bluuebunny/crossref_metadata_2025_split huggingface dataset:
Assessment of the transmural unipolar electrogram morphology change radius during contact force-guided pulmonary vein isolation using the VISITAG™ Module and CARTOREPLAY™
Authors: Tomlinson David R. , Stevens Kara N. , Streeter Adam J. 
Published: March 2018

AbstractAimsTo investigate the radius of transmural (TM) ablation effect at the left atrial posterior wall (LAPW) during contact force (CF)-guided pulmonary vein isolation (PVI), using pure R unipolar electrogram (UE) morphology change – a histologically validated marker of radiofrequency (RF)-induced TM atrial ablation.MethodsFollowing PVI in 24 consecutive patients (30W, continuous RF), VISITAG™ Module and CARTOREPLAY™ (Biosense Webster Inc.) RF and UE data at left and right-sided LAPW annotated sites 1 and 2 were analysed.ResultsAcutely durable PVI without spontaneous / dormant recovery was achieved following 15s and 10-11s RF, at sites 1 and 2, respectively (p<0.0001). At site 1, RS UE morphology was noted pre-ablation, with RF-induced pure R UE morphology change in 47/48 (98%). Left and right-sided second RF site annotation was at 5.8mm and 5.2mm from site 1 respectively (p=0.64), yet immediate pure R UE morphology was noted in 35/48 (73%). For second-annotated sites, 30 demonstrated inter-ablation site transition time ≤17ms; pure R UE morphology was noted at annotation onset in 22/30 (73%), with overall median time to pure R morphology change significantly shorter than at site 1 – 0.0s, versus 4.1s and 5.3s, for left and right-sided first-annotated LAPW sites, respectively (p<0.0001).ConclusionWhen the first and second-annotated LAPW RF sites were within 7mm, 73% second-annotated sites demonstrated immediate pure R UE morphology change. These analyses support a paradigm of shorter RF duration at immediately adjacent sites during continuous RF application, and may usefully inform the further development of “tailored” approaches towards CF-guided PVI.What’s known?The VISITAG™ Module and CARTOREPLAY™ permit investigations into the tissue effects of RF energy deliveryin vivo, via objective annotation methodology and retrospective evaluation of histologically validated unipolar electrogram (UE) criteria for transmural (TM) atrial ablation.Greater RF energy effect is seen at left compared to right-sided first-annotated left atrial posterior wall (LAPW) sites during pulmonary vein isolation (PVI).What’s new?Following ∼15s RF delivery at first-annotated LAPW sites and aiming for ≤6mm inter-ablation site distance during continuous RF delivery, 73% second-annotated sites demonstrated immediate TM UE morphology change.At second-annotated sites, ∼10s RF resulted in acutely durable PVI in all. Greater left-sided RF energy effect was observed, not explained by differences in RF duration, mean CF or catheter position stability.The radius of TM RF effect may be determined at the LAPW following CF and VISITAG™ Module-guided PVI.
Mechanistic description of spatial processes using integrative modelling of noise-corrupted imaging data
Authors: Hross Sabrina , Theis Fabian J. , Sixt Michael , Hasenauer Jan 
Published: March 2018

AbstractSpatial patterns are ubiquitous on the subcellular, cellular and tissue level, and can be studied using imaging techniques such as light and fluorescence microscopy. Imaging data provide quantitative information about biological systems, however, mechanisms causing spatial patterning often remain illusive. In recent years, spatio-temporal mathematical modelling helped to overcome this problem. Yet, outliers and structured noise limit modelling of whole imaging data, and models often consider spatial summary statistics. Here, we introduce an integrated data-driven modelling approach that can cope with measurement artefacts and whole imaging data. Our approach combines mechanistic models of the biological processes with robust statistical models of the measurement process. The parameters of the integrated model are calibrated using a maximum likelihood approach. We used this integrated modelling approach to studyin vivogradients of the chemokine (C-C motif) ligand 21 (CCL21). CCL21 gradients guide dendritic cells and are important in the adaptive immune response. Using artificial data, we verified that the integrated modelling approach provides reliable parameter estimates in the presence of measurement noise and that bias and variance of these estimates are reduced compared to conventional approaches. The application to experimental data allowed the parameterisation and subsequent refinement of the model using additional mechanisms. Among others, model-based hypothesis testing predicted lymphatic vessel dependent concentration of heparan sulfate, the binding partner of CCL21. The selected model provided an accurate description of the experimental data and was partially validated using published data. Our findings demonstrate that integrated statistical modelling of whole imaging data is computationally feasible and can provide novel biological insights.
A Bayesian sequential learning framework to parameterise continuum models of melanoma invasion into human skin
Authors: Browning Alexander P , Haridas Parvathi , Simpson Matthew J 
Published: March 2018

