Production of 3-O-xylosyl quercetin in Escherichia coli

Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/?pgi, E. coli BL21(DE3)/?zwf, E. coli BL21(DE3)/?pgi?zwf, and E. coli BL21(DE3)/?pgi?zwf?ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/?pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/?pgi?zwf?ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.     

Sex Specific Differences in Fetal Middle Cerebral Artery and Umbilical Venous Doppler

Background

The incidence of several adverse pregnancy outcomes including fetal growth restriction are higher in pregnancies where the fetus is male, leading to suggestions that placental insufficiency is more common in these fetuses. Placental insufficiency associated with fetal growth restriction may be identified by multi-vessel Doppler assessment, but little evidence exists regarding sex specific differences in these Doppler indices or placental function. This study aims to investigate sex specific differences in fetal and placental perfusion and to correlate these changes with intra-partum outcome.

Methods and Findings

This is a prospective cohort study. We measured Doppler indices of 388 term pregnancies immediately prior to the onset of active labour (≤3 cm dilatation). Fetal sex was unknown at the time of the ultrasound assessment. Information from the ultrasound scan was not made available to clinical staff. Case notes and electronic records were reviewed following delivery. We report significantly lower Middle Cerebral artery pulsatility index (1.34 vs. 1.43, p = 0.004), Middle Cerebral artery peak velocity (53.47 cm/s vs. 58.10 cm/s, p = <0.001), and Umbilical venous flow/kg (56 ml/min/kg vs. 61 ml/min/kg, p = 0.02) in male fetuses. These differences however, were not associated with significant differences in intra-partum outcome.

Conclusion

Sex specific differences in feto-placental perfusion indices exist. Whilst the physiological relevance of these is currently unknown, the identification of these differences adds to our knowledge of the physiology of male and female fetuses in utero. A number of disease processes have now been shown to have an association with changes in fetal haemodynamics in-utero, as well as having a sex bias, making further investigation of the sex specific differences present during fetal life important. Whilst the clinical application of these findings is currently limited, the results from this study do provide further insight into the gender specific circulatory differences present in the fetal period.     

Role of H2AX in DNA damage response and human cancers

H2AX, the evolutionarily conserved variant of histone H2A, has been identified as one of the key histones to undergo various post-translational modifications in response to DNA double-strand breaks (DSBs). By virtue of these modifications, that include acetylation, phosphorylation and ubiquitination, H2AX marks the damaged DNA double helix, facilitating local recruitment and retention of DNA repair and chromatin remodeling factors to restore genomic integrity. These modifications are essential for effective DSB repair, so is their removal for cell, to recover from checkpoint arrest. Because of these vital roles during DSB signaling and also its activation during early cancer stages, H2AX is emerging as an intriguing gene in tumor biology, supported further by frequent deletion of the region harboring this gene. This review focuses on the insights gained from recent studies on dynamic regulation of H2AX in DSB repair. Also, posing future challenges in the area of chromatin reorganization and retention of epigenetic signature post-DSB-repair with implication of its haploinsufficiency in human cancers.     

Determining bioclimatic space of Himalayan alder for agroforestry systems in Nepal

Himalayan alder species are proven to be very useful in traditional as well as contemporary agroforestry practice. These nitrogen-fixing trees are also useful in the land restoration. Therefore, understanding the distribution of Himalayan alder and the potential zone for plantation is meaningful in the agroforestry sector. Suitable climatic zones of Alnus spp. were modelled in MaxEnt software using a subset of least correlated bioclimatic variables for current conditions (1950-2000), topographic variables (DEM derived) and Landuse Landcover (LULC) data. We generated several models and selected the best model against random models using ANOVA and t-test. The environmental variables that best explained the current distribution of the species were identified and used to project into the future. For future projections, ensemble scenarios of climate change projection derived from the results of 19 Earth System Models (ESM) were used. Our model revealed that the most favorable conditions for Alnus nepalensis are in central Nepal in the moist north-west facing slope, whereas for Alnus nitida they are in western Nepal. The major climatic factor that contributes to Alnus species distribution in Nepal appears to be precipitation during the warmest quarter for A. nepalensis and precipitation during the driest quarter for A. nitida. Future projections revealed changes in the probability distribution of these species, as well as where they need conservation and where they can be planted. Also, our model predicts that the distribution of Alnus spp. in hilly regions will remain unchanged, and therefore may represent sites that can be used to revitalize traditional agroforestry systems and extract source material for land restoration.     

<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.     

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.     

An E3 ubiquitin ligase prevents ectopic localization of the centromeric histone H3 variant via the centromere targeting domain

Proper centromere function is critical to maintain genomic stability and to prevent aneuploidy, a hallmark of tumors and birth defects. A conserved feature of all eukaryotic centromeres is an essential histone H3 variant called CENP-A that requires a centromere targeting domain (CATD) for its localization. Although proteolysis prevents CENP-A from mislocalizing to euchromatin, regulatory factors have not been identified. Here, we identify an E3 ubiquitin ligase called Psh1 that leads to the degradation of Cse4, the budding yeast CENP-A homolog. Cse4 overexpression is toxic to psh1Δ cells and results in euchromatic localization. Strikingly, the Cse4 CATD is a key regulator of its stability and helps Psh1 discriminate Cse4 from histone H3. Taken together, we propose that the CATD has a previously unknown role in maintaining the exclusive localization of Cse4 by preventing its mislocalization to euchromatin via Psh1-mediated degradation.