Sexuality Generates Diversity in the Aflatoxin Gene Cluster: Evidence on a Global Scale

Aflatoxins are produced by Aspergillus flavus and A. parasiticus in oil-rich seed and grain crops and are a serious problem in agriculture, with aflatoxin B₁ being the most carcinogenic natural compound known. Sexual reproduction in these species occurs between individuals belonging to different vegetative compatibility groups (VCGs). We examined natural genetic variation in 758 isolates of A. flavus, A. parasiticus and A. minisclerotigenes sampled from single peanut fields in the United States (Georgia), Africa (Benin), Argentina (Córdoba), Australia (Queensland) and India (Karnataka). Analysis of DNA sequence variation across multiple intergenic regions in the aflatoxin gene clusters of A. flavus, A. parasiticus and A. minisclerotigenes revealed significant linkage disequilibrium (LD) organized into distinct blocks that are conserved across different localities, suggesting that genetic recombination is nonrandom and a global occurrence. To assess the contributions of asexual and sexual reproduction to fixation and maintenance of toxin chemotype diversity in populations from each locality/species, we tested the null hypothesis of an equal number of MAT1-1 and MAT1-2 mating-type individuals, which is indicative of a sexually recombining population. All samples were clone-corrected using multi-locus sequence typing which associates closely with VCG. For both A. flavus and A. parasiticus, when the proportions of MAT1-1 and MAT1-2 were significantly different, there was more extensive LD in the aflatoxin cluster and populations were fixed for specific toxin chemotype classes, either the non-aflatoxigenic class in A. flavus or the B₁-dominant and G₁-dominant classes in A. parasiticus. A mating type ratio close to 1∶1 in A. flavus, A. parasiticus and A. minisclerotigenes was associated with higher recombination rates in the aflatoxin cluster and less pronounced chemotype differences in populations. This work shows that the reproductive nature of the population (more sexual versus more asexual) is predictive of aflatoxin chemotype diversity in these agriculturally important fungi.     

Kinetic Partitioning Modulates Human Telomere DNA G-Quadruplex Structural Polymorphism

Telomeres are specialized chromatin structures found at the end of chromosomes and are crucial to the maintenance of eukaryotic genome stability. Human telomere DNA is comprised of the repeating sequence (T₂AG₃)_n, which is predominantly double-stranded but terminates with a 3’ single-stranded tail. The guanine-rich tail can fold into secondary structures known as a G-quadruplexes (GQs) that may exist as a polymorphic mixture of anti-parallel, parallel, and several hybrid topological isomers. Using single-molecule Förster resonance energy transfer (smFRET), we have reconstructed distributions of telomere DNA GQ conformations generated by an in situ refolding protocol commonly employed in single-molecule studies of GQ structure, or using a slow cooling DNA annealing protocol typically used in the preparation of GQ samples for ensemble biophysical analyses. We find the choice of GQ folding protocol has a marked impact on the observed distributions of DNA conformations under otherwise identical buffer conditions. A detailed analysis of the kinetics of GQ folding over timescales ranging from minutes to hours revealed the distribution of GQ structures generated by in situ refolding gradually equilibrates to resemble the distribution generated by the slow cooling DNA annealing protocol. Interestingly, conditions of low ionic strength, which promote transient GQ unfolding, permit the fraction of folded DNA molecules to partition into a distribution that more closely approximates the thermodynamic folding equilibrium. Our results are consistent with a model in which kinetic partitioning occurs during in situ folding at room temperature in the presence of K⁺ ions, producing a long-lived non-equilibrium distribution of GQ structures in which the parallel conformation predominates on the timescale of minutes. These results suggest that telomere DNA GQ folding kinetics, and not just thermodynamic stability, likely contributes to the physiological ensemble GQ structures.     

