<Emphasis Type="Italic">Escherichia coli</Emphasis> modular coculture system for resveratrol glucosides production

In bio-based fermentation, the overall bioprocess efficiency is significantly affected by the metabolic burden associated with the expression of complete biosynthetic pathway as well as precursor and cofactor generating enzymes into a single microbial cell. To attenuate such burden by compartmentalizing the enzyme expression, recently synthetic biologists have used coculture or poly-culture techniques for biomolecules synthesis. In this paper, coculture system of two metabolically engineered Escherichia coli populations were employed which comprises upstream module expressing two enzymes converting para-coumaric acid into resveratrol and the downstream module expressing glucosyltransferase to convert the resveratrol into its glucosidated forms; polydatin and resveratroloside. Upon optimization of the initial inoculum ratio of two E. coli populations, 92 mg resveratrol glucosides/L (236 µM) was produced i.e. achieving 84% bioconversion from 280 µM of p-coumaric acid in 60 h by 3 L fed batch fermentor. This is the report of applying coculture system to produce resveratrol glucosides by expressing the aglycone formation pathway and sugar dependent pathway into two different cells.     

Patient-Reported Outcomes for Quality of Life Assessment in Atrial Fibrillation: A Systematic Review of Measurement Properties

BackgroundAtrial fibrillation is a large and growing burden across all types of healthcare. Both incidence and prevalence are expected to double in the next 20 years, with huge impact on hospital admissions, costs and patient quality of life. Patient wellbeing determines the management strategy for atrial fibrillation, including the use of rhythm control therapy and the clinical success of heart rate control. Hence, evaluation of quality of life is an emerging and important part of the assessment of patients with atrial fibrillation. Although a number of questionnaires to assess quality of life in atrial fibrillation are available, a comprehensive overview of their measurement properties is lacking.InterpretationGiven the low ratings for many measurement properties, no single questionnaire can be recommended, although AFEQT performed strongest. Further studies to robustly assess reliability, validity and responsiveness of AF-specific quality of life questionnaires are required. This review consolidates the current evidence for quality of life assessment in patients with atrial fibrillation and identifies priority areas for future research.     

All-Cause Mortality of Low Birthweight Infants in Infancy,Childhood, and Adolescence: Population Study of England and Wales

BackgroundLow birthweight (LBW) is associated with increased mortality in infancy, but its association with mortality in later childhood and adolescence is less clear. We investigated the association between birthweight and all-cause mortality and identified major causes of mortality for different birthweight groups.ConclusionsLBW is associated with infant and later child and adolescent mortality, with perinatal factors and congenital malformations explaining many of the deaths. By understanding and ameliorating the influences of upstream exposures such as maternal smoking and deprivation, later mortality can be decreased by reducing the delivery of vulnerable infants with LBW.     

Production of 7-O-methyl aromadendrin, a medicinally valuable flavonoid, in Escherichia coli

7-O-Methyl aromadendrin (7-OMA) is an aglycone moiety of one of the important flavonoid-glycosides found in several plants, such as Populus alba and Eucalyptus maculata, with various medicinal applications. To produce such valuable natural flavonoids in large quantity, an Escherichia coli cell factory has been developed to employ various plant biosynthetic pathways. Here, we report the generation of 7-OMA from its precursor, p-coumaric acid, in E. coli for the first time. Primarily, naringenin (NRN) (flavanone) synthesis was achieved by feeding p-coumaric acid and reconstructing the plant biosynthetic pathway by introducing the following structural genes: 4-coumarate-coenzyme A (CoA) ligase from Petroselinum crispum, chalcone synthase from Petunia hybrida, and chalcone isomerase from Medicago sativa. In order to increase the availability of malonyl-CoA, a critical precursor of 7-OMA, genes for the acyl-CoA carboxylase α and β subunits (nfa9890 and nfa9940), biotin ligase (nfa9950), and acetyl-CoA synthetase (nfa3550) from Nocardia farcinica were also introduced. Thus, produced NRN was hydroxylated at position 3 by flavanone-3-hydroxylase from Arabidopsis thaliana, which was further methylated at position 7 to produce 7-OMA in the presence of 7-O-methyltransferase from Streptomyces avermitilis. Dihydrokaempferol (DHK) (aromadendrin) and sakuranetin (SKN) were produced as intermediate products. Overexpression of the genes for flavanone biosynthesis and modification pathways, along with malonyl-CoA overproduction in E. coli, produced 2.7 mg/liter (8.9 μM) 7-OMA upon supplementation with 500 μM p-coumaric acid in 24 h, whereas the strain expressing only the flavanone modification enzymes yielded 30 mg/liter (99.2 μM) 7-OMA from 500 μM NRN in 24 h.     

Myogenic contraction in rat skeletal muscle arterioles: smooth muscle membrane potential and Ca(2+) signaling

