Performance of factor IX extended half-life product measurements in external quality control assessment programs

Background: Patients with hemophilia B are increasingly treated with extended half-life (EHL) factor IX (FIX) concentrates. For the laboratory, introduction of these EHL concentrates presents a major challenge. To understand the variation in FIX activity levels, all available diagnostic assays need to be directly compared. Methods: The ECAT, UKNEQAS, and RCPAQAP have collaboratively performed a global survey to evaluate the quality of FIX measurements using FIX deficient plasma samples spiked with recombinant FIX (rFIX), rFIXFP, rFIXFc, and N9-GP to levels at typical FIX trough (6 IU/dL) and peak levels (60 IU/dL). Participants were asked to use their routine protocols, using one-stage assays (OSA) or chromogenic assays (CA). Results: In samples spiked with 6 IU/dL product, median (25%-75% range) FIX activity levels (OSA), were 8.0 IU/dL (7.0-9.2) for rFIX, 6.0 IU/dL (4.0-7.1) for rFIXFP, 6.6 IU/dL (5.5-8.0) for rFIXFc, and 4.9 IU/dL (3.5-8.4) for N9-GP. In samples spiked with 60 IU/dL, FIX activity levels measured (using OSA) was 63.0 IU/dL (59.9-67.0) for rFIX, 42.5 IU/dL (28.2-47.0) for rFIXFP, 50.0 IU/dL (45.0-55.0) for rFIXFc, and 34.0 IU/dL (24.8-67.5) for N9-GP. Considerable differences were observed between reagents for all samples. With CA, there was also quite some variation, but no differences between reagents. Conclusion: Large variation is observed in the measurement of FIX activity levels after administration of rFIX and EHL FIX products. For N9-GP, most silica-based assays show especially high levels. It is essential to standardize and improve reliability of measurements of these concentrates as diagnosis and treatment monitoring is based on these results

The coalition for conservation genetics: Working across organizations to build capacity and achieve change in policy and practice

Abstract The Coalition for Conservation Genetics (CCG) brings together four eminent organizations with the shared goal of improving the integration of genetic information into conservation policy and practice. We provide a historical context of conservation genetics as a field and reflect on current barriers to conserving genetic diversity, highlighting the need for collaboration across traditional divides, international partnerships, and coordinated advocacy. We then introduce the CCG and illustrate through examples how a coalition approach can leverage complementary expertise and improve the organizational impact at multiple levels. The CCG has proven particularly successful at implementing large synthesis-type projects, training early-career scientists, and advising policy makers. Achievements to date highlight the potential for the CCG to make effective contributions to practical conservation policy and management that no one “parent” organization could achieve on its own. Finally, we reflect on the lessons learned through forming the CCG, and our vision for the future

T cell shape is orchestrated by PDZ-containing proteins: Parallels with epithelial polarity

The Cre/loxP system is facilitates excision of DNA sequences located between two loxP sites by the Cre recombinase (Cre) of Bacteriophage P1. Generation of different tissue-specific Cre transgenic mice, which can be used for conditional gene inactivation in specific tissues, is an ideal tool for studying gene function in different tissues in mice. The winged-helix transcription factor, Foxa2, is essential for development of the node and notochord. In mouse, Foxa2 expression is observed in the node, notochord, floor plate and gut epithelium during development. In order to facilitate genetic studies of notochord development, we have used a 520 bp element (mNE) upstream of the Foxa2 gene, which directs specific expression in the node and notochord to generate a mouse line, mNE-Cre, in which the Cre gene was placed under the regulatory control of the mNE and linked to an IRES-lacZ reporter. Staining for b-galactocidase (b-gal) activity revealed that the Cre transgene was expressed from E7.5 to E9.5 specifically in the notochord. Moreover, crossing of the mNE-Cre transgenic mice with the loxP mouse reporter line, Z/EG, resulted in enhanced green fluorescent protein (EGFP) signal specifically in the node and notochord from E8.0 to E9.5. The mNE-Cre mice have been used to conditionally inactivate Smoothened (Smo) in the notochord, and preliminary data revealed that loss of both alleles of Smo in the notochord resulted in embryonic lethality. The mNE-Cre transgenic mice are therefore a useful resource for conditional gene manipulation in the notochord in mice.link_to_subscribed_fulltex