Immune modulation of effector CD4+ and regulatory T cell function by sorafenib in patients with hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a difficult to treat cancer characterized by poor tumor immunity with only one approved systemic drug, sorafenib. If novel combination treatments are to be developed with immunological agents, the effects of sorafenib on tumor immunity are important to understand. In this study, we investigate the impact of sorafenib on the CD4+CD25? effector T cells (Teff) and CD4+CD25+ regulatory T cells (Tregs) from patients with HCC. We isolated Teff and Treg from peripheral mononuclear cells of HCC patients to determine immune reactivity by thymidine incorporation, ELISA and flow cytometry. Teff cultured alone or with Treg were supplemented with different concentrations of sorafenib. The effects of sorafenib on Teff responses were dose-dependent. Pharmacologic doses of sorafenib decreased Teff activation by down regulating CD25 surface expression. In contrast, sub-pharmacologic concentrations of sorafenib resulted in Teff activation. These low doses of sorafenib in the Teff cultures led to a significant increase in Teff proliferation, IL2 secretion and up-regulation of CD25 expression on the cell surface. In addition, low doses of sorafenib in the suppression Teff/Treg cocultures restored Teff responses by eliminating Treg suppression. The loss of Treg suppressive function correlated with an increase in IL2 and IL6 secretion. Our findings show that sub-pharmacologic doses of sorafenib impact subsets of T cells differently, selectively increasing Teff activation while blocking Treg function. In conclusion, this study describes novel immune activating properties of low doses of sorafenib by promoting immune responsiveness in patients with HCC.     

Ecosystem engineering,environmental decay and environmental states of landscapes

Although environmental modification by ecosystem engineers influences species distributions and abundances and ecological process rates, general determinants of the environmental states of engineered landscapes are not well understood. Here we develop a general, spatially implicit model of engineered landscapes that includes parameters driving engineer populations (demographics, environmental modification) and environmental decay. We show that average environmental states and heterogeneities of landscapes are the result of a balance between parameters determining engineering rates and decay rates that can be expressed as a net engineering ratio (NER). This ratio highlights the need to include environmental decay in ecosystem engineering studies. Moreover, it defines a significant engineer as one that can alter the environment despite decay and generates expectations for different kinds of effects on the engineer, other species and ecological processes depending on ratio values. Finally, it suggests that, in general, decay places limits as to what can be inferred about engineer population dynamics from environmental dynamics and vice versa.     

Activity of the Arabidopsis RD29A and RD29B promoter elements in soybean under water stress

The constitutive and drought-induced activities of the Arabidopsis thaliana RD29A and RD29B promoters were monitored in soybean (Glycine max (L.) Merr.] via fusions with the visual marker gene β-glucuronidase (GUS). Physiological responses of soybean plants were monitored over 9 days of water deprivation under greenhouse conditions. Data were used to select appropriate time points to monitor the activities of the respective promoter elements. Qualitative and quantitative assays for GUS expression were conducted in root and leaf tissues, from plants under well-watered and dry-down conditions. Both RD29A and RD29B promoters were significantly activated in soybean plants subjected to dry-down conditions. However, a low level of constitutive promoter activity was also observed in both root and leaves of plants under well-watered conditions. GUS expression was notably higher in roots than in leaves. These observations suggest that the respective drought-responsive regulatory elements present in the RD29X promoters may be useful in controlling targeted transgenes to mitigate abiotic stress in soybean, provided the transgene under control of these promoters does not invoke agronomic penalties with leaky expression when no abiotic stress is imposed.     

Teaching ethnic and racial relations: some comments on Michael Banton

Banton's critique of race and ethnic relations highlights differences between the U.K. and the U.S.A. The interplay between ethnic politics and the teaching of the subject in the universities reflects differing reactions to the demise of Marxism, the impact of globalization and the growth of transnationalism. In America, anthropology, economics, cultural studies and conflict resolution have had a mixed impact on the field, but an examination of textbooks over the past thirty years - as well as Banton's prolific scholarship - suggests that most of the core problems remain the same.     

