ERK/ribosomal S6 kinase (RSK) signaling positively regulates death receptor 5 expression through co-activation of CHOP and Elk1

Death receptor 5 (DR5) is a death domain-containing transmembrane receptor that triggers apoptosis upon binding to its ligand or when overexpressed. Its expression is induced by certain small molecule drugs, including celecoxib, through mechanisms that have not been fully elucidated. The current study has revealed a novel ERK/ribosomal S6 kinase (RSK)-dependent mechanism that regulates DR5 expression primarily using celecoxib as a DR5 inducer. Both C/EBP homologous protein (CHOP) and Elk1 are required for celecoxib-induced DR5 expression based on promoter deletion and mutation analysis and siRNA-mediated gene silencing results. Co-expression of both CHOP and Elk1 exhibited enhanced effects on increasing DR5 promoter activity and DR5 expression, indicating that CHOP and Elk1 co-operatively regulate DR5 expression. Because Elk1 is an ERK-regulated protein, we accordingly found that celecoxib increased the levels of phosphorylated ERK1/2, RSK2, and Elk1. Inhibition of either ERK signaling with a MEK inhibitor or ERK1/2 siRNA, or RSK2 signaling with an RSK2 inhibitor or RSK2 siRNA abrogated DR5 up-regulation by celecoxib as well as other agents. Moreover, these inhibitions suppressed celecoxib-induced CHOP up-regulation. Thus, ERK/RSK-dependent, CHOP and Elk1-mediated mechanisms are critical for DR5 induction. Additionally, celecoxib increased CHOP promoter activity in an ATF4-dependent manner, and siRNA-mediated blockade of ATF4 abrogated both CHOP induction and DR5 up-regulation, indicating that ATF4 is involved in celecoxib-induced CHOP and DR5 expression. Collectively, we conclude that small molecules such as celecoxib induce DR5 expression through activating ERK/RSK signaling and subsequent Elk1 activation and ATF4-dependent CHOP induction.     

A novel metagenome-derived β-galactosidase: gene cloning, overexpression, purification and characterization

A novel β-galactosidase gene, zd410, was isolated by screening a soil metagenomic library. Sequence analysis revealed that zd410 encodes a protein of 672 amino acids with a predicted molecular weight of 78.6 kDa. The recombinant ZD410 was expressed and purified in Pichia pastoris, with a yield of ca. 300 mg from 1 L culture. The purified enzyme displayed optimal activity at 38°C and pH 7.0. Given that the enzyme had 54% of the maximal activity at 20°C and 11% of the maximal activity at close to 0°C, ZD410 was regarded as a cold-adapted β-galactosidase. ZD410 displays high enzymatic activity for its synthetic substrate-ONPG (o-nitrophenyl-β-d-galactopyranoside, 243 U/mg) and its natural substrate-lactose (25.4 U/mg), while its activity was slightly stimulated by addition of Na⁺, K⁺, or Ca²⁺ at low concentrations. ZD410 is a good candidate of β-galactosidases for food industry after further study.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Isolation and characterization of eleven microsatellite loci in small abalone, Haliotis diversicolor Reeve

Eleven novel microsatellite markers were isolated from small abalone, Haliotis diversicolor Reeve. These loci were tested on 22 individuals from two different geographic populations. We identified a total of 162 alleles from the 11 microsatellite loci. All of the loci were highly polymorphic. Polymorphism information content (PIC) is ranging from 0.7276 to 0.9163. Observed and expected heterozygosities ranged from 0.2727 to 1.0000 and from 0.7738 to 0.9429, respectively. Three loci deviated significantly from Hardy–Weinberg equilibrium. No pairs of loci displayed linkage disequilibrium. These polymorphic markers will be used to analyze population structure, genetic diversity and construct a genetic linkage map. Xin Zhan and Hai-Yan Hu contribute equally to this study.     

