Unusual DNA/RNA structures of the C9orf72 repeat may participate in repeat expansions or pathogenesis of amyotrophic lateral sclerosis and frontotemporal dementia. Expanded repeats are CpG methylated with unknown consequences. Typically, quadruplex structures form by G-rich but not complementary C-rich strands. Using CD, UV and electrophoresis, we characterized the structures formed by (GGGGCC)8 and (GGCCCC)8 strands with and without 5-methylcytosine (5mCpG) or 5-hydroxymethylcytosine (5hmCpG) methylation. All strands formed heterogenous mixtures of structures, with features of quadruplexes (at pH 7.5, in K+, Na+ or Li+), but no feature typical of i-motifs. C-rich strands formed quadruplexes, likely stabilized by G•C•G•C-tetrads and C•C•C•C-tetrads. Unlike G•G•G•G-tetrads, some G•C•G•C-tetrad conformations do not require the N7-Guanine position, hence C9orf72 quadruplexes still formed when N7-deazaGuanine replace all Guanines. 5mCpG and 5hmCpG increased and decreased the thermal stability of these structures. hnRNPK, through band-shift analysis, bound C-rich but not G-rich strands, with a binding preference of unmethylated > 5hmCpG > 5mCpG, where methylated DNA-protein complexes were retained in the wells, distinct from unmethylated complexes. Our findings suggest that for C-rich sequences interspersed with G-residues, one must consider quadruplex formation and that methylation of quadruplexes may affect epigenetic processes.

Transcription Activator-Like Effectors (TALEs) of Xanthomonas bacteria are programmable DNA binding proteins with unprecedented target specificity. Comparative studies into TALE repeat structure and function are hindered by the limited sequence variation among TALE repeats. More sequence-diverse TALE-like proteins are known from Ralstonia solanacearum (RipTALs) and Burkholderia rhizoxinica (Bats), but RipTAL and Bat repeats are conserved with those of TALEs around the DNA-binding residue. We study two novel marine-organism TALE-like proteins (MOrTL1 and MOrTL2), the first to date of non-terrestrial origin. We have assessed their DNA-binding properties and modelled repeat structures. We found that repeats from these proteins mediate sequence specific DNA binding conforming to the TALE code, despite low sequence similarity to TALE repeats, and with novel residues around the BSR. However, MOrTL1 repeats show greater sequence discriminating power than MOrTL2 repeats. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. Additionally, comparing MOrTL repeats with those of other TALE-likes suggests a common evolutionary origin for the TALEs, RipTALs and Bats.

RNA G-quadruplexes (G4s) are one of the key components of the transcriptome that act as efficient post-transcriptional regulatory elements in living cells. To conduct further studies of the unique biological functions of RNA G4s, techniques need to be developed that can efficiently recognize RNA G4 structures under various conditions, in fixed cells and living cells, as well as in vitro. This paper presents the development of such a method, a new technique using a cyanine dye called CyT, which can detect both canonical and non-canonical RNA G4 structures from test tubes to living human cells. The ability of CyT to distinguish between G4 and nonG4 RNA offers a promising tool for future RNA G4-based biomarker discovery and potential diagnostic applications.

A perisinusoidal niche for extramedullary haematopoiesis in the spleen

Nature 527, 7579 (2015). doi:10.1038/nature15530

Authors: Christopher N. Inra, Bo O. Zhou, Melih Acar, Malea M. Murphy, James Richardson, Zhiyu Zhao & Sean J. Morrison

Haematopoietic stresses mobilize haematopoietic stem cells (HSCs) from the bone marrow to the spleen and induce extramedullary haematopoiesis (EMH). However, the cellular nature of the EMH niche is unknown. Here we assessed the sources of the key niche factors, SCF (also known as KITL) and

