bluuebunny/crossref_metadata_2025_split huggingface dataset:
Halogen-aromatic π interactions modulate inhibitor residence time
Authors: Heroven Christina , Georgi Victoria , Ganotra Gaurav K. , Brennan Paul E. , Wolfreys Finn , Wade Rebecca C. , Fernández-Montalván Amaury E. , Chaikuad Apirat , Knapp Stefan 
Published: January 2018

ABSTRACTProlonged drug residence times may result in longer lasting drug efficacy, improved pharmacodynamic properties and “kinetic selectivity” over off-targets with fast drug dissociation rates. However, few strategies have been elaborated to rationally modulate drug residence time and thereby to integrate this key property into the drug development process. Here, we show that the interaction between a halogen moiety on an inhibitor and an aromatic residue in the target protein can significantly increase inhibitor residence time. By using the interaction of the serine/threonine kinase haspin with 5-iodotubercidin (5-iTU) derivatives as a model for an archetypal active state (type I) kinase-inhibitor binding mode, we demonstrate that inhibitor residence times markedly increase with the size and polarizability of the halogen atom. This key interaction is dependent on the interactions with an aromatic residue in the gate keeper position and we observe this interaction in other kinases with an aromatic gate keeper residue. We provide a detailed mechanistic characterization of the halogen-aromatic π interactions in the haspin-inhibitor complexes by means of kinetic, thermodynamic, and structural measurements along with binding energy calculations. Since halogens are frequently used in drugs and aromatic residues are often present in the binding sites of proteins, our results provide a compelling rationale for introducing aromatic-halogen interactions to prolong drug-target residence times.
Harnessing a Novel P450 Fatty Acid Decarboxylase fromMacrococcus caseolyticusfor Microbial Biosynthesis of Odd Chain Terminal Alkenes
Authors: Lee Jong-won , Niraula Narayan P. , Trinh Cong T. 
Published: January 2018

ABSTRACTAlkenes are industrially important platform chemicals with broad applications. In this study, we report a microbial conversion route for direct biosynthesis of medium and long chain terminal alkenes from fermentable sugars by harnessing a novel P450 fatty acid (FA) decarboxylase fromMacrococcus caseolyticus(OleTMC). We first characterized OleTMCand demonstrated itsin vitroH2O2-independent activities towards linear and saturated C10:0-C18:0 FAs, with the highest activity for C16:0 and C18:0 FAs. Combining protein homology modeling,in silicoresidue mutation analysis, and docking simulation with direct experimental evidence, we elucidated the underlying mechanism for governing the observed substrate preference of OleTMC, which depends on the size of FA binding pocket, not the catalytic site. Next, we engineered the terminal alkene biosynthesis pathway, consisting of an engineeredE. colithioesterase (TesA*) and OleTMC, and introduced this pathway intoE. colifor direct terminal alkene biosynthesis from glucose. The recombinant strainE. coliEcNN101 produced a total of 17.78 ± 0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% ± 0.5% C11 alkene, 12.7% ± 2.2% C13 alkene, 82.7% ± 1.7% C15 alkene, and 3.7% ± 0.8% C17 alkene, and a yield of 0.87 ± 0.03 (mg/g) on glucose after 48 h in baffled shake flasks. To improve the terminal alkene production, we identified and overcame the electron transfer limitation in OleTMC, by introducing a two-component redox system, consisting of a putidaredoxin reductase CamA and a putidaredoxin CamB fromPseudomonas putida,into EcNN101, and demonstrated the terminal alkene production increased ∼2.8 fold after 48 h. Overall, this study provides a better understanding of the function of P450 FA decarboxylases and helps guide future protein and metabolic engineering for enhanced microbial production of target designer alkenes in a recombinant host.
Caenorhabditis elegansheterochromatin factor SET-32 plays an essential role in transgenerational establishment of nuclear RNAi-mediated epigenetic silencing
Authors: Kalinava Natallia , Ni Julie Zhouli , Gajic Zoran , Ushakov Helen , Gu Sam Guoping 
Published: January 2018

