADPH oxidase 2/4, and the quantified data of the Western blot results were shown (D, E; n = 6). Data were given as the mean ± SD; * P

    Techniques Used: Expressing, Immunofluorescence, Staining, Fluorescence, Software, Mouse Assay, Western Blot

    33) Product Images from "DNase Pretreatment of Master Mix Reagents Improves the Validity of Universal 16S rRNA Gene PCR Results"

    Article Title: DNase Pretreatment of Master Mix Reagents Improves the Validity of Universal 16S rRNA Gene PCR Results

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.41.4.1763-1765.2003

    Influence of DNase I dosages on the sensitivity of PCR assays. Rate of positive PCR results at the detection limit are given depending on DNase I dosages for assays I (A), II (B), and III (C) (for details, see text). The threshold dosage of DNase I that was required for the complete elimination of false-positive PCR results is underlined. Amounts of DNase I increasing stepwise reduced the sensitivity of the PCRs.
    Figure Legend Snippet: Influence of DNase I dosages on the sensitivity of PCR assays. Rate of positive PCR results at the detection limit are given depending on DNase I dosages for assays I (A), II (B), and III (C) (for details, see text). The threshold dosage of DNase I that was required for the complete elimination of false-positive PCR results is underlined. Amounts of DNase I increasing stepwise reduced the sensitivity of the PCRs.

    Techniques Used: Polymerase Chain Reaction

    DNase I dosages required to improve universal PCR results. Percentages of PCR runs delivering valid (white bars), false-positive (grey bars), and false-negative (black bars) results depending on DNase I dosages are shown for assays I (A), II (B), and III (C) (for details, see text). Complete elimination of false-positive results and significant increases in valid results were achieved with highly varying amounts of DNase I (0.1 through 70 IU). The asterisks mark the significantly high proportion of valid PCR results obtained with DNase pretreatment compared to the results from PCR runs without DNase pretreatment.
    Figure Legend Snippet: DNase I dosages required to improve universal PCR results. Percentages of PCR runs delivering valid (white bars), false-positive (grey bars), and false-negative (black bars) results depending on DNase I dosages are shown for assays I (A), II (B), and III (C) (for details, see text). Complete elimination of false-positive results and significant increases in valid results were achieved with highly varying amounts of DNase I (0.1 through 70 IU). The asterisks mark the significantly high proportion of valid PCR results obtained with DNase pretreatment compared to the results from PCR runs without DNase pretreatment.

    Techniques Used: Polymerase Chain Reaction

    34) Product Images from "Regulation of Glucose Metabolism in Pseudomonas"

    Article Title: Regulation of Glucose Metabolism in Pseudomonas

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.014555

    DNase I footprinting of promoters regulated by HexR. Assays were done with P zwf DNA operator ( A ) and of edd / gap -1 promoter ( B ). Lane 1 contains a control without HexR, lanes 2 and 3 contain DNA incubated with 0.1 and 3 μ m concentrations of purified
    Figure Legend Snippet: DNase I footprinting of promoters regulated by HexR. Assays were done with P zwf DNA operator ( A ) and of edd / gap -1 promoter ( B ). Lane 1 contains a control without HexR, lanes 2 and 3 contain DNA incubated with 0.1 and 3 μ m concentrations of purified

    Techniques Used: Footprinting, Incubation, Purification

    35) Product Images from "Stability and Function of Secondary Th1 Memory Cells Is Dependent on the Nature of the Secondary Stimulus"

    Article Title: Stability and Function of Secondary Th1 Memory Cells Is Dependent on the Nature of the Secondary Stimulus

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1200244

    Heterologous rechallenge boosts the frequency of tissue homing Th1 effector memory cells. Lymphocytes were isolated from perfused livers following digestion in Collagenase B and DNAse I. Total numbers of CD4 +  IFNγ-producing cells were calculated
    Figure Legend Snippet: Heterologous rechallenge boosts the frequency of tissue homing Th1 effector memory cells. Lymphocytes were isolated from perfused livers following digestion in Collagenase B and DNAse I. Total numbers of CD4 + IFNγ-producing cells were calculated

