Many conserved noncoding sequences function as transcriptional enhancers that regulate gene expression. Here, we report that protein-coding DNA also frequently contains enhancers functioning at the transcriptional level. We tested the enhancer activity of 31 protein-coding exons, which we chose based on strong sequence conservation between zebrafish and human, and occurrence in developmental genes, using a Tol2 transposable GFP reporter assay in zebrafish. For each exon we measured GFP expression in hundreds of embryos in 10 anatomies via a novel system that implements the voice-recognition capabilities of a cellular phone. We find that 24/31 (77%) exons drive GFP expression compared to a minimal promoter control, and 14/24 are anatomy-specific (expression in four anatomies or less). GFP expression driven by these coding enhancers frequently overlaps the anatomies where the host gene is expressed (60%), suggesting self-regulation. Highly conserved coding sequences and highly conserved noncoding sequences do not significantly differ in enhancer activity (coding: 24/31 vs. noncoding: 105/147) or tissue-specificity (coding: 14/24 vs. noncoding: 50/105). Furthermore, coding and noncoding enhancers display similar levels of the enhancer-related histone modification H3K4me1 (coding: 9/24 vs noncoding: 34/81). Meanwhile, coding enhancers are over three times as likely to contain an H3K4me1 mark as other exons of the host gene. Our work suggests that developmental transcriptional enhancers do not discriminate between coding and noncoding DNA and reveals widespread dual functions in protein-coding DNA.

INTRODUCTION: Although WHO declared the world moving into the post-pandemic period on August 10, 2010, influenza A(H1N1) 2009 virus continued to circulate globally. Its impact was expected to continue during the 2010-11 influenza season. This study describes the nationwide surveillance findings of the pandemic and post-pandemic influenza periods in Taiwan and assesses the impact of influenza A(H1N1) 2009 during the post-pandemic period. METHODS: The Influenza Laboratory Surveillance Network consisted of 12 contract laboratories for collecting and testing samples with acute respiratory tract infections. Surveillance of emergency room visits and outpatient department visits for influenza-like illness (ILI) were conducted using the Real-Time Outbreak and Disease Surveillance system and the National Health Insurance program data, respectively. Hospitalized cases with severe complications and deaths were reported to the National Notifiable Disease Surveillance System. RESULTS: During the 2009-10 influenza season, pandemic A(H1N1) 2009 was the predominant circulating strain and caused 44 deaths. However, the 2010-11 influenza season began with A(H3N2) being the predominant circulating strain, changing to A(H1N1) 2009 in December 2010. Emergency room and outpatient department ILI surveillance displayed similar trends. By March 31, 2011, there were 1,751 cases of influenza with severe complications; 50.1% reported underlying diseases. Of the reported cases, 128 deaths were associated with influenza. Among these, 93 (72.6%) were influenza A(H1N1) 2009 and 30 (23.4%) A(H3N2). Compared to the pandemic period, during the immediate post-pandemic period, increased number of hospitalizations and deaths were observed, and the patients were consistently older. CONCLUSIONS: Reemergence of influenza A(H1N1) 2009 during the 2010-11 influenza season had an intense activity with age distribution shift. To further mitigate the impact of future influenza epidemics, Taiwan must continue its multifaceted influenza surveillance systems, remain flexible with antiviral use policies, and revise the vaccine policies to include the population most at risk.

Cholestasis is frequently related to endotoxemia and inflammatory response. Our previous investigation revealed a significant increase in plasma endotoxin and CD14 levels during biliary atresia. We therefore propose that lipopolysacharides (LPS) may stimulate CD14 production in liver cells and promote the removal of endotoxins. The aims of this study are to test the hypothesis that CD14 is upregulated by LPS and investigate the pathophysiological role of CD14 production during cholestasis. Using Western blotting, qRT-PCR, and promoter activity assay, we demonstrated that LPS was associated with a significant increase in CD14 and MD2 protein and mRNA expression and CD14 promoter activity in C9 rat hepatocytes but not in the HSC-T6 hepatic stellate cell line in vitro. To correlate CD14 expression and endotoxin sensitivity, in vivo biliary LPS administration was performed on rats two weeks after they were subjected to bile duct ligation (BDL) or a sham operation. CD14 expression and endotoxin levels were found to significantly increase after LPS administration in BDL rats. These returned to basal levels after 24 h. In contrast, although endotoxin levels were increased in sham-operated rats given LPS, no increase in CD14 expression was observed. However, mortality within 24 h was more frequent in the BDL animals than in the sham-operated group. In conclusion, cholestasis and LPS stimulation were here found to upregulate hepatic CD14 expression, which may have led to increased endotoxin sensitivity and host proinflammatory reactions, causing organ failure and death in BDL rats.

