Management of broodstock and quality control of fish seed in Hungary

Common carp (Cyprinus carpio) breeding has a long tradition in Hungary. However, recent economic changes in Eastern Europe and new developments in aquaculture necessitated the need for ensuring quality of the brood stock used in hatcheries and the legal and institutional frameworks needed to implement the program. In addition to good research and development programs and gene banking, it became essential to establish an appropriate legal framework, organize, coordinate and control breeding activities, and provide financial support. It was a major breakthrough for carp breeding when C.carpio was recognized as one of the cultivated animals in the Animal Breeding Act in 1993. The Carp Breeding Section of the Hungarian Fish Producers Association plays an important role in carp breeding programs. Thirteen breeding farms of the Carp Breeding Section have 24 certified C.carpio varieties. In Hungary, about 80 % of the seed used as stocking for commercial production are from high quality certified breeders

Lessons from the breeding program on common carp in Hungary

Common carp is one of the most important cultured freshwater fish species in the world. Its production in freshwater areas is the second largest in Europe after rainbow trout. Common carp production in Europe was 146,845 t in 2004 (FAO Fishstat Plus 2006). Common carp production is concentrated mainly in Central and Eastern Europe. In Hungary, common carp has been traditionally cultured in earthen ponds since the late 19th century, following the sharp drop in catches from natural waters, due to the regulation of main river systems. Different production technologies and unintentional selection methods resulted in a wide variety of this species. Just before the intensification of rearing technology and the exchange of stocking materials among fish farms (early sixties), ôlandracesö of carp were collected from practically all Hungarian fish farms into a live gene bank at the Research Institute for Fisheries, Aquaculture and Irrigation (HAKI) at Szarvas (Bakos and Gorda 1995; Bakos and Gorda 2001). In order to provide highly productive hybrids for production purposes starting from 1964, different strains and crosses between Hungarian landraces were created and tested. During the last 40 years, approximately 150 two-, three-, and four-line hybrids were produced. While developing parental lines, methods of individual selection, inbreeding, backcrossing of lines, gynogenesis and sex reversal were used. This breeding program resulted in three outstanding hybrids: ôSzarvas 215 mirrorö and ôSzarvas P31 scalyö for pond production, and ôSzarvas P34 scalyö for angling waters. Besides satisfying the needs of industry, the live gene bank helped to conserve the biological diversity of Hungarian carp landraces. Fifteen Hungarian carp landraces are still maintained today in the gene bank. Through exchange programs fifteen foreign carp strains were added to the collection from Central and Eastern Europe, as well as Southeast Asia (Bakos and Gorda 2001). Besides developing the methodology to maintain live specimens in the gene bank, the National Carp Breeding Program has been initiated in cooperation with all the key stakeholders in Hungary, namely the National Association of Fish Producers (HOSZ), the National Institute for Agricultural Quality Control (OMMI), and the Research Institute for Fisheries, Aquaculture and Irrigation (HAKI). In addition, methodologies or technologies for broodstock management and carp performance testing have been developed. This National Carp Breeding Program is being implemented successfully since the mid-1990s.Biotechnology, Genetics, Food fish, Genetic drift, Genetic diversity, Aquatic animals, DNA, Selective breeding, Breeding success, Research programmes Cyprinus carpio

The iQ200 Microscopic Analyzer is valuable tool for evaluation of urinary sediment at transplanted patients

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Fluorescent Transgenic Zebrafish Tg(nkx2.2a:mEGFP) Provides a Highly Sensitive Monitoring Tool for Neurotoxins

10.1371/journal.pone.0055474PLoS ONE82

The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2

Hepatocyte nuclear factor 4 alpha (HNF4alpha) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4alpha is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4alpha. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4alpha. However, based on our previous results we hypothesized that HNF4alpha is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4alpha in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4alpha leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4alpha. Accordingly, we have observed decreasing but not disappearing binding of HNF4alpha to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4alpha chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4a gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4alpha-dependent hepatic gene expression

Peptide Ligands Incorporated into the Threefold Spike Capsid Domain to Re-Direct Gene Transduction of AAV8 and AAV9 In Vivo

Efficiency and specificity of viral vectors are vital issues in gene therapy. Insertion of peptide ligands into the adeno-associated viral (AAV) capsid at receptor binding sites can re-target AAV2-derived vectors to alternative cell types. Also, the use of serotypes AAV8 and -9 is more efficient than AAV2 for gene transfer to certain tissues in vivo. Consequently, re-targeting of these serotypes by ligand insertion could be a promising approach but has not been explored so far. Here, we generated AAV8 and -9 vectors displaying peptides in the threefold spike capsid domain. These peptides had been selected from peptide libraries displayed on capsids of AAV serotype 2 to optimize systemic gene delivery to murine lung tissue and to breast cancer tissue in PymT transgenic mice (PymT). Such peptide insertions at position 590 of the AAV8 capsid and position 589 of the AAV9 capsid changed the transduction properties of both serotypes. However, both peptides inserted in AAV8 did not result in the same changes of tissue tropism as they did in AAV2. While the AAV2 peptides selected on murine lung tissue did not alter tropism of serotypes 8 and -9, insertion of the AAV2-derived peptide selected on breast cancer tissue augmented tumor gene delivery in both serotypes. Further, this peptide mediated a strong but unspecific in vivo gene transfer for AAV8 and abrogated transduction of various control tissues for AAV9. Our findings indicate that peptide insertion into defined sites of AAV8 and -9 capsids can change and improve their efficiency and specificity compared to their wild type variants and to AAV2, making these insertion sites attractive for the generation of novel targeted vectors in these serotypes

Expression and In Vivo Rescue of Human ABCC6 Disease-Causing Mutants in Mouse Liver

Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application