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.     

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.     

Measles Contributes to Rheumatoid Arthritis: Evidence from Pathway and Network Analyses of Genome-Wide Association Studies

Growing evidence from epidemiological studies indicates the association between rheumatoid arthritis (RA) and measles. However, the exact mechanism for this association is still unclear now. We consider that the strong association between both diseases may be caused by shared genetic pathways. We performed a pathway analysis of large-scale RA genome-wide association studies (GWAS) dataset with 5,539 cases and 20,169 controls of European descent. Meanwhile, we evaluated our findings using previously identified RA loci, protein-protein interaction network and previous results from pathway analysis of RA and other autoimmune diseases GWAS. We confirmed four pathways including Cytokine-cytokine receptor interaction, Jak-STAT signaling, T cell receptor signaling and Cell adhesion molecules. Meanwhile, we highlighted for the first time the involvement of Measles and Intestinal immune network for IgA production pathways in RA. Our results may explain the strong association between RA and measles, which may be caused by the shared genetic pathway. We believe that our results will be helpful for future genetic studies in RA pathogenesis and may significantly assist in the development of therapeutic strategies.     

Identification of MiRNA from Eggplant (Solanum melongena L.) by Small RNA Deep Sequencing and Their Response to Verticillium dahliae Infection

MiRNAs are a class of non-coding small RNAs that play important roles in the regulation of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data, including eggplant (Solanum melongena L.). To identify miRNAs in eggplant and their response to Verticillium dahliae infection, a fungal pathogen for which clear understanding of infection mechanisms and effective cure methods are currently lacking, we deep-sequenced two small RNA (sRNA) libraries prepared from mock-infected and infected seedlings of eggplants. Specifically, 30,830,792 reads produced 7,716,328 unique miRNAs representing 99 known miRNA families that have been identified in other plant species. Two novel putative miRNAs were predicted with eggplant ESTs. The potential targets of the identified known and novel miRNAs were also predicted based on sequence homology search. It was observed that the length distribution of obtained sRNAs and the expression of 6 miRNA families were obviously different between the two libraries. These results provide a framework for further analysis of miRNAs and their role in regulating plant response to fungal infection and Verticillium wilt in particular.     

Investigation of the binding network of IGF-I on the cavity surface of IGFBP4

Insulin-like growth factor-binding proteins (IGFBPs) control bioactivity and distribution of insulin-like growth factors (IGFs) through high-affinity complex of IGFBP and IGF. To get more insight into the binding interaction of IGF system, the site-directed mutagenesis and force-driving desorption methods were employed to study the interaction mechanism of IGFBP4 and IGF-I by molecular dynamics (MD) simulation. In IGF-I, residues Gly7 to Asp12 were found to be the hot spots and they mainly anchored on the N-domain of IGFBP4. The contact area, the shape and size of protein, the surroundings of the binding site, the hydrophobic and electrostatic interaction between the two proteins worked as a complex network to regulate the protein-protein interaction. It was also found that the unfolding of the helix was not inevitable in the mutant, and it could be regulated by careful selection of the substituted amino acid.

The tumourigenicity of iPS cells and their differentiated derivates

Induced pluripotent stem cell (iPSC) provides a promising seeding cell for regenerative medicine. However, iPSC has the potential to form teratomas after transplantation. Therefore, it is necessary to evaluate the tumorigenic risks of iPSC and all its differentiated derivates prior to use in a clinical setting. Here, murine iPSCs were transduced with dual reporter gene consisting of monomeric red fluorescent protein (mRFP) and firefly luciferase (Fluc). Undifferentiated iPSCs, iPSC derivates from induced differentiation (iPSC‐derivates), iPSC‐derivated cardiomyocyte (iPSC‐CMs) were subcutaneously injected into the back of nude mice. Non‐invasive bioluminescence imaging (BLI) was longitudinally performed at day 1, 7, 14 and 28 after transplantation to track the survival and proliferation of transplanted cells. At day 28, mice were killed and grafts were explanted to detect teratoma formation. The results demonstrated that transplanted iPSCs, iPSC‐derivates and iPSC‐CMs survived in receipts. Both iPSCs and iPSC‐derivates proliferated dramatically after transplantation, while only slight increase in BLI signals was observed in iPSC‐CM transplanted mice. At day 28, teratomas were detected in both iPSCs and iPSC‐derivates transplanted mice, but not in iPSC‐CM transplanted ones. In vitro study showed the long‐term existence of pluripotent cells during iPSC differentiation. Furthermore, when these cells were passaged in feeder layers as undifferentiated iPSCs, they would recover iPSC‐like colonies, indicating the cause for differentiated iPSC's tumourigenicity. Our study indicates that exclusion of tumorigenic cells by screening in addition to lineage‐specific differentiation is necessary prior to therapeutic use of iPSCs.