bluuebunny/crossref_metadata_2025_split huggingface dataset:
Potential high-frequency off-target mutagenesis induced by CRISPR/Cas9 in Arabidopsis and its prevention
Authors: Zhang Qiang , Xing Hui-Li , Wang Zhi-Ping , Zhang Hai-Yan , Yang Fang , Wang Xue-Chen , Chen Qi-Jun 
Published: November 2017

AbstractSpecificity of CRISPR/Cas9 tools has been a major concern along with the reports of their successful applications. We report unexpected observations of high frequency off-target mutagenesis induced by CRISPR/Cas9 in T1 Arabidopsis mutants although the sgRNA was predicted to have a high specificity score. We also present evidence that the off-target effects were further exacerbated in the T2 progeny. To prevent the off-target effects, we tested and optimized two strategies in Arabidopsis, including introduction of a mCherry cassette for a simple and reliable isolation of Cas9-free mutants and the use of highly specific mutant SpCas9 variants. Optimization of the mCherry vectors and subsequent validation found that fusion of tRNA with the mutant rather than the original sgRNA scaffold significantly improves editing efficiency. We then examined the editing efficiency of eight high-specificity SpCas9 variants in combination with the improved tRNA-sgRNA fusion strategy. Our results suggest that highly specific SpCas9 variants require a higher level of expression than their wild-type counterpart to maintain high editing efficiency. Additionally, we demonstrate that T-DNA can be inserted into the cleavage sites of CRISPR/Cas9 targets with high frequency. Altogether, our results suggest that in plants, continuous attention should be paid to off-target effects induced by CRISPR/Cas9 in current and subsequent generations, and that the tools optimized in this report will be useful in improving genome editing efficiency and specificity in plants and other organisms.
Morphological changes of plasma membrane and protein assembly during clathrin-mediated endocytosis
Authors: Yoshida Aiko , Sakai Nobuaki , Uekusa Yoshitsugu , Imaoka Yuka , Itagaki Yoshitsuna , Suzuki Yuki , Yoshimura Shige H. 
Published: November 2017

AbstractClathrin-mediated endocytosis (CME) proceeds through a series of morphological changes of the plasma membrane induced by a number of protein components. Although the spatiotemporal assembly of these proteins has been elucidated by fluorescence-based techniques, the protein-induced morphological changes of the plasma membrane have not been fully clarified in living cells. Here we visualize membrane morphology together with protein localizations during CME by utilizing high-speed atomic force microscopy combined with a confocal laser scanning unit. The plasma membrane starts to invaginate ~30 seconds after clathrin starts to assemble, and the aperture diameter increases as clathrin accumulates. Actin rapidly accumulates around the pit and induces a small membrane swelling, which within 30 seconds rapidly covers the pit irreversibly. Inhibition of actin turnover abolishes the swelling and induces a reversible open-close motion of the pit, indicating that actin dynamics are necessary for efficient and irreversible pit closure at the end of the CME.
Incipient signs of genetic differentiation among African elephant populations in fragmenting miombo ecosystems in south-western Tanzania
Authors: Lobora Alex L. , Nahonyo Cuthbert L. , Munishi Linus K. , Caro Tim , Foley Charles , Beale Colin M. , Eggert Lori S. 
Published: November 2017

AbstractHabitat fragmentation plays a major role in the reduction of genetic diversity among wildlife populations. The African savannah elephant population of the Ruaha-Rungwa and Katavi-Rukwa ecosystems in south-western Tanzania, comprises one of the world’s largest remaining elephant populations, but is increasingly threatened by loss of connectivity and poaching for ivory. We investigate whether there are incipient signs of genetic isolation (loss of heterozygosity) within the younger cohort as a result of habitat loss between the two ecosystems. To investigate the genetic structure of populations, we compared the genotypes for 11 microsatellite loci in the western (n = 81 individuals from Katavi-Rukwa), central (n = 36 individuals from Lukwati and Piti), and eastern populations (n = 193, individuals from Ruaha-Rungwa). We found evidence of significant genetic differentiation among the three populations, but the levels were low, suggesting recent divergence. Furthermore, we identified weak isolation by distance, suggesting higher gene flow among nearer individuals with samples within 50km of each other being more genetically similar to one another than beyond. Although sample sizes were small, a further analysis of genetic differences across populations and in separate age classes revealed evidence of increasing genetic structure among younger age classes across the landscape. In a long-lived species with overlapping generations, it takes a long time to develop genetic substructure even when there are substantial obstacles to migration. Thus, in these recently fragmented populations, inbreeding (and the loss of heterozygosity) may be less of an immediate concern than demography (the loss of adults due to illegal hunting).
Trends and variation in Prescribing of Low-Priority Medicines Identified by NHS England: A Cross-Sectional Study and Interactive Data Tool in English Primary Care
Authors: Walker Alex J , Curtis Helen J , Bacon Seb , Croker Richard , Goldacre Ben 
Published: November 2017

