Robust Quantification of Polymerase Chain Reactions Using Global Fitting

BACKGROUND: Quantitative polymerase chain reactions (qPCR) are used to monitor relative changes in very small amounts of DNA. One drawback to qPCR is reproducibility: measuring the same sample multiple times can yield data that is so noisy that important differences can be dismissed. Numerous analytical methods have been employed that can extract the relative template abundance between samples. However, each method is sensitive to baseline assignment and to the unique shape profiles of individual reactions, which gives rise to increased variance stemming from the analytical procedure itself. PRINCIPAL FINDINGS: We developed a simple mathematical model that accurately describes the entire PCR reaction profile using only two reaction variables that depict the maximum capacity of the reaction and feedback inhibition. This model allows quantification that is more accurate than existing methods and takes advantage of the brighter fluorescence signals from later cycles. Because the model describes the entire reaction, the influences of baseline adjustment errors, reaction efficiencies, template abundance, and signal loss per cycle could be formalized. We determined that the common cycle-threshold method of data analysis introduces unnecessary variance because of inappropriate baseline adjustments, a dynamic reaction efficiency, and also a reliance on data with a low signal-to-noise ratio. SIGNIFICANCE: Using our model, fits to raw data can be used to determine template abundance with high precision, even when the data contains baseline and signal loss defects. This improvement reduces the time and cost associated with qPCR and should be applicable in a variety of academic, clinical, and biotechnological settings

Rapid Depletion of Target Proteins Allows Identification of Coincident Physiological Responses

Targeted protein degradation is a powerful tool that can be used to create unique physiologies depleted of important factors. Current strategies involve modifying a gene of interest such that a degradation peptide is added to an expressed target protein and then conditionally activating proteolysis, either by expressing adapters, unmasking cryptic recognition determinants, or regulating protease affinities using small molecules. For each target, substantial optimization may be required to achieve a practical depletion, in that the target remains present at a normal level prior to induction and is then rapidly depleted to levels low enough to manifest a physiological response. Here, we describe a simplified targeted degradation system that rapidly depletes targets and that can be applied to a wide variety of proteins without optimizing target protease affinities. The depletion of the target is rapid enough that a primary physiological response manifests that is related to the function of the target. Using ribosomal protein Si as an example, we show that the rapid depletion of this essential translation factor invokes concomitant changes to the levels of several mRNAs, even before appreciable cell division has occurred

Carbon on the Northwest European Shelf: Contemporary Budget and Future Influences

A carbon budget for the northwest European continental shelf seas (NWES) was synthesized using available estimates for coastal, pelagic and benthic carbon stocks and flows. Key uncertainties were identified and the effect of future impacts on the carbon budget were assessed. The water of the shelf seas contains between 210 and 230 Tmol of carbon and absorbs between 1.3 and 3.3 Tmol from the atmosphere annually. Off-shelf transport and burial in the sediments account for 60–100 and 0–40% of carbon outputs from the NWES, respectively. Both of these fluxes remain poorly constrained by observations and resolving their magnitudes and relative importance is a key research priority. Pelagic and benthic carbon stocks are dominated by inorganic carbon. Shelf sediments contain the largest stock of carbon, with between 520 and 1600 Tmol stored in the top 0.1 m of the sea bed. Coastal habitats such as salt marshes and mud flats contain large amounts of carbon per unit area but their total carbon stocks are small compared to pelagic and benthic stocks due to their smaller spatial extent. The large pelagic stock of carbon will continue to increase due to the rising concentration of atmospheric CO2, with associated pH decrease. Pelagic carbon stocks and flows are also likely to be significantly affected by increasing acidity and temperature, and circulation changes but the net impact is uncertain. Benthic carbon stocks will be affected by increasing temperature and acidity, and decreasing oxygen concentrations, although the net impact of these interrelated changes on carbon stocks is uncertain and a major knowledge gap. The impact of bottom trawling on benthic carbon stocks is unique amongst the impacts we consider in that it is widespread and also directly manageable, although its net effect on the carbon budget is uncertain. Coastal habitats are vulnerable to sea level rise and are strongly impacted by management decisions. Local, national and regional actions have the potential to protect or enhance carbon storage, but ultimately global governance, via controls on emissions, has the greatest potential to influence the long-term fate of carbon stocks in the northwestern European continental shelf

Detection of HIV-1 dual infections in highly exposed treated patients

<p>Abstract</p> <p>Background</p> <p>Genetic characterization of HIV-1 in Argentina has shown that BF recombinants predominate among heterosexuals and injecting drug users, while in men who have sex with men the most prevalent form is subtype B.</p> <p>Objectives</p> <p>The aim of this work was to investigate the presence of HIV dual infections in HIV-infected individuals with high probability of reinfection</p> <p>Study design</p> <p>Blood samples were collected from 23 HIV positive patients with the risk of reinfection from Buenos Aires. A fragment of the HIV gene <it>pol </it>was amplified and phylogenetic analyses were performed. Antiretroviral drug resistance patterns of all the sequences were analyzed.</p> <p>Results</p> <p>Five dual infections were detected with four patients coinfected with subtype B and BF recombinants and one patient was coinfected with two BF recombinants presenting different recombination patterns. Prolonged infection with a stable clinical condition was observed in the five individuals. Resistance mutation patterns were different between the predominant and the minority strains.</p> <p>Conclusions</p> <p>Our results show that HIV dual infection can occur with closely related subtypes, and even with different variants of the same recombinant form in certain populations. Clinical observations showed neither aggressive disease progression nor impact on the resistance patterns in the dually-infected patients.</p

Genomewide Association Study of Statin-Induced Myopathy in Patients Recruited Using the UK Clinical Practice Research Datalink.

Statins can be associated with myopathy. We have undertaken a genomewide association study (GWAS) to discover and validate genetic risk factors for statin-induced myopathy in a "real-world" setting. One hundred thirty-five patients with statin myopathy recruited via the UK Clinical Practice Research Datalink were genotyped using the Illumina OmniExpress Exome version 1.0 Bead Chip and compared with the Wellcome Trust Case-Control Consortium (n = 2,501). Nominally statistically significant single nucleotide polymorphism (SNP) signals in the GWAS (P T in the SLCO1B1 gene) SNP was genomewide significant in the severe myopathy (creatine kinase > 10 × upper limit of normal or rhabdomyolysis) group (P = 2.55 × 10-9 ; odds ratio 5.15; 95% confidence interval 3.13-8.45). The association with SLCO1B1 was present for several statins and replicated in the independent validation cohorts. The data highlight the role of SLCO1B1 c.521C>T SNP as a replicable genetic risk factor for statin myopathy. No other novel genetic risk factors with a similar effect size were identified

A cell topography-based mechanism for ligand discrimination by the T cell receptor.

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.This work was funded by The Wellcome Trust, the UK Medical Research Council, the UK Biotechnology and Biological Sciences Research Council and Cancer Research UK. We thank the Wolfson Imaging Centre, University of Oxford, for access to their microscope facility. We would like to thank the Wellcome Trust for the Sir Henry Dale Fellowship of R.A.F. (WT101609MA), the Royal Society for the University Research Fellowship of S.F.L. (UF120277) and acknowledge a GSK Professorship (D.K.). We are also grateful to Doug Tischer (UCSF, US) and Muaz Rushdi (Georgia Tech, US) for their critical comments on the manuscript

Bright spots as climate‐smart marine spatial planning tools for conservation and blue growth

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Redefining professional identity: the voice of a language teacher in a context of collaborative learning

Following a narrative and biographic approach, in this study, we present the case of an in-service language teacher and her professional learning trajectory in the context of the project ‘Languages and education: constructing and sharing train- ing’. This project aimed at the construction of a collaborative teacher education context for learning and transformation of experiences, views and practices in language education, and involved teachers, teacher educators and researchers. Based on a single case study, the analysis tries to disclose the teacher’s discur- sive displacements as hints of professional transformation while she reinterprets the learning taking place in the collaborative education process. The signs of change are visible in the way she constructs meanings regarding her professional identity, re-identifies her mission as a language teacher and reconsiders her pro- fessional identity. Finally, we reflect upon how collaborative teacher education scenarios may foster teachers’ personal professional learning and renewed self- images

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure