A Trifluoromethyl Analog of Verbenachalcone Promotes Neurite Outgrowth and Cell Proliferation of NeuroScreen-1 Cells

Past research has shown that natural products of plant and marine origins and their congeners enhance the actions of neuritogenic factors of the central nervous system (CNS) such as nerve growth factor (NGF). However, the role of fluorine substitutions in their structure–activity relationship (SAR) has not been explored. We have synthesized a trifluoromethyl analog of verbenachalcone (VC), a pharmacologically active natural compound previously shown to potentiate NGF activity. This analog, designated C278, enhances neurite outgrowth and proliferation of NeuroScreen-1™ (NS-1) cells, a subclone of PC12 pheochromocytoma cells. C278 increases the percentage of neurite bearing cells in the presence of suboptimal doses of NGF in comparison with controls treated with NGF alone. In addition, C278 stimulates cell growth in reduced serum and serum-free cell culture conditions based on our observation of increases in cell number and metabolic assessment with MTT reduction and resazurin assays. The addition of C278 partially restored inhibition of NGF-induced neurite outgrowth by the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 and U0126. Short-term sequential exposure of cells to U0126, C278, and NGF enhanced phosphorylation of extracellular signal-regulated kinase (ERK) in comparison with cells treated with only the MEK inhibitor and NGF. C278 also attenuated cell growth arrest caused by exposure to PD98059, U0126 and the phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002 but did not alter phosphorylation of Akt, a classic downstream target of PI3K during cell survival. These data suggest that C278 promotes NGF-dependent neurite outgrowth in NS-1 cells through a MEK signaling pathway by a mechanism that alters short-term activation of ERK. In contrast, C278 promotes PI3K-mediated survival independently of Akt phosphorylation.     

A new cytosolic pathway from a Parkinson disease-associated kinase, BRPK/PINK1: activation of AKT via mTORC2

Accumulating evidence indicates that dysfunction of mitochondria is a common feature of Parkinson disease. Functional loss of a familial Parkinson disease-linked gene, BRPK/PINK1 (PINK1), results in deterioration of mitochondrial functions and eventual neuronal cell death. A mitochondrial chaperone protein has been shown to be a substrate of PINK1 kinase activity. In this study, we demonstrated that PINK1 has another action point in the cytoplasm. Phosphorylation of Akt at Ser-473 was enhanced by overexpression of PINK1, and the Akt activation was crucial for protection of SH-SY5Y cells from various cytotoxic agents, including oxidative stress. Enhanced Akt phosphorylation was not due to activation of phosphatidylinositol 3-kinase but due to activation of mammalian target of rapamycin complex 2 (mTORC2) by PINK1. Rictor, a specific component of mTORC2, was phosphorylated by overexpression of PINK1. Furthermore, overexpression of PINK1 enhanced cell motility. These results indicate that PINK1 exerts its cytoprotective function not only in mitochondria but also in the cytoplasm through activation of mTORC2.     

TRP14 Inhibits Osteoclast Differentiation via Its Catalytic Activity

We previously reported the inhibitory role of thioredoxin-related protein of 14 kDa (TRP14), a novel disulfide reductase, in nuclear factor-κB (NF-κB) activation, but its biological function has remained to be explored. Here, we evaluated the role of TRP14 in the differentiation and function of osteoclasts (OCs), for which NF-κB and cellular redox regulation have been known to be crucial, using RAW 264.7 macrophage cells expressing wild-type TRP14 or a catalytically inactive mutant, as well as its small interfering RNA. TRP14 depletion enhanced OC differentiation, actin ring formation, and bone resorption, as well as the accumulation of reactive oxygen species (ROS). TRP14 depletion promoted the activation of NF-κB, c-Jun NH₂-terminal kinase, and p38, the expression of c-Fos, and the consequent induction of nuclear factor of activated T cell, cytoplasmic 1 (NFATc1), a key determinant of osteoclastogenesis. However, pretreatment with N-acetylcysteine or diphenylene iodonium significantly reduced the OC differentiation, as well as the ROS accumulation and NF-κB activation, that were enhanced by TRP14 depletion. Furthermore, receptor activator of NF-κB ligand (RANKL)-induced ROS accumulation, NF-κB activation, and OC differentiation were inhibited by the ectopic expression of wild-type TRP14 but not by its catalytically inactive mutant. These results suggest that TRP14 regulates OC differentiation and bone resorption through its catalytic activity and that enhancing TRP14 may present a new strategy for preventing bone resorption diseases.     

