An immortalized drug-resistant cell line established from 12-13-day mouse embryos for the propagation of human embryonic stem cells

Human embryonic stem (ES) cells are usually co-cultivated with supporting cells consisting of short-term cultures of fibroblasts (not an immortalized line) in a medium lacking serum. This method has promoted important progress in the field, but suffers from certain disadvantages. By serial cultivation for 27 consecutive transfers and about 63 cell generations, we have evolved an immortalized line from fibroblastic cells of 12-13-day mouse embryos. This line (MMM) supports the multiplication of H9 cells better than the 3T3 line. It supports the growth of H9 cells as well as do available short-term fibroblast cultures, but maintains more effectively the stem cell character of the H9 cells, judging by their better retention of Oct4. We have made MMM cells resistant to blasticidin and zeocin, the most efficient antibiotics for selection of stable transformants. In the presence of zeocin, the resistant MMM were able to support multiplication and selection of ES cells transfected with an exogenous gene encoding zeocin resistance.     

Optimized submerged batch fermentation strategy for systems scale studies of metabolic switching in Streptomyces coelicolor A3(2)

Background

Given the complex mechanisms underlying biochemical processes systems biology researchers tend to build ever increasing computational models. However, dealing with complex systems entails a variety of problems, e.g. difficult intuitive understanding, variety of time scales or non-identifiable parameters. Therefore, methods are needed that, at least semi-automatically, help to elucidate how the complexity of a model can be reduced such that important behavior is maintained and the predictive capacity of the model is increased. The results should be easily accessible and interpretable. In the best case such methods may also provide insight into fundamental biochemical mechanisms.

Results

We have developed a strategy based on the Computational Singular Perturbation (CSP) method which can be used to perform a "biochemically-driven" model reduction of even large and complex kinetic ODE systems. We provide an implementation of the original CSP algorithm in COPASI (a COmplex PAthway SImulator) and applied the strategy to two example models of different degree of complexity - a simple one-enzyme system and a full-scale model of yeast glycolysis.

Conclusion

The results show the usefulness of the method for model simplification purposes as well as for analyzing fundamental biochemical mechanisms. COPASI is freely available at http://www.copasi.org.     

Nutritional regulation and kinetics of flocculosin synthesis by Pseudozyma flocculosa

This study sought to identify the factors and conditions that affected production of the antifungal glycolipid flocculosin by the biocontrol agent Pseudozyma flocculosa. For this purpose, different parameters known or reported to influence glycolipid release in fungi were tested. Concentration of the start-up inoculum was found to play an important role in flocculosin production, as the optimal level increased productivity by as much as tenfold. Carbon availability and nitrogen source (i.e., organic vs inorganic) both had a direct influence on the metabolism of P. flocculosa, leading to flocculosin synthesis. In general, if conditions were conducive for production of the glycolipid, carbon availability appeared to be the only limiting factor. On the other hand, if yeast extract was supplied as nitrogen source, fungal biomass was immediately stimulated to the detriment of flocculosin synthesis. Unlike other reports of glycolipid release by yeast-like fungi, inorganic nitrogen starvation did not trigger production of flocculosin. The relationship between the factors influencing flocculosin production in vitro and the conditions affecting the release of the molecule by P. flocculosa in its natural habitat appears to be linked to the availability of a suitable and plentiful food source for the biocontrol agent.     

CD154: the atherosclerotic risk factor in rheumatoid arthritis?

Atherosclerosis, now regarded as a chronic inflammatory disease of the arterial wall, and its clinical manifestations have increasingly been associated with rheumatoid arthritis (RA), supporting the notion that autoimmune diseases and vascular disorders share common etiological features. Indeed, evidence pertaining to this matter indicates that inflammation and its multiple components are the driving force behind the pathogenesis of these disorders. Interestingly, CD154 and its receptors have emerged as major players in the development of RA and atherosclerosis, which raises the possibility that this axis may represent an important biological link between both complications. Indeed, CD154 signaling elicits critical inflammatory responses that are common to the pathogenesis of both diseases. Here, we provide an overview of the traditional and disease-related interrelations between RA and vascular abnormalities, while focusing on CD154 as a potential mediator in the development of atherosclerotic events in RA patients.     

