Differential Growth of and Nanoscale TiO2 Accumulation in Tetrahymena thermophila by Direct Feeding versus Trophic Transfer from Pseudomonas aeruginosa

Nanoscale titanium dioxide (TiO₂) is increasingly used in consumer goods and is entering waste streams, thereby exposing and potentially affecting environmental microbes. Protozoans could either take up TiO₂ directly from water and sediments or acquire TiO₂ during bactivory (ingestion of bacteria) of TiO₂-encrusted bacteria. Here, the route of exposure of the ciliated protozoan Tetrahymena thermophila to TiO₂ was varied and the growth of, and uptake and accumulation of TiO₂ by, T. thermophila were measured. While TiO₂ did not affect T. thermophila swimming or cellular morphology, direct TiO₂ exposure in rich growth medium resulted in a lower population yield. When TiO₂ exposure was by bactivory of Pseudomonas aeruginosa, the T. thermophila population yield and growth rate were lower than those that occurred during the bactivory of non-TiO₂-encrusted bacteria. Regardless of the feeding mode, T. thermophila cells internalized TiO₂ into their food vacuoles. Biomagnification of TiO₂ was not observed; this was attributed to the observation that TiO₂ appeared to be unable to cross the food vacuole membrane and enter the cytoplasm. Nevertheless, our findings imply that TiO₂ could be transferred into higher trophic levels within food webs and that the food web could be affected by the decreased growth rate and yield of organisms near the base of the web.     

DEFENSE TRAITS OF LARVAL DROSOPHILA MELANOGASTER EXHIBIT GENETICALLY BASED TRADE‐OFFS AGAINST DIFFERENT SPECIES OF PARASITOIDS

Populations of Drosophila melanogaster face significant mortality risks from parasitoid wasps that use species‐specific strategies to locate and survive in hosts. We tested the hypothesis that parasitoids with different strategies select for alternative host defense characteristics and in doing so contribute to the maintenance of fitness variation and produce trade‐offs among traits. We characterized defense traits of Drosophila when exposed to parasitoids with different host searching behaviors (Aphaereta sp. and Leptopilina boulardi). We used host larvae with different natural alleles of the gene Dopa decarboxylase (Ddc), a gene controlling the production of dopamine and known to influence the immune response against parasitoids. Previous population genetic analyses indicate that our focal alleles are maintained by balancing selection. Genotypes exhibited a trade‐off between the immune response against Aphaereta sp. and the ability to avoid parasitism by L. boulardi. We also identified a trade‐off between the ability to avoid parasitism by L. boulardi and larval competitive ability as indicated by differences in foraging and feeding behavior. Genotypes differed in dopamine levels potentially explaining variation in these traits. Our results highlight the potential role of parasitoid biodiversity on host fitness variation and implicate Ddc as an antagonistic pleiotropic locus influencing larval fitness traits.     

Identification of Pauses during Formation of HIV-1 Virus Like Particles

HIV Gag polymerizes on the plasma membrane to form virus like particles (VLPs) that have similar diameters to wild-type viruses. We use multicolor, dual-penetration depth, total internal reflection fluorescence microscopy, which corrects for azimuthal movement, to image the assembly of individual VLPs from the time of nucleation to the recruitment of VPS4 (a component of the endosomal sorting complexes required for transport, and which marks the final stage of VLP assembly). Using a mathematical model for assembly and maximum-likelihood comparison of fits both with and without pauses, we detect pauses during Gag polymerization in 60% of VLPs. Pauses range from 2 to 20 min, with an exponentially distributed duration that is independent of cytosolic Gag concentration. VLPs assembled with late domain mutants of Gag (which do not recruit the key endosomal sorting complexes required for transport proteins Alix or TSG101) exhibit similar pause distributions. These pauses indicate that a single rate-limiting event is required for continuation of assembly. We suggest that pauses are either related to incorporation of defects in the hexagonal Gag lattice during VLP assembly or are caused by shortcomings in interactions of Gag with essential and still undefined cellular components during formation of curvature on the plasma membrane.     

N- versus O-alkylation: Utilizing NMR methods to establish reliable primary structure determinations for drug discovery

A classic synthetic issue that remains unresolved is the reaction that involves the control of N- versus O-alkylation of ambident anions. This common chemical transformation is important for medicinal chemists, who require predictable and reliable protocols for the rapid synthesis of inhibitors. The uncertainty of whether the product(s) are N- and/or O-alkylated is common and can be costly if undetermined. Herein, we report an NMR-based strategy that focuses on distinguishing inhibitors and intermediates that are N- or O-alkylated. The NMR strategy involves three independent and complementary methods. However, any combination of two of the methods can be reliable if the third were compromised due to resonance overlap or other issues. The timely nature of these methods (HSQC/HMQC, HMBC. ROESY, and ¹³C shift predictions) allows for contemporaneous determination of regioselective alkylation as needed during the optimization of synthetic routes.     

Synthesis and optimization of a novel series of HCV NS3 protease inhibitors: 4-Arylproline analogs

In this report we describe the synthesis and evaluation of diverse 4-arylproline analogs as HCV NS3 protease inhibitors. Introduction of this novel P2 moiety opened up new SAR and, in combination with a synthetic approach providing a versatile handle, allowed for efficient exploitation of this novel series of NS3 protease inhibitors. Multiple structural modifications of the aryl group at the 4-proline, guided by structural analysis, led to the identification of analogs which were very potent in both enzymatic and cell based assays. The impact of this systematic SAR on different drug properties is reported.     