T cell shape and function is orchestrated by a network of T polarity proteins

Loss of polarity is one of the earliest hallmarks of epithelial neoplasia and leads to aberrant communication between the epithelial cell and its microenvironment. Understanding how deregulation of polarity occurs and how it may contribute to tumour formation is a new and unexplored area of cancer research. Genetic screens in Drosophila have identified scribble, discs large (dlg) and lethal giant larvae (lgl) as key epithelial polarity regulators with mutation in any of these genes resulting in loss of polarity, overproliferation and multilayering of epithelial cells leading to 3D-tumourous overgrowth, and in the presence of activated Ras, invasion and metastasis. We have recently described the human homologue of Scribble and demonstrated using complementation studies in Drosophila that expression of human Scribble can also regulate polarity and proliferation. Evidence from cancer patients suggests that Scribble and Dlg could act as a tumour suppressor in some epithelial cancers with, in many cases, low levels of Scribble or Dlg correlating with increased tumour invasiveness and malignancy. We have undertaken a detailed functional analysis of Scribble in mammalian epithelial cells and mice mutant for Scribble and uncovered a critical role for Scribble in the regulation of epithelial polarity required for directed migration during development and wound healing in vivo. In addition, we show that impaired human Scribble can function together with activated Ras to lead to increased invasion and tumourigenesis. Therefore, Scribble can act to promote or inhibit migration dependent on the cellular context. We propose that Scribble and other polarity regulators may be key signalling molecules involved in a new pathway regulating epithelial tumour progression in mammals

A mutant tat protein provides strong protection from HIV-1 infection in human CD4+ T cells

Here we show potent inhibition of HIV-1 replication in a human T cell line and primary human CD4+ cells by expressing a single antiviral protein. Nullbasic is a mutant form of the HIV-1 Tat protein that was previously shown to strongly inhibit HIV-1 replication in nonhematopoietic cell lines by targeting three steps of HIV-1 replication: reverse transcription, transport of viral mRNA, and trans-activation of HIV-1 gene expression. Here we investigated gene delivery of Nullbasic, using lentiviral and retroviral vectors. Although Nullbasic could be delivered by lentiviral vectors to target cells, transduction efficiencies were sharply reduced primarily because of negative effects on reverse transcription mediated by Nullbasic. However, Nullbasic did not inhibit transduction of HEK293T cells by a murine leukemia virus (MLV)-based retroviral vector. Therefore, MLV-based virus-like particles were used to transduce and express Nullbasic-EGFP or EGFP in Jurkat cells, a human leukemia T cell line, and Nullbasic-ZsGreen1 or ZsGreen1 in primary human CD4+ cells. HIV-1 replication kinetics were similar in parental Jurkat and Jurkat-EGFP cells, but were strongly attenuated in Jurkat-Nullbasic-EGFP cells. Similarly, virus replication in primary CD4+ cells expressing a Nullbasic-ZsGreen1 fusion protein was inhibited by approximately 8-to 10-fold. These experiments demonstrate the potential of Nullbasic, which has unique inhibitory activity, as an antiviral agent against HIV-1 infection

First circumglobal assessment of southern hemisphere humpback whale mitochondrial genetic variation and implications for management

The description of genetic population structure over a species' geographic range can provide insights into its evolutionary history and also support effective management efforts. Assessments for globally distributed species are rare, however, requiring significant international coordination and collaboration. The global distribution of demographically discrete populations for the humpback whale Megaptera novaeangliae is not fully known, hampering the definition of appropriate management units. Here, we present the first circumglobal assessment of mitochondrial genetic population structure across the species' range in the Southern Hemisphere and Arabian Sea. We combine new and existing data from the mitochondrial (mt)DNA control region that resulted in a 311 bp consensus sequence of the mtDNA control region for 3009 individuals sampled across 14 breeding stocks and subpopulations currently recognized by the International Whaling Commission. We assess genetic diversity and test for genetic differentiation and also estimate the magnitude and directionality of historic matrilineal gene flow between putative populations. Our results indicate that maternally directed site fidelity drives significant genetic population structure between breeding stocks within ocean basins. However, patterns of connectivity differ across the circumpolar range, possibly as a result of differences in the extent of longitudinal movements on feeding areas. The number of population comparisons observed to be significantly differentiated were found to diminish at the subpopulation scale when nucleotide differences were examined, indicating that more complex processes underlie genetic structure at this scale. It is crucial that these complexities and uncertainties are afforded greater consideration in management and regulatory efforts