Sequence-level population simulations over large genomic regions

Simulation is an invaluable tool for investigating the effects of various population genetics modeling assumptions on resulting patterns of genetic diversity, and for assessing the performance of statistical techniques, for example those designed to detect and measure the genomic effects of selection. It is also used to investigate the effectiveness of various design options for genetic association studies. Backward-in-time simulation methods are computationally efficient and have become widely used since their introduction in the 1980s. The forward-in-time approach has substantial advantages in terms of accuracy and modeling flexibility, but at greater computational cost. We have developed flexible and efficient simulation software and a rescaling technique to aid computational efficiency that together allow the simulation of sequence-level data over large genomic regions in entire diploid populations under various scenarios for demography, mutation, selection, and recombination, the latter including hotspots and gene conversion. Our forward evolution of genomic regions (FREGENE) software is freely available from www.ebi.ac.uk/projects/BARGEN together with an ancillary program to generate phenotype labels, either binary or quantitative. In this article we discuss limitations of coalescent-based simulation, introduce the rescaling technique that makes large-scale forward-in-time simulation feasible, and demonstrate the utility of various features of FREGENE, many not previously available.     

Toxoplasma gondii scavenges host-derived lipoic acid despite its de novo synthesis in the apicoplast

In contrast to other eukaryotes, which manufacture lipoic acid, an essential cofactor for several vital dehydrogenase complexes, within the mitochondrion, we show that the plastid (apicoplast) of the obligate intracellular protozoan parasite Toxoplasma gondii is the only site of de novo lipoate synthesis. However, antibodies specific for protein-attached lipoate reveal the presence of lipoylated proteins in both, the apicoplast and the mitochondrion of T. gondii. Cultivation of T. gondii-infected cells in lipoate-deficient medium results in substantially reduced lipoylation of mitochondrial (but not apicoplast) proteins. Addition of exogenous lipoate to the medium can rescue this effect, showing that the parasite scavenges this cofactor from the host. Exposure of T. gondii to lipoate analogues in lipoate-deficient medium leads to growth inhibition, suggesting that T. gondii might be auxotrophic for this cofactor. Phylogenetic analyses reveal the secondary loss of the mitochondrial lipoate synthase gene after the acquisition of the plastid. Our studies thus reveal an unexpected metabolic deficiency in T. gondii and raise the question whether the close interaction of host mitochondria with the parasitophorous vacuole is connected to lipoate supply by the host.     

Heparan sulfate mimicry: a synthetic glycoconjugate that recognizes the heparin binding domain of interferon-gamma inhibits the cytokine activity

Cell-associated heparan sulfate (HS) is endowed with the remarkable ability to bind numerous proteins. As such, it represents a unique system that integrates signaling from circulating ligands with cellular receptors. This polysaccharide is extraordinary complex, and examples that define the structure-function relationship of HS are limited. In particular, it remains difficult to understand the structures by which HS interact with proteins. Among them, interferon-gamma (IFNgamma), a dimeric cytokine, binds to a complex oligosaccharide motif encompassing a N-acetylated glucosamine-rich domain and two highly sulfated sequences, each of which binds to one IFNgamma monomer. Based on this template, we have synthesized a set of glycoconjugate mimetics and evaluated their ability to interact with IFNgamma. One of these molecules, composed of two authentic N-sulfated octasaccharides linked to each other through a 50-Angstroms-long spacer termed 2O(10), displays high affinity for the cytokine and inhibits IFNgamma-HS binding with an IC(50) of 35-40 nm. Interestingly, this molecule also inhibits the binding of IFNgamma to its cellular receptor. Thus, in addition to its ability to delocalize the cytokine from cell surface-associated HS, this compound has direct anti-IFNgamma activity. Altogether, our results represent the first synthetic HS-like molecule that targets a cytokine, strongly validating the HS structural determinants for IFNgamma recognition, providing a new strategy to inhibit IFNgamma in a number of diseases in which the cytokine has been identified as a target, and reinforcing the view that it is possible to create"tailor-made"sequences based on the HS template to isolate therapeutic activities.     