Ubiquitous time variability of integrated stellar populations

Nature 527, 7579 (2015). doi:10.1038/nature15731

Authors: Charlie Conroy, Pieter G. van Dokkum & Jieun Choi

Long-period variable stars arise in the final stages of the asymptotic giant branch phase of stellar evolution. They have periods of up to about 1,000 days and amplitudes that can exceed a factor of three in the I-band flux. These stars pulsate predominantly in their fundamental mode, which is a function of mass and radius, and so the pulsation periods are sensitive to the age of the underlying stellar population. The overall number of long-period variables in a population is directly related to their lifetimes, which is difficult to predict from first principles because of uncertainties associated with stellar mass-loss and convective mixing. The time variability of these stars has not previously been taken into account when modelling the spectral energy distributions of galaxies. Here we construct time-dependent stellar population models that include the effects of long-period variable stars, and report the ubiquitous detection of this expected ‘pixel shimmer’ in the massive metal-rich galaxy M87. The pixel light curves display a variety of behaviours. The observed variation of 0.1 to 1 per cent is very well matched to the predictions of our models. The data provide a strong constraint on the properties of variable stars in an old and metal-rich stellar population, and we infer that the lifetime of long-period variables in M87 is shorter by approximately 30 per cent compared to predictions from the latest stellar evolution models.

Abstract

The maximum stable set problem is NP-hard, even when restricted to triangle-free graphs. In particular, one cannot expect a polynomial time algorithm deciding if a bull-free graph has a stable set of size k, when k is part of the instance. Our main result in this paper is to show the existence of an FPT algorithm when we parameterize the problem by the solution size k. A polynomial kernel is unlikely to exist for this problem. We show however that our problem has a polynomial size Turing-kernel. More precisely, the hard cases are instances of size \({O}(k^5)\) . As a byproduct, if we forbid odd holes in addition to the bull, we show the existence of a polynomial time algorithm for the stable set problem. We also prove that the chromatic number of a bull-free graph is bounded by a function of its clique number and the maximum chromatic number of its triangle-free induced subgraphs. All our results rely on a decomposition theorem for bull-free graphs due to Chudnovsky which is modified here, allowing us to provide extreme decompositions, adapted to our computational purpose.

Abstract

Directed evolution is a powerful tool for engineering protein function. The process of directed evolution involves iterative rounds of sequence diversification followed by assaying activity of variants and selection. The range of sequence variants and linked activities generated in the course of an evolution are a rich information source for investigating relationships between sequence and function. Key residue positions determining protein function, combinatorial contributors to activity and even potential functional mechanisms have been revealed in directed evolutions. The recent application of high throughput sequencing substantially increases the information that can be retrieved from directed evolution experiments. Combined with computational analysis this additional sequence information has allowed high-resolution analysis of individual residue contributions to activity. These developments promise to significantly enhance the depth of insight that experimental evolution provides into mechanisms of protein function.

Abstract

Change in folding kinetics of globular proteins upon point mutation is crucial to a wide spectrum of biological research, such as protein misfolding, toxicity and aggregations. Here we seek to address whether residue-level co-evolutionary information of globular proteins can be informative to folding rate changes upon point-mutations. Generating residue-level co-evolutionary networks of globular proteins, we analyze three parameters: relative co-evolution order (rCEO), network density (ND) and characteristic path length (CPL). A point-mutation is considered to be equivalent to a node deletion of this network and respective percentage changes in rCEO, ND, CPL are found linearly correlated (0.84, 0.73 and −0.61 respectively) with experimental folding rate changes. The three parameters predict the folding rate change upon a point-mutation with 0.031, 0.045 and 0.059 standard errors respectively. This article is protected by copyright. All rights reserved.

by Gabriel Madirolas, Gonzalo G. de Polavieja

Groups can make precise collective estimations in cases like the weight of an object or the number of items in a volume. However, in others tasks, for example those requiring memory or mental calculation, subjects often give estimations with large deviations from factual values. Allowing members of the group to communicate their estimations has the additional perverse effect of shifting individual estimations even closer to the biased collective estimation. Here we show that this negative effect of social interactions can be turned into a method to improve collective estimations. We first obtained a statistical model of how humans change their estimation when receiving the estimates made by other individuals. We confirmed using existing experimental data its prediction that individuals use the weighted geometric mean of private and social estimations. We then used this result and the fact that each individual uses a different value of the social weight to devise a method that extracts the subgroups resisting social influence. We found that these subgroups of individuals resisting social influence can make very large improvements in group estimations. This is in contrast to methods using the confidence that each individual declares, for which we find no improvement in group estimations. Also, our proposed method does not need to use historical data to weight individuals by performance. These results show the benefits of using the individual characteristics of the members in a group to better extract collective wisdom.

