Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Plasmid Preparation, Recombinant, PCR Cloning, Expressing, Mutagenesis, Amplification, Clone Assay

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Analysis of the genetic locus affected in the A. baumannii ATCC 19606T 2010 insertion derivative. (A) Genetic map of the locus harboring the secA gene (ORF 2), which was truncated due to a transposon insertion near its 3′ end (vertical black arrow), and the ORF1 and ORF 3 flanking coding regions. The horizontal arrows represent the location and direction of transcription of predicted coding regions. The locations of the KpnI (K) and PvuII (P) restriction sites used to confirm the EZ::TN insertion site are indicated. The three long black horizontal bars represent cloned DNA regions used as probes in Southern blotting (pMU734), to complement the 2010 insertion derivative (pMU472), or to overproduce a His-tagged SecA derivative (pMU481). The short black horizontal bar flanked by primer numbers indicate the 5′-end secA region used to test transcriptional expression by qRT-PCR. (B and C) Southern blotting of KpnI-PvuII-digested total DNA isolated from the ATCC 19606T parental strain (lanes 2) and the 2010 derivative (lanes 3) probed with pMU734 (B) or the aph gene (C). (D) Western blotting of cytoplasmic proteins isolated from the ATCC 19606T parental strain (lane 1) and the 2010 derivative (lane 2) probed with anti-SecA antibodies. The white arrow indicates the truncated SecA protein produced in the 2010 insertion derivative.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Clone Assay, Southern Blot, Expressing, Quantitative RT-PCR, Isolation, Western Blot, Produced

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Transcriptional analysis of the secA gene in ATCC 19606T and 2010 cells. qRT-PCR was used to detect secA transcripts produced in the ATCC 19606T parental strain and the isogenic 2010 secA insertion mutant using the primers 3976 and 3977, which anneal to the 5′ end of the this gene, using recA expression for normalization. The error bars represent the standard error (SE) of the mean of data collected from three independent biological samples.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Quantitative RT-PCR, Produced, Mutagenesis, Expressing

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Growth of A. baumannii ATCC 19606T and 2010 under different culture conditions. (A) Growth curves of the ATCC 19606T parental strain and the 2010 derivative incubated in LB broth. The OD600 of 1-ml samples taken from a 50-ml LB broth culture was determined hourly for 8 h. (B) Growth of the ATCC 19606T parental strain, the 2010 mutant, its complemented derivative 2010.C, and the t6 BauA mutant incubated in LB broth in the absence of DIP or the presence of 100 μM or 200 μM DIP. Bacterial growth (OD600) was determined after overnight incubation (12 to 14 h) at 37°C in a shaking incubator set at 200 rpm. The error bars show standard errors (SEs) of the means from an assay done in triplicate. Inset, detection of BauA in outer membrane fractions isolated from 2010 (lane 1) and 2010.C (lane 2) cells. SDS-PAGE size-fractionated proteins were probed with anti-BauA polyclonal antibodies.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Incubation, Mutagenesis, Membrane, Isolation, SDS Page

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Cross-feeding bioassays and HPLC analysis of iron-restricted culture supernatants. (A to C) Cross-feeding bioassays were done with LB agar plates containing 200 μM DIP and seeded with 2010 (A), t6 acinetobactin uptake-deficient mutant (B), or s1 acinetobactin synthesis-deficient mutant (C) overnight-cultured bacteria. Sterile filter discs impregnated with 10 μl of 10 mM FeCl3, sterile distilled water, purified acinetobactin (Ab), or cell-free supernatants from 2010 overnight cultures, which contained a subinhibitory concentration of DIP, were deposited on the surface of each plate. Plates were incubated overnight at 37°C before results were recorded. The error bars show the standard errors (SEs) of the means from an assay performed in triplicate. (D) HPLC analysis of M9 minimal medium supernatants from ATCC 19606T (19606), 2010, and t6 overnight cultures. The elution peaks corresponding to acinetobactin (Ab) and the precursor DHBA are indicated. HPLC of sterile M9 minimal medium was used as a negative control.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Mutagenesis, Cell Culture, Bacteria, Sterility, Purification, Concentration Assay, Incubation, Negative Control

