Development of antiviral carbon quantum dots that target the Japanese encephalitis virus envelope protein.

Japanese encephalitis is a mosquito-borne disease caused by the Japanese encephalitis virus (JEV) that is prevalent in Asia and the Western Pacific. Currently, there is no effective treatment for Japanese encephalitis. Curcumin (Cur) is a compound extracted from the roots of Curcuma longa, and many studies have reported its antiviral and anti-inflammatory activities. However, the high cytotoxicity and very low solubility of Cur limit its biomedical applications. In this study, Cur carbon quantum dots (Cur-CQDs) were synthesized by mild pyrolysis-induced polymerization and carbonization, leading to higher water solubility and lower cytotoxicity, as well as superior antiviral activity against JEV infection. We found that Cur-CQDs effectively bound to the E protein of JEV, preventing viral entry into the host cells. In addition, after continued treatment of JEV with Cur-CQDs, a mutant strain of JEV was evolved that did not support binding of Cur-CQDs to the JEV envelope. Using transmission electron microscopy, biolayer interferometry, and molecular docking analysis, we revealed that the S123R and K312R mutations in the E protein play a key role in binding Cur-CQDs. The S123 and K312 residues are located in structural domains II and III of the E protein, respectively, and are responsible for binding to receptors on and fusing with the cell membrane. Taken together, our results suggest that the E protein of flaviviruses represents a potential target for the development of CQD-based inhibitors to prevent or treat viral infections.

Meis1, Hi1α, and GATA1 are integrated into a hierarchical regulatory network to mediate primitive erythropoiesis.

During development, erythroid cells are generated by two waves of hematopoiesis. In zebrafish, primitive erythropoiesis takes place in the intermediate cell mass region, and definitive erythropoiesis arises from the aorta-gonad mesonephros. TALE-homeoproteins Meis1 and Pbx1 function upstream of GATA1 to specify the erythroid lineage. Embryos lacking Meis1 or Pbx1 have weak gata1 expression and fail to produce primitive erythrocytes. Nevertheless, the underlying mechanism of how Meis1 and Pbx1 mediate gata1 transcription in erythrocytes remains unclear. Here we show that Hif1α acts downstream of Meis1 to mediate gata1 expression in zebrafish embryos. Inhibition of Meis1 expression resulted in suppression of hif1a expression and abrogated primitive erythropoiesis, while injection with in vitro-synthesized hif1α mRNA rescued gata1 transcription in Meis1 morphants and recovered their erythropoiesis. Ablation of Hif1α expression either by morpholino knockdown or Crispr-Cas9 knockout suppressed gata1 transcription and abrogated primitive erythropoiesis. Results of chromatin immunoprecipitation assays showed that Hif1α associates with hypoxia-response elements located in the 3'-flanking region of gata1 during development, suggesting that Hif1α regulates gata1 expression in vivo. Together, our results indicate that Meis1, Hif1α, and GATA1 indeed comprise a hierarchical regulatory network in which Hif1α acts downstream of Meis1 to activate gata1 transcription through direct interactions with its cis-acting elements in primitive erythrocytes.

Gene Ontology based housekeeping gene selection for RNA-seq normalization.

RNA-seq analysis provides a powerful tool for revealing relationships between gene expression level and biological function of proteins. In order to identify differentially expressed genes among various RNA-seq datasets obtained from different experimental designs, an appropriate normalization method for calibrating multiple experimental datasets is the first challenging problem. We propose a novel method to facilitate biologists in selecting a set of suitable housekeeping genes for inter-sample normalization. The approach is achieved by adopting user defined experimentally related keywords, GO annotations, GO term distance matrices, orthologous housekeeping gene candidates, and stability ranking of housekeeping genes. By identifying the most distanced GO terms from query keywords and selecting housekeeping gene candidates with low coefficients of variation among different spatio-temporal datasets, the proposed method can automatically enumerate a set of functionally irrelevant housekeeping genes for pratical normalization. Novel and benchmark testing RNA-seq datasets were applied to demostrate that different selections of housekeeping gene lead to strong impact on differential gene expression analysis, and compared results have shown that our proposed method outperformed other traditional approaches in terms of both sensitivity and specificity. The proposed mechanism of selecting appropriate houskeeping genes for inter-dataset normalization is robust and accurate for differential expression analyses.