    Techniques Used: Isolation

    36) Product Images from "Promoter Scanning for Transcription Inhibition with DNA-Binding Polyamides"

    Article Title: Promoter Scanning for Transcription Inhibition with DNA-Binding Polyamides

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.6.1723-1733.2002

    Inhibition of the ternary TBP-TFIIA-DNA complex by polyamide 3. (A) DNase I footprint of U−2 DNA in the presence of DNA alone (lane 2); TBP (lane 3); TBP plus TFIIA (lane 4); TBP, TFIIA, and polyamide 3 at 3, 10, 15, 20, 30, and 50 nM (lanes 5 to 10, respectively). All reaction components were incubated simultaneously for 30 min prior to digestion with DNase. Lane 1, G+A sequencing ladder; lane 11, 50 nM polyomide 3, no TBP or TFIIA. The location of the TATA box and polyamide-binding site are indicated. (B) Graphical representation of inhibition plotted as the fraction of DNA bound (normalized to the fraction of TBP bound in the absence of polyamide) versus polyamide concentration.
    Figure Legend Snippet: Inhibition of the ternary TBP-TFIIA-DNA complex by polyamide 3. (A) DNase I footprint of U−2 DNA in the presence of DNA alone (lane 2); TBP (lane 3); TBP plus TFIIA (lane 4); TBP, TFIIA, and polyamide 3 at 3, 10, 15, 20, 30, and 50 nM (lanes 5 to 10, respectively). All reaction components were incubated simultaneously for 30 min prior to digestion with DNase. Lane 1, G+A sequencing ladder; lane 11, 50 nM polyomide 3, no TBP or TFIIA. The location of the TATA box and polyamide-binding site are indicated. (B) Graphical representation of inhibition plotted as the fraction of DNA bound (normalized to the fraction of TBP bound in the absence of polyamide) versus polyamide concentration.

    Techniques Used: Inhibition, Incubation, Sequencing, Binding Assay, Concentration Assay

    Inhibition of TBP binding to the TATA box by polyamide 3. (A) DNase I footprint of the radiolabeled D+6 DNA in the presence of DNA alone (lane 2); 40 nM polyamide 3 (lane 3); TBP (lane 4); and TBP and polyamide 3 at 3, 5, 10, 15, 20, 30, and 40 nM (lanes 5 to 11, respectively). Lane 1, G+A sequencing ladder. (B) Co-occupancy of TBP and polyamide 3. DNase I footprint of D+10 in the presence of DNA alone (lane 2); 40 nM polyamide 3 (lane 3); TBP (lane 4); and TBP and polyamide 3 at 3, 5, 10, 20, 30, and 40 nM (lanes 5 to 10, respectively). Lane 1, G+A sequencing ladder.
    Figure Legend Snippet: Inhibition of TBP binding to the TATA box by polyamide 3. (A) DNase I footprint of the radiolabeled D+6 DNA in the presence of DNA alone (lane 2); 40 nM polyamide 3 (lane 3); TBP (lane 4); and TBP and polyamide 3 at 3, 5, 10, 15, 20, 30, and 40 nM (lanes 5 to 11, respectively). Lane 1, G+A sequencing ladder. (B) Co-occupancy of TBP and polyamide 3. DNase I footprint of D+10 in the presence of DNA alone (lane 2); 40 nM polyamide 3 (lane 3); TBP (lane 4); and TBP and polyamide 3 at 3, 5, 10, 20, 30, and 40 nM (lanes 5 to 10, respectively). Lane 1, G+A sequencing ladder.

