Sustained expression of MCP-1 induced low wall shear stress loading in conjunction with turbulent flow on endothelial cells of intracranial aneurysm

Shear stress was reported to regulate the expression of AC007362, but its underlying mechanisms remain to be explored. In this study, to isolate endothelial cells of blood vessels, unruptured and ruptured intracranial aneurysm (IA) tissues were collected from IA patients. Subsequently, quantitative real-time PCR (qRT-PCR), Western blot and luciferase assay were performed to investigate the relationships between AC007362, miRNAs-493 and monocyte chemoattractant protein-1 (MCP-1) in human umbilical vein endothelial cells (HUVECs) exposed to shear stress. Reduced representation bisulphite sequencing (RRBS) was performed to assess the level of DNA methylation in AC007362 promoter. Accordingly, AC007362 and MCP-1 were significantly up-regulated while miR-493 was significantly down-regulated in HUVECs exposed to shear stress. AC007362 could suppress the miR-493 expression and elevate the MCP-1 expression, and miR-493 was shown to respectively target AC007362 and MCP-1. Moreover, shear stress in HUVECs led to the down-regulated DNA methyltransferase 1 (DNMT1), as well as the decreased DNA methylation level of AC007362 promoter. Similar results were also observed in ruptured IA tissues when compared with unruptured IA tissues. In conclusion, this study presented a deep insight into the operation of the regulatory network of AC007362, miR-493 and MCP-1 upon shear stress. Under shear stress, the expression of AC007362 was enhanced by the inhibited promoter DNA methylation, while the expression of MCP-1 was enhanced by sponging the expression of miR-493.     

Crystal structure of Drosophila melanogaster tryptophan 2,3-dioxygenase reveals insights into substrate recognition and catalytic mechanism

Tryptophan 2,3-dioxygenase (TDO) catalyzes the oxidative cleavage of the indole ring of l-tryptophan to N-formylkynurenine in the kynurenine pathway, and is considered as a drug target for cancer immunotherapy. Here, we report the first crystal structure of a eukaryotic TDO from Drosophila melanogaster (DmTDO) in complex with heme at 2.7 Å resolution. DmTDO consists of an N-terminal segment, a large domain and a small domain, and assumes a tetrameric architecture. Compared with prokaryotic TDOs, DmTDO contains two major insertion sequences: one forms part of the heme-binding site and the other forms a large portion of the small domain. The small domain which is unique to eukaryotic TDOs, interacts with the active site of an adjacent monomer and plays a role in the catalysis. Molecular modeling and dynamics simulation of DmTDO-heme-Trp suggest that like prokaryotic TDOs, DmTDO adopts an induced-fit mechanism to bind l-Trp; in particular, two conserved but flexible loops undergo conformational changes, converting the active site from an open conformation to a closed conformation. The functional roles of the key residues involved in recognition and binding of the heme and the substrate are verified by mutagenesis and kinetic studies. In addition, a modeling study of DmTDO in complex with the competitive inhibitor LM10 provides useful information for further inhibitor design. These findings reveal insights into the substrate recognition and the catalysis of DmTDO and possibly other eukaryotic TDOs and shed lights on the development of effective anti-TDO inhibitors.     

Stochastic modelling of wall stresses in abdominal aortic aneurysms treated by a gene therapy

A stochastic mechanical model using the membrane theory was used to simulate the in vivo mechanical behaviour of abdominal aortic aneurysms (AAAs) in order to compute the wall stresses after stabilisation by gene therapy. For that, both length and diameter of AAAs rats were measured during their expansion. Four groups of animals, control and treated by an endovascular gene therapy during 3 or 28 days were included. The mechanical problem was solved analytically using the geometric parameters and assuming the shape of aneurysms by a ‘parabolic–exponential curve’. When compared to controls, stress variations in the wall of AAAs for treated arteries during 28 days decreased, while they were nearly constant at day 3. The measured geometric parameters of AAAs were then investigated using probability density functions (pdf) attributed to every random variable. Different trials were useful to define a reliable confidence region in which the probability to have a realisation is equal to 99%. The results demonstrated that the error in the estimation of the stresses can be greater than 28% when parameters uncertainties are not considered in the modelling. The relevance of the proposed approach for the study of AAA growth may be studied further and extended to other treatments aimed at stabilisation AAAs, using biotherapies and pharmacological approaches.     

