Rhesus macaques infected with lymphocytic choriomeningitis virus (LCMV) provide a model for human Lassa fever. Disease begins with flu-like symptoms and progresses rapidly with fatal consequences. Previously, we profiled the blood transcriptome of LCMV-infected monkeys (M. Djavani et al J. Virol. 2007) showing distinct pre-viremic and viremic stages that discriminated virulent from benign infections. In the present study, changes in liver gene expression from macaques infected with virulent LCMV-WE were compared to gene expression in uninfected monkeys as well as to monkeys that were infected but not diseased.

A method of loop-mediated isothermal amplification (LAMP) was employed to develop a rapid and simple detection system for porcine parvovirus (PPV) DNA. The amplification could be finished in 45 min under isothermal condition at 62 degrees C by employing a set of four primers targeting VP2 gene of PPV. LAMP assay showed higher sensitivity than PCR, with a detection limit of 5 copies of PPV genomic DNA per reaction. No cross reactivity was observed from the samples of other related viruses including canine parvovirus, parvovirus B19, porcine circovirus type 1, porcine circovirus type 2 and porcine peudorabies virus. The detection rate of PPV LAMP for 125 clinical samples was 97.6% and appeared higher than that of PCR method. The result indicated the potential usefulness of the technique as a simple, rapid procedure for the detection of PPV.

Genome-wide association studies (GWAS) have linked common single nucleotide polymorphisms (SNPs) on chromosome 9p21 near the INK4/ARF (CDKN2A/B) tumor suppressor locus with risk of atherosclerotic diseases and type 2 diabetes mellitus. To explore the mechanism of this association, we investigated whether expression of proximate transcripts (p16(INK4a), p15(INK4b), ARF, ANRIL and MTAP) correlate with genotype of representative 9p21 SNPs.

DNA-reactive B cells play a central role in systemic lupus erythematosus (SLE); DNA antibodies precede clinical disease and in established disease correlate with renal inflammation and contribute to dendritic cell activation and high levels of type 1 interferon. A number of central and peripheral B cell tolerance mechanisms designed to control the survival, differentiation and activation of autoreactive B cells are thought to be disturbed in patients with SLE. The characterization of DNA-reactive B cells has, however, been limited by their low frequency in peripheral blood. Using a tetrameric configuration of a peptide mimetope of DNA bound by pathogenic anti-DNA antibodies, we can identify B cells producing potentially pathogenic DNA-reactive antibodies. We, therefore, characterized the maturation and differentiation states of peptide, (ds) double stranded DNA cross-reactive B cells in the peripheral blood of lupus patients and correlated these with clinical disease activity. Flow cytometric analysis demonstrated a significantly higher frequency of tetramer-binding B cells in SLE patients compared to healthy controls. We demonstrated the existence of a novel tolerance checkpoint at the transition of antigen-naïve to antigen-experienced. We further demonstrate that patients with moderately active disease have more autoreactive B cells in both the antigen-naïve and antigen-experienced compartments consistent with greater impairment in B cell tolerance in both early and late checkpoints in these patients than in patients with quiescent disease. This methodology enables us to gain insight into the development and fate of DNA-reactive B cells in individual patients with SLE and paves the way ultimately to permit better and more customized therapies.

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with diverse cytoprotective effects, and reported to have an important role in angiogenesis recently. Here we investigated whether HO-1 transduced by mesenchymal stem cells (MSCs) can induce angiogenic effects in infarcted myocardium. HO-1 was transfected into cultured MSCs using an adenoviral vector. 1 × 106 Ad-HO-1-transfected MSCs (HO-1-MSCs) or Ad-Null-transfected MSCs (Null-MSCs) or PBS was respectively injected into rat hearts intramyocardially at 1 h post-myocardial infarction. The results showed that HO-1-MSCs were able to induce stable expression of HO-1 in vitro and in vivo. The capillary density and expression of angiogenic growth factors, VEGF and FGF2 were significantly enhanced in HO-1-MSCs-treated hearts compared with Null-MSCs-treated and PBS-treated hearts. However, the angiogenic effects of HO-1 were abolished by treating the animals with HO inhibitor, zinc protoporphyrin. The myocardial apoptosis was marked reduced with significantly reduced fibrotic area in HO-1-MSCs-treated hearts; Furthermore, the cardiac function and remodeling were also significantly improved in HO-1-MSCs-treated hearts. Our current findings support the premise that HO-1 transduced by MSCs can induce angiogenic effects and improve heart function after acute myocardial infarction.

