Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Formoterol (FORM) suppressed EPO‐induced VSMC senescence in mouse aorta. A) GO enrichment for the intersection genes. B) KEGG enrichment for the intersection genes. C) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of γH2AX (red particles) and αSMA (specific for SMCs, green particles) in ApoE −/− mice receiving vehicle, EPO and EPO+ medium‐dose formoterol treatment, respectively (scale bars = 10 µm). D) Quantitative analysis of γH2AX/αSMC colocalization in (C) ( n = 6 per group). E) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of SIRT1 (green particles) and αSMA (specific for SMCs, red particles) in ApoE −/− mice receiving vehicle, EPO and EPO+medium‐dose formoterol treatment, respectively. (scale bars = 10 µm). F) Quantitative analysis of γH2AX/αSMC colocalization in (E) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, One‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Formoterol ameliorated aortic senescence via β2AR. A) Representative immunofluorescent analysis of the abdominal aortic sections for detecting the colocalization (yellow particles) of β2AR (green particles) and αSMA (specific for SMCs, red particles) in the aorta of ApoE −/− mice (scale bars = 10 µm). B) Representative western blot analysis of protein expression of SIRT1 and P21 in VSMCs treated with vehicle, EPO, EPO+0.01 nmol mL −1 formoterol, EPO+0.1 nmol mL −1 formoterol, EPO+1 nmol mL −1 formoterol and EPO+10 nmol mL −1 formoterol, respectively. C,D) Quantitative analysis of western blot in (B) ( n = 6 per group). E) Representative sections of SA‐β‐gal staining of VSMCs transfected with β2AR siRNA (siβ2AR) or negative control siRNA (siNC) and treated with vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. (scale bars = 10 µm). F) Quantitative analysis of percentage of VSMCs with positive SA‐β‐gal staining in (E) ( n = 6 per group). G) Representative western blot analysis of protein expression of SIRT1, P21 in VSMCs transfected with β2AR siβ2AR or siNC and treated with vehicle, EPO and EPO + 0.1 nmol mL −1 formoterol, respectively. H‐I) Quantitative analysis of western blot in (G) ( n = 6 per group). J) Representative images of immunostaining of SIRT1 in VSMCs transfected with β2AR siβ2AR or siNC and treated with vehicle, EPO, and EPO+ 0.1 nmol mL −1 formoterol, respectively. K) Quantitative analysis of colocalization of dapi/SIRT1 in VSMCs in(G) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, Two‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Western Blot, Expressing, Staining, Transfection, Negative Control, Immunostaining, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: cAMP‐regulated the effect of formoterol on VSMC senescence. A) Quantification of intracellular cAMP levels in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. B) Representative images of SA‐β‐gal staining of VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. (scale bars = 10 µm). C) Quantitative analysis of SA‐β‐gal staining in (B) ( n = 6 per group). D) Representative western blot assay of SIRT1, P21 in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. E‐F) Quantitative analysis of protein expression of SIRT1, P21 protein levels by western blot in (D), ( n = 6 per group). G) Representative images of immunostaining of SIRT1 in VSMC treated with mAC inhibitor SQ22536 (80 µM) or control solution DMSO receiving vehicle, EPO and EPO+0.1 nmol mL −1 formoterol, respectively. H) Quantitative analysis of colocalization of dapi/SIRT1 in (G) ( n = 6 per group). * p < 0.05, ** p < 0.01, *** p < 0.001, Two‐way ANOVA followed by Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Control, Staining, Western Blot, Expressing, Immunostaining, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Casitas B‐lineage lymphoma (CBL) was essential for EPO‐induced VSMC senescence. A) Representative immunofluorescent analysis of the colocalization (yellow particles) of CBL (red particles) and SIRT1 (green particles) in VSMC receiving vehicle and EPO treatment respectively (scale bars = 10 µm). B) Quantitative analysis of CBL/SIRT1 colocalization in (A) ( n = 6 per group). C) Representative western blot analysis of protein expression of SIRT1 and P21 in VSMC transfected with siCBL or negative control siRNA (siNC) and treated with vehicle and EPO respectively. D‐E) Quantitative analysis of western blot in (C) ( n = 6 per group). F) Representative sections of SA‐β‐gal staining of VSMC transfected with CBL siRNA (siCBL) or negative control siRNA (siNC) and treated with vehicle and EPO respectively (scale bars = 10 µm). G) Quantitative analysis of the percentage of VSMC with positive SA‐β‐gal staining in (F) ( n = 6 per group). ** p < 0.01, *** p < 0.001, unpaired two‐tailed Student's t‐tests with Welch's correction were applied in (B). The other data were analyzed via Two‐way ANOVA followed by the Tukey test for post hoc comparison, mean ± SEM.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Western Blot, Expressing, Transfection, Negative Control, Staining, Two Tailed Test, Comparison

