The Pseudomonas aeruginosa exotoxin A translocation domain facilitates the routing of CPP-protein cargos to the cytosol of eukaryotic cells.

The use of cell-penetrating peptides (CPPs), such as polyarginine, has been shown to facilitate the import of drugs and other cargos into cells. However, a major obstacle limiting their use as delivery agents is their entrapment following internalization into endocytic vesicles, leading to either their recycling out of cells or their degradation in lysosomes. To address this challenge, we fused a CPP sequence to the translocation domain of Pseudomonas aeruginosa exotoxin A (ETA) to facilitate the endosomal escape of imported CPP-containing protein constructs. Specifically, a fusion protein incorporating ten arginines linked to residues 253 to 412 of ETA (ETA(253-412)) was tested for its ability to effectively route a protein cargo (enhanced green fluorescent protein, eGFP) to the cytosol of cells. Using flow cytometry and fluorescence live-cell imaging, we observed a 5-fold improvement of cellular uptake as well as a 40-fold increase in cytosolic delivery of the CPP-ETA(253-412)-eGFP construct in relation to CPP-eGFP. Furthermore, analysis of intracellular routing events indicated that the incorporation of ETA(253-412) within the CPP-containing protein fusion construct avoided lysosomal degradation by re-directing the construct from early endosomes to the ER lumen and finally to the cytosol. Studies using inhibitors of vesicular transport confirmed that the ER lumen is a key compartment reached by the CPP-ETA(253-412)-eGFP construct before accessing the cytosol. Together, these findings suggest that incorporating a CPP motif and the ETA translocation domain into protein constructs can facilitate their cytosolic delivery.

Left ventricular diastolic mechanical dyssynchrony and associated clinical outcomes in children with dilated cardiomyopathy.

BACKGROUND: We investigated diastolic mechanical dyssynchrony and its relation to clinical status in pediatric dilated cardiomyopathy (DCM). METHODS AND RESULTS: We calculated a diastolic and systolic dyssynchrony index (standard deviation of time to peak tissue early diastolic/systolic velocity in 12 left ventricular segments) in 33 children with DCM and 46 control subjects. A threshold to diagnose diastolic dyssynchrony was determined, and cardiac function and clinical outcomes were compared between DCM patients with and without diastolic dyssynchrony. Left ventricular wall motion was more synchronized in diastole than in systole. The diastolic dyssynchrony index was significantly higher in children with DCM than in control subjects (28.1+/-18.1 versus 9.1+/-3.8 ms, P<0.0001). A 17-ms threshold indicated the presence of diastolic dyssynchrony. Patients who died or underwent transplantation had greater diastolic dyssynchrony (diastolic dyssynchrony index 37.9+/-20.5 versus 22.1+/-13.8 ms, P=0.01), and the rate of transplant-free survival appeared to be worse for DCM patients with diastolic dyssynchrony than for patients with synchronous DCM (hazard ratio 2.98, P=0.11; hazard ratio adjusted for disease duration 2.95, P=0.17). CONCLUSIONS: Left ventricular diastolic mechanical dyssynchrony is common in pediatric DCM, especially in patients who subsequently experience transplantation or death, and may be associated with a decreased length of transplantation-free survival.