PURPOSE High-mobility group box 1 protein (HMGB1) has been reported to be a potent proangiogenic factor induced by inflammatory stress. In this study, we explore the role of HMGB1 in advanced glycation end products (AGEs)-induced vascular endothelial growth factor A (VEGF-A) production in rat retinal ganglion cell line 5 (RGC-5) cells. METHODS The VEGF-A protein and mRNA levels in conditioned medium of RGC-5 cells incubated with AGE-modified BSA (AGE-BSA) were examined with real-time PCR and enzyme-linked immunosorbent assay (ELISA), and BSA-treated cells were used as controls. The expression of HMGB1, c-Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase (ERK), and p38 mitogen-activated protein kinase (p38 MAPK) was assessed with immunofluorescence and western blot analysis. Reactive oxidative species (ROS) were detected with flow cytometry measurements of peroxide-dependent oxidation of 2'-7'-dichlorofluorescein-diacetate (DCFH-DA). N-Acetyl-L-cysteine (NAC), glycyrrhizin (GZ), and SP600125 were used to block ROS, HMGB1, and JNK, respectively. RESULTS Compared with the BSA controls, the RGC-5 cells incubated with AGE-BSA showed a dose- and time-dependent increase in VEGF-A mRNA and VEGF-A protein secretion in the supernatant, with the highest levels achieved at 24 h. AGE-BSA stimulated a significant release of HMGB1 in the supernatant and a significant increase of intracellular ROS production at 3 h. NAC blocked HMGB1 production in a dose-dependent manner. Blocking with GZ, NAC, and JNK significantly suppressed AGE-induced VEGF-A production. CONCLUSIONS HMGB1 is implicated in the production of VEGF-A in retinal ganglion cell line-5 (RGC-5). Blocking HMGB1, ROS, or the JNK pathway may attenuate VEGF-A production, suggesting HMGB1 and related signaling molecules play a role in diabetic retinopathy.

Obstructive jaundice (OJ) increases the risk of liver injury and sepsis, leading to increased mortality. Cholestatic liver injury is associated with a downregulation of hepcidin expression levels. In fact, hepcidin has an important antimicrobial effect, especially against Escherichia coli. It is unknown whether supplementing recombinant hepcidin is effective in alleviating cholestasis-induced liver injury and mortality in mice with superimposed sepsis. A mouse model of cholestasis was developed using extrahepatic bile duct ligation for 3 days. In addition, sepsis due to E. coli 0111:B4 lipopolysaccharide (LPS) was induced in the model. The serum levels of total bilirubin, AST, ALT, and LDH and the mRNA levels of IL-1β, TNF-α, and MCP-1 in the liver were significantly higher in the OJ mice receiving LPS than in the sham-operated mice receiving LPS. Compared to the OJ mice receiving LPS, the hepcidin-pretreated OJ mice receiving LPS showed a significant decrease in the above mentioned parameters, as well as a reversal in the downregulation of LC3B-II and upregulation of cleaved caspase-3; this, in turn, led to significantly decreased lethality in 24h. In conclusion, these results indicate that hepcidin pretreatment significantly reduced hepatic proinflammatory cytokine expression and liver injury, leading to reduced early lethality in OJ mice receiving LPS. Enhanced autophagy and reduced apoptosis may account for the protective effects of hepcidin.

Phyllanthus urinaria (P. urinaria), in this study, was used for the treatment of human osteosarcoma cells, which is one of the tough malignancies with few therapeutic modalities. Herein, we demonstrated that P. urinaria inhibited human osteosarcoma 143B cells growth through an apoptotic extrinsic pathway to activate Fas receptor/ligand expression. Both intracellular and mitochondrial reactive oxygen species were increased to lead to alterations of mitochondrial membrane permeability and Bcl-2 family including upregulation of Bid, tBid, and Bax and downregulation of Bcl-2. P. urinaria triggered an intrinsic pathway and amplified the caspase cascade to induce apoptosis of 143B cells. However, upregulation of both intracellular and mitochondrial reactive oxygen species and the sequential membrane potential change were less pronounced in the mitochondrial respiratory-defective 143Bρ(0) cells compared with the 143B cells. This study offers the evidence that mitochondria are essential for the anticancer mechanism induced by P. urinaria through both extrinsic and intrinsic pathways.

PURPOSE: Kawasaki disease (KD) is a systemic febrile vasculitis complicated by coronary artery lesions (CAL). Anemia is common in patients with KD and is associated with a prolonged duration of active inflammation. Hepcidin is a central modulator of inflammation-associated anemia, acting via control of iron absorption and a direct inhibitory effect on erythropoiesis. The aims of this study were to investigate the role of inflammation-induced hepcidin in the development of anemia, the occurrence of CAL formation, and IVIG treatment response in patients with KD. METHODS: Eighty-six KD patients and 30 febrile controls were enrolled. Levels of interleukin (IL)-6 and serum hepcidin were measured in sera by enzyme-linked immunosorbent assay. Hemoglobin and serum iron levels were also measured. RESULTS: Hemoglobin and iron levels were lower in KD patients than in controls (p < 0.001 and p = 0.009, respectively). Serum hepcidin and IL-6 levels were higher in KD patients than in controls (both p < 0.001) before intravenous immunoglobulin (IVIG) treatment. After IVIG treatment, serum hepcidin, IL-6, and hemoglobin levels decreased significantly (all p < 0.001). In addition, the serum hepcidin levels before IVIG treatment were negatively correlated with hemoglobin levels after IVIG treatment (R = -0.188, p = 0.046) and positively correlated with the changes of hemoglobin levels after IVIG treatment (R = 0.269, p = 0.015). Furthermore, serum hepcidin levels were negatively correlated with serum iron levels (R = -0.412, p = 0.002), which were positively correlated with hemoglobin levels (R = 0.210, p = 0.045). Additionally, the change of hepcidin levels was associated with IVIG treatment response and the occurrence of CAL formation. CONCLUSIONS: Inappropriately raised hepcidin levels impair iron metabolism and are associated with decreased hemoglobin levels in KD patients. Inflammation-induced hepcidin is associated with the development of anemia and disease outcomes in patients with KD.

BACKGROUND It has been increasingly appreciated that coding sequences harbor regulatory sequence motifs in addition to encoding for protein. These sequence motifs are expected to be overrepresented in nucleotide sequences bound by a common protein or small RNA. However, detecting overrepresented motifs has been difficult because of interference by constraints at the protein level. Sampling-based approaches to solve this problem based on codon-shuffling have been limited to exploring only an infinitesimal fraction of the sequence space and by their use of parametric approximations. RESULTS We present a novel O(N(log N)2)-time algorithm, CodingMotif, to identify nucleotide-level motifs of unusual copy number in protein-coding regions. Using a new dynamic programming algorithm we are able to exhaustively calculate the distribution of the number of occurrences of a motif over all possible coding sequences that encode the same amino acid sequence, given a background model for codon usage and dinucleotide biases. Our method takes advantage of the sparseness of loci where a given motif can occur, greatly speeding up the required convolution calculations. Knowledge of the distribution allows one to assess the exact non-parametric p-value of whether a given motif is over- or under- represented. We demonstrate that our method identifies known functional motifs more accurately than sampling and parametric-based approaches in a variety of coding datasets of various size, including ChIP-seq data for the transcription factors NRSF and GABP. CONCLUSIONS CodingMotif provides a theoretically and empirically-demonstrated advance for the detection of motifs overrepresented in coding sequences. We expect CodingMotif to be useful for identifying motifs in functional genomic datasets such as DNA-protein binding, RNA-protein binding, or microRNA-RNA binding within coding regions. A software implementation is available at http://bioinformatics.bc.edu/chuanglab/codingmotif.tar.

Adverse events following pandemic (H1N1) 2009 vaccines ("2009 H1N1 vaccines") in Taiwan were passively reported to the National Adverse Drug Reaction Reporting System. To evaluate the completeness of spontaneous reporting, cases of death, Guillain-Barré syndrome (GBS), convulsion, Bell's palsy, and idiopathic thrombocytopenic purpura (ITP) after 2009 H1N1 vaccination that occurred between November 1, 2009 and August 31, 2010 were selected from the National Adverse Drug Reaction Reporting System (NADRRS) database and an additionally constructed nationwide large-linked database (LLDB), and matched on a unique personal identifier, date of vaccination (within ±7 days), and date of diagnosis (within ±7 days). Overall, matches occurred between the two data sources included 21 for death, 5 for GBS, 19 for convulsion, 22 for Bell's palsy, and 5 for ITP. The Chapman capture-recapture estimated spontaneous reporting completeness within 0-42 days of vaccination was 4% for death, 71% for GBS, 3% for convulsion, 9% for Bell's palsy, and 15% for ITP. For the interval ≥43 days after vaccination, reporting completeness was 0.1% for death, 14% for GBS, 0.1% for convulsion,<0.1% for Bell's palsy, and 0% for ITP. The estimated-to-expected ratio for Bell's palsy in the interval 0-42 days after vaccination was 1.48 (95% CI 1.11-1.98). Reporting completeness was higher for GBS than other adverse events after 2009 H1N1 vaccination. Linking the NADRRS to existing data sources in a capture-recapture analysis can be considered as an alternative to enhance Taiwan's postlicensure safety assessment of other routine vaccines. Nevertheless, the possibility of an increased risk for Bell's palsy detected by capture-recapture analyses needs further evaluation by controlled studies.

ABSTRACT: BACKGROUND: The evolution of gene expression is a challenging problem in evolutionary biology, for which accurate, well-calibrated measurements and methods are crucial. RESULTS: We quantified gene expression with whole-transcriptome sequencing in four diploid, prototrophic strains of Saccharomyces species grown under the same condition to investigate the evolution of gene expression. We found that variation in expression is gene-dependent with large variations in each gene's expression between replicates of the same species, confounding the identification of genes differentially expressed across species. To address this, we developed a statistical approach to establish significance bounds for inter-species differential expression in RNA-Seq data based on the variance measured across biological replicates. This metric estimates the combined effects of technical and environmental variance, as well as Poisson sampling noise by isolating each component. Despite a paucity of large expression changes, we found a strong correlation between the variance of gene expression change and species divergence (R2=0.90). CONCLUSION: We provide an improved methodology for measuring gene expression changes in evolutionary diverged species using RNA Seq, where experimental artifacts can mimic evolutionary effects.