Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)

The presence of acetate exceeding 5 g/L is a major concern during E. coli fermentation due to its inhibitory effect on cell growth, thereby limiting high-density cell culture and recombinant protein production. Hence, engineered E. coli strains with enhanced acetate tolerance would be valuable for these bioprocesses. In this work, the acetate tolerance of E. coli was much improved by rewiring its global regulator cAMP receptor protein (CRP), which is reported to regulate 444 genes. Error-prone PCR method was employed to modify crp and the mutagenesis libraries (~3×10⁶) were subjected to M9 minimal medium supplemented with 5–10 g/L sodium acetate for selection. Mutant A2 (D138Y) was isolated and its growth rate in 15 g/L sodium acetate was found to be 0.083 h^-1, much higher than that of the control (0.016 h^-1). Real-time PCR analysis via OpenArray^® system revealed that over 400 CRP-regulated genes were differentially expressed in A2 with or without acetate stress, including those involved in the TCA cycle, phosphotransferase system, etc. Eight genes were chosen for overexpression and the overexpression of uxaB was found to lead to E. coli acetate sensitivity.     

Sequencing and Characterisation of Complete Mitogenome DNA for Rasbora hobelmani (Cyprinidae) with Phylogenetic Consideration

Journal of Ichthyology - The Kottelat rasbora Rasbora hobelmani is a small ray-finned fish categorized under the genus Rasbora in the Cyprinidae family. In this study, the complete mitogenome...     

Reprogramming of sugar transport pathways in Escherichia coli using a permeabilized SecY protein-translocation channel

In the initial step of sugar metabolism, sugar-specific transporters play a decisive role in the passage of sugars through plasma membranes into cytoplasm. The SecY complex (SecYEG) in bacteria forms a membrane channel responsible for protein translocation. The present work shows that permeabilized SecY channels can be used as nonspecific sugar transporters in Escherichia coli. SecY with the plug domain deleted allowed the passage of glucose, fructose, mannose, xylose, and arabinose, and, with additional pore-ring mutations, facilitated lactose transport, indicating that sugar passage via permeabilized SecY was independent of sugar stereospecificity. The engineered E. coli showed rapid growth on a wide spectrum of monosaccharides and benefited from the elimination of transport saturation, improvement in sugar tolerance, reduction in competitive inhibition, and prevention of carbon catabolite repression, which are usually encountered with native sugar uptake systems. The SecY channel is widespread in prokaryotes, so other bacteria may also be engineered to utilize this system for sugar uptake. The SecY channel thus provides a unique sugar passageway for future development of robust cell factories for biotechnological applications.     

Integrated bioinformatics analysis revealed the regulation of angiogenesis by tumor cells in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality, metastasis accounts for most of the cases. Angiogenesis plays an important role in cancer metastasis, but how tumor cells affect the function of endothelial cells by dictating their microRNA (miRNA) expression remains largely unknown.Differentially expressed miRNAs (DEMs) were identified through dataset downloaded from the Gene Expression Omnibus (GEO) database and analyzed by GEO2R. We then used online tools to obtain potential targets of candidate miRNAs and functional enrichment analysis, as well as the protein-protein interaction (PPI). Finally, the function of miR-302c-3p was validated through in vitro assay.In the current study, we found that HCC cells altered miRNA expression profiles of human umbilical vein endothelial cells (HUVECs) and miR-302c-3p was the most down-regulated miRNA in HUVECs when they were co-cultured with HCC-LM3 cells. Functional enrichment analysis of the candidate targets revealed that these genes were involved in epigenetic regulation of gene expression, in particular, cytosine methylation. In addition, PPI network demonstrated distinct roles of genes targeted by miR-302c-3p. Importantly, inhibition of angiogenesis, migration and permeability by the most down-regulated miR-302c-3p in HUVECs was confirmed in vitro. These findings brought us novel insight into the regulation of angiogenesis by HCC cells and provided potential targets for the development of therapeutic strategies.     

Molecular Dynamics Study on Protein and it's Water Structure at High Pressure

Abstract

We have performed NPT molecular dynamics simulations (Langevin Piston Method) on two types of solvated proteins-‘denaturation-unfavorable’ protein (insulin) and ‘denaturation-favorable protein’ (ribonuclease A) at high pressure (from 1 bar up to 20 kbar). The method is based on the extended system formalism introduced by Andersen, where the deterministic equations of motion for the piston degree of freedom are replaced by Langevin equation. We report the structural changes of proteins (ribonuclease A and insulin) and water molecules through radius of gyration, solvent accessible surface area, hydrogen bond pattern, and the topology of water clusters connected by the hydrogen bonded circular network. The solvent accessibility of ribonuclease A is mainly decreased by hydrophilic residues rather than hydrophobic residues under high pressure. From the results of hydrogen bond analysis, we have found that α-helix is more stable than β-sheet under high pressure. In addition, from the analysis of the water cluster, we have observed that for ribonuclease A, 5-membered ring structure is more favorable than 6-membered ring at higher pressure. However, for insulin, the ratio of 5 to 6-ring is constant over the pressure ranges for which we have performed MD simulation. This indicates that the water structure around insulin does not change under high pressure.     

