bluuebunny/crossref_metadata_2025_split huggingface dataset:
CRISPR adenine and cytosine base editors with reduced RNA off-target activities
Authors: Grünewald Julian , Zhou Ronghao , Iyer Sowmya , Lareau Caleb A. , Garcia Sara P. , Aryee Martin J. , Keith Joung J. 
Published: May 2019

AbstractCRISPR-guided DNA base editors enable the efficient installation of targeted single-nucleotide changes. Cytosine or adenine base editors (CBEs or ABEs), which are fusions of cytidine or adenosine deaminases to CRISPR-Cas nickases, can efficiently induce DNA C-to-T or A-to-G alterations in DNA, respectively1-4. We recently demonstrated that both the widely used CBE BE3 (harboring a rat APOBEC1 cytidine deaminase) and the optimized ABEmax editor can induce tens of thousands of guide RNA-independent, transcriptome-wide RNA base edits in human cells with high efficiencies5. In addition, we showed the feasibility of creating SElective Curbing of Unwanted RNA Editing (SECURE)-BE3 variants that exhibit substantially reduced unwanted RNA editing activities while retaining robust and more precise on-target DNA editing5. Here we describe structure-guided engineering of SECURE-ABE variants that not only possess reduced off-target RNA editing with comparable on-target DNA activities but are also the smallest Streptococcus pyogenes Cas9 (SpCas9) base editors described to date. In addition, we tested CBEs composed of cytidine deaminases other than APOBEC1 and found that human APOBEC3A (hA3A) cytidine deaminase CBE induces substantial transcriptome-wide RNA base edits with high efficiencies. By contrast, a previously described “enhanced” A3A (eA3A) cytidine deaminase CBE or a human activation-induced cytidine deaminase (hAID) CBE induce substantially reduced or near background levels of RNA edits. In sum, our work describes broadly useful SECURE-ABE and -CBE base editors and reinforces the importance of minimizing RNA editing activities of DNA base editors for research and therapeutic applications.
Emergence and evolution of the ERM proteins and merlin in metazoans
Authors: Shabardina V. , Kashima Y. , Suzuki Y. , Makalowski W. 
Published: May 2019

AbstractEzrin, radixin, moesin, and merlin are the cytoskeletal proteins which functions are specific to metazoans. They participate in cell cortex rearrangements, including cell-cell contact formation, and play important role in cancer progression. Here we perform a comprehensive phylogenetic analysis of the proteins spanning 87 species. The results describe a possible mechanism of the protein family origin in the root of Metazoa, paralogs diversification in vertebrates and acquisition of novel functions, including tumor suppression. In addition, a merlin paralog, present in most of vertebrates, but lost in mammals, has been described. We also highlight the set of amino acid variations within the conserved motifs as the candidates for determining physiological differences between the ERM paralogs.
Contact sensitivity in paediatric patients with atopic dermatitis in Laos
Authors: Wootton Catriona I. , Sodaly Mong K. , Billamay Somxay X. , English John S.C. , Mayfong Mayxay 
Published: May 2019

AbstractBackgroundDermatological services in Laos, South East Asia are limited mainly to the capital and patch testing is currently not available, so no data exists regarding the common cutaneous allergens in this population.ObjectivesThe aim of this study was to document common allergens in paediatric patients with atopic dermatitis attending the allergy clinic in the capital, Vientiane.Patients/Materials/MethodsFifty paediatric patients with atopic dermatitis were patch tested using TRUE Test® panels 1 to 3 (35 allergens). Readings were taken at Days 2 and 4.ResultsTwenty-six positive patch tests were recorded on Day 4 in 15 children (30%). The most common allergens were: gold (18%), nickel (10%), formaldehyde (6%) and p-Phenylenediamine (6%). Other positive allergens were potassium dichromate (2%), cobalt dichloride (2%), Bronopol (2%), paraben mix (2%), fragrance mix 1 (2%) and neomycin (2%). The majority of the patients with positive reactions were female.ConclusionsThis study represents the first documented patch test results in the Lao population. It is hoped that these findings will help clinicians to advise the families of children with atopic dermatitis on common allergens to avoid and inform future work on contact dermatitis in this population.
PIME: a package for discovery of novel differences among microbial communities
Authors: Roesch Luiz Fernando W. , Dobbler Priscila Thiago , Pylro Victor Satler , Kolaczkowski Bryan , Drew Jennifer C. , Triplett Eric W. 
Published: May 2019

AbstractMassive sequencing of genetic markers, such as the 16S rRNA gene for prokaryotes, allows the comparative analysis of diversity and abundance of whole microbial communities. However, the data used for profiling microbial communities is usually low in signal and high in noise preventing the identification of real differences among treatments. PIME (Prevalence Interval for Microbiome Evaluation) fills this gap by removing those taxa that may be high in relative abundance in just a few samples but have a low prevalence overall. The reliability and robustness of PIME were compare against the existing methods and verified by a number of approaches using 16S rRNA independent datasets. To remove the noise, PIME filters microbial taxa not shared in a per treatment prevalence interval starting at 5% with increments of 5% at each filtering step. For each prevalence interval, hundreds of decision trees are calculated to predict the likelihood of detecting differences in treatments. The best prevalence-filtered dataset is user-selected by choosing the prevalence interval that keeps the majority of the 16S rRNA reads in the dataset and shows the lowest error rate. To obtain the likelihood of introducing bias while building prevalence-filtered datasets, an error detection step based in random permutations is also included. A reanalysis of previews published datasets with PIME uncovered previously missed microbial associations improving the ability to detect important organisms, which may be masked when only relative abundance is considered.
Conditional silencing of H-2Dbclass I molecule expression on dendritic cells modulates the protective and pathogenic kinetics of virus-antigen specific CD8 T cell responses during Theiler’s Virus infection
Authors: Tritz Zachariah P. , Orozco Robin C. , Malo Courtney S. , Yokanovich Lila T , Ayasoufi Katayoun , Fain Cori E. , Khadka Roman H. , Settell Megan L. , Hansen Mike J. , Jin Fang , Johnson Aaron J 
Published: May 2019

ABSTRACTTheiler’s murine encephalomyelitis virus (TMEV) infection of the central nervous system is rapidly cleared in C57BL/6 mice by an anti-viral CD8 T cell response restricted by the MHC class I molecule, H-2Db. While the CD8 T cell response against neurotropic viruses is well characterized, the identity and function of the antigen presenting cell(s) involved in this process is(are) less well defined. To address this gap in knowledge, we developed a novel C57BL/6 H-2Dbconditional knockout mouse that expresses an H-2Dbtransgene in which the transmembrane domain locus is flanked by LoxP sites. We crossed these H-2DbLoxP mice with MHC class I-deficient mice expressing Cre-recombinase under either the CD11c or LysM promoter in order to silence H-2Dbrestricted antigen presentation predominantly in dendritic cells or macrophages, respectively. Upon challenge with intracranial TMEV infection, we observe that CD11c+ APCs are critical for early priming of CD8 T cells against the immunodominant TMEV peptide VP2121-130 presented in the context of the H-2Dbmolecule. This stands in stark contrast to later time points post TMEV infection where CD11c+ APCs appear dispensable for the activation of antigen-specific T cells; the functionality of these late-arising antiviral CD8 T cells is reflected in the restoration of viral control at later time points. These late-arising CD8 T cells also retain their capacity to induce blood-brain barrier disruption. In contrast, when H-2Dbrestricted antigen presentation was selectively silenced in LysM+ APCs there was no overt impact on the priming of Db:VP2121-130 epitope-specific CD8 T cells, although a modest reduction in immune cell entry into the CNS was observed. This work establishes a model system which enables critical dissection of MHC class I restricted antigen presentation to T cells, revealing cell specific and temporal features involved in the generation of antiviral CD8 T cell responses. Employing this novel system, we established CD11c+ cells as a pivotal driver of acute, but not later-arising, antiviral CD8 T cell responses against the TMEV immunodominant epitope VP2121-130, with functional implications both for T cell-mediated viral control and immunopathology.
Differential impact of self and environmental antigens on the ontogeny and maintenance of CD4+ T cell memory
Authors: Hogan Thea , Nowicka Maria , Cownden Daniel , Pearson Claire , Yates Andrew J. , Seddon Benedict 
Published: May 2019

