Differentiation of liver cells from human primordial germ cell-derived progenitors

In previous studies, progenitor embryoid body-derived (EBD) cells have been derived from human embryonic germ cells. These cells express lineage markers of three primary germ layers, although their potential to produce true fetal cells of various types has yet to be tested. To this end, we have transplanted EBD cells into the fetal sheep liver. We show that these cells respond appropriately to environmental cues and give rise to hepatocytes and well-structured bile ducts. These results suggest that EBD cells are relatively uncommitted early progenitors capable of effective incorporation and differentiation in vivo. The ability to generate functional liver cells makes EBD cells potentially useful for cell therapy.     

Vaspin protects mouse mesenchymal stem cells from oxidative stress-induced apoptosis through the MAPK/p38 pathway

Molecular and Cellular Biochemistry - The aim of the work was to study the influence of vaspin on oxidative stress-induced apoptosis of mouse mesenchymal stem cells (MSCs). MSCs originated from...     

A Small Molecule Inhibitor of Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) Inhibits Repair of Interstrand DNA Cross-link,Enhances DNA Double Strand Break,and Sensitizes Cancer Cells to Cisplatin

Small molecule inhibitors of proliferating cell nuclear antigen (PCNA)/PCNA interacting protein box (PIP-Box) interactions, including T2 amino alcohol (T2AA), inhibit translesion DNA synthesis. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to the surface adjacent to the subunit interface of the homotrimer of PCNA in addition to the PIP-box binding cavity. Because this site is close to Lys-164, which is monoubiquitinated by RAD18, we postulated that T2AA would affect monoubiquitinated PCNA interactions. Binding of monoubiquitinated PCNA and a purified pol η fragment containing the UBZ and PIP-box was inhibited by T2AA in vitro. T2AA decreased PCNA/pol η and PCNA/REV1 chromatin colocalization but did not inhibit PCNA monoubiquitination, suggesting that T2AA hinders interactions of pol η and REV1 with monoubiquitinated PCNA. Interstrand DNA cross-links (ICLs) are repaired by mechanisms using translesion DNA synthesis that is regulated by monoubiquitinated PCNA. T2AA significantly delayed reactivation of a reporter plasmid containing an ICL. Neutral comet analysis of cells receiving T2AA in addition to cisplatin revealed that T2AA significantly enhanced formation of DNA double strand breaks (DSBs) by cisplatin. T2AA promoted colocalized foci formation of phospho-ATM and 53BP1 and up-regulated phospho-BRCA1 in cisplatin-treated cells, suggesting that T2AA increases DSBs. When cells were treated by cisplatin and T2AA, their clonogenic survival was significantly less than that of those treated by cisplatin only. These findings show that the inhibitors of monoubiquitinated PCNA chemosensitize cells by inhibiting repair of ICLs and DSBs.     

Effects of rearing conditions on the parasitism of Tetrastichus brontispae on its pupal host Octodonta nipae

Octodonta nipae (Maulik) (Coleoptera: Chrysomelidae) is currently a serious invasive pest of palm plants in southern China. Although previous studies reported that Tetrastichus brontispae Ferriere (Hymenoptera: Eulophidae) could successfully parasitize the pupae of O. nipae, little is known of the potential for T. brontispae to control this pest. To understand the interaction between T. brontispae and O. nipae, the effects of parasitoid feeding regimes, host age and density, parasitoid age and density, and cold storage on the parasitism of T. brontispae were investigated. Our results indicate that a supply of either glucose or sucrose increased the longevity and fecundity of T. brontispae. The parasitoid preferred one-day-old host pupae and its fecundity increased with increasing host or parasitoid female density. Extended cold storage negatively affected the parasitism of T. brontispae. These results demonstrate how to efficiently raise T. brontispae and will contribute to the effective management of O. nipae.     

Decreased levels of active SMAD2 correlate with poor prognosis in gastric cancer

Background

TGF-β plays a dual role in the progression of human cancer. During the early stages of carcinogenesis, TGF-β functions as a tumor suppressor. During the late stages of tumor development, however, TGF-β can promote tumor growth and metastasis. A shift in Smad2/3 phosphorylation from the carboxy terminus to linker sites is a key event determining biological function of TGF-β in colorectal and hepatocellular carcinoma. In the present study, we investigated the potential role of differential Smad2/3 phosphorylation in gastric adenocarcinoma.

