Integration-free iPS cells engineered using human artificial chromosome vectors

Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.     

The effectiveness of RNAi in Caenorhabditis elegans is maintained during spaceflight

Background

Overcoming spaceflight-induced (patho)physiologic adaptations is a major challenge preventing long-term deep space exploration. RNA interference (RNAi) has emerged as a promising therapeutic for combating diseases on Earth; however the efficacy of RNAi in space is currently unknown.

Methods

Caenorhabditis elegans were prepared in liquid media on Earth using standard techniques and treated acutely with RNAi or a vector control upon arrival in Low Earth Orbit. After culturing during 4 and 8 d spaceflight, experiments were stopped by freezing at −80°C until analysis by mRNA and microRNA array chips, microscopy and Western blot on return to Earth. Ground controls (GC) on Earth were simultaneously grown under identical conditions.

Results

After 8 d spaceflight, mRNA expression levels of components of the RNAi machinery were not different from that in GC (e.g., Dicer, Argonaute, Piwi; P>0.05). The expression of 228 microRNAs, of the 232 analysed, were also unaffected during 4 and 8 d spaceflight (P>0.05). In spaceflight, RNAi against green fluorescent protein (gfp) reduced chromosomal gfp expression in gonad tissue, which was not different from GC. RNAi against rbx-1 also induced abnormal chromosome segregation in the gonad during spaceflight as on Earth. Finally, culture in RNAi against lysosomal cathepsins prevented degradation of the muscle-specific α-actin protein in both spaceflight and GC conditions.

Conclusions

Treatment with RNAi works as effectively in the space environment as on Earth within multiple tissues, suggesting RNAi may provide an effective tool for combating spaceflight-induced pathologies aboard future long-duration space missions. Furthermore, this is the first demonstration that RNAi can be utilised to block muscle protein degradation, both on Earth and in space.     

Regulation of macrophage differentiation and polarization by group IVC phospholipase A₂

Although the cellular function of group IVC phospholipase A(2) (IVC-PLA(2)) remains to be understood, the expression of IVC-PLA(2) in human monocytic THP-1 cells was increased during phorbol ester-induced macrophage differentiation. We therefore examined the role of IVC-PLA(2) in macrophage differentiation using THP-1 cells. Two THP-1 cell lines stably expressing IVC-PLA(2)-specific shRNA were established. Differentiation and maturation into macrophages on treatment with phorbol ester were facilitated by knockdown of IVC-PLA(2) without the compensatory induction of mRNA expression for other group IV and VI PLA(2)s. Furthermore, the enhancement of macrophage differentiation by IVC-PLA(2)-knockdown were abolished by treatment with lysophosphatidylcholine, a metabolite of phospholipids generated by PLA(2)-mediated hydrolysis, indicating that PLA(2) activity is necessary for the inhibition of macrophage differentiation by IVC-PLA(2). Additionally, we found that the differentiation into classically activated M1 macrophage was superior in IVC-PLA(2)-knockdown cells, whereas the differentiation into alternatively activated M2 macrophage was suppressed by IVC-PLA(2)-knockdown. These findings suggest that IVC-PLA(2) is involved in regulations of macrophage differentiation and macrophage polarization.     

A congenital muscular dystrophy with mitochondrial structural abnormalities caused by defective de novo phosphatidylcholine biosynthesis

Congenital muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized clinically by muscle weakness and hypotonia in early infancy. A number of genes harboring causative mutations have been identified, but several cases of congenital muscular dystrophy remain molecularly unresolved. We examined 15 individuals with a congenital muscular dystrophy characterized by early-onset muscle wasting, mental retardation, and peculiar enlarged mitochondria that are prevalent toward the periphery of the fibers but are sparse in the center on muscle biopsy, and we have identified homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB). This is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. In muscle of three affected individuals with nonsense mutations, choline kinase activities were undetectable, and phosphatidylcholine levels were decreased. We identified the human disease caused by disruption of a phospholipid de novo biosynthetic pathway, demonstrating the pivotal role of phosphatidylcholine in muscle and brain.     

