Genetic Analysis of Ligation-Induced Neointima Formation in an F2 Intercross of C57BL/6 and FVB/N Inbred Mouse Strains

Objective

Proliferation and migration of vascular smooth muscle cells (SMCs) are central for arterial diseases including atherosclerosis and restenosis. We hypothesized that the underlying mechanisms may be modeled by carotid ligation in mice. In FVB/N inbred mice, ligation leads to abundant neointima formation with proliferating media-derived SMCs, whereas in C57BL/6 mice hardly any neointima is formed. In the present study, we aimed to identify the chromosomal location of the causative gene variants in an F2 intercross between these two mouse strains.

Methods and Results

The neointimal cross-sectional area was significantly different between FVB/N, C57BL/6 and F1 female mice 4 weeks after ligation. Carotid artery ligation and a genome scan using 800 informative SNP markers were then performed in 157 female F2 mice. Using quantitative trait loci (QTL) analysis, we identified suggestive, but no genome-wide significant, QTLs on chromosomes 7 and 12 for neointimal cross-sectional area and on chromosome 14 for media area. Further analysis of the cross revealed 4 QTLs for plasma cholesterol, which combined explained 69% of the variation among F2 mice.

Conclusions

We identified suggestive QTLs for neointima and media area after carotid ligation in an intercross of FVB/N and C57BL/6 mice, but none that reached genome-wide significance indicating a complex genetic architecture of the traits. Genome-wide significant QTLs for total cholesterol levels were identified on chromosomes 1, 3, 9, and 12.     

Modulation of Sweet Taste by Umami Compounds via Sweet Taste Receptor Subunit hT1R2

Although the five basic taste qualities—sweet, sour, bitter, salty and umami—can be recognized by the respective gustatory system, interactions between these taste qualities are often experienced when food is consumed. Specifically, the umami taste has been investigated in terms of whether it enhances or reduces the other taste modalities. These studies, however, are based on individual perception and not on a molecular level. In this study we investigated umami-sweet taste interactions using umami compounds including monosodium glutamate (MSG), 5’-mononucleotides and glutamyl-dipeptides, glutamate-glutamate (Glu-Glu) and glutamate-aspartic acid (Glu-Asp), in human sweet taste receptor hT1R2/hT1R3-expressing cells. The sensitivity of sucrose to hT1R2/hT1R3 was significantly attenuated by MSG and umami active peptides but not by umami active nucleotides. Inhibition of sweet receptor activation by MSG and glutamyl peptides is obvious when sweet receptors are activated by sweeteners that target the extracellular domain (ECD) of T1R2, such as sucrose and acesulfame K, but not by cyclamate, which interact with the T1R3 transmembrane domain (TMD). Application of umami compounds with lactisole, inhibitory drugs that target T1R3, exerted a more severe inhibitory effect. The inhibition was also observed with F778A sweet receptor mutant, which have the defect in function of T1R3 TMD. These results suggest that umami peptides affect sweet taste receptors and this interaction prevents sweet receptor agonists from binding to the T1R2 ECD in an allosteric manner, not to the T1R3. This is the first report to define the interaction between umami and sweet taste receptors.     

Five hTRPA1 Agonists Found in Indigenous Korean Mint,Agastache rugosa

Transient receptor potential ankyrin1 (TRPA1) and transient receptor potential vanilloid 1 (TRPV1) are members of the TRP superfamily of structurally related, nonselective cation channels and mediators of several signaling pathways. Previously, we identified methyl syringate as an hTRPA1 agonist with efficacy against gastric emptying. The aim of this study was to find hTRPA1 and/or hTRPV1 activators in Agastache rugosa (Fisch. et Meyer) O. Kuntze (A.rugosa), commonly known as Korean mint to improve hTRPA1-related phenomena. An extract of the stem and leaves of A.rugosa (Labiatae) selectively activated hTRPA1 and hTRPV1. We next investigated the effects of commercially available compounds found in A.rugosa (acacetin, 4-allylanisole, p-anisaldehyde, apigenin 7-glucoside, L-carveol, β-caryophyllene, trans-p-methoxycinnamaldehyde, methyl eugenol, pachypodol, and rosmarinic acid) on cultured hTRPA1- and hTRPV1-expressing cells. Of the ten compounds, L-carveol, trans-p-methoxycinnamaldehyde, methyl eugenol, 4-allylanisole, and p-anisaldehyde selectively activated hTRPA1, with EC50 values of 189.1±26.8, 29.8±14.9, 160.2±21.9, 1535±315.7, and 546.5±73.0 μM, respectively. The activities of these compounds were effectively inhibited by the hTRPA1 antagonists, ruthenium red and HC-030031. Although the five active compounds showed weaker calcium responses than allyl isothiocyanate (EC₅₀=7.2±1.4 μM), our results suggest that these compounds from the stem and leaves of A.rugosa are specific and selective agonists of hTRPA1.     

Efficient Nash Equilibrium Resource Allocation Based on Game Theory Mechanism in Cloud Computing by Using Auction

One of the most complex issues in the cloud computing environment is the problem of resource allocation so that, on one hand, the cloud provider expects the most profitability and, on the other hand, users also expect to have the best resources at their disposal considering the budget constraints and time. In most previous work conducted, heuristic and evolutionary approaches have been used to solve this problem. Nevertheless, since the nature of this environment is based on economic methods, using such methods can decrease response time and reducing the complexity of the problem. In this paper, an auction-based method is proposed which determines the auction winner by applying game theory mechanism and holding a repetitive game with incomplete information in a non-cooperative environment. In this method, users calculate suitable price bid with their objective function during several round and repetitions and send it to the auctioneer; and the auctioneer chooses the winning player based the suggested utility function. In the proposed method, the end point of the game is the Nash equilibrium point where players are no longer inclined to alter their bid for that resource and the final bid also satisfies the auctioneer’s utility function. To prove the response space convexity, the Lagrange method is used and the proposed model is simulated in the cloudsim and the results are compared with previous work. At the end, it is concluded that this method converges to a response in a shorter time, provides the lowest service level agreement violations and the most utility to the provider.     

Differential Impact of Constrictive Physiology after Pericardiocentesis in Malignancy Patients with Pericardial Effusion

Background

Echocardiographic signs of constrictive physiology (CP) after pericardiocentesis are frequently observed in malignancy patients. The purpose of the current study was to explore whether features of CP after pericardiocentesis have prognostic impact in malignancy patients with pericardial effusion (PE).

Methods

We retrospectively reviewed 467 consecutive patients who underwent pericardiocentesis at our institution from January 2006 to May 2014. Among them, 205 patients with advanced malignancy who underwent comprehensive echocardiography after the procedure comprised the study population. Co-primary end points were all-cause mortality (ACM) and repeated drainage (RD) for PE. Patients were divided into four subgroups according to cytologic result for malignant cells and CP (positive cytology with negative CP, both positive, both negative, and negative cytology with positive CP).

Results

CP after pericardiocentesis was present in 106 patients (50%) at median 4 days after the procedure. During median follow-up of 208 days, ACM and RD occurred in 162 patients (79%) and 29 patients (14%), respectively. Cox regression analysis revealed that independent predictors for ACM were male gender and positive cytology (all, p < 0.05). For RD, predictors were positive cytology, the absence of cardiac tamponade, and negative CP after pericardiocentesis (all, p < 0.05). When the patients were divided into four subgroups, patients with negative cytology and positive CP demonstrated the most favorable survival (hazard ratio [HR]: 0.39, p = 0.005) and the lowest RD rates (HR: 0.07, p = 0.012).

Conclusion

CP after pericardiocentesis is common, but does not always imply poor survival or the need for RD in patients with advanced malignancies. On the contrary, the presence of CP in patients with negative cytology conferred the most favorable survival and the lowest rate of RD. Comprehensive echocardiographic evaluation for CP after pericardiocentesis would be helpful for predicting prognosis in patients with advanced malignancies.     

Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (K_D ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC₅₀ typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical.     

