Recursive construction of perfect DNA molecules from imperfect oligonucleotides

Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error-prone oligonucleotides are recursively combined in vitro, forming error-prone DNA molecules; error-free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms

Only one independent genetic association with rheumatoid arthritis within the KIAA1109-TENR-IL2-IL21 locus in Caucasian sample sets: confirmation of association of rs6822844 with rheumatoid arthritis at a genome-wide level of significance

INTRODUCTION: The single nucleotide polymorphism (SNP) rs6822844 within the KIAA1109-TENR-IL2-IL21 gene cluster has been associated with rheumatoid arthritis (RA). Other variants within this cluster, including rs17388568 that is not in linkage disequilibrium (LD) with rs6822844, and rs907715 that is in moderate LD with rs6822844 and rs17388568, have been associated with a number of autoimmune phenotypes, including type 1 diabetes (T1D). Here we aimed to: one, confirm at a genome-wide level of significance association of rs6822844 with RA and, two, evaluate whether or not there were effects independent of rs6822844 on RA at the KIAA1109-TENR-IL2-IL21 locus. METHODS: A total of 842 Australasian RA patients and 1,115 controls of European Caucasian ancestry were genotyped for rs6822844, rs17388568 and rs907715. Meta-analysis of these data with published and publicly-available data was conducted using STATA. RESULTS: No statistically significant evidence for association was observed in the Australasian sample set for rs6822844 (odds ratio (OR)=0.95 (0.80 to 1.12), P=0.54), or rs17388568 (OR=1.03 (0.90 to 1.19), P=0.65) or rs907715 (OR=0.98 (0.86 to 1.12), P=0.69). When combined in a meta-analysis using data from a total of 9,772 cases and 10,909 controls there was a genome-wide level of significance supporting association of rs6822844 with RA (OR=0.86 (0.82 to 0.91), P=8.8x10(-8), P=2.1x10(-8) including North American Rheumatoid Arthritis Consortium data). Meta-analysis of rs17388568, using a total of 6,585 cases and 7,528 controls, revealed no significant association with RA (OR=1.03, (0.98 to 1.09); P=0.22) and meta-analysis of rs907715 using a total of 2,689 cases and 4,045 controls revealed a trend towards association (OR=0.93 (0.87 to 1.00), P=0.07). However, this trend was not independent of the association at rs6822844. CONCLUSIONS: The KIAA1109-TENR-IL2-IL21 gene cluster, that encodes an interleukin (IL-21) that plays an important role in Th17 cell biology, is the 20th locus for which there is a genome-wide (P ≤ 5x10(-8)) level of support for association with RA. As for most other autoimmune diseases, with the notable exception of T1D, rs6822844 is the dominant association in the locus. The KIAA1109-TENR-IL2-IL21 locus also confers susceptibility to other autoimmune phenotypes with a heterogeneous pattern of association

The phenotypic spectrum associated with OTX2 mutations in humans

OBJECTIVE: The transcription factor OTX2 is implicated in ocular, craniofacial, and pituitary development. DESIGN: We aimed to establish the contribution of OTX2 mutations in congenital hypopituitarism patients with/without eye abnormalities, study functional consequences, and establish OTX2 in the human brain, with a view to investigating the mechanism of action. METHODS: We screened patients from the UK (n=103), international centers (n=24), and Brazil (n=282); 145 were within the septo-optic dysplasia spectrum, and 264 had no eye phenotype. Transactivation ability of OTX2 variants was analysed in murine hypothalamic GT1-7 neurons. In situ hybridization was performed on human embryonic brain sections. Genetically engineered mice were generated with a series of C-terminal OTX2 variants. RESULTS: Two chromosomal deletions and six haploinsufficient mutations were identified in individuals with eye abnormalities; an affected relative of one patient harboured the same mutation without an ocular phenotype. OTX2 truncations led to significant transactivation reduction. A missense variant was identified in another patient without eye abnormalities, however studies revealed it was most likely not causative. In the mouse, truncations proximal to aa219 caused anophthalmia, while distal truncations and the missense variant were tolerated. During human embryogenesis, OTX2 was expressed in the posterior pituitary, retina, ear, thalamus, choroid plexus, and partially in the hypothalamus, but not in the anterior pituitary. CONCLUSIONS: OTX2 mutations are rarely associated with hypopituitarism in isolation without eye abnormalities, and may be variably penetrant, even within the same pedigree. Our data suggest that the endocrine phenotypes in patients with OTX2 mutations are of hypothalamic origin

Mapping gene associations in human mitochondria using clinical disease phenotypes