Sip1, a Conserved AP-1 Accessory Protein,Is Important for Golgi/Endosome Trafficking in Fission Yeast

We had previously identified the mutant allele of apm1⁺ that encodes a homolog of the mammalian μ 1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex and demonstrated that the AP-1 complex plays a role in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. Here, we isolated a mutant allele of its4⁺/sip1⁺, which encodes a conserved AP-1 accessory protein. The its4-1/sip1-i4 mutants and apm1 -deletion cells exhibited similar phenotypes, including sensitivity to the calcineurin inhibitor FK506, Cl⁻ and valproic acid as well as various defects in Golgi/endosomal trafficking and cytokinesis. Electron micrographs of sip1-i4 mutants revealed vacuole fragmentation and accumulation of abnormal Golgi-like structures and secretory vesicles. Overexpression of Apm1 suppressed defective membrane trafficking in sip1-i4 mutants. The Sip1-green fluorescent protein (GFP) co-localized with Apm1-mCherry at Golgi/endosomes, and Sip1 physically interacted with each subunit of the AP-1 complex. We found that Sip1 was a Golgi/endosomal protein and the sip1-i4 mutation affected AP-1 localization at Golgi/endosomes, thus indicating that Sip1 recruited the AP-1 complex to endosomal membranes by physically interacting with each subunit of this complex. Furthermore, Sip1 is required for the correct localization of Bgs1/Cps1, 1,3-β-D-glucan synthase to polarized growth sites. Consistently, the sip1-i4 mutants displayed a severe sensitivity to micafungin, a potent inhibitor of 1,3-β-D-glucan synthase. Taken together, our findings reveal a role for Sip1 in the regulation of Golgi/endosome trafficking in coordination with the AP-1 complex, and identified Bgs1, required for cell wall synthesis, as the new cargo of AP-1-dependent trafficking.     

N-Acetylglucosaminyltransferase V regulates TGF-β response in hepatic stellate cells and the progression of steatohepatitis

N-Acetylglucosaminyltransferase V (GnT-V), catalyzing β1-6 branching in asparagine-linked oligosaccharides, is one of the most important glycosyltransferases involved in tumor metastasis and carcinogenesis. Although the expression of GnT-V is induced in chronic liver diseases, the biological meaning of GnT-V in the diseases remains unknown. The aim of this study was to investigate the effects of GnT-V on the progression of chronic hepatitis, using GnT-V transgenic (Tg) mice fed a high fat and high cholesterol (HFHC) diet, an experimental model of murine steatohepatitis. Although enhanced hepatic lymphocytes infiltration and fibrosis were observed in wild-type (WT) mice fed the HFHC diet, they were dramatically prevented in Tg mice. In addition, the gene expression of inflammatory Th1 cytokines in the liver was significantly decreased in Tg mice than WT mice. Inhibition of liver fibrosis was due to the dysfunction of hepatic stellate cells (HSCs), which play pivotal roles in liver fibrosis through the production of transforming growth factor (TGF)-β1. Although TGF-β1 signaling was enhanced in Tg mouse-derived HSCs (Tg-HSCs) compared with WT mouse-derived HSCs (WT-HSCs), collagen expression was significantly reduced in Tg-HSCs. As a result from DNA microarray, cyclooxygenase-2 (COX2) expression, known as a negative feedback signal for TGF-β1, was significantly elevated in Tg-HSCs compared with WT-HSCs. Prostaglandin E2 (PGE2), the product of COX2, production was also significantly elevated in Tg-HSCs. COX2 inhibition by celecoxib decreased PGE2 and increased collagen expression in Tg-HSCs. In conclusion, GnT-V prevented steatohepatitis progression through modulating lymphocyte and HSC functions.     

Distribution of putative denitrifying methane oxidizing bacteria in sediment of a freshwater lake, Lake Biwa

Methane oxidation coupled to denitrification is mediated by 'Candidatus Methylomirabilis oxyfera', which belongs to the candidate phylum NC10. The distribution of putative denitrifying methane-oxidizing bacteria related to "M. oxyfera" was investigated in a freshwater lake, Lake Biwa, Japan. In the surface layer of the sediment from a profundal site, a phylotype closely related to "M. oxyfera" was most frequently detected among NC10 bacteria in PCR analysis of the 16S rRNA gene. In the sediment, sequences related to "M. oxyfera" were also detected in a pmoA gene library. The presence of NC10 bacteria was also confirmed by catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR indicated that the abundance of the "M. oxyfera"-related phylotype was higher in the upper layers of the profundal sediment. The horizontal distribution of the putative methanotrophs in lake sediment was also analyzed by DGGE, which revealed that their occurrence was restricted to deep water areas. These results agreed with those in a previous study of another freshwater lake, and suggested that the upper layer of the profundal sediments is the main habitat for denitrifying methanotrophs.