UniQua: a universal signal processor for MS-based qualitative and quantitative proteomics applications.

Recent developments in high resolution mass spectrometry (HR-MS) technology have ushered proteomics into a new era. However, the importance of using a common, open data platform for signal processing of HR-MS spectra has not been sufficiently addressed. In this study, a MS signal processor was developed to facilitate data integration from different instruments and different proteomics approaches into a unified platform without compromising protein identification and quantitation performance. This processor supports parallel processing capability which allows full utilization of computing resources to speed up signal processing performance to >1 gigabytes/min. The storage space occupied by the processed MS data can be reduced to ~10%, which helps the analysis and management of large quantities of data from comprehensive proteomics studies. For quantitation at the MS level, processing accuracy is improved and processing time for ASAPRatio is reduced to ~50%. For quantitation at the MS/MS level, accurate reporter ion ratios from different instruments can be directly determined by the processed MS/MS spectra and reported in the Mascot search result directly without using specialized iTRAQ software.

Regulation of zebrafish CYP3A65 transcription by AHR2.

CYP3A proteins are the most abundant CYPs in the liver and intestines, and they play a pivotal role in drug metabolism. In mammals, CYP3A genes are induced by various xenobiotics through processes mediated by PXR. We previously identified zebrafish CYP3A65 as a CYP3A ortholog that is constitutively expressed in gastrointestinal tissues, and is upregulated by treatment with dexamethasone, rifampicin or tetrachlorodibenzo-p-dioxin (TCDD). However, the underlying mechanism of TCDD-mediated CYP3A65 transcription is unclear. Here we generated two transgenic zebrafish, Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP), which contain 2.1 and 5.4 kb 5' flanking sequences, respectively, of the CYP3A65 gene upstream of EGFP. Both transgenic lines express EGFP in larval gastrointestinal tissues in a pattern similar to that of the endogenous CYP3A65 gene. Moreover, EGFP expression can be significantly induced by TCDD exposure during the larval stage. In addition, EGFP expression can be stimulated by kynurenine, a putative AHR ligand produced during tryptophan metabolism. AHRE elements in the upstream regulatory region of the CYP3A65 gene are indispensible for basal and TCDD-induced transcription. Furthermore, the AHR2 DNA and ligand-binding domains are required to mediate effective CYP3A65 transcription. AHRE sequences are present in the promoters of many teleost CYP3 genes, but not of mammalian CYP3 genes, suggesting that AHR/AHR2-mediated transcription is likely a common regulatory mechanism for teleost CYP3 genes. It may also reflect the different environments that terrestrial and aquatic organisms encounter.

Distinctive microbial community and genome structure in coastal seawater from a human-made port and nearby offshore island in northern Taiwan facing the Northwestern Pacific Ocean.

