Establishment and maintenance of human embryonic stem cell lines on human feeder cells derived from uterine endometrium under serum-free condition

Human embryonic stem (hES) cells are usually established and maintained on mouse embryonic fibroblast (MEFs) feeder layers. However, it is desirable to develop human feeder cells because animal feeder cells are associated with risks such as viral infection and/or pathogen transmission. In this study, we attempted to establish new hES cell lines using human uterine endometrial cells (hUECs) to prevent the risks associated with animal feeder cells and for their eventual application in cell-replacement therapy. Inner cell masses (ICMs) of cultured blastocysts were isolated by immunosurgery and then cultured on mitotically inactivated hUEC feeder layers. Cultured ICMs formed colonies by continuous proliferation and were allowed to proliferate continuously for 40, 50, and 55 passages. The established hES cell lines (Miz-hES-14, -15, and -9, respectively) exhibited typical hES cells characteristics, including continuous growth, expression of specific markers, normal karyotypes, and differentiation capacity. The hUEC feeders have the advantage that they can be used for many passages, whereas MEF feeder cells can only be used as feeder cells for a limited number of passages. The hUECs are available to establish and maintain hES cells, and the high expression of embryotrophic factors and extracellular matrices by hUECs may be important to the efficient growth of hES cells. Clinical applications require the establishment and expansion of hES cells under stable xeno-free culture systems.     

The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14

ULK1 (unc-51 like autophagy activating kinase 1), the key mediator of MTORC1 signaling to autophagy, regulates early stages of autophagosome formation in response to starvation or MTORC1 inhibition. How ULK1 regulates the autophagy induction process remains elusive. Here, we identify that ATG13, a binding partner of ULK1, mediates interaction of ULK1 with the ATG14-containing PIK3C3/VPS34 complex, the key machinery for initiation of autophagosome formation. The interaction enables ULK1 to phosphorylate ATG14 in a manner dependent upon autophagy inducing conditions, such as nutrient starvation or MTORC1 inhibition. The ATG14 phosphorylation mimics nutrient deprivation through stimulating the kinase activity of the class III phosphatidylinositol 3-kinase (PtdIns3K) complex and facilitates phagophore and autophagosome formation. By monitoring the ATG14 phosphorylation, we determined that the ULK1 activity requires BECN1/Beclin 1 but not the phosphatidylethanolamine (PE)-conjugation machinery and the PIK3C3 kinase activity. Monitoring the phosphorylation also allowed us to identify that ATG9A is required to suppress the ULK1 activity under nutrient-enriched conditions. Furthermore, we determined that ATG14 phosphorylation depends on ULK1 and dietary conditions in vivo. These results define a key molecular event for the starvation-induced activation of the ATG14-containing PtdIns3K complex by ULK1, and demonstrate hierarchical relations between the ULK1 activation and other autophagy proteins involved in phagophore formation.     

Modulation of immunogenicity of poly(sarcosine) displayed on various nanoparticle surfaces due to different physical properties

Poly(sarcosine) displayed on polymeric micelle is reported to trigger a T cell‐independent type2 reaction with B1a cells in the mice to produce IgM and IgG3 antibodies. In addition to polymeric micelle, three kinds of vesicles displaying poly(sarcosine) on surface were prepared here to evaluate the amounts and avidities of IgM and IgG3, which were produced in mice, to correlate them with physical properties of the molecular assemblies. The largest amount of IgM was produced after twice administrations of a polymeric micelle of 35 nm diameter ( G1 ). On the other hand, the production amount of IgG3 became the largest after twice administrations of G3 (vesicle of 229 nm diameter) or G4 (vesicle of 85 nm diameter). The augmented avidity of IgG3 after the twice administrations compared with that at the single administration was the highest with G3 . These differences in immune responses are discussed in terms of surface density of poly(sarcosine) chains, nanoparticle size, hydrophobic component of poly(L‐lactic acid) or (Leu‐ or Val‐Aib)_n, and membrane elasticity of the nanoparticles. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Fecal microbiota and diets of muskox female adults and calves

In mammals, the gut microbiome is vertically transmitted during maternal lactation at birth. In this study, we investigated the gut microbiome and diets of muskox, a large herbivore inhabiting in the high Arctic. We compared the microbiota composition using bacterial 16S rRNA gene sequencing and diets using stable isotope analysis of muskox feces of six female adults and four calves on Ella Island, East Greenland. Firmicutes were the most abundant bacterial phylum in both the adults and calves, comprising 94.36% and 94.03%, respectively. Significant differences were observed in the relative abundance of the two Firmicutes families. The adults were primarily dominated by Ruminococcaceae (73.90%), and the calves were dominated by both Ruminococcaceae (56.25%) and Lachnospiraceae (24.00%). Stable isotope analysis of the feces in the study area revealed that both adults and calves had similar ranges of ¹³C and ¹⁵N, likely derived from the dominant diet plants. Despite their similar diets, the different gut microbiome compositions in muskox adults and calves indicate that the gut microbiome of the calves may not be fully colonized to the extent of that of the adults.     

NMR study of in vivo RIF-1 tumors: Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances

Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-1) tumors grown in mice have been analyzed by multinuclear NMR spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the ³¹P resonances observed in vivo to specific phosphorus-containing metabolites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the ³¹P-NMR spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. HPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The ¹H and ¹³C resonance assignments of the majority of metabolites present in RIF-1 extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vivo and in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.     

Control of the pericentrosomal H2O2 level by peroxiredoxin I is critical for mitotic progression

Proteins associated with the centrosome play key roles in mitotic progression in mammalian cells. The activity of Cdk1-opposing phosphatases at the centrosome must be inhibited during early mitosis to prevent premature dephosphorylation of Cdh1—an activator of the ubiquitin ligase anaphase-promoting complex/cyclosome—and the consequent premature degradation of mitotic activators. In this paper, we show that reversible oxidative inactivation of centrosome-bound protein phosphatases such as Cdc14B by H₂O₂ is likely responsible for this inhibition. The intracellular concentration of H₂O₂ increases as the cell cycle progresses. Whereas the centrosome is shielded from H₂O₂ through its association with the H₂O₂-eliminating enzyme peroxiredoxin I (PrxI) during interphase, the centrosome-associated PrxI is selectively inactivated through phosphorylation by Cdk1 during early mitosis, thereby exposing the centrosome to H₂O₂ and facilitating inactivation of centrosome-bound phosphatases. Dephosphorylation of PrxI by okadaic acid–sensitive phosphatases during late mitosis again shields the centrosome from H₂O₂ and thereby allows the reactivation of Cdk1-opposing phosphatases at the organelle.     

Potential Interaction of Plasmodium falciparum Hsp60 and Calpain

After invasion of red blood cells, malaria matures within the cell by degrading hemoglobin avidly. For enormous protein breakdown in trophozoite stage, many efficient and ordered proteolysis networks have been postulated and exploited. In this study, a potential interaction of a 60-kDa Plasmodium falciparum (Pf)-heat shock protein (Hsp60) and Pf-calpain, a cysteine protease, was explored. Pf-infected RBC was isolated and the endogenous Pf-Hsp60 and Pf-calpain were determined by western blot analysis and similar antigenicity of GroEL and Pf-Hsp60 was determined with anti-Pf-Hsp60. Potential interaction of Pf-calpain and Pf-Hsp60 was determined by immunoprecipitation and immunofluorescence assay. Mizoribine, a well-known inhibitor of Hsp60, attenuated both Pf-calpain enzyme activity as well as P. falciparum growth. The presented data suggest that the Pf-Hsp60 may function on Pf-calpain in a part of networks during malaria growth.