Edesign: Primer and Enhanced Internal Probe Design Tool for Quantitative PCR Experiments and Genotyping Assays

Analytical PCR experiments preferably use internal probes for monitoring the amplification reaction and specific detection of the amplicon. Such internal probes have to be designed in close context with the amplification primers, and may require additional considerations for the detection of genetic variations. Here we describe Edesign, a new online and stand-alone tool for designing sets of PCR primers together with an internal probe for conducting quantitative real-time PCR (qPCR) and genotypic experiments. Edesign can be used for selecting standard DNA oligonucleotides like for instance TaqMan probes, but has been further extended with new functions and enhanced design features for Eprobes. Eprobes, with their single thiazole orange-labelled nucleotide, allow for highly sensitive genotypic assays because of their higher DNA binding affinity as compared to standard DNA oligonucleotides. Using new thermodynamic parameters, Edesign considers unique features of Eprobes during primer and probe design for establishing qPCR experiments and genotyping by melting curve analysis. Additional functions in Edesign allow probe design for effective discrimination between wild-type sequences and genetic variations either using standard DNA oligonucleotides or Eprobes. Edesign can be freely accessed online at http://www.dnaform.com/edesign2/, and the source code is available for download.     

Heterologous Expression Screens in Nicotiana benthamiana Identify a Candidate Effector of the Wheat Yellow Rust Pathogen that Associates with Processing Bodies

Rust fungal pathogens of wheat (Triticum spp.) affect crop yields worldwide. The molecular mechanisms underlying the virulence of these pathogens remain elusive, due to the limited availability of suitable molecular genetic research tools. Notably, the inability to perform high-throughput analyses of candidate virulence proteins (also known as effectors) impairs progress. We previously established a pipeline for the fast-forward screens of rust fungal candidate effectors in the model plant Nicotiana benthamiana. This pipeline involves selecting candidate effectors in silico and performing cell biology and protein-protein interaction assays in planta to gain insight into the putative functions of candidate effectors. In this study, we used this pipeline to identify and characterize sixteen candidate effectors from the wheat yellow rust fungal pathogen Puccinia striiformis f sp tritici. Nine candidate effectors targeted a specific plant subcellular compartment or protein complex, providing valuable information on their putative functions in plant cells. One candidate effector, {"type":"entrez-protein","attrs":{"text":"PST02549","term_id":"1371656274"}}PST02549, accumulated in processing bodies (P-bodies), protein complexes involved in mRNA decapping, degradation, and storage. {"type":"entrez-protein","attrs":{"text":"PST02549","term_id":"1371656274"}}PST02549 also associates with the P-body-resident ENHANCER OF mRNA DECAPPING PROTEIN 4 (EDC4) from N. benthamiana and wheat. We propose that P-bodies are a novel plant cell compartment targeted by pathogen effectors.     

Hand Preference for a Novel Bimanual Coordinated Task During Termite Feeding in Wild Western Gorillas (<Emphasis Type="Italic">Gorilla gorilla gorilla</Emphasis>)

Although the level of handedness in humans varies cross-culturally, humans are generally described as right-handed, which has been considered a uniquely human trait. Recently, captive chimpanzees (Pan troglodytes) have been shown to exhibit right-hand preference when performing bimanual but not unimanual tasks. Less clear is whether this pattern also occurs in wild chimpanzees and other African apes. Using videos (N = 49) of six wild western gorillas (Gorilla gorilla gorilla) feeding on termites at the Mondika Research Center (Republic of Congo), we tested whether they exhibit hand preference when performing unimanual, i.e., reaching for termite mound pieces; bimanual, i.e., “termite tapping”: rhythmically shaking a piece of termite mound with the dominant hand and collecting the termites in the other hand tasks; or hand transfer prior to bimanual tasks, i.e., transferring a piece of termite mound from one hand to the other. All individuals exhibited exclusive hand preference when performing the bimanual tasks, with five of six gorillas preferring the right hand. Conversely, most individuals did not show any manual preference during the unimanual task. In addition, hand preference during hand transfer revealed clear hand dominance of similar strength and direction of those shown for the bimanual task, suggesting that this measure is as sensitive as the bimanual task itself. Thus, we propose “termite feeding” as a novel task to be considered in future hand-preference studies in wild western gorillas. Our results are in concordance with those for chimpanzees and captive gorillas showing hemispheric specialization for bimanual actions in apes.     