Isolation and characterization of kinetoplast DNA and RNA of Phytomonas davidi

Kinetoplast DNA networks were isolated from stationary-phase culture forms of Phytomonas davidi. The networks banded in CsCl at a density of 1.699 g/ml and consisted of covalently closed circular molecules. The networks were sensitive to shear forces and exhibited several discrete sedimenting components in neutral and alkaline sucrose. Closed monomeric minicircles were isolated from sonicated networks by alkaline band sedimentation. Closed monomers showed a heterogeneous banding pattern on electrophoresis in acrylamide-agarose gels and had sedimentation coefficients of 20.5 S in alkaline sucrose and 11 S in neutral sucrose. The mean minicircle molecular weight as measured by cospreading with φXRF II was 0.70 × 10⁶ or 1064 nucleotide pairs. Minicircles exhibited a sequence microheterogeneity as evidenced by restriction enzyme analysis, melting analysis, and renaturation kinetics. Network maxicircles were evidenced by the appearance of high molecular weight fragments after restriction with several enzymes and by the existence of supertwisted “edge loops” extending out from the periphery of networks. The maxicircle molecular weight was estimated to be approximately 24 × 10⁶. A purified kinetoplast-mitochondrion fraction was found to contain 9 and 12 S RNA species that comigrated with L. tarentolae 9 and 12 S kinetoplast RNAs.     

Glutamate Oxidation by Soybean Cotyledon and Leaf Mitochondria

Mitochondria purified from cotyledons of soybean seedlings fiveto ten days old have the capacity to rapidly oxidize glutamate(measured as glutamate dependent oxygen consumption). This capacitywas greatest at ten days after planting but was very low priorto emergence of cotyledons from the vermiculite and during senescence.Solubilized glutamate dehydrogenase activity, on the other hand,was substantial at two days after planting, peaked at sevendays, then declined and rose again during senescence. It issuggested that mitochondrial glutamate oxidation plays a rolein reserve mobilization and amino acid metabolism during seedlinggrowth. Leaf mitochondria and those from senescing cotyledonscould not sustain rapid rates of glutamate oxidation despiteready oxidation of other substrates and high solubilized glutamatedehydrogenase activity, suggesting an alternative role for theenzyme in these tissues. Possible controlling factors are discussed. ² Present address, Garvan Institute, Darlinghurst, N. S. W.,Australia. ³ Permanent address, Department de Biologia Vegetal, Facultatde Biologia, Universitat de Barcelona, Barcelona, Spain. (Received May 6, 1988; Accepted August 3, 1988)     

Is the epithelium-derived inhibitory factor in guinea-pig trachea a prostanoid?

To examine further the possible prostanoid involvement in the influence of the epithelium on guinea-pig tracheal smooth muscle responsiveness, we have analyzed the effects of LTD₄, methacholine and histamine on the level of airway smooth muscle tone and on the amounts of PGE_2α and PGI₂ (determined by radioimmunoassay) in the presence and absence of the epithelium. Removal of the epithelium increased the sensitivity of guinea-pig trachea to the contractile effects of LTD₄, methacholine and histamine. LTD₄ (3–100 nM), methacoline (0.1–10 μM) or histamine (0.3–30 μM) did not increase prostanoid release above control values in either the presence or absence of the epithelium. The unstimulated release of PGE₂ and PGF_2α but not PGI₂, was decreased in tissues lacking epithelium. Indomethacin (1 μM) reduced the baseline tone to a smaller extent in the absence of epithelium. In the presence but not the absence of the epithelium, indomethacin increased the sensitivity of preparations to the contractile effect of methacholine. The results support the postulate of an epithelium-derived inhibitory factor modulating guinea-pig tracheal smooth muscle responsiveness. The identity of this factor is not known but is not PGI₂ and is unlikely to be PGF_2α or PGE₂. However, the possibility remains that the basal release of PGE₂ and/or PGF_2α derived from the epithelium may markedly affect the responsiveness of guinea-pig tracheal smooth muscle. Furthermore, the epithelium is a significant source of PGE₂ and PGF_2α which may be involved in the maintenance of baseline tone.     