Allostery of P-selectin lectin (Lec) domain followed by an epithelial growth factor (EGF)-like domain is essential for its biological functionality, but the underlying pathways have not been well understood. Here the molecular dynamics simulations were performed on the crystallized structures to visualize the dynamic conformational change for state 1 (S1) or state 2 (S2) Lec domain with respective bent (B) or extended (E) EGF orientation. Simulations illustrated that both S1 and S2 conformations were unable to switch from one to another directly. Instead, a novel S1' conformation was observed from S1 when crystallized B-S1 or reconstructed "E-S1" structure was employed, which was superposed well with that of equilibrated S1 Lec domain alone. It was also indicated that the corresponding allosteric pathway from S1 to S1' conformation started with the separation between residues Q30 and K67 and terminated with the release of residue N87 from residue C109. These results provided an insight into understanding the structural transition and the structure-function relationship of P-selectin allostery.

Interleukin 17 (IL-17) plays critical roles in the pathogenesis of various autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE). How the signals triggered by this powerful inflammatory cytokine are controlled to avoid abnormal inflammatory responses is not well understood. In this study, we report that TRAF3 is a receptor proximal negative regulator of IL-17 receptor (IL-17R) signaling. TRAF3 greatly suppressed IL-17-induced NF-κB and mitogen-activated protein kinase activation and subsequent production of inflammatory cytokines and chemokines. Mechanistically, the binding of TRAF3 to IL-17R interfered with the formation of the receptor signaling activation complex IL-17R-Act1-TRAF6, resulting in suppression of downstream signaling. TRAF3 markedly inhibited IL-17-induced expression of inflammatory cytokine and chemokine genes in vivo and consequently delayed the onset and greatly reduced the incidence and severity of EAE. Thus, TRAF3 is a negative regulator of IL-17R proximal signaling.

Hand, foot and mouth disease (HFMD), a common contagious disease that usually affects children, is normally mild but can have life-threatening manifestations. It can be caused by enteroviruses, particularly Coxsackieviruses and human enterovirus 71 (HEV71) with highly variable clinical manifestations. In the spring of 2008, a large, unprecedented HFMD outbreak in Fuyang city of Anhui province in the central part of southeastern China resulted in a high aggregation of fatal cases. In this study, epidemiologic and clinical investigations, laboratory testing, and genetic analyses were performed to identify the causal pathogen of the outbreak. Of the 6,049 cases reported between 1 March and 9 May of 2008, 3023 (50%) were hospitalized, 353 (5.8%) were severe and 22 (0.36%) were fatal. HEV71 was confirmed as the etiological pathogen of the outbreak. Phylogenetic analyses of entire VP1 capsid protein sequence of 45 Fuyang HEV71 isolates showed that they belong to C4a cluster of the C4 subgenotype. In addition, genetic recombinations were found in the 3D region (RNA-dependent RNA polymerase, a major component of the viral replication complex of the genome) between the Fuyang HEV71 strain and Coxsackievirus A16 (CV-A16), resulting in a recombination virus. In conclusion, an emerging recombinant HEV71 was responsible for the HFMD outbreak in Fuyang City of China, 2008.

Mammalian Ras genes regulate diverse cellular processes including proliferation and differentiation and are frequently mutated in human cancers. Tumor development in response to Ras activation varies between different tissues and the molecular basis for these variations are poorly understood. The murine lens and cornea have a common embryonic origin and arise from adjacent regions of the surface ectoderm. Activation of the fibroblast growth factor (FGF) signaling pathway induces the corneal epithelial cells to proliferate and the lens epithelial cells to exit the cell cycle. The molecular mechanisms that regulate the differential responses of these two related tissues have not been defined. We have generated transgenic mice that express a constitutively active version of human H-Ras in their lenses and corneas.

In this study, we calculated the relative synonymous codon usage (RSCU) values and codon usage bias (CUB) values to implement a comparative analysis of codon usage pattern of open reading frames (ORFs) which belong to the two main genotypes of porcine reproductive and respiratory syndrome virus (PRRSV). By analysis of synonymous codon usage values in each ORF of PRRSV, the optimal codons for most amino acids were all C or G-ended codons except GAU for Asp, CAU for His, UUU for Phe and CCU for Pro. The synonymous codon usage patterns in different ORFs of PRRSV were different and genetically conserved. Among them, ORF1a, ORF4, ORF5 and ORF7 could cluster these strains into the two main serotypes (EU and US). Due to mutational pressure, compositional constraint played an important role in shaping the synonymous codon usage pattern in different ORFs, and the synonymous codon usage diversity in ORFs was correlated with gene function. The degree of CUB for some particular amino acids under strong selection pressure probably served as a potential genetic marker for each ORF in PRRSV. However, gene length and translational selection in nature had no effect on the synonymous codon usage pattern in PRRSV. These conclusions could not only offer an insight into the synonymous codon usage pattern and differentiation of gene function, but also assist in understanding the discrepancy of evolution among ORFs in PRRSV.