Effects of anti‐human T lymphocyte immune globulins in patients: new or old

Multiple studies demonstrated that anti‐human T lymphocyte immune globulins (ATG) can decrease the incidence of acute and chronic graft rejection in cell or organ transplants. However, further in‐depth study indicates that different subgroups may benefit from either different regimes or alteration of them. Studies among renal transplant patients indicate that low immunological risk patients may not gain the same amount of benefit and thus tilt the risk versus benefit consideration. This may hold true for low immunological risk patients receiving other organ transplants and would be worth further investigation. The recovery time of T cells and natural killer (NK) cells also bears consideration and the impact that it has on the severity and incidence of opportunistic infections closely correlated with the dosage of ATG. The use of lower doses of ATG in combination with other induction medications may offer a solution. The finding that ATG may lose efficacy in cases of multiple transplants or re‐transplants in the case of heart transplants may hold true for other transplantations. This may lead to reconsideration of which induction therapies would be most beneficial in the clinical setting. These studies on ATG done on different patient groups will naturally not be applicable to all, but the evidence accrued from them as a whole may offer us new and different perspectives on how to approach and potentially solve the clinical question of how to best reduce the mortality associated with chronic host‐versus‐graft disease.     

Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis

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Chemical modifications of imidazole-containing alkoxyamines increase C–ON bond homolysis rate: Effects on their cytotoxic properties in glioblastoma cells

Previously, we described alkoxyamines bearing a pyridine ring as new pro-drugs with low molecular weights and theranostic activity. Upon chemical stimulus, alkoxyamines undergo homolysis and release free radicals, which can, reportedly, enhance magnetic resonance imaging and trigger cancer cell death. In the present study, we describe the synthesis and the anti-cancer activity of sixteen novel alkoxyamines that contain an imidazole ring. Activation of the homolysis was conducted by protonation and/or methylation. These new molecules displayed cytotoxic activities towards human glioblastoma cell lines, including the U251-MG cells that are highly resistant to the conventional chemotherapeutic agent Temozolomide. We further showed that the biological activities of the alkoxyamines were not only related to their half-life times of homolysis. We lastly identified the alkoxyamine (RS/SR)-4a, with both a high antitumour activity and favourable logD_7.4 and pK_a values, which make it a robust candidate for blood-brain barrier penetrating therapeutics against brain neoplasia.     

Looking to the future: key points for sustainable management of northern Great Plains grasslands

The grasslands of the northern Great Plains (NGP) region of North America are considered endangered ecosystems and priority conservation areas yet have great ecological and economic importance. Grasslands in the NGP are no longer self‐regulating adaptive systems. The challenges to these grasslands are widespread and serious (e.g. climate change, invasive species, fragmentation, altered disturbance regimes, and anthropogenic chemical loads). Because the challenges facing the region are dynamic, complex, and persistent, a paradigm shift in how we approach restoration and management of the grasslands in the NGP is imperative. The goal of this article is to highlight four key points for land managers and restoration practitioners to consider when planning management or restoration actions. First, we discuss the appropriateness of using historical fidelity as a restoration or management target because of changing climate, widespread pervasiveness of invasive species, the high level of fragmentation, and altered disturbance regimes. Second, we highlight ecosystem resilience and long‐term population persistence as alternative targets. Third, because the NGP is so heavily impacted with anthropogenic chemical loading, we discuss the risks of ecological traps and extinction debt. Finally, we highlight the importance of using adaptive management and having patience during restoration and management. Consideration of these four points will help management and restoration of grasslands move toward a more successful and sustainable future. Although we specifically focus on the NGP of North America, these same issues and considerations apply to grasslands and many other ecosystems globally.