by Benjamin D. Heavner, Nathan D. Price

We have compared 12 genome-scale models of the Saccharomyces cerevisiae metabolic network published since 2003 to evaluate progress in reconstruction of the yeast metabolic network. We compared the genomic coverage, overlap of annotated metabolites, predictive ability for single gene essentiality with a selection of model parameters, and biomass production predictions in simulated nutrient-limited conditions. We have also compared pairwise gene knockout essentiality predictions for 10 of these models. We found that varying approaches to model scope and annotation reflected the involvement of multiple research groups in model development; that single-gene essentiality predictions were affected by simulated medium, objective function, and the reference list of essential genes; and that predictive ability for single-gene essentiality did not correlate well with predictive ability for our reference list of synthetic lethal gene interactions (R = 0.159). We conclude that the reconstruction of the yeast metabolic network is indeed gradually improving through the iterative process of model development, and there remains great opportunity for advancing our understanding of biology through continued efforts to reconstruct the full biochemical reaction network that constitutes yeast metabolism. Additionally, we suggest that there is opportunity for refining the process of deriving a metabolic model from a metabolic network reconstruction to facilitate mechanistic investigation and discovery. This comparative study lays the groundwork for developing improved tools and formalized methods to quantitatively assess metabolic network reconstructions independently of any particular model application, which will facilitate ongoing efforts to advance our understanding of the relationship between genotype and cellular phenotype.

Cell fate: Transition loses its invasive edge

Nature 527, 7579 (2015). doi:10.1038/nature16313

Authors: Shyamala Maheswaran & Daniel A. Haber

Two studies provide evidence that epithelial tumour cells do not need to transition to a mesenchymal-cell state to form metastases, but that this process does contribute to drug resistance. See Article p.472 & Letter p.525

Epithelial-to-mesenchymal transition is not required for lung metastasis but contributes to chemoresistance

Nature 527, 7579 (2015). doi:10.1038/nature15748

Authors: Kari R. Fischer, Anna Durrans, Sharrell Lee, Jianting Sheng, Fuhai Li, Stephen T. C. Wong, Hyejin Choi, Tina El Rayes, Seongho Ryu, Juliane Troeger, Robert F. Schwabe, Linda T. Vahdat, Nasser K. Altorki, Vivek Mittal & Dingcheng Gao

The role of epithelial-to-mesenchymal transition (EMT) in metastasis is a longstanding source of debate, largely owing to an inability to monitor transient and reversible EMT phenotypes in vivo. Here we establish an EMT lineage-tracing system to monitor this process in mice, using a mesenchymal-specific

Extremely metal-poor stars from the cosmic dawn in the bulge of the Milky Way

Nature 527, 7579 (2015). doi:10.1038/nature15747

Authors: L. M. Howes, A. R. Casey, M. Asplund, S. C. Keller, D. Yong, D. M. Nataf, R. Poleski, K. Lind, C. Kobayashi, C. I. Owen, M. Ness, M. S. Bessell, G. S. Da Costa, B. P. Schmidt, P. Tisserand, A. Udalski, M. K. Szymański, I. Soszyński, G. Pietrzyński, K. Ulaczyk, Ł. Wyrzykowski, P. Pietrukowicz, J. Skowron, S. Kozłowski & P. Mróz

The first stars are predicted to have formed within 200 million years after the Big Bang, initiating the cosmic dawn. A true first star has not yet been discovered, although stars with tiny amounts of elements heavier than helium (‘metals’) have been found in the outer regions (‘halo’) of the Milky Way. The first stars and their immediate successors should, however, preferentially be found today in the central regions (‘bulges’) of galaxies, because they formed in the largest over-densities that grew gravitationally with time. The Milky Way bulge underwent a rapid chemical enrichment during the first 1–2 billion years, leading to a dearth of early, metal-poor stars. Here we report observations of extremely metal-poor stars in the Milky Way bulge, including one star with an iron abundance about 10,000 times lower than the solar value without noticeable carbon enhancement. We confirm that most of the metal-poor bulge stars are on tight orbits around the Galactic Centre, rather than being halo stars passing through the bulge, as expected for stars formed at redshifts greater than 15. Their chemical compositions are in general similar to typical halo stars of the same metallicity although intriguing differences exist, including lower abundances of carbon.

