Metabolic profiling and enhanced production of phytosterols by elicitation with methyl jasmonate and silver nitrate in whole plant cultures of Lemna paucicostata

In this study, the effects of methyl jasmonate (MJ) and silver nitrate (SN) treatment on metabolic profiles and yields of phytosterols such as campesterol, stigmasterol, and β-sitosterol in whole plant cultures of Lemna paucicostata were investigated using gas chromatography–mass spectrometry coupled with multivariate statistical analysis. The MJ and SN treatments retarded the growth of L. paucicostata plants, while they enhanced the yields of three phytosterols, compared to control. Higher yields of phytosterols were attained at day 28 compared to day 42. Moreover, stigmasterol yield was the highest at 0.85 mg/g from day 28 plants grown under MJ + SN co-treated culture. Among the various metabolites, the levels of palmitic and stearic acids, which might participate in a defense mechanism, were higher in the MJ + SN condition than in control. To determine the optimal timing of MJ + SN addition, MJ + SN was added on days 21, 28, and 35 after inoculation. The total yield and productivity of phytosterol reached maximum levels when the MJ + SN was added at day 35. The highest productivity of stigmasterol (6.08 mg/L) was also achieved when MJ + SN was added on day 35.     

Toward the production of flavone-7-O-β-d-glucopyranosides using Arabidopsis glycosyltransferase in Escherichia coli

Flavonoid glycosides are highly attractive targets due to their dominant roles in clinical, cosmetic production and in the food industry. In this research, an Escherichia coli strain bearing the reconstructed uridine-diphosphate glucose (UDP-glucose) pathway cassette and a putative glycosyltransferase from Arabidopsis thaliana, was developed as a host for the production of apigenin-7-O-β-d-glucoside (APG) and baicalein-7-O-β-d-glucoside (BCG) from exogenously supplied flavone aglycones (apigenin and baicalein, respectively). In order to improve the yield, genetic engineering of E. coli strains for optimization of intracellular UDP-glucose generation, as well as media optimization were carried out. The production was scaled up using a fed batch fermentation, and the maximal yield of products reached 90.88 μM (39.28 mg L^?1) and 76.82 μM (33.19 mg L^?1) of APG and BCG, respectively. And, the maximum bioconversion rate corresponded to 90.88% and 76.82% of apigenin and baicalein, respectively.     

Airway epithelial cells initiate the allergen response through transglutaminase 2 by inducing IL-33 expression and a subsequent Th2 response

Background

Transglutaminase 2 (TG2) is a post-translational protein-modifying enzyme that catalyzes the transamidation reaction, producing crosslinked or polyaminated proteins. Increased TG2 expression and activity have been reported in various inflammatory conditions, such as rheumatoid arthritis, inflammation-associated pulmonary fibrosis, and autoimmune encephalitis. In particular, TG2 from epithelial cells is important during the initial inflammatory response in the lung. In this study, we evaluated the role of TG2 in the pathogenesis of allergic asthma, particularly whether TG2 affects initial activation signaling leading to Th2 differentiation against antigens.

Methods

We induced allergic asthma by ovalbumin sensitization and intranasal challenge in wild-type (WT) BALB/c and TG2-deficient mice. Broncheoalveolar lavage fluid cells and intracellular cytokine production were analyzed by flow cytometry. Interleukin (IL)-33 and TG2 expression in lung epithelial cells was detected by confocal microscopy.

Results

Airway responsiveness was attenuated in TG2-deficient mice compared to that in the WT control. In addition, recruitment of eosinophils and Th2 and Th17 differentiation decreased in TG2-deficient mice. Treatment with cysteamine, a transglutaminase inhibitor, also reduced airway hypersensitivity, inflammatory cell recruitment, and T helper cell differentiation. TG2-deficient mice showed reduced IL-33 expression following induction of allergic asthma compared to those in the WT control.

Conclusions

We found that pulmonary epithelial cells damaged by allergens triggered TG2-mediated IL-33 expression leading to type 2 responses by recruiting both innate and adaptive arms of the immune system.     

