Journal: Journal of Advanced Research

Article Title: Specifically targeted antimicrobial peptides synergize with bacterial-entrapping peptide against systemic MRSA infections

doi: 10.1016/j.jare.2024.01.023

Figure Lengend Snippet: Design, screening, and antimicrobial activity of AMPs obtained from traversal design and machine learning. a, Flow chart of AMP design. b, c, Confusion matrix and ROC curve of machine learning model (WeightedEnsemble_L3; ROC-AUC = 0.87; accuracy = 72 %). d, MIC of AMPs against gram-positive bacteria ( S. aureus ATCC25923 (Sa), S. aureus USA300 (MRSA), and E. faecalis ATCC29212, B. subtilis ATCC6633) and gram-positive bacteria ( E. coli ATCC25922, P. aeruginosa ATCC15442, A. baumannii ATCC19606). Each test was performed on four independent experiments. Dark orange represents greater growth. e, Violin plots of the in vitro effect of propensity on aggregation (left) and liner moment (right) on MIC for S. aureus. f, Violin plots of the effect of hydrophobic moment (left), hydrophobicity (middle), and net charge (right) on antimicrobial activity levels. Data were obtained from DBAASP database, while significant differences between the independent groups were determined using a non-parametric Mann–Whitney U test. g, CD spectrum of P18 in water, hydrophobic, and anionic membrane environment. All CD measurements were performed with three technical replicates. The helix and sheet content of P18 shown in . h, Helical wheel projection (left) and 3D structure (right) of P18. i–l, NPN (n = 4), PI (n = 3, statistical plots shown in ), DiSC 3 -5 assays (n = 5), and DNA binding assays revealed the effects of P18 on cell wall permeabilization, membrane permeability, depolarization, and DNA binding, respectively. Dosage-dependent increase of signals was observed. Data are presented as the mean ± SD. m, n, TEM (m) and SEM (n) images of untreated or 2 × MIC P18-treated Sa and MRSA. Scale bars, 0.5 or 1 μm. Experiments in l–n were performed in triplicate with similar results; one representative image is shown. Sa, S. aureus ATCC25923; MRSA, S. aureus USA300. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: These clones were selected based on their binding affinity for S. aureus and lack of reactivity with shielding antigens from other species like E. coli ATCC 25922, A. baumannii ATCC 19606, and P. aeruginosa ATCC 15442 ( f–h and ).

Techniques: Activity Assay, Bacteria, In Vitro, MANN-WHITNEY, Membrane, Binding Assay, Permeability

Journal: Journal of Advanced Research

Article Title: Specifically targeted antimicrobial peptides synergize with bacterial-entrapping peptide against systemic MRSA infections

doi: 10.1016/j.jare.2024.01.023

Figure Lengend Snippet: Dual activity of P1P18 and P18E6. a , Fluorescence image of Sa and MRSA treated with 2 × MIC FITC-P1P18 or FITC-P18E6 and 10 μM PI. b , Fluorescence image of TRITC-Sa or TRITC-MRSA mixed with five bacterial strains (namely, E. faecalis , B. subtilis , E. coli , P. aeruginosa , and A. baumannii ) treated with 2 × MIC FITC-P1P18 or FITC-P18E6. Sa, S. aureus ATCC25923; MRSA, S. aureus USA300.

Article Snippet: These clones were selected based on their binding affinity for S. aureus and lack of reactivity with shielding antigens from other species like E. coli ATCC 25922, A. baumannii ATCC 19606, and P. aeruginosa ATCC 15442 ( f–h and ).

