Interaction of HIF and USF signaling pathways at human genes flanked by hypoxia-response elements and e-box palindromes

Deprivation of oxygen is a main characteristic of solid human tumors. It was discovered in the 90’s that a novel transcription factor termed Hypoxia Inducible Factor 1 (HIF-1), α subunit (HIF-1α) gradually accumulated during markedly reduced oxygen partial pressures (pO2), a state otherwise known as hypoxia. So far, the heterodimeric HIF-1 α/β- complex has been implicated in targeting more than 70 genes involved in cell metabolism, cell cycle and proliferation, apoptosis and angiogenesis. As a consequence, HIF-1 is instrumental in promoting tumor growth and survival in most experimental models. To date, control of the HIF pathway via the O2-requiring hydroxylations of specific alpha-subunit proline and asparagine residues, leading to the factors’ ubiquitylation and proteasome catalyzed degradation and suppressing its interaction with vital co-activator proteins in (re)oxygenated cells, respectively, is relatively well understood. In contrast, much remains to be discovered in regard to critical oxygen-independent controls that also impinge on HIF-1 signaling in tumor cells. A previous study had documented the binding of HIF-1 to two of three hypoxia response cis-elements (HREs) within the promoter of the globin-2 gene of Daphnia magna (phb2), as a strict requirement for the reporter’s maximal hypoxic activation in transfected hepatoma (Hep3B) cancer cells. However, binding of an unknown and constitutive transcription factor to the third phb2 motif, a CACGTG E-box palindrome, in human hepatoma cells (Hep3B), significantly weakened the HIF/HRE-mediated hypoxic induction. This observation suggested that CACGTG-complexes might function to fine-tune or inhibit HIF-driven gene responses in cancer cells and prompted a two-fold objective for the present work: a) was to identify this CACGTG-binding transcription factor in different human cancer cells, and b) to study the co-regulation of human genes through HIF-1 and this CACGTG- mediated signaling pathway in vitro and in vivo. As a first step of this PhD thesis, gel supershift and oligonucleotide pull-down assays were used and consistently and reproducibly identified the basic-helix-loop-helix/leucine zipper (bHLH/ZIP) upstream stimulatory factors 1 and 2 (USF1 and 2) as the major phb2 CACGTG binding factors in human hepatoma cells (Hep3B), human cervical carcinoma cells (HeLa) and human breast carcinoma cells (MCF7). Next, a genome-wide computational scan for human HRE/E-box promoters, i.e. those containing the CACGTG palindrome adjacent to or overlapping with a HRE, was carried out and retrieved with lactate dehydrogenase A (LDHA), Bcl2/adenovirus E1B 19kD-interacting protein 3 (BNIP3), 4E-binding protein 1 (4EBP1) and vascular endothelial growth factor C (VEGFC) etc. as known hypoxia targets. Having verified the human-mouse-rat conservation of this HRE/E-box constellation with regard to the following selected HRE/E-box genes, we either received as generous gifts (BNIP3) or generated ourselves (LDHA, 4EBP1 and melanocortin 1 receptor MC1R) HRE/E- box promoter-luciferase reporter plasmids, along with HIF-1 (i.e. prolyl hydroxylase domain 2 PHD2) or USF-specific (i.e. tyrosinase TYR) reporter controls, to systematically investigate the interaction of HIF-1 and USF pathways at DNA level. Of these four HRE/E-box candidate genes only LDHA and BNIP3 reporter revealed a prominent hypoxia-mediated up-regulation in Hep3B, HeLa and MCF7 cells. Towards the co-regulation of LDHA and BNIP3 promoters by HIF-1 and USFs, co-overexpression of HIF-1α and USF plasmids revealed a significant attenuation of the HIF-dependent hypoxic up-regulation