AbstractBackgroundRoutine accessible audit of prescribing data presents significant opportunities to identify cost-saving opportunities. NHS England recently announced a consultation seeking to discourage use of medicines it considers to be low-value. We set out to produce an interactive data resource to show savings in each NHS general practice, and to assess the current use of these medicines, their change in use over time, and the extent and reasons for variation in such prescribing.ResultsThe total NHS spend on all low-value medicines identified by NHS England was £153.5m in the last year, across 5.8m prescriptions (mean £26 per prescription). Among individual medications, liothyronine had the highest prescribing cost at £29.6m, followed by trimipramine (£20.2m) and gluten-free foods (£18.7m). Over time, the overall total number of low-value prescriptions decreased, but the cost increased, although this varied greatly between medications. Annual practice level spending varied widely (median, £2,262 per thousand patients, IQR £1,439 to £3,298). The proportion of patients over 65 showed the strongest association with low-value prescribing; CCG was also strongly associated. Our interactive data tool was deployed to OpenPrescribing.net where monthly updated figures and graphs can be viewed.ConclusionsPrescribing of low-value medications is extensive but varies widely by medication, geographic area and individual practice. Despite a fall in prescription numbers, the overall cost of prescribing for low-value items has risen. Prescribing behaviour is clustered by CCG, which may represent variation in medicines optimisation efficiency, or in some cases access inequality.AbbreviationsGPGeneral PracticeNHSNational Health ServiceNICENational Institute for Health and Care ExcellencePCAPrescription cost AnalysisQOFQuality Outcomes Framework
Uneven Data Quality and the Earliest Occupation of Europe – The Case of Untermassfeld (Germany)
Authors: Roebroeks Wil , Gaudzinski-Windheuser Sabine , Baales Michael , Kahlke Ralf-Dietrich 
Published: November 2017

AbstractThe database regarding the earliest occupation of Europe has increased significantly in quantity and quality of data points over the last two decades, mainly through the addition of new sites as a result of long-term systematic excavations and large-scale prospections of Early and early Middle Pleistocene exposures. The site distribution pattern suggests an ephemeral presence of hominins in the south of Europe from around one million years ago, with occasional short northward expansions along the western coastal areas when temperate conditions permitted. From around 600,000-700,000 years ago Acheulean artefacts appear in Europe and somewhat later hominin presence seems to pick up, with more sites and now some also present in colder climatic settings. It is again only later, around 350,000 years ago, that the first sites show up in more continental, central parts of Europe, east of the Rhine. A series of recent papers on the Early Pleistocene palaeontological site of Untermassfeld (Germany) makes claims that are of great interest for studies of earliest Europe and are at odds with the described pattern: the papers suggest that Untermassfeld has yielded stone tools and humanly modified faunal remains, evidence for a one million years old hominin presence in European continental mid-latitudes, and additional evidence that hominins were well-established in Europe already around that time period. Here we evaluate these claims and demonstrate that these studies are severely flawed in terms of data on provenance of the materials studied and in the interpretation of faunal remains and lithics as testifying to a hominin presence at the site. In actual fact any reference to the Untermassfeld site as an archaeological one is unwarranted. Furthermore, it is not the only European Early Pleistocene site where inferred evidence for hominin presence is problematic. The strength of the spatiotemporal patterns of hominin presence and absence depend on the quality of the data points we work with, and data base maintenance, including critical evaluation of new sites, is crucial to advance our knowledge of the expansions and contractions of hominin ranges during the Pleistocene.
The amyloidogenicity of the influenza virus PB1-derived peptide sheds light on its antiviral activity
Authors: Zabrodskaya Yana A. , Lebedev Dmitry V. , Egorova Marja A. , Shaldzhyan Aram A. , Shvetsov Alexey V. , Kuklin Alexander I. , Vinogradova Daria S. , Klopov Nikolay V. , Matusevich Oleg V. , Cheremnykh Taisiia A. , Dattani Rajeev , Egorov Vladimir V. 
Published: November 2017