    Techniques Used: Inhibition, Binding Assay, Sequencing

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    Roche dnase i
    Real-time PCR quantitation of DNase I-resistant genomes after incubation with nuclear extract or cytoplasmic extract. A total of 10 10 particles of purified AAV2/2-hF.IX vector were incubated for 30 min at 37°C with 50 μg of nuclear extract, cytoplasmic extract, or the same volume of buffer alone, before digesting with <t>DNase</t> I for a further hour at 37°C (final volume, 100 μl). (A) DNase I-resistant VG in 5 μl were quantified by real-time PCR. Error bars show the standard error of the mean of four samples per group. (B) Twenty microliters of the remaining sample was analyzed by Western blotting with the anti-VP1,2,3 antibody to detect the integrity of the capsid proteins following incubation with nuclear or cytoplasmic extract. Lanes 1 and 2, AAV particles incubated with buffer only; lanes 3 to 6, AAV particles incubated with cytoplasmic extract; lanes 7 to 10, AAV particles incubated with nuclear extract.
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    Real-time PCR quantitation of DNase I-resistant genomes after incubation with nuclear extract or cytoplasmic extract. A total of 10 10 particles of purified AAV2/2-hF.IX vector were incubated for 30 min at 37°C with 50 μg of nuclear extract, cytoplasmic extract, or the same volume of buffer alone, before digesting with DNase I for a further hour at 37°C (final volume, 100 μl). (A) DNase I-resistant VG in 5 μl were quantified by real-time PCR. Error bars show the standard error of the mean of four samples per group. (B) Twenty microliters of the remaining sample was analyzed by Western blotting with the anti-VP1,2,3 antibody to detect the integrity of the capsid proteins following incubation with nuclear or cytoplasmic extract. Lanes 1 and 2, AAV particles incubated with buffer only; lanes 3 to 6, AAV particles incubated with cytoplasmic extract; lanes 7 to 10, AAV particles incubated with nuclear extract.

    Journal: Journal of Virology

    Article Title: Rapid Uncoating of Vector Genomes Is the Key to Efficient Liver Transduction with Pseudotyped Adeno-Associated Virus Vectors

    doi: 10.1128/JVI.78.6.3110-3122.2004

    Figure Lengend Snippet: Real-time PCR quantitation of DNase I-resistant genomes after incubation with nuclear extract or cytoplasmic extract. A total of 10 10 particles of purified AAV2/2-hF.IX vector were incubated for 30 min at 37°C with 50 μg of nuclear extract, cytoplasmic extract, or the same volume of buffer alone, before digesting with DNase I for a further hour at 37°C (final volume, 100 μl). (A) DNase I-resistant VG in 5 μl were quantified by real-time PCR. Error bars show the standard error of the mean of four samples per group. (B) Twenty microliters of the remaining sample was analyzed by Western blotting with the anti-VP1,2,3 antibody to detect the integrity of the capsid proteins following incubation with nuclear or cytoplasmic extract. Lanes 1 and 2, AAV particles incubated with buffer only; lanes 3 to 6, AAV particles incubated with cytoplasmic extract; lanes 7 to 10, AAV particles incubated with nuclear extract.

    Article Snippet: One half was digested with 50 U of DNase I as described above, while the other half was left undigested.

    Techniques: Real-time Polymerase Chain Reaction, Quantitation Assay, Incubation, Purification, Plasmid Preparation, Western Blot

    Immunoprecipitation of intact AAV2/2 particles from purified liver nuclei. Liver nuclei purified at different time points after intraportal injection of 5 × 10 11 VG of AAV2/2-hF.IX16 into C57BL/6 mice were incubated with DNase I for 1 h at 37°C. (A and B) Intact AAV capsids were immunoprecipitated from solubilized, DNase I-treated nuclei using the A20 antibody (A) or heparin-Sepharose beads (B). Immunoprecipitated capsids were boiled for 5 min in alkaline buffer, and the released VG were separated on a 1% alkaline agarose gel, blotted, and probed with a sequence-specific probe. (C) Control immunoprecipitations were performed with the anti-VP1,2,3 antibody, which recognizes dissociated, but not intact, capsid proteins. Positive and negative controls were performed for immunoprecipitations with all antibodies. For the positive control, purified nuclei were spiked with 10 9 VG of AAV2/2-hF.IX.16 prior to solubilization (lane 6). For the negative control, solubilized nuclei were spiked with 10 10 VG of AAV-hF.IX16 DNA extracted from purified AAV2/2-hF.IX16 particles (lane 5). Lanes 1 to 4 show copy number standards (purified AAV2/2-hF.IX particles boiled in alkaline buffer prior to loading). Lanes 7 to 15 represent individual mice. (D) Ten microliters of supernatant from solubilized nuclei (from a total volume of 1 ml for each mouse) removed prior to immunoprecipitation was also boiled in alkaline buffer and loaded on a gel.