γ-Herpesvirus reactivation differentially stimulates epitope-specific CD8 T cell responses

The γ-herpesviruses are characterized by their ability to establish lifelong latency. Subsequent immune suppression leads to viral reactivation from latency and the onset of a variety of pathologies, including lymphoproliferative disease and cancers. CD8 T cells play a key role in preventing reactivation of latent virus. Therefore, to develop effective therapeutic immune strategies, it is essential to understand the maintenance of CD8 T cell responses during latency. Because the γ-herpesviruses are highly species-specific and mice cannot be infected with the human pathogens, EBV or Kaposi's sarcoma-associated herpesvirus, we have used a natural rodent γ-herpesvirus experimental infection model, γ-herpesvirus-68. In this report, we show that during long-term latent infection, naive CD8 T cells are recruited into the ongoing immune response in an epitope-specific manner. When virus reactivation is induced in vivo, the recruitment of CD8 T cells for some, but not all, epitopes is enhanced. The variation in recruitment is not due to differences in epitope presentation. We also show that CD8 T cells that are newly stimulated during reactivation are functionally impaired compared with acutely stimulated cells in terms of cytokine production. Thus, our results demonstrate unexpected complexity in the response of CD8 T cells specific for different viral epitopes that were stimulated during acute infection, quiescent latency, and reactivation.     

In silico identification and Bayesian phylogenetic analysis of multiple new mammalian kallikrein gene families

Kallikrein gene families have been identified previously in genomes of the human, the mouse, and the rat, and individual kallikrein-like genes have been found in many more species. This study presents the in silico identification of kallikrein gene families in the recently sequenced genomes of four additional mammalian species, the chimpanzee, the dog, the pig, and the opossum. Phylogenies were constructed with gene sequences from all seven mammalian families, using Bayesian analysis, which clarified the evolutionary relationships between these genes. Individual gene sequences, as well as concatenated constructs of multiple sequences, were used. Fifteen kallikrein genes were located in the chimpanzee (Pan troglodytes) genome, while only 14 were identified in the canine (Canis familiaris) genome as no orthologue to human KLK3 was found. Thirteen genes were identified from the pig (Sus scrofa) genome, which lacked homologues to KLK2 and KLK3, and 11 genes, orthologous to human KLK5 through KLK15, were found in the opossum (Monodelphis domestica) genome. No kallikrein genes were identified from the available genome sequences of the chicken (Gallus gallus) or African clawed frog (Xenopus tropicalis). Within the family of kallikreins several subfamilies were suggested by phylogenetic analysis. One consisted of KLK4, KLK5, and KLK14; another of KLK9, KLK11, and KLK15; a third of KLK10 and KLK12; a fourth of KLK6 and KLK13; and finally one of KLK8 and the classical kallikreins (KLK1, KLK2, and KLK3).     

Mucosal Immunization of Lactating Female Rhesus Monkeys with a Transmitted/Founder HIV-1 Envelope Induces Strong Env-Specific IgA Antibody Responses in Breast Milk

We previously demonstrated that vaccination of lactating rhesus monkeys with a DNA prime/vector boost strategy induces strong T-cell responses but limited envelope (Env)-specific humoral responses in breast milk. To improve vaccine-elicited antibody responses in milk, hormone-induced lactating rhesus monkeys were vaccinated with a transmitted/founder (T/F) HIV Env immunogen in a prime-boost strategy modeled after the moderately protective RV144 HIV vaccine. Lactating rhesus monkeys were intramuscularly primed with either recombinant DNA (n = 4) or modified vaccinia virus Ankara (MVA) poxvirus vector (n = 4) expressing the T/F HIV Env C.1086 and then boosted twice intramuscularly with C.1086 gp120 and the adjuvant MF59. The vaccines induced Env-binding IgG and IgA as well as neutralizing and antibody-dependent cellular cytotoxicity (ADCC) responses in plasma and milk of most vaccinated animals. Importantly, plasma neutralization titers against clade C HIV variants MW965 (P = 0.03) and CAP45 (P = 0.04) were significantly higher in MVA-primed than in DNA-primed animals. The superior systemic prime-boost regimen was then compared to a mucosal-boost regimen, in which animals were boosted twice intranasally with C.1086 gp120 and the TLR 7/8 agonist R848 following the same systemic prime. While the systemic and mucosal vaccine regimens elicited comparable levels of Env-binding IgG antibodies, mucosal immunization induced significantly stronger Env-binding IgA responses in milk (P = 0.03). However, the mucosal regimen was not as potent at inducing functional IgG responses. This study shows that systemic MVA prime followed by either intranasal or systemic protein boosts can elicit strong humoral responses in breast milk and may be a useful strategy to interrupt postnatal HIV-1 transmission.     

Correction to: Predator‑based selection and the impact of edge sympatry on components of coral snake mimicry

Evolutionary Ecology -