of the BNIP3 luciferase reporter by exogenous USF1 and 2a in Hep3B and HeLa cells. Similarly, the endogenous HIF-dependent hypoxic induction of LDHA was significantly reduced by over-expressed USF1 and 2a in MCF7 cells. To further evaluate the specificity of the expression manipulation of either HIF or USF on this regulation of LDHA reporter, a stable USF2a knockdown MCF7 clone was generated by short-hairpin RNA (shRNA) technology while a stable HIF-1α knockdown MCF7 clone was kindly provided to us by Dr. D. Stiehl (group of Prof. R. Wenger, University Zurich). Luciferase assay in these stable MCF7 knockdown clones revealed a reduction of LDHA promoter activity upon silencing of USF2a transcription cells and an independent transactivation of LDHA promoter by HIF-1 and USF cascades. We also confirmed the competitive effect on the BNIP3 regulation by HIF-1α and USFs signaling in Hep3B cells by transiently silencing HIF-1α, USF1 or USF2a expression through specific siRNAs. The following luciferase assay revealed for the USF1 knockdown a significantly increased hypoxic activity of the BNIP3 promoter-reporter. In vivo binding of HIF and USF within the promoters of LDHA and BNIP3 was shown by chromatin immunoprecipitation (ChIP) assays for Hep3B, HeLa and MCF7 cells. Regarding the BNIP3 promoter, the ChIP assay revealed chief occupancy in deoxygenated cells by HIF-1 along with the weak and constitutive attachment of USF1 and 2a to this DNA. Remarkably, some HIF-1α had successfully escaped proteolytic degradation since we detected it bound, as heterodimer, to the BNIP3 promoter even in normoxic cells. The native LDHA promoter (region: -2533/-2376 from translation start site) was dominated by bound HIF-1 in hypoxic MCF7 cells, while USF1 and USF2a co-occupied this regulatory DNA during periods of normoxia (Hep3B, MCF7). The latter finding implied upstream stimulatory factors as physiological drivers of aerobic glycolysis in cancer cells (Warburg effect). We also noted that during hypoxia, both USFs maintained their association with this DNA, which offers additional support of the notion that maximal LDHA transcription during low pO2 arises from the cooperative transcriptional control by HIF-1 and USF1/2a. Additional gel supershift assays mapped the exact in vitro HIF-1 and USF binding sites within the promoter of LDHA to the CACGTG motif (-2465/-2460; HIF-1 and USFs), the CACGTG palindrome at -2367/-2362 (USFs only) and the GACGTG HRE at -2353/-2345 (HIF-1 only). Thus, distinct binding sites in the LDHA promoter might facilitate the independent transactivation of the gene by both signaling pathways. In contrast, HIF-1 and USFs were found to have either identical or overlapping binding locations within the DNA surrounding the -251/-246 HRE in the BNIP3 promoter, again in line with a mutually displacing, competitive mode of interaction at DNA level. Currently, I am extending this study to human melanoma cells, since in these cells HIF-1 and USF pathways are induced by physiological stimuli, i.e. hypoxia and tanning- response conferring ultraviolet radiation, respectively. This will allow to examine the response of co-regulated BNIP3 and LDHA promoters in melanoma cells that have been subjected to hypoxia plus UV dual stimulation. Eine negative Bilanz zwischen der Sauerstoffversorgung und dem Sauerstoffverbrauch ist ein klinisch signifikantes Charakteristikum der meisten bösartigen Tumore. Die resultierende O2-Mangelsituation (Hypoxie), die sich lokal im Tumorgewebe ausbilden kann, wird von den Zellen primär durch einen Transkriptionsfaktor registriert. Dieser Hypoxie- induzierbare Faktor 1 (HIF-1) wird bei reduziertem Sauerstoffpartialdruck (pO2) aktiviert und kann DNA in Form eines, aus