    Journal: Journal of Virology

    Article Title: Rapid Uncoating of Vector Genomes Is the Key to Efficient Liver Transduction with Pseudotyped Adeno-Associated Virus Vectors

    doi: 10.1128/JVI.78.6.3110-3122.2004

    Figure Lengend Snippet: Immunoprecipitation of intact AAV2/2 particles from purified liver nuclei. Liver nuclei purified at different time points after intraportal injection of 5 × 10 11 VG of AAV2/2-hF.IX16 into C57BL/6 mice were incubated with DNase I for 1 h at 37°C. (A and B) Intact AAV capsids were immunoprecipitated from solubilized, DNase I-treated nuclei using the A20 antibody (A) or heparin-Sepharose beads (B). Immunoprecipitated capsids were boiled for 5 min in alkaline buffer, and the released VG were separated on a 1% alkaline agarose gel, blotted, and probed with a sequence-specific probe. (C) Control immunoprecipitations were performed with the anti-VP1,2,3 antibody, which recognizes dissociated, but not intact, capsid proteins. Positive and negative controls were performed for immunoprecipitations with all antibodies. For the positive control, purified nuclei were spiked with 10 9 VG of AAV2/2-hF.IX.16 prior to solubilization (lane 6). For the negative control, solubilized nuclei were spiked with 10 10 VG of AAV-hF.IX16 DNA extracted from purified AAV2/2-hF.IX16 particles (lane 5). Lanes 1 to 4 show copy number standards (purified AAV2/2-hF.IX particles boiled in alkaline buffer prior to loading). Lanes 7 to 15 represent individual mice. (D) Ten microliters of supernatant from solubilized nuclei (from a total volume of 1 ml for each mouse) removed prior to immunoprecipitation was also boiled in alkaline buffer and loaded on a gel.

    Article Snippet: One half was digested with 50 U of DNase I as described above, while the other half was left undigested.

    Techniques: Immunoprecipitation, Purification, Injection, Mouse Assay, Incubation, Agarose Gel Electrophoresis, Sequencing, Positive Control, Negative Control

    Southern blot analysis of rAAV VG extracted from purified liver nuclei without preincubation with DNase I (A), or after incubation with DNase I for 5 h at 37°C (B). Vector forms in DNA samples extracted 3 weeks after injection of pseudotyped AAV-hF.IX16 vectors are shown. Forty micrograms of undigested total DNA (A) or the equivalent volume of DNase I-treated sample (B) was separated on a 1% agarose gel, blotted, and probed with a vector sequence-specific probe. Lanes 1 and 2, 1- and 0.1-VG/DGE standards, respectively (40 μg of DNA extracted from naïve mice was spiked with a 6.4-kb plasmid containing the AAVhF.IX16 sequence and then digested with Sac I prior to loading). A total of 10 7 VG of AAV-hF.IX16 extracted from purified vector stock was denatured by boiling for 5 min in the presence of formamide and loaded in lane 3 as a size marker for ss AAV-hF.IX genomes. Lanes 5 to 15 represent individual mice. Arrowheads indicate the different molecular forms of the AAV-hF.IX16 genome. ds indicates either relaxed circular, supercoiled circular, or linear ds forms. c, concatemers.