Development of uniform density control with self-assembled colloidal gold nanoparticles on a modified silicon substrate

Here, we present a simple method for controlling the density of Au nanoparticles (Au NPs) on a modified silicon substrate, by destabilizing the colloidal Au NPs with 3-mercaptopropyltrimethoxylsilane (3-MPTMS) for microelectromechanical-system-based applications to reduce tribological issues. A silicon surface was pretreated with a 3-MPTMS solution, immediately after which thiolated Au NPs were added to it, resulting in their uniform deposition on the silicon substrate. Without any material property change of the colloidal Au NPs, we observed the formation of large clusters Au NPs on the modified silicon surface. Analysis by scanning electron microscopy with energy dispersive X-ray spectroscopy indicated that the addition of 3-MPTMS resulted in an alternation of the chemical characteristics of the solution. Atomic force microscopy imaging supported the notion that silicon surface modification is the most important factor on tribological properties of materials along with ligand-modified Au NPs. The density of Au NPs on a silicon surface was significantly dependent on several factors, including the concentration of colloidal Au NPs, deposition time, and concentration of 3-MPTMS solution, while temperature range which was used throughout experiment was determined to have no significant effect. A relatively high density of Au NPs forms on the silicon surface as the concentrations of Au NPs and 3-MPTMS are increased. In addition, the maximum deposition of Au NPs on silicon wafer was observed at 3 h, while the effects of temperature variation were minimal.     

Bis-class: a new classification tool of methylation status using bayes classifier and local methylation information

Background

Whole genome sequencing of bisulfite converted DNA (‘methylC-seq’) method provides comprehensive information of DNA methylation. An important application of these whole genome methylation maps is classifying each position as a methylated versus non-methylated nucleotide. A widely used current method for this purpose, the so-called binomial method, is intuitive and straightforward, but lacks power when the sequence coverage and the genome-wide methylation level are low. These problems present a particular challenge when analyzing sparsely methylated genomes, such as those of many invertebrates and plants.

Results

We demonstrate that the number of sequence reads per position from methylC-seq data displays a large variance and can be modeled as a shifted negative binomial distribution. We also show that DNA methylation levels of adjacent CpG sites are correlated, and this similarity in local DNA methylation levels extends several kilobases. Taking these observations into account, we propose a new method based on Bayesian classification to infer DNA methylation status while considering the neighborhood DNA methylation levels of a specific site. We show that our approach has higher sensitivity and better classification performance than the binomial method via multiple analyses, including computational simulations, Area Under Curve (AUC) analyses, and improved consistencies across biological replicates. This method is especially advantageous in the analyses of sparsely methylated genomes with low coverage.

Conclusions

Our method improves the existing binomial method for binary methylation calls by utilizing a posterior odds framework and incorporating local methylation information. This method should be widely applicable to the analyses of methylC-seq data from diverse sparsely methylated genomes. Bis-Class and example data are provided at a dedicated website (http://bibs.snu.ac.kr/software/Bisclass).

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-608) contains supplementary material, which is available to authorized users.     