Pollution in human-made fishing ports caused by petroleum from boats, dead fish, toxic chemicals, and effluent poses a challenge to the organisms in seawater. To decipher the impact of pollution on the microbiome, we collected surface water from a fishing port and a nearby offshore island in northern Taiwan facing the Northwestern Pacific Ocean. By employing 16S rRNA gene amplicon sequencing and whole-genome shotgun sequencing, we discovered that Rhodobacteraceae, Vibrionaceae, and Oceanospirillaceae emerged as the dominant species in the fishing port, where we found many genes harboring the functions of antibiotic resistance (ansamycin, nitroimidazole, and aminocoumarin), metal tolerance (copper, chromium, iron and multimetal), virulence factors (chemotaxis, flagella, T3SS1), carbohydrate metabolism (biofilm formation and remodeling of bacterial cell walls), nitrogen metabolism (denitrification, N2 fixation, and ammonium assimilation), and ABC transporters (phosphate, lipopolysaccharide, and branched-chain amino acids). The dominant bacteria at the nearby offshore island (Alteromonadaceae, Cryomorphaceae, Flavobacteriaceae, Litoricolaceae, and Rhodobacteraceae) were partly similar to those in the South China Sea and the East China Sea. Furthermore, we inferred that the microbial community network of the cooccurrence of dominant bacteria on the offshore island was connected to dominant bacteria in the fishing port by mutual exclusion. By examining the assembled microbial genomes collected from the coastal seawater of the fishing port, we revealed four genomic islands containing large gene-containing sequences, including phage integrase, DNA invertase, restriction enzyme, DNA gyrase inhibitor, and antitoxin HigA-1. In this study, we provided clues for the possibility of genomic islands as the units of horizontal transfer and as the tools of microbes for facilitating adaptation in a human-made port environment.

Comparative Transcriptome Analysis of Organ-Specific Adaptive Responses to Hypoxia Provides Insights to Human Diseases.

The common carp is a hypoxia-tolerant fish, and the understanding of its ability to live in low-oxygen environments has been applied to human health issues such as cancer and neuron degeneration. Here, we investigated differential gene expression changes during hypoxia in five common carp organs including the brain, the gill, the head kidney, the liver, and the intestine. Based on RNA sequencing, gene expression changes under hypoxic conditions were detected in over 1800 genes in common carp. The analysis of these genes further revealed that all five organs had high expression-specific properties. According to the results of the GO and KEGG, the pathways involved in the adaptation to hypoxia provided information on responses specific to each organ in low oxygen, such as glucose metabolism and energy usage, cholesterol synthesis, cell cycle, circadian rhythm, and dopamine activation. DisGeNET analysis showed that some human diseases such as cancer, diabetes, epilepsy, metabolism diseases, and social ability disorders were related to hypoxia-regulated genes. Our results suggested that common carp undergo various gene regulations in different organs under hypoxic conditions, and integrative bioinformatics may provide some potential targets for advancing disease research.

Computational methods for discovering gene networks from expression data.

Designing and conducting experiments are routine practices for modern biologists. The real challenge, especially in the post-genome era, usually comes not from acquiring data, but from subsequent activities such as data processing, analysis, knowledge generation and gaining insight into the research question of interest. The approach of inferring gene regulatory networks (GRNs) has been flourishing for many years, and new methods from mathematics, information science, engineering and social sciences have been applied. We review different kinds of computational methods biologists use to infer networks of varying levels of accuracy and complexity. The primary concern of biologists is how to translate the inferred network into hypotheses that can be tested with real-life experiments. Taking the biologists' viewpoint, we scrutinized several methods for predicting GRNs in mammalian cells, and more importantly show how the power of different knowledge databases of different types can be used to identify modules and subnetworks, thereby reducing complexity and facilitating the generation of testable hypotheses.

Characterization of a salt-tolerant xylanase from Thermoanaerobacterium saccharolyticum NTOU1.

A xylanase gene was PCR-cloned from Thermoanaerobacterium saccharolyticum and expressed in Escherichia coli. The xylanase (XynA) consisted of a signal peptide, glycoside hydrolase family 10 domains, carbohydrate-binding modules, and surface layer homology domains. It was optimally active at 70-73°C and at pH 5-7. It had enhanced activity with NaCl with optimal activity at 0.4 M but was tolerant up to 2 M NaCl. The thermostable and salt-tolerant properties of this xylanase suggest that it may be useful for industrial applications.

Enhanced Over-Representation Analysis for the Differential Regulation of Birc5a and HIF2α-Knockdown Approaches.

