Squalene epoxidase (SE) gene related to triterpenoid biosynthesis assists to select elite genotypes in medicinal plant: Cyclocarya paliurus (Batal.) Iljinskaja.

Triterpenoids, known for their anti-inflammatory, anticancer, and hypoglycemic properties, are the major bioactive components in Cyclocarya paliurus (Batal.) Iljinskaja. Selecting elite individuals with high triterpenoids content is the basis of C. paliurus industry for medicinal use. In this study, seasonal variation patterns of total triterpenoids and five triterpene monomers accumulation for three groups with different total triterpenoid contents (TTC; H: 59.74-64.03 mg g-1; M: 47.66-57.08 mg g-1, and L: 35.26-42.22 mg g-1) were surveyed. Seasonal expression dynamics of 6 key genes relevant to triterpenoids biosynthesis, including HMGR, DXR, SQS, SE, LUS, and ß-AS, were described by quantitative real-time PCR (qRT-PCR) for three groups. The expression levels of HMGR, SE, LUS, and ß-AS genes in group H were higher than in groups M and L. In addition, Pearson correlation analysis showed that they were significantly positively correlated with triterpene accumulation, and the expression level of SE gene not only was significantly correlated with downstream genes, but also exhibited a linear relationship with TTC, especially in September. These results suggest that SE gene could serve as an effective make for screening elite individuals with high TTC from the germplasm of C. paliurus for medicinal use. Further testing on randomly selected individuals in next September proved the feasibility and reliability of SE gene in assisted selection. Also, we successfully cloned the full-length cDNA of SE. Thus, our work provides an efficient way to attain superior genotypes to develop medicinal industry of C. paliurus in practice.

Screening and engineering of high-activity promoter elements through transcriptomics and red fluorescent protein visualization in Rhodobacter sphaeroides.

The versatile photosynthetic α-proteobacterium Rhodobacter sphaeroides, has recently been extensively engineered as a novel microbial cell factory (MCF) to produce pharmaceuticals, nutraceuticals, commodity chemicals and even hydrogen. However, there are no well-characterized high-activity promoters to modulate gene transcription during the engineering of R. sphaeroides. In this study, several native promoters from R. sphaeroides JDW-710 (JDW-710), an industrial strain producing high levels of co-enzyme Q10 (Q10) were selected on the basis of transcriptomic analysis. These candidate promoters were then characterized by using gusA as a reporter gene. Two native promoters, P rsp _ 7571 and P rsp _ 6124 , showed 620% and 800% higher activity, respectively, than the tac promoter, which has previously been used for gene overexpression in R. sphaeroides. In addition, a P rsp _ 7571 -derived synthetic promoter library with strengths ranging from 54% to 3200% of that of the tac promoter, was created on the basis of visualization of red fluorescent protein (RFP) expression in R. sphaeroides. Finally, as a demonstration, the synthetic pathway of Q10 was modulated by the selected promoter T334* in JDW-710; the Q10 yield in shake-flasks increased 28% and the production reached 226 mg/L. These well-characterized promoters should be highly useful in current synthetic biology platforms for refactoring the biosynthetic pathway in R. sphaeroides-derived MCFs.

RNA in situ hybridization and expression of related genes regulating the accumulation of triterpenoids in Cyclocarya paliurus.

Cyclocarya paliurus (Batal.) Iljinskaja, a woody medicinal species in the Juglandaceae, grows extensively in subtropical areas of China. Triterpenoids in the leaves have health-promoting effects, including hypoglycemic and hypolipidemic activities. To understand triterpenoid biosynthesis, transport and accumulation in C. paliurus during the growing season, gene cloning, gene expression and RNA in situ hybridization of related genes were used, and accumulation was examined in various organs. The complete coding sequences (CDSs) of three genes, CpHMGR, CpDXR and CpSQS, were obtained from GenBank and RACE. RNA in situ hybridization signals of the three genes mainly occurred in the epidermis, palisade tissue, phloem and xylem of leaf, shoot and root, with the signals generally consistent with the accumulation of metabolites in tissues, except in the xylem. Both gene expression and triterpenoid accumulations showed seasonal variations in all organs. However, total triterpenoid content in the leaves was significantly higher than that in the shoots, with the maximum in shoots in August and in leaves in October. According to Pearson correlation analysis, triterpenoid accumulation in the leaves was significantly positively related with the relative expression of CpSQS. However, the relation between gene expression and accumulation was dependent on the role of the gene in the pathway as well as on the plant organ. The results suggested that most of the intermediates catalyzed by CpHMGR and CpDXR in young shoots and roots were used in growth and flowering in the spring, whereas subsequent triterpenoid biosynthesis in the downstream catalyzed by CpSQS mainly occurred in the leaves by using transferred and in situ intermediates as substrates. Thus, this study provides a reference to improve triterpenoid accumulation in future C. paliurus plantations.