A Modified Valley Restrained Monte Carlo Method to Efficiently Search the Low Energy Structures of Peptides

Abstract

A new Monte Carlo sampling scheme, namely the Modified Valley Restrained Monte Carlo procedure, is used to obtain the global energy minimum conformations for polypeptides, such as Met-enkephalin and Melittin. For each peptide, we found close agreement with previous results from both theoretical and experimental studies. The simple idea for controlling the step size according to the Valley Function, provides useful suggestions in searching the global energy minimum structures, and furthermore helps solve the multiple minima problem.     

Histamine synergistically promotes bFGF‐induced angiogenesis by enhancing VEGF production via H1 receptor

Histamine, a major mediator present in mast cells that is released into the extracellular milieu upon degranulation, is well known to possess a wide range of biological activities in several classic physiological and pathological processes. However, whether and how it participates in angiogenesis remains obscure. In the present study, we observed its direct and synergistic action with basic fibroblast growth factor (bFGF), an important inducer of angiogenesis, on in vitro angiogenesis models of endothelial cells. Data showed that histamine (0.1, 1, 10 µM) itself was absent of direct effects on the processes of angiogenesis, including the proliferation, migration, and tube formation of endothelial cells. Nevertheless, it could concentration‐dependently enhance bFGF‐induced angiogenesis as well as production of vascular endothelial growth factor (VEGF) from endothelial cells. The synergistic effect of histamine on VEGF production could be reversed by pretreatments with diphenhydramine (H1‐receptor antagonist), SB203580 (selective p38 mitogen‐activated protein kinase (MAPK) inhibitor) and L ‐NAME (nitric oxide synthase (NOS) inhibitor), but not with cimetidine (H2‐receptor antagonist) and indomethacin (cyclooxygenase (COX) inhibitor). Moreover, histamine could augment bFGF‐incuced phosphorylation and degradation of IκBα, a key factor accounting for the activation and translocation of nuclear factor κB (NF‐κB) in endothelial cells. These findings indicated that histamine was able to synergistically augment bFGF‐induced angiogenesis, and this action was linked to VEGF production through H1‐receptor and the activation of endothelial nitric oxide synthase (eNOS), p38 MAPK, and IκBα in endothelial cells. J. Cell. Biochem. 114: 1009–1019, 2013. © 2012 Wiley Periodicals, Inc.     

Effect of mesenchymal stem cells on inhibiting airway remodeling and airway inflammation in chronic asthma

Previous studies proved that bone marrow‐derived mesenchymal stem cells (BMSCs) could improve a variety of immune‐mediated disease by its immunomodulatory properties. In this study, we investigated the effect on airway remodeling and airway inflammation by administrating BMSCs in chronic asthmatic mice. Forty‐eight female BALB/c mice were randomly distributed into PBS group, BMSCs treatment group, BMSCs control group, and asthmatic group. The levels of cytokine and immunoglobulin in serum and bronchoalveolar lavage fluid were detected by enzyme‐linked immunosorbent assay. The number of CD4⁺CD25⁺regulatory T cells and morphometric analysis was determined by flow cytometry, hematoxylin‐eosin, immunofluorescence staining, periodic‐acid Schiff, and masson staining, respectively. We found that airway remodeling and airway inflammation were evident in asthmatic mice. Moreover, low level of IL‐12 and high levels of IL‐13, IL‐4, OVA‐specific IgG1, IgE, and IgG2a and the fewer number of CD4⁺CD25⁺regulatory T cells were present in asthmatic group. However, transplantation of BMSCs significantly decreased airway inflammation and airway remodeling and level of IL‐4, OVA‐specific IgE, and OVA‐specific IgG1, but elevated level of IL‐12 and the number of CD4 + CD25 + regulatory T cells in asthma (P < 0.05). However, BMSCs did not contribute to lung regeneration and had no significant effect on levels of IL‐10, IFN‐Y, and IL‐13. In our study, BMSCs engraftment prohibited airway inflammation and airway remodeling in chronic asthmatic group. The beneficial effect of BMSCs might involved the modulation imbalance cytokine toward a new balance Th1–Th2 profiles and up‐regulation of protective CD4 + CD25 + regulatory T cells in asthma, but not contribution to lung regeneration. J. Cell. Biochem. 114: 1595–1605, 2013. © 2013 Wiley Periodicals, Inc.     

Microsatellite genetic variation in the Chinese endemic Eucommia ulmoides (Eucommiaceae): implications for conservation

Eucommia ulmoides, a traditional Chinese medicinal plant, is endangered as a consequence of long‐term and widespread harvest in the late 20th century. It has been widely cultivated as a source of herbal medicine and for use in the organic chemical industry in China. In this study, eight microsatellite markers were applied to investigate genetic diversity in E. ulmoides. Three hundred individuals from one semi‐wild population and nine cultivated populations across its main production area were collected. A high level of genetic diversity at population levels (H_E = 0.716) was observed. The highly outcrossed mating system, high longevity of E. ulmoides and seed admixture may be responsible for high genetic variation within populations. A genetic bottleneck was observed in one population. Populations were only slightly differentiated from one another (F_ST = 0.063); this was also supported by AMOVA, which revealed that 94.05% of the total variation resided within populations. This is probably attributable to long‐distance gene flow mediated by the exchange of seeds by local farmers. Implications of these results for the conservation of genetic resources of E. ulmoides are discussed. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 775–785.