Efficient and accurate analysis of microRNA using a specific extension sequence

Molecular Biology Reports - We present here on an innovative assay for detecting miRNAs using a uniquely designed specific extension sequence that provides high efficiency and accuracy. This assay...     

Structural and biochemical analysis of mammalian methionine sulfoxide reductase B2

Methionine sulfoxide reductases are antioxidant enzymes that repair oxidatively damaged methionine residues in proteins. Mammals have three members of the methionine-R-sulfoxide reductase family, including cytosolic MsrB1, mitochondrial MsrB2, and endoplasmic reticulum MsrB3. Here, we report the solution structure of reduced Mus musculus MsrB2 using high resolution nuclear magnetic resonance (NMR) spectroscopy. MsrB2 is a β-strand rich globular protein consisting of eight antiparallel β-strands and three N-terminal α-helical segments. The latter secondary structure elements represent the main structural difference between mammalian MsrB2 and MsrB1. Structural comparison of mammalian and bacterial MsrB structures indicates that the general topology of this MsrB family is maintained and that MsrB2 more resembles bacterial MsrBs than MsrB1. Structural and biochemical analysis supports the catalytic mechanism of MsrB2 that, in contrast to MsrB1, does not involve a resolving cysteine (Cys). pH dependence of catalytically relevant residues in MsrB2 was accessed by NMR spectroscopy and the pK(a) of the catalytic Cys162 was determined to be 8.3. In addition, the pH-dependence of MsrB2 activity showed a maximum at pH 9.0, suggesting that deprotonation of the catalytic Cys is a critical step for the reaction. Further mobility analysis showed a well-structured N-terminal region, which contrasted with the high flexibility of this region in MsrB1. Our study highlights important structural and functional aspects of mammalian MsrB2 and provides a unifying picture for structure-function relationships within the MsrB protein family.     

Inhibition of ganglioside GD1a synthesis suppresses the differentiation of human mesenchymal stem cells into osteoblasts

In this study, we investigated the regulatory role of ganglioside GD1a in the differentiation of osteoblasts from human mesenchymal stem cells (hMSCs) by using lentivirus-containing short hairpin (sh)RNA to knockdown ST3 β-galactoside α-2, 3-sialyltransferase 2 (ST3Gal II) mRNA expression. After hMSCs were infected for 72 h with the lentivirus constructed with ST3Gal II shRNAs, the puromycin-resistant cells were selected and subcultured to produce hMSCs with ST3Gal II mRNA knockdown. The hMSCs established from human dental papilla abundantly expressed CD44 and CD105, but not CD45 and CD117. Osteoblasts that differentiated from normal hMSCs showed a significant increase in alkaline phosphatase (ALP) activity and ganglioside GD1a expression level compared with those in hMSCs. Lentiviral infection of hMSCs successfully induced a marked inhibition of ST3Gal II mRNA expression and caused a significant decrease in ALP activity and ganglioside GD1a expression. During osteoblastic differentiation, the increased ALP activity remarkably reduced by suppression of ganglioside GD1a expression by ST3Gal II shRNA. Ganglioside GD1a and ALP were mainly expressed in the cell body of hMSCs and osteoblasts with colocalization. The phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 mitogen-activated protein (MAP) kinase and epidermal growth factor receptor (EGFR) was significantly reduced in the osteoblasts that had differentiated from the hMSCs with ST3Gal II mRNA knockdown. These results suggest that ganglioside GD1a plays an important role in the regulation of osteoblastic differentiation of hMSCs through the activation of ERK 1/2 MAP kinase and EGFR.     

Complete mitochondrial genome sequence of the Korean water deer Hydropotes inermis argyropus (Cervidae, Capreolinae)

The complete mitochondrial genome sequence of Hydropotes inermis argyropus consists of 13 protein-coding, 22 tRNA, and two rRNA genes, and 1 control region (CR). Three overlaps among the 13 protein-coding genes were found: ATP8/ATP6, ND4L/ND4, and ND5/ND6. The CR was located between the tRNA-Pro and tRNA-Phe genes and is 928 bp in length. The typical conserved domains, such as TAS and CSB, were identified in the CR.     

