Aarne Fleischer

The anti‐apoptotic molecules Bcl‐xL and Bcl‐w target protein phosphatase 1α to Bad

Bcl‐xL and Bcl‐w specifically interact with PP1α and Bad. A phosphatase activity sensitive to okadaic acid was detected in Bcl‐xL, Bcl‐w and Bad immunoprecipitates. Serine phosphorylation of Bcl‐xL and Bcl‐w correlates with the number of trimolecular complexes formed. Depletion of Bcl‐xL and Bcl‐w decreases the remaining Bad‐associated phosphatase activity and association of protein phosphatase 1 (PP1)α to Bad. Bcl‐xL and Bcl‐w contain the R/K X V/I X F consensus motif shared by PP1 targeting subunits. This motif, in addition to F X X R X R motif, is involved in binding of Bcl‐xL and Bcl‐w to PP1α. Disruption of Bcl‐xL/PP1α or Bcl‐w/PP1α association strongly decreases Bad‐associated phosphataseactivity and stability of trimolecular complexes. These results suggest that Bcl‐xL and Bcl‐w are PP1α targeting subunits and this trimolecular complex may be involved in the control of apoptosis.

Segregation of Bad from lipid rafts is implicated in the induction of apoptosis

Many molecules relocate subcellularly in cells undergoing apoptosis. Using coimmunoprecipitation experiments we demonstrate that Bad is not associated to 14-3-3 protein, suggesting a new mechanism for the control of the proapoptotic role of Bad. Here we show, by confocal microscopy and cellular fractionation, that Bad is attached to lipid rafts in IL-4-stimulated cells and thymocytes while associated with mitochondria in IL-4-deprived cells. Disruption of lipid rafts by methyl-β-cyclodextrin treatment induces segregation of Bad from rafts, which correlates with apoptosis. Our results suggest that the interaction of Bad with rafts is a dynamic process regulated by IL-4 and involved in the control of apoptosis.

The Association of Aiolos Transcription Factor and Bcl-xL Is Involved in the Control of Apoptosis

We have analyzed the mechanism implicated in the control of the anti-apoptotic role of Bcl-xL. We show that IL-4 deprivation induces apoptosis, but does not modulate Bcl-xL expression. Because Bcl-xL does not promote cell survival in the absence of IL-4, we investigate the mechanism by which Bcl-xL was unable to inhibit apoptosis. Using yeast two-hybrid system, coimmunoprecipitation, and indirect immunofluorescence techniques, we found that Bcl-xL interacts with the transcription factor Aiolos in IL-4-stimulated cells, increasing upon IL-4 deprivation. IL-4 does not promote translocation of Aiolos or Bcl-xL, but induces tyrosine phosphorylation of Aiolos, which is required for dissociation from Bcl-xL. Transfection experiments confirm that cells overexpressing Bcl-xL are able to prevent apoptosis in the absence of IL-4. On the contrary, cells that overexpress Bcl-xL and Aiolos are unable to block apoptosis in the absence of IL-4. We propose a model for the regulation of the Bcl-xL anti-apoptotic role via Aiolos.

Generation of Mouse and Human Induced Pluripotent Stem Cells (iPSC) from Primary Somatic Cells

