Fwd shuttles Rab11 to the cleavage furrow by both kinase-dependent and -independent mechanisms.

In the embryonic heart, electrical impulses propagate in a unidirectional manner from the sinus venosus and appear to be involved in cardiogenesis. In this work, aligned and random polyaniline/polyetersulfone (PANI/PES) nanofibrous scaffolds doped by Camphor-10-sulfonic acid (β) (CPSA) were fabricated via electrospinning and used to conduct electrical impulses in a unidirectional and multidirectional fashion, respectively. A bioreactor was subsequently engineered to apply electrical impulses to cells cultured on PANI/PES scaffolds. We established cardiovascular disease-specific induced pluripotent stem cells (CVD-iPSCs) from the fibroblasts of patients undergoing cardiothoracic surgeries. The CVD-iPSCs were seeded onto the scaffolds, cultured in cardiomyocyte-inducing factors, and exposed to electrical impulses for 1 h/day, over a 15-day time period in the bioreactor. The application of the unidirectional electrical stimulation to the cells significantly increased the number of cardiac Troponin T (cTnT+) cells in comparison to multidirectional electrical stimulation using random fibrous scaffolds. This was confirmed by real-time polymerase chain reaction for cardiac-related transcription factors (NKX2.5, GATA4, and NPPA) and a cardiac-specific structural gene (TNNT2). Here we report for the first time that applying electrical pulses in a unidirectional manner mimicking the unidirectional wave of electrical stimulation in the heart, could increase the derivation of cardiomyocytes from CVD-iPSCs.

Objective-The goal of this study was to characterize the factors and conditions required for smooth muscle cell (SMC)-directed differentiation of Sox2 ϩ multipotent rat and human skin-derived precursors (SKPs) and to define whether they represent a source of fully functional vascular SMCs for applications in vivo. Methods and Results-We found that rat SKPs can differentiate almost exclusively into SMCs by reducing serum concentrations to 0.5% to 2% and plating them at low density. Human SKPs derived from foreskin required the addition of transforming growth factor-␤1 or -␤3 to differentiate into SMCs, but they did so even in the absence of serum. SMC formation was confirmed by quantitative reverse transcription-polymerase chain reaction, immunocytochemistry, and fluorescence-activated cell sorting, with increased expression of smoothelin-B and little to no expression of telokin or smooth muscle ␥-actin, together indicating that SKPs differentiated into vascular rather than visceral SMCs. Rat and human SKP-derived SMCs were able to contract in vitro and also wrap around and support new capillary and larger blood vessel formation in angiogenesis assays in vivo. Conclusion-SKPs are Sox2ϩ progenitors that represent an attainable autologous source of stem cells that can be easily differentiated into functional vascular SMCs in defined serum-free conditions without reprogramming. SKPs represent a clinically viable cell source for potential therapeutic applications in neovascularization. (Arterioscler Thromb Vasc Biol. 2011;31:2938-2948.)

Background Schizosaccharomyces pombe pik1 encodes a phosphatidylinositol 4-kinase, reported to bind Cdc4, but not Cdc4G107S.Principal FindingsGene deletion revealed that pik1 is essential. In cells with pik1 deleted, ectopic expression of a loss-of-function allele, created by fusion to a temperature-sensitive dihydrofolate reductase, allowed normal cell proliferation at 25°C. At 36°C, cells arrested with abnormally thick, misplaced or supernumerary septa, indicating a defect late in septation. In addition to being Golgi associated, ectopically expressed GFP-tagged Pik1 was observed at the medial cell plane late in cytokinesis. New alleles, created by site-directed mutagenesis, were expressed ectopically. Lipid kinase and Cdc4-binding activity assays were performed. Pik1D709A was kinase-dead, but bound Cdc4. Pik1R838A did not bind Cdc4, but was an active kinase. Genomic integration of these substitutions in S. pombe and complementation studies in Saccharomyces cerevisiae pik1-101 cells revealed that D709 is essential in both cases while R838 is dispensable. In S. pombe, ectopic expression of pik1 was dominantly lethal; while, pik1D709A,R838A was innocuous, pik1R838A was almost innocuous, and pik1D709A produced partial lethality and septation defects. The pik1 ectopic expression lethal phenotype was suppressed in cdc4G107S. Thus, D709 is essential for kinase activity and septation.ConclusionsPik1 kinase activity is required for septation. The Pik1 R838 residue is required for important protein-protein interactions, possibly with Cdc4.