AbstractEpigenetic inheritance contributes fundamentally to transgenerational physiology and fitness. Mechanistic understanding of RNA-mediated chromatin modification and transgenerational epigenetic inheritance, which inC. eleganscan be triggered by exogenous double-stranded RNA (exo-dsRNA) or facilitated by endogenous small interfering RNAs (endo-siRNAs), has mainly been limited to the post-initiation phases of silencing. Indeed, the dynamic process by which nuclear RNAi engages a transcriptionally active target, before the repressive state is stably established, remains largely a mystery. Here we found that the onset of exo-dsRNA-induced nuclear RNAi is a transgenerational process, and that establishment requires SET-32, one of the three putative histone methyltransferases (HMTs) that are required for H3K9me3 deposition at the nuclear RNAi targets. We also performed multigenerational whole-genome analyses to examine the establishment of silencing at endogenous targets of germline nuclear RNAi. The nuclear Argonaute protein HRDE-1 is essential for the maintenance of nuclear RNAi. Repairing a loss-of-function mutation inhrde-1by CRISPR restored the silencing of endogenous targets in animals carrying wild typeset-32. However, for numerous endogenous targets, repairing thehrde-1mutation in aset-32;hrde-1double mutant failed to restore their silencing states in up to 20 generations after thehrde-1repair, using a similar genome editing approach. We found that despite a prominent role in the establishment of silencing, however,set-32is completely dispensable for the maintenance of silencing once HRDE-1-dependent gene repression is established. Our study indicates that: 1) establishment and maintenance of siRNA-guided transcriptional repression are two distinct processes with different genetic requirements; and 2) the rate-limiting step of the establishment phase is a transgenerational, chromatin-based process. In addition, our study reveals a novel paradigm in which a heterochromatin factor primarily functions to promote the establishment of transgenerational silencing, expanding mechanistic understanding of the well-recognized role of heterochromatin in epigenetic maintenance.
Neural circuitry of a polycystin-mediated hydrodynamic startle response for predator avoidance
Authors: Bezares-Calderón Luis A. , Berger Jürgen , Jasek Sanja , Verasztó Csaba , Mendes Sara , Gühmann Martin , Almeda Rodrigo , Shahidi Réza , Jékely Gáspár 
Published: January 2018

AbstractStartle responses triggered by aversive stimuli including predators are widespread across animals. These coordinated whole-body actions require the rapid and simultaneous activation of a large number of muscles. Here we study a startle response in a planktonic larva to understand the whole-body circuit implementation of the behavior. Upon encountering water vibrations, larvae of the annelidPlatynereisclose their locomotor cilia and simultaneously contract the body and raise the parapodia. The startle response is mediated by collar receptor neurons expressing the polycystins PKD1-1 and PKD2-1. CRISPR-generatedPKD1-1andPKD2-1mutant larvae do not startle and fall prey to a copepod predator at a higher rate. Reconstruction of the whole-body connectome of the collar-receptor-cell circuitry revealed converging feedforward circuits to the ciliary bands and muscles. The wiring diagram suggests circuit mechanisms for the intersegmental and left-right coordination of the response. Our results reveal how polycystin-mediated mechanosensation can trigger a coordinated whole-body effector response involved in predator avoidance.Short SummaryThe neuronal circuitry of thePlatynereisstartle response links polycystin-dependent hydrodynamic sensors to muscle and ciliary effector cells
Phylodynamic model adequacy using posterior predictive simulations
Authors: Duchene Sebastian , Bouckaert Remco , Duchene David A. , Stadler Tanja , Drummond Alexei J. 
Published: January 2018

AbstractRapidly evolving pathogens, such as viruses and bacteria, accumulate genetic change at a similar timescale over which their epidemiological processes occur, such that it is possible to make inferences about their infectious spread using phylogenetic time-trees. For this purpose it is necessary to choose a phylodynamic model. However, the resulting inferences are contingent on whether the model adequately describes key features of the data. Model adequacy methods allow formal rejection of a model if it cannot generate the main features of the data. We present TreeModelAdequacy (TMA), a package for the popular BEAST2 software, that allows assessing the adequacy of phylodynamic models. We illustrate its utility by analysing phylogenetic trees from two viral outbreaks of Ebola and H1N1influenza. The main features of the Ebola data were adequately described by the coalescent exponential-growth model, whereas the H1N1influenza data was best described by the birth-death SIR model.
Genome-wide prediction of bacterial effectors across six secretion system types using a feature-based supervised learning framework
Authors: Dhroso Andi , Eidson Samantha , Korkin Dmitry 
Published: January 2018

AbstractGram-negative bacteria are responsible for hundreds of millions infections worldwide, including the emerging hospital-acquired infections and neglected tropical diseases in the third-world countries. Finding a fast and cheap way to understand the molecular mechanisms behind the bacterial infections is critical for efficient diagnostics and treatment. An important step towards understanding these mechanisms is discovering bacterial effectors, the proteins secreted into the host through one of the six common secretion system types. Unfortunately, current effector prediction methods are designed to specifically target one of three secretion systems, and no accurate “secretion system-agnostic” method is available.Here, we present PREFFECTOR, a computational feature-based approach to discover effectors in Gram-negative bacteria without prior knowledge on bacterial secretion system(s) or cryptic secretion signals. Our approach was first evaluated using several assessment protocols on a manually curated, balanced dataset of experimentally determined effectors across all six secretion systems as well as non-effector proteins. The evaluation revealed high accuracy of the top performing classifiers in PREFFECTOR, with the small false positive discovery rate across all six secretion systems. Our method was also applied to four bacteria that had limited knowledge on virulence factors or secreted effectors. PREFFECTOR web-server is freely available at: http://korkinlab.org/preffector.
Lost in Space: The Impact of Traditional Neuroimaging Methods on the Spatial Localization of Cortical Areas
Authors: Coalson Timothy S. , Van Essen David C. , Glasser Matthew F. 
Published: January 2018

AbstractLocalizing human brain functions is a long-standing goal in systems neuroscience. Towards this goal, neuroimaging studies have traditionally used volume-based smoothing, registered data to volume-based standard spaces, and reported results relative to volume-based parcellations. A novel 360-area surface-based cortical parcellation was recently generated using multimodal data from the Human Connectome Project (HCP), and a volume-based version of this parcellation has frequently been requested for use with traditional volume-based analyses. However, given the major methodological differences between traditional volumetric and HCP-style processing, the utility and interpretability of such an altered parcellation must first be established. By starting from automatically generated individual-subject parcellations and processing them with different methodological approaches, we show that traditional processing steps, especially volume-based smoothing and registration, substantially degrade cortical area localization when compared to surface-based approaches. We also show that surface-based registration using features closely tied to cortical areas, rather than to folding patterns alone, improves the alignment of areas, and that the benefits of high resolution acquisitions are largely unexploited by traditional volume-based methods. Quantitatively, we show that the most common version of the traditional approach has spatial localization that is only 35% as good as the best surface-based method as assessed with two objective measures (peak areal probabilities and ‘captured area fraction’ for maximum probability maps). Finally, we demonstrate that substantial challenges exist when attempting to accurately represent volume-based group analysis results on the surface, which has important implications for the interpretability of studies, both past and future, that use these volume-based methods.Significance StatementMost human brain imaging studies have traditionally used low-resolution images, inaccurate methods of cross-subject alignment, and extensive blurring. Recently, a high-resolution approach with more accurate alignment and minimized blurring was used by the Human Connectome Project to generate a multi-modal map of human cortical areas in hundreds of individuals. Starting from this data, we systematically compared these two approaches, showing that the traditional approach is nearly three times worse than the HCP’s improved approach in two objective measures of spatial localization of cortical areas. Further, we demonstrate considerable challenges in comparing data across the two approaches, and, as a result, argue that there is an urgent need for the field to adopt more accurate methods of data acquisition and analysis.
Frequent sgRNA-barcode Recombination in Single-cell Perturbation Assays
Authors: Xie Shiqi , Cooley Anne , Armendarez Daniel , Zhou Pei , Hon Gary C. 
Published: January 2018

AbstractSimultaneously detecting CRISPR-based perturbations and induced transcriptional changes in the same cell is a powerful approach to unraveling genome function. Several lentiviral approaches have been developed, some of which rely on the detection of distally located genetic barcodes as an indirect proxy of sgRNA identity. Since barcodes are often several kilobases from their corresponding sgRNAs, viral recombination-mediated swapping of barcodes and sgRNAs is feasible. Using a self-circularization-based sgRNA-barcode library preparation protocol, we estimate the recombination rate to be ~50% and we trace this phenomenon to the pooled viral packaging step. Recombination is random, and decreases the signal-to-noise ratio of the assay. Our results suggest that alternative approaches can increase the throughput and sensitivity of single-cell perturbation assays.
Individual differences in functional brain connectivity predict temporal discounting preference in the transition to adolescence
Authors: Anandakumar Jeya , Mills Kathryn L. , Earl Eric , Irwin Lourdes , Miranda-Dominguez Oscar , Demeter Damion V. , Walton-Weston Alexandra , Karalunas Sarah , Nigg Joel , Fair Damien A. 
Published: January 2018

