Synthesis and antimalarial evaluation of a screening library based on a tetrahydroanthraquinone natural product scaffold

As part of a research program aimed at discovering new antimalarial leads from Australian macrofungi a unique fungi-derived prefractionated library was screened against a chloroquine-sensitive Plasmodium falciparum line (3D7) using a radiometric growth inhibition assay. A library fraction derived from a Cortinarius species displayed promising antimalarial activity. UV-guided fractionation on the CH₂Cl₂/MeOH extract from this fungus resulted in the isolation of four known compounds: (1S,3R)-austrocortirubin (1), (1S,3S)-austrocortirubin (2), 1-deoxyaustrocortirubin (3), and austrocortinin (4). Compound 2 was used as a natural product scaffold in the parallel solution-phase synthesis of a small library of N-substituted tetrahydroanthraquinones (5–15). All compounds (1–15) were tested in vitro against P. falciparum 3D7 parasites and (1S,3S)-austrocortirubin (2), the major fungal constituent, was shown to be the most active compound with an IC₅₀ of 1.9 μM. This compound displayed moderate cytotoxicity against neonatal foreskin fibroblast (NFF) cells with an IC₅₀ of 15.6 μM.     

A single nucleotide polymorphism assay for the identification of unisexual Ambystoma salamanders

Unisexual (all female) salamanders in the genus Ambystoma are animals of variable ploidy (2N‐5N) that reproduce via a unique system of ‘leaky’ gynogenesis. As a result, these salamanders have a diverse array of nuclear genome combinations from up to five sexual species: the blue‐spotted (A. laterale), Jefferson (A. jeffersonianum), smallmouth (A. texanum), tiger (A. tigrinum) and streamside (A. barbouri) salamanders. Identifying the genome complement, or biotype, is a critical first step in addressing a broad range of ecological and evolutionary questions about these salamanders. Previous work relied upon genome‐related differences in allele size distributions for specific microsatellite loci, but overlap in these distributions among different genomes makes definitive identification and ploidy determination in unisexuals difficult or impossible. Here, we develop the first single nucleotide polymorphism assay for the identification of unisexual biotypes, based on species‐specific nucleotide polymorphisms in noncoding DNA loci. Tests with simulated and natural unisexual DNA samples show that this method can accurately identify genome complement and estimate ploidy, making this a valuable tool for assessing the genome composition of unisexual samples.     

Facilitation and Competition among Invasive Plants: A Field Experiment with Alligatorweed and Water Hyacinth

Ecosystems that are heavily invaded by an exotic species often contain abundant populations of other invasive species. This may reflect shared responses to a common factor, but may also reflect positive interactions among these exotic species. Armand Bayou (Pasadena, TX) is one such ecosystem where multiple species of invasive aquatic plants are common. We used this system to investigate whether presence of one exotic species made subsequent invasions by other exotic species more likely, less likely, or if it had no effect. We performed an experiment in which we selectively removed exotic rooted and/or floating aquatic plant species and tracked subsequent colonization and growth of native and invasive species. This allowed us to quantify how presence or absence of one plant functional group influenced the likelihood of successful invasion by members of the other functional group. We found that presence of alligatorweed (rooted plant) decreased establishment of new water hyacinth (free-floating plant) patches but increased growth of hyacinth in established patches, with an overall net positive effect on success of water hyacinth. Water hyacinth presence had no effect on establishment of alligatorweed but decreased growth of existing alligatorweed patches, with an overall net negative effect on success of alligatorweed. Moreover, observational data showed positive correlations between hyacinth and alligatorweed with hyacinth, on average, more abundant. The negative effect of hyacinth on alligatorweed growth implies competition, not strong mutual facilitation (invasional meltdown), is occurring in this system. Removal of hyacinth may increase alligatorweed invasion through release from competition. However, removal of alligatorweed may have more complex effects on hyacinth patch dynamics because there were strong opposing effects on establishment versus growth. The mix of positive and negative interactions between floating and rooted aquatic plants may influence local population dynamics of each group and thus overall invasion pressure in this watershed.     

Multi-Tissue Microarray Analysis Identifies a Molecular Signature of Regeneration

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.     

High Resolution Size Analysis of Fetal DNA in the Urine of Pregnant Women by Paired-End Massively Parallel Sequencing

Background

Fetal DNA in maternal urine, if present, would be a valuable source of fetal genetic material for noninvasive prenatal diagnosis. However, the existence of fetal DNA in maternal urine has remained controversial. The issue is due to the lack of appropriate technology to robustly detect the potentially highly degraded fetal DNA in maternal urine.

Methodology

We have used massively parallel paired-end sequencing to investigate cell-free DNA molecules in maternal urine. Catheterized urine samples were collected from seven pregnant women during the third trimester of pregnancies. We detected fetal DNA by identifying sequenced reads that contained fetal-specific alleles of the single nucleotide polymorphisms. The sizes of individual urinary DNA fragments were deduced from the alignment positions of the paired reads. We measured the fractional fetal DNA concentration as well as the size distributions of fetal and maternal DNA in maternal urine.

Principal Findings

Cell-free fetal DNA was detected in five of the seven maternal urine samples, with the fractional fetal DNA concentrations ranged from 1.92% to 4.73%. Fetal DNA became undetectable in maternal urine after delivery. The total urinary cell-free DNA molecules were less intact when compared with plasma DNA. Urinary fetal DNA fragments were very short, and the most dominant fetal sequences were between 29 bp and 45 bp in length.

Conclusions

With the use of massively parallel sequencing, we have confirmed the existence of transrenal fetal DNA in maternal urine, and have shown that urinary fetal DNA was heavily degraded.     

Localization of the bacterial agent of juvenile oyster disease (Roseovarius crassostreae) within affected eastern oysters (Crassostrea virginica)

The bacterium Roseovarius crassostreae causes seasonal mortalities among commercially produced eastern oysters (Crassostrea virginica) grown in the Northeastern United States. Phylogenetically, the species belongs to a major lineage of marine bacteria (the Roseobacter clade), within which Roseovarius crassostreae is the only known pathogen to be isolated in laboratory culture. The objective of the current study was to determine the location and nature of R. crassostreae interactions with oysters affected by juvenile oyster disease (JOD). Scanning electron microscopy of diseased individuals revealed abundant colonization of the inner shell surfaces by bacteria which were morphologically similar to R. crassostreae. The same types of cells were also observed on and within layers of host-derived conchiolin on the inner valves. Most bacterial cells were alive as determined by the use of a fluorescent viability stain. Further, most were clearly attached at the cell poles, which is consistent with the ability of R. crassostreae to express polar fimbriae. When material from the pallial fluid, soft tissue and inner valve surfaces was cultured, the highest numbers of R. crassostreae were recovered from the inner valves. These samples also contained the greatest abundance of R. crassostreae as a percentage of total colonies. Cloning and sequencing of 16S rRNA genes provided culture-independent evidence of the numerical dominance of R. crassostreae among the bacterial consortia associated with the inner shell surfaces of JOD-affected animals. The ability of R. crassostreae to colonize shell and conchiolin is consistent with the described JOD-pathology and may aid the bacteria in avoiding hemocyte-mediated killing.     

Observations on Antricola ticks: small nymphs feed on mammalian hosts and have a salivary gland structure similar to ixodid ticks

Ticks use bloodmeals as a source of nutrients and energy to molt and survive until the next meal and to oviposit, in the case of females. However, only the larvae of some tick species are known to feed upon bats; females are obligatorily autogenous, and nymphal stages are believed to not feed. We investigated the presence of blood in a natural population of nymphal Antricola delacruzi ticks collected from bat guano; their ability to feed upon laboratory hosts; and the microscopic structure of both salivary glands and gut. DNA amplification of gut contents of freshly collected material was positive for a mammal in 4 of 11 first instar nymphs, but we were unsuccessful in the amplification of host bloodmeal DNA from late instar nymphs. All early nymphal stages (n = 10) fed on rabbits, and host DNA was detected and sequenced from gut contents. However, all the large nymphs (n = 10) rejected feeding, and host DNA remained undetected in these ticks. All stages of A. delacruzi have salivary glands similar in morphology to the ixodid agranular Type I salivary gland acini and to granular Type II or Type B acini. All stages of A. delacruzi had a similar gut structure, consisting of digestive cells in the basal portion that contained hematin granules. Neither regenerative nor secretory cell traces were observed in the sections of gut.     

c-Rel is essential for the development of innate and T cell-induced colitis

Inflammatory bowel disease is a chronic inflammatory response of the gastrointestinal tract mediated in part by an aberrant response to intestinal microflora. Expression of IL-23 subunits p40 and p19 within cells of the innate immune system plays a central role in the development of lower bowel inflammation in response inflammatory challenge. The NF-kappaB subunit c-Rel can regulate expression of IL-12/23 subunits suggesting that it could have a critical role in mediating the development of chronic inflammation within the lower bowel. In this study, we have analyzed the role of c-Rel within the innate immune system in the development of lower bowel inflammation, in two well-studied models of murine colitis. We have found that the absence of c-Rel significantly impaired the ability of Helicobacter hepaticus to induce colitis upon infection of RAG-2-deficient mice, and ameliorated the ability of CD4(+)CD45RB(high) T cells to induce disease upon adoptive transfer into RAG-deficient mice. The absence of c-Rel interfered with the expression of IL-12/23 subunits both in cultured primary macrophages and within the colon. Thus, c-Rel plays a critical role in regulating the innate inflammatory response to microflora within the lower bowel, likely through its ability to modulate expression of IL-12/23 family members.     

Myelin-reactive type B T cells and T cells specific for low-affinity MHC-binding myelin peptides escape tolerance in HLA-DR transgenic mice

Genes of the MHC show the strongest genetic association with multiple sclerosis (MS), but the underlying mechanisms have remained unresolved. In this study, we asked whether the MS-associated MHC class II molecules, HLA-DRB1*1501, HLA-DRB5*0101, and HLA-DRB1*0401, contribute to autoimmune CNS demyelination by promoting pathogenic T cell responses to human myelin basic protein (hMBP), using three transgenic (Tg) mouse lines expressing these MHC molecules. Unexpectedly, profound T cell tolerance to the high-affinity MHC-binding hMBP82-100 epitope was observed in all Tg mouse lines. T cell tolerance to hMBP82-100 was abolished upon back-crossing the HLA-DR Tg mice to MBP-deficient mice. In contrast, T cell tolerance was incomplete for low-affinity MHC-binding hMBP epitopes. Furthermore, hMBP82-100-specific type B T cells escaped tolerance in HLA-DRB5*0101 Tg mice. Importantly, T cells specific for low-affinity MHC-binding hMBP epitopes and hMBP82-100-specific type B T cells were highly encephalitogenic. Collectively, the results show that MS-associated MHC class II molecules are highly efficient at inducing T cell tolerance to high-affinity MHC-binding epitope, whereas autoreactive T cells specific for the low-affinity MHC-binding epitopes and type B T cells can escape the induction of T cell tolerance and may promote MS.