In this study, we examined whether matrine could inhibit the differentiation of 3T3-L1 preadipocytes and further explored the possible inhibitory mechanisms. Evidenced by Oil Red O staining and AdipoRed assay, matrine dose-dependently inhibited lipid accumulation at concentrations of 125, 250 and 500 microg/ml. At molecular level, the expression of transcription factors, PPARgamma and C/EBPalpha, was reduced by matrine during adipogenesis. After treatment for 6 days, the mRNA levels of adipocyte-specific genes, such as aP2, LPL, adiponectin and leptin, were also down-regulated by matrine in a dose-dependent manner. Moreover, 500 microg/ml matrine inhibited the phosphorylation of ERK1/2 at the early stage of differentiation. Our results indicate that inhibition of 3T3-L1 preadipocyte differentiation by matrine is associated with the suppression of ERK1/2 phosphorylation. Thus, matrine has the potential to be an alternative natural product for the treatment of obesity.

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin alpha2beta1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.

High myopia, which is extremely prevalent in the Chinese population, is one of the leading causes of blindness in the world. Genetic factors play a critical role in the development of the condition. To identify the genetic variants associated with high myopia in the Han Chinese, we conducted a genome-wide association study (GWAS) of 493,947 SNPs in 1088 individuals (419 cases and 669 controls) from a Han Chinese cohort and followed up on signals that were associated with p < 1.0 × 10(-4) in three independent cohorts (combined, 2803 cases and 5642 controls). We identified a significant association between high myopia and a variant at 13q12.12 (rs9318086, combined p = 1.91 × 10(-16), heterozygous odds ratio = 1.32, and homozygous odds ratio = 1.64). Furthermore, five additional SNPs (rs9510902, rs3794338, rs1886970, rs7325450, and rs7331047) in the same linkage disequilibrium (LD) block with rs9318086 also proved to be significantly associated with high myopia in the Han Chinese population; p values ranged from 5.46 × 10(-11) to 6.16 × 10(-16). This associated locus contains three genes-MIPEP, C1QTNF9B-AS1, and C1QTNF9B. MIPEP and C1QTNF9B were found to be expressed in the retina and retinal pigment epithelium (RPE) and are more likely than C1QTNF9B-AS1 to be associated with high myopia given the evidence of retinal signaling that controls eye growth. Our results suggest that the variants at 13q12.12 are associated with high myopia.

The effects of hormonal and dietary stimuli on hepatic glucose and lipid homeostasis include regulation of gene expression. Berberine, an effective compound in certain Chinese medicinal herbs, has been reported to lower plasma glucose and lipid levels in diabetic and hypercholesterolemic patients. We hypothesized that it may affect the expression of hepatic genes involved in glucose and lipid metabolism. The effects of berberine hydrochloride on viability, gene expression, and activation of AMP activated protein kinase (AMPK) in primary hepatocytes from Sprague-Dawley (SD), Zucker lean (ZL) or fatty (ZF) rats were examined with MTT assay, real-time PCR, and western blotting, respectively. Berberine hydochloride at 50 µM or higher caused cytotoxic effects on hepatocytes. In SD and ZL hepatocytes, it induced Gck and suppressed G6pc expression at 10 and 25 µM, but not as potent as 1 nM insulin. Its effects on Pck1, and insulin-regulated Gck and G6pc expression depended on the hepatocyte sources and the dosage used. In ZF hepatocytes, it increased Gck, and suppressed Pck1 and G6pc expression without insulin. Its effects on Gck and G6pc, but not Pck1 expression, were additive with insulin. Berberine hydrochloride at 25 µM attenuated insulin-suppressed Pck1 (ZL/ZF cells), and insulin-induced Srebp-1c expression (SD/ZL/ZF cells), suggesting modulation of insulin action. Berberine hydrochloride did not alter these genes' mRNA stability. Its treatment caused a dose-dependent increase of phosphorylation of AMPKα, and its substrate, acetyl-CoA carboxylase, in primary hepatocytes. We conclude that berberine hydrochloride regulated the transcription of hepatic genes involved in glucose and fatty acid metabolism.