Techniques: Activity Assay, Fluorescence

Journal: Journal of Advanced Research

Article Title: Specifically targeted antimicrobial peptides synergize with bacterial-entrapping peptide against systemic MRSA infections

doi: 10.1016/j.jare.2024.01.023

Figure Lengend Snippet: Entrapment property of SP5. a, Schematic description of SP5 design. SP5 uses P18 as a template to reduce net charge and improve propensity to in vitro aggregation. b, MIC of the HAMPs against the tested pathogens. Each test was performed in four independent experiments. c, CD spectrum (left) and helical wheel projections (right) of SP5. The helix and sheet content of SP5 listed in . d, SP5 and HAMPs agglutinated 10 7 CFU Sa and MRSA within 10 s rather than E. coli . e, TEM of SP5 in water (Scale bar: 100 nm) and hydrophobic environment (Scale bar: 5 μm) at a concentration of 128 μg/mL. f, Concentration-dependent self-assembly and CACs of SP5. g, Particle size of SP5 at a concentration of 128 μg/mL. The data in f and g represent the average of three technical replicates. h, i, Antimicrobial (h) and agglutination (i) capability of SP5 against MRSA (n = 3 biological replicates). Data are shown as the mean ± SD. j, Growth inhibition checkerboards of SP5 in combination with P18E6. Dark blue represents greater growth. The data represent the average of three biological replicates. k, SEM images of MRSA treated without or with SP5 and SP5 + P18E6. Scale bar: 2 μm. l, Fluorescence image of MRSA treated with SP5 (details are shown in ). Scale bar: 2 or 10 μm. Experiments in d, e, and k were performed in triplicate with similar results; one representative image is shown. m–o, Mice and organs were imaged using in vivo imaging system at 12 h post I.P. 5 mg/kg FITC-P18E6 or FITC-SP5; the intestine showed strong signals compared with other organs. n = 3 mice for per group. Statistical plots are shown in . (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: These clones were selected based on their binding affinity for S. aureus and lack of reactivity with shielding antigens from other species like E. coli ATCC 25922, A. baumannii ATCC 19606, and P. aeruginosa ATCC 15442 ( f–h and ).

Techniques: In Vitro, Concentration Assay, Agglutination, Inhibition, Fluorescence, In Vivo Imaging

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: ( A ) Predicted hairpins involving the sequestration of the SD sequence by the aSD1 motif. Prediction of these secondary structures were carried out on the aapA3 -Tr transcript (see for visualizing the whole structure) using the same software as in , but with no additional constraint. The A28C mutation (dark orange) generates an extra G-C base-pair; in the A40T mutant (purple), two additional A-U pairs stabilize the hairpin; the A40T antagonist mutation A33T is shown in red; the SD and aSD1 sequences are shown in turquoise and yellow, respectively. ( B ) (left) PCR constructs used to assess the SD sequestering structure by transformation assay. (right) For each transformation with the indicated PCR constructs, the number of Str R obtained per total number of transformed cells was calculated and plotted on a log scale. Error bars represent s.d; n = 3 biological replicates. (***p<0.0001; *p=0.001 according to unpaired t -test). ( C ) Graph representing the RNase H assays. The position of the oligonucleotides (FA644 for WT and A40T; FA651 for A33T/A40T; and FA652 for A28C, see ) used in the RNase H protection assay is indicated by a black arrow along the first 45 nucleotides of the aapA3 mRNA . 30 fmol of internally labeled WT and mutated aapA3 -Tr transcripts were incubated with 0 to 100 pmoles of each specific DNA oligonucleotide and subjected to digestion by E. coli RNase H1. Digestion products were analyzed on an 8% PAA denaturing gel . Substrate consumption was quantified as relative substrate band intensity, 100% corresponding to the intensity obtained in absence of oligonucleotide. Error bars represent the s.d; n = 2 technical replicates. 10.7554/eLife.47549.018 Figure 5—source data 1. Raw data to determine the transformation efficiency in . 10.7554/eLife.47549.019 Figure 5—source data 2. Numerical values of the graph shown in in .

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Sequencing, Software, Mutagenesis, Construct, Transformation Assay, Labeling, Incubation

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: 30 fmol of internally labeled RNA were used. DNA oligonucleotides were used to a final concentration of 0 to 10 μM. Reactions were incubated for 30 min at 30°C in the presence or absence (C, control) of 0.25 U E. coli RNase H1. Reactions were stopped with 10 μl of 2X Loading Buffer and products were analyzed in an 8% denaturing PAA gel. See for relative band quantification. P1 and P2 indicate the two RNase H-oligonucleotide-mediated RNA cleavage products.