Hypoxia-inducible factors (HIFs) and survivin (Birc5) genes are often considered important cancer drug targets for molecularly targeted therapy, as both genes play important roles in the cellular differentiation and development of neuronal cells. Pathway enrichment analysis is predominantly applied when interpreting the correlated behaviors of activated gene clusters. Traditional enrichment analysis is evaluated via p-values only, regardless of gene expression fold-change levels, gene locations, and possible hidden interactions within a pathway. Here, we combined these factors to retrieve significant pathways, as compared with traditional approaches. We performed RNA-seq analyses on Birc5a and HIF2α knocked down in zebrafish during the embryogenesis stage. Regarding Birc5a, two additional biological pathways, sphingolipid metabolism and herpes simplex infection, were identified; whereas for HIF2α, four biological pathways were re-identified, including ribosome biogenesis in eukaryotes, proteasome, purine metabolism, and complement and coagulation cascades. Our proposed approaches identified additional significant pathways directly related to cell differentiation or cancer, also providing comprehensive mechanisms for designing further biological experiments.

REMUS: a tool for identification of unique peptide segments as epitopes.

We provide a 'R(E)MUS' (reinforced merging techniques for unique peptide segments) web server for identification of the locations and compositions of unique peptide segments from a set of protein family sequences. Different levels of uniqueness are determined according to substitutional relationship in the amino acids, frequency of appearance and biological properties such as priority for serving as candidates for epitopes where antibodies recognize. R(E)MUS also provides interactive visualization of 3D structures for allocation and comparison of the identified unique peptide segments. Accuracy of the algorithm was found to be 70% in terms of mapping a unique peptide segment as an epitope. The R(E)MUS web server is available at http://biotools.cs.ntou.edu.tw/REMUS and the PC version software can be freely downloaded either at http://bioinfo.life.nthu.edu.tw/REMUS or http://spider.cs.ntou.edu.tw/BioTools/REMUS. User guide and working examples for PC version are available at http://spider.cs.ntou.edu.tw/BioTools/REMUS-DOCS.html, and details of the proposed algorithm can be referred to the documents as described previously [H. T. Chang, T. W. Pai, T. C. Fan, B. H. Su, P. C. Wu, C. Y. Tang, C. T. Chang, S. H. Liu and M. D. T. Chang (2006) BMC Bioinformatics, 7, 38 and T. W. Pai, B. H. Su, P. C. Wu, M. D. T. Chang, H. T. Chang, T. C. Fan and S. H. Liu (2006) J. Bioinform. Comput. Biol., 4, 75-92].

Differences in substrate specificity of V. cholerae FabH enzymes suggest new approaches for the development of novel antibiotics and biofuels.

Vibrio cholerae, the causative pathogen of the life-threatening infection cholera, encodes two copies of ß-ketoacyl-acyl carrier protein synthase III (vcFabH1 and vcFabH2). vcFabH1 and vcFabH2 are pathogenic proteins associated with fatty acid synthesis, lipid metabolism, and potential applications in biofuel production. Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl-CoA and CoA thioesters with short acyl chains, similar to that observed for FabH homologs found in most gram-negative bacteria. vcFabH2 prefers medium chain-length acyl-CoA thioesters, particularly octanoyl-CoA, which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH1 and six vcFabH2 structures determined in either apo form or in complex with acetyl-CoA/octanoyl-CoA indicate that the substrate-binding pockets of vcFabH1 and vcFabH2 are of different sizes, accounting for variations in substrate chain-length specificity. An unusual and unique feature of vcFabH2 is its C-terminal fragment that interacts with both the substrate-entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against V. cholerae. Additionally, the distinctive substrate preference of FabH2 in V. cholerae and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production.

Functional enrichment analysis based on long noncoding RNA associations.