Comparison and quantitative verification of mapping algorithms for whole-genome bisulfite sequencing

Coupling bisulfite conversion with next-generation sequencing (Bisulfite-seq) enables genome-wide measurement of DNA methylation, but poses unique challenges for mapping. However, despite a proliferation of Bisulfite-seq mapping tools, no systematic comparison of their genomic coverage and quantitative accuracy has been reported. We sequenced bisulfite-converted DNA from two tissues from each of two healthy human adults and systematically compared five widely used Bisulfite-seq mapping algorithms: Bismark, BSMAP, Pash, BatMeth and BS Seeker. We evaluated their computational speed and genomic coverage and verified their percentage methylation estimates. With the exception of BatMeth, all mappers covered >70% of CpG sites genome-wide and yielded highly concordant estimates of percentage methylation (r² ≥ 0.95). Fourfold variation in mapping time was found between BSMAP (fastest) and Pash (slowest). In each library, 8–12% of genomic regions covered by Bismark and Pash were not covered by BSMAP. An experiment using simulated reads confirmed that Pash has an exceptional ability to uniquely map reads in genomic regions of structural variation. Independent verification by bisulfite pyrosequencing generally confirmed the percentage methylation estimates by the mappers. Of these algorithms, Bismark provides an attractive combination of processing speed, genomic coverage and quantitative accuracy, whereas Pash offers considerably higher genomic coverage.     

Validating Solution Ensembles from Molecular Dynamics Simulation by Wide-Angle X-ray Scattering Data

Wide-angle x-ray scattering (WAXS) experiments of biomolecules in solution have become increasingly popular because of technical advances in light sources and detectors. However, the structural interpretation of WAXS profiles is problematic, partly because accurate calculations of WAXS profiles from structural models have remained challenging. In this work, we present the calculation of WAXS profiles from explicit-solvent molecular dynamics (MD) simulations of five different proteins. Using only a single fitting parameter that accounts for experimental uncertainties because of the buffer subtraction and dark currents, we find excellent agreement to experimental profiles both at small and wide angles. Because explicit solvation eliminates free parameters associated with the solvation layer or the excluded solvent, which would require fitting to experimental data, we minimize the risk of overfitting. We further find that the influence from water models and protein force fields on calculated profiles are insignificant up to q ≈ 15 nm^?1. Using a series of simulations that allow increasing flexibility of the proteins, we show that incorporating thermal fluctuations into the calculations significantly improves agreement with experimental data, demonstrating the importance of protein dynamics in the interpretation of WAXS profiles. In addition, free MD simulations up to one microsecond suggest that the calculated profiles are highly sensitive with respect to minor conformational rearrangements of proteins, such as an increased flexibility of a loop or an increase of the radius of gyration by  <  1%. The present study suggests that quantitative comparison between MD simulations and experimental WAXS profiles emerges as an accurate tool to validate solution ensembles of biomolecules.     

Inflammatory Stress Increases Hepatic CD36 Translational Efficiency via Activation of the mTOR Signalling Pathway

Inflammatory stress is an independent risk factor for the development of non-alcoholic fatty liver disease (NAFLD). Although CD36 is known to facilitate long-chain fatty acid uptake and contributes to NAFLD progression, the mechanisms that link inflammatory stress to hepatic CD36 expression and steatosis remain unclear. As the mammalian target of rapamycin (mTOR) signalling pathway is involved in CD36 translational activation, this study was undertaken to investigate whether inflammatory stress enhances hepatic CD36 expression via mTOR signalling pathway and the underlying mechanisms. To induce inflammatory stress, we used tumour necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) stimulation of the human hepatoblastoma HepG2 cells in vitro and casein injection in C57BL/6J mice in vivo. The data showed that inflammatory stress increased hepatic CD36 protein levels but had no effect on mRNA expression. A protein degradation assay revealed that CD36 protein stability was not different between HepG2 cells treated with or without TNF-α or IL-6. A polysomal analysis indicated that CD36 translational efficiency was significantly increased by inflammatory stress. Additionally, inflammatory stress enhanced the phosphorylation of mTOR and its downstream translational regulators including p70S6K, 4E-BP1 and eIF4E. Rapamycin, an mTOR-specific inhibitor, reduced the phosphorylation of mTOR signalling pathway and decreased the CD36 translational efficiency and protein level even under inflammatory stress resulting in the alleviation of inflammatory stress-induced hepatic lipid accumulation. This study demonstrates that the activation of the mTOR signalling pathway increases hepatic CD36 translational efficiency, resulting in increased CD36 protein expression under inflammatory stress.     