Methodology

Immunohistochemical staining with anti-P-Smad2/3C and P-Smad2/3L antibodies was performed on 130 paraffin-embedded gastric adenocarcinoma specimens. The relationship between P-Smad2/3C and P-Smad2/3L immunohistochemical score and clinicopathologic characteristics of patients was analyzed. Real time PCR was used to measure mRNA expression of Smad2 and Smad3 in cancer and surrounding non-tumor tissue.

Principal Findings

No significant P-Smad2L and/or P-Smad3L positive staining was detected in the majority of specimens (positive staining in 18/130 samples). Positive P-Smad2/3L staining was not associated with a decrease in carboxyterminal phosphorylation staining. Loss of P-Smad2C remarkably correlated with depth of tumor infiltration and poor differentiation of cancer cells in patients with gastric cancer. No correlation was detectable between P-Smad3C and clinicopathologic characteristics of gastric adenocarcinoma. However, co-staining analysis revealed that P-Smad3C co-localised with α-SMA and collagen I in gastric cancer cells, indicating a potential link between P-Smad3C and epithelial-to-mesenchymal transition of cancer. Real time PCR demonstrated reduced mRNA expression of Smad2 in gastric cancer when compared with surrounding non-tumor tissue in 15/16 patients.

Conclusions

Loss of P-Smad2C tightly correlated with cancer invasion and poor differentiation in gastric cancer. Contrary to colorectal and hepatocellular carcinoma, canonical carboxy-terminal phosphorylation, but not linker phosphorylation, of Smad2 is critical for gastric cancer.     

Assessment of protoxin composition of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains by use of polyacrylamide gel block and mass spectrometry

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Zujiao Fu and Yunjun Sun contributed equally to this work.     

Efficient transfer of two large secondary metabolite pathway gene clusters into heterologous hosts by transposition

Horizontal gene transfer by transposition has been widely used for transgenesis in prokaryotes. However, conjugation has been preferred for transfer of large transgenes, despite greater restrictions of host range. We examine the possibility that transposons can be used to deliver large transgenes to heterologous hosts. This possibility is particularly relevant to the expression of large secondary metabolite gene clusters in various heterologous hosts. Recently, we showed that the engineering of large gene clusters like type I polyketide/nonribosomal peptide pathways for heterologous expression is no longer a bottleneck. Here, we apply recombineering to engineer either the epothilone (epo) or myxochromide S (mchS) gene cluster for transpositional delivery and expression in heterologous hosts. The 58-kb epo gene cluster was fully reconstituted from two clones by stitching. Then, the epo promoter was exchanged for a promoter active in the heterologous host, followed by engineering into the MycoMar transposon. A similar process was applied to the mchS gene cluster. The engineered gene clusters were transferred and expressed in the heterologous hosts Myxococcus xanthus and Pseudomonas putida. We achieved the largest transposition yet reported for any system and suggest that delivery by transposon will become the method of choice for delivery of large transgenes, particularly not only for metabolic engineering but also for general transgenesis in prokaryotes and eukaryotes.     

Molecular mechanisms of anti-oxidant and anti-aging effects induced by convallatoxin in Caenorhabditis elegans

Convallatoxin is widely used as a cardiac glycoside in acute and chronic congestive heart-failure and paroxysmal tachycardia, with many effects and underlying protective mechanisms on inflammation and cellular proliferation. However, convallatoxin has not been investigated in its antioxidant effects and lifespan extension in Caenorhabditis elegans. In this study, we found that convallatoxin (20?μM) could significantly prolong the lifespan of wild-type C. elegans up to 16.3% through daf-16, but not sir-2.1 signalling and increased thermotolerance and resistance to paraquat-induced oxidative stress. Convallatoxin also improved pharyngeal pumping, locomotion, reduced lipofuscin accumulation and reactive oxygen species levels in C. elegans, which were attributed to hormesis, free radical-scavenging effects in vivo, and up-regulation of stress resistance-related proteins, such as SOD-3 and HSP-16.1. Furthermore, aging-associated genes daf-16, sod-3, and ctl-2 also appeared to contribute to the stress-resistance effect of convallatoxin. In summary, this study demonstrates that convallatoxin can protect against heat and oxidative stress and extend the lifespan of C. elegans, pointing it as a potential novel drug for retarding the aging process in humans.