BACKGROUND: Differential gene expression analysis using RNA-seq data is a popular approach for discovering specific regulation mechanisms under certain environmental settings. Both gene ontology (GO) and KEGG pathway enrichment analysis are major processes for investigating gene groups that participate in common biological responses or possess related functions. However, traditional approaches based on differentially expressed genes only detect a few significant GO terms and pathways, which are frequently insufficient to explain all-inclusive gene regulation mechanisms. METHODS: Transcriptomes of survivin (birc5) gene knock-down experimental and wild-type control zebrafish embryos were sequenced and assembled, and a differential expression (DE) gene list was obtained for traditional functional enrichment analysis. In addition to including DE genes with significant fold-change levels, we considered additional associated genes near or overlapped with differentially expressed long noncoding RNAs (DE lncRNAs), which may directly or indirectly activate or inhibit target genes and play important roles in regulation networks. Both the original DE gene list and the additional DE lncRNA-associated genes were combined to perform a comprehensive overrepresentation analysis. RESULTS: In this study, a total of 638 DE genes and 616 DE lncRNA-associated genes (lncGenes) were leveraged simultaneously in searching for significant GO terms and KEGG pathways. Compared to the traditional approach of only using a differential expression gene list, the proposed method of employing DE lncRNA-associated genes identified several additional important GO terms and KEGG pathways. In GO enrichment analysis, 60% more GO terms were obtained, and several neuron development functional terms were retrieved as complete annotations. We also observed that additional important pathways such as the FoxO and MAPK signaling pathways were retrieved, which were shown in previous reports to play important roles in apoptosis and neuron development functions regulated by the survivin gene. CONCLUSIONS: We demonstrated that incorporating genes near or overlapped with DE lncRNAs into the DE gene list outperformed the traditional enrichment analysis method for effective biological functional interpretations. These hidden interactions between lncRNAs and target genes could facilitate more comprehensive analyses.

Differential evolutionary conservation of motif modes in the yeast protein interaction network.

BACKGROUND: The importance of a network motif (a recurring interconnected pattern of special topology which is over-represented in a biological network) lies in its position in the hierarchy between the protein molecule and the module in a protein-protein interaction network. Until now, however, the methods available have greatly restricted the scope of research. While they have focused on the analysis in the resolution of a motif topology, they have not been able to distinguish particular motifs of the same topology in a protein-protein interaction network. RESULTS: We have been able to assign the molecular function annotations of Gene Ontology to each protein in the protein-protein interactions of Saccharomyces cerevisiae. For various motif topologies, we have developed an algorithm, enabling us to unveil one million "motif modes", each of which features a unique topological combination of molecular functions. To our surprise, the conservation ratio, i.e., the extent of the evolutionary constraints upon the motif modes of the same motif topology, varies significantly, clearly indicative of distinct differences in the evolutionary constraints upon motifs of the same motif topology. Equally important, for all motif modes, we have found a power-law distribution of the motif counts on each motif mode. We postulate that motif modes may very well represent the evolutionary-conserved topological units of a protein interaction network. CONCLUSION: For the first time, the motifs of a protein interaction network have been investigated beyond the scope of motif topology. The motif modes determined in this study have not only enabled us to differentiate among different evolutionary constraints on motifs of the same topology but have also opened up new avenues through which protein interaction networks can be analyzed.

Ultrastrong trapping of VEGF by graphene oxide: Anti-angiogenesis application.

Angiogenesis is the process of formation of new blood vessels, which is essential to human biology, and also plays a crucial role in several pathologies such as tumor growth and metastasis, exudative age-related macular degeneration, and ischemia. Vascular endothelial growth factor (VEGF), in particular, VEGF-A165 is the most important pro-angiogenic factor for angiogenesis. Thus, blocking the interaction between VEGFs and their receptors is considered an effective anti-angiogenic strategy. We demonstrate for that first time that bovine serum albumin-capped graphene oxide (BSA-GO) exhibits high stability in physiological saline solution and possesses ultrastrong binding affinity towards VEGF-A165 [dissociation constant (Kd) ∼3 × 10-12 M], which is at least five orders of magnitude stronger than that of high-abundant plasma proteins such as human serum albumin, fibrinogen, transferrin, and immunoglobulin G. Due to the surprising binding specificity of BSA-GO for VEGF-A165 in complex plasma fluid, we have also studied the anti-angiogenic effects in vitro and in vivo. Results show that BSA-GO not only effectively inhibits the proliferation, migration and tube formation of human umbilical vein endothelial cells, but also strongly disturbs the physiological process of angiogenesis in chick chorioallantoic membrane and blocks VEGF-A165-induced blood vessel formation in rabbit corneal neovascularization. Our findings indicate that GO nanomaterials can potentially act as therapeutic anti-angiogenic agents via ultrastrong VEGF adsorption and its activity suppression.