Journal: Advanced Science

Article Title: Medium‐Dose Formoterol Attenuated Abdominal Aortic Aneurysm Induced by EPO via β2AR/cAMP/SIRT1 Pathway

doi: 10.1002/advs.202306232

Figure Lengend Snippet: Schematic diagram showing the mechanism of therapeutic effects of medium‐dose formoterol on EPO‐induced AAA. Formoterol binds to β2AR and activates cAMP, which increases SIRT1 protein expression, leading to suppressed VSMC senescence induced by EPO. In contrast, SIRT1 is downregulated by EPO via activation of CBL, resulting in aggravated VSMC senescence. Thus, medium‐dose formoterol attenuated EPO‐induced AAA via β2AR/cAMP/SIRT1 pathways, which provides a promising medication for the treatment of AAA.

Article Snippet: [ ] In the fourth part of the in vitro experiments, to detect the effect of EPO on CBL/SIRT1 colocalization, VSMC were treated with vehicle (PBS) and 5IU mL −1 EPO (287‐TC‐500, R&D Systems, USA), respectively.

Techniques: Expressing, Activation Assay

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 1 Agarose gel electrophoresis analysis of DMOMP. Notes: Molecular weight (1 kb) marker (lane 1), native phCMV1 vector (lane 2), native phCMV1 MOMP (lane 3), phCMV1 vector (4239 bp) restricted BamH 1 and Not 1 (lane 4), phCMV1 MOMP positive clones (4212 bp and 1134 bp) restricted BamH 1 and Not 1 (lanes 5 and 6) and phCMV1 MOMP clones (lanes 7 and 8) restricted Pst 1 (4823 bp and 531 bp).

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: Agarose Gel Electrophoresis, Molecular Weight, Marker, Plasmid Preparation, Clone Assay

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 3 Size and morphological assessments of nanoparticles by SEM and TEM. SEM analyses of CNP (A), DMCNP (B), and TEM analysis of CNP (C) and DMCNP (D). Note: A drop of the nanoparticles was deposited on a copper grid for TEM, or a glass slide mounted on a stub for SEM. Abbreviations: CNP, phosphate buffered saline encapsulated in chitosan nanoparticles; DMOMP, DNA of the major outer membrane protein of C. trachomatis DMOMP; DMCNP, DMOMP encapsulated in chitosan nanoparticles.

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: Saline, Membrane

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 4 Cumulative DNA release and electrophoretic analysis of released DMOMP. (A) Analysis of encapsulated DMOMP released from DMCNP was evaluated using similar intestinal fluid (SIF, pH 7.0) and similar gastrointestinal fluid (SGF, pH 2.0). Samples were collected at each designated time-interval (2 hours, day 1, 2, 4, 5 and 7) and the released DNA measured using NanoDrop at 260 nm. Each bar represents the mean ± standard deviation of triplicate samples. (B) The released products from each time- point were precipitated with 5 M NaCl and amplified by PCR and amplicons subjected to agarose gel electrophoresis for verification of the specific DMOMP product. Notes: Lanes indicate the sequential collection of amplicons as follows: lane 1 (1 kb marker), lane 2 (2 hours), lane 3 (day 1), lane 4 (day 2), lane 5 (day 4), lane 6 (day 5), lane 7 (day 7) and lane 8 (positive DMOMP clone) with an expected size of 1154 bp. Abbreviations: DMOMP, DNA of the major outer membrane protein of C. trachomatis; DMCNP, DMOMP encapsulated in chitosan nanoparticles; hrs, hours; PBS, phosphate buffered saline; PCR, polymerase chain reaction; SGF, similar gastrointestinal fluid; SIF, similar intestinal fluid.

