Metabolic pathway optimization using ribosome binding site variants and combinatorial gene assembly

The genes encoding the mevalonate-based farnesyl pyrophosphate (FPP) biosynthetic pathway were encoded in two operons and expressed in Escherichia coli to increase the production of sesquiterpenes. Inefficient translation of several pathway genes created bottlenecks and led to the accumulation of several pathway intermediates, namely, mevalonate and FPP, and suboptimal production of the sesquiterpene product, amorphadiene. Because of the difficulty in choosing ribosome binding sites (RBSs) to optimize translation efficiency, a combinatorial approach was used to choose the most appropriate RBSs for the genes of the lower half of the mevalonate pathway (mevalonate to amorphadiene). RBSs of various strengths, selected based on their theoretical strengths, were cloned 5′ of the genes encoding mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, and amorphadiene synthase. Operons containing one copy of each gene and all combinations of RBSs were constructed and tested for their impact on growth, amorphadiene production, enzyme level, and accumulation of select pathway intermediates. Pathways with one or more inefficiently translated enzymes led to the accumulation of pathway intermediates, slow growth, and low product titers. Choosing the most appropriate RBS combination and carbon source, we were able to reduce the accumulation of toxic metabolic intermediates, improve growth, and improve the production of amorphadiene approximately fivefold. This work demonstrates that balancing flux through a heterologous pathway and maintaining steady growth are key determinants in optimizing isoprenoid production in microbial hosts.     

ABCC8 genetic variants and risk of diabetes mellitus

Diabetes mellitus (DM) is a major health problem worldwide and it will rapidly increase. This disease is characterized by hyperglycemia caused by defects in insulin secretion, insulin action or both. DM has three types: T1DM, T2M and gestational DM (GDM), of them T2DM is more frequent. Multiple genes and their interactions are involved in insulin secretion pathway. Sulfonylurea receptor encoded by ABCC8 gene, together with inward-rectifier potassium ion channel (Kir6.2) regulates insulin secretion by ATP-sensitive K⁺ (KATP) channel located in the plasma membranes. Disruption of these molecules by different mutations is responsible for risk of DM. Several single nucleotide polymorphisms (SNPs) of ABCC8 gene and their interaction are involved in pathogenicity of DM. This review summarizes the current evidence of contribution of ABC8 genetic variants to the development of DM.     

Divergent pro-inflammatory profile of human dendritic cells in response to commensal and pathogenic bacteria associated with the airway microbiota

Recent studies using culture-independent methods have characterized the human airway microbiota and report microbial communities distinct from other body sites. Changes in these airway bacterial communities appear to be associated with inflammatory lung disease, yet the pro-inflammatory properties of individual bacterial species are unknown. In this study, we compared the immune stimulatory capacity on human monocyte-derived dendritic cells (DCs) of selected airway commensal and pathogenic bacteria predominantly associated with lungs of asthma or COPD patients (pathogenic Haemophillus spp. and Moraxella spp.), healthy lungs (commensal Prevotella spp.) or both (commensal Veillonella spp. and Actinomyces spp.). All bacteria were found to induce activation of DCs as demonstrated by similar induction of CD83, CD40 and CD86 surface expression. However, asthma and COPD-associated pathogenic bacteria provoked a 3-5 fold higher production of IL-23, IL-12p70 and IL-10 cytokines compared to the commensal bacteria. Based on the differential cytokine production profiles, the studied airway bacteria could be segregated into three groups (Haemophilus spp. and Moraxella spp. vs. Prevotella spp. and Veillonella spp. vs. Actinomyces spp.) reflecting their pro-inflammatory effects on DCs. Co-culture experiments found that Prevotella spp. were able to reduce Haemophillus influenzae-induced IL-12p70 in DCs, whereas no effect was observed on IL-23 and IL-10 production. This study demonstrates intrinsic differences in DC stimulating properties of bacteria associated with the airway microbiota.     