AbstractThe influenza virus polymerase complex is a promising target for new antiviral drug development. It is known that, within the influenza virus polymerase complex, the PB1 subunit region from the 1st to the 25th amino acid residues has to be is in an alpha-helical conformation for proper interaction with the PA subunit. We have previously shown that PB1(6–13) peptide at low concentrations is able to interact with the PB1 subunit N-terminal region in a peptide model which shows aggregate formation and antiviral activity in cell cultures.In this paper, it was shown that PB1(6–13) peptide is prone to form the amyloid-like fibrillar aggregates. The peptide homo-oligomerization kinetics were examined, and the affinity and characteristic interaction time of PB1(6–13) peptide monomers and the influenza virus polymerase complex PB1 subunit N-terminal region were evaluated by the SPR and TR-SAXS methods. Based on the data obtained, a hypothesis about the PB1(6–13) peptide mechanism of action was proposed: the peptide in its monomeric form is capable of altering the conformation of the PB1 subunit N-terminal region, causing a change from an alpha helix to a beta structure. This conformational change disrupts PB1 and PA subunit interaction and, by that mechanism, the peptide displays antiviral activity.Graphical abstract
GPseudoRank: a permutation sampler for single cell orderings
Authors: Strauß Magdalena E , Reid John E , Wernisch Lorenz 
Published: November 2017

AbstractMotivationA number of pseudotime methods have provided point estimates of the ordering of cells for scRNA-seq data. A still limited number of methods also model the uncertainty of the pseudotime estimate. However, there is still a need for a method to sample from complicated and multi-modal distributions of orders, and to estimate changes in the amount of the uncertainty of the order during the course of a biological development, as this can support the selection of suitable cells for the clustering of genes or for network inference.ResultsIn an application to a microarray data set our proposed method, GPseudoRank, identifies two modes of the distribution, each of them corresponding to point estimates of orders obtained by a different established method. In an application to scRNA-seq data we demonstrate the potential of GPseudoRank to identify phases of lower and higher pseudotime uncertainty during a biological process. GPseudoRank also correctly identifies cells precocious in their antiviral response.Availability and implementationOur method is available on github: https://github.com/magStra/GPseudoRank.Contactmagdalena.strauss@mrc-bsu.cam.ac.ukSupplementary informationSupplementary materials are available.
Mechanical strain can increase segment number in live chick embryos
Authors: Nelemans Ben K. A. , Schmitz Manuel , Tahir Hannan , Merks Roeland M. H. , Smit Theodoor H. 
Published: November 2017

AbstractPhysical cues, experienced during early embryonic development, can influence species-specific vertebral numbers. Here we show that mechanical stretching of live chicken embryos can induce the formation of additional somites and thereby modify early segmental patterning. Stretching deforms the somites, and results in a cellular reorganization that forms stable daughter somites. Cells from the somite core thereby undergo mesenchymal-to-epithelial transitions (MET), thus meeting the geometrical demand for more border cells. Using a Cellular Potts Model, we suggest that this MET occurs through lateral induction by the existing epithelial cells. Our results indicate that self-organizing properties of the somitic mesoderm generate phenotypic plasticity that allows it to cope with variations in the mechanical environment. This plasticity may provide a novel mechanism for explaining how vertebral numbers in species may have increased during evolution. Additionally, by preventing the formation of transitional vertebrae, these self-organization qualities of somites may be selectively advantageous.
Lamins organize the global three-dimensional genome from the nuclear periphery
Authors: Zheng Xiaobin , Hu Jiabiao , Yue Sibiao , Kristiani Lidya , Kim Miri , Sauria Michael , Taylor James , Kim Youngjo , Zheng Yixian 
Published: November 2017

