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.     

Suppression of ceramide-induced cell death by hepatitis C virus core protein

The hepatitis C virus (HCV) core protein is believed to be one of viral proteins that are capable of preventing virus-infected cell death upon various stimuli. But, the effect of the HCV core protein on apoptosis that is induced by various stimuli is contradictory. We examined the possibility that the HCV core protein affects the ceramide-induced cell death in cells expressing the HCV core protein through the sphingomyelin pathway. Cell death that is induced by C(2)-ceramide and bacterial sphingomyelinase was analyzed in 293 cells that constitutively expressed the HCV core protein and compared with 293 cells that were stably transfected only with the expression vector. The HCV core protein inhibited the cell death that was induced by these reagents. The protective effects of the HCV core protein on ceramide-induced cell death were reflected by the reduced expression of p21(WAF1/Cip1/Sid1) and the sustained expression of the Bcl-2 protein in the HCV core-expressing cells with respect to the vector-transfected cells. These results suggest that the HCV core protein in 293 cells plays a role in the modulation of the apoptotic response that is induced by ceramide. Also, the ability of the HCV core protein to suppress apoptosis might have important implications in understanding the pathogenesis of the HCV infection.     

Comparison of PERV genomic locations between Asian and European pigs

Xenotransplantation from pigs provides a possible solution to the shortage of human organs for allotransplantation. Porcine endogenous retroviruses (PERVs) are a possible obstacle to using porcine organs in addition to the immunological barriers. Three main types of PERVs (A, B and C) have been previously investigated in diverse pig breeds. To examine the copy numbers of PERVs and their genomic locations in the Korean native pig genome, we screened a BAC (Bacterial Artificial Chromosome) library with PERV-specific protease primers for initial recognition of PERV-positive clones and three sets of envelope-specific primers for the identification of PERV types. A total of 45 PERV-positive clones, nine PERV-A and 36 PERV-B, have been identified from the library screening and the BAC contigs were constructed using the primers designed from BAC end sequences (BESs). These primers were also used for SCH (Somatic Cell Hybrid) and RH (Radiation Hybrid) mapping of the PERV-positive clones. The results indicate that 45 PERV-positive BAC clones belong to nine contigs and a singleton. SCH and IMpRH (INRA-Minnesota Porcine Radiation Hybrid) mapping results indicated that there are at least eight separate PERV genomic locations, consisting of three PERV-A and five PERV-B. One contig could not be mapped, and two contigs are closely located on SSC7. Southern blotting indicates there may be up to 15 additional sites. Further investigation of these clones will contribute to a general strategy to generate PERV-free lines of pigs suitable for xenotransplantation.     

CCL5/CCR5 axis promotes the motility of human oral cancer cells

CCL5 (previously called RANTES) is in the CC‐chemokine family and plays a crucial role in the migration and metastasis of human cancer cells. On the other hand, the effect of CCL5 is mediated via CCR receptor. RT‐PCR and flow cytometry studies demonstrated CCR5 but not CCR1 and CCR3 mRNA in oral cancer cell lines, especially higher in those with high invasiveness (SCC4) as compared with lower levels in HSC3 cells and SCC9 cells. Stimulation of oral cancer cells with CCL5 directly increased the migration and metalloproteinase‐9 (MMP‐9) production. MMP‐9 small interfering RNA inhibited the CCL5‐induced MMP‐9 expression and thereby significantly inhibited the CCL5‐induced cell migration. Activations of phospholipase C (PLC), protein kinase Cδ (PKCδ), and NF‐κB pathways after CCL5 treatment was demonstrated, and CCL5‐induced expression of MMP‐9 and migration activity was inhibited by the specific inhibitor of PLC, PKCδ, and NF‐κB cascades. In addition, migration‐prone sublines demonstrate that cells with increasing migration ability had more expression of MMP‐9, CCL5, and CCR5. Taken together, these results indicate that CCL5/CCR5 axis enhanced migration of oral cancer cells through the increase of MMP‐9 production. J. Cell. Physiol. 220: 418–426, 2009. © 2009 Wiley‐Liss, Inc.     

Heterotic patterns in rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.): I. Crosses between spring and Chinese semi-winter lines

Chinese semi-winter rapeseed is genetically diverse from Canadian and European spring rapeseed. This study was conducted to evaluate the potential of semi-winter rapeseed for spring rapeseed hybrid breeding, to assess the genetic effects involved, and to estimate the correlation of parental genetic distance (GD) with hybrid performance, heterosis, general combining ability (GCA) and specific combining ability (SCA) in crosses between spring and semi-winter rapeseed lines. Four spring male sterile lines from Germany and Canada as testers were crossed with 13 Chinese semi-winter rapeseed lines to develop 52 hybrids, which were evaluated together with their parents and commercial hybrids for seed yield and oil content in three sets of field trials with 8 environments in Canada and Europe. The Chinese parental lines were not adapted to local environmental conditions as demonstrated by poor seed yields per se. However, the hybrids between the Chinese parents and the adapted spring rapeseed lines exhibited high heterosis for seed yield. The average mid-parent heterosis was 15% and ca. 50% of the hybrids were superior to the respective hybrid control across three sets of field trials. Additive gene effects mainly contributed to hybrid performance since the mean squares of GCA were higher as compared to SCA. The correlation between parental GD and hybrid performance and heterosis was found to be low whereas the correlation between GCA_{(f + m)} and hybrid performance was high and significant in each set of field trials, with an average of r = 0.87 for seed yield and r = 0.89 for oil content, indicating that hybrid performance can be predicted by GCA_{(f + m)}. These results demonstrate that Chinese semi-winter rapeseed germplasm has a great potential to increase seed yield in spring rapeseed hybrid breeding programs in Canada and Europe.     

