Congruence between environmental parameters, morphology and genetic structure in Australia’s most widely distributed eucalypt, Eucalyptus camaldulensis

Eucalyptus camaldulensis is one of the most widely utilised eucalypts. It is also the only eucalypt that occurs across the Australian continent, playing a key ecological role as fauna habitat and in riverbank stabilisation. Despite its ecological and economic importance, uncertainty remains regarding the delineation of genetic and morphological variants. Nine hundred and ninety trees from 97 populations, representing the species’ geographic range were genotyped using 15 microsatellite loci and patterns of diversity compared with restriction fragment length polymorphisms in 29 of these populations. Both markers showed that despite having a riverine distribution, downstream seed dispersal has had less influence than geographic distance on dispersal patterns. Spatial patterns in the distribution of microsatellite genotypes were compared with environmental parameters and boundaries defined by river systems, drainage basins and proposed subspecies. Significant genetic differences among populations within river systems indicated that rivers should not be treated as a single genetic entity in conservation or breeding programmes. Strong geographic trends were evident with 40% of variation in genetic diversity explained by latitude and moisture index. Isolation by distance and significant correlations between genetic distance and environmental parameters for most loci suggest historical factors have had more influence than selection on current patterns of distribution of genetic diversity. Geographic structuring of molecular variation, together with congruence between genetic and morphological variation indicate that E. camaldulensis should be treated as a number of subspecies rather than a single variable taxon. High levels of genetic diversity and geographic trends in the distribution of variation provide a firm basis for further exploration of the species’ genetic resources.     

Structure and functional implications of a complex containing a segment of U6 RNA bound by a domain of Prp24

U6 RNA plays a critical role in pre-mRNA splicing. Assembly of U6 into the spliceosome requires a significant structural rearrangement and base-pairing with U4 RNA. In the yeast Saccharomyces cerevisiae, this process requires the essential splicing factor Prp24. We present the characterization and structure of a complex containing one of Prp24''s four RNA recognition motif (RRM) domains, RRM2, and a fragment of U6 RNA. NMR methods were used to identify the preferred U6 binding sequence of RRM2 (5′-GAGA-3′), measure the affinity of the interaction, and solve the structure of RRM2 bound to the hexaribonucleotide AGAGAU. Interdomain contacts observed between RRM2 and RRM3 in a crystal structure of the free protein are not detectable in solution. A structural model of RRM1 and RRM2 bound to a longer segment of U6 RNA is presented, and a partial mechanism for Prp24''s annealing activity is proposed.     

Codon usage bias and the evolution of influenza A viruses. Codon Usage Biases of Influenza Virus

Background  

The influenza A virus is an important infectious cause of morbidity and mortality in humans and was responsible for 3 pandemics in the 20^th century. As the replication of the influenza virus is based on its host's machinery, codon usage of its viral genes might be subject to host selection pressures, especially after interspecies transmission. A better understanding of viral evolution and host adaptive responses might help control this disease.     

Enaminones 9. Further studies on the anticonvulsant activity and potential type IV phosphodiesterase inhibitory activity of substituted vinylic benzamides

Structure-activity relationship studies were employed to synthesize a series of 3- and 3,4-substituted benzamides from 3-amino-2-cyclohexenones. An improved method for the synthesis of benzamides from 3-amino-2-cyclohexenones is presented which provided significantly higher yields (71-79%) for the reported compounds. NMR and X-ray structural analyses were undertaken to note the possible intra- and intermolecular interactions of the synthesized analogs. Molecular modeling studies were used to determine the minimized configuration and were compared to their X-ray structures for correlation. These new entities were evaluated as potential anticonvulsants and type IV phosphodiesterase inhibitors (PDE4).     

Lipid body formation during maturation of human mast cells

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.     