Phosphate limitation increases coenzyme Q10 production in industrial Rhodobacter sphaeroides HY01.

Coenzyme Q10 (CoQ10) is an important component of the respiratory chain in humans and some bacteria. As a high-value-added nutraceutical antioxidant, CoQ10 has excellent capacity to prevent cardiovascular disease. The content of CoQ10 in the industrial Rhodobacter sphaeroides HY01 is hundreds of folds higher than normal physiological levels. In this study, we found that overexpression or optimization of the synthetic pathway failed CoQ10 overproduction in the HY01 strain. Moreover, under phosphate- limited conditions (decreased phosphate or in the absence of inorganic phosphate addition), CoQ10 production increased significantly by 12% to220 mg/L, biomass decreased by 12%, and the CoQ10 productivity of unit cells increased by 27%. In subsequent fed-batch fermentation, CoQ10 production reached 272 mg/L in the shake-flask fermentation and 1.95 g/L in a 100-L bioreactor under phosphate limitation. Furthermore, to understand the mechanism associated with CoQ10 overproduction under phosphate- limited conditions, the comparatve transcriptome analysis was performed. These results indicated that phosphate limitation combined with glucose fed-batch fermentation represented an effective strategy for CoQ10 production in the HY01. Phosphate limitation induced a pleiotropic effect on cell metabolism, and that improved CoQ10 biosynthesis efficiency was possibly related to the disturbance of energy metabolism and redox potential.

Epigenome-wide study for the offspring exposed to maternal HBV infection during pregnancy, a pilot study.

BACKGROUND & AIM: Hepatitis B virus (HBV) can be transmitted to infants, and is related to infants' later disease risk. Epigenetic change (such as DNA methylation) may be mechanism underlying the relationship. In this study, we aimed to investigate whether prenatal HBV infection could alter DNA methylation status in newborns. METHOD: We selected 12 neonates with intrauterine HBV infection whose mothers were HBsAg-positive during pregnancy, relative to 12 HBV-free neonates with HBsAg-negative mothers. The pattern of genome-wide DNA methylation in the umbilical cord blood was investigated by Illumina Infinium Human Methylation 450K BeadChip. RESULT: The average level of global methylation in infected neonates exposed to maternal HBV infection was not significantly different from controls. However, after adjusting for multiple comparisons, we found differential significance in the cases group compared to the controls for 663 CpG sites, associated with 534 genes. Among these sites, 53.85% (357/663) had decreased methylation (ΔM < 0) and 46.15% (306/663) had increased methylation (ΔM > 0). The average percentage change (Δß) in methylation ranged from -46% to 36%. Validated by pyrosequencing, we identified 4 significantly differentially methylated CpG sites in the KLHL35 gene and additional CpGs for the CPT1B gene. These genes play a role in the development of hepatocellular and colorectal carcinoma and fatty acid oxidation, suggesting the candidature of these genes in HBV related disease. CONCLUSION: Prenatal HBV exposure, even without malformation or preterm birth, may alter the epigenome profile in newborns. We identified a set of genes with differentially methylated CpG sites presented in the cord blood of HBV-infected newborns with HBsAg-positive mothers, demonstrating that DNA methylation status at birth can be used as a biomarker of prenatal exposure. These DNA methylation differences suggest a possible role for epigenetic processes in neonatal development in response to prenatal HBV exposure.

Author Correction: Levels of Faecal Calprotectin and Magnetic Resonance Enterocolonography Correlate with Severity of Small Bowel Crohn's Disease: A Retrospective Cohort Study.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Eosinophilic gastroenteritis presenting as upper gastrointestinal hematoma and ulcers after endoscopic biopsy: A case report and literature review.

RATIONALE: Eosinphilic gastroenteritis (EG) is a gastrointestinal disorder characterized by eosinophilic infiltration with various manifestations. The diagnosis is usually confirmed by an endoscopic biopsy, which is considered a safe and routine procedure for the majority. PATIENT CONCERNS: We report a 54-year-old male who was presented with intermittent periumbilical pain and melena, and only revealed verrucous gastritis by endoscopy. DIAGNOSES: The patient's condition worsened two days after the endoscopic biopsy, and another endoscopy found hematoma and ulcers in upper gastrointestinal tract. He was diagnosed with EG by the pathological analysis of biopsy specimen. INTERVENTIONS: Oral methylprednisolone and Montelukast were prescribed. OUTCOMES: The patient got remission after initiation of the treatment. LESSONS: This case highlights an extremely rare but potentially severe complication of endoscopic biopsies in patients with EG. Physicians should be cautious with hematoma or ulceration, and consider it in such patients who undergo this procedure.

Levels of Faecal Calprotectin and Magnetic Resonance Enterocolonography Correlate with Severity of Small Bowel Crohn's Disease: A Retrospective Cohort Study.

Few studies have evaluated the usefulness of fecal calprotectin (FC) or magnetic resonance enterography (MRE) in diagnosing active Crohn's disease (CD) of the small bowel. In the study, we investigated the reliability of FC and MRE in assessing the activity of ileal CD and further explored the relationship between levels of FC and MRE scores. A total of 221 patients were diagnosed with ileal or ileo-colitis CD in our department between July 2012 and October 2016. The global magnetic resonance index of activity (MaRIA) correlated with the simple endoscopic score for CD (SES-CD) (r = 0.527, P = 0.005). When analysed segment-by-segment, a significant correlation was still observed (r = 0.590, P < 0.001). The SES-CD correlated closest with FC (r = 0.503), followed by CRP (r = 0.461), ESR (0.377) and the CDAI (r = 0.320). In receiver operating characteristic (ROC) analyses, the FC cut-off value of mucosal healing was 213.1 µg/g, with 76.1% sensitivity and 66.7% specificity. As for MaRIA, a cut-off value of 6.8 for each segment provided a sensitivity of 100% and a specificity of 79.2%. No agreement between MaRIA and FC levels was found. In conclusion, a combination of FC levels and MaRIA could be effective in monitoring mucosal activity in patients with small bowel CD.

Thymoquinone Alleviates the Experimental Diabetic Peripheral Neuropathy by Modulation of Inflammation.

Thymoquinone has been reported to exhibit antioxidant and anti-inflammatory effects. Inflammation plays an important role in pathogenesis of diabetic peripheral neuropathy. This study investigated the effects of TQ on proliferation and apoptosis of Schwann cells exposed to high glucose conditions and electrophysiological and morphological changes of the sciatic nerve in a DPN rat model as well as relevant inflammatory mechanism. Cell proliferation and apoptosis of Schwann cells were measured using the Cell Counting Kit-8 and flow cytometry. DPN model was established in streptozotocin-induced diabetic rats. Nerve conduction velocity was measured before and after treatment. Morphologic changes were observed by H&E staining and transmission electron microscopy. COX-2, IL-1ß, IL-6, and Caspase-3 expression was investigated by western blotting and Bio-Plex Pro(TM) Assays. Finally, TQ alleviated the inhibition of Schwann cell proliferation and protected against Schwann cell apoptosis. It improved nerve conduction velocity, and alleviated the DPN-induced morphological changes and demyelination of the sciatic nerve. COX-2, IL-1ß, IL-6 and Caspase-3 expression in sciatic nerve or isolated cultured Schwann cells, were also decreased by TQ. These results indicate TQ has a protective effect on peripheral nerves in a DPN rat model. The mechanism may be mediated partly by the modulation of the inflammatory reaction.

Hyperactivity and impaired attention in Gamma aminobutyric acid transporter subtype 1 gene knockout mice.