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Labeling, Concentration Assay, Incubation, Control

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: ( A ) The cell lysate of the T78C strain was subjected to ultracentrifugation through a sucrose gradient. RNA was analyzed as in . The different transcripts aapA3 -FL, aapA3 -Tr, and 5S rRNA (loading control) are indicated. M+D, monosomes + disomes. ( B ) Prediction of the secondary structure involving the second aSD sequence (aSD2). Prediction was carried out on the biologically relevant mRNA ( aapA3 -Tr) (see ). No additional constrains were used. The location of the oligonucleotide used in this assay (FA633) is shown by a black arrow in . The T78C mutation is shown in dark blue, SD sequence in turquoise, anti-SD sequence in yellow and start codon in green. ( C ) A typical RNase H protection assay is shown. A total of 30 fmol of internally labeled aapA3- FL and aapA3 -Tr RNA (WT or T78C) were incubated in presence of 0 to 100 pmoles of DNA oligonucleotide (FA633) and subjected to digestion by E. coli RNase H1. Lane C contains only the labeled substrate in absence of the enzyme. Two digestion products, P1 and P2, are indicated. ( D ) Substrate consumption was quantified as the relative substrate band intensity and plotted as a function of DNA oligonucleotide concentration. Error bars represent the s.d; n = 2 technical replicates. 10.7554/eLife.47549.024 Figure 6—source data 1. Numerical values of the graph shown in in .

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Control, Sequencing, Mutagenesis, Labeling, Incubation, Concentration Assay

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: 2D structure was predicted using the RNAfold Web Server with no additional constraints. VARNA was used for drawing. Translation start and stop codons are highlighted in green and red, respectively. Shine-Dalgarno (SD) and anti-SD (aSD) sequences are shown in turquoise and yellow, respectively. The suppressor mutation T78C is highlighted in dark blue. The predicted delta G value (kcal/mol) is shown under the structure. The small black arrow indicates the binding site of the oligonucleotide used in the RNase H assay shown in .

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Mutagenesis, Binding Assay, Rnase H Assay

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet:

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Plasmid Preparation, Bacteria, DNA Extraction, Recombinant, Sequencing, Software

Journal:

Article Title: Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells

doi: 10.1073/pnas.241308598

Figure Lengend Snippet: S. typhimurium-regulated expression of CCL20 gene in epithelial cells. Caco-2 cells in Transwell cultures were infected apically for 45 min with S. typhimurium ATCC14028 (moi = 100), washed, and incubated for the indicated times in gentamicin-supplemented medium. (a) Transcriptional activation of CCL20 gene: Total RNA was extracted and reverse transcribed. CCL20 mRNA levels were quantified by using real-time PCR and 18S rRNA amplicons as standards. Values were expressed as relative increase of CCL20 mRNA quantity compared with noninfected Caco-2 cells. (b) Secretion of CCL20 chemokine in basal culture medium. CCL20 concentration was measured by CCL20-specific ELISA on cell culture medium of Caco-2 cells.

Article Snippet: Microplates coated with 3 μg/ml of human CCL20-specific mAb (clone 67310.111; R & D Systems) were used to capture CCL20 in culture medium.

Techniques: Expressing, Infection, Incubation, Activation Assay, Reverse Transcription, Real-time Polymerase Chain Reaction, Concentration Assay, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal:

Article Title: Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells

doi: 10.1073/pnas.241308598

Figure Lengend Snippet: Pathogen-specific induction of CCL20 transcription in epithelial cells. Monolayers of Caco-2 cells were exposed apically for 45 min to bacterial strains (moi = 100) and incubated for 2.5 h in gentamicin-containing medium. CCL20 expression was quantified by real-time RT-PCR. ATCC14028 was used as a positive control of CCL20 induction. Results are representative of at least 2 independent experiments. CCL20 transcription was analyzed after exposure to laboratory E. coli DH5α (a); bacteria from human colon flora, E. coli EMO, B. vulgatus, and B. bifidum (b); and enteroinvasive bacteria, S. enteritidis SE857 and L. monocytogenes LO28 (c).

