ALBERT

Description: Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase ( HPRT ), we monitor the relative utilization of three DSBR pathways following cleavage by I-Sce I or CRISPR/Cas9 nucleases. For I-Sce I, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-Sce I and Cas9 induced markedly different DSBR profiles. Also, using an I-Sce I-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-Sce I derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.

Description: DNA double-strand breaks (DSBs) are toxic lesions, which if improperly repaired can result in cell death or genomic instability. DSB repair is usually facilitated by the classical non-homologous end joining (C-NHEJ), or homologous recombination (HR) pathways. However, a mutagenic alternative NHEJ pathway, microhomology-mediated end joining (MMEJ), can also be deployed. While MMEJ is suppressed by C-NHEJ, the relationship between HR and MMEJ is less clear. Here, we describe a role for HR genes in suppressing MMEJ in human cells. By monitoring DSB mis-repair using a sensitive HPRT assay, we found that depletion of HR proteins, including BRCA2, BRCA1 or RPA, resulted in a distinct mutational signature associated with significant increases in break-induced mutation frequencies, deletion lengths and the annealing of short regions of microhomology (2–6 bp) across the break-site. This signature was dependent on CtIP, MRE11, POLQ and PARP, and thus indicative of MMEJ. In contrast to CtIP or MRE11, depletion of BRCA1 resulted in increased partial resection and MMEJ, thus revealing a functional distinction between these early acting HR factors. Together these findings indicate that HR factors suppress mutagenic MMEJ following DSB resection.

Description: Horseshoe crabs are xiphosuran chelicerates, the sister group to arachnids. As such, they are important for understanding the most recent common ancestor of Euchelicerata and the evolution and diversification of Arthropoda. Limulus polyphemus is the most investigated of the four extant species of horseshoe crabs, and the structure and function of its visual system have long been a major focus of studies critical for understanding the evolution of visual systems in arthropods. Likewise, studies of genes encoding Limulus opsins, the protein component of the visual pigments, are critical for understanding opsin evolution and diversification among chelicerates, where knowledge of opsins is limited, and more broadly among arthropods. In the present study, we sequenced and assembled a high quality nuclear genomic sequence of L. polyphemus and used these data to annotate the full repertoire of Limulus opsins. We conducted a detailed phylogenetic analysis of Limulus opsins, including using gene structure and synteny information to identify relationships among different opsin classes. We used our phylogeny to identify significant genomic events that shaped opsin evolution and therefore the visual system of Limulus . We also describe the tissue expression patterns of the 18 opsins identified and show that transcripts encoding a number, including a peropsin, are present throughout the central nervous system. In addition to significantly extending our understanding of photosensitivity in Limulus and providing critical insight into the genomic evolution of horseshoe crab opsins, this work provides a valuable genomic resource for addressing myriad questions related to xiphosuran physiology and arthropod evolution.

Description: Clusters are geographic concentrations of industries related by knowledge, skills, inputs, demand and/or other linkages. There is an increasing need for cluster-based data to support research, facilitate comparisons of clusters across regions and support policymakers in defining regional strategies. This article develops a novel clustering algorithm that systematically generates and assesses sets of cluster definitions (i.e., groups of closely related industries). We implement the algorithm using 2009 data for U.S. industries (six-digit NAICS), and propose a new set of benchmark cluster definitions that incorporates measures of inter-industry linkages based on co-location patterns, input–output links, and similarities in labor occupations. We also illustrate the algorithm’s ability to compare alternative sets of cluster definitions by evaluating our new set against existing sets in the literature. We find that our proposed set outperforms other methods in capturing a wide range of inter-industry linkages, including the grouping of industries within the same three-digit NAICS.

Description: Many massive galaxies at the centres of relaxed galaxy clusters and groups have vast reservoirs of warm (~10 000 K) and cold (100 K) gas. In many such low-redshift systems this gas is lifted into the hot interstellar medium in filamentary structures, which are long lived and are typically not forming stars. Two important questions are how far do these reservoirs cool and if cold gas is abundant what is the cause of the low star formation efficiency? Heating and excitation of the filaments from collisions and mixing of hot particles in the surrounding X-ray gas describes well the optical and near infrared line ratios observed in the filaments. In this paper we examine the theoretical properties of dense, cold clouds emitting in the far infrared and sub-millimetre through the bright lines of [C  ii ] 157 μm , [O  i ] 63 μm and CO, exposed to such energetic ionizing particles. We find that optical depth effects and thermal pressure support alone cannot account for the line ratios; however, a very modest additional pressure support can fit the observed [O  i ] 63 μm/[C  ii ] 157 μm line ratios by decreasing the density of the gas. This may also help stabilize the filaments against collapse leading to the low rates of star formation. We make predictions for the line ratios expected from cold gas under these conditions and present diagnostic diagrams for comparison with further observations. We provide our code as an Appendix.

