Linear amplification for deep sequencing

Linear amplification for deep sequencing (LADS) is an amplification method that produces representative libraries for Illumina next-generation sequencing within 2 d. The method relies on attaching two different sequencing adapters to blunt-end repaired and A-tailed DNA fragments, wherein one of the adapters is extended with the sequence for the T7 RNA polymerase promoter. Ligated and size-selected DNA fragments are transcribed in vitro with high RNA yields. Subsequent cDNA synthesis is initiated from a primer complementary to the first adapter, ensuring that the library will only contain full-length fragments with two distinct adapters. Contrary to the severely biased representation of AT- or GC-rich fragments in standard PCR-amplified libraries, the sequence coverage in T7-amplified libraries is indistinguishable from that of nonamplified libraries. Moreover, in contrast to amplification-free methods, LADS can generate sequencing libraries from a few nanograms of DNA, which is essential for all applications in which the starting material is limited.     

Malaria: Could its unusual epigenome be the weak spot?

The epigenetic contribution to the regulation and maintenance of gene expression patterns by histone modifications is well established in eukaryotes. In Plasmodium falciparum, the mechanisms and factors regulating gene expression during progression through its infected red blood cell cycle (iRBC) and underlying mutually exclusive expression of antigenic variation genes involved in immune evasion are far from understood. Recently, the first comprehensive analyses of the P. falciparum chromatin landscape at different iRBC stages have been performed. These studies uncovered the existence of well-defined heterochromatic regions within a generally euchromatic epigenome. Notably, silencing of genes encoding for virulence determinants such as var genes, appears to be orchestrated by the concerted action of the Sir2 and HP1 orthologs and the presence of the histone mark, H3K9me3. Epigenetic speciation could make the parasite exquisitely vulnerable to epigenetic drug treatment, unless this deadly parasite still has a number of tricks up his sleeves.     

Correlations among salivary testosterone, mood, and selective attention to threat in humans.

An experiment was designed to investigate the relation among salivary testosterone, mood, and selective attention to threat. The participant group consisted of 32 nonclinical subjects (16 men and 16 women). Individuals completed the Profile Of Mood States (POMS) and performed a pictorial emotional Stroop task measuring selective attention to angry faces. Anticipating a time lag between testosterone (as measured in saliva) and cognitive emotional behavior, multiple time-coursed saliva samples were taken preceding the assessment of questionnaire and task for every subject. In both sexes, salivary testosterone was significantly related to mood (i.e., anger and tension) and selective attention to angry faces when saliva samples were taken 6 h before questionnaire and task assessment. Research on the relation between testosterone and human behavior might benefit by taking into account time lags between the behavioral manifestations and the continuously changing levels of testosterone.     

Assessing changes to ecosystem structure and function following invasion by Spartina alterniflora and Phragmites australis: a meta-analysis

Biological invasions resulting from anthropogenic activities are one of the greatest threats to maintaining ecosystem functioning and native biodiversity. Invasions are especially problematic when the invading species behaves as an ecosystem engineer that is capable of transforming ecosystem structure, function, and community dynamics. Of particular concern is the spread of emergent wetland grasses whose root systems alter hydrology and structural stability of soils, modify ecosystem functions, and change community dynamics and species richness. To address the threats posed to ecosystems across the globe, management practices focus on the control and removal of invasive grasses. However, it remains unclear how severely invasive grasses alter ecosystem functions and whether alterations persist after invasive grass removal, limiting our ability to determine if management practices are truly sufficient to fully restore ecosystems. Here, we conducted a meta-analysis to quantify ecological alterations and the efficacy of management following the invasion of Spartina alterniflora and Phragmites australis, two common and pervasive invaders in coastal wetlands. Our results indicate that S. alterniflora and P. australis significantly alter measures of ecosystem functioning and organismal abundance. Invaded ecosystems had significant elevations in abiotic carbon and nitrogen fixation and uptake in areas with invasive grasses, with differential photosynthetic pathways of these two grass species further explaining carbon fluxes. Moreover, evidence from our analyses indicates that management practices may not adequately promote recovery from invasion, but more data are needed to fully assess management efficacy. We call for future studies to conduct pairwise comparisons between uninvaded, invaded, and managed systems and provide research priorities.