Article Snippet: Microplates coated with 3 μg/ml of human CCL20-specific mAb (clone 67310.111; R & D Systems) were used to capture CCL20 in culture medium.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Positive Control, Bacteria

Journal:

Article Title: Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells

doi: 10.1073/pnas.241308598

Figure Lengend Snippet: Salmonella induction factor for CCL20 expression is a heat-stable secreted protein. Polarized Caco-2 cells were exposed apically for 45 min to bacteria (moi = 100) (a). Then, cells were incubated for 2.5 h in gentamicin-supplemented medium. Alternatively, cells were exposed for 3.25 h to bacterial products (b and c). Activation of CCL20 gene transcription was quantified by real-time RT-PCR. Results are representative of at least 3 independent experiments. (a) Induction of CCL20 transcription does not depend on Salmonella-mediated invasion. (b) LPS-independent CCL20 transcription. Epithelial cells were treated apically or basally with 10 μg/ml of LPS from S. typhimurium. (c) Induction factor is a Salmonella-secreted protein. Cells were exposed apically to 100 μl of supernatants from S. typhimurium, heat-treated supernatant, or trypsin-digested and heat-treated supernatant. LB broth treated in the same conditions was used as control.

Article Snippet: Microplates coated with 3 μg/ml of human CCL20-specific mAb (clone 67310.111; R & D Systems) were used to capture CCL20 in culture medium.

Techniques: Expressing, Bacteria, Incubation, Activation Assay, Quantitative RT-PCR

Journal:

Article Title: Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells

doi: 10.1073/pnas.241308598

Figure Lengend Snippet: Salmonella flagellins are inducing factors of CCL20 and IL-8 transcription in epithelial cells. Polarized Caco-2 cells were treated apically with bacteria (moi = 100) or flagellin. CCL20 and IL-8 gene transcription was quantified by real-time RT-PCR (a and b). Results are representative of at least 3 independent experiments. (a) Cells were infected for 45 min with S. enteritidis, the fliC mutant SEFK32, or SEFK32(pRP2) (complemented with the FliC flagellin of S. typhimurium) and incubated for 2.5 h in gentamicin-containing medium. (b) Dose-dependent induction of CCL20 and IL-8 expression by flagellin. Cells were exposed apically for 3.25 h to purified S. typhimurium FliC flagellin at the indicated concentrations. (c) Flagellin expression in Salmonella strains. Supernatants (0.5 ml) from Salmonella cultures or purified S. typhimurium FliC flagellin (1 μg) were analyzed after SDS/PAGE by Coomassie blue staining (Upper) and by immunoblotting (Lower) with flagellin-specific Ab. Arrow and asterisk indicate the position of flagellins from ATCC14028 (52 kDa) and SE857 (56 kDa), respectively. Agglutination with flagellin-specific Ab was performed on bacteria grown in the same conditions.

Article Snippet: Microplates coated with 3 μg/ml of human CCL20-specific mAb (clone 67310.111; R & D Systems) were used to capture CCL20 in culture medium.

Techniques: Bacteria, Quantitative RT-PCR, Infection, Mutagenesis, Incubation, Expressing, Purification, SDS Page, Staining, Western Blot, Agglutination

Journal:

Article Title: Flagellin stimulation of intestinal epithelial cells triggers CCL20-mediated migration of dendritic cells

doi: 10.1073/pnas.241308598

Figure Lengend Snippet: Immature DCs migrate in response to medium from flagellin-treated epithelial cells. rhCCL20 (7 ng/ml), control medium, or basal medium of untreated or of flagellin-treated Caco-2 cells (7 ng/ml of CCL20) were used in migration assays of immature DCs. When specified, CCL20-specific mAb was mixed with medium 30 min before assay to neutralize CCL20. Results are representative of 2 independent experiments.