AbstractWe present a novel framework to parameterise a mathematical model of cell invasion that describes how a population of melanoma cells invades into human skin tissue. Using simple experimental data extracted from complex experimental images, we estimate three model parameters:(i) the melanoma cell proliferation rate,λ; (ii) the melanoma cell diffusivity,D; and (iii)δ, a constant that determines the rate that melanoma cells degrade the skin tissue. The Bayesian sequential learning frame-work involves a sequence of increasingly-sophisticated experimental data from:(i) a spatially uniform cell proliferation assay; (ii) a two-dimensional circular barrier assay; and, (iii) a three-dimensional invasion assay. The Bayesian sequential learning approach leads to well-defined parameter estimates. In contrast, taking a naive approach that attempts to estimate all parameters from a single set of images from the same experiment fails to produce meaningful results. Overall our approach to inference is simple-to-implement, computationally efficient, and well-suited for many cell biology phenomena that can be described by low dimensional continuum models using ordinary differential equations and partial differential equations. We anticipate that this Bayesian sequential learning framework will be relevant in other biological contexts where it is challenging to extract detailed, quantitative biological measurements from experimental images and so we must rely on using relatively simple measurements from complex images.
Implementation of CRISPR-Cas13a system in fission yeast and its repurposing for precise RNA editing
Authors: Jing Xinyun , Xie Bingran , Chen Longxian , Zhang Niubing , Jiang Yiyi , Qin Hang , Hao Pei , Yang Sheng , Li Xuan 
Published: March 2018

AbstractIn contrast to genome editing that introduces genetic changes at DNA level, disrupting or editing genes’ transcripts provides a distinctive approach to perturb a genetic system, offering benefits complementary to classic genetic approaches. To develop a new toolset for manipulation of RNA, we first implemented a member of type VI CRISPR systems, Cas13a from Leptotrichia shahii (LshCas13a) in Schizosaccharomyces pombe, an important model organism employed by biologists to study key cellular mechanisms conserved from yeast to humans. While it was shown to knock down targeted endogenous genes’ transcripts, differently from previous studies in E. coli, no collateral cleavage of other non-specific RNA by activated Cas13a-crRNA complex was detected in fission yeast. Second, we engineered a RNA-editing system by tethering an inactive form of LshCas13a (dCas13) to the catalytic domain of human Adenosine Deaminase Act on RNA 2 (hADAR2d), which was shown to be programmable with crRNA to target messenger RNAs and precisely edit specific nucleotide residues. We optimized the system parameters using a dual-florescence reporter and demonstrated its utility in editing of randomly selected endogenous genes’ transcripts. Our engineered RNA-editing system enables a new toolset for transcriptomic manipulation that is widely applicable in basic genetic and biotechnological research.
Efficient genome editing ofMagnetospirillum magneticumAMB-1 by CRISPR-Cas9 system for analyzing magnetotactic behavior
Authors: Chen Haitao , Zhang Sheng-Da , Chen Linjie , Cai Yao , Zhang Wei-Jia , Song Tao , Wu Long-Fei 
Published: March 2018