Identification of the N-Terminal Domain of the Influenza Virus PA Responsible for the Suppression of Host Protein Synthesis

Cellular protein synthesis is suppressed during influenza virus infection, allowing for preferential production of viral proteins. To explore the impact of polymerase subunits on protein synthesis, we coexpressed enhanced green fluorescent protein (eGFP) or luciferase together with each polymerase component or NS1 of A/California/04/2009 (Cal) and found that PA has a significant impact on the expression of eGFP and luciferase. Comparison of the suppressive activity on coexpressed proteins between various strains revealed that avian virus or avian-origin PAs have much stronger activity than human-origin PAs, such as the one from A/WSN/33 (WSN). Protein synthesis data suggested that reduced expression of coexpressed proteins is not due to PA''s reported proteolytic activity. A recombinant WSN containing Cal PA showed enhanced host protein synthesis shutoff and induction of apoptosis. Further characterization of the PA fragment indicated that the N-terminal domain (PANt), which includes the endonuclease active site, is sufficient to suppress cotransfected gene expression. By characterizing various chimeric PANts, we found that multiple regions of PA, mainly the helix α4 and the flexible loop of amino acids 51 to 74, affect the activity. The suppressive effect of PANt cDNA was mainly due to PA-X, which was expressed by ribosomal frameshifting. In both Cal and WSN viruses, PA-X showed a stronger effect than the corresponding PANt, suggesting that the unique C-terminal sequences of PA-X also play a role in suppressing cotransfected gene expression. Our data indicate strain variations in PA gene products, which play a major role in suppression of host protein synthesis.     

The Effect of Genomic Inversions on Estimation of Population Genetic Parameters from SNP Data

In recent years it has emerged that structural variants have a substantial impact on genomic variation. Inversion polymorphisms represent a significant class of structural variant, and despite the challenges in their detection, data on inversions in the human genome are increasing rapidly. Statistical methods for inferring parameters such as the recombination rate and the selection coefficient have generally been developed without accounting for the presence of inversions. Here we exploit new software for simulating inversions in population genetic data, invertFREGENE, to assess the potential impact of inversions on such methods. Using data simulated by invertFREGENE, as well as real data from several sources, we test whether large inversions have a disruptive effect on widely applied population genetics methods for inferring recombination rates, for detecting selection, and for controlling for population structure in genome-wide association studies (GWAS). We find that recombination rates estimated by LDhat are biased downward at inversion loci relative to the true contemporary recombination rates at the loci but that recombination hotspots are not falsely inferred at inversion breakpoints as may have been expected. We find that the integrated haplotype score (iHS) method for detecting selection appears robust to the presence of inversions. Finally, we observe a strong bias in the genome-wide results of principal components analysis (PCA), used to control for population structure in GWAS, in the presence of even a single large inversion, confirming the necessity to thin SNPs by linkage disequilibrium at large physical distances to obtain unbiased results.     

Using multilevel models to identify drivers of landscape‐genetic structure among management areas

Landscape genetics offers a powerful approach to understanding species' dispersal patterns. However, a central obstacle is to account for ecological processes operating at multiple spatial scales, while keeping research outcomes applicable to conservation management. We address this challenge by applying a novel multilevel regression approach to model landscape drivers of genetic structure at both the resolution of individuals and at a spatial resolution relevant to management (i.e. local government management areas: LGAs) for the koala (Phascolartos cinereus) in Australia. Our approach allows for the simultaneous incorporation of drivers of landscape‐genetic relationships operating at multiple spatial resolutions. Using microsatellite data for 1106 koalas, we show that, at the individual resolution, foliage projective cover (FPC) facilitates high gene flow (i.e. low resistance) until it falls below approximately 30%. Out of six additional land‐cover variables, only highways and freeways further explained genetic distance after accounting for the effect of FPC. At the LGA resolution, there was significant variation in isolation‐by‐resistance (IBR) relationships in terms of their slopes and intercepts. This was predominantly explained by the average resistance distance among LGAs, with a weaker effect of historical forest cover. Rates of recent landscape change did not further explain variation in IBR relationships among LGAs. By using a novel multilevel model, we disentangle the effect of landscape resistance on gene flow at the fine resolution (i.e. among individuals) from effects occurring at coarser resolutions (i.e. among LGAs). This has important implications for our ability to identify appropriate scale‐dependent management actions.