Gender-Specific Metabolomic Profiling of Obesity in Leptin-Deficient ob/ob Mice by 1H NMR Spectroscopy

Despite the numerous metabolic studies on obesity, gender bias in obesity has rarely been investigated. Here, we report the metabolomic analysis of obesity by using leptin-deficient ob/ob mice based on the gender. Metabolomic analyses of urine and serum from ob/ob mice compared with those from C57BL/6J lean mice, based on the ¹H NMR spectroscopy in combination with multivariate statistical analysis, revealed clear metabolic differences between obese and lean mice. We also identified 48 urine and 22 serum metabolites that were statistically significantly altered in obese mice compared to lean controls. These metabolites are involved in amino acid metabolism (leucine, alanine, ariginine, lysine, and methionine), tricarbocylic acid cycle and glucose metabolism (pyruvate, citrate, glycolate, acetoacetate, and acetone), lipid metabolism (cholesterol and carnitine), creatine metabolism (creatine and creatinine), and gut-microbiome-derived metabolism (choline, TMAO, hippurate, p-cresol, isobutyrate, 2-hydroxyisobutyrate, methylamine, and trigonelline). Notably, our metabolomic studies showed distinct gender variations. The obese male mice metabolism was specifically associated with insulin signaling, whereas the obese female mice metabolism was associated with lipid metabolism. Taken together, our study identifies the biomarker signature for obesity in ob/ob mice and provides biochemical insights into the metabolic alteration in obesity based on gender.     

Identification of Pyrus Single Nucleotide Polymorphisms (SNPs) and Evaluation for Genetic Mapping in European Pear and Interspecific Pyrus Hybrids

We have used new generation sequencing (NGS) technologies to identify single nucleotide polymorphism (SNP) markers from three European pear (Pyrus communis L.) cultivars and subsequently developed a subset of 1096 pear SNPs into high throughput markers by combining them with the set of 7692 apple SNPs on the IRSC apple Infinium® II 8K array. We then evaluated this apple and pear Infinium® II 9K SNP array for large-scale genotyping in pear across several species, using both pear and apple SNPs. The segregating populations employed for array validation included a segregating population of European pear (‘Old Home’בLouise Bon Jersey’) and four interspecific breeding families derived from Asian (P. pyrifolia Nakai and P. bretschneideri Rehd.) and European pear pedigrees. In total, we mapped 857 polymorphic pear markers to construct the first SNP-based genetic maps for pear, comprising 78% of the total pear SNPs included in the array. In addition, 1031 SNP markers derived from apple (13% of the total apple SNPs included in the array) were polymorphic and were mapped in one or more of the pear populations. These results are the first to demonstrate SNP transferability across the genera Malus and Pyrus. Our construction of high density SNP-based and gene-based genetic maps in pear represents an important step towards the identification of chromosomal regions associated with a range of horticultural characters, such as pest and disease resistance, orchard yield and fruit quality.     

Chromosomes in the Porcine First Polar Body Possess Competence of Second Meiotic Division within Enucleated MII Stage Oocytes

To determine whether chromosomes in the porcine first polar body (PB1) can complete the second meiotic division and subsequently undergo normal pre-implantation embryonic development, we examined the developmental competence of PB1 chromosomes injected into enucleated MII stage oocytes by nuclear transfer method (chromosome replacement group, CR group). After parthenogenetic activation (PA) or in vitro fertilization (IVF), the cleavage rate of reconstructed oocytes in the IVF group (CR-IVF group, 36.4 ± 3.2%) and PA group (CR-PA group, 50.8 ± 4.2%) were significantly lower than that of control groups in which normal MII oocytes were subjected to IVF (MII-IVF group, 75.8 ± 1.5%) and PA (MII-PA group, 86.9 ± 3.7%). Unfertilized rates was significantly higher in the CR-IVF group (48.6 ± 3.3%) than in the MII-IVF group (13.1 ± 3.4%). The blastocyst formation rate was 8.3 ± 1.9% in the CR-PA group, whereas no blastocyst formation was observed in the CR-IVF group. To produce tetraploid parthenogenetic embryos, intact MII stage oocytes injected with PB1chromosomes were electrically stimulated, treated with 7.5 μg/mL cytochalasin B for 3 h (MII oocyte + PB1 + CB group), and then cultured without cytochalasin B. The average cleavage rate of reconstructed oocytes was 72.5% (48 of 66), and the blastocyst formation rate was 18.7% (9 of 48). Chromosome analysis showed similar proportions of haploid and diploid cells in the control (normal MII oocytes) and CR groups after PA; overall, 23.6% of blastocysts were tetraploid in the MII oocyte + PB1 + CB group. These results demonstrate that chromosomes in PB1 can participate in normal pre-implantation embryonic development when injected into enucleated MII stage oocytes, and that tetraploid PA blastocysts are produced (although at a low proportion) when PB1 chromosomes are injected into intact MII stage oocytes.     

Prevalence of and Factors Associated with Albuminuria in the Korean Adult Population: The 2011 Korea National Health and Nutrition Examination Survey

Background

Microalbuminuria is associated with increased risk of renal disease and cardiovascular diseases even in non-diabetic subjects. High incidence rates of microalbuminuria have been found in a number of population-based studies. However, the prevalence and risk factors associated with microalbuminuria in the general population in Korea are unclear.

