Pathological changes following femoral artery thrombosis.

<p>(A. Femoral artery thrombosis without thrombolytic therapy (observation of thrombus morphology in femoral artery thrombosis). B, C. A completely dissolved femoral artery thrombus after thrombolytic therapy. D, E, F. A partially dissolved femoral artery thrombus after thrombolytic therapy. G, H. An undissolved femoral artery thrombus after thrombolytic therapy.)</p

Classification of experimental groups based on three factors (n = 72).

<p>Classification of experimental groups based on three factors (n = 72).</p

ANOVA analysis of femoral artery blood flow at different time point after thrombolysis compared to the baseline blood flow.

<p>ANOVA analysis of femoral artery blood flow at different time point after thrombolysis compared to the baseline blood flow.</p

Schematic diagram of the femoral artery thrombosis model.

<p>Pulsed Doppler flowmetry was used to continuously monitor arterial blood flow. When a thrombus had formed, the pulse Doppler blood flow meter displayed values < 0.05 l/min, while the two-dimensional and color Doppler ultrasound confirmed the presence of an occlusive thrombus.</p

The recanalization rate of femoral artery after thrombolytic therapy at various time points (%) (n = 72).

<p>The recanalization rate of femoral artery after thrombolytic therapy at various time points (%) (n = 72).</p

Arterial blood flow monitored by Doppler flow during thrombolysis (L/min) (n = 72).

<p>Arterial blood flow monitored by Doppler flow during thrombolysis (L/min) (n = 72).</p

Data_Sheet_1_Functional evaluation of constructed pseudo-endogenous microRNA-targeted myocardial ultrasound nanobubble.PDF

BackgroundStem cell transplantation is one of the treatment methods for acute myocardial infarction (AMI). MicroRNA-1 contributes to the study of the essential mechanisms of stem cell transplantation for treating AMI by targeted regulating the myocardial microenvironment after stem cell transplantation at the post-transcriptional level. Thus, microRNA-1 participates in regulating the myocardial microenvironment after stem cell transplantation, a promising strategy for the Stem cell transplantation treatment of AMI. However, the naked microRNA-1 synthesized is extremely unstable and non-targeting, which can be rapidly degraded by circulating RNase. Herein, to safely and effectively targeted transport the naked microRNA-1 synthesized into myocardial tissue, we will construct pseudo-endogenous microRNA-targeted myocardial ultrasound nanobubble pAd-AAV-9/miRNA-1 NB and evaluate its characteristics, targeting, and function.MethodsThe pAd-AAV-9/miRNA-1 gene complex was linked to nanobubble NBs by the “avidin-biotin bridging” method to prepare cardiomyocyte-targeted nanobubble pAd-AAV-9/miRNA-1 NB. The shape, particle size, dispersion, and stability of nanobubbles and the connection of pAd-AAV-9/miRNA-1 gene complex to nanobubble NB were observed. The virus loading efficiency was determined, and the myocardium-targeting imaging ability was evaluated using contrast-enhanced ultrasound imaging in vivo. The miRNA-1 expression level in myocardial tissue and other vital organs ex vivo of SD rats was considered by Q-PCR. Also, the cytotoxic effects were assessed.ResultsThe particle size of NBs was 504.02 ± 36.94 nm, and that of pAd-AAV-9/miRNA-1 NB was 568.00 ± 37.39 nm. The particle size and concentration of pAd-AAV-9/miRNA-1 NBs did not change significantly within 1 h at room temperature (p > 0.05). pAd-AAV-9/miRNA-1 NB had the highest viral load rate of 86.3 ± 2.2% (p  0.05).ConclusionThe pAd-AAV-9/miRNA-1 NB constructed in this study could carry naked miRNA-1 synthesized in vitro for targeted transport into myocardial tissue successfully and had sound contrast-enhanced imaging effects in vivo.</p

A multiple-sequence alignment of the amino acid sequence in GJA8 from different species.

<p>The alignment data indicates that the Glu at the 201st amino acid position is highly conserved among many species (indicated by an arrow).</p

Functional analysis of GJA8 mutation.

<p>(A) Western blotting of wild-type and mutant GJA8. Hela cells were transfected with GJA8WT and GJA8E201K expression plasmids, respectively and western blotting was performed with GFP antibody. (B) Subcellular localization of GJA8. GFP-GJA8 expression constructs containing wild-type or mutant were transfected into Hela cells using Lipofectamine 2000. Localization of wild-type and mutant GJA8 GFP-fusion protein in transfected Hela cells was viewed using a fluorescent microscope. (C) Induction of GJA8E201K protein expression decreased the number of cells. Phase contrast photomicrographs from cells containing wild-type or mutant GJA8. GJA8WT (left panel) and GJA8E201K (right panel) cells were observed using a phase contrast microscope after transfection for wild-type and mutant GJA8 expression plasmids for 72 hours. Bar, 100 μm. (D) Graph shows the quantitation of these data as mean ± S.E.M. of the number of cells containing wild-type or mutant GJA8, respectively. *, p<0.05; **, p<0.01 (n = 3). (E) Induction of GJA8E201K protein expression decreased the viability of cells by MTT assay. Cells were transfected with wild-type and mutant GJA8 vectors for 3 days and cell viability was tested using MTT assay.</p

Partial sequence of GJA8 at exon2.

<p>A: Sequence of affected individual. In panel A, the heterozygous mutation c.601G>A was identified in all affected participants. B: Sequence of unaffected individual.</p