Whole genome sequencing of enriched chloroplast DNA using the Illumina GAII platform

<p>Abstract</p> <p>Background</p> <p>Complete chloroplast genome sequences provide a valuable source of molecular markers for studies in molecular ecology and evolution of plants. To obtain complete genome sequences, recent studies have made use of the polymerase chain reaction to amplify overlapping fragments from conserved gene loci. However, this approach is time consuming and can be more difficult to implement where gene organisation differs among plants. An alternative approach is to first isolate chloroplasts and then use the capacity of high-throughput sequencing to obtain complete genome sequences. We report our findings from studies of the latter approach, which used a simple chloroplast isolation procedure, multiply-primed rolling circle amplification of chloroplast DNA, Illumina Genome Analyzer II sequencing, and de novo assembly of paired-end sequence reads.</p> <p>Results</p> <p>A modified rapid chloroplast isolation protocol was used to obtain plant DNA that was enriched for chloroplast DNA, but nevertheless contained nuclear and mitochondrial DNA. Multiply-primed rolling circle amplification of this mixed template produced sufficient quantities of chloroplast DNA, even when the amount of starting material was small, and improved the template quality for Illumina Genome Analyzer II (hereafter Illumina GAII) sequencing. We demonstrate, using independent samples of karaka (<it>Corynocarpus laevigatus</it>), that there is high fidelity in the sequence obtained from this template. Although less than 20% of our sequenced reads could be mapped to chloroplast genome, it was relatively easy to assemble complete chloroplast genome sequences from the mixture of nuclear, mitochondrial and chloroplast reads.</p> <p>Conclusions</p> <p>We report successful whole genome sequencing of chloroplast DNA from karaka, obtained efficiently and with high fidelity.</p

The social life of infants in the context of infectious disease transmission; social contacts and mixing patterns of the very young.

Insight into how humans interact helps further understanding of the transmission of infectious diseases. For diseases such as pertussis, infants are at particular risk for severe outcomes. To understand the contact pattern of infants, especially those too young to be vaccinated, we sent contact diaries to a representative sample of 1000 mothers in the United Kingdom. We received 115 responses with a total of 758 recorded contacts. The average number of daily contacts for an infant was 6.68 overall and 5.7 for those aged ≤ 10 weeks. Of the latter, 2.1 (37%) contacts were with non-household members and were >15 minutes duration, suggesting that a cocooning programme may miss a substantial proportion of exposures leading to disease transmission. The least contact was between adolescents and infants. Thus the impact of adolescent (pertussis) vaccination on infants would likely be limited, unless it reduces transmission to other age groups whose contact with infants is greater

The Coordinated Action of MYB Activators and Repressors Controls Proanthocyanidin and Anthocyanin Biosynthesis in Vaccinium

Vaccinium berries are regarded as “superfoods” owing to their high concentrations of anthocyanins, flavonoid metabolites that provide pigmentation and positively affect human health. Anthocyanin localization differs between the fruit of cultivated highbush blueberry (V. corymbosum) and wild bilberry (V. myrtillus), with the latter having deep red flesh coloration. Analysis of comparative transcriptomics across a developmental series of blueberry and bilberry fruit skin and flesh identified candidate anthocyanin regulators responsible for this distinction. This included multiple activator and repressor transcription factors (TFs) that correlated strongly with anthocyanin production and had minimal expression in blueberry (non-pigmented) flesh. R2R3 MYB TFs appeared key to the presence and absence of anthocyanin-based pigmentation; MYBA1 and MYBPA1.1 co-activated the pathway while MYBC2.1 repressed it. Transient overexpression of MYBA1 in Nicotiana benthamiana strongly induced anthocyanins, but this was substantially reduced when co-infiltrated with MYBC2.1. Co-infiltration of MYBC2.1 with MYBA1 also reduced activation of DFR and UFGT, key anthocyanin biosynthesis genes, in promoter activation studies. We demonstrated that these TFs operate within a regulatory hierarchy where MYBA1 activated the promoters of MYBC2.1 and bHLH2. Stable overexpression of VcMYBA1 in blueberry elevated anthocyanin content in transgenic plants, indicating that MYBA1 is sufficient to upregulate the TF module and activate the pathway. Our findings identify TF activators and repressors that are hierarchically regulated by SG6 MYBA1, and fine-tune anthocyanin production in Vaccinium. The lack of this TF module in blueberry flesh results in an absence of anthocyanins.publishedVersio