Synthesis of biocompatible PEG-Based star polymers with cationic and degradable core for siRNA delivery

Star polymers with poly(ethylene glycol) (PEG) arms and a degradable cationic core were synthesized by the atom transfer radical copolymerization (ATRP) of poly(ethylene glycol) methyl ether methacrylate macromonomer (PEGMA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), and a disulfide dimethacrylate (cross-linker, SS) via an "arm-first" approach. The star polymers had a diameter ~15 nm and were degraded under redox conditions by glutathione treatment into individual polymeric chains due to cleavage of the disulfide cross-linker, as confirmed by dynamic light scattering. The star polymers were cultured with mouse calvarial preosteoblast-like cells, embryonic day 1, subclone 4 (MC3T3-E1.4) to determine biocompatibility. Data suggest star polymers were biocompatible, with ≥ 80% cell viability after 48 h of incubation even at high concentration (800 μg/mL). Zeta potential values varied with N/P ratio confirming complexation with siRNA. Successful cellular uptake of the star polymers in MC3T3-E1.4 cells was observed by confocal microscopy and flow cytometry after 24 h of incubation.     

Genome-Wide Analysis of Human Metapneumovirus Evolution

Human metapneumovirus (HMPV) has been described as an important etiologic agent of upper and lower respiratory tract infections, especially in young children and the elderly. Most of school-aged children might be introduced to HMPVs, and exacerbation with other viral or bacterial super-infection is common. However, our understanding of the molecular evolution of HMPVs remains limited. To address the comprehensive evolutionary dynamics of HMPVs, we report a genome-wide analysis of the eight genes (N, P, M, F, M2, SH, G, and L) using 103 complete genome sequences. Phylogenetic reconstruction revealed that the eight genes from one HMPV strain grouped into the same genetic group among the five distinct lineages (A1, A2a, A2b, B1, and B2). A few exceptions of phylogenetic incongruence might suggest past recombination events, and we detected possible recombination breakpoints in the F, SH, and G coding regions. The five genetic lineages of HMPVs shared quite remote common ancestors ranging more than 220 to 470 years of age with the most recent origins for the A2b sublineage. Purifying selection was common, but most protein genes except the F and M2-2 coding regions also appeared to experience episodic diversifying selection. Taken together, these suggest that the five lineages of HMPVs maintain their individual evolutionary dynamics and that recombination and selection forces might work on shaping the genetic diversity of HMPVs.     

Independent Relationship between Amyloid Precursor Protein (APP) Dimerization and γ-Secretase Processivity

Altered production of β-amyloid (Aβ) from the amyloid precursor protein (APP) is closely associated with Alzheimer’s disease (AD). APP has a number of homo- and hetero-dimerizing domains, and studies have suggested that dimerization of β-secretase derived APP carboxyl terminal fragment (CTFβ, C99) impairs processive cleavage by γ-secretase increasing production of long Aβs (e.g., Aβ1-42, 43). Other studies report that APP CTFβ dimers are not γ-secretase substrates. We revisited this issue due to observations made with an artificial APP mutant referred to as 3xK-APP, which contains three lysine residues at the border of the APP ectodomain and transmembrane domain (TMD). This mutant, which dramatically increases production of long Aβ, was found to form SDS-stable APP dimers, once again suggesting a mechanistic link between dimerization and increased production of long Aβ. To further evaluate how multimerization of substrate affects both initial γ-secretase cleavage and subsequent processivity, we generated recombinant wild type- (WT) and 3xK-C100 substrates, isolated monomeric, dimeric and trimeric forms of these proteins, and evaluated both ε-cleavage site utilization and Aβ production. These show that multimerization significantly impedes γ-secretase cleavage, irrespective of substrate sequence. Further, the monomeric form of the 3xK-C100 mutant increased long Aβ production without altering the initial ε-cleavage utilization. These data confirm and extend previous studies showing that dimeric substrates are not efficient γ-secretase substrates, and demonstrate that primary sequence determinants within APP substrate alter γ-secretase processivity.     

The Australian giant fleas Macropsylla Rothschild, 1905 (Siphonaptera: Macropsyllidae: Macropsyllinae), their identification,evolution, ecology,and conservation biology

Systematic Parasitology - Macropsylla Rothschild, 1905 is an endemic Australian flea genus represented by two species: M. hercules Rothschild, 1905 and M. novaehollandiae Hastriter, 2002. However,...