Culture under low physiological oxygen conditions improves the stemness and quality of induced pluripotent stem cells.

The ex vivo expansion of stem cells under low physiological oxygen (O2 ) conditions has been demonstrated to improve the stemness and genomic stability of the cells. We investigated whether low-oxygen culture would be beneficial for the culture of induced pluripotent stem (iPS) cells. Two human iPS cell lines (201B7 and 253G1) were used for the experiments. Cells expanded from a single colony of each cell line were initiated for culture in 2.5% O2 , 5% O2 , or 20% O2 and maintained for 2 months in parallel. The levels of intracellular and mitochondrial reactive oxygen species did not differ between the cells cultured under different conditions. More colonies of uniformly smaller size were observed at 2.5% and 5% O2 than at 20% O2 . All of these iPS colonies that expanded under the various oxygen conditions stained positively for Oct3/4, Nanog, SSEA-4, and ALP. However, Western blot analysis showed that the iPS cells cultured at 2.5% and 5% O2 expressed significantly more Nanog but less 53BP1 than those cultured at 20% O2 . Data from an array CGH showed no significant chromosomal abnormalities, although some genes involved in cellular and metabolic processes were amplified in the low oxygen culture, particularly at 2.5% O2 . Our data suggest that low physiological oxygen culture could improve the stemness and quality of iPS cells, a result that might be associated with the amplification of genes involved in metabolic and cellular processes. Long-term culture will be necessary to confirm whether low physiological oxygen levels also improve genomic stability.

Autophagy is involved in anti-viral activity of pentagalloylglucose (PGG) against Herpes simplex virus type 1 infection in vitro.

Pentagalloylglucose (PGG) is a natural polyphenolic compound with broad-spectrum anti-viral activity, however, the mechanisms underlying anti-viral activity remain undefined. In this study, we investigated the effects of PGG on anti-viral activity against Herpes simplex virus type 1 (HSV-1) associated with autophagy. We found that the PGG anti-HSV-1 activity was impaired significantly in MEF-atg7-/- cells (autophagy-defective cells) derived from an atg7-/- knockout mouse. Transmission electron microscopy revealed that PGG-induced autophagosomes engulfed HSV-1 virions. The mTOR signaling pathway, an essential pathway for the regulation of autophagy, was found to be suppressed following PGG treatment. Data presented in this report demonstrated for the first time that autophagy induced following PGG treatment contributed to its anti-HSV activity in vitro.

Antiviral activity and possible mechanisms of action of pentagalloylglucose (PGG) against influenza A virus.

Influenza A virus (IAV) infection is a major public health threat leading to significant morbidity and mortality. The emergence of drug-resistant virus strains highlights the urgent need to develop novel antiviral drugs with alternative modes of action. Pentagalloylglucose (PGG), a naturally occurring polyphenolic compound, possesses a broad spectrum of biological activities. In this study, we found that PGG has anti-influenza-virus activity, and investigated its possible mechanism(s) of action in vitro. Both pre-incubation of virus prior to infection and post-exposure of infected cells with PGG significantly inhibited virus yields. Influenza-virus-induced hemagglutination of chicken red blood cells was inhibited by PGG treatment, suggesting that PGG can inhibit IAV infection by interacting with the viral hemagglutinin. PGG did not affect viral protein synthesis or nuclear transport of viral nucleoprotein (NP) but greatly reduced plasma membrane accumulation of NP protein at the late stage of the replication cycle. Furthermore, PGG significantly reduced virus budding and progeny virus release from infected cells. This study revealed for the first time that PGG can inhibit IAV replication with a dual mode of action and offers new insights into its underlying mechanisms of antiviral action.

Proteomic analysis reveals novel binding partners of MIP-T3 in human cells.

MIP-T3 (microtubule-interacting protein associated with TRAF3) is a microtubule-interacting protein that evolutionarily conserved from worms to humans, but whose cellular functions remains unknown. To get insight into the functions of MIP-T3, we set out to identify MIP-T3 interacting proteins by immunoprecipitation in human embryonic kidney 293 cells and MS analysis. As the results, a total of 34 proteins were identified and most of them were novel MIP-T3 putative partners. The MIP-T3-associated proteins could be grouped into nine clusters based on their molecule functions, including cytoskeleton, chaperone, nucleic acid binding, kinase and so on. Three MIP-T3-interacted proteins - actin, HSPA8 and tubulin - were further confirmed by reciprocal coimmunoprecipitations and colocalization analysis. The interaction of MIP-T3 with both actin filaments and microtubule suggested that MIP-T3 may play an important role in regulation of cytoskeleton dynamics in cells. Our results therefore not only uncover a large number of MIP-T3-associated proteins that possess a variety of cellular functions, but also provide new research directions for the study of the functions of MIP-T3.