Painted flowers: Eluta generates pigment patterning in Antirrhinum

*In the early 1900s, Erwin Baur established Antirrhinum majus as a model system, identifying and characterising numerous flower colour variants. This included Picturatum /Eluta, which restricts the accumulation of magenta anthocyanin pigments, forming bullseye markings on the flower face. *We identified the gene underlying the Eluta locus by transposon-tagging, using an Antirrhinum line that spontaneously lost the non-suppressive el phenotype. A candidate MYB repressor gene at this locus contained a CACTA transposable element. We subsequently identified plants where this element excised, reverting to a suppressive Eluta phenotype. El alleles inhibit expression of anthocyanin biosynthetic genes, confirming it to be a regulatory locus. The modes of action of Eluta were investigated by generating stable transgenic tobacco lines, biolistic transformation of Antirrhinum petals and by promoter activation/repression assays. *Eluta competes with MYB activators for promoter cis-elements, and also by titrating essential co-factors (bHLH proteins) to reduce transcription of target genes. Eluta restricts the pigmentation established by the R2R3-MYB factors, Rosea and Venosa, with greatest repression on those parts of the petals where Eluta is most highly expressed. *Baur questioned the origin of heredity units determining flower colour variation in cultivated A. majus. Our findings support introgression from wild species into cultivated varieties. <br/

Analysing and Recommending Options for Maintaining Universal Coverage with Long-Lasting Insecticidal Nets: The Case of Tanzania in 2011.

Tanzania achieved universal coverage with long-lasting insecticidal nets (LLINs) in October 2011, after three years of free mass net distribution campaigns and is now faced with the challenge of maintaining high coverage as nets wear out and the population grows. A process of exploring options for a continuous or "Keep-Up" distribution system was initiated in early 2011. This paper presents for the first time a comprehensive national process to review the major considerations, findings and recommendations for the implementation of a new strategy. Stakeholder meetings and site visits were conducted in five locations in Tanzania to garner stakeholder input on the proposed distribution systems. Coverage levels for LLINs and their decline over time were modelled using NetCALC software, taking realistic net decay rates, current demographic profiles and other relevant parameters into consideration. Costs of the different distribution systems were estimated using local data. LLIN delivery was considered via mass campaigns, Antenatal Care-Expanded Programme on Immunization (ANC/EPI), community-based distribution, schools, the commercial sector and different combinations of the above. Most approaches appeared unlikely to maintain universal coverage when used alone. Mass campaigns, even when combined with a continuation of the Tanzania National Voucher Scheme (TNVS), would produce large temporal fluctuations in coverage levels; over 10 years this strategy would require 63.3 million LLINs and a total cost of $444 million USD. Community mechanisms, while able to deliver the required numbers of LLINs, would require a massive scale-up in monitoring, evaluation and supervision systems to ensure accurate application of identification criteria at the community level. School-based approaches combined with the existing TNVS would reach most Tanzanian households and deliver 65.4 million LLINs over 10 years at a total cost of$449 million USD and ensure continuous coverage. The cost of each strategy was largely driven by the number of LLINs delivered. The most cost-efficient strategy to maintain universal coverage is one that best optimizes the numbers of LLINs needed over time. A school-based approach using vouchers targeting all students in Standards 1, 3, 5, 7 and Forms 1 and 2 in combination with the TNVS appears to meet best the criteria of effectiveness, equity and efficiency

MYBA From Blueberry (Vaccinium Section Cyanococcus) Is a Subgroup 6 Type R2R3MYB Transcription Factor That Activates Anthocyanin Production

The Vaccinium genus in the family Ericaceae comprises many species, including the fruit-bearing blueberry, bilberry, cranberry, huckleberry, and lingonberry. Commercially, the most important are the blueberries (Vaccinium section Cyanococcus), such as Vaccinium corymbosum (northern highbush blueberry), Vaccinium virgatum (rabbiteye blueberry), and Vaccinium angustifolium (lowbush blueberry). The rising popularity of blueberries can partly be attributed to their “superfood” status, with an increasing body of evidence around human health benefits resulting from the fruit metabolites, particularly products of the phenylpropanoid pathway such as anthocyanins. Activation of anthocyanin production by R2R3-MYB transcription factors (TFs) has been characterized in many species, but despite recent studies on blueberry, cranberry, and bilberry, no MYB anthocyanin regulators have been reported for Vaccinium. Indeed, there has been conjecture that at least in bilberry, MYB TFs divergent to the usual type are involved. We report identification of MYBA from blueberry, and show through sequence analysis and functional studies that it is homologous to known anthocyanin-promoting R2R3-MYBs of subgroup 6 of the MYB superfamily. In transient assays, MYBA complemented an anthocyanin MYB mutant of Antirrhinum majus and, together with a heterologous bHLH anthocyanin regulator, activated anthocyanin production in Nicotiana benthamiana. Furthermore anthocyanin accumulation and anthocyanin structural gene expression (assayed by qPCR and RNA-seq analyses) correlated with MYBA expression, and MYBA was able to transactivate the DFR promoter from blueberry and other species. The RNA-seq data also revealed a range of other candidate genes involved in the regulation of anthocyanin production in blueberry fruit. The identification of MYBA will help to resolve the regulatory mechanism for anthocyanin pigmentation in the Vaccinium genus. The sequence information should also prove useful in developing tools for the accelerated breeding of new Vaccinium cultivars

A high-resolution map of human evolutionary constraint using 29 mammals.