Anticancer activity of TTAC-0001, a fully human anti-vascular endothelial growth factor receptor 2 (VEGFR-2/KDR) monoclonal antibody,is associated with inhibition of tumor angiogenesis

Vascular endothelial growth factor (VEGF) and its receptors are considered the primary cause of tumor-induced angiogenesis. Specifically, VEGFR-2/kinase insert domain receptor (KDR) is part of the major signaling pathway that plays a significant role in tumor angiogenesis, which is associated with the development of various types of tumor and metastasis. In particular, KDR is involved in tumor angiogenesis as well as cancer cell growth and survival. In this study, we evaluated the therapeutic potential of TTAC-0001, a fully human antibody against VEGFR-2/KDR. To assess the efficacy of the antibody and pharmacokinetic (PK) relationship in vivo, we tested the potency of TTAC-0001 in glioblastoma and colorectal cancer xenograft models. Antitumor activity of TTAC-0001 in preclinical models correlated with tumor growth arrest, induction of tumor cell apoptosis, and inhibition of angiogenesis. We also evaluated the combination effect of TTAC-0001 with a chemotherapeutic agent in xenograft models. We were able to determine the relationship between PK and the efficacy of TTAC-0001 through in vivo single-dose PK study. Taken together, our data suggest that targeting VEGFR-2 with TTAC-0001 could be a promising approach for cancer treatment.     

Pathway Analysis Based on a Genome-Wide Association Study of Polycystic Ovary Syndrome

Background

Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders in women of reproductive age, and it is affected by both environmental and genetic factors. Although the genetic component of PCOS is evident, studies aiming to identify susceptibility genes have shown controversial results. This study conducted a pathway-based analysis using a dataset obtained through a genome-wide association study (GWAS) to elucidate the biological pathways that contribute to PCOS susceptibility and the associated genes.

Methods

We used GWAS data on 636,797 autosomal single nucleotide polymorphisms (SNPs) from 1,221 individuals (432 PCOS patients and 789 controls) for analysis. A pathway analysis was conducted using meta-analysis gene-set enrichment of variant associations (MAGENTA). Top-ranking pathways or gene sets associated with PCOS were identified, and significant genes within the pathways were analyzed.

Results

The pathway analysis of the GWAS dataset identified significant pathways related to oocyte meiosis and the regulation of insulin secretion by acetylcholine and free fatty acids (all nominal gene-set enrichment analysis (GSEA) P-values < 0.05). In addition, INS, GNAQ, STXBP1, PLCB3, PLCB2, SMC3 and PLCZ1 were significant genes observed within the biological pathways (all gene P-values < 0.05).

Conclusions

By applying MAGENTA pathway analysis to PCOS GWAS data, we identified significant pathways and candidate genes involved in PCOS. Our findings may provide new leads for understanding the mechanisms underlying the development of PCOS.     

Incidence of Alternaria Species Associated with Watermelon Leaf Blight in Korea

Alternaria leaf blight is one of the most common diseases in watermelon worldwide. In Korea, however, the Alternaria species causing the watermelon leaf blight have not been investigated thoroughly. A total of 16 Alternaria isolates was recovered from diseased watermelon leaves with leaf blight symptoms, which were collected from 14 fields in Korea. Analysis of internal transcribed spacer (ITS) region, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and RNA polymerase II second largest subunit (RPB2) were not competent to differentiate the Alternaria isolates. On the contrary, analysis of amplicon size of the histone H3 (HIS3) gene successfully differentiated the isolates into three Alternaria subgroups, and further sequence analysis of them identified three Alternaria spp. Alternaria tenuissima, A. gaisen, and A. alternata. Representative Alternaria isolates from three species induced dark brown leaf spot lesions on detached watermelon leaves, indicating that A. tenuissima, A. gaisen, and A. alternata are all causal agents of Alternaria leaf blight. Our results indicate that the Alternaria species associated watermelon leaf blight in Korea is more complex than reported previously. This is the first report regarding the population structure of Alternaria species causing watermelon leaf blight in Korea.     

DNA Methylation Effects on Tetra-Nucleosome Compaction and Aggregation

DNA CpG methylation has been associated with chromatin compaction and gene silencing. Whether DNA methylation directly contributes to chromatin compaction remains an open question. In this study, we used fluorescence fluctuation spectroscopy (FFS) to evaluate the compaction and aggregation of tetra-nucleosomes containing specific CpG patterns and methylation levels. The compactness of both unmethylated and methylated tetra-nucleosomes is dependent on DNA sequences. Specifically, methylation of the CpG sites located in the central dyad and the major grooves of DNA seem to have opposite effects on modulating the compactness of tetra-nucleosomes. The interactions among tetra-nucleosomes, however, seem to be enhanced because of DNA methylation independent of sequence contexts. Our finding can shed light on understanding the role of DNA methylation in determining nucleosome positioning pattern and chromatin compactness.