pH/enzyme dual sensitive and nucleus-targeting dendrimer nanoparticles to enhance the antitumour activity of doxorubicin.

Direct delivery of drugs into the nucleus is a promising nanotechnology therapy, since the nucleus is one of the most important organelles controlling cell proliferation and apoptosis. Here, we report a nucleus-targeting nanocarrier for nuclear drug delivery using a pH/enzyme dual sensitive strategy. The specific ligand PGM (PKKKRKV-GFLG-Mp), composed of nuclear localization sequence (PKKKRKV), enzyme-sensitive tetrapeptide (Gly-Phe-Leu-Gly, GFLG), and pH-sensitive molecules morpholine (Mp), was modified on poly (amidoamine) (PAMAM) by maleimide active polyethylene glycol ester (NHS-PEG-MAL) to form PAMAM-PEG-PGM. Doxorubicin (DOX) was loaded into the cavity of PAMAM to prepare DOX/PAMAM-PEG-PGM. In vitro release study suggested DOX release from DOX/PAMAM-PEG-PGM nanoparticles followed pH and enzyme-triggered manner. In vitro studies showed DOX/PAMAM-PEG-PGM nanoparticles could not only promote cell internalization through the charge switching of morpholine, but also achieve nuclear internalization by the mediation of composite formed by NLS and importin α/ß receptor. Further, employing H22 tumour-bearing BALB/c mice as a model, the systemic distribution and anticancer effects of nanoparticles were studied in vivo. The results indicated the nanoparticles could preferentially accumulate in the tumour site in vivo, and the tumour inhibition rate was 88.47%. In short, the nanoparticles developed could be promising in application to nucleus-targeting therapy to enhance antitumour activity.

Investigation of Citrinin and Pigment Biosynthesis Mechanisms in Monascus purpureus by Transcriptomic Analysis.

Monascus purpureus YY-1 is widely used in food colorant production in China. Our previous study clearly illustrated the whole-genome data for YY-1 and provided useful insight into evolutionary research and industrial applications. However, the presence of citrinin, which has nephrotoxic, hepatotoxic, and carcinogenic activities, has attracted attention to the safety of Monascus products. In an effort to reduce the harmful effects of citrinin in Monascus-related products, a random mutant of M. purpureus YY-1 with low citrinin production (designated as "winter") was obtained in this study. To analyze the biosynthesis and regulation mechanisms of pigment and citrinin, a transcriptomic analysis of the M. purpureus YY-1 and winter strains was performed. Comparative transcriptomic analysis reveals pksCT, the essential gene for citrinin synthesis, showed a low expression level in M. purpureus YY-1 and winter, which suggested there might be isoenzymes in M. purpureus YY-1 that were responsible for the citrinin synthesis during evolution. In addition, changes in transcription factor expression may also influence the network regulating the citrinin synthesis pathway in M. purpureus. Moreover, the yields of pigments produced by the winter mutant were significantly increased. Repressing the central carbon metabolism and improving the acetyl-CoA pool can contribute to a high pigment yield, and enhanced NADPH regeneration can also lead to the metabolic flux of pigment production in M. purpureus. Investigations into the biosynthesis and regulation of citrinin and pigment production in M. purpureus will enhance our knowledge of the mechanisms behind the biosynthesis of fungal secondary metabolites.

Hyaluronan-CD44 interaction promotes growth of decidual stromal cells in human first-trimester pregnancy.

Hyaluronan (HA) and its receptor CD44 are expressed at the maternal-fetal interface, but its role in early pregnancy remains unclear. Here, we found that primary decidual stromal cells (DSCs) continuously secreted HA and expressed its receptor CD44. Pregnancy-associated hormones up-regulated HA synthetase (HAS) 2 transcription and HA release from DSCs. High molecular weight-HA (HMW-HA), but not medium molecular weight (MMW-HA) or low molecular weight (LMW-HA), promoted proliferation and inhibited apoptosis of DSCs in a CD44-dependent manner. The in-cell Western analysis revealed HMW-HA activated PI3K/AKT and mitogen-activated protein kinase (MAPK)/ERK1/2 signaling pathways time-dependently. Blocking these pathways by specific inhibitor LY294002 or U0126 abrogated HMW-HA-regulated DSc proliferation and apoptosis. Finally, we have found that HA content, HA molecular weight, HAS2 mRNA level, and CD44 expression were significantly decreased in DSCs from unexplained miscarriage compared with the normal pregnancy. Collectively, our results indicate that higher level and greater molecular mass of HA at maternal-fetal interface contributes to DSc growth and maintenance of DSCs in human early pregnancy.