AbstractLamins are structural components of the nuclear lamina (NL) that regulate genome organization and gene expression, but the mechanism remains unclear. Using Hi-C, we show that lamins maintain proper interactions among the topologically associated chromatin domains (TADs) but not their overall architecture. Combining Hi-C with fluorescence in situ hybridization (FISH) and analyses of lamina-associated domains (LADs), we reveal that lamin loss causes expansion or detachment of specific LADs in mouse ES cells. The detached LADs disrupt 3D interactions of both LADs and interior chromatin. 4C and epigenome analyses further demonstrate that lamins maintain the active and repressive chromatin domains among different TADs. By combining these studies with transcriptome analyses, we found a significant correlation between transcription changes and the changes of active and inactive chromatin domain interactions. These findings provide a foundation to further study how the nuclear periphery impacts genome organization and transcription in development and NL-associated diseases.HighlightsLamin loss does not affect the overall TAD structure but alters TAD-TAD interactionsLamin null ES cells exhibit decondensation or detachment of specific LAD regionsExpansion and detachment of LADs can alter genome-wide 3D chromatin interactionsAltered chromatin domain interactions are correlated with altered transcription
Protein Moonlighting Revealed by Non-Catalytic Phenotypes of Yeast Enzymes
Authors: Espinosa-Cantú Adriana , Ascencio Diana , Herrera-Basurto Selene , Xu Jiewei , Roguev Assen , Krogan Nevan J. , DeLuna Alexander 
Published: November 2017

ABSTRACTA single gene can partake in several biological processes, and therefore gene deletions can lead to different—sometimes unexpected—phenotypes. However, it is not always clear whether such pleiotropy reflects the loss of a unique molecular activity involved in different processes or the loss of a multifunctional protein. Here, usingSaccharomyces cerevisiaemetabolism as a model, we systematically test the null hypothesis that enzyme phenotypes depend on a single annotated molecular function, namely their catalysis. We screened a set of carefully selected genes by quantifying the contribution of catalysis to gene-deletion phenotypes under different environmental conditions. While most phenotypes were explained by loss of catalysis, 30% could be readily complemented by a catalytically-inactive enzyme. Such non-catalytic phenotypes were frequent in the Alt1 and Bat2 transaminases and in the isoleucine/valine-biosynthetic enzymes Ilv1 and Ilv2, suggesting novel "moonlighting" activities in these proteins. Furthermore, differential genetic-interaction profiles of gene-deletion and catalytic mutants indicated thatILV1is functionally associated to regulatory processes, specifically to chromatin modification. Our systematic study shows that gene-loss phenotypes and their genetic interactions are frequently not driven by the loss of an annotated catalytic function, underscoring the moonlighting nature of cellular metabolism.
Comparison of the 3D-IR - BTFE method and the conventional method in the head MRI contrast
Authors: Hirahara Daisuke 
Published: November 2017

AbstractMRI using gadolinium contrast media is useful in diagnosis; however, nephrogenic systemic fibrosis is a serious side effect of gadolinium exposure. Moreover, it turns out that gadolinium deposits in the brain. This has escalated the necessity for a suitable method to use gadolinium contrast media. I developed a new imaging method that had excellent contrast. This study examined the usefulness of that new imaging method and found the method is highly effective.
Successful aggregation of Tau protein labelled on its native cysteines
Authors: Radli Martina , Verdonschot Romy E , Ferrari Luca , Rüdiger Stefan GD 
Published: November 2017

AbstractThe formation of fibrillar tangles of the Tau protein is crucial in the development of Alzheimer’s disease. Biophysical methods based on labelling of the cysteines of Tau with fluorescence dyes would allow to study fibril formation with an ‘internal eye’. However, the two native cysteines of Tau at positions 291 and 322 are located in the repeat domain, which is involved in forming the fibrils. The contribution of both cysteines to this process is unclear. Here we show that blocking natural cysteines using large fluorescent dyes does not interfere with Tau fibrillation so that FRET can be used to follow structural changes during the process. We anticipate that cysteine-labelled Tau enables following structural rearrangements during fibril formation in detail. This may also allow to monitor the effect of drugs, small molecules and proteins on the process.
Local and global Cdc42 GEFs for fission yeast cell polarity are coordinated by microtubules and the Tea1/Tea4/Pom1 axis
Authors: Tay Ye Dee , Leda Marcin , Goryachev Andrew B. , Sawin Kenneth E. 
Published: November 2017