Objectives

The present study was performed to estimate the prevalence of microalbuminuria and investigate the associated risk factors in the general adult population using the Fifth Korea National Health and Nutrition Examination Survey (KNHANES V-2) data from 2011.

Methods

A total of 5,202 participants (mean age, 45.6 years; men, 2,337; women, 2,865) were included in the analysis. Microalbuminuria was evaluated in participants of KNHANES V-2 based on the urine albumin–creatinine ratio. Estimated glomerular filtration rate was calculated using the Modification of Diet in Renal Disease study equation.

Results

The weighted prevalence of microalbuminuria was 5.2% (95% CI, 4.4–6.1) in the general population. The prevalence of albuminuria is increased with age. After adjustment for age and sex, the presence of albuminuria was associated with increased waist circumference, systolic and diastolic blood pressure, aspartate aminotransferase, triglyceride, fasting plasma glucose, and the presence of hypertension and diabetes. In logistic regression analyses, older age, female sex, diabetes, hypertension, and serum aspartate aminotransferase were independently associated with the presence of albuminuria.

Conclusion

The prevalence of microalbuminuria was found to be 5.2%, and conventional risk factors for cardiovascular diseases are closely related to the presence of microalbuminuria in Korea. Microalbuminuria may be a useful marker to identify individuals with increased risk of cardiovascular disease.     

Dynamic Chromatin Remodeling Mediated by Polycomb Proteins Orchestrates Pancreatic Differentiation of Human Embryonic Stem Cells

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Highlights? Pancreatic lineage progression is governed by PcG-dependent chromatin remodeling ? A temporal chromatin signature predicts regulators of pancreatic development ? Endocrine cells differentiated from hESCs in vivo are similar to native human islets ? In vitro-produced malfunctioning endocrine cells exhibit aberrant chromatin structure     

hESC Expansion and Stemness Are Independent of Connexin Forty-Three-Mediated Intercellular Communication between hESCs and hASC Feeder Cells

Background

Human embryonic stem cells (hESCs) are a promising and powerful source of cells for applications in regenerative medicine, tissue engineering, cell-based therapies, and drug discovery. Many researchers have employed conventional culture techniques using feeder cells to expand hESCs in significant numbers, although feeder-free culture techniques have recently been developed. In regard to stem cell expansion, gap junctional intercellular communication (GJIC) is thought to play an important role in hESC survival and differentiation. Indeed, it has been reported that hESC-hESC communication through connexin 43 (Cx43, one of the major gap junctional proteins) is crucial for the maintenance of hESC stemness during expansion. However, the role of GJIC between hESCs and feeder cells is unclear and has not yet been reported.

Methodology/Principal Findings

This study therefore examined whether a direct Cx43-mediated interaction between hESCs and human adipose-derived stem cells (hASCs) influences the maintenance of hESC stemness. Over 10 passages, hESCs cultured on a layer of Cx43-downregulated hASC feeder cells showed normal morphology, proliferation (colony growth), and stemness, as assessed by alkaline phosphatase (AP), OCT4 (POU5F1-Human gene Nomenclature Database), SOX2, and NANOG expression.

Conclusions/Significance

These results demonstrate that Cx43-mediated GJIC between hESCs and hASC feeder cells is not an important factor for the conservation of hESC stemness and expansion.     

Combination of Entner-Doudoroff Pathway with MEP Increases Isoprene Production in Engineered Escherichia coli

Embden-Meyerhof pathway (EMP) in tandem with 2-C-methyl-D-erythritol 4-phosphate pathway (MEP) is commonly used for isoprenoid biosynthesis in E. coli. However, this combination has limitations as EMP generates an imbalanced distribution of pyruvate and glyceraldehyde-3-phosphate (G3P). Herein, four glycolytic pathways—EMP, Entner-Doudoroff Pathway (EDP), Pentose Phosphate Pathway (PPP) and Dahms pathway were tested as MEP feeding modules for isoprene production. Results revealed the highest isoprene production from EDP containing modules, wherein pyruvate and G3P were generated simultaneously; isoprene titer and yield were more than three and six times higher than those of the EMP module, respectively. Additionally, the PPP module that generates G3P prior to pyruvate was significantly more effective than the Dahms pathway, in which pyruvate production precedes G3P. In terms of precursor generation and energy/reducing-equivalent supply, EDP+PPP was found to be the ideal feeding module for MEP. These findings may launch a new direction for the optimization of MEP-dependent isoprenoid biosynthesis pathways.