Description: : We describe BiopLib, a mature C programming library for manipulating protein structure, and BiopTools, a set of command-line tools which exploit BiopLib. The library also provides a small number of functions for handling protein sequence and general purpose programming and mathematics. BiopLib transparently handles PDBML (XML) format and standard PDB files. BiopTools provides facilities ranging from renumbering atoms and residues to calculation of solvent accessibility. Availability and implementation: BiopLib and BiopTools are implemented in standard ANSI C. The core of the BiopLib library is a reliable PDB parser that handles alternate occupancies and deals with compressed PDB files and PDBML files automatically. The library is designed to be as flexible as possible, allowing users to handle PDB data as a simple list of atoms, or in a structured form using chains, residues and atoms. Many of the BiopTools command-line tools act as filters, taking a PDB (or PDBML) file as input and producing a PDB (or PDBML) file as output. All code is open source and documented using Doxygen. It is provided under the GNU Public Licence and is available from the authors’ web site or from GitHub. Contact: andrew@bioinf.org.uk

Description: When modelling an ionized plasma, all spectral synthesis codes need the thermally averaged free–free Gaunt factor defined over a very wide range of parameter space in order to produce an accurate prediction for the spectrum. Until now no data set exists that would meet these needs completely. We have therefore produced a table of relativistic Gaunt factors over a much wider range of parameter space than has ever been produced before. We present tables of the thermally averaged Gaunt factor covering the range 10 log 2  = –6 to 10 and 10 log u  = –16 to 13 for all atomic numbers Z  = 1 through 36. The data were calculated using the relativistic Bethe–Heitler–Elwert (BHE) approximation and were subsequently merged with accurate non-relativistic results in those parts of the parameter space where the BHE approximation is not valid. These data will be incorporated in the next major release of the spectral synthesis code cloudy . We also produced tables of the frequency integrated Gaunt factor covering the parameter space 10 log 2  = –6 to +10 for all values of Z between 1 and 36. All the data presented in this paper are available online.

Description: Turbulence is a key ingredient for the evolution of the intracluster medium, whose properties can be predicted with high-resolution numerical simulations. We present initial results on the generation of solenoidal and compressive turbulence in the intracluster medium during the formation of a small-size cluster using highly resolved, non-radiative cosmological simulations, with a refined monitoring in time. In this first of a series of papers, we closely look at one simulated cluster whose formation was distinguished by a merger around z  ~ 0.3. We separate laminar gas motions, turbulence and shocks with dedicated filtering strategies and distinguish the solenoidal and compressive components of the gas flows using Hodge–Helmholtz decomposition. Solenoidal turbulence dominates the dissipation of turbulent motions (~95 per cent) in the central cluster volume at all epochs. The dissipation via compressive modes is found to be more important (~30 per cent of the total) only at large radii (≥0.5 r vir ) and close to merger events. We show that enstrophy (vorticity squared) is good proxy of solenoidal turbulence. All terms ruling the evolution of enstrophy (i.e. baroclinic, compressive, stretching and advective terms) are found to be significant, but in amounts that vary with time and location. Two important trends for the growth of enstrophy in our simulation are identified: first, enstrophy is continuously accreted into the cluster from the outside, and most of that accreted enstrophy is generated near the outer accretion shocks by baroclinic and compressive processes. Secondly, in the cluster interior vortex, stretching is dominant, although the other terms also contribute substantially.