OBJECTIVES: Attention-deficit hyperactivity disorder (ADHD) is a common neurobehavioural disorder. It is conceivable that Gamma aminobutyric acid (GABA) neurotransmission is implicated in the pathophysiology of ADHD. This study investigated the effect of GABA transporter 1 (GAT-1) on the anxiety-like behaviours and cognitive function in knockout mice. METHODS: In all, 20 adult male mice were divided into two groups: wild-type (WT) group and GAT-1-/- group. The open field test, elevated O-maze (EZM) and Morris water maze were used to evaluate behavioural traits relevant to ADHD. RESULTS: Compared with WT mice, the GAT-1-/- mice travelled longer and displayed an enhanced kinematic velocity with the significant reduction of rest time in the open field test (p<0.05). The EZM showed that GAT-1-/- mice displayed a significant increase in total entries into the open sectors and the closed sectors compared with the WT mice. The WT mice showed shorter latencies after the training session (p<0.01), whereas the GAT-1-/- mice made no difference during probe test, the GAT-1-/- mice spent less time in the target quadrant (p<0.01). CONCLUSION: Our results demonstrated that GAT-1-/- mice have phenotypes of hyperactivity, impaired sustained attention and learning deficiency, and the performance of GAT-1-/- mice is similar to ADHD symptoms. So, the study of the GAT-1-/- mice may provide new insights into the mechanisms and the discovery of novel therapeutics for the treatment of ADHD.

[Levels of phthalate internal exposure levels in pregnant women and influencing factors].

OBJECTIVE: To investigate the levels and influencing factors of phthalate internal exposure in pregnant women (gestation age ≤ 16 weeks). METHODS: During April to June in 2013, 1 020 pregnant women (gestation age ≤ 16 weeks) who had established the maternal care manual were recruited in maternal and child health hospital of Siming District, Xiamen city. Participators were asked to complete a questionnaire to obtain information on socio-demographic characteristics, lifestyle behaviors, and antenatal examination and to provide a urine sample. Finally, 998 pregnant women who provided a urine sample and completed the questionnaire were enrolled. Adopting systematic sampling method, 100 ones were selected randomly among 998 pregnant women. High performance liquid chromatography-electrospray ionization-tandern mass was used to determine the concentration of five phthalate monoesters in each urine, including mono-n-methyl phthalate (MMP), mono-ethyl phthalate (MEP), mono-butyl phthalate (MBP), mono-benzyl phthalate (MBzP), mono-ethylhexyl phthalate (MEHP). Based on the measurements and questionnaire data, multivariate logistic regression was used to analyze the association between the phthalate monoester levels and potential influential factors. RESULTS: The detection rates of MMP, MEP, MBP, MBzP and MEHP in 100 pregnant urine samples were 94%, 93%, 87%, 83%, 99%, respectively. And the urinary median uncorrected concentrations of MMP, MEP, MBP, MBzP and MEHP in 100 urine samples were 20.56, 17.62, 10.15, 2.03, and 5.12 ng/ml, respectively. Specific gravity-corrected concentration were 20.81, 20.36, 12.88, 2.58, 5.00 ng/ml, respectively. The results of multivariate logistic regression analysis indicated that: education degree was negatively associated with urinary concentration of MMP, MEP, MBP, MBzP and MEHP, OR (95% CI) were 0.495 (0.253-0.966), 0.380 (0.191-0.755), 0.379 (0.186-0.774), 0.401 (0.196-0.819), 0.373(0.183-0.762), respectively. Participants who had hair permed and dyed during pregnancy had higher urinary level of MBP and MBzP, OR (95% CI) were 12.867 (1.240-133.525), 15.982 (1.367-186.911), respectively; Participants who use cosmetics during pregnancy had higher urinary level of MEP and MBP, OR (95% CI) were 2.977 (1.012-8.757), 4.440 (1.485-13.272), respectively; plastic bottled water consumption was positively associated with urinary concentrations of MEP and MEHP, OR (95% CI) were 3.780 (1.417-10.083), 2.699 (1.039-7.010), respectively; annual household income was negatively associated with urinary concentration of MMP, OR (95% CI) was 0.597 (0.372-0.959); individuals who took medications during pregnancy had higher urinary level of MEHP than non-takers, OR (95% CI) was 4.853 (1.084-21.732). CONCLUSION: Pregnant women whose gestation age was less than 16 weeks are generally exposed to phthalate. Phthalate internal exposure levels are significantly associated with most measured factors and the influencing factors with different phthalates internal exposure levels are different.