Chemokine CCL28 induces apoptosis of decidual stromal cells via binding CCR3/CCR10 in human spontaneous abortion.

Spontaneous abortion is the most common complication of pregnancy. Immune activation and the subsequent inflammation-induced tissue injury are often observed at the maternal-fetal interface as the final pathological assault in recurrent spontaneous abortion. However, the precise mechanisms responsible for spontaneous abortion involving inflammation are not fully understood. Chemokine CCL28 and its receptors CCR3 and CCR10 are important regulators in inflammatory process. Here, we examined the expression of CCL28 and its receptors in decidual stromal cells (DSCs) by immunochemistry and flow cytometry (FCM), and compared their expression level in DSCs from normal pregnancy versus spontaneous abortion, and their relationship to inflammatory cytokines production by DSCs. We further analyzed regulation of the pro-inflammatory cytokines on CCL28 expression in DSCs by real-time polymerase chain reaction, In-cell Western and FCM. The effects of CCL28-CCR3/CCR10 interaction on DSC apoptosis was investigated by Annexin V staining and FCM analysis or DAPI staining and nuclear morphology. Higher levels of the inflammatory cytokines interleukin (IL)-1ß, IL-17A and tumor necrosis factor-α, and increased CCR3/CCR10 expression were observed in DSCs from spontaneous abortion compared with normal pregnancy. Treatment with inflammatory cytokines differently affected CCL28 and CCR3/CCR10 expression in DSCs. Human recombinant CCL28 promoted DSC apoptosis, which was eliminated by pretreatment with neutralizing antibodies against CCR3/CCR10 and CCL28. However, CCL28 did not affect DSC growth. These results suggest that the inflammation-promoted up-regulation of CCL28 and its receptors interaction in DSCs is involved in human spontaneous abortion via inducing DSC apoptosis.

Sphingomonas ginsengisoli sp. nov. and Sphingomonas sediminicola sp. nov.

Two novel bacteria, designated strains Gsoil 634(T) and Dae 20(T), were isolated in South Korea from soil of a ginseng field and freshwater sediment, respectively and were characterized by a polyphasic approach to clarify their taxonomic positions. Phylogenetic analysis based on 16S rRNA gene sequences indicated that, although they probably represented two distinct species (indicated by a sequence similarity of 96.6 %), both strain Gsoil 634(T) and strain Dae 20(T) belonged to the genus Sphingomonas and were most closely related to 'Sphingomonas humi' PB323 (97.8 % and 96.7 % sequence similarity, respectively), Sphingomonas kaistensis PB56(T) (96.8 % and 96.7 %), Sphingomonas astaxanthinifaciens TDMA-17(T) (96.6 % and 95.4 %) and Sphingomonas jaspsi TDMA-16(T) (95.6 % and 95.8 %). For both novel strains, the major ubiquinone was Q-10, the major polyamine was homospermidine, the major cellular fatty acids included summed feature 7 (C(18 : 1)ω7c, C(18 : 1)ω9t and/or C(18 : 1)ω12t), C(17 : 1)ω6c and C(16 : 0), and the polar lipids included sphingoglycolipid. These chemotaxonomic data supported the affiliation of both strains to the genus Sphingomonas. However, the DNA-DNA relatedness value between strain Gsoil 634(T) and 'Sphingomonas humi' PB323(T) was 31 %. Moreover, the results of physiological and biochemical tests allowed the phenotypic differentiation of strains Gsoil 634(T) and Dae 20(T) from established members of the genus Sphingomonas. Based on these data, the two isolates represent two novel species in the genus Sphingomonas, for which the names Sphingomonas ginsengisoli sp. nov. (type strain Gsoil 634(T) = KCTC 12630(T) = DSM 18094(T) = LMG 23739(T)) and Sphingomonas sediminicola sp. nov. (type strain Dae 20(T)  = KCTC 12629(T) = DSM 18106(T) = LMG 23592(T)) are proposed.