ABSTRACTThe conserved Rho-family GTPase Cdc42 plays a central role in eukaryotic cell polarity. The rod-shaped fission yeastSchizosaccharomyces pombehas two Cdc42 guanine-nucleotide exchange factors (GEFs), Scd1 and Gef1, but little is known about how they are coordinated in polarized growth. Although the microtubule cytoskeleton is normally not required for polarity maintenance in fission yeast, here we show that whenscdlfunction is compromised, disruption of microtubules or the polarity landmark proteins Tea1, Tea4, or Pom1 leads to isotropic rather than polarized growth. Surprisingly, this isotropic growth is due to spatially inappropriate activity of Gef1, which is a cytosolic protein rather than a membrane-associated protein at cell tips like Scd1. Microtubules and the Tea1/Tea4/Pom1 axis counteract inappropriate Gef1 activity by regulating the localization of the Cdc42 GTPase-activating protein Rga4. Our results thus demonstrate coordination of “local” (Scd1) and “global” (Gef1) Cdc42 GEFs via microtubules and microtubule-dependent polarity landmarks.
Two-photon photoactivated voltage imaging in tissue with an Archaerhodopsin-derived reporter
Authors: Chien Miao-Ping , Brinks Daan , Adam Yoav , Bloxham William , Kheifets Simon , Cohen Adam E. 
Published: November 2017

AbstractRobust voltage imaging in tissue remains a technical challenge. Existing combinations of genetically encoded voltage indicators (GEVIs) and microscopy techniques cannot simultaneously achieve sufficiently high voltage sensitivity, background rejection, and time resolution for highresolution mapping of sub-cellular voltage dynamics in intact brain tissue. We developed a pooled high-throughput screening approach to identify Archaerhodopsin mutants with unusual photophysical properties. After screening ~105 cells, we identified a novel GEVI, NovArch, whose 1-photon near infrared fluorescence is reversibly enhanced by weak 2-photon excitation. Because the 2-photon excitation acts catalytically rather than stoichiometrically, high fluorescence signals, optical sectioning, and high time resolution are achieved simultaneously, at modest 2-photon laser power. We developed a microscopy system optimized for NovArch imaging in tissue. The combination of protein and optical engineering enhanced signal contrast sufficiently to enable optical mapping of back-propagating action potentials in dendrites in acute mouse brain slice.
C11orf70 mutations causing primary ciliary dyskinesia disrupt a conserved step in the intraflagellar transport-dependent assembly of multiple axonemal dyneins
Authors: Fassad Mahmoud R. , Shoemark Amelia , Borgne Pierrick le , Koll France , Patel Mitali , Dixon Mellisa , Hayward Jane , Richardson Charlotte , Frost Emily , Jenkins Lucy , Cullup Thomas , Chung Eddie MK , Lemullois Michel , Aubusson-Fleury Anne , Hogg Claire , Mitchell David R. , Tassin Anne-Marie , Mitchison Hannah M. 
Published: November 2017

AbstractPrimary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disorder characterized by destructive respiratory disease and laterality abnormalities due to randomised left-right body asymmetry. PCD is mostly caused by mutations affecting components of the core axoneme structure of motile cilia that are essential for cilia movement. In addition, there is a growing group of PCD genes that encode proteins essential for the assembly of the ciliary dynein motors and the active transport process that delivers them from their cytoplasmic assembly site into the axoneme. We screened a cohort of affected individuals for disease-causing mutations using a targeted next generation sequencing panel and identified 2 unrelated families (3 affected children) with mutations in the uncharacterized C11orf70 gene. The affected children share a consistent PCD phenotype from early life with laterality defects and immotile respiratory cilia displaying combined loss of inner and outer dynein arms (IDA+ODA). Phylogenetic analysis shows C11orf70 is highly conserved, distributed across species similarly to proteins involved in the intraflagellar transport (IFT)-dependant assembly of axonemal dyneins. Paramecium C11orf70 RNAi knockdown led to combined loss of ciliary IDA+ODA with reduced cilia beating and swim velocity. Fluorescently tagged C11orf70 in Paramecium and Chlamydomonas localises mainly in the cytoplasm with a small amount in the ciliary component, its abundance in the axoneme being IFT-dependant. During ciliogenesis, C11orf70 accumulates at the ciliary tips in a similar distribution to the IFT-B protein IFT46. In summary, C11orf70 is essential for IFT-dependant assembly of dynein arms and C11orf70 mutations cause defective cilia motility and PCD.