Measurement of Neutralizing Serum Antibodies of Patients Vaccinated with Human Papillomavirus L1 or L2-Based Immunogens Using Furin-Cleaved HPV Pseudovirions

Antibodies specific for neutralizing epitopes in either Human papillomavirus (HPV) capsid protein L1 or L2 can mediate protection from viral challenge and thus their accurate and sensitive measurement at high throughput is likely informative for monitoring response to prophylactic vaccination. Here we compare measurement of L1 and L2-specific neutralizing antibodies in human sera using the standard Pseudovirion-Based Neutralization Assay (L1-PBNA) with the newer Furin-Cleaved Pseudovirion-Based Neutralization Assay (FC-PBNA), a modification of the L1-PBNA intended to improve sensitivity towards L2-specific neutralizing antibodies without compromising assay of L1-specific responses. For detection of L1-specific neutralizing antibodies in human sera, the FC- PBNA and L1-PBNA assays showed similar sensitivity and a high level of correlation using WHO standard sera (n = 2), and sera from patients vaccinated with Gardasil (n = 30) or an experimental human papillomavirus type 16 (HPV16) L1 VLP vaccine (n = 70). The detection of L1-specific cross-neutralizing antibodies in these sera using pseudovirions of types phylogenetically-related to those targeted by the L1 virus-like particle (VLP) vaccines was also consistent between the two assays. However, for sera from patients (n = 17) vaccinated with an L2-based immunogen (TA-CIN), the FC-PBNA was more sensitive than the L1-PBNA in detecting L2-specific neutralizing antibodies. Further, the neutralizing antibody titers measured with the FC-PBNA correlated with those determined with the L2-PBNA, another modification of the L1-PBNA that spacio-temporally separates primary and secondary receptor engagement, as well as the protective titers measured using passive transfer studies in the murine genital-challenge model. In sum, the FC-PBNA provided sensitive measurement for both L1 VLP and L2-specific neutralizing antibody in human sera. Vaccination with TA-CIN elicits weak cross-protective antibody in a subset of patients, suggesting the need for an adjuvant.     

Crosstalk between Adipocytes and Immune Cells in Adipose Tissue Inflammation and Metabolic Dysregulation in Obesity

Recent findings, notably on adipokines and adipose tissue inflammation, have revised the concept of adipose tissues being a mere storage depot for body energy. Instead, adipose tissues are emerging as endocrine and immunologically active organs with multiple effects on the regulation of systemic energy homeostasis. Notably, compared with other metabolic organs such as liver and muscle, various inflammatory responses are dynamically regulated in adipose tissues and most of the immune cells in adipose tissues are involved in obesity-mediated metabolic complications, including insulin resistance. Here, we summarize recent findings on the key roles of innate (neutrophils, macrophages, mast cells, eosinophils) and adaptive (regulatory T cells, type 1 helper T cells, CD8 T cells, B cells) immune cells in adipose tissue inflammation and metabolic dysregulation in obesity. In particular, the roles of natural killer T cells, one type of innate lymphocyte, in adipose tissue inflammation will be discussed. Finally, a new role of adipocytes as antigen presenting cells to modulate T cell activity and subsequent adipose tissue inflammation will be proposed.     

Histone Demethylase Jumonji AT-rich Interactive Domain 1B (JARID1B) Controls Mammary Gland Development by Regulating Key Developmental and Lineage Specification Genes

The JmjC domain-containing H3K4 histone demethylase jumonji AT-rich interactive domain 1B (JARID1B) (also known as KDM5B and PLU1) is overexpressed in breast cancer and is a potential target for breast cancer treatment. To investigate the in vivo function of JARID1B, we developed Jarid1b^−/− mice and characterized their phenotypes in detail. Unlike previously reported Jarid1b^−/− strains, the majority of these Jarid1b^−/− mice were viable beyond embryonic and neonatal stages. This allowed us to further examine phenotypes associated with the loss of JARID1B in pubertal development and pregnancy. These Jarid1b^−/− mice exhibited decreased body weight, premature mortality, decreased female fertility, and delayed mammary gland development. Related to these phenotypes, JARID1B loss decreased serum estrogen level and reduced mammary epithelial cell proliferation in early puberty. In mammary epithelial cells, JARID1B loss diminished the expression of key regulators for mammary morphogenesis and luminal lineage specification, including FOXA1 and estrogen receptor α. Mechanistically, JARID1B was required for GATA3 recruitment to the Foxa1 promoter to activate Foxa1 expression. These results indicate that JARID1B positively regulates mammary ductal development through both extrinsic and cell-autonomous mechanisms.