The Relationship Between Insulin Resistance and CpG Island Methylation of LMNA Gene in Polycystic Ovary Syndrome

We sought to investigate the relationship between the changes of CpG island methylation status of LMNA gene and insulin resistance in polycystic ovary syndrome (PCOS) patients. The genome-wide methylation microarray screening was done in three PCOS cases of insulin resistance and one case of a normal woman. The PCOS insulin resistance-related genes were identified as indicated by the results of gene chip screening. Then, 24 cases of insulin-resistant PCOS patients and 24 cases of normal individuals were studied to identify the effects of the candidate genes using genome-wide study of DNA from the peripheral blood analyzed by MassARRAY^®EpiTYPER? DNA methylation analysis technique. We found that the methylation status of CpG island in the promoter area of LMNA gene was changed. The 20 CG sites in CpG island of LMNA gene were examined using case control experiment among which 12 CpG sites differed significantly (P < 0.05) between two groups while the remaining eight CpG sites differed non-significantly. We, therefore, concluded that the changes in the hypermethylation status of CpG island of LMNA gene were related to the insulin resistance in PCOS patients, indicating that this gene may be involved in the regulation of PCOS-associated insulin resistance.     

Cloning of the crustacean hyperglycemic hormone and evidence for molt-inhibiting hormone within the central nervous system of the blue crab Portunus pelagicus

The crustacean X-organ–sinus gland (XO–SG) complex controls molt-inhibiting hormone (MIH) production, although extra expression sites for MIH have been postulated. Therefore, to explore the expression of MIH and distinguish between the crustacean hyperglycemic hormone (CHH) superfamily, and MIH immunoreactive sites (ir) in the central nervous system (CNS), we cloned a CHH gene sequence for the crab Portunus pelagicus (Ppel-CHH), and compared it with crab CHH-type I and II peptides. Employing multiple sequence alignments and phylogenic analysis, the mature Ppel-CHH peptide exhibited residues common to both CHH-type I and II peptides, and a high degree of identity to the type-I group, but little homology between Ppel-CHH and Ppel-MIH (a type II peptide). This sequence identification then allowed for the use of MIH antisera to further confirm the identity and existence of a MIH-ir 9 kDa protein in all neural organs tested by Western blotting, and through immunohistochemistry, MIH-ir in the XO, optic nerve, neuronal cluster 17 of the supraesophageal ganglion, the ventral nerve cord, and cell cluster 22 of the thoracic ganglion. The presence of MIH protein within such a diversity of sites in the CNS, and external to the XO–SG, raises new questions concerning the established mode of MIH action.     

Studies on the Effect of Thymine-Mercury-Thymine Stem as a Structural or Functional Motif in DNAzymes

T-Hg-T base pair formation has been demonstrated to be compatible with duplex DNA context, with considerable thermal stability contribution. Here, the T-Hg-T stem in two small DNAzymes 8–17 and 10–23 was studied for its structural and functional roles. The recognition arm 5′ to the cleavage site of 10–23 DNAzyme complex and the stem in the catalytic loop of 8–17 DNAzyme could be replaced by consecutive T-Hg-T stem of different length. The linear relationship between the activity of the complex 10–23DZ-6T+D19–6T and the concentration of Hg²⁺ demonstrated that the T-Hg-T stem contributes thermal stability of the recognition arm binding. The effect of T-Hg-T stem in the catalytic core of 8–17 DNAzyme and the position-dependent effect in 10–23 DNAzyme demonstrated that T-Hg-T base pair is not compatible with canonical base pairs in playing the functions of nucleic acids.     

Epithelial cell adhesion molecule (EpCAM) is associated with prostate cancer metastasis and chemo/radioresistance via the PI3K/Akt/mTOR signaling pathway

Prostate cancer (CaP) is the second leading malignancy in men. The role of epithelial cell adhesion molecule (EpCAM), also known as CD326, in CaP progression and therapeutic resistance is still uncertain. Here, we aimed to investigate the roles of EpCAM in CaP metastasis and chemo/radioresistance. Expression of EpCAM in CaP cell lines and human CaP tissues was assessed using immunofluorescence and immunohistochemistry, respectively. EpCAM was knocked down (KD) in PC-3, DU145 and LNCaP-C4-2B cells using small interfering RNA (siRNA), and KD results were confirmed by confocal microscope, Western blotting and quantitative real time polymerase chain reaction (qRT-PCR). Cell growth was evaluated by proliferation and colony formation assays. The invasive potential was assessed using a matrigel chamber assay. Tumorigenesis potential was measured by a sphere formation assay. Chemo-/radiosensitivity were measured using a colony formation assay. Over-expression of EpCAM was found in primary CaP tissues and lymph node metastases including cancer cells and surrounding stromal cells. KD of EpCAM suppressed CaP proliferation and invasive ability, reduced sphere formation, enhanced chemo-/radiosensitivity, and down-regulated E-cadherin, p-Akt, p-mTOR, p-4EBP1 and p-S6K expression in CaP cells. Our findings suggest that EpCAM plays an important role in CaP proliferation, invasion, metastasis and chemo-/radioresistance associated with the activation of the PI3K/Akt/mTOR signaling pathway and is a novel therapeutic target to sensitize CaP cells to chemo-/radiotherapy.     

VRAA: virtualized resource auction and allocation based on incentive and penalty

Virtualization is widely used in cloud computing environments to efficiently manage resources, but it also raises several challenges. One of them is the fairness issue of resource allocation among virtual machines. Traditional virtualized resource allocation approaches distribute physical resources equally without taking into account the actual workload of each virtual machine and thus often leads to wasting. In this paper, we propose a virtualized resource auction and allocation model (VRAA) based on incentive and penalty to correct this wasting problem. In our approach, we use Nash equilibrium of cooperative games to fairly allocate resources among multiple virtual machines to maximize revenue of the system. To illustrate the effectiveness of the proposed approach, we then apply the basic laws of auction gaming to investigate how CPU allocation and contention can affect applications’ performance (i.e., response time), and its effect on CPU utilization. We find that in our VRAA model, the fairness index is high, and the resource allocation is closely proportional to the actual workloads of the virtual machines, so the wasting of resources is reduced. Experiment results show that our model is general, and can be applied to other virtualized non-CPU resources.     

Biochemical Characterization of a First Fungal Esterase from Rhizomucor miehei Showing High Efficiency of Ester Synthesis

Background

Esterases with excellent merits suitable for commercial use in ester production field are still insufficient. The aim of this research is to advance our understanding by seeking for more unusual esterases and revealing their characterizations for ester synthesis.

Methodology/Principal Findings

A novel esterase-encoding gene from Rhizomucor miehei (RmEstA) was cloned and expressed in Escherichia coli. Sequence analysis revealed a 975-bp ORF encoding a 324-amino-acid polypeptide belonging to the hormone-sensitive lipase (HSL) family IV and showing highest similarity (44%) to the Paenibacillus mucilaginosus esterase/lipase. Recombinant RmEstA was purified to homogeneity: it was 34 kDa by SDS-PAGE and showed optimal pH and temperature of 6.5 and 45°C, respectively. The enzyme was stable to 50°C, under a broad pH range (5.0–10.6). RmEstA exhibited broad substrate specificity toward p-nitrophenol esters and short-acyl-chain triglycerols, with highest activities (1,480 U mg⁻¹ and 228 U mg⁻¹) for p-nitrophenyl hexanoate and tributyrin, respectively. RmEstA efficiently synthesized butyl butyrate (92% conversion yield) when immobilized on AOT-based organogel.

Conclusion

RmEstA has great potential for industrial applications. RmEstA is the first reported esterase from Rhizomucor miehei.