A Sensitive Branched DNA HIV-1 Signal Amplification Viral Load Assay with Single Day Turnaround

Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) (“Versant Assay”) currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16–18 h to 2.5 h, composition of only the “Lysis Diluent” solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements

Evolution favors protein mutational robustness in sufficiently large populations

BACKGROUND: An important question is whether evolution favors properties such as mutational robustness or evolvability that do not directly benefit any individual, but can influence the course of future evolution. Functionally similar proteins can differ substantially in their robustness to mutations and capacity to evolve new functions, but it has remained unclear whether any of these differences might be due to evolutionary selection for these properties. RESULTS: Here we use laboratory experiments to demonstrate that evolution favors protein mutational robustness if the evolving population is sufficiently large. We neutrally evolve cytochrome P450 proteins under identical selection pressures and mutation rates in populations of different sizes, and show that proteins from the larger and thus more polymorphic population tend towards higher mutational robustness. Proteins from the larger population also evolve greater stability, a biophysical property that is known to enhance both mutational robustness and evolvability. The excess mutational robustness and stability is well described by existing mathematical theories, and can be quantitatively related to the way that the proteins occupy their neutral network. CONCLUSIONS: Our work is the first experimental demonstration of the general tendency of evolution to favor mutational robustness and protein stability in highly polymorphic populations. We suggest that this phenomenon may contribute to the mutational robustness and evolvability of viruses and bacteria that exist in large populations

Systematic Conservation Planning in the Face of Climate Change: Bet-Hedging on the Columbia Plateau

Systematic conservation planning efforts typically focus on protecting current patterns of biodiversity. Climate change is poised to shift species distributions, reshuffle communities, and alter ecosystem functioning. In such a dynamic environment, lands selected to protect today's biodiversity may fail to do so in the future. One proposed approach to designing reserve networks that are robust to climate change involves protecting the diversity of abiotic conditions that in part determine species distributions and ecological processes. A set of abiotically diverse areas will likely support a diversity of ecological systems both today and into the future, although those two sets of systems might be dramatically different. Here, we demonstrate a conservation planning approach based on representing unique combinations of abiotic factors. We prioritize sites that represent the diversity of soils, topographies, and current climates of the Columbia Plateau. We then compare these sites to sites prioritized to protect current biodiversity. This comparison highlights places that are important for protecting both today's biodiversity and the diversity of abiotic factors that will likely determine biodiversity patterns in the future. It also highlights places where a reserve network designed solely to protect today's biodiversity would fail to capture the diversity of abiotic conditions and where such a network could be augmented to be more robust to climate-change impacts

Mediator and cohesin connect gene expression and chromatin architecture

Transcription factors control cell-specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. Here we report that mediator and cohesin physically and functionally connect the enhancers and core promoters of active genes in murine embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with cohesin, which can form rings that connect two DNA segments. The cohesin-loading factor Nipbl is associated with mediator–cohesin complexes, providing a means to load cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by mediator and cohesin. Mediator and cohesin co-occupy different promoters in different cells, thus generating cell-type-specific DNA loops linked to the gene expression program of each cell.National Institutes of Health (U.S.) (Fellowship)Canadian Institutes of Health Research (Research Fellowship)National Institutes of Health (U.S.) (Grant R01 HG002668

Sildenafil, a phosphodiesterase type 5 inhibitor, enhances the antidepressant activity of amitriptyline but not desipramine, in the forced swim test in mice

The cholinergic theory of depression highlights the involvement of muscarinic acetylcholine receptors in the neurobiology of mood disorders. The present study was designed to investigate the effect of sildenafil, a phosphodiesterase type 5 inhibitor which exhibits cholinomimetic properties, alone and in combination with scopolamine in the forced swim test in mice. Moreover, we assessed the ability of sildenafil to modify the antidepressant activity of two tricyclic antidepressants with distinct cholinolytic activity, amitriptyline and desipramine. Swim sessions were conducted by placing mice in glass cylinders filled with water for 6 min and the duration of behavioral immobility during the last 4 min of the test was evaluated. Locomotor activity was measured with photoresistor actimeters. To evaluate the potential pharmacokinetic interaction between amitriptyline and sildenafil, brain and serum concentrations of amitriptyline were determined by HPLC. Sildenafil (1.25–20 mg/kg) as well as scopolamine (0.5 mg/kg) and its combination with sildenafil (1.25 mg/kg) did not affect the total immobility time duration. However, joint administration of scopolamine with sildenafil at doses of 2.5 and 5 mg/kg significantly reduced immobility time as compared to control group. Moreover, co-administration of scopolamine with sildenafil at the highest dose (5 mg/kg) significantly decreased immobility time as compared to scopolamine-treated group. Sildenafil (1.25, 2.5 and 5 mg/kg) significantly enhanced the antidepressant activity of amitriptyline (5 mg/kg). No changes in anti-immobility action of desipramine (20 mg/kg) in combination with sildenafil (5, 10 and 20 mg/kg) were observed. Sildenafil did not affect amitriptyline level in both brain and serum. In conclusion, the present study suggests that sildenafil may enhance the activity of antidepressant drugs which exhibit cholinolytic activity