    Journal: Journal of Virology

    Article Title: Rapid Uncoating of Vector Genomes Is the Key to Efficient Liver Transduction with Pseudotyped Adeno-Associated Virus Vectors

    doi: 10.1128/JVI.78.6.3110-3122.2004

    Figure Lengend Snippet: Southern blot analysis of rAAV VG extracted from purified liver nuclei without preincubation with DNase I (A), or after incubation with DNase I for 5 h at 37°C (B). Vector forms in DNA samples extracted 3 weeks after injection of pseudotyped AAV-hF.IX16 vectors are shown. Forty micrograms of undigested total DNA (A) or the equivalent volume of DNase I-treated sample (B) was separated on a 1% agarose gel, blotted, and probed with a vector sequence-specific probe. Lanes 1 and 2, 1- and 0.1-VG/DGE standards, respectively (40 μg of DNA extracted from naïve mice was spiked with a 6.4-kb plasmid containing the AAVhF.IX16 sequence and then digested with Sac I prior to loading). A total of 10 7 VG of AAV-hF.IX16 extracted from purified vector stock was denatured by boiling for 5 min in the presence of formamide and loaded in lane 3 as a size marker for ss AAV-hF.IX genomes. Lanes 5 to 15 represent individual mice. Arrowheads indicate the different molecular forms of the AAV-hF.IX16 genome. ds indicates either relaxed circular, supercoiled circular, or linear ds forms. c, concatemers.

    Article Snippet: One half was digested with 50 U of DNase I as described above, while the other half was left undigested.

    Techniques: Southern Blot, Purification, Incubation, Plasmid Preparation, Injection, Agarose Gel Electrophoresis, Sequencing, Mouse Assay, Marker

    Real-time PCR quantitation of the proportions of DNase I-resistant AAV genomes localized within the nucleus over time. Numbers of AAV genomes were quantified in total DNA extracted from purified liver nuclei prepared at different time points after intraportal injection of 10 11  VG of pseudotyped AAV-hF.IX16 vectors into C57BL/6 mice. Solid black bars indicate the total number of nuclear-localized AAV genomes at each time point (expressed as copy numbers per DGE), and open bars indicate the relative number of DNase I-resistant AAV genomes. The ratio of DNase I-resistant to total nuclear-localized VG at each time point is expressed as a percentage above each graph.  n  = 4 mice per vector per time point. Error bars show standard errors of the means.

    Journal: Journal of Virology

    Article Title: Rapid Uncoating of Vector Genomes Is the Key to Efficient Liver Transduction with Pseudotyped Adeno-Associated Virus Vectors

    doi: 10.1128/JVI.78.6.3110-3122.2004

    Figure Lengend Snippet: Real-time PCR quantitation of the proportions of DNase I-resistant AAV genomes localized within the nucleus over time. Numbers of AAV genomes were quantified in total DNA extracted from purified liver nuclei prepared at different time points after intraportal injection of 10 11 VG of pseudotyped AAV-hF.IX16 vectors into C57BL/6 mice. Solid black bars indicate the total number of nuclear-localized AAV genomes at each time point (expressed as copy numbers per DGE), and open bars indicate the relative number of DNase I-resistant AAV genomes. The ratio of DNase I-resistant to total nuclear-localized VG at each time point is expressed as a percentage above each graph. n = 4 mice per vector per time point. Error bars show standard errors of the means.

    Article Snippet: One half was digested with 50 U of DNase I as described above, while the other half was left undigested.

    Techniques: Real-time Polymerase Chain Reaction, Quantitation Assay, Purification, Injection, Mouse Assay, Plasmid Preparation

    B-cell factors binding in vitro to the HRE of MMTV. DNase I footprinting analysis with nuclear extracts of the M12 B-cell line shows two protected sites, fp1 and fp2 (arrowheads), not seen with nuclear extracts of the fibroblastic Ltk − cell line. A DNA fragment comprising the sequences from the Sty I restriction site at positions −303 to +133, where a synthetic Bam HI linker was inserted, was 5′ end labeled at the Sty ]).