AbstractThe transition from childhood to adolescence is marked by distinct changes in behavior, including how one values waiting for a large reward compared to receiving an immediate, yet smaller, reward. While previous research has emphasized the relationship between this preference and age, it is also proposed that this behavior is related to circuitry between valuation and cognitive control systems. In this study, we examined how age and intrinsic functional connectivity strength within and between these neural systems relate to changes in discounting behavior across the transition into adolescence. We used mixed-effects modeling and linear regression to assess the contributions of age and connectivity strength in predicting discounting behavior. First, we identified relevant connections in a longitudinal sample of 64 individuals who completed MRI scans and behavioral assessments 2-3 times across ages 7-15 years (137 scans). We then repeated the analysis in a separate, cross-sectional, sample of 84 individuals (7-13 years). Both samples showed an age-related increase in preference for waiting for larger rewards. Connectivity strength within and between valuation and cognitive control systems accounted for further variance not explained by age. These results suggest that individual differences in functional neural organization can account for behavioral changes typically associated with age.
Effects of Parathyroid Hormone, Alendronate and Odanacatib on the mineralisation process in intracortical and endocortical Haversian bone of ovariectomized rabbits
Authors: Vrahnas Christina , Buenzli Pascal R , Pearson Thomas A , Pennypacker Brenda L , Tobin Mark J , Bambery Keith R , Duong Le T , Sims Natalie A 
Published: January 2018

AbstractAlthough cortical bone strength depends on optimal bone composition, the influences of standard therapeutic agents for osteoporosis on bone mineral accrual in cortical bone are not understood. This study compared effects on cortical bone composition of two current therapeutic approaches for osteoporosis: the anti-resorptive bisphosphonate alendronate (ALN), and anabolic intermittent parathyroid hormone (PTH). The experimental anti-resorptive cathepsin K inhibitor, odanacatib (ODN) which inhibits resorption without inhibiting bone formation, was also tested.To determine effects of these agents on Haversian remodeling and mineral accrual, we compared ALN (100μg/kg/2xweek), PTH(1-34) (15μg/kg, 5x/week) and ODN (7.5μM/day) administered for 10 months commencing 6 months after ovariectomy (OVX) in skeletally mature rabbits by histomorphometry. We used synchrotron-based Fourier-transform infrared microspectroscopy (sFTIRM), coupled to fluorochrome labelling, to measure maturation of the cortical matrixin situat both endocortical and intracortical sites of bone formation.PTH and ODN, but not ALN, treatment increased bone toughness, and PTH treatment stimulated bone formation, not only on endocortical and periosteal bone, but also in intracortical pores. In Sham and OVX rabbits, normal matrix maturation was observed at both endocortical and intracortical sites including: mineral accrual (increasing mineral:matrix), carbonate substitution (carbonate:mineral) and collagen molecular compaction (amide I:II)in situin endocortical and intracortical bone. ALN treatment reduced bone formation on these surfaces. In ALN-treated bone, while intracortical bone matured normally, endocortical bone did not show a significant increase in mineral:matrix. ODN treatment resulted in slower mineral accrual and limited carbonate substitution. While PTH-treatment did not modify matrix maturation in endocortical bone, the initial stages of mineral accrual were slower in intracortical bone.In conclusion, these three classes of therapy have differing effects on both bone formation, and the process of bone matrix maturation. ALN suppresses bone formation, and the normal process of matrix maturation in endocortical bone. ODN does not suppress bone formation, but limits mineral accrual. PTH stimulates bone formation, and the matrix formed matures normally in endocortical bone. The ability of PTH treatment to stimulate bone formation in intracortical bone may provide a novel additional mechanism by which PTH increases bone strength.
Regulation of global translation during the cell cycle
Authors: Stonyte Vilte , Boye Erik , Grallert Beáta 
Published: January 2018

AbstractIt is generally accepted that global translation varies during the cell cycle and is low in mitosis. However, addressing this issue is challenging because it involves cell synchronization, which evokes stress responses which, in turn, affect translation rates. Here we have used two approaches to measure global translation rates in different cell-cycle phases. First, synchrony in different cell-cycle phases was obtained involving the same stress, by using temperature-sensitive mutants. Second, translation and DNA content were measured by flow cytometry in exponentially growing, single cells. We found no major variation in global translation rates through the cell cycle in either fission-yeast or mammalian cells. We also measured phosphorylation of eukaryotic initiation factor-2α, an event thought to downregulate global translation in mitosis. In contrast with the prevailing view, eIF2α phosphorylation correlated poorly with downregulation of general translation and ectopically induced eIF2α phosphorylation inhibited general translation only at high levels.
An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein
Authors: Dewari Pooran Singh , Southgate Benjamin , Mccarten Katrina , Monogarov German , O’Duibhir Eoghan , Quinn Niall , Tyrer Ashley , Plumb Colin , Blin Carla , Finch Rebecca , Bressan Raul Bardini , Morrison Gillian , Jacobi Ashley M. , Behlke Mark A. , Kriegsheim Alex von , Tomlinson Simon , Krijgsveld Jeroen , Pollard Steven M. 
Published: January 2018

AbstractCRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5-30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain tumour-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of sixty different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.