Rational design of ornithine decarboxylase with high catalytic activity for the production of putrescine

Putrescine finds wide industrial applications in the synthesis of polymers, pharmaceuticals, agrochemicals, and surfactants. Owing to economic and environmental concerns, the microbial production of putrescine has attracted a great deal of attention, and ornithine decarboxylase (ODC) is known to be a key enzyme in the biosynthetic pathway. Herein, we present the design of ODC from Escherichia coli with high catalytic efficiency using a structure-based rational approach. Through a substrate docking into the model structure of the enzyme, we first selected residues that might lead to an increase in catalytic activity. Of the selected residues that are located in the α-helix and the loops constituting the substrate entry site, a mutational analysis of the single mutants identified two key residues, I163 and E165. A combination of two single mutations resulted in a 62.5-fold increase in the catalytic efficiency when compared with the wild-type enzyme. Molecular dynamics simulations of the best mutant revealed that the substrate entry site becomes more flexible through mutations, while stabilizing the formation of the dimeric interface of the enzyme. Our approach can be applied to the design of other decarboxylases with high catalytic efficiency for the production of various chemicals through bio-based processes.     

Modulation of cytokeratin expression during in vitro cultivation of human hepatic stellate cells: evidence of transdifferentiation from epithelial to mesenchymal phenotype

The embryonal origin of hepatic stellate cells (HSCs), the principal cells in hepatic fibrogenesis, is still intriguing. To explore the origin and the differentiation of HSCs, we studied the expression of cytokeratin 18 (CK18) and 19 (CK19), the standard markers of simple epithelial cells, in cultured human HSCs. Hepatic stellate cells were isolated from five normal human livers. In immunofluorescence staining, both clone C-51 anti-CK18 antibody and clone RCK108 anti-CK19 antibody labeled almost all stellate cells in the primary culture. Double immunofluorescence staining for cytokeratin/vimentin and cytokeratin/alpha-smooth muscle actin detected by confocal laser scanning microscopy clearly demonstrated the localization of cytokeratin immunoreactivity in human HSCs. During subsequent cultivation of human HSCs to the tenth passage, immunocytochemical staining and western blot analysis demonstrated gradually decreasing profiles of CK18 and CK19 expression. The progressive reduction of cytokeratin expression was further confirmed in a culture of clone cells originated from a single HSC. In conclusion, both CK18 and CK19 are expressed in cultured human HSCs, and the extent of their expression decreases gradually during prolonged cultivation. Our results suggest that HSCs may be of epithelial origin, and that they undergo the transdifferentiation from epithelial to mesenchymal phenotype during an activation process in vitro.     

An improved transformation protocol for studying gene expression in hairy roots of sugar beet (Beta vulgaris L.)

A transformation protocol, based on co-inoculation with two strains of Agrobacterium, Agrobacterium tumefaciens LBA4404 and A. rhizogenes 15834 containing a binary vector with the GUS gene, was established for the induction of transgenic hairy roots from sugar beet (Beta vulgaris L.) explants. It resulted in marked improvement in the formation of hairy roots and the integration of the binary vector T-DNA into the host genome. Of 250 inoculated sugar beet hypocotyls, 84% yielded hairy roots 5–7 days after inoculation, of which 70% were co-transformed with the binary vector T-DNA. To determine stable expression of alien genes in hairy roots, the nematode resistance gene Hs1 ^pro-1 was used as a reporter gene. In addition, molecular marker analysis was applied to monitor stable incorporation of a translocation from the wild beet B. procumbens. The molecular analysis and the nematode (Heterodera schachtii) resistance test in vitro demonstrated that the genomic structure and the expression of the Hs1 ^pro-1 -mediated nematode resistance were well-maintained in all hairy root cultures even after repeated sub-culture. Received: 25 November 1997 / Revision received: 26 May 1998 / Accepted: 15 June 1998     

Combinatorial polymer electrospun matrices promote physiologically-relevant cardiomyogenic stem cell differentiation

Myocardial infarction results in extensive cardiomyocyte death which can lead to fatal arrhythmias or congestive heart failure. Delivery of stem cells to repopulate damaged cardiac tissue may be an attractive and innovative solution for repairing the damaged heart. Instructive polymer scaffolds with a wide range of properties have been used extensively to direct the differentiation of stem cells. In this study, we have optimized the chemical and mechanical properties of an electrospun polymer mesh for directed differentiation of embryonic stem cells (ESCs) towards a cardiomyogenic lineage. A combinatorial polymer library was prepared by copolymerizing three distinct subunits at varying molar ratios to tune the physicochemical properties of the resulting polymer: hydrophilic polyethylene glycol (PEG), hydrophobic poly(ε-caprolactone) (PCL), and negatively-charged, carboxylated PCL (CPCL). Murine ESCs were cultured on electrospun polymeric scaffolds and their differentiation to cardiomyocytes was assessed through measurements of viability, intracellular reactive oxygen species (ROS), α-myosin heavy chain expression (α-MHC), and intracellular Ca(2+) signaling dynamics. Interestingly, ESCs on the most compliant substrate, 4%PEG-86%PCL-10%CPCL, exhibited the highest α-MHC expression as well as the most mature Ca(2+) signaling dynamics. To investigate the role of scaffold modulus in ESC differentiation, the scaffold fiber density was reduced by altering the electrospinning parameters. The reduced modulus was found to enhance α-MHC gene expression, and promote maturation of myocyte Ca(2+) handling. These data indicate that ESC-derived cardiomyocyte differentiation and maturation can be promoted by tuning the mechanical and chemical properties of polymer scaffold via copolymerization and electrospinning techniques.