Analysis of progressively overexpressed genes in tumorigenesis of colorectal cancers using cDNA microarray.

The identification of differentially expressed genes has important implications in understanding the biology of colorectal tumorigenesis and progression, as well as developing new diagnostic and therapeutic strategies. In this study, cDNA microarray technology was used to identify colorectal tumor-related functional genes, which are overexpressed continuously from colorectal adenoma to adenocarcinoma. A set of 23 genes with progressive overexpression in the development of colorectal cancer (CRC) was identified by cDNA microarray, then analyzed by sequencing and Northern blot analysis. Validation of our array results was simultaneously performed by exploring the SAGEmap database. Furthermore, the gradually over-expressed genes from adenoma to adenocarcinoma were validated by Northern blot analysis with additional samples from three patients with synchronous colorectal adenocarcinoma and adenoma and four patients with CRC. Of these 23 genes, one was a function-unknown gene, designated as Homo sapiens chromosome 21q22.1 anonymous mRNA sequence (Genbank accession no. AF003738), and 22 were function-known genes. Searching through the Gene Ontology Browser at the Cancer Genome Analysis Project website revealed that the biological roles of these 22 function-known genes are involved in cell motility, cell adhesion, chemokine activity, signal transduction, cytoskeleton organization, proteolysis, apoptosis, and cell proliferation. The genes identified in the present study offer valuable information on colorectal carcinogenesis and metastasis, and represent a potential source of novel targets for new strategies for CRC diagnosis and therapy.

Stochastic simulation of notch signaling reveals novel factors that mediate the differentiation of neural stem cells.

Notch signaling controls cell fate decisions and regulates multiple biological processes, such as cell proliferation, differentiation, and apoptosis. Computational modeling of the deterministic simulation of Notch signaling has provided important insight into the possible molecular mechanisms that underlie the switch from the undifferentiated stem cell to the differentiated cell. Here, we constructed a stochastic model of a Notch signaling model containing Hes1, Notch1, RBP-Jk, Mash1, Hes6, and Delta. mRNA and protein were represented as a discrete state, and 334 reactions were employed for each biochemical reaction using a graphics processing unit-accelerated Gillespie scheme. We employed the tuning of 40 molecular mechanisms and revealed several potential mediators capable of enabling the switch from cell stemness to differentiation. These effective mediators encompass different aspects of cellular regulations, including the nuclear transport of Hes1, the degradation of mRNA (Hes1 and Notch1) and protein (Notch1), the association between RBP-Jk and Notch intracellular domain (NICD), and the cleavage efficiency of the NICD. These mechanisms overlap with many modifiers that have only recently been discovered to modulate the Notch signaling output, including microRNA action, ubiquitin-mediated proteolysis, and the competitive binding of the RBP-Jk-DNA complex. Moreover, we identified the degradation of Hes1 mRNA and nuclear transport of Hes1 as the dominant mechanisms that were capable of abolishing the cell state transition induced by other molecular mechanisms.

Identifying significant associations of orthologous simple sequence repeats with gene ontologies.