Nuclear genes encode most mitochondrial proteins, and their mutations cause diverse and debilitating clinical disorders. To date, 1,200 of these mitochondrial genes have been recorded, while no standardized catalog exists of the associated clinical phenotypes. Such a catalog would be useful to develop methods to analyze human phenotypic data, to determine genotype-phenotype relations among many genes and diseases, and to support the clinical diagnosis of mitochondrial disorders. Here we establish a clinical phenotype catalog of 174 mitochondrial disease genes and study associations of diseases and genes. Phenotypic features such as clinical signs and symptoms were manually annotated from full-text medical articles and classified based on the hierarchical MeSH ontology. This classification of phenotypic features of each gene allowed for the comparison of diseases between different genes. In turn, we were then able to measure the phenotypic associations of disease genes for which we calculated a quantitative value that is based on their shared phenotypic features. The results showed that genes sharing more similar phenotypes have a stronger tendency for functional interactions, proving the usefulness of phenotype similarity values in disease gene network analysis. We then constructed a functional network of mitochondrial genes and discovered a higher connectivity for non-disease than for disease genes, and a tendency of disease genes to interact with each other. Utilizing these differences, we propose 168 candidate genes that resemble the characteristic interaction patterns of mitochondrial disease genes. Through their network associations, the candidates are further prioritized for the study of specific disorders such as optic neuropathies and Parkinson disease. Most mitochondrial disease phenotypes involve several clinical categories including neurologic, metabolic, and gastrointestinal disorders, which might indicate the effects of gene defects within the mitochondrial system. The accompanying knowledgebase (http://www.mitophenome.org/) supports the study of clinical diseases and associated genes

Genome-wide meta-analysis of cerebral white matter hyperintensities in patients with stroke.

OBJECTIVE: For 3,670 stroke patients from the United Kingdom, United States, Australia, Belgium, and Italy, we performed a genome-wide meta-analysis of white matter hyperintensity volumes (WMHV) on data imputed to the 1000 Genomes reference dataset to provide insights into disease mechanisms. METHODS: We first sought to identify genetic associations with white matter hyperintensities in a stroke population, and then examined whether genetic loci previously linked to WMHV in community populations are also associated in stroke patients. Having established that genetic associations are shared between the 2 populations, we performed a meta-analysis testing which associations with WMHV in stroke-free populations are associated overall when combined with stroke populations. RESULTS: There were no associations at genome-wide significance with WMHV in stroke patients. All previously reported genome-wide significant associations with WMHV in community populations shared direction of effect in stroke patients. In a meta-analysis of the genome-wide significant and suggestive loci (p < 5 × 10(-6)) from community populations (15 single nucleotide polymorphisms in total) and from stroke patients, 6 independent loci were associated with WMHV in both populations. Four of these are novel associations at the genome-wide level (rs72934505 [NBEAL1], p = 2.2 × 10(-8); rs941898 [EVL], p = 4.0 × 10(-8); rs962888 [C1QL1], p = 1.1 × 10(-8); rs9515201 [COL4A2], p = 6.9 × 10(-9)). CONCLUSIONS: Genetic associations with WMHV are shared in otherwise healthy individuals and patients with stroke, indicating common genetic susceptibility in cerebral small vessel disease

Impacts of climate change on plant diseases – opinions and trends

There has been a remarkable scientific output on the topic of how climate change is likely to affect plant diseases in the coming decades. This review addresses the need for review of this burgeoning literature by summarizing opinions of previous reviews and trends in recent studies on the impacts of climate change on plant health. Sudden Oak Death is used as an introductory case study: Californian forests could become even more susceptible to this emerging plant disease, if spring precipitations will be accompanied by warmer temperatures, although climate shifts may also affect the current synchronicity between host cambium activity and pathogen colonization rate. A summary of observed and predicted climate changes, as well as of direct effects of climate change on pathosystems, is provided. Prediction and management of climate change effects on plant health are complicated by indirect effects and the interactions with global change drivers. Uncertainty in models of plant disease development under climate change calls for a diversity of management strategies, from more participatory approaches to interdisciplinary science. Involvement of stakeholders and scientists from outside plant pathology shows the importance of trade-offs, for example in the land-sharing vs. sparing debate. Further research is needed on climate change and plant health in mountain, boreal, Mediterranean and tropical regions, with multiple climate change factors and scenarios (including our responses to it, e.g. the assisted migration of plants), in relation to endophytes, viruses and mycorrhiza, using long-term and large-scale datasets and considering various plant disease control methods

The impact of parenthood on environmental attitudes and behaviour: a longitudinal investigation of the legacy hypothesis

Willingness to engage in sustainable actions may be limited by the psychological distance of climate change. In this study, we test the legacy hypothesis, which holds that having children leads parents to consider the legacy left to offspring in respect of environmental quality. Using the Understanding Society dataset, a longitudinal survey representative of the UK population (n = 18,176), we assess how having children may change people’s individual environmental attitudes and behaviour. Results indicate that having a new child is associated with a small decrease in the frequency of a few environmental behaviours. Only parents with already high environmental concern show a small increase in the desire to act more sustainably after the birth of their first child. Overall, the results do not provide evidence in support of the legacy hypothesis in terms of individual-level environmental attitudes and behaviours. We argue that the transition to parenthood is a time where concern is prioritised on the immediate wellbeing of the child and not on the future environmental threats

Natural Killer Cell Mediated Cytotoxic Responses in the Tasmanian Devil

The Tasmanian devil (Sarcophilus harrisii), the world's largest marsupial carnivore, is under threat of extinction following the emergence of an infectious cancer. Devil facial tumour disease (DFTD) is spread between Tasmanian devils during biting. The disease is consistently fatal and devils succumb without developing a protective immune response. The aim of this study was to determine if Tasmanian devils were capable of forming cytotoxic antitumour responses and develop antibodies against DFTD cells and foreign tumour cells. The two Tasmanian devils immunised with irradiated DFTD cells did not form cytotoxic or humoral responses against DFTD cells, even after multiple immunisations. However, following immunisation with xenogenic K562 cells, devils did produce cytotoxic responses and antibodies against this foreign tumour cell line. The cytotoxicity appeared to occur through the activity of natural killer (NK) cells in an antibody dependent manner. Classical NK cell responses, such as innate killing of DFTD and foreign cancer cells, were not observed. Cells with an NK-like phenotype comprised approximately 4 percent of peripheral blood mononuclear cells. The results of this study suggest that Tasmanian devils have NK cells with functional cytotoxic pathways. Although devil NK cells do not directly recognise DFTD cancer cells, the development of antibody dependent cell-mediated cytotoxicity presents a potential pathway to induce cytotoxic responses against the disease. These findings have positive implications for future DFTD vaccine research

A computational study of the influence of surface roughness on material strength

In machine component stress analysis, it usually assumed that the geometry specified in CAD provides a fair representation of the geometry of the real component. While in particular circumstances, tolerance information, such as minimum thickness of a highly stressed region, might be taken into consideration, there is no standard practice for the representation of surface quality. It is known that surface roughness significantly influences fatigue life, but for this to be useful in the context of life prediction, there is a need to examine the nature of surface roughness and determine how best to characterise it. Non-smooth geometry can be represented in mathematics by fractals or other methods, but for a representation to have a practical value for a manufactured component, it is necessary to accept that there is a lower limit to surface profile measurement resolution. Resolution and mesh refinement also play a part in any computational analysis undertaken to assess surface profile effects: in the analyses presented, a nominal axi-symmetric geometry has been taken, with a finite non-smooth region on the boundary. Various surface roughness representations are modelled, and the significance of the characterized surface roughness type is investigated. It is shown that the applied load gives rise to a nominally uni-axial stress state of 90% of the yield, although surface roughness features have the effect of modifying the load path, and give rise to localized regions of plasticity near to the surface. The material of the test model is assumed to be elasto-plastic, and the development and evolution of plastic zones formed within the geometry are shown for multiple load cycles

A Phenotypic Profile of the Candida albicans Regulatory Network

Candida albicans is a normal resident of the gastrointestinal tract and also the most prevalent fungal pathogen of humans. It last shared a common ancestor with the model yeast Saccharomyces cerevisiae over 300 million years ago. We describe a collection of 143 genetically matched strains of C. albicans, each of which has been deleted for a specific transcriptional regulator. This collection represents a large fraction of the non-essential transcription circuitry. A phenotypic profile for each mutant was developed using a screen of 55 growth conditions. The results identify the biological roles of many individual transcriptional regulators; for many, this work represents the first description of their functions. For example, a quarter of the strains showed altered colony formation, a phenotype reflecting transitions among yeast, pseudohyphal, and hyphal cell forms. These transitions, which have been closely linked to pathogenesis, have been extensively studied, yet our work nearly doubles the number of transcriptional regulators known to influence them. As a second example, nearly a quarter of the knockout strains affected sensitivity to commonly used antifungal drugs; although a few transcriptional regulators have previously been implicated in susceptibility to these drugs, our work indicates many additional mechanisms of sensitivity and resistance. Finally, our results inform how transcriptional networks evolve. Comparison with the existing S. cerevisiae data (supplemented by additional S. cerevisiae experiments reported here) allows the first systematic analysis of phenotypic conservation by orthologous transcriptional regulators over a large evolutionary distance. We find that, despite the many specific wiring changes documented between these species, the general phenotypes of orthologous transcriptional regulator knockouts are largely conserved. These observations support the idea that many wiring changes affect the detailed architecture of the circuit, but not its overall output