einer α und β Untereinheit bestehenden, heterodimeren Komplexes binden. Erwiesenermassen kontrolliert HIF-1 in hypoxischen Zellen die Transkription von mehr als 70 Genen. Das veränderte Abschreiben dieser Zielgene zieht zelluläre Anpassungen innerhalb des Stoffwechsels, Zellzyklus, Apoptose und Angiogenese nach sich. In den meisten Tumormodellen fungiert ein aktiver HIF Signalweg daher als Protagonist von zellulärem Wachstum, Hypoxietoleranz, Metastaseneigung und Therapieresistenz. Dank jüngerer Arbeiten ist die O2-abhängige Kontrolle von HIF-1 und dem nahe verwandten HIF-2 Komplex relativ gut verstanden. Von zentraler Bedeutung für diese Kontrolle sind enzymatische Hydroxylierungen einzelner Proline und Asparagine in den HIF- 1α und HIF-2α Untereinheiten. Als Folge dieser Signale werden bei einem pO2 Anstieg beide HIF-α Faktoren nicht nur transkriptionell inaktiviert, sondern auch ubiquitinyliert und proteolytisch abgebaut. Diese O2-verbrauchenden post-translationalen Hydroxylierungen von HIF-1α/-2α bedingen, dass in normoxischen oder re-oxygenierenden Zellen der HIF Signalweg sehr schnell zum Erliegen kommt. Für eine effektivere Bekämpfung von HIF in einem chronisch desoxygenierten Milieu, müssen jedoch auch die Hydroxylierungs- unabhängigen Kontrollen dieses Signalwegs noch eingehender erforscht und therapeutisch einbezogen werden. Eine frühere Arbeit befasste sich mit der hypoxisch-induzierbaren Expression von Globingenen, insbesondere des hb2 Gens, in dem planktonischen Süsswasserkrebs („Wasserfloh“) Daphnia magna. Im Rahmen sogenannter heterologer Transfektionen von humanen Hepatomazellen (Hep3B Zellen) wurde festgestellt, dass die kooperative Bindung von HIF-1 an zwei von drei funktionellen HIF-1 Bindungsstellen (hypoxia-response element, HRE) im Promoter von hb2 (phb2) für die maximale hypoxische Induktion eines vom Promoter abgeleiteten Luziferase (LUZ) Reportergen Plasmids unbedingt erforderlich war. Ein O2-unabhängiger (konstitutiver) Faktor hingegen interagierte mit der dritten Bindungsstelle, einem CACGTG Palindrom, im phb2/LUZ Konstrukt. Nach seiner Bindung an das Palindrom war der CACGTG Komplex in der Lage die HIF/HRE-vermittelte Aktivierung des Daphnia phb2 Reportergens deutlich zu schwächen. Möglicherweise liegt die Funktion dieses CACGTG-Komplexes in Hep3B Zellen in der Feinregulierung der durch HIF-1 gesteuerten Genaktivität. Ziel der vorliegenden Arbeit war es daher diesen unbekannten Bindungskomplex in verschiedenen Krebszelllinien zu identifizieren und die Regulation humaner Gene als Folge der Wechselwirkung von HRE- und CACGTG- Komplexen zu untersuchen. Zur Identifikation des unbekannten Faktors kamen electrophoretic mobility shift assays (EMSA) und Oligonukleotid Pull-down Techniken zum Einsatz. Upstream Stimulatory Faktor 1 und 2 (USF1, USF2), ihrerseits Mitglieder der bHLH-leucine zipper (bHLH/ZIP) Transkriptionsfaktorfamilie, konnten als die primären, phb2-CACGTG bindende Faktoren in menschlichen Hepatoma- (Hep3B), Zervikalkarzinom- (HeLa) und Brustkarzinomzellen (MCF7) identifiziert werden. Zur weiteren Übertragbarkeit dieser Befunde auf menschliche Tumorzellen, wurde ein bioinformatischer Screen des humanen Genoms durchgeführt, um sogenannte HRE/E-box Genkandidaten zu erfassen. Humane HRE/E-box Gene tragen, phb2- ähnlich, CACGTG-Palindrome und HIF-bindende HREs unmittelbar benachbart in ihrem Promoter. Mehrere bekannte Hypoxie Zielgene waren unter den HRE/E-box Kandidaten, u.a.: Laktat Dehydrogenase A (LDHA), 4E-bindendes Protein 1 (4EBP1) und der vaskuläre endotheliale Wachstumsfaktor C (VEGFC). Die jeweiligen Palindrom und HRE Motive sind in den Promotoren der homologen Mensch-, Maus- und Rattengene sämtlich konserviert und damit von wahrscheinlicher