Description: The upper Tonian Chuar, Uinta Mountain, and middle Pahrump (ChUMP) groups of present-day western Laurentia collectively record the early breakup of Rodinia, large-scale perturbations in the carbon cycle, and eukaryotic evolution, all of which preceded the onset of global glaciation by tens of millions of years. The spectacularly preserved and shale-rich Chuar Group of the Grand Canyon Supergroup stands out as one of the best global records of this time period, particularly for paleobiology. A new U-Pb age of 782 Ma on detrital zircons ( n = 14 young grains) from the underlying Nankoweap Formation refines the Chuar Group’s maximum depositional age to younger than 782 Ma. A new 40 Ar/ 39 Ar age of 764 ± 16 Ma (2) from K-feldspar within early diagenetic marcasite nodules from the upper Chuar Group (Awatubi Member) helps calibrate the rich Chuar microfossil record and constrain the large-magnitude shift in 13 C org (up to 18; referred to here as the Awatubi positive carbon-isotope excursion or APCIE) to between ca. 764 and ca. 742 Ma, the date of an ash near the top of the Chuar Group. In addition to the maximum depositional age of ca. 782 Ma, U-Pb detrital zircon analyses ( n = 826 grains) on sandstone beds from the underlying Nankoweap Formation indicate the presence of multiple older Laurentian age peaks. The similarity of detrital zircon populations and sedimentary character to that of the overlying Chuar Group ( n = 764 grains) suggests that the Nankoweap Formation should be included as the lowermost unit in the Chuar Group. This revised geochronological framework indicates a 300 Ma unconformity between the Chuar Group (including the Nankoweap Formation) and the underlying 1.1 Ga Cardenas Basalt of the Unkar Group. Chuar Group detrital zircon populations share similarities with those of the Uinta Mountain Group and especially the middle Pahrump Group, including ca. 780 Ma grains. Biostratigraphic correlation using microfossils enhances the ChUMP connection and shows a trend of higher acritarch diversity in the lower Chuar and Uinta Mountain groups, and the presence of vase-shaped microfossils in the upper intervals of all three ChUMP units. Comparisons of 13 C org and 13 C carb among ChUMP successions suggest a combination of local and regional controls. Thus, ChUMP successions are coeval within the 780–740 Ma range, show similar fossil and C-isotope trends, and derived sediments from similar Laurentian sources or source types. In light of recent age constraints and compiled paleontology in other Neoproterozoic basins, our high-resolution correlation of ChUMP successions can be extended to the Callison Lake dolostone of NW Canada and the Akademikerbreen-Polarisbreen groups of Svalbard. Biostratigraphic correlation with poorly age-constrained strata such as the Akademikerbreen-Polarisbreen groups and, farther afield, the Visingsö Group of Baltica suggests that ChUMP units record continentwide—and perhaps global—evolutionary patterns. The 13 C org and 13 C carb values in the Chuar Group and its equivalents in Canada and Svalbard show broadly similar trends, including the APCIE, suggesting that 13 C org values from organic-rich shale record variations in the C-isotope composition of late Tonian oceans. Intracratonic basins and contiguous rift margins of ChUMP age are inferred to have been important locations for microbial productivity and significant organic carbon burial that induced large positive shifts in 13 C and changes in global carbon balance prior to the onset of snowball Earth.

Description: Type 2 brittle cornea syndrome (BCS2) is an inherited connective tissue disease with a devastating ocular phenotype caused by mutations in the transcription factor PR domain containing 5 ( PRDM5 ) hypothesized to exert epigenetic effects through histone and DNA methylation. Here we investigate clinical samples, including skin fibroblasts and retinal tissue from BCS2 patients, to elucidate the epigenetic role of PRDM5 and mechanisms of its dysregulation in disease. First we report abnormal retinal vascular morphology in the eyes of two cousins with BCS2 ( PRDM5 exons 9–14) using immunohistochemistry, and mine data from skin fibroblast expression microarrays from patients with PRDM5 mutations p.Arg590* and exons 9–14, as well as from a PRDM5 ChIP-sequencing experiment. Gene ontology analysis of dysregulated PRDM5-target genes reveals enrichment for extracellular matrix (ECM) genes supporting vascular integrity and development. Q-PCR and ChIP-qPCR confirm upregulation of critical mediators of ECM stability in vascular structures ( COL13A1 , COL15A1 , NTN1 , CDH5 ) in patient fibroblasts. We identify H3K9 di-methylation (H3K9me2) at these PRDM5-target genes in fibroblasts, and demonstrate that the BCS2 mutation p.Arg83Cys diminishes interaction of PRDM5 with repressive complexes, including NuRD complex protein CHD4, and the repressive chromatin interactor HP1BP3, by co-immunoprecipitation combined with mass spectrometry. We observe reduced heterochromatin protein 1 binding protein 3 (HP1BP3) staining in the retinas of two cousins lacking exons 9–14 by immunohistochemistry, and dysregulated H3K9me2 in skin fibroblasts of three patients (p.Arg590*, p.Glu134* and exons 9–14) by western blotting. These findings suggest that defective interaction of PRDM5 with repressive complexes, and dysregulation of H3K9me2, play a role in PRDM5-associated disease.