Cyclosporine A enhances Th2 bias at the maternal-fetal interface in early human pregnancy with aid of the interaction between maternal and fetal cells.

Our previous study has demonstrated that cyclosporine A (CsA) administration in vivo induces Th2 bias at the maternal-fetal interface, leading to improved murine pregnancy outcomes. Here, we investigated how CsA treatment in vitro induced Th2 bias at the human maternal-fetal interface in early pregnancy. The cell co-culture in vitro in different combination of component cells at the maternal-fetal interface was established to investigate the regulation of CsA on cytokine production from the interaction of these cells. It was found that interferon (IFN)-γ was produced only by decidual immune cells (DICs), and not by trophoblasts or decidual stromal cells (DSCs); all these cells secreted interleukin (IL)-4, IL-10, and tumor necrosis factor (TNF)-α. Treatment with CsA completely blocked IFN-γ production in DICs and inhibited TNF-α production in all examined cells. CsA increased IL-10 and IL-4 production in trophoblasts co-cultured with DSCs and DICs although CsA treatment did not affect IL-10 or IL-4 production in any of the cells when cultured alone. These results suggest that CsA promotes Th2 bias at the maternal-fetal interface by increasing Th2-type cytokine production in trophoblasts with the aid of DSCs and DICs, while inhibiting Th1-type cytokine production in DICs and TNF-α production in all investigated cells. Our study might be useful in clinical therapeutics for spontaneous pregnancy wastage and other pregnancy complications.

Effect of suppressor of cytokine signaling 2 (SOCS2) on fat metabolism induced by growth hormone (GH) in porcine primary adipocyte.

SOCS2, a member of suppressor of cytokine signaling (SOCS) family, is a negative regulator of the signal pathway Janus kinase/signal transducers and activators of transcription (JAK/STAT). Growth hormone (GH) could stimulate lipolysis in adipose tissue. To demonstrate the specific influence of SOCS2 on porcine adipocytes differentiation and lipid metabolism induced by GH, we induced porcine primary adipocytes with 500 ng/ml GH and then tested the triglyceride (TG) accumulation and mRNA expressions of crucial genes in lipid metabolism like peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid synthase (FAS), adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), SOCS2 and SOCS3. Then we retested these genes expressions in different time point after further treatment that over expressed SOCS2 in primary adipocytes and treated with 500 ng/ml GH. Results showed 500 ng/ml GH significantly restrained the porcine primary adipocytes differentiation. Specifically, 0.5 h after the induction with GH, accumulation of TG began to increase, and turned down since 8 h after. GH could promote PPARγ and FAS expressions during earlier stage (0-1 h), restrain from 4 h. However, ATGL and HSL mRNA expressions were stabile increasing. The expression of SOCS2 increased steadily after GH stimulation while SOCS3 expression was instantaneous rise. Overexpression of SOCS2 significantly decreased GH-induced the increase of PPARγ, FAS, ATGL and HSL mRNA expressions in earlier stage (0-1 h), as well as FAS and ATGL protein expression. Otherwise SOCS2 overexpression significantly decreased signal transducers and activators of transcription 3 (STAT3), signal transducers and activators of transcription 5 (STAT5) mRNA expressions and tyrosine phosphorylation levels with GH stimulation. At the same time SOCS3 mRNA kept in a lower level in Ad-SOCS2 transfected adipocytes. In conclusion, SOCS2 might be an important negative regulator of GH signaling in porcine adipocytes, which would provide the ground work for the mechanism of SOCS2 regulation fat metabolism.