AbstractLaboratory mice develop populations of circulating memory CD4+ T cells in the absence of overt infection. We have previously shown that these populations are replenished from naive precursors at high levels throughout life (Gossel et al., 2017). However, the nature, relative importance and timing of the forces generating these cells remain unclear. Here, we tracked the generation of memory CD4+ T cell subsets in mice housed in facilities differing in their ‘dirtiness’. We found evidence for sequential naive to central memory to effector memory development, and confirmed that both memory subsets are heterogeneous in their rates of turnover. We also inferred that early exposure to self and environmental antigens establishes persistent memory populations at levels determined largely, though not exclusively, by the dirtiness of the environment. After the first few weeks of life, however, these populations are continuously supplemented by new memory cells at rates that are independent of environment.
Human cardiac fibrosis-on-a-chip model recapitulates disease hallmarks and can serve as a platform for drug screening
Authors: Mastikhina Olya , Moon Byeong-Ui , Williams Kenneth , Hatkar Rupal , Gustafson Dakota , Sun Xuetao , Koo Margaret , Lam Alan Y.L. , Sun Yu , Fish Jason E. , Young Edmond W.K. , Nunes Sara S. 
Published: May 2019

AbstractWhile interstitial fibrosis plays a significant role in heart failure, our understanding of disease progression in humans is limited. To address this limitation, we have engineered a cardiac-fibrosis-on-a-chip model consisting of a microfabricated device with live force measurement capabilities using co-cultured human cardiac fibroblasts and pluripotent stem cell-derived cardiomyocytes. Transforming growth factor-β was used as a trigger for fibrosis. Here, we have reproduced the classic hallmarks of fibrosis-induced heart failure including high collagen deposition, increased tissue stiffness, BNP secretion, and passive tension. Force of contraction was significantly decreased in fibrotic tissues that displayed a transcriptomic signature consistent with human cardiac fibrosis/heart failure. Treatment with an anti-fibrotic drug decreased tissue stiffness and BNP secretion, with corresponding changes in the transcriptomic signature. This model represents an accessible approach to study human heart failure in vitro, and allows for testing anti-fibrotic drugs while facilitating the real-time assessment of cardiomyocyte function.
Functional tunability from a distance: Rheostat positions influence allosteric coupling between two distant binding sites
Authors: Wu Tiffany , Swint-Kruse Liskin , Fenton Aron W. 
Published: May 2019

AbstractFor protein mutagenesis, a common expectation is that important positions will behave like on/off “toggle” switches (i.e., a few substitutions act like wildtype, most abolish function). However, there exists another class of important positions that manifests a wide range of functional outcomes upon substitution: “rheostat” positions. Previously, we evaluated rheostat positions located near the allosteric binding sites for inhibitor alanine (Ala) and activator fructose-1,6-bisphosphate (Fru-1,6-BP) in human liver pyruvate kinase. When substituted with multiple amino acids, many positions demonstrated moderate rheostat effects on allosteric coupling between effector binding and phosphoenolpyruvate (PEP) binding in the active site. Nonetheless, the combined outcomes of all positions sampled the full range of possible allosteric coupling (full tunability). However, that study only evaluated allosteric tunability of “local” positions, i.e., positions were located near the binding sites of the allosteric ligand being assessed. Here, we evaluated tunability of allosteric coupling when mutated sites were distant from the allosterically-coupled binding sites. Positions near the Ala binding site had rheostat outcomes on allosteric coupling between Fru-1,6-BP and PEP binding. In contrast, positions in the Fru-1,6-BP site exhibited modest effects on coupling between Ala and PEP binding. Analyzed in aggregate, both PEP/Ala and PEP/Fru-1,6-BP coupling were again fully tunable by amino acid substitutions at this limited set of distant positions. Furthermore, some positions exhibited rheostatic control over multiple parameters and others exhibited rheostatic effects on one parameter and toggle control over a second. These findings highlight challenges in efforts to both predict/interpret mutational outcomes and engineer functions into proteins.
Systematic discovery of Short Linear Motifs decodes calcineurin phosphatase signaling
Authors: Wigington Callie P. , Roy Jagoree , Damle Nikhil P. , Yadav Vikash K. , Blikstad Cecilia , Resch Eduard , Wong Cassandra J. , Mackay Douglas R. , Wang Jennifer T. , Krystkowiak Izabella , Bradburn Devin , Tsekitsidou Eirini , Hong Su Hyun , Kaderali Malika Amyn , Xu Shou-Ling , Stearns Tim , Gingras Anne-Claude , Ullman Katharine S. , Ivarsson Ylva , Davey Norman E. , Cyert Martha S. 
Published: May 2019

SummaryShort linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLiM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxIxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins – structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.
Physiotherapist confidence level in mobilising stroke patients after decompressive hemicraniectomy: are helmets useful?
Authors: Budhdeo Sanjay , Meek Toby , Cosco Theodore D , Turaga Sanchit , Chari Aswin , Sharma Nikhil 
Published: May 2019

AbstractIntroductionDecompressive hemicraniectomy is a lifesaving measure in malignant middle cerebral artery infarction; however, this leaves patients with a skull defect. There is variability of helmet use in this patient group across Britain. We aimed to examine whether (1) specialist physiotherapist were more confident mobilising a patient with hemiparesis and skull defect than a non-specialist physiotherapist (2) non-specialist and specialist physiotherapists would be more comfortable mobilising this patient with a helmet as opposed to without a helmet.MethodsWe carried out a cross-sectional online survey of specialist physiotherapists and non-specialist physiotherapists in Britain. Recruitment was through mailing lists. Physiotherapists were asked to rank their confidence level on a 5-point Likert scale of mobilising an example patient with and without a helmet. They were also asked about the number of additional therapists needed to safely mobilise the patient.Findings96 physiotherapists completed the survey; 44 were specialists and 52 were non-specialists. Specialist physiotherapists felt more comfortable mobilising patients (mean difference = 0.68, p < 0.001). Non-specialist physiotherapists felt significantly more comfortable mobilising patients with a helmet (mean difference = 0.96, p value < 0.001), as did specialist physiotherapists (mean difference = 0.68, p value < 0.001). There was no difference in confidence level arising from helmet use between the two groups (p = 0.72).ConclusionsUse of helmets may allow specialist and non-specialist physiotherapists to feel more comfortable when mobilising stroke patients post-decompressive hemicraniectomy. Consideration should be made by hospitals and health systems for the provision of helmets this patient group, to maximise functional gains.
Generating kinetic environments to study dynamic cellular processes in single cells
Authors: Thiemicke Alexander , Jashnsaz Hossein , Li Guoliang , Neuert Gregor 
Published: May 2019

AbstractCells of any organism are consistently exposed to changes over time in their environment. The kinetics by which these changes occur are critical for the cellular response and fate decision. It is therefore important to control the temporal changes of extracellular stimuli precisely to understand biological mechanisms in a quantitative manner. Most current cell culture and biochemical studies focus on instant changes in the environment and therefore neglect the importance of kinetic environments. To address these shortcomings, we developed two experimental methodologies to precisely control the environment of single cells. These methodologies are compatible with standard biochemistry, molecular, cell and quantitative biology assays. We demonstrate applicability by obtaining time series and time point measurements in both live and fixed cells. We demonstrate the feasibility of the methodology in yeast and mammalian cell culture in combination with widely used assays such as flow cytometry, time-lapse microscopy and single-molecule RNA Fluorescent in-situ Hybridization. Our experimental methodologies are easy to implement in most laboratory settings and allows the study of kinetic environments in a wide range of assays and different cell culture conditions.
Automated cell boundary and 3D nuclear segmentation of cells in suspension
Authors: Kesler Benjamin , Li Guoliang , Thiemicke Alexander , Venkat Rohit , Neuert Gregor 
Published: May 2019

AbstractTo characterize cell types, cellular functions and intracellular processes, an understanding of the differences between individual cells is required. Although microscopy approaches have made tremendous progress in imaging cells in different contexts, the analysis of these imaging data sets is a long-standing, unsolved problem. The few robust cell segmentation approaches that exist often rely on multiple cellular markers and complex time-consuming image analysis. Recently developed deep learning approaches can address some of these challenges, but they require tremendous amounts of data and well-curated reference data sets for algorithm training. We propose an alternative experimental and computational approach, called CellDissect, in which we first optimize specimen preparation and data acquisition prior to image processing to generate high quality images that are easier to analyze computationally. By focusing on fixed suspension and dissociated adherent cells, CellDissect relies only on widefield images to identify cell boundaries and nuclear staining to automatically segment cells in two dimensions and nuclei in three dimensions. This segmentation can be performed on a desktop computer or a computing cluster for higher throughput. We compare and evaluate the accuracy of different nuclear segmentation approaches against manual expert cell segmentation for different cell lines acquired with different imaging modalities.
FGF signalling plays similar roles in development and regeneration of the skeleton in the brittle starAmphiura filiformis
Authors: Czarkwiani Anna , Dylus David V. , Carballo Luisana , Oliveri Paola 
Published: May 2019

