'Footprinting' proteins on DNA with peroxonitrous acid.

The peroxonitrite anion (ONOO-) is a stable species in alkaline solution that quickly generates a strong oxidant at neutral pH. A convenient procedure for the preparation of ONOOK has been developed based on the procedure of Keith & Powell [(1969) J. Chem. Soc. A, 90], which when added to a sample of duplex DNA buffered at neutral pH rapidly generates a strong oxidant capable of nonspecifically cleaving the DNA present. We show that this solution containing ONOOK can be used to hydroxyl radical footprint the binding the cl-repressor (cl) of phage lambda with the right operator, OR. In addition, we show that the individual-site binding isotherms determined by quantitative DNase I, Fe-EDTA and ONOOK footprinting are identical within experimental error. The identical isotherms obtained with the three different reagents with greatly differing sampling times indicates that the sampling time of the footprinting probe need not be short relative to the kinetic dissociation constants that govern protein-DNA interactions.     

Ocean warming and acidification have complex interactive effects on the dynamics of a marine fungal disease

Diseases threaten the structure and function of marine ecosystems and are contributing to the global decline of coral reefs. We currently lack an understanding of how climate change stressors, such as ocean acidification (OA) and warming, may simultaneously affect coral reef disease dynamics, particularly diseases threatening key reef-building organisms, for example crustose coralline algae (CCA). Here, we use coralline fungal disease (CFD), a previously described CCA disease from the Pacific, to examine these simultaneous effects using both field observations and experimental manipulations. We identify the associated fungus as belonging to the subphylum Ustilaginomycetes and show linear lesion expansion rates on individual hosts can reach 6.5 mm per day. Further, we demonstrate for the first time, to our knowledge, that ocean-warming events could increase the frequency of CFD outbreaks on coral reefs, but that OA-induced lowering of pH may ameliorate outbreaks by slowing lesion expansion rates on individual hosts. Lowered pH may still reduce overall host survivorship, however, by reducing calcification and facilitating fungal bio-erosion. Such complex, interactive effects between simultaneous extrinsic environmental stressors on disease dynamics are important to consider if we are to accurately predict the response of coral reef communities to future climate change.     

Virus genome dynamics under different propagation pressures: reconstruction of whole genome haplotypes of west nile viruses from NGS data

Background

Extensive focus is placed on the comparative analyses of consensus genotypes in the study of West Nile virus (WNV) emergence. Few studies account for genetic change in the underlying WNV quasispecies population variants. These variants are not discernable in the consensus genome at the time of emergence, and the maintenance of mutation-selection equilibria of population variants is greatly underestimated. The emergence of lineage 1 WNV strains has been studied extensively, but recent epidemics caused by lineage 2 WNV strains in Hungary, Austria, Greece and Italy emphasizes the increasing importance of this lineage to public health. In this study we explored the quasispecies dynamics of minority variants that contribute to cell-tropism and host determination, i.e. the ability to infect different cell types or cells from different species from Next Generation Sequencing (NGS) data of a historic lineage 2 WNV strain.

Results

Minority variants contributing to host cell membrane association persist in the viral population without contributing to the genetic change in the consensus genome. Minority variants are shown to maintain a stable mutation-selection equilibrium under positive selection, particularly in the capsid gene region.

Conclusions

This study is the first to infer positive selection and the persistence of WNV haplotype variants that contribute to viral fitness without accompanying genetic change in the consensus genotype, documented solely from NGS sequence data. The approach used in this study streamlines the experimental design seeking viral minority variants accurately from NGS data whilst minimizing the influence of associated sequence error.     

An active transposable element, Herves, from the African malaria mosquito Anopheles gambiae

Transposable elements have proven to be invaluable tools for genetically manipulating a wide variety of plants, animals, and microbes. Some have suggested that they could be used to spread desirable genes, such as refractoriness to Plasmodium infection, through target populations of Anopheles gambiae, thereby disabling the mosquito's ability to transmit malaria. To achieve this, a transposon must remain mobile and intact after the initial introduction into the genome. Endogenous, active class II transposable elements from An. gambiae have not been exploited as gene vectors/drivers because none have been isolated. We report the discovery of an active class II transposable element, Herves, from the mosquito An. gambiae. Herves is a member of a distinct subfamily of hAT elements that includes the hopper-we element from Bactrocera dorsalis and B. cucurbitae. Herves was transpositionally active in mobility assays performed in Drosophila melanogaster S2 cells and developing embryos and was used as a germ-line transformation vector in D. melanogaster. Herves displays an altered target-site preference from the distantly related hAT elements, Hermes and hobo. Herves is also present in An. arabiensis and An. merus with copy numbers similar to that found in An. gambiae. Preliminary data from an East African population are consistent with the element being transpositionally active in mosquitoes.     

Sedation at Sea of Entangled North Atlantic Right Whales (Eubalaena glacialis) to Enhance Disentanglement

Background

The objective of this study was to enhance removal of fishing gear from right whales (Eubalaena glacialis) at sea that evade disentanglement boat approaches. Titrated intra muscular injections to achieve sedation were undertaken on two free swimming right whales.