The comparison of related genomes has emerged as a powerful lens for genome interpretation. Here we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and locate constrained elements covering ∼4.2% of the genome. We use evolutionary signatures and comparisons with experimental data sets to suggest candidate functions for ∼60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements and more than 1,000 primate- and human-accelerated elements. Overlap with disease-associated variants indicates that our findings will be relevant for studies of human biology, health and disease

Dried blood spot UHPLC-MS/MS analysis of oseltamivir and oseltamivircarboxylate—a validated assay for the clinic

The neuraminidase inhibitor oseltamivir (Tamiflu®) is currently the first-line therapy for patients with influenza virus infection. Common analysis of the prodrug and its active metabolite oseltamivircarboxylate is determined via extraction from plasma. Compared with these assays, dried blood spot (DBS) analysis provides several advantages, including a minimum sample volume required for the measurement of drugs in whole blood. Samples can easily be obtained via a simple, non-invasive finger or heel prick. Mainly, these characteristics make DBS an ideal tool for pediatrics and to measure multiple time points such as those needed in therapeutic drug monitoring or pharmacokinetic studies. Additionally, DBS sample preparation, stability, and storage are usually most convenient. In the present work, we developed and fully validated a DBS assay for the simultaneous determination of oseltamivir and oseltamivircarboxylate concentrations in human whole blood. We demonstrate the simplicity of DBS sample preparation, and a fast, accurate and reproducible analysis using ultra high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer. A thorough validation on the basis of the most recent FDA guidelines for bioanalytical method validation showed that the method is selective, precise, and accurate (≤15% RSD), and sensitive over the relevant clinical range of 5–1,500 ng/mL for oseltamivir and 20–1,500 ng/mL for the oseltamivircarboxylate metabolite. As a proof of concept, oseltamivir and oseltamivircarboxylate levels were determined in DBS obtained from healthy volunteers who received a single oral dose of Tamiflu®

Protest Cycles and Political Process: American Peace Movements in the Nuclear Age

Since the dawn of the nuclear age small groups of activists have consistently protested both the content of United States national security policy, and the process by which it is made. Only occasionally, however, has concern about nuclear weapons spread beyond these relatively marginal groups, generated substantial public support, and reached mainstream political institutions. In this paper, I use histories of peace protest and analyses of the inside of these social movements and theoretical work on protest cycles to explain cycles of movement engagement and quiescence in terms of their relation to external political context, or the "structure of political opportunity." I begin with a brief review of the relevant literature on the origins of movements, noting parallels in the study of interest groups. Building on recent literature on political opportunity structure, I suggest a theoretical framework for understanding the lifecycle of a social movement that emphasizes the interaction between activist choices and political context, proposing a six-stage process through which challenging movements develop. Using this theoretical framework I examine the four cases of relatively broad antinuclear weapons mobilization in postwar America. I conclude with a discussion of movement cycles and their relation to political alignment, public policy, and institutional politics.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/68552/2/10.1177_106591299304600302.pd

Arabidopsis leucine-rich repeat receptor–like kinase NILR1 is required for induction of innate immunity to parasitic nematodes

Plant-parasitic nematodes are destructive pests causing losses of billions of dollars annually. An effective plant defence against pathogens relies on the recognition of pathogen-associated molecular patterns (PAMPs) by surface-localised receptors leading to the activation of PAMP-triggered immunity (PTI). Extensive studies have been conducted to characterise the role of PTI in various models of plant-pathogen interactions. However, far less is known about the role of PTI in roots in general and in plant-nematode interactions in particular. Here we show that nematode-derived proteinaceous elicitor/s is/are capable of inducing PTI in Arabidopsis in a manner dependent on the common immune co-receptor BAK1. Consistent with the role played by BAK1, we identified a leucine-rich repeat receptor-like kinase, termed NILR1 that is specifically regulated upon infection by nematodes. We show that NILR1 is essential for PTI responses initiated by nematodes and nilr1 loss-of-function mutants are hypersusceptible to a broad category of nematodes. To our knowledge, NILR1 is the first example of an immune receptor that is involved in induction of basal immunity (PTI) in plants or in animals in response to nematodes. Manipulation of NILR1 will provide new options for nematode control in crop plants in future