Simple Sequence Repeats (SSRs), also known as microsatellites, regulate gene functions. SSR mutations in a disease gene may cause various genetic disorders. To identify putative functional SSRs, a web-based system, Gene Ontology SSR Hierarchy (GOSH), was developed to facilitate discovery of significant associations between SSRs and Gene Ontology (GO) terms. Using the GO hierarchy term structure, GOSH assists users with selecting functional or biological gene subsets. Significant SSR patterns are retrieved and identified via comprehensive overrepresentation analysis within a target gene subset and by comparing results with orthologous genes. Pattern relationships between different biological subsets or supersets can be observed by using the GO hierarchy structure directly. GOSH also supports GO searching through identified significant SSR patterns and all GO terms possessing such patterns are listed for consultation. GOSH is the first comprehensive and efficient online mining tool for discovering significant orthologous SSR patterns in GO terms and is available at http://gosh.cs.ntou.edu.tw/.

Hypoxia-inducible factor 2 alpha is essential for hepatic outgrowth and functions via the regulation of leg1 transcription in the zebrafish embryo.

The liver plays a vital role in metabolism, detoxification, digestion, and the maintenance of homeostasis. During development, the vertebrate embryonic liver undergoes a series of morphogenic processes known as hepatogenesis. Hepatogenesis can be separated into three interrelated processes: endoderm specification, hepatoblast differentiation, and hepatic outgrowth. Throughout this process, signaling molecules and transcription factors initiate and regulate the coordination of cell proliferation, apoptosis, differentiation, intercellular adhesion, and cell migration. Hifs are already recognized to be essential in embryonic development, but their role in hepatogenesis remains unknown. Using the zebrafish embryo as a model organism, we report that the lack of Hif2-alpha but not Hif1-alpha blocks hepatic outgrowth. While Hif2-alpha is not involved in hepatoblast specification, this transcription factor regulates hepatocyte cell proliferation during hepatic outgrowth. Furthermore, we demonstrated that the lack of Hif2-alpha can reduce the expression of liver-enriched gene 1 (leg1), which encodes a secretory protein essential for hepatic outgrowth. Additionally, exogenous mRNA expression of leg1 can rescue the small liver phenotype of hif2-alpha morphants. We also showed that Hif2-alpha directly binds to the promoter region of leg1 to control leg1 expression. Interestingly, we discovered overrepresented, high-density Hif-binding sites in the potential upstream regulatory sequences of leg1 in teleosts but not in terrestrial mammals. We concluded that hif2-alpha is a key factor required for hepatic outgrowth and regulates leg1 expression in zebrafish embryos. We also proposed that the hif2-alpha-leg1 axis in liver development may have resulted from the adaptation of teleosts to their environment.

Molecular evolution of multiple-level control of heme biosynthesis pathway in animal kingdom.

Adaptation of enzymes in a metabolic pathway can occur not only through changes in amino acid sequences but also through variations in transcriptional activation, mRNA splicing and mRNA translation. The heme biosynthesis pathway, a linear pathway comprised of eight consecutive enzymes in animals, provides researchers with ample information for multiple types of evolutionary analyses performed with respect to the position of each enzyme in the pathway. Through bioinformatics analysis, we found that the protein-coding sequences of all enzymes in this pathway are under strong purifying selection, from cnidarians to mammals. However, loose evolutionary constraints are observed for enzymes in which self-catalysis occurs. Through comparative genomics, we found that in animals, the first intron of the enzyme-encoding genes has been co-opted for transcriptional activation of the genes in this pathway. Organisms sense the cellular content of iron, and through iron-responsive elements in the 5' untranslated regions of mRNAs and the intron-exon boundary regions of pathway genes, translational inhibition and exon choice in enzymes may be enabled, respectively. Pathway product (heme)-mediated negative feedback control can affect the transport of pathway enzymes into the mitochondria as well as the ubiquitin-mediated stability of enzymes. Remarkably, the positions of these controls on pathway activity are not ubiquitous but are biased towards the enzymes in the upstream portion of the pathway. We revealed that multiple-level controls on the activity of the heme biosynthesis pathway depend on the linear depth of the enzymes in the pathway, indicating a new strategy for discovering the molecular constraints that shape the evolution of a metabolic pathway.