Article Snippet: Microplates coated with 3 μg/ml of human CCL20-specific mAb (clone 67310.111; R & D Systems) were used to capture CCL20 in culture medium.

Techniques: Migration

Journal: Foods

Article Title: A Dual-Recognition Electrochemical Sensor Using Bacteria-Imprinted Polymer and Concanavalin A for Sensitive and Selective Detection of Escherichia coli O157:H7

doi: 10.3390/foods14071099

Figure Lengend Snippet: Schematic illustration for the fabrication process and operational principle of the electrochemical sensor for E. coli O157:H7 detection.

Article Snippet: The bacterial species employed in this study were E. coli O157:H7 (ATCC 43889), Salmonella paratyphi B ( S. Paratyphi B , CMCC 50094), Staphylococcus aureus ( S. aureus , ATCC 25923), Listeria monocytogenes ( L. monocytogenes , ATCC 19115) and Escherichia coli O6 ( E. coli O6, ATCC 25922).

Techniques:

Journal: Foods

Article Title: A Dual-Recognition Electrochemical Sensor Using Bacteria-Imprinted Polymer and Concanavalin A for Sensitive and Selective Detection of Escherichia coli O157:H7

doi: 10.3390/foods14071099

Figure Lengend Snippet: ( A ) DPV of E. coli O157:H7 in PBS (0–10 5 CFU mL −1 ). ( B ) Calibration curve of Δ I against logarithmic concentration of E. coli O157:H7.

Article Snippet: The bacterial species employed in this study were E. coli O157:H7 (ATCC 43889), Salmonella paratyphi B ( S. Paratyphi B , CMCC 50094), Staphylococcus aureus ( S. aureus , ATCC 25923), Listeria monocytogenes ( L. monocytogenes , ATCC 19115) and Escherichia coli O6 ( E. coli O6, ATCC 25922).

Techniques: Concentration Assay

Journal: Foods

Article Title: A Dual-Recognition Electrochemical Sensor Using Bacteria-Imprinted Polymer and Concanavalin A for Sensitive and Selective Detection of Escherichia coli O157:H7

doi: 10.3390/foods14071099

Figure Lengend Snippet: ( A ) DPV current response of the sensor toward E. coli O157:H7 and various control bacteria. ( B ) Bar graph showing the statistical analysis of sensor selectivity.

Article Snippet: The bacterial species employed in this study were E. coli O157:H7 (ATCC 43889), Salmonella paratyphi B ( S. Paratyphi B , CMCC 50094), Staphylococcus aureus ( S. aureus , ATCC 25923), Listeria monocytogenes ( L. monocytogenes , ATCC 19115) and Escherichia coli O6 ( E. coli O6, ATCC 25922).

Techniques: Control, Bacteria

Journal: Foods

Article Title: A Dual-Recognition Electrochemical Sensor Using Bacteria-Imprinted Polymer and Concanavalin A for Sensitive and Selective Detection of Escherichia coli O157:H7

doi: 10.3390/foods14071099

Figure Lengend Snippet: Test of E. coli O157:H7 in milk ( n = 3).

Article Snippet: The bacterial species employed in this study were E. coli O157:H7 (ATCC 43889), Salmonella paratyphi B ( S. Paratyphi B , CMCC 50094), Staphylococcus aureus ( S. aureus , ATCC 25923), Listeria monocytogenes ( L. monocytogenes , ATCC 19115) and Escherichia coli O6 ( E. coli O6, ATCC 25922).