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: Standard Deviation, Amplification, Agarose Gel Electrophoresis, Marker, Membrane, Saline, Polymerase Chain Reaction

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 5 Stability studies of DMOMP in chitosan nanoparticles. (A) Electrophoretic analysis of CNP protection of encapsulated DMOMP after incubation with chitosanase, DNAase I, and restriction enzymes. Both DMCNP and DMOMP (1 mg/mL) were subjected to enzymatic digestion with NdeI and SalI in combination with, or without chitosanase or with DNAase 1. Lane 1, (1 kb molecular marker), lane 2 (trapped nanoparticle in well), lane 3 (released DMOMP in red box), lanes 4 and 5 (degraded released DMOMP), lane 6 (blank), lane 7 (1 kb marker), and lane 8 (DMOMP positive clone). (B) pH stability of encapsulated DMOMP. Notes: DMCNP was added to individual micro-centrifuge tubes and adjusted to various pH values (2, 4, 6, 8, 9, 10, 10.5 and 12) followed by incubation at 37°C on a shaker for 30 minutes. Lanes 1 and 10 are 1 kb marker. All samples were analyzed by agarose gel electrophoresis and visualized using the ChemiImager gel documentation system. Abbreviations: CNP, phosphate buffered saline encapsulated in chitosan nanoparticles; DMOMP, DNA of the major outer membrane protein of C. trachomatis; DMCNP, DMOMP encapsulated in chitosan nanoparticles; RE, restriction.

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: Incubation, Marker, Agarose Gel Electrophoresis, Saline, Membrane

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 7 In vitro expression of MOMP protein in transfected Cos-7 cells. Cos-7 cells (1 × 106 cells/well) were transfected by electroporation with DMOMP (A) and DMCNP (B) at concentrations of 2, 5 and 10 µg. Notes: Transfected cells were incubated for 48 hrs at 37°C, fixed and blocked prior to incubation with goat anti-C. trachomatis polyclonal antibodies followed by a secondary FITC rabbit anti-Goat IgG (H+L) antibody. Immunofluorescence of cells were visualized using a Nikon Eclipse Ti-U microscope. Abbreviations: DMOMP, DNA of the major outer membrane protein of C. trachomatis; DMCNP, DMOMP encapsulated in chitosan nanoparticles; MOMP, major outer membrane protein of C. trachomatis.

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: In Vitro, Expressing, Transfection, Electroporation, Incubation, Immunofluorescence, Microscopy, Membrane

Journal: International Journal of Nanomedicine

Article Title: Formulation, characterization, and expression of a recombinant MOMP Chlamydia trachomatis DNA vaccine encapsulated in chitosan nanoparticles

doi: 10.2147/ijn.s42723

Figure Lengend Snippet: Figure 8 Expression of MOMP at the protein and gene transcript levels. (A) Cos- 7 cells were transfected as described in Figure 7, immunostained (positive MOMP fluorescence cells) and then mounted with DAPI (blue nuclei stain) combined with an anti-fade mounting solution. (B) Bright-field visualization of Cos-7 cell monolayer showing the MOMP expressed protein. Red circle (positive MOMP fluorescence cells) shows expression of the MOMP protein. (C) Confirmation of expressed MOMP protein by western blot. Cos-7 cells (4 × 105 cells/well) were transfected with DMCNP or phCMV1 vector using Lipofectamine and incubated at 37°C for 48 hours. Cell lysates were collected, run on an SDS-PAGE gel, transferred onto a PVDF membrane and probed using anti-MOMP polyclonal antibodies followed by an Alexa fluor 680 secondary antibody. The bound antibody was viewed using LI-COR Odyssey imaging apparatus. (D) In vitro expression of MOMP gene transcript. Notes: RNA samples were extracted from mouse thigh muscles and spleens reversed transcribed to cDNA and then subjected to RT-PCR amplification of the MOMP gene transcript using MOMP specific primers. Lane 1 (MW marker), lanes 2 and 7 (DMOMP positive clones), lane 3 (thigh muscle of DMCNP mice), lane 4 (thigh muscle of PBS mice), lane 5 (spleen from DMCNP mice), and lane 6 (spleen from PBS mice). Red rectangle shows positive MOMP gene transcripts. Abbreviations: DMCNP, DMOMP encapsulated in chitosan nanoparticles; DMOMP, DNA of the major outer membrane protein of C. trachomatis; MOMP, major outer membrane protein of C. trachomatis; PVDF, polyvinylidene difluoride.

Article Snippet: Polyclonal antibodies against C. trachomatis (MOMP) were purchased from Fitzgerald Industries International (Acton, MA, USA), with FITC rabbit anti-goat antibodies IgG (H+L) being purchased from Southern Biotech (Birmingham, AL, USA).

Techniques: Expressing, Transfection, Fluorescence, Staining, Western Blot, Plasmid Preparation, Incubation, SDS Page, Membrane, Imaging, In Vitro, Muscles, Reverse Transcription Polymerase Chain Reaction, Amplification, Marker, Clone Assay