Non-Canonical Smads Phosphorylation Induced by the Glutamate Release Inhibitor,Riluzole, through GSK3 Activation in Melanoma

Riluzole, an inhibitor of glutamate release, has shown the ability to inhibit melanoma cell xenograft growth. A phase 0 clinical trial of riluzole as a single agent in patients with melanoma resulted in involution of tumors associated with inhibition of both the mitogen-activated protein kinase (MAPK) and phophoinositide-3-kinase/AKT (PI3K/AKT) pathways in 34% of patients. In the present study, we demonstrate that riluzole inhibits AKT-mediated glycogen synthase kinase 3 (GSK3) phosphorylation in melanoma cell lines. Because we have demonstrated that GSK3 is involved in the phosphorylation of two downstream effectors of transforming growth factor beta (TGFβ), Smad2 and Smad3, at their linker domain, our aim was to determine whether riluzole could induce GSK3β-mediated linker phosphorylation of Smad2 and Smad3. We present evidence that riluzole increases Smad2 and Smad3 linker phosphorylation at the cluster of serines 245/250/255 and serine 204 respectively. Using GSK3 inhibitors and siRNA knock-down, we demonstrate that the mechanism of riluzole-induced Smad phosphorylation involved GSK3β. In addition, GSK3β could phosphorylate the same linker sites in vitro. The riluzole-induced Smad linker phosphorylation is mechanistically different from the Smad linker phosphorylation induced by TGFβ. We also demonstrate that riluzole-induced Smad linker phosphorylation is independent of the expression of the metabotropic glutamate receptor 1 (GRM1), which is one of the glutamate receptors whose involvement in human melanoma has been documented. We further show that riluzole upregulates the expression of INHBB and PLAU, two genes associated with the TGFβ signaling pathway. The non-canonical increase in Smad linker phosphorylation induced by riluzole could contribute to the modulation of the pro-oncogenic functions of Smads in late stage melanomas.     

Genetic variants of microRNA-146a gene: an indicator of systemic lupus erythematosus susceptibility,lupus nephritis,and disease activity

Genetic variations of microRNA encoding genes influence various sorts of diseases by modifying the expression or activity of microRNAs. MicroRNA 146a is an epigenetic regulator of immune response through controlling the type I interferon (IFN) and nuclear factor kappa B (NF-κB) pathways. Genetic variations of microRNA 146a impact the susceptibility to systemic lupus erythematosus (SLE) and its clinical presentations. This study aimed to investigate the polymorphisms of microRNA-146a gene (rs2431697 and rs57095329) in patients with SLE and its association with disease activity. Sixty-five patients with SLE and 40 apparently healthy controls were enrolled in this study. Patients were subjected to history taking, clinical examination, and disease activity evaluation by SLEDAI score. The microRNA-146a variants were determined by allele discrimination real-time PCR method in all participants. We found a statistically significant association between rs2431697 T allele and SLE (P-value?<?0.05), but there was no significant association between rs57095329 and SLE. The T/T genotype of microRNA-146a rs2431697 was associated with lupus nephritis, higher disease activity, and autoantibodies production. The microRNA-146a rs2431697 T allele could be a potential risk factor that contributes to SLE susceptibility, development of lupus nephritis, and disease activity.

     

Extracellular matrix production by mouse 3T3-F442A cells during adipose differentiation in culture

When cultured 3T3-F442A cells undergo adipose differentiation, they produce extracellular matrix (ECM) that is not present in undifferentiated cells. This ECM stains strongly with ruthenium red, tannic acid and with Alcian blue at both pH 1 and 2.5, showing histochemical characteristics similar to sulphated and non-sulphated glycosaminoglycans. Under the electron microscope, ECM was observed bound to the cell surface and in the intercellular space; it was composed of fibrils of several thicknesses with attached granules and fibrous long-spacing forms of collagen. In addition, adipocytes were observed as rounded cells interconnected with the ECM fibrils, thus giving rise to fat cell clusters similar to the adipocyte lobules found in adipose tissue. Since fat cell clusters in culture emerge by clonal expansion of one adipose precursor cell, we suggest that this ECM can keep daughter adipocytes interconnected during differentiation. ECM production by adipocytes might have some significance for the formation of fat cell lobules in vivo.     

Identification of the phosphoglycerate dehydrogenase isoform EDA9 as the essential gene for embryo and male gametophyte development in Arabidopsis

Three different pathways of serine (Ser) biosynthesis have been described in plants: the Glycolate pathway, which is part of the Photorespiratory pathway, and 2 non-Photorespiratory pathways, the Glycerate and the Phosphorylated pathways. The Phosphorylated Pathway of Ser Biosynthesis (PPSB) has been known to exist since the 1950s, but its biological relevance was not revealed until quite recently when the last enzyme of the pathway, the Phosphoserine Phosphatase, was functionally characterized. In the associated study¹, we characterized a family of genes coding for putatite phosphoglycerate dehydrogenases (PGDH, 3-PGDH, and EDA9), the first enzyme of the PPSB. A metabolomics study using overexpressing plants indicated that all PGDH family genes were able to regulate Ser homeostasis but only lacking of EDA9 expression caused drastic developmental defects. We provided genetic and molecular evidence for the essential role of EDA9 for embryo and pollen development. Here, some new insights into the physiological/molecular function of PPSB and Ser are presented and discussed.