Design,synthesis and biological evaluation of novel aminothiazoles as antiviral compounds acting against human rhinovirus

We describe here the design, synthesis and biological evaluation of antiviral compounds acting against human rhinovirus (HRV). A series of aminothiazoles demonstrated pan-activity against the HRV genotypes screened and productive structure–activity relationships. A comprehensive investigational library was designed and performed allowing the identification of potent compounds with lower molecular weight and improved ADME profile. 31d-1, 31d-2, 31f showed good exposures in CD-1 mice. The mechanism of action was discovered to be a host target: the lipid kinase phosphatidylinositol 4-kinase III beta (PI4KIIIß). The identification of the pan-HRV active compound 31f combined with a structurally distinct literature compound T-00127-HEV1 allowed the assessment of target related tolerability of inhibiting this kinase for a short period of time in order to prevent HRV replication.     

A comparison of pedigree‐ and DNA‐based measures for identifying inbreeding depression in the critically endangered Attwater's Prairie‐chicken

The primary goal of captive breeding programmes for endangered species is to prevent extinction, a component of which includes the preservation of genetic diversity and avoidance of inbreeding. This is typically accomplished by minimizing mean kinship in the population, thereby maintaining equal representation of the genetic founders used to initiate the captive population. If errors in the pedigree do exist, such an approach becomes less effective for minimizing inbreeding depression. In this study, both pedigree‐ and DNA‐based methods were used to assess whether inbreeding depression existed in the captive population of the critically endangered Attwater's Prairie‐chicken (Tympanuchus cupido attwateri), a subspecies of prairie grouse that has experienced a significant decline in abundance and concurrent reduction in neutral genetic diversity. When examining the captive population for signs of inbreeding, variation in pedigree‐based inbreeding coefficients (f_pedigree) was less than that obtained from DNA‐based methods (f_DNA). Mortality of chicks and adults in captivity were also positively correlated with parental relatedness (r_DNA) and f_DNA, respectively, while no correlation was observed with pedigree‐based measures when controlling for additional variables such as age, breeding facility, gender and captive/release status. Further, individual homozygosity by loci (HL) and parental r_DNA values were positively correlated with adult mortality in captivity and the occurrence of a lethal congenital defect in chicks, respectively, suggesting that inbreeding may be a contributing factor increasing the frequency of this condition among Attwater's Prairie‐chickens. This study highlights the importance of using DNA‐based methods to better inform management decisions when pedigrees are incomplete or errors may exist due to uncertainty in pairings.     

Assessing the cryptic invasion of a domestic conspecific: American mink in their native range

Control of invasions is facilitated by their early detection, but this may be difficult when invasions are cryptic due to similarity between invaders and native species. Domesticated conspecifics offer an interesting example of cryptic invasions because they have the ability to hybridize with their native counterparts, and can thus facilitate the introgression of maladaptive genes. We assessed the cryptic invasion of escaped domestic American mink (Neovison vison) within their native range. Feral mink are a known alien invader in many parts of the world, but invasion of their native range is not well understood. We genetically profiled 233 captive domestic mink from different farms in Ontario, Canada and 299 free‐ranging mink from Ontario, and used assignments tests to ascertain genetic ancestries of free‐ranging animals. We found that 18% of free‐ranging mink were either escaped domestic animals or hybrids, and a tree regression showed that these domestic genotypes were most likely to occur south of a latitude of 43.13°N, within the distribution of mink farms in Ontario. Thus, domestic mink appear not to have established populations in Ontario in locations without fur farms. We suspect that maladaptation of domestic mink and outbreeding depression of hybrid and introgressed mink have limited their spread. Mink farm density and proximity to mink farms were not important predictors of domestic genotypes but rather, certain mink farms appeared to be important sources of escaped domestic animals. Our results show that not all mink farms are equal with respect to biosecurity, and thus that the spread of domestic genotypes can be mitigated by improved biosecurity.     

Rapid Genome Mapping in Nanochannel Arrays for Highly Complete and Accurate De Novo Sequence Assembly of the Complex Aegilops tauschii Genome

Next-generation sequencing (NGS) technologies have enabled high-throughput and low-cost generation of sequence data; however, de novo genome assembly remains a great challenge, particularly for large genomes. NGS short reads are often insufficient to create large contigs that span repeat sequences and to facilitate unambiguous assembly. Plant genomes are notorious for containing high quantities of repetitive elements, which combined with huge genome sizes, makes accurate assembly of these large and complex genomes intractable thus far. Using two-color genome mapping of tiling bacterial artificial chromosomes (BAC) clones on nanochannel arrays, we completed high-confidence assembly of a 2.1-Mb, highly repetitive region in the large and complex genome of Aegilops tauschii, the D-genome donor of hexaploid wheat (Triticum aestivum). Genome mapping is based on direct visualization of sequence motifs on single DNA molecules hundreds of kilobases in length. With the genome map as a scaffold, we anchored unplaced sequence contigs, validated the initial draft assembly, and resolved instances of misassembly, some involving contigs <2 kb long, to dramatically improve the assembly from 75% to 95% complete.