     

Pharmacokinetics of the beta-carboline norharman in man.

In healthy subjects, pharmacokinetics were characterised using single oral and sublingual administrations of the beta-carboline norharman. For this purpose, norharman levels in blood plasma were measured up to 90-105 min after both routes of administration. Dose proportionality of three different single oral doses of norharman (7, 65 and 110 microg/kg) administered as 0.52 and 5 mg capsules was evaluated at 8 time points. Peak levels were attained at 30 min after the oral load of norharman. Mean relative availabilities determined by the area under the curve (AUC) procedure were 14.3 and 98.0 nmol.min/l after oral dosing of 7 and 65 microg/kg, respectively. AUC values in women were 3-4 times higher than in men. Sublingual dosing of 6.5 and 13 microg/kg norharman encapsulated in 5 mg of cyclodextrins resulted in a much higher mean AUC and a more rapid absorption. Mean AUC after sublingual administration of 6.5 microg/kg was 929.8 nmol.min/kg and plasma levels were maximal 10-15 min after norharman was given. Moreover, apparently no sex difference was found using this way of application. Norharman disappeared from the plasma with half-lifes of 25-35 min, irrespective of the route of administration. Even at the highest measured norharman levels of 53 nmol/l plasma, no behavioral effects were observed. In addition, the subjects did neither report any effects nor any side-effects during the experiment. This is the first study in which the kinetics of ingested norharman have been measured in humans.     

MBD2/NuRD and MBD3/NuRD, two distinct complexes with different biochemical and functional properties

The human genome contains a number of methyl CpG binding proteins that translate DNA methylation into a physiological response. To gain insight into the function of MBD2 and MBD3, we first applied protein tagging and mass spectrometry. We show that MBD2 and MBD3 assemble into mutually exclusive distinct Mi-2/NuRD-like complexes, called MBD2/NuRD and MBD3/NuRD. We identified DOC-1, a putative tumor suppressor, as a novel core subunit of MBD2/NuRD as well as MBD3/NuRD. PRMT5 and its cofactor MEP50 were identified as specific MBD2/NuRD interactors. PRMT5 stably and specifically associates with and methylates the RG-rich N terminus of MBD2. Chromatin immunoprecipitation experiments revealed that PRMT5 and MBD2 are recruited to CpG islands in a methylation-dependent manner in vivo and that H4R3, a substrate of PRMT, is methylated at these loci. Our data show that MBD2/NuRD and MBD3/NuRD are distinct protein complexes with different biochemical and functional properties.     

Plasmodium falciparum centromeres display a unique epigenetic makeup and cluster prior to and during schizogony

Centromeres are essential for the faithful transmission of chromosomes to the next generation, therefore being essential in all eukaryotic organisms. The centromeres of Plasmodium falciparum, the causative agent of the most severe form of malaria, have been broadly mapped on most chromosomes, but their epigenetic composition remained undefined. Here, we reveal that the centromeric histone variant PfCENH3 occupies a 4–4.5 kb region on each P. falciparum chromosome, which is devoid of pericentric heterochromatin but harbours another histone variant, PfH2A.Z. These CENH3 covered regions pinpoint the exact position of the centromere on all chromosomes and revealed that all centromeric regions have similar size and sequence composition. Immunofluorescence assay of PfCENH3 strongly suggests that P. falciparum centromerescluster to a single nuclear location prior to and during mitosis and cytokinesis but dissociate soon after invasion. In summary, we reveal a dynamic association of Plasmodium centromeres, which bear a unique epigenetic signature and conform to a strict structure. These findings suggest that DNA‐associated and epigenetic elements play an important role in centromere establishment in this important human pathogen.