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Detection of acinetobactin transport proteins and transcripts. (A) Genetic organization of the bauDCEBA operon coding for acinetobactin transport functions. (B to D) SDS-PAGE of size-fractionated outer membrane (B) and cytoplasmic membrane (C and D) proteins, which were isolated from ATCC 19606T and 2010 cells cultured in LB broth or LB broth containing 100 μM DIP, were blotted onto nitrocellulose filters and probed with anti-BauA (B), anti-BauB (C), or anti-BauE (D) antibodies. The positions of molecular mass markers are shown on the left-hand side of each blot. (E to G) Agarose gel electrophoresis of bauA (E), bauB (F), and bauD (G) RT-PCR amplicons using as the template total RNA isolated from ATCC 19606T and 2010 cells cultured in LB broth or LB broth containing 100 μM DIP. Lane M, molecular size marker.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: SDS Page, Membrane, Isolation, Cell Culture, Agarose Gel Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Proteins differentially produced by the ATCC 19606T parental strain and the 2010 secA isogenic mutant. Proteins were grouped based on their predicted cellular functions. Numbers between parentheses indicate the number of proteins included in each functional category.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Produced, Mutagenesis, Functional Assay

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Growth of the ATCC 19606T parental strain and the 2010 SecA isogenic mutant with different salt concentrations. Experiments were done using 96-well plates containing 200 μl LB broth that were inoculated with 20 μl of overnight cultures and then incubated at 37°C in a plate-shaking incubator. The OD600 was determined hourly for 11 h. The error bars show the standard errors (SEs) of the means from an assay done in triplicate.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Mutagenesis, Incubation

Journal: Infection and Immunity

Article Title: Role of the Carboxy Terminus of SecA in Iron Acquisition, Protein Translocation, and Virulence of the Bacterial Pathogen Acinetobacter baumannii

doi: 10.1128/IAI.02925-14

Figure Lengend Snippet: Role of SecA in virulence. G. mellonella caterpillars (n = 30) were injected with 1 × 105 bacteria of the ATCC 19606T parental strain (19606), the t6 bauA insertion derivative, or the 2010 secA insertion derivative in the absence (A) or the presence (B) of 50 μM FeCl3. Negative controls included noninjected caterpillars or caterpillars injected with comparable volumes of PBS or PBS plus 50 μM FeCl3. Caterpillar death was determined daily for 5 days during incubation at 37°C in darkness.

Article Snippet: Detection by Southern blotting of a single 2.4-kb KpnI-PvuII restriction fragment, the size of which was increased by 2 kb because of the transposon insertion in the 2010 derivative ( and ), indicates that this is the only ATCC 19606 T coding region disrupted by the transposition insertion.

Techniques: Injection, Bacteria, Incubation

Journal: Aging and disease

Article Title: Enhancement of Mesenchymal Stem Cell-Driven Bone Regeneration by Resveratrol-Mediated SOX2 Regulation

doi: 10.14336/ad.2018.0802

Figure Lengend Snippet: Figure 5. RSV induction improves bone healing potential of 6w-MSCs. (A) Critical-sized calvarial defects (8-mm diameter) in rats were covered with fibrin glue, except for defect control treatment. Eight weeks after implantation, bone regeneration was measured by micro-computed tomography. A representative image is shown. (B) This graph shows the bone volume per mm3 (right panel) (n = 10). *, p < 0.05 compared to defect. #, p < 0.05 compared to 6w-MSCs treated with vehicle. (C) Hematoxylin and eosin staining was performed to observe new bone formation. The arrows show the edges of the host bone and line with asterisks indicates newly regenerated bone. Scale bar = 500 μm. (D) To confirm whether the newly regenerated bone was derived from a human origin, immunohistochemistry was performed using antibodies specific to human vimentin. The arrows indicate tissue derived from a human origin. Scale bar = 20 μm. (E) To confirm whether the transplanted 6w-MSCs contributed to bone regeneration of calvarial defects, immunohistochemistry was performed using antibodies against SIRT1, SOX2, RUNX2, and OCN as well as antibodies specific to human vimentin. The nucleus was stained with DAPI, and human VIMENTIN was stained with FITC-conjugated secondary antibody. SIRT1, SOX2, RUNX2, and OCN were stained with phycoerythrin (PE, red)-conjugated secondary antibody. Scale bar = 50 μm. (F) Effect of 6w-MSCs with RSV induction on tumorigenicity in 5-week-old female BALB/C nude mice. (G) Effect on the growth of MKN-74 cells, 6w-MSCs with vehicle induction, and 6w-MSCs with RSV induction xenografted in nude mice, showing no tumor growth in both 6w-MSC groups. (H) G-banding chromosome karyotype from 6w-MSCs with vehicle or RSV induction for 6 weeks. 6w-MSCs with 12th to 14th passages were used for animal experiments.

Article Snippet: Phycoerythrin-conjugated goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology) and FITC-conjugated goat anti-mouse secondary antibodies were used to visualize the primary antibodies.

Techniques: Control, Micro-CT, Staining, Derivative Assay, Immunohistochemistry