In this study, an abundant (A+U)% and low codon bias were revealed in duck hepatitis virus type 1 (DHV-1) and the new serotype strains isolated from Taiwan, South Korea and Mainland China (DHV-N). The general correlation between base composition and codon usage bias suggests that mutational pressure rather than natural selection is the main factor that determines the codon usage bias in these samples. By comparative analysis of the codon usage patterns of 40 ORFs of DHV, we found that all of DHV-1 strains grouped in genotype C; the DHV-N strains isolated in South Korea and China clustered into genotypes B; and the DHV-N strains isolated from Taiwan clustered into genotypes A. The findings revealed that more than one subtype of DHV-1 circulated in East Asia. Furthermore, the results of phylogenetic analyses based on RSCU values and Clustal W method indicated obvious phylogenetic congruities. This suggested that better genome consistency of DHV may exist in nature and phylogenetic analyses based on RSCU values maybe a good method in classifying genotypes of the virus. Our work might give some clues to the features and some evolutionary information of DHV.

DNA methylation plays critical roles in transcriptional regulation and chromatin remodeling. Differentially methylated regions (DMRs) have important implications for development, aging and diseases. Therefore, genome-wide mapping of DMRs across various temporal and spatial methylomes is important in revealing the impact of epigenetic modifications on heritable phenotypic variation. We present a quantitative approach, quantitative differentially methylated regions (QDMRs), to quantify methylation difference and identify DMRs from genome-wide methylation profiles by adapting Shannon entropy. QDMR was applied to synthetic methylation patterns and methylation profiles detected by methylated DNA immunoprecipitation microarray (MeDIP-chip) in human tissues/cells. This approach can give a reasonable quantitative measure of methylation difference across multiple samples. Then DMR threshold was determined from methylation probability model. Using this threshold, QDMR identified 10,651 tissue DMRs which are related to the genes enriched for cell differentiation, including 4740 DMRs not identified by the method developed by Rakyan et al. QDMR can also measure the sample specificity of each DMR. Finally, the application to methylation profiles detected by reduced representation bisulphite sequencing (RRBS) in mouse showed the platform-free and species-free nature of QDMR. This approach provides an effective tool for the high-throughput identification of potential functional regions involved in epigenetic regulation.

Exposure to cigarette smoke is a major risk factor for head and neck squamous cell carcinoma (HNSCC). We have previously established a chronic cigarette smoke extract (CSE)-treated human oral normal keratinocyte model, demonstrating an elevated frequency of mitochondrial mutations in CSE treated cells. Using this model we further characterized the mechanism by which chronic CSE treatment induces increased cellular proliferation.

Synthesis of selenoproteins depends on decoding of the UGA stop codon as the amino acid selenocysteine (Sec). This process requires the presence of a Sec insertion sequence element (SECIS) in the 3'-untranslated region of selenoprotein mRNAs and its interaction with the SECIS binding protein 2 (SBP2). In humans, mutations in the SBP2-encoding gene Sec insertion sequence binding protein 2 (SECISBP2) that alter the amino acid sequence or cause splicing defects lead to abnormal thyroid hormone metabolism. Herein, we present the first in silico and in vivo functional characterization of alternative splicing of SECISBP2. We report a complex splicing pattern in the 5'-region of human SECISBP2, wherein at least eight splice variants encode five isoforms with varying N-terminal sequence. One of the isoforms, mtSBP2, contains a mitochondrial targeting sequence and localizes to mitochondria. Using a minigene-based in vivo splicing assay we characterized the splicing efficiency of several alternative transcripts, and show that the splicing event that creates mtSBP2 can be modulated by antisense oligonucleotides. Moreover, we show that full-length SBP2 and some alternatively spliced variants are subject to a coordinated transcriptional and translational regulation in response to ultraviolet type A irradiation-induced stress. Overall, our data broadens the functional scope of a housekeeping protein essential to selenium metabolism.

Seed oil accumulates primarily as triacylglycerol (TAG). While the biochemical pathway for TAG biosynthesis is known, its regulation remains unclear. Previous research identified microsomal diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) as controlling a rate-limiting step in the TAG biosynthesis pathway. Of note, overexpression of DGAT1 results in substantial increases in oil content and seed size. To further analyze the global consequences of manipulating DGAT1 levels during seed development, a concerted transcriptome and metabolome analysis of transgenic B. napus prototypes was performed.

MicroRNAs are key regulators of many biological processes, including cell differentiation. Here we show that during human monocyte-macrophage differentiation, expression of the microRNAs miR-223, miR-15a and miR-16 decreased considerably, which led to higher expression of the serine-threonine kinase IKKalpha in macrophages. In macrophages, higher IKKalpha expression in conjunction with stabilization of the kinase NIK induced larger amounts of p52. Because of low expression of the transcription factor RelB in untreated macrophages, high p52 expression repressed basal transcription of both canonical and noncanonical NF-kappaB target genes. However, proinflammatory stimuli in macrophages resulted in greater induction of noncanonical NF-kappaB target genes. Thus, a decrease in certain microRNAs probably prevents macrophage hyperactivation yet primes the macrophage for certain responses to proinflammatory stimuli.