Funktionalität. Nach erfolgter Klonierung dieser HRE/E-box Promotoren als LUZ Plasmide (i. LDHA; ii. 4EBP1; iii. Bcl2/adenovirus E1B 19kD- interagierendes Protein 3, BNIP3; iv. Melanocortin 1 Rezeptor, MC1R), untersuchte eine erste Serie von Reportergen Transfektionen die Hypoxie-vermittelte Induktion sämtlicher HRE/E- box Konstrukte in Hep3B, HeLa und MCF7 Zellen. Niedrige Sauerstoffkonzentrationen (1% O2/16h) aktivierten von den getesteten Plasmiden lediglich die LDHA und BNIP3 Reporter in allen drei Zelllinien. Bezüglich einer HIF/USF-Ko-Regulation von LDHA- und BNIP3- Promotoren, wurden HIF-1α, USF1 und 2a in Hep3B, HeLa und MCF7 Zellen überexprimiert. Diese Ko-Transfektionen zeigten, dass die HIF-1α-abhängige hypoxische Erhöhung der BNIP3-Promotor-Aktivität signifikant durch exogenes USF1 und USF2a, nicht aber durch USF2b, abgeschwächt wurde. Ferner unterdrückte die Überexpression USF1/2a in MCF7 Zellen die endogene hypoxische Induktion von LDHA. Um die spezifischen Einflüsse von HIF-1 oder USF1/2a auf ein HRE/E-box Konstrukt unabhängig von Überexpressionen zu prüfen, wurden stabile USF2a und HIF-1α shRNA knockdown (kd) Klone in MCF7 Zellen generiert. Der MCF7 HIF-1α kd Klon wurde uns freundlicherweise durch Dr. D. Stiehl (Gruppe Prof. Wenger, Univ. Zürich) überlassen. Die folgenden Luziferase-Versuche zeigten, dass die Aktivität des LDHA-Promoters in kd USF2a Klonen unter Normoxie reduziert war und dass die HIF-1 und USF Kaskaden im LDHA- Promoter größtenteils unabhängig voneinander agierten. Umgekehrt bestätigten Versuchsreihen mit transientem (siRNA basierenden) knockdown von HIF-1α, USF1 und USF2a in Hep3B Zellen die Kompetition beider Signalwege auf Ebene des BNIP3-Promoters. Die hypoxische Aktivität des BNIP3 Reportergens war signifikant in Hep3B Zellen erhöht, die mit siRNAs gegen USF1, nicht aber gegen USF2a, transfiziert wurden. Um sicher zu stellen, dass die HIF-1 und USF1/2a Signalwege auch unter physiologischen Bedingungen auf die Bindungsstellen im LDHA- und BNIP3-Promotor konvergieren, wurden Chromatin Immunopräzipitation- (ChIP) und EMSA-Experimente durchgeführt. Diese Arbeiten zeigten für BNIP3, dass USF1 und 2a schwach und konstitutiv an dieselbe Stelle, die schon als funktionelle HRE erkannt wurde, binden können. Interaktion von HIF-1 mit der BNIP3 HRE war der dominierende Einfluss in hypoxischen Zellen. Allerdings wurde HIF-1, gebunden am BNIP3 Promoter, auch in normoxischen Kernen detektiert. Ferner binden HIF-1, USF1 und USF2a unter physiologischen Bedingungen an den LDHA Promoter (ChIP-Daten). Während die regulatorischen LDHA Elemente unter hohem pO2 vornehmlich durch USF1/2a besetzt waren, überwog der HIF-1 Besatz in hypoxischen Zellen (MCF7). Die USF Faktoren behielten allerdings ihre Bindung am LDHA Promoter auch unter Hypoxie bei, was gegen eine Verdrängung durch HIF-1 spricht und einen weiteren Nachweis für die unabhängige HIF/USF Bindung an die LDHA Motive liefert. Die EMSA- Versuche zeigten, dass HIF-1 und USF1/2a vornehmlich an ihre eigenen cis-Elemente im LDHA Promoter binden, d.h. hier, relativ zum BNIP3 Gen, nur eine schwache Konkurrenz zwischen HIF/HRE und USF/Palindrom Komplexen vorherrscht. Allem Anschein nach korreliert die Präsenz individueller Bindungsstellen mit der größtenteils unabhängigen Konvergenz von HIF-1 und USF Signalwegen auf das LDHA Gen. Demgegenüber resultiert aus der Konkurrenz von HIF-1 und USFs um eine gemeinsame Bindungssequenz im BNIP3 Promoter die dargestellte negative Wechselwirkung beider Signalwege auf Genebene. In fortlaufenden Experimenten in humanen Melanoma Zellen wird zurzeit die Ko- Regulation der LDHA- und BNIP3-Expression durch HIF-1 und USFs durch strikt physiologische Stimuli (Hypoxie HIF-1; UV USFs) erforscht