AbstractMagnetotactic bacteria are a diverse group of microorganisms with the ability to use geomagnetic fields for direction sensing. This magnetotactic behavior can help microorganisms move towards favorable habitats for optimal growth and reproduction. Highly efficient genomic editing is very useful for a comprehensive understanding of the magnetotactic mechanism at the molecular level. In this study, we adapted an engineered CRISPR-cas9 system for efficient inactivation of gene in a widely used magnetotactic bacteria model strain,Magnetospirillum magneticumAMB-1. By combining an engineered nuclease-deficient Cas9 and single-guide RNA, a CRISPR interference system was successfully developed to silenceamb0994expression. More importantly, we succeeded in the construction of a singleamb0994gene deletion mutant using CRISPR-Cas9 with approximate 60-fold high efficiency compared to classical homology double-crossing replacement procedure. This mutant synthesized normally the magnetosomes, but reacted quicker and with less time than the wild-type strain to abrupt magnetic field reversals. A dynamics simulation by modelingM. magneticumAMB-1 cell as an ellipsoid showed that the difference of the motions between wild andΔamb0994is due to flagellar influence. The behavior observation being consistent with dynamics simulation indicated that Amb0994 is involved in the cellular response to magnetic torque change via controlling flagella. Besides the contribution to a better understanding of the magnetotaxis mechanism, this study demonstrates the CRISPR system as a useful genetic toolbox for high-efficiency genome editing in magnetotactic bacteria.
Conversion of a soluble protein into a potent chaperonein vivo
Authors: Kwon Soon Bin , Ryu Kisun , Son Ahyun , Jeong Hotcherl , Lim Keo-Heun , Kim Kyun-Hwan , Seong Baik L. , Choi Seong Il 
Published: March 2018

AbstractProtein-folding assistance and aggregation inhibition by cellular factors are largely understood in the context of molecular chaperones. As an alternative and complementary model, we previously proposed that, in general, soluble cellular macromolecules including chaperones with large excluded volume and surface charges exhibit the intrinsic chaperone activity to prevent aggregation of their connected polypeptides, irrespective of the connection types, and thus to aid productive protein folding. As a proof of concept, we here demonstrated that a model soluble protein with an inactive protease domain robustly exerted chaperone activity toward various proteins harboring a short protease-recognition tag of 7 residues inEscherichia coli. The chaperone activity of this protein was similar or even superior to that of representativeE. colichaperonesin vivo. Furthermore,in vitrorefolding experiments confirmed thein vivoresults. Our findings revealed that a soluble protein exhibits the intrinsic chaperone activity, which is manifested, upon binding to aggregation-prone proteins. This study gives new insights into the ubiquitous chaperoning role of cellular macromolecules in protein-folding assistance and aggregation inhibition underlying the maintenance of protein solubility and proteostasisin vivo.
Synthetic standards combined with error and bias correction improves the accuracy and quantitative resolution of antibody repertoire sequencing in human naïve and memory B cells
Authors: Friedensohn Simon , Lindner John M. , Cornacchione Vanessa , Iazeolla Mariavittoria , Miho Enkelejda , Zingg Andreas , Meng Simon , Traggiai Elisabetta , Reddy Sai T. 
Published: March 2018

ABSTRACTHigh-throughput sequencing of immunoglobulin repertoires (Ig-seq) is a powerful method for quantitatively interrogating B cell receptor sequence diversity. When applied to human repertoires, Ig-seq provides insight into fundamental immunological questions, and can be implemented in diagnostic and drug discovery projects. However, a major challenge in Ig-seq is ensuring accuracy, as library preparation protocols and sequencing platforms can introduce substantial errors and bias that compromise immunological interpretation. Here, we have established an approach for performing highly accurate human Ig-seq by combining synthetic standards with a comprehensive error and bias correction pipeline. First, we designed a set of 85 synthetic antibody heavy chain standards (in vitro transcribed RNA) to assess correction workflow fidelity. Next, we adapted a library preparation protocol that incorporates unique molecular identifiers (UIDs) for error and bias correction which, when applied to the synthetic standards, resulted in highly accurate data. Finally, we performed Ig-seq on purified human circulating B cell subsets (naïve and memory), combined with a cellular replicate sampling strategy. This strategy enabled robust and reliable estimation of key repertoire features such as clonotype diversity, germline segment and isotype subclass usage, and somatic hypermutation (SHM). We anticipate that our standards and error and bias correction pipeline will become a valuable tool for researchers to validate and improve accuracy in human Ig-seq studies, thus leading to potentially new insights and applications in human antibody repertoire profiling.
Allosteric receptor modulation of FFA2R turns natural agonists into potent activators of the superoxide generating neutrophil NADPH-oxidase
Authors: Mårtensson Jonas , Holdfeldt André , Sundqvist Martina , Gabl Michael , Björkman Lena , Forsman Huamei , Dahlgren Claes 
Published: March 2018

AbstractAcetate, agonist for the free fatty acid receptor 2 (FFA2R/GPR43), triggers an increase in the cytosolic concentration of free Ca2+in neutrophils without any assembly of the superoxide generating NADPH-oxidase. We show that the phenylacetamide compound 58 (Cmp58; (S)-2-(4-chlorophenyl)-3,3-dimethyl-N-(5-phenylthiazol-2-yl)butanamide, lacking a direct activating effect on neutrophils, acts as a positive allosteric FFA2R modulator that turns acetate into a potent activating agonist that triggers an assembly of the NADPH-oxidase. The NADPH-oxidase activity could be further increased in neutrophils treated with the pro-inflammatory cytokine TNF. Many neutrophil chemoattractant receptors are stored in secretory organelles but no FFA2R mobilization was induced in neutrophils treated with TNF. The receptor selectivity was demonstrated through the inhibition of the neutrophil response induced by the combined action of acetate and Cmp58 by the FFA2R antagonist CATPB. Allosteric modulators that positively co-operate with natural FFA2R agonists and prime neutrophils in their response to such agonists, may serve as good tools for further unraveling the physiological functions of the FFA2R and its involvement in various diseases. In this study, allosteric modulation of FFA2R is introduced as a novel receptor selective mechanism to prime neutrophils to produce increased amounts of reactive oxygen species.
Depleting Trim28 in adult mice is well tolerated and reduces levels of α-synuclein and tau
Authors: Rousseaux Maxime W.C. , Revelli Jean-Pierre , Vázquez-Vélez Gabriel E. , Kim Ji-Yoen , Craigen Evelyn , Gonzales Kristyn , Beckinghausen Jaclyn , Zoghbi Huda Y. 
Published: March 2018

AbstractAlzheimer’s and Parkinson’s disease are late onset neurodegenerative diseases that will require therapy over decades to mitigate the effects of disease-driving proteins such tau and α-synuclein (α-Syn). We recently found that TRIM28 regulates the levels and toxicity of α-Syn and tau (Rousseaux et al., 2016), however, how TRIM28 regulates α-Syn and whether its chronic inhibition later in life is safe remained unknown. Here, we show that TRIM28 mediates the SUMOylation of α-Syn and tau, and that genetic suppression of Trim28 in adult mice is compatible with life. We were surprised to see that mice lacking Trim28 in adulthood do not exhibit behavioral or pathological phenotypes, and importantly, adult reduction of TRIM28 results in a decrease of α-Syn and tau levels. These results suggest that deleterious effects from TRIM28 depletion are limited to development and that its inhibition adulthood provides a potential path for modulating α-Syn and tau levels.
Avian keratin disorder of Alaska black-capped chickadees is associated with Poecivirus infection
Authors: Zylberberg Maxine , Van Hemert Caroline , Handel Colleen M. , DeRisi Joseph L. 
Published: March 2018

