bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single molecule analysis of effects of non-canonical guide RNAs and specificity-enhancing mutations on Cas9-induced DNA unwinding
Authors: Okafor Ikenna C. , Singh Digvijay , Wang Yanbo , Jung Minhee , Wang Haobo , Mallon John , Bailey Scott , Lee Jungjoon K. , Ha Taekjip 
Published: May 2019

ABSTRACTCas9 has made a wide range of genome engineering applications possible. However, its specificity continues to be a challenge. Non-canonical gRNAs and new engineered variants of Cas9 have been developed to improve specificity but at the cost of the on-target activity. DNA unwinding is the primary checkpoint before cleavage by Cas9 and was shown to be made more sensitive to sequence mismatches by specificity-enhancing mutations in Cas9. Here we performed single-molecule FRET-based DNA unwinding experiments using various combinations of non-canonical gRNAs and different Cas9s. All engineered Cas9s were less promiscuous than wild type when canonical gRNA was used but HypaCas9 had much-reduced on-target unwinding. Cas9-HF1 and eCas9 showed the best balance between low promiscuity and high on-target activity with canonical gRNA. When extended gRNAs with one or two guanines added were used, Sniper1-Cas9 showed the lowest promiscuity while maintaining high on-target activity. Truncated gRNA generally reduced unwinding and adding a non-matching guanine to the 5’ end of gRNA influenced unwinding in a sequence-context dependent manner. Our results are consistent with cell-based cleavage data and provide a mechanistic understanding of how various Cas9/gRNA combinations perform in genome engineering.
Transcriptome-wide association study of attention deficit hyperactivity disorder identifies associated genes and phenotypes
Authors: Liao Calwing , Laporte Alexandre D. , Spiegelman Dan , Akçimen Fulya , Joober Ridha , Dion Patrick A. , Rouleau Guy A. 
Published: May 2019

AbstractAttention deficit/hyperactivity disorder (ADHD) is one of the most common neurodevelopmental psychiatric disorders. Previous studies have shown that the disorder is highly heritable and associated with several different risk-taking behaviors. Additionally, brain-imaging studies have identified various brain regions such as the cerebellum and frontal cortex to be altered in ADHD. Large genome-wide association studies (GWAS) have identified several loci associated with ADHD. However, understanding the biological relevance of these genetic loci has proven to be difficult. Here, we conducted the largest ADHD transcriptome-wide association study (TWAS) to date consisting of 19,099 cases and 34,194 controls and identified 9 transcriptome-wide significant hits. We successfully demonstrate that several previous GWAS hits can be largely explained by expression. Probabilistic causal fine-mapping of TWAS signals prioritized KAT2B with a posterior probability of 0.467 in the dorsolateral prefrontal cortex and TMEM161B with a posterior probability of 0.838 in the amygdala. Furthermore, pathway enrichments identified dopaminergic and norepinephrine pathways, which are highly relevant for ADHD. Finally, we used the top eQTLs associated with the TWAS genes to identify phenotypes relevