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.     

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.     

Inhibition of in vivo Slicer activity of Argonaute protein 1 by the viral 2b protein independent of its dsRNA‐binding function

The 2b protein of Cucumber mosaic virus (CMV) has several unique properties, such as targeting to the nucleolus and interaction with both Argonautes (AGOs) and short and long double‐stranded RNA (dsRNA). We have recently uncoupled the domain requirements for dsRNA binding and nucleolar targeting from the physical interactions with AGO proteins, and have found that the direct 2b–AGO interaction is sufficient to inhibit the in vitro AGO1 Slicer function independent of the other biochemical properties of 2b. Because the AGO binding activity of 2b is not required for its suppressor function in vivo, this raises the question of whether in vivo 2b–AGO interaction is possible to inhibit the in vivo AGO Slicer function. In this study, by taking advantage of a technology for the production of artificial trans‐acting small interfering RNA (tasiRNA), a process uniquely associated with AGO1‐mediated in vivo Slicer activity, we demonstrated that the expression of the 2b protein in planta interfered with the production of tasiRNA. Through further detailed analysis with deletion mutants of 2b proteins, we found that the inhibition of in vivo AGO1 Slicer function required the nucleolar localization signal (NoLS), in addition to the AGO‐binding domain, of the 2b protein. Our finding demonstrates that in vivo 2b–AGO1 interaction is sufficient to inhibit AGO1 Slicer function independent of the dsRNA‐binding activity of the 2b protein.     

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.     

Structure of the Catalytic Domain of EZH2 Reveals Conformational Plasticity in Cofactor and Substrate Binding Sites and Explains Oncogenic Mutations

Polycomb repressive complex 2 (PRC2) is an important regulator of cellular differentiation and cell type identity. Overexpression or activating mutations of EZH2, the catalytic component of the PRC2 complex, are linked to hyper-trimethylation of lysine 27 of histone H3 (H3K27me3) in many cancers. Potent EZH2 inhibitors that reduce levels of H3K27me3 kill mutant lymphoma cells and are efficacious in a mouse xenograft model of malignant rhabdoid tumors. Unlike most SET domain methyltransferases, EZH2 requires PRC2 components, SUZ12 and EED, for activity, but the mechanism by which catalysis is promoted in the PRC2 complex is unknown. We solved the 2.0 Å crystal structure of the EZH2 methyltransferase domain revealing that most of the canonical structural features of SET domain methyltransferase structures are conserved. The site of methyl transfer is in a catalytically competent state, and the structure clarifies the structural mechanism underlying oncogenic hyper-trimethylation of H3K27 in tumors harboring mutations at Y641 or A677. On the other hand, the I-SET and post-SET domains occupy atypical positions relative to the core SET domain resulting in incomplete formation of the cofactor binding site and occlusion of the substrate binding groove. A novel CXC domain N-terminal to the SET domain may contribute to the apparent inactive conformation. We propose that protein interactions within the PRC2 complex modulate the trajectory of the post-SET and I-SET domains of EZH2 in favor of a catalytically competent conformation.     

Ginsenoside Rg-1 Protects Retinal Pigment Epithelium (RPE) Cells from Cobalt Chloride (CoCl2) and Hypoxia Assaults

Severe retinal ischemia causes persistent visual impairments in eye diseases. Retinal pigment epithelium (RPE) cells are located near the choroidal capillaries, and are easily affected by ischemic or hypoxia. Ginsenoside Rg-1 has shown significant neuroprotective effects. This study was performed to test the cytoprotective effect of ginsenoside Rg-1 in RPE cells against hypoxia and cobalt chloride (CoCl₂) assaults, and to understand the underlying mechanisms. We found that Rg-1 pre-administration significantly inhibited CoCl₂- and hypoxia-induced RPE cell death and apoptosis. Reactive oxygen specisis (ROS)-dependent p38 and c-Jun NH(2)-terminal kinases (JNK) MAPK activation was required for CoCl₂-induced RPE cell death, and Rg-1 pre-treatment significantly inhibited ROS production and following p38/JNK activation. Further, CoCl₂ suppressed pro-survival mTOR complex 1 (mTORC1) activation in RPE cells through activating of AMP-activated protein kinase (AMPK), while Rg-1 restored mTORC1 activity through inhibiting AMPK activation. CoCl₂-induced AMPK activation was also dependent on ROS production, and anti-oxidant N-acetylcysteine (NAC) prevented AMPK activation and RPE cell death by CoCl₂. Our results indicated that Rg-1 could be further investigated as a novel cell-protective agent for retinal ischemia.