Techniques: Milk

Journal: Frontiers in Microbiology

Article Title: Fis Regulates Type III Secretion System by Influencing the Transcription of exsA in Pseudomonas aeruginosa Strain PA14

doi: 10.3389/fmicb.2017.00669

Figure Lengend Snippet: Fis is required for T3SS gene expression and bacterial cytotoxicity. (A) Relative mRNA levels of T3SS genes exoU, pcrV, exsC, exsA . Total RNA was isolated from bacteria grown with or without 5 mM EGTA and relative mRNA levels of these genes were determined by quantitative real-time PCR. Data represents the mean ± standard deviation from three samples. * p < 0.05; ** p < 0.01; *** p < 0.001 by Student's t -test. (B) PA14, fis ::Tn mutant and fis ::Tn/ att7 :: fis carrying an exoU -His driven by its native promoter (P exoU - exoU -His) were grown at 37°C with or without 5 mM EGTA for 3 h. Proteins samples from equal amounts of protein were separated by SDS-PAGE and the ExoU-His levels were determined by western blotting analysis using an anti-His antibody. (C) Bacterial cytotoxicity on HeLa cells. HeLa cells were infected with indicated strains at a MOI of 50 for 3 h. The bacterial cytotoxicity was determined by the LDH release assay. Error bars indicate standard deviations of triplicate assays. ** p < 0.01 by Student's t -test. (D) Relative mRNA levels of T3SS genes during lung infection. Mice were infected intranasally with indicated strains. 6 hpi, bacteria from BALF were collected, followed by RNA isolation. Relative mRNA levels of exoU, pcrV, exsC , and exsA were determined by quantitative real-time PCR. Data represents the mean ± standard deviation from three independent experiments. * p < 0.05; ** p < 0.01 by Student's t -test.

Article Snippet: The target proteins were hybridized with a rabbit monoclonal anti-His antibody (CST, USA) or a mouse monoclonal anti-Flag antibody (Sigma, USA).

Techniques: Gene Expression, Isolation, Bacteria, Real-time Polymerase Chain Reaction, Standard Deviation, Mutagenesis, SDS Page, Western Blot, Infection, Lactate Dehydrogenase Assay

Journal: Frontiers in Microbiology

Article Title: Fis Regulates Type III Secretion System by Influencing the Transcription of exsA in Pseudomonas aeruginosa Strain PA14

doi: 10.3389/fmicb.2017.00669

Figure Lengend Snippet: Fis is required for the activation of the T3SS. (A) The strain Δ fis /pMMB67EH- fis -His was grown at 37°C with indicated concentrations of IPTG to an OD 600 of 1.0. Proteins samples from equal amounts of protein were separated by SDS-PAGE and the Fis-His levels were determined by western blotting analysis using an anti-His antibody. (B) Relative mRNA levels of T3SS genes. Total RNA of indicated strains was isolated from bacteria grown with indicated concentrations of IPTG and mRNA levels of T3SS genes were determined by quantitative real time PCR. Data represents the mean ± standard deviation from three samples. * p < 0.05; ** p < 0.01; *** p < 0.001 by Student's t -test. (C) Bacterial cytotoxicity on HeLa cells. HeLa cells were infected with indicated strains with indicated concentration of IPTG at a MOI of 50 for 3 h. The bacterial cytotoxicity was determined by the LDH release assay. Error bars indicate standard deviations of triplicate assays. ** p < 0.01, by Student's t -test. (D) HeLa cells were infected with indicated strains at a MOI of 50 for 3 h. The bacterial cytotoxicity was determined by the LDH release assay. Error bars indicate standard deviations of triplicate assays. ** p < 0.01; *** p < 0.001 by Student's t -test. (E) Mice were inoculated intranasally with 1 × 10 7 CFU bacteria of indicated strains. 14 hpi, mice were sacrificed and lungs were isolated and homogenized. Bacterial loads were determined by serial dilution and plating. The central bar indicates the mean, and error bars indicate standard error of the mean. *** p < 0.001 by the Mann-Whitney test.

Article Snippet: The target proteins were hybridized with a rabbit monoclonal anti-His antibody (CST, USA) or a mouse monoclonal anti-Flag antibody (Sigma, USA).