Methodology/Principal Findings

Following initial trials with beached whales, a sedation protocol was developed for right whales. Mass was estimated from sighting and necropsy data from comparable right whales. Midazolam (0.01 to 0.025 mg/kg) was first given alone or with meperidine (0.17 to 0.25 mg/kg) either once or four times over two hours to whale #1102 by cantilevered pole syringe. In the last attempt on whale #1102 there appeared to be a mild effect in 20–30 minutes, with duration of less than 2 hours that included exhalation before the blowhole fully cleared the water. Boat avoidance, used as a measure of sedation depth, was not reduced. A second severely entangled animal in 2009, whale #3311, received midazolam (0.03 mg/kg) followed by butorphanol (0.03 mg/kg) an hour later, delivered ballistically. Two months later it was then given midazolam (0.07 mg/kg) and butorphanol (0.07 mg/kg) simultaneously. The next day both drugs at 0.1 mg/kg were given as a mixture in two darts 10 minutes apart. The first attempt on whale #3311 showed increased swimming speed and boat avoidance was observed after a further 20 minutes. The second attempt on whale #3311 showed respiration increasing mildly in frequency and decreasing in strength. The third attempt on whale #3311 gave a statistically significant increase in respiratory frequency an hour after injection, with increased swimming speed and marked reduction of boat evasion that enabled decisive cuts to entangling gear.

Conclusions/Significance

We conclude that butorphanol and midazolam delivered ballistically in appropriate dosages and combinations may have merit in future refractory free swimming entangled right whale cases until other entanglement solutions are developed.     

Side-to-side differences in anterior cruciate ligament volume in healthy control subjects

Examination of anterior cruciate ligament (ACL) anatomy is of great interest both in studying injury mechanisms and surgical reconstruction. However, after a typical acute ACL rupture it is not possible to measure the dimensions of the ACL itself due to concomitant or subsequent degeneration of the remaining ligamentous tissue. The contralateral ACL may be an appropriate surrogate for measuring anatomical dimensions, but it remains unknown whether side-to-side differences preclude using the contralateral as a valid surrogate for the ruptured ACL. This study examined whether the ACL volume is significantly different between the left and right knees of uninjured subjects. ACL volumes were calculated for the left and right sides of 28 individuals using a previously validated MRI-based method. The mean ACL volume was not significantly different (p=0.2331) between the two sides in this population. Side-to-side ACL volume was also well correlated (correlation=0.91, p<0.0001). The results of this study show that the volume of the contralateral ACL is a valid surrogate measure for a missing ACL on the injured side. This non-invasive, in vivo technique for measuring ACL volume may prove useful in future large-scale comprehensive studies of potential risk factors for ACL rupture, in quantifying potential loading effects on ACL size as a prophylactic measure against ACL rupture, and in the use of ACL volume as a screening tool for assessing risk of injury.     

Rainfall-driven sex-ratio genes in African buffalo suggested by correlations between Y-chromosomal haplotype frequencies and foetal sex ratio

Background  

The Y-chromosomal diversity in the African buffalo (Syncerus caffer) population of Kruger National Park (KNP) is characterized by rainfall-driven haplotype frequency shifts between year cohorts. Stable Y-chromosomal polymorphism is difficult to reconcile with haplotype frequency variations without assuming frequency-dependent selection or specific interactions in the population dynamics of X- and Y-chromosomal genes, since otherwise the fittest haplotype would inevitably sweep to fixation. Stable Y-chromosomal polymorphism due one of these factors only seems possible when there are Y-chromosomal distorters of an equal sex ratio, which act by negatively affecting X-gametes, or Y-chromosomal suppressors of a female-biased sex ratio. These sex-ratio (SR) genes modify (suppress) gamete transmission in their own favour at a fitness cost, allowing for stable polymorphism.     

Full-genome scan for linkage in 50 families segregating the bipolar affective disease phenotype

A genome scan of approximately 12-cM initial resolution was done on 50 of a set of 51 carefully ascertained unilineal multiplex families segregating the bipolar affective disorder phenotype. In addition to standard multipoint linkage analysis methods, a simultaneous-search algorithm was applied in an attempt to surmount the problem of genetic heterogeneity. The results revealed no linkage across the genome. The results exclude monogenic models and make it unlikely that two genes account for the disease in this sample. These results support the conclusion that at least several hundred kindreds will be required in order to establish linkage of susceptibility loci to bipolar disorder in heterogeneous populations.     

Swine in biomedical research: creating the building blocks of animal models

The opportunities for utilizing swine biomedical models are immense, particularly in models that address lifestyle issues (nutrition, stress, alcohol, drugs of abuse, etc.). However, in order to fully capitalize upon the promise, there needs to be a more general recognition of these cofactors, such as nutrition, as key modulators of phenotype via genomic, epigenetic, and postgenomic mechanisms. Furthermore, increased interactions between nutrition scientists and clinical and fundamental researchers in other disciplines, including developmental biology, immunology, neuroscience, oncology, and cardiovascular and gastrointestinal physiology, are required. Closing discussions focused on the need for future conferences at more frequent intervals to support interactions between the various disciplines. This was especially critical because of the global distribution of investigators.