Endoscopic Biopsy in Gastrointestinal Neuroendocrine Neoplasms: A Retrospective Study

Background

Gastrointestinal neuroendocrine neoplasms (GI-NENs) are often located in the deep mucosa or submucosa, and the efficacy of endoscopic biopsy for diagnosis and treatment of GI-NENs is not fully understood.

Objective

The current study analyzed GI-NENs, especially those diagnosed pathologically and resected endoscopically, and focused on the biopsy and cold biopsy forceps polypectomy (CBP) to analyze their roles in diagnosing and treating GI-NENs.

Methods

Clinical data of all GI-NENs were reviewed from January 2006 to March 2012. Histopathology was used to diagnose GI-NENs, which were confirmed by immunohistochemistry.

Results

67.96% GI-NENs were diagnosed pathologically by endoscopy. Only 26.21% were diagnosed pathologically by biopsies before treatment. The diagnostic rate was significantly higher in polypoid (76.47%) and submucosal lesions (68.75%), than in ulcerative lesions (12.00%). However, biopsies were only taken in 56.31% patients, including 51.52% of polypoid lesions, 35.56% of submucosal lesions and 100.00% of ulcerative lesions. Endoscopic resection removed 61.76% of GI-NENs, including six by CBP, 14 by snare polypectomy with electrocauterization, 28 by endoscopic mucosal resection (EMR) and 15 by endoscopic submucosal dissection (ESD). 51.52% polypoid GI-NENs had infiltrated the submucosa under microscopic examination. CBP had a significantly higher rate of remnant (33.33%) than snare polypectomy with electrocauterization, EMR and ESD (all 0.00%).

Conclusions

Biopsies for all polypoid and submucosal lesions will improve pre-operative diagnosis. The high rate of submucosal infiltration of polypoid GI-NENs determined that CBP was inadequate in the treatment of GI-NENs. Diminutive polypoid GI-NENs that disappeared after CBP had a high risk of remnant and should be closely followed up over the long term.     

Precipitation Regime Shift Enhanced the Rain Pulse Effect on Soil Respiration in a Semi-Arid Steppe

The effect of resource pulses, such as rainfall events, on soil respiration plays an important role in controlling grassland carbon balance, but how shifts in long-term precipitation regime regulate rain pulse effect on soil respiration is still unclear. We first quantified the influence of rainfall event on soil respiration based on a two-year (2006 and 2009) continuously measured soil respiration data set in a temperate steppe in northern China. In 2006 and 2009, soil carbon release induced by rainfall events contributed about 44.5% (83.3 g C m⁻²) and 39.6% (61.7 g C m⁻²) to the growing-season total soil respiration, respectively. The pulse effect of rainfall event on soil respiration can be accurately predicted by a water status index (WSI), which is the product of rainfall event size and the ratio between antecedent soil temperature to moisture at the depth of 10 cm (r ² = 0.92, P<0.001) through the growing season. It indicates the pulse effect can be enhanced by not only larger individual rainfall event, but also higher soil temperature/moisture ratio which is usually associated with longer dry spells. We then analyzed a long-term (1953–2009) precipitation record in the experimental area. We found both the extreme heavy rainfall events (>40 mm per event) and the long dry-spells (>5 days) during the growing seasons increased from 1953–2009. It suggests the shift in precipitation regime has increased the contribution of rain pulse effect to growing-season total soil respiration in this region. These findings highlight the importance of incorporating precipitation regime shift and its impacts on the rain pulse effect into the future predictions of grassland carbon cycle under climate change.