The structural basis for RNA specificity and Ca2+ inhibition of an RNA-dependent RNA polymerase

The RNA-dependent RNA polymerase of bacteriophage phi6 transcribes mRNA from the three segments of the dsRNA viral genome. We have cocrystallized RNA oligonucleotides with the polymerase, revealing the mode of binding of RNA templates. This binding is somewhat different from that previously seen for DNA oligomers, leading to additional RNA-protein hydrogen bonds, consistent with a preference for RNA. Activation of the RNA/polymerase complex by the addition of substrate and Mg2+ initiates a single round of reaction within the crystal to form a dead-end complex that partially collapses within the enzyme active site. By replacing Mg2+ with Ca2+, we have been able to capture the inhibited complex which shows distortion that explains the structural basis for the inhibition of such polymerases by Ca2+.     

Assessment of RainDrop BS-seq as a method for large-scale,targeted bisulfite sequencing

We present a systematic assessment of RainDrop BS-seq, a novel method for large-scale, targeted bisulfite sequencing using microdroplet-based PCR amplification coupled with next-generation sequencing. We compared DNA methylation levels at 498 target loci (1001 PCR amplicons) in human whole blood, osteosarcoma cells and an archived tumor tissue sample. We assessed the ability of RainDrop BS-seq to accurately measure DNA methylation over a range of DNA quantities (from 10 to 1500 ng), both with and without whole-genome amplification (WGA) following bisulfite conversion. DNA methylation profiles generated using at least 100 ng correlated well (median R = 0.92) with those generated on Illumina Infinium HumanMethylation450 BeadChips, currently the platform of choice for epigenome-wide association studies (EWAS). WGA allowed for testing of samples with a starting DNA amount of 10 and 50 ng, although a reduced correlation was observed (median R = 0.79). We conclude that RainDrop BS-seq is suitable for measuring DNA methylation levels using nanogram quantities of DNA, and can be used to study candidate epigenetic biomarker loci in an accurate and high-throughput manner, paving the way for its application to routine clinical diagnostics.     

Links between plant and fungal communities across a deforestation chronosequence in the Amazon rainforest

Understanding the interactions among microbial communities, plant communities and soil properties following deforestation could provide insights into the long-term effects of land-use change on ecosystem functions, and may help identify approaches that promote the recovery of degraded sites. We combined high-throughput sequencing of fungal rDNA and molecular barcoding of plant roots to estimate fungal and plant community composition in soil sampled across a chronosequence of deforestation. We found significant effects of land-use change on fungal community composition, which was more closely correlated to plant community composition than to changes in soil properties or geographic distance, providing evidence for strong links between above- and below-ground communities in tropical forests.     

Methylome analysis identifies a Wilms tumor epigenetic biomarker detectable in blood

Background

Wilms tumor is the most common pediatric renal malignancy and there is a clinical need for a molecular biomarker to assess treatment response and predict relapse. The known mutated genes in this tumor type show low mutation frequencies, whereas aberrant methylation at 11p15 is by far the most common aberration. We therefore analyzed the epigenome, rather than the genome, to identify ubiquitous tumor-specific biomarkers.

Results

Methylome analysis of matched normal kidney and Wilms tumor identifies 309 preliminary methylation variable positions which we translate into three differentially methylated regions (DMRs) for use as tumor-specific biomarkers. Using two novel algorithms we show that these three DMRs are not confounded by cell type composition. We further show that these DMRs are not methylated in embryonic blastema but are intermediately methylated in Wilms tumor precursor lesions. We validate the biomarker DMRs using two independent sample sets of normal kidney and Wilms tumor and seven Wilms tumor histological subtypes, achieving 100% and 98% correct classification, respectively. As proof-of-principle for clinical utility, we successfully use biomarker DMR-2 in a pilot analysis of cell-free circulating DNA to monitor tumor response during treatment in ten patients.

Conclusions

These findings define the most common methylated regions in Wilms tumor known to date which are not associated with their embryonic origin or precursor stage. We show that this tumor-specific methylated DNA is released into the blood circulation where it can be detected non-invasively showing potential for clinical utility.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0434-y) contains supplementary material, which is available to authorized users.