The present studies examined relationships between intraluminal pressure, membrane potential (E(m)), and myogenic tone in skeletal muscle arterioles. Using pharmacological interventions targeting Ca(2+) entry/release mechanisms, these studies also determined the role of Ca(2+) pathways and E(m) in determining steady-state myogenic constriction. Studies were conducted in isolated and cannulated arterioles under zero flow. Increasing intraluminal pressure (0-150 mmHg) resulted in progressive membrane depolarization (-55.3 +/- 4.1 to -29.4 +/- 0.7 mV) that exhibited a sigmoidal relationship between extent of myogenic constriction and E(m). Thus, despite further depolarization, at pressures >70 mmHg, little additional vasoconstriction occurred. This was not due to an inability of voltage-operated Ca(2+) channels to be activated as KCl (75 mM) evoked depolarization and vasoconstriction at 120 mmHg. Nifedipine (1 microM) and cyclopiazonic acid (30 microM) significantly attenuated established myogenic tone, whereas inhibition of inositol 1,4,5-trisphosphate-mediated Ca(2+) release/entry by 2-aminoethoxydiphenylborate (50 microM) had little effect. Combinations of the Ca(2+) entry blockers with the sarcoplasmic reticulum (SR) inhibitor caused a total loss of tone, suggesting that while depolarization-mediated Ca(2+) entry makes a significant contribution to myogenic tone, an interaction between Ca(2+) entry and SR Ca(2+) release is necessary for maintenance of myogenic constriction. In contrast, none of the agents, in combination or alone, altered E(m), demonstrating the downstream role of Ca(2+) mobilization relative to changes in E(m). Large-conductance Ca(2+)-activated K(+) channels modulated E(m) to exert a small effect on myogenic tone, and consistent with this, skeletal muscle arterioles appeared to show an inherently steep relationship between E(m) and extent of myogenic tone. Collectively, skeletal muscle arterioles exhibit complex relationships between E(m), Ca(2+) availability, and myogenic constriction that impact on the tissue's physiological function.     

Synergic Activation of Toll-Like Receptor (TLR) 2/6 and 9 in Response to Ureaplasma parvum & urealyticum in Human Amniotic Epithelial Cells

Ureaplasma species are the most frequently isolated microorganisms inside the amniotic cavity and have been associated with spontaneous abortion, chorioamnionitis, premature rupture of the membranes (PROM), preterm labour (PL) pneumonia in neonates and bronchopulmonary dysplasia in neonates. The mechanisms by which Ureaplasmas cause such diseases remain unclear, but it is believed that inappropriate induction of inflammatory responses is involved, triggered by the innate immune system. As part of its mechanism of activation, the innate immune system employs germ-lined encoded receptors, called pattern recognition receptors (PRRs) in order to “sense” pathogens. One such family of PRRs are the Toll like receptor family (TLR). In the current study we aimed to elucidate the role of TLRs in Ureaplasma-induced inflammation in human amniotic epithelial cells. Using silencing, as well as human embryonic kidney (HEK) transfected cell lines, we demonstrate that TLR2, TLR6 and TLR9 are involved in the inflammatory responses against Ureaplasma parvum and urealyticum serovars. Ureaplasma lipoproteins, such as Multiple Banded antigen (MBA), trigger responses via TLR2/TLR6, whereas the whole bacterium is required for TLR9 activation. No major differences were observed between the different serovars. Cell activation by Ureaplasma parvum and urealyticum seem to require lipid raft function and formation of heterotypic receptor complexes comprising of TLR2 and TLR6 on the cell surface and TLR9 intracellularly.     

Proteomic approach to enhance doxorubicin production in <Emphasis Type="Italic">panK</Emphasis>-integrated <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> ATCC 27952

Biosynthesis of polyketide compounds depends upon the starter and extender units of coenzyme A derivatives of carboxylic acids present in the host organism. To increase the coenzyme A (CoA) pool, pantothenate kinase (panK) gene from Escherichia coli was integrated into S. peucetius ATCC 27952 (panK-integrated strain, BG200), which resulted in increase in aglycone polyketide ε-rhodomycinone (RHO), but decrease in the desired product, i.e., doxorubicin (DXR). To reduce RHO accumulation by synthesizing daunorubicin (DNR) from RHO more efficiently, glycosyltransferase (dnrQS) was overexpressed (pIBR25::dnrQS in panK-integrated strain, BG201). However, DnrQS overexpression still resulted in less production of DXR compared with the parental strain. To understand the results in detail by investigating the proteome changes in the panK-integrated strain, two-dimensional (2D) gel electrophoresis was performed. Among the several proteins that are up- or downregulated in BG200, efflux protein DrrA was our main target of interest, because it is directly related to DXR/DNR production in S. peucetius. DXR transporter DrrAB was additionally introduced in BG200 to enhance secretion of toxic DXR. Compared with S. peucetius ATCC 27952, BG204 (pIBR25::drrAB in panK-integrated strain), produced two times higher amount of DXR, which is 9.4-fold higher than that of panK-integrated strain BG200. The results show that the proteomic approach is quite useful in host development of Streptomyces and understanding cell physiology for antibiotic production.     

Identification and characterization of <Emphasis Type="Italic">gerPI</Emphasis> and <Emphasis Type="Italic">gerPII</Emphasis> involved in epoxidation and hydroxylation of dihydrochalcolactone in <Emphasis Type="Italic">Streptomyces</Emphasis> species KCTC 0041BP

The macrolide antibiotics are biosynthesized by initial assembly of a macrolactone ring, followed by a series of post-polyketide (PKS) modifications. In general, the additional hydroxyl or epoxy groups are installed by cytochrome P450 enzymes, improving the bioactivity profile through structural diversification of natural products. The biosynthetic gene cluster for the 16-membered macrolide antibiotic dihydrochalcomycin (DHC) has been cloned from Streptomyces sp. KCTC 0041BP. Three cytochrome P450 genes are found in the DHC biosynthetic gene (ger) cluster. Two P450 enzymes were characterized from this cluster. Disruption of gerPI accumulated predominantly 12,13-de-epoxydihydrochalcomycin while disruption of gerPII accumulated 8-dehydroxy-12,13-de-epoxydihydrochalcomycin; DHC production was abolished in both cases. The results suggest that GerPII P450 catalyzes hydroxylation at the C₈ position followed by an epoxidation reaction catalyzed by GerPI P450 at the C₁₂–C₁₃ position.