CD4 T Cell Immunity Is Critical for the Control of Simian Varicella Virus Infection in a Nonhuman Primate Model of VZV Infection

Primary infection with varicella zoster virus (VZV) results in varicella (more commonly known as chickenpox) after which VZV establishes latency in sensory ganglia. VZV can reactivate to cause herpes zoster (shingles), a debilitating disease that affects one million individuals in the US alone annually. Current vaccines against varicella (Varivax) and herpes zoster (Zostavax) are not 100% efficacious. Specifically, studies have shown that 1 dose of varivax can lead to breakthrough varicella, albeit rarely, in children and a 2-dose regimen is now recommended. Similarly, although Zostavax results in a 50% reduction in HZ cases, a significant number of recipients remain at risk. To design more efficacious vaccines, we need a better understanding of the immune response to VZV. Clinical observations suggest that T cell immunity plays a more critical role in the protection against VZV primary infection and reactivation. However, no studies to date have directly tested this hypothesis due to the scarcity of animal models that recapitulate the immune response to VZV. We have recently shown that SVV infection of rhesus macaques models the hallmarks of primary VZV infection in children. In this study, we used this model to experimentally determine the role of CD4, CD8 and B cell responses in the resolution of primary SVV infection in unvaccinated animals. Data presented in this manuscript show that while CD20 depletion leads to a significant delay and decrease in the antibody response to SVV, loss of B cells does not alter the severity of varicella or the kinetics/magnitude of the T cell response. Loss of CD8 T cells resulted in slightly higher viral loads and prolonged viremia. In contrast, CD4 depletion led to higher viral loads, prolonged viremia and disseminated varicella. CD4 depleted animals also had delayed and reduced antibody and CD8 T cell responses. These results are similar to clinical observations that children with agammaglobulinemia have uncomplicated varicella whereas children with T cell deficiencies are at increased risk of progressive varicella with significant complications. Moreover, our studies indicate that CD4 T cell responses to SVV play a more critical role than antibody or CD8 T cell responses in the control of primary SVV infection and suggest that one potential mechanism for enhancing the efficacy of VZV vaccines is by eliciting robust CD4 T cell responses

A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome

Citation: Chapman, J. A., Mascher, M., Buluç, A., Barry, K., Georganas, E., Session, A., . . . Rokhsar, D. S. (2015). A whole-genome shotgun approach for assembling and anchoring the hexaploid bread wheat genome. Genome Biology, 16(1). doi:10.1186/s13059-015-0582-8Polyploid species have long been thought to be recalcitrant to whole-genome assembly. By combining high-throughput sequencing, recent developments in parallel computing, and genetic mapping, we derive, de novo, a sequence assembly representing 9.1 Gbp of the highly repetitive 16 Gbp genome of hexaploid wheat, Triticum aestivum, and assign 7.1 Gb of this assembly to chromosomal locations. The genome representation and accuracy of our assembly is comparable or even exceeds that of a chromosome-by-chromosome shotgun assembly. Our assembly and mapping strategy uses only short read sequencing technology and is applicable to any species where it is possible to construct a mapping population. © 2015 Chapman et al. licensee BioMed Central.Additional Authors: Muehlbauer, G. J.;Stein, N.;Rokhsar, D. S

Time domains of the hypoxic ventilatory response in ectothermic vertebrates

Over a decade has passed since Powell et al. (Respir Physiol 112:123–134, 1998) described and defined the time domains of the hypoxic ventilatory response (HVR) in adult mammals. These time domains, however, have yet to receive much attention in other vertebrate groups. The initial, acute HVR of fish, amphibians and reptiles serves to minimize the imbalance between oxygen supply and demand. If the hypoxia is sustained, a suite of secondary adjustments occur giving rise to a more long-term balance (acclimatization) that allows the behaviors of normal life. These secondary responses can change over time as a function of the nature of the stimulus (the pattern and intensity of the hypoxic exposure). To add to the complexity of this process, hypoxia can also lead to metabolic suppression (the hypoxic metabolic response) and the magnitude of this is also time dependent. Unlike the original review of Powell et al. (Respir Physiol 112:123–134, 1998) that only considered the HVR in adult animals, we also consider relevant developmental time points where information is available. Finally, in amphibians and reptiles with incompletely divided hearts the magnitude of the ventilatory response will be modulated by hypoxia-induced changes in intra-cardiac shunting that also improve the match between O2 supply and demand, and these too change in a time-dependent fashion. While the current literature on this topic is reviewed here, it is noted that this area has received little attention. We attempt to redefine time domains in a more ‘holistic’ fashion that better accommodates research on ectotherms. If we are to distinguish between the genetic, developmental and environmental influences underlying the various ventilatory responses to hypoxia, however, we must design future experiments with time domains in mind