The Social Influence Enhanced by the Mass Media Broadcasting in Innovation Diffusion

Based on the Bass model and the micro-model of mass media network, we propose an agent-based threshold approach including the mutual interaction between social relationship network and mass media network. Taking the heterogeneity of individuals into account, it is found that the direct advertisement from mass media broadcasting will attract the potential adopters greatly at the beginning of innovation diffusion. In the middle of diffusion process, the previous adopters formalize a positive feedback to the potential adopters via mass media broadcasting. The social collective effect can be strongly enhanced if the potential adopter prefer to mass media broadcasting. Furthermore, it is found that the complexity of the social relationship network may postpone the social collective effect

Morganella morganii, a non-negligent opportunistic pathogen

AbstractMorganella morganii belongs to the tribe Proteeae of the Enterobacteriaceae family. This species is considered as an unusual opportunistic pathogen that mainly causes post-operative wound and urinary tract infections. However, certain clinical M. morganii isolates present resistance to multiple antibiotics by carrying various resistant genes (such as blaNDM-1, and qnrD1), thereby posing a serious challenge for clinical infection control. Moreover, virulence evolution makes M. morganii an important pathogen. Accumulated data have demonstrated that M. morganii can cause various infections, such as sepsis, abscess, purple urine bag syndrome, chorioamnionitis, and cellulitis. This bacterium often results in a high mortality rate in patients with some infections. M. morganii is considered as a non-negligent opportunistic pathogen because of the increased levels of resistance and virulence. In this review, we summarized the epidemiology of M. morganii, particularly on its resistance profile and resistant genes, as well as the disease spectrum and risk factors for its infection

Photodynamic Therapy Combined with Terbinafine Against Chromoblastomycosis and the Effect of PDT on Fonsecaea monophora In Vitro

Chromoblastomycosis, a chronic fungal infection of skin and subcutaneous tissue caused by dematiaceous fungi, is associated with low cure and high relapse rates. Among all factors affecting clinical outcome, etiological agents have an important position. In southern China, Fonsecaea pedrosoi and Fonsecaea monophora are main causative agents causing Chromoblastomycosis. We treated one case of chromoblastomycosis by photodynamic therapy (PDT) of 5-aminolevulinic acid (ALA) irradiation combined with terbinafine 250 mg a day. The lesions were improved after two sessions of ALA-PDT treatment, each including nine times, at an interval of 1 week, combined with terbinafine 250 mg/day oral, and clinical improvement could be observed. In the following study, based on the clinical treatment, the effect of PDT and antifungal drugs on this isolate was detected in vitro. It showed sensitivity to terbinafine, itraconazole or voriconazole, and PDT inhibited the growth. Both the clinic and experiments in vitro confirm the good outcome of ALA-PDT applied in the inhibition of F. monophora. It demonstrated that combination of antifungal drugs with ALA-PDT arises as a promising alternative method for the treatment of these refractory cases of chromoblastomycosis.National Natural Science Foundation (China) (81371747