Techniques: Activation Assay, SDS Page, Western Blot, Isolation, Bacteria, Real-time Polymerase Chain Reaction, Standard Deviation, Infection, Concentration Assay, Lactate Dehydrogenase Assay, Serial Dilution, MANN-WHITNEY

Journal: Frontiers in Microbiology

Article Title: Fis Regulates Type III Secretion System by Influencing the Transcription of exsA in Pseudomonas aeruginosa Strain PA14

doi: 10.3389/fmicb.2017.00669

Figure Lengend Snippet: Role of Fis in the regulation of P exsA . (A) Diagram of the P exsA -lacZ transcriptional fusion. (B) PA14 and the fis ::Tn mutant carrying the P exsA -lacZ fusion reporter or empty vector (promoterless lacZ ) were grown at 37°C with or without 5 mM EGTA for 3 h. The values (Miller units) are the means of three experiments. ns, not significant by Student's t -test. (C) Diagram of the P exsA -exsA- His construct. The exsA open reading frame with its upstream 300 bp region was fused with a 6 × His tag at the C-terminus. (D) PA14 and the fis ::Tn mutant carrying an exsA -His driven by its native promoter (P exsA - exsA -His) were grown at 37°C with or without 5 mM EGTA for 3 h. Proteins samples from equal amounts of protein were separated by SDS-PAGE. The ExsA-His levels were determined by western blotting analysis with an anti-His antibody.

Article Snippet: The target proteins were hybridized with a rabbit monoclonal anti-His antibody (CST, USA) or a mouse monoclonal anti-Flag antibody (Sigma, USA).

Techniques: Mutagenesis, Plasmid Preparation, Construct, SDS Page, Western Blot

Journal: Frontiers in Microbiology

Article Title: Fis Regulates Type III Secretion System by Influencing the Transcription of exsA in Pseudomonas aeruginosa Strain PA14

doi: 10.3389/fmicb.2017.00669

Figure Lengend Snippet: exsA transcription relies mainly on P exsC . (A) Relative mRNA levels of exsA . Total RNA of indicated strains was isolated from bacteria grown with or without 5 mM EGTA and mRNA levels of exsA were determined by quantitative real time PCR. Data represents the mean ± standard deviation from three samples. *** p < 0.001 by Student's t -test. (B) Relative mRNA levels of exsC . Total RNA of indicated strains was isolated from bacteria grown with or without 5 mM EGTA and mRNA levels of exsC were determined by quantitative real time PCR. Data represents the mean ± standard deviation from three samples. *** p < 0.001 by Student's t -test. (C) PA14, fis ::Tn mutant, PA14 T0T1 or fis ::Tn T0T1 carrying an exoU -His driven by its native promoter (P exoU - exoU -His) were grown at 37°C with or without 5 mM EGTA for 3 h. Proteins samples from equal amounts of protein were separated by SDS-PAGE. ExoU-His levels were determined by western blotting analysis using an anti-His antibody.

Article Snippet: The target proteins were hybridized with a rabbit monoclonal anti-His antibody (CST, USA) or a mouse monoclonal anti-Flag antibody (Sigma, USA).

Techniques: Isolation, Bacteria, Real-time Polymerase Chain Reaction, Standard Deviation, Mutagenesis, SDS Page, Western Blot

Journal: Nanomaterials

Article Title: Photocatalytic Inactivation of Plant Pathogenic Bacteria Using TiO 2 Nanoparticles Prepared Hydrothermally

doi: 10.3390/nano10091730

Figure Lengend Snippet: Antibacterial activity of A310C and P25 TiO 2 photocatalysts against Xanthomonas arboricola pv. juglandis , Allorhizobium vitis , Erwinia amylovora , and Pseudomonas syringae under UV-A irradiation. The concentration of aqueous TiO 2 dispersions was 0.5 mg/mL. Before UV-A irradiation, dispersions containing bacteria were kept in the dark for 15 min.

Article Snippet: For the pathogen inactivation study, four Gram-negative bacterium species such as the ATCC 49946 strain Erwinia amylovora (American Type Culture Collection, Manassas, VA, USA), the DSMZ 1049 strain Xanthomonas arboricola pv. juglandis (DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), the DC3000 strain Pseudomonas syringae pv. tomato and the Allorhizobium vitis isolates were used.

Techniques: Activity Assay, Irradiation, Concentration Assay, Bacteria