Theory of inbreeding and covariances between relatives under full-sib mating in diploids.

A generation matrix theory of full-sib mating is developed in which 13 mating "classes" are distinguished according to identity of genes in individuals mated and identity of genotypes as belonging to homozygous, parental, or offspring sets. The 13 times 13 matrix reveals some properties of the full-sib mating system not shown by previous work. The eigenvalues and a set of eigenvectors for the generation matrix, and the general solution for the frequencies of mating classes among descendants of an original mating of genotypes ab times cd, are given. The genotypic array of descendants in an arbitrary generation is also given. A new formula is derived for the coefficient of inbreeding in generation n + m in terms of coefficients of inbreeding in earlier generations. An algorithm is presented for calculating the probability of a given situation of identity of alleles carried by two individuals given only the indices of their own respective generations and the generation of their most recent common ancestor. The application of such probabilities to obtaining covariances between relatives in a full-sib mating system, under the assumptions of independence and non-interaction among loci, is illustrated. All results are shown to agree with previous work in special cases. All possible full sib, generation n - 1 parent-generation n + m offspring, and generation n uncle-generation n + m nephew covariances for 1 less than n + m less than or equal to 8 are obtained using the given algorithm.     

An apparatus for anesthetizing small laboratory animals.

A simple apparatus was designed for simultaneous general anesthesia of a number of small laboratory animals with spontaneous respiration. Anesthesia is consistent over many hr. The apparatus consists of an oxygen tank and vaporizer, a glass distribution bottle, and a non-return circuit of separate inspiratory and expiratory valves for each animal. The equipment is inexpensive to construct and can be rapidly adjusted for exact control of gas flow and anesthetic depth.     

Reporter mice for isolating and auditing cell type‐specific extracellular vesicles in vivo

Extracellular vesicles (EVs) are abundant, lipid‐enclosed vectors that contain nucleic acids and proteins, they can be secreted from donor cells and freely circulate, and they can be engulfed by recipient cells thus enabling systemic communication between heterotypic cell types. However, genetic tools for labeling, isolating, and auditing cell type‐specific EVs in vivo, without prior in vitro manipulation, are lacking. We have used CRISPR‐Cas9‐mediated genome editing to generate mice bearing a CD63‐emGFP^{loxP/stop/loxP} knock‐in cassette that enables the specific labeling of circulating CD63⁺ vesicles from any cell type when crossed with lineage‐specific Cre recombinase driver mice. As proof‐of‐principle, we have crossed these mice with Cdh5‐Cre^ERT2 mice to generate CD63^emGFP+ vasculature. Using these mice, we show that developing vasculature is marked with emerald GFP (emGFP) following tamoxifen administration to pregnant females. In adult mice, quiescent vasculature and angiogenic vasculature (in tumors) is also marked with emGFP. Moreover, whole plasma‐purified EVs contain a subpopulation of emGFP⁺ vesicles that are derived from the endothelium, co‐express additional EV (e.g., CD9 and CD81) and endothelial cell (e.g., CD105) markers, and they harbor specific miRNAs (e.g., miR‐126, miR‐30c, and miR‐125b). This new mouse strain should be a useful genetic tool for generating cell type‐specific, CD63⁺ EVs that freely circulate in serum and can subsequently be isolated and characterized using standard methodologies.     

The Synthesis and Biological activity of a Highly Selective Adenosine A2a. Receptor Agonist

Abstract

Three novel nucleosides 1, 2, and 3 were prepared that contained side chains at the 2-position of adenosine. Compound 1 was shown to be the most selective A_2a receptor agonist reported to date having an A₁/A₂ ratio of 2400. In addition, compound 1 was shown to reduce blood pressure in rats and dogs with only minimal effects on heart rate.     

The endosomal trafficking regulator LITAF controls the cardiac Nav1.5 channel via the ubiquitin ligase NEDD4-2