ABSTRACTBackgroundAvian keratin disorder (AKD) is an epizootic of debilitating beak deformities, first documented in black-capped chickadees (Poecile atricapillus) in Alaska during the late 1990s. Similar deformities have now been recorded in dozens of species of birds across multiple continents. Despite this, the etiology of AKD has remained elusive, making it difficult to assess the impacts of this disease on wild populations. We previously identified an association between infection with a novel picornavirus, Poecivirus, and AKD in a small cohort of black-capped chickadees.MethodsTo test if the association between Poecivirus and AKD holds in a lager study population, we used targeted PCR followed by Sanger sequencing to screen 124 symptomatic and asymptomatic black-capped chickadees for Poecivirus infection. We further compared the efficacy of multiple non-terminal field sampling methods (buccal swabs, cloacal swabs, fecal samples, and blood samples) for Poecivirus screening. Finally, we used both in situ hybridization and a strand-specific expression assay to localize Poecivirus to beak tissue of AKD-positive individuals and to determine if virus is actively replicating in beak tissue.ResultsPoecivirus was detected in 29/29 (100%) individuals with AKD, but only 9/95 (9.5%) asymptomatic individuals with apparently normal beaks (p < 0.0001). We found that cloacal swabs are the most sensitive of these sample types for detecting Poecivirus in birds with AKD, but that buccal swabs should be combined with cloacal swabs in evaluating the infection status of asymptomatic birds. Finally, we used both in situ hybridization and a strand-specific expression assay to localize Poecivirus to beak tissue of AKD-positive individuals and to provide evidence of active viral replication.ConclusionThe data presented here show a strong, statistically significant relationship between Poecivirus infection and AKD, and provide evidence that Poecivirus is indeed an avian virus, infecting and actively replicating in beak tissue of AKD-affected BCCH. Taken together, these data corroborate and extend the evidence for a potential causal association between Poecivirus and AKD in the black-capped chickadee. Poecivirus continues to warrant further investigation as a candidate agent of AKD.
High-throughput antibody engineering in mammalian cells by CRISPR/Cas9-mediated homology-directed mutagenesis
Authors: Mason Derek M , Weber Cédric R , Parola Cristina , Meng Simon M , Greiff Victor , Kelton William J , Reddy Sai T 
Published: March 2018

ABSTRACTAntibody engineering is performed to improve therapeutic properties by directed evolution, usually by high-throughput screening of phage or yeast display libraries. Engineering antibodies in mammalian cells offers advantages associated with expression in their final therapeutic format (full-length glycosylated IgG), however, the inability to express large and diverse libraries severely limits their potential throughput. To address this limitation, we have developed homology-directed mutagenesis (HDM), a novel method which extends the concept of CRISPR/Cas9-mediated homology-directed repair (HDR). HDM leverages oligonucleotides with degenerate codons to generate site-directed mutagenesis libraries in mammalian cells. By improving HDM efficiency (>35-fold) and combining mammalian display screening with next-generation sequencing (NGS), we validated this approach can be used for key applications in antibody engineering at high-throughput: rational library construction, novel variant discovery, affinity maturation, and deep mutational scanning (DMS). We anticipate that HDM will be a valuable tool for engineering and optimizing antibodies in mammalian cells, and eventually enable directed evolution of other complex proteins and cellular therapeutics.
Heterogeneity of the Frequency Domain Patterns in Persistent Atrial Fibrillation
Authors: Iravanian Shahriar , Langberg Jonathan J 
Published: March 2018

AbstractBackgroundPersistent atrial fibrillation (AF) has remained a challenging clinical problem. The mechanisms of persistent AF are still subject to debate. Both a single mother-rotor with fibrillatory conduction and multiple meandering spiral waves have been proposed to explain persistent AF. Previous frequency domain studies have reported the presence of dominant frequency (DF) gradient (a marker of single mother-rotor) in paroxysmal, but not persistent AF.Methods and ResultsWe performed temporally-dense high-resolution frequency domain analysis of 10-40 minutes segments of intracardiac signals recorded in 24 patients undergoing ablation of persistent AF. We observed two predominant patterns. The expected signature of the mother-rotor mechanism was observed in 38% of the patients. The frequency pattern in 54% consisted of two or more distinct frequency peaks with no obvious gradient, which is consistent with multiple separate primary spiral waves in electrophysiologically heterogeneous areas of atria. The average measured number of rotors per case was 1.71 ± 0.32, which provides a lower limit on the actual number of rotors. The single-zone pattern was exclusively seen in patients who were on a membrane-active antiarrhythmic medication at the time of ablation (P < 0.005).ConclusionsAF is a heterogeneous disorder. High-frequency resolution analysis is a useful tool to detect the underlying mechanisms of AF and to classify it into patterns consistent with a single mother-rotor vs. multiple meandering wavelets.