    Journal: Journal of Virology

    Article Title: Synergistic Action of GA-Binding Protein and Glucocorticoid Receptor in Transcription from the Mouse Mammary Tumor Virus Promoter

    doi:

    Figure Lengend Snippet: B-cell factors binding in vitro to the HRE of MMTV. DNase I footprinting analysis with nuclear extracts of the M12 B-cell line shows two protected sites, fp1 and fp2 (arrowheads), not seen with nuclear extracts of the fibroblastic Ltk − cell line. A DNA fragment comprising the sequences from the Sty I restriction site at positions −303 to +133, where a synthetic Bam HI linker was inserted, was 5′ end labeled at the Sty ]).

    Article Snippet: Samples without nuclear extracts received a 20-fold dilution of DNase I for 2 or 5 min. After proteinase K digestion, extraction, and precipitation , the DNA was separated by electrophoresis on 6% denaturing polyacrylamide gels , fixed, and dried prior to autoradiography at −80°C with intensifying screens.

    Techniques: Binding Assay, In Vitro, Footprinting, Labeling

    Diagram of experimental design. 1st: Sample collection. 2nd: Deoxyribonuclease I (DNase) treatment. 3rd: DNA isolation. 4th: Downstream applications. 5th: Comparative analysis.

    Journal: American Journal of Rhinology & Allergy

    Article Title: Dead or alive: Deoxyribonuclease I sensitive bacteria and implications for the sinus microbiome

    doi: 10.2500/ajra.2016.30.4278

    Figure Lengend Snippet: Diagram of experimental design. 1st: Sample collection. 2nd: Deoxyribonuclease I (DNase) treatment. 3rd: DNA isolation. 4th: Downstream applications. 5th: Comparative analysis.

    Article Snippet: Duplicate samples were placed in separate, sterile tubes and immerged in sufficient diluted DNase I buffer per manufacture standard protocol (Roche, Basel, Switzerland); 10 U/μL DNase I (Roche) was added to one sample in each pair.

    Techniques: DNA Extraction

    Immunofluorescence microscopy of GAPDH in fixed biofilms. Untreated and proteinase K- or DNase I-treated biofilms were fixed and probed with a primary anti-GAPDH antibody and a secondary anti-rabbit antibody conjugated to Alexa Fluor 488 (green), and cell nuclei were strained with DAPI (blue). Size bars, 10 μm.

    Journal: Journal of Bacteriology

    Article Title: An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus

    doi: 10.1128/JB.00726-15

    Figure Lengend Snippet: Immunofluorescence microscopy of GAPDH in fixed biofilms. Untreated and proteinase K- or DNase I-treated biofilms were fixed and probed with a primary anti-GAPDH antibody and a secondary anti-rabbit antibody conjugated to Alexa Fluor 488 (green), and cell nuclei were strained with DAPI (blue). Size bars, 10 μm.

    Article Snippet: Also in agreement with our model were results showing that the integrity of the biofilm remained sensitive to DNase I for the entire period of biofilm formation; indeed, maximal sensitivity was seen at 23 h. Conversely, proteinase K sensitivity decreased with time ( ).

    Techniques: Immunofluorescence, Microscopy

    Quantification of eDNA by qPCR. (A) Quantification of eDNA in untreated and proteinase K- or DNase I-treated biofilms by qPCR. eDNA was quantified in biofilm medium supernatant (pH approximately 4.5 to 5) following resuspension after remaining cells were removed by filtration. (B) Quantification of eDNA release upon suspension of biofilms in PBS at pH 5 or 7.5. (C) Quantification of eDNA release of untreated and DNase I- or proteinase K-treated biofilms upon suspension in PBS at pH 7.5 with or without washing. The amount of eDNA measured was normalized to that in unwashed, untreated cells. For panels B and C, eDNA was quantified after biofilms were resuspended in PBS and cells were removed by centrifugation and filtration. For all experiments, primers for the gene for gyrase A ( gyrA ) were used and dilutions of genomic  S. aureus  DNA were included to calculate absolute DNA concentrations. Average values and standard deviations of at least three independent experiments with three biological and three technical replicates are shown. Significant differences were calculated with Student's  t  test. Not significant (ns),  P >  0.05; *,  P  ≤ 0.05; ***,  P  ≤ 0.001.