The mechanism of poly-galloyl-glucoses preventing Influenza A virus entry into host cells.

Hemagglutinin (HA) is essential for Influenza A virus infection, but its diversity of subtypes presents an obstacle to developing broad-spectrum HA inhibitors. In this study, we investigated the molecular mechanisms by which poly-galloyl glucose (pGG) analogs inhibit influenza hemagglutinin (HA) in vitro and in silico. We found that (1) star-shaped pGG analogs exhibit HA-inhibition activity by interacting with the conserved structural elements of the receptor binding domain (RBD); (2) HA inhibition depends on the number of galloyl substituents in a pGG analog; the best number is four; and when PGG binds with two HA trimers at their conserved receptor binding domains (loop 130, loop 220, and 190-α-helix), PGG acts as a molecular glue by aggregating viral particles so as to prevent viral entry into host cells (this was revealed via an in silico simulation on the binding of penta-galloyl-glucose (PGG) with HA). pGGs are also effective on a broad-spectrum influenza A subtypes (including H1, H3, H5, H7); this suggests that pGG analogs can be applied to most influenza A subtypes as a prophylactic against influenza viral infections.

Effects of antioxidants on the quality and genomic stability of induced pluripotent stem cells.

Effects of antioxidants on the quality and genomic stability of induced pluripotent stem (iPS) cells were investigated with two human iPS cell lines (201B7 and 253G1). Cells used in this study were expanded from a single colony of each cell line with the addition of proprietary antioxidant supplement or homemade antioxidant cocktail in medium, and maintained in parallel for 2 months. The cells grew well in all culture conditions and kept "stemness". Although antioxidants modestly decreased the levels of intracellular reactive oxygen species, there were no differences in the expression of 53BP1 and pATM, two critical molecules related with DNA damage and repair, under various culture conditions. CGH analysis showed that the events of genetic aberrations were decreased only in the 253G1 iPS cells with the addition of homemade antioxidant cocktail. Long-term culture will be necessary to confirm whether low dose antioxidants improve the quality and genomic stability of iPS cells.

The C-terminus of MIP-T3 protein is required for ubiquitin-proteasome-mediated degradation in human cells.

The intraflagellar transport (IFT) complex is essential for the formation and functional maintenance of eukaryotic cilia which play a vital role in development and tissue homeostasis. However, the biochemical characteristics and precise functions of IFT proteins remain unknown. Here, we report that MIP-T3, a human microtubule-interacting protein recently identified as a novel conserved component of the IFT complex, is an easily degradable protein in human cell lines. Protein degradation is mediated by the ubiquitin-proteasome system, and the C-terminus is required for ubiquitination and proteasome-mediated degradation of MIP-T3 protein. This study provides the first evidence for regulation of IFT protein stability.

[Efficient purification of recombinant human NDPK-A in pilot-scale].

To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) efficiently in pilot scale, cells of rhNDPK-A producing E. coli were homogenized by high pressure under 4 degrees C, 950 Pa. The insoluble debris was removed by microfiltration and the soluble portion was concentrated by ultrafiltration. The resulted crude sample was loaded on DEAE-sepharose Fast Flow. The target fraction was collected and then load on Cibacron Blue 3GA Sepharose CL-4B. Eluted with buffer containing ATP from the AC column, rhNDPK-A was polished with ultrafiltration. The results showed that after homogenized 2 rounds, 1500g cells of E. coli brought crude sample containing 47.6g NDPK-A. Treated with microfiltration and ultrafiltration, 27.3g of NDPK-A were recovered from this bacteria homogenate. After 2-step purification with column chromatography and then polished with ultrafiltration, 17.2 g rhNDPK-A were collected with purity of 96.3%. The recovery of the whole purification process was 36.2%, and the productivity of rhNDPK-A was 1.15 g per 100 g wet cells. Comparing the recovery of each purification step, it was found that the recovery of polish is higher than that of affinity chromatography, which is higher than that of ion exchange chromatography. The limit step was the process of sample pretreatment among the 4 purification steps. Combine with the fermentation results reported before, it was deduced that the productivity of rhNDPK-A was 510 mg/L. In conclusion, an easily controlled purification condition with high yield provides material for the translation researches of NDPK; In addition, it was suggested the crucial step determine the recovery of non-secretive recombinant proteins might be the process of sample pretreatment, not be the process of column chromatography.