AbstractRegeneration is an adult developmental process considered to be an epiphenomenon of embryonic development. Although several studies have shown that various embryonic genes are expressed during regeneration, there have been no large-scale, direct and functional comparative studies between the development and regeneration of a specific structure in one animal. Here, we use the brittle starAmphiura filiformisto characterise the role of the FGF signalling pathway during skeletal development and regeneration. In both processes, we find the ligands expressed in ectodermal cells flanking underlying mesodermal cells, and the receptors expressed specifically by these skeletogenic cells. Perturbation of FGF but not VEGF signalling during skeletogenesis completely inhibited skeleton formation in both embryogenesis and regeneration, without affecting other key developmental processes like cell migration or proliferation. Transcriptome-wide differential analysis identified a highly similar cohort of skeletogenic differentiation genes downstream of the FGF signalling pathway, whereas upstream transcription factors involved in the initial specification of the skeletogenic lineage where unaffected. Comparison to the sea urchin indicated that many of the affected genes are associated with differentiation. Moreover, several genes showed no homology to a cohort from other species, leading to the discovery of brittle star specific, downstream skeletogenic genes. In conclusion, our results show that the FGF pathway is crucial for skeletogenesis in the brittle star, as it is in other deuterostomes, and for the first time provide evidence for the re-deployment of a gene regulatory module during both regeneration and development.
Validation study of a diffusion MRI derived vessel density biomarker for detecting viral hepatitis-b induced liver fibrosis
Authors: Xiao Ben-Heng , Huang Hua , Wang Li-Fei , Qiu Shi-Wen , Guo Sheng-Wen , Wáng Yì Xiáng J. 
Published: May 2019

AbstractAimLiver vessel density can be evaluated by an imaging biomarker DDVD (diffusion derived vessel density): DDVD/area(b0b2) = Sb0/ROIarea0 – Sb2/ROIarea2, where Sb0 and Sb2 refer to the liver signal whenbis 0 or 2 (s/mm2); ROIarea0 and ROIarea2 refer to the region-of-interest onb= 0 or 2 images; and Sb2 may be replaced by Sb15 (b=15). This concept was validated in this study.Materials and MethodsLiver diffusion images were acquired at 1.5T. For a scan-rescan repeatability study of 6 subjects,b-values of 0 and 2 were used. The validation study composed of 26 healthy volunteers and 19 consecutive suspected chronic viral hepatitis-b patients, and diffusion images with 16b-values of 0, 2, 4, 7, 10, 15, 20, 30, 46, 60, 72, 100, 150, 200, 400, 600 were acquired. Four patients did not have liver fibrosis, and the rest were four stage-1, three stage-2, four stage 3, and one stage-4 patients respectively.ResultsIntraclass correlation coefficient for repeatability was 0.994 for DDVD/area(Sb0Sb2), and 0.978 for DDVD/area(Sb0Sb15). In the validation study, DDVD/area(Sb0Sb2) and area(Sb0Sb15) were 14.80±3.06 and 26.58±3.97 for healthy volunteers, 10.51±1.51 and 20.15±2.21 for stage 1-2 fibrosis patients, and 9.42±0.87 and 19.42±1.89 for stage 3-4 fibrosis patients. For 16 patients where IVIM analysis was performed, a combination of DDVD/area, PF, and Dfast achieved the best differentiation for non-fibrotic livers and fibrotic livers. DDVD/area were weakly correlated with PF or Dfast.ConclusionBoth DDVD/area(Sb0Sb2) and area(Sb0Sb15) are useful imaging biomarker to separate fibrotic and non-fibrotic livers, with fibrotic livers had lower measurements.