    Journal: Journal of Bacteriology

    Article Title: An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus

    doi: 10.1128/JB.00726-15

    Figure Lengend Snippet: Quantification of eDNA by qPCR. (A) Quantification of eDNA in untreated and proteinase K- or DNase I-treated biofilms by qPCR. eDNA was quantified in biofilm medium supernatant (pH approximately 4.5 to 5) following resuspension after remaining cells were removed by filtration. (B) Quantification of eDNA release upon suspension of biofilms in PBS at pH 5 or 7.5. (C) Quantification of eDNA release of untreated and DNase I- or proteinase K-treated biofilms upon suspension in PBS at pH 7.5 with or without washing. The amount of eDNA measured was normalized to that in unwashed, untreated cells. For panels B and C, eDNA was quantified after biofilms were resuspended in PBS and cells were removed by centrifugation and filtration. For all experiments, primers for the gene for gyrase A ( gyrA ) were used and dilutions of genomic S. aureus DNA were included to calculate absolute DNA concentrations. Average values and standard deviations of at least three independent experiments with three biological and three technical replicates are shown. Significant differences were calculated with Student's t test. Not significant (ns), P > 0.05; *, P ≤ 0.05; ***, P ≤ 0.001.

    Article Snippet: Also in agreement with our model were results showing that the integrity of the biofilm remained sensitive to DNase I for the entire period of biofilm formation; indeed, maximal sensitivity was seen at 23 h. Conversely, proteinase K sensitivity decreased with time ( ).

    Techniques: Real-time Polymerase Chain Reaction, Filtration, Centrifugation

    Quantitative assays of biofilms undergoing different treatments at different time points. Proteinase K, DNase I, or water was added to biofilms at inoculation (bars 0), after 7 h of incubation (bars 7), or after 23 h of incubation (bars 23). The OD 600 s of the washed resuspended biofilm, the two combined PBS washes, and the growth medium were measured after 24 h of incubation.

    Journal: Journal of Bacteriology

    Article Title: An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus

    doi: 10.1128/JB.00726-15

    Figure Lengend Snippet: Quantitative assays of biofilms undergoing different treatments at different time points. Proteinase K, DNase I, or water was added to biofilms at inoculation (bars 0), after 7 h of incubation (bars 7), or after 23 h of incubation (bars 23). The OD 600 s of the washed resuspended biofilm, the two combined PBS washes, and the growth medium were measured after 24 h of incubation.

    Article Snippet: Also in agreement with our model were results showing that the integrity of the biofilm remained sensitive to DNase I for the entire period of biofilm formation; indeed, maximal sensitivity was seen at 23 h. Conversely, proteinase K sensitivity decreased with time ( ).

    Techniques: Incubation

    SDS-PAGE of matrix proteins released from biofilm cells and corresponding cell lysates. (A) Untreated and DNase I-treated biofilms were resuspended and incubated in PBS at pH 5 or 7.5, and cells were removed by centrifugation and filtration. Proteins in the resulting supernatant were concentrated by TCA precipitation and separated by SDS-PAGE. (B) Pelleted cells from biofilms resuspended in PBS at pHs 5 and 7.5 were lysed with zirconium beads, and whole-cell lysates were separated by SDS-PAGE. Molecular size markers are shown on the left.

    Journal: Journal of Bacteriology

    Article Title: An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus

    doi: 10.1128/JB.00726-15

    Figure Lengend Snippet: SDS-PAGE of matrix proteins released from biofilm cells and corresponding cell lysates. (A) Untreated and DNase I-treated biofilms were resuspended and incubated in PBS at pH 5 or 7.5, and cells were removed by centrifugation and filtration. Proteins in the resulting supernatant were concentrated by TCA precipitation and separated by SDS-PAGE. (B) Pelleted cells from biofilms resuspended in PBS at pHs 5 and 7.5 were lysed with zirconium beads, and whole-cell lysates were separated by SDS-PAGE. Molecular size markers are shown on the left.