Beneficial Evaluation of Residential Dual Water Supply System

The scientific development and utilization of regional unconventional water resources and the implementation of high-quality water supply at the county level is beneficial both in the water resources allocation and the water shortage alleviation. It is also important in improving the human society as well as protecting the ecological environment, which shows significant economic, social and environmental benefits. The beneficial evaluation analysis of the development and utilization of regional unconventional water resources and the potential of dual water supply are conducted in this study. The reasonable beneficial evaluation of the utilization of unconventional water resources and regional dual water supply system are of great significance to the regional effective water sources allocation and water supply method

Differential Characteristics of Viral siRNAs between Leaves and Roots of Wheat Plants Naturally Infected with Wheat Yellow Mosaic Virus, a Soil-Borne Virus

RNA silencing is an important innate antiviral defense in plants. Soil-borne plant viruses naturally infect roots via soil-inhabiting vectors, but it is unclear how antiviral RNA silencing responds to virus infection in this particular tissue. In this study, viral small interfering RNA (siRNA) profiles from leaves and roots of wheat plants naturally infected with a soil-borne virus, wheat yellow mosaic virus (WYMV, genus Bymovirus), were analyzed by deep sequencing. WYMV siRNAs were much more abundant in roots than leaves, which was positively correlated with the accumulation of viral RNA. WYMV siRNAs in leaves and roots were predominantly 21- and 22-nt long and equally derived from the positive- and negative-strands of the viral genome. WYMV siRNAs from leaves and roots differed in distribution pattern along the viral genome. Interestingly, compared to siRNAs from leaves (and most other reports), those from roots obviously had a lower A/U bias at the 5′-terminal nucleotide. Moreover, the expression of Dicer-like genes upon WYMV infection were differently regulated between leaves and roots. Our data suggest that RNA silencing in roots may operate differently than in leaves against soil-borne virus invasion

Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity

BACKGROUND: Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome. RESULTS: After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51 N-6-methyladenines and 152 N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi(-) mutant of Escherichia coli DH5α as a host bacterium to construct a shotgun library. CONCLUSIONS: This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-803) contains supplementary material, which is available to authorized users

Virulence Determinants Are Required for Brain Abscess Formation Through Staphylococcus aureus Infection and Are Potential Targets of Antivirulence Factor Therapy

Bacterial brain abscesses (BAs) are difficult to treat with conventional antibiotics. Thus, the development of alternative therapeutic strategies for BAs is of high priority. Identifying the virulence determinants that contribute to BA formation induced by Staphylococcus aureus would improve the effectiveness of interventions for this disease. In this study, RT-qPCR was performed to compare the expression levels of 42 putative virulence determinants of S. aureus strains Newman and XQ during murine BA formation, ear colonization, and bacteremia. The alterations in the expression levels of 23 genes were further confirmed through specific TaqMan RT-qPCR. Eleven S. aureus genes that persistently upregulated expression levels during BA infection were identified, and their functions in BA formation were confirmed through isogenic mutant experiments. Bacterial loads and BA volumes in mice infected with isdA, isdC, lgt, hla, or spa deletion mutants and the hla/spa double mutant strain were lower than those in mice infected with the wild-type Newman strain. The therapeutic application of monoclonal antibodies against Hla and SpA decreased bacterial loads and BA volume in mice infected with Newman. This study provides insights into the virulence determinants that contribute to staphylococcal BA formation and a paradigm for antivirulence factor therapy against S. aureus infections

A chromosomelevel genome assembly of the Asian arowana, Scleropages formosus

Asian arowana (Scleropages formosus), an ancient teleost belonging to the Order Osteoglossomorpha, has been a valuable ornamental fish with some varieties. However, its biological studies and breeding germplasm have been remarkably limited by the lack of a reference genome. To solve these problems, here we report high-quality genome sequences of three common varieties of Asian arowana (the golden, red and green arowana). We firstly generated a chromosome-level genome assembly of the golden arowana, on basis of the genetic linkage map constructed with the restriction site-associated DNA sequencing (RAD-seq). In addition, we obtained draft genome assemblies of the red and green varieties. Finally, we annotated 22,016, 21,256 and 21,524 protein-coding genes in the genome assemblies of golden, red and green varieties respectively. Our data were deposited in publicly accessible repositories to promote biological research and molecular breeding of Asian arowana