Cellular reprogramming consists of the conversion of differentiated cells into pluripotent cells; the so-called induced Pluripotent Stem Cells. iPSC are amenable to in vitro manipulation and, in theory, direct production of any differentiated cell type. Furthermore, iPSC can be obtained from sick individuals and subsequently used for disease modeling, drug discovery and regenerative treatments. iPSC production was first achieved by transducing, with the use of retroviral vectors, four specific transcription factors: Oct4, Klf4, Sox2 and c-Myc (OKSM), into primary cells in culture Takahashi and Yamanaka, (Cell 126(4):663–676, 2006). Many alternative protocols have since been proposed: repeated transfections of expression plasmids containing the four pluripotency-associated genes Okita et al. (Science 322(5903):949–953, 2008), lentiviral delivery of the four factors Sommer et al. (Stem Cells 27(3):543–549, 2009), Sendai virus delivery Fusaki et al. (Proceedings of the Japan Academy. Series B, Physical and Biological Sciences 85(8):348–362, 2009), removal of the reprogramming vectors by ‘piggyBac’ transposition Woltjen et al. (Nature 458(7239):766–770, 2009); Kaji et al. (Nature 458(7239):771–775, 2009), Cre-recombinase excisable viruses Soldner et al. (Cell 136(5):964–977, 2009), episomal vectors Yu et al. (Science 324(5928):797–801, 2009), cell-penetrating reprogramming proteins Zhou et al. (Stem Cells 4(5):381–384, 2009), mammalian artificial chromosomes Hiratsuka et al. (PLoS One 6(10):e25961, 2011) synthetically modified mRNAs Warren et al. (Scientific Reports 2:657, 2012), miRNA Anokye-Danso et al. (Cell Stem Cell 8(4):376–388, 2009); however, although some of these methods are commercially available, in general they still need to attain the reproducibility and reprogramming efficiency required for routine applications Mochiduki and Okita (Biotechnol Journal 7(6):789–797, 2012). Herein we explain, in four detailed protocols, the isolation of mouse and human somatic cells and their reprogramming into iPSC. All-encompassing instructions, not previously published in a single document, are provided for mouse and human iPSC colony isolation and derivation. Although mouse and human iPSC share similarities in the cellular reprogramming process and culture, both cell types need to be handled differently.

Pulmonary delivery of tobramycin-loaded nanostructured lipid carriers for Pseudomonas aeruginosa infections associated with cystic fibrosis.

Among the pathogens that affect cystic fibrosis (CF) patients, Pseudomonas aeruginosa is the most prevalent. As a way to fight against this infection, nanotechnology has emerged over the last decades as a promising alternative to overcome resistance to antibiotics in infectious diseases. The goal of this work was to elaborate and characterize lipid nanoparticles for pulmonary delivery of tobramycin. Tobramycin-loaded nanostructured lipid carriers (Tb-NLCs) were prepared by hot melt homogenization technique. In addition, nanoparticles labeled with infrared dye (IR-NLCs) were used to investigate their in vivo performance after pulmonary administration. Tb-NLCs displayed a mean diameter size around 250 nm, high drug encapsulation (93%) and sustained release profile. Tb-NLCs showed to be active against clinically isolated P. aeruginosa. Moreover, Tb-NLCs did not decrease cell viability and were able to overcome an artificial mucus barrier in the presence of mucolytics agents. During the in vivo assay, IR-NLCs were administered to several mice by the intratracheal route using a Penn Century device. Next, the biodistribution of the nanoparticles was analyzed at different time points showing a wide nanosystem distribution in the lungs. Altogether, tobramycin-loaded NLCs seem to us an encouraging alternative to the currently available CF therapies.

Generation of two induced pluripotent stem cells lines from Mucopolysaccharydosis IIIA patient: IMEDEAi004-A and IMEDEAi004-B

Mucoplysaccharydosis IIIA (MPSIIIA) is the most severe form of Sanfilippo syndrome. Skin fibroblasts from a MPSIIIA compound heterozygous (E447K/R245H) patient were nucleofected with four OriP/EBNA1-based episomal plasmids containing: OCT3/4, SOX2, KLF4, L-Myc, LIN28, BCL-xL and shp53. The two iPSCs lines generated carry both sulfamidase enzyme (SGSH) mutations, are free of plasmid integration, have normal karyotype, express pluripotency-associated markers and are able to differentiate into the three germ layers.

Generation of two induced pluripotent stem cell (iPSC) lines from p.F508del Cystic Fibrosis patients

Cystic Fibrosis (CF) is a monogenic, lethal disease caused by mutations in the cystic fibrosis transmembrane conductance (CFTR) gene. Here we report the production of CF-iPS cell lines from two different p.F508del homozygous female patients (Table 1). Two different primary cell types, skin fibroblasts and keratinocytes, were transfected with retroviral cocktails containing four: c-MYC, KLF4, OCT4 and SOX2 (MKOS) or three: KLF4, OCT4 and SOX2 (KOS) reprogramming factors. Two fibroblast-derived MKOS lines are described in the main text. The lines carry the p.F508del mutation, have a normal karyotype, express pluripotency markers and are able to differentiate into the three germ layers.