A comparison of serum androgens in pre-eclamptic and normotensive pregnant women during the third trimester of pregnancy

Objective: To compare the serum androgens level during the third trimester of pregnancy between normotensive and pre-eclamptic women. Method: A case-control study was performed on 64 pregnant women with the gestational age of 28-34 weeks. 32 women were pre-eclamptic (case group), and 32 women were normotensive till term gestation (control group). The serum level of androgens including sex hormone binding globulin (SHBG), total and free testosterone, androstenedione (ADD), and dehydroepiandrosterone sulfate (DHEA-S), were compared between the two groups. Results: The women of the two groups had no statistically significant difference according to age, gestational age, BMI (body mass index), parity and fetal sex. Serum level of SHBG (90.86 ± 9.30 vs. 55.86 ± 8.02 nmol/l, p = 0.02), total testosterone (3.70 ± 0.57 vs. 2.06 ± 0.24 ng/ml, p = 0.01), free testosterone (1.28 ± 0. 17 vs. 0. 74 ± 0.07 pg/ml, p = 0.01), and ADD (2.47 ± 0.10 vs. 2.17 ± 0.10 ng/ml, p = 0.04), was higher in the pre-eclamptic women. However, there was no difference between the two groups for DHEA-S (0.75 ± 0.18 vs. 0.51 ± 0.08 μg/ml, p = 0.19). Conclusion: Serum androgen levels during third trimester of pregnancy are higher in pre-eclamptic women and this may propose an effect of androgens in the pathogenesis of pre-eclampsia.     

Nitric oxide-dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition

Endothelial nitric oxide synthase (eNOS)-mediated NO production plays a critical role in the regulation of vascular function and pathophysiology. Caveolin-1 (Cav-1) binding to eNOS holds eNOS in an inactive conformation; however, the mechanism of Cav-1-mediated inhibition of activated eNOS is unclear. Here the role of Src-dependent Cav-1 phosphorylation in eNOS negative feedback regulation is investigated. Using fluorescence resonance energy transfer (FRET) and coimmunoprecipitation analyses, we observed increased interaction between eNOS and Cav-1 following stimulation of endothelial cells with thrombin, vascular endothelial growth factor, and Ca(2+) ionophore A23187, which is corroborated in isolated perfused mouse lung. The eNOS/Cav-1 interaction is blocked by eNOS inhibitor L-N(G)-nitroarginine methyl ester (hydrochloride) and Src kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3, 4-d] pyrimidine. We also observe increased binding of phosphomimicking Y14D-Cav-1 mutant transduced in human embryonic kidney cells overexpressing eNOS and reduced Ca(2+)-induced NO production compared to cells expressing the phosphodefective Y14F-Cav-1 mutant. Finally, Src FRET biosensor, eNOS small interfering RNA, and NO donor studies demonstrate NO-induced Src activation and Cav-1 phosphorylation at Tyr-14, resulting in increased eNOS/Cav-1 interaction and inhibition of eNOS activity. Taken together, these data suggest that activation of eNOS promotes Src-dependent Cav-1-Tyr-14 phosphorylation and eNOS/Cav-1 binding, that is, eNOS feedback inhibition.     

Inhibition of GSK‐3β enhances neural differentiation in unrestricted somatic stem cells

GSK‐3β is a key molecule in several signalling pathways, including the Wnt/β‐catenin signalling pathway. There is increasing evidence suggesting Wnt/β‐catenin signalling is involved in the neural differentiation of embryonic, somatic and neural stem cells. However, a large body of evidence indicates that this pathway maintains stem cells in a proliferative state. To address this controversy, we have investigated whether the Wnt/β‐catenin pathway is present and involved in the neural differentiation of newly introduced USSCs (unrestricted somatic stem cells). Our results indicate that the components of Wnt/β‐catenin signalling are present in undifferentiated USSCs. We also show that the treatment of neurally induced USSCs with BIO (6‐bromoindirubin‐3′‐oxime), a specific GSK‐3β inhibitor and Wnt activator, for 5 and 10 days results in increased expression of a general neuronal marker (β‐tubulin III). Moreover, the expression of pGSK‐3β and stabilized β‐catenin increased by BIO in neurally induced USSCs, indicates that the Wnt pathway is activated and functional in these cells. Thus, inhibition of GSK‐3β in USSCs enhances their neural differentiation, which suggests a positive role of the Wnt/β‐catenin signalling pathway towards neural fate.     