    Article Snippet: Also in agreement with our model were results showing that the integrity of the biofilm remained sensitive to DNase I for the entire period of biofilm formation; indeed, maximal sensitivity was seen at 23 h. Conversely, proteinase K sensitivity decreased with time ( ).

    Techniques: SDS Page, Incubation, Centrifugation, Filtration, TCA Precipitation

    Quantification of clump sizes from phase-contrast microscopy images of untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of DNA (DNA from S. aureus [SA] or salmon sperm [Sal] at 24 μg ml −1 ) after DNase I and proteinase K treatment of biofilms. Clumps of 1 to 4, 5 to 10, 11 to 20, or > 20 cells were identified. Data represent average values of three experiments with standard deviations of quantifications of 20 randomly picked microscopic fields per experiment. Significantly more clumps of > 20 cells were detected in untreated biofilms than in proteinase K- or DNase I-treated biofilms, as well as in DNase I-treated samples with added DNA (Sal or SA) versus only DNase I-treated biofilms. Significant differences were calculated with Student's t test. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001.

    Journal: Journal of Bacteriology

    Article Title: An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus

    doi: 10.1128/JB.00726-15

    Figure Lengend Snippet: Quantification of clump sizes from phase-contrast microscopy images of untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of DNA (DNA from S. aureus [SA] or salmon sperm [Sal] at 24 μg ml −1 ) after DNase I and proteinase K treatment of biofilms. Clumps of 1 to 4, 5 to 10, 11 to 20, or > 20 cells were identified. Data represent average values of three experiments with standard deviations of quantifications of 20 randomly picked microscopic fields per experiment. Significantly more clumps of > 20 cells were detected in untreated biofilms than in proteinase K- or DNase I-treated biofilms, as well as in DNase I-treated samples with added DNA (Sal or SA) versus only DNase I-treated biofilms. Significant differences were calculated with Student's t test. *, P ≤ 0.05; ***, P ≤ 0.001; ****, P ≤ 0.0001.

    Article Snippet: Also in agreement with our model were results showing that the integrity of the biofilm remained sensitive to DNase I for the entire period of biofilm formation; indeed, maximal sensitivity was seen at 23 h. Conversely, proteinase K sensitivity decreased with time ( ).

    Techniques: Microscopy

    Microscopic images of clumping of resuspended cells from untreated biofilm and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of exogenous DNA to DNase I- and proteinase K-treated biofilms. (A) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase. (B) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from biofilms treated with DNase I or proteinase K, with subsequent addition of exogenous DNA from S. aureus (SA) or salmon sperm (Sal) at 24 μg ml −1 .

    Journal: Journal of Bacteriology

    Article Title: An Electrostatic Net Model for the Role of Extracellular DNA in Biofilm Formation by Staphylococcus aureus

    doi: 10.1128/JB.00726-15

    Figure Lengend Snippet: Microscopic images of clumping of resuspended cells from untreated biofilm and biofilms treated with DNase I, proteinase K, or RNase plus the effect of addition of exogenous DNA to DNase I- and proteinase K-treated biofilms. (A) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from untreated biofilms and biofilms treated with DNase I, proteinase K, or RNase. (B) Phase-contrast microscopy images of biofilm cells, resuspended in growth medium, from biofilms treated with DNase I or proteinase K, with subsequent addition of exogenous DNA from S. aureus (SA) or salmon sperm (Sal) at 24 μg ml −1 .

    Article Snippet: Also in agreement with our model were results showing that the integrity of the biofilm remained sensitive to DNase I for the entire period of biofilm formation; indeed, maximal sensitivity was seen at 23 h. Conversely, proteinase K sensitivity decreased with time ( ).

    Techniques: Microscopy