Single-molecule sequencing of the desiccation-tolerant grass Oropetium thomaeum

Nature 527, 7579 (2015). doi:10.1038/nature15714

Authors: Robert VanBuren, Doug Bryant, Patrick P. Edger, Haibao Tang, Diane Burgess, Dinakar Challabathula, Kristi Spittle, Richard Hall, Jenny Gu, Eric Lyons, Michael Freeling, Dorothea Bartels, Boudewijn Ten Hallers, Alex Hastie, Todd P. Michael & Todd C. Mockler

Plant genomes, and eukaryotic genomes in general, are typically repetitive, polyploid and heterozygous, which complicates genome assembly. The short read lengths of early Sanger and current next-generation sequencing platforms hinder assembly through complex repeat regions, and many draft and reference genomes are fragmented, lacking skewed GC and repetitive intergenic sequences, which are gaining importance due to projects like the Encyclopedia of DNA Elements (ENCODE). Here we report the whole-genome sequencing and assembly of the desiccation-tolerant grass Oropetium thomaeum. Using only single-molecule real-time sequencing, which generates long (>16 kilobases) reads with random errors, we assembled 99% (244 megabases) of the Oropetium genome into 625 contigs with an N50 length of 2.4 megabases. Oropetium is an example of a ‘near-complete’ draft genome which includes gapless coverage over gene space as well as intergenic sequences such as centromeres, telomeres, transposable elements and rRNA clusters that are typically unassembled in draft genomes. Oropetium has 28,466 protein-coding genes and 43% repeat sequences, yet with 30% more compact euchromatic regions it is the smallest known grass genome. The Oropetium genome demonstrates the utility of single-molecule real-time sequencing for assembling high-quality plant and other eukaryotic genomes, and serves as a valuable resource for the plant comparative genomics community.

Epithelial-to-mesenchymal transition is dispensable for metastasis but induces chemoresistance in pancreatic cancer

Nature 527, 7579 (2015). doi:10.1038/nature16064

Authors: Xiaofeng Zheng, Julienne L. Carstens, Jiha Kim, Matthew Scheible, Judith Kaye, Hikaru Sugimoto, Chia-Chin Wu, Valerie S. LeBleu & Raghu Kalluri

Diagnosis of pancreatic ductal adenocarcinoma (PDAC) is associated with a dismal prognosis despite current best therapies; therefore new treatment strategies are urgently required. Numerous studies have suggested that epithelial-to-mesenchymal transition (EMT) contributes to early-stage dissemination of cancer cells and is pivotal for invasion and metastasis of PDAC. EMT is associated with phenotypic conversion of epithelial cells into mesenchymal-like cells in cell culture conditions, although such defined mesenchymal conversion (with spindle-shaped morphology) of epithelial cells in vivo is rare, with quasi-mesenchymal phenotypes occasionally observed in the tumour (partial EMT). Most studies exploring the functional role of EMT in tumours have depended on cell-culture-induced loss-of-function and gain-of-function experiments involving EMT-inducing transcription factors such as Twist, Snail and Zeb1 (refs 2, 3, 7, 8, 9, 10). Therefore, the functional contribution of EMT to invasion and metastasis remains unclear, and genetically engineered mouse models to address a causal connection are lacking. Here we functionally probe the role of EMT in PDAC by generating mouse models of PDAC with deletion of Snail or Twist, two key transcription factors responsible for EMT. EMT suppression in the primary tumour does not alter the emergence of invasive PDAC, systemic dissemination or metastasis. Suppression of EMT leads to an increase in cancer cell proliferation with enhanced expression of nucleoside transporters in tumours, contributing to enhanced sensitivity to gemcitabine treatment and increased overall survival of mice. Collectively, our study suggests that Snail- or Twist-induced EMT is not rate-limiting for invasion and metastasis, but highlights the importance of combining EMT inhibition with chemotherapy for the treatment of pancreatic cancer.