Fusion Protein Strategy to Increase Expression and Solubility of Hypervariable Region of VP2 Protein of Infectious Bursal Disease Virus in Escherichia coli

Infectious bursal disease is one of the most important viral diseases in the young chickens. VP2 protein is the major host protective immunogen of the virus. A hypervariable region is present in VP2 protein (hvVP2) that contains immunodominant epitops. The high hydrophobicity of hvVP2 region causes protein aggregation in Escherichia coli (E. coli). The objective of the present study was to improve the expression and the solubility of the hvVP2 protein in E. coli. The effects of fusion partners on the solubility of hvVP2 protein were studied. The protein was expressed in forms of unfused and N-terminally fused to GST and NusA. The results showed that the unfused hvVP2 protein was expressed in very low level. But, N-terminally fused hvVP2 protein to GST (glutathione-S-transferase) and NusA (N utilization substance A) showed significantly enhanced protein expression. The fusion of GST and hvVP2 was produced in aggregated form while in the presence of NusA, the hvVP2 protein was expressed in a soluble form. The NusA-hvVP2 protein was detected by a neutralizing monoclonal antibody, 1A6, in antigen-capture ELISA. In conclusion, the NusA protein is a suitable fusion partner to improve expression and solubility of the hvVP2 protein in E. coli.     

The anti-apoptotic effect of IGF-1 on tissue resident stem cells is mediated via PI3-kinase dependent secreted frizzled related protein 2 (Sfrp2) release

Previous studies suggest that IGF-1 may be used as an adjuvant to stem cell transfer in order to improve cell engraftment in ischemic tissue. In the current study, we investigated the effect of IGF-1 on serum deprivation and hypoxia induced stem cell apoptosis and the possible mechanisms involved. Exposure of adipose tissue derived stem cells (ASCs) to serum deprivation and hypoxia resulted in significant apoptosis in ASC which is partially prevented by IGF-1. IGF-1’s anti-apoptotic effect was abolished in ASCs transfected with Sfrp2 siRNA but not by the control siRNA. Using Western blot analysis, we demonstrated that serum deprivation and hypoxia reduced the expression of nuclear β-catenin, which is reversed by IGF-1. IGF-1’s effect on β-catenin expression was abolished by the presence of PI3-kinase inhibitor LY294002 or in ASCs transfected with Sfrp2 siRNA. These results suggest that IGF-1, through the release of the Sfrp2, contributes to cell survival by stabilizing β-catenin.     

Wheat plants containing an <Emphasis Type="Italic">Osmotin</Emphasis> gene show enhanced ability to produce roots at high NaCl concentration

The genetically engineered osmotin gene located in a binary vector was transferred by shotgun to an Iranian wheat cultivar (Triticum aestivum, cv. Marvdasht). Screening of transgenic plants was carried out by using both molecular and phenotypic approaches. Transgenic lines were studied for their ability to tolerate salinity stress at various concentrations of NaCl. Transgenic line of cv. Marvdasht exhibited a high ability to produce roots at the concentration of 250 mM NaCl. In this research, a resistant Iranian cv. Zarrin and nontransgenic lines of cv. Marvdasht were also compared for their root length under salinity condition. Results showed that transgenic Marvdasht lines are significantly more tolerant than nontransgenic ones and very similar to naturally resistant Zarrin in this aspect. This text was submitted by the authors in English.