The QT interval is a recording of cardiac electrical activity. Previous genome-wide association studies identified genetic variants that modify the QT interval upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor-α factor), a protein encoding a regulator of endosomal trafficking. However, it was not clear how LITAF might impact cardiac excitation. We investigated the effect of LITAF on the voltage-gated sodium channel Nav1.5, which is critical for cardiac depolarization. We show that overexpressed LITAF resulted in a significant increase in the density of Nav1.5-generated voltage-gated sodium current I_Na and Nav1.5 surface protein levels in rabbit cardiomyocytes and in HEK cells stably expressing Nav1.5. Proximity ligation assays showed co-localization of endogenous LITAF and Nav1.5 in cardiomyocytes, whereas co-immunoprecipitations confirmed they are in the same complex when overexpressed in HEK cells. In vitro data suggest that LITAF interacts with the ubiquitin ligase NEDD4-2, a regulator of Nav1.5. LITAF overexpression down-regulated NEDD4-2 in cardiomyocytes and HEK cells. In HEK cells, LITAF increased ubiquitination and proteasomal degradation of co-expressed NEDD4-2 and significantly blunted the negative effect of NEDD4-2 on I_Na. We conclude that LITAF controls cardiac excitability by promoting degradation of NEDD4-2, which is essential for removal of surface Nav1.5. LITAF-knockout zebrafish showed increased variation in and a nonsignificant 15% prolongation of action potential duration. Computer simulations using a rabbit-cardiomyocyte model demonstrated that changes in Ca²⁺ and Na⁺ homeostasis are responsible for the surprisingly modest action potential duration shortening. These computational data thus corroborate findings from several genome-wide association studies that associated LITAF with QT interval variation.     

Adoptive Transfer of Lymphocytes Isolated from Simian Immunodeficiency Virus SIVmac239Δnef-Vaccinated Macaques Does Not Affect Acute-Phase Viral Loads but May Reduce Chronic-Phase Viral Loads in Major Histocompatibility Complex-Matched Recipients

The live attenuated simian immunodeficiency virus (SIV) SIVmac239Δnef is the most effective SIV/human immunodeficiency virus (HIV) vaccine in preclinical testing. An understanding of the mechanisms responsible for protection may provide important insights for the development of HIV vaccines. Leveraging the uniquely restricted genetic diversity of Mauritian cynomolgus macaques, we performed adoptive transfers between major histocompatibility complex (MHC)-matched animals to assess the role of cellular immunity in SIVmac239Δnef protection. We vaccinated and mock vaccinated donor macaques and then harvested between 1.25 × 10⁹ and 3.0 × 10⁹ mononuclear cells from multiple tissues for transfer into 12 naive recipients, followed by challenge with pathogenic SIVmac239. Fluorescently labeled donor cells were detectable for at least 7 days posttransfer and trafficked to multiple tissues, including lung, lymph nodes, and other mucosal tissues. There was no difference between recipient macaques'' peak or postpeak plasma viral loads. A very modest difference in viral loads during the chronic phase between vaccinated animal cell recipients and mock-vaccinated animal cell recipients did not reach significance (P = 0.12). Interestingly, the SIVmac239 challenge virus accumulated escape mutations more rapidly in animals that received cells from vaccinated donors. These results may suggest that adoptive transfers influenced the course of infection despite the lack of significant differences in the viral loads among animals that received cells from vaccinated and mock-vaccinated donor animals.     

The WISER metadatabase: the key to more than 100 ecological datasets from European rivers, lakes and coastal waters

In ecological sciences, the role of metadata (i.e. key information about a dataset) to make existing datasets visible and discoverable has become increasingly important. Within the EU-funded WISER project (Water bodies in Europe: Integrative Systems to assess Ecological status and Recovery), we designed a metadatabase to allow scientists to find the optimal data for their analyses. An online questionnaire helped to collect metadata from the data providers and an online query tool (http://www.wiser.eu/results/meta-database/) facilitated data evaluation. The WISER metadatabase currently holds information on 114 datasets (22 river, 71 lake, 1 general freshwater and 20 coastal/transitional datasets), which also can be accessed by external scientists. We evaluate if generally used metadata standards (e.g. Darwin Core, ISO 19115, CSDGM, EML) are suitable for such specific purposes as WISER and suggest at least the linkage with standard metadata fields. Furthermore, we discuss whether the simple metadata documentation is enough for others to reuse a dataset and why there is still reluctance to publish both metadata and primary research data (i.e. time and financial constraints, misuse of data, abandoning intellectual property rights). We emphasise that metadata publication has major advantages as it makes datasets detectable by other scientists and generally makes a scientist’s work more visible.     

Suppression of Hepcidin Expression and Iron Overload Mediate Salmonella Susceptibility in Ankyrin 1 ENU-Induced Mutant

Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1^Ity16/Ity16 mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1^Ity16/Ity16 mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1^Ity16/Ity16, the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1^+/Ity16 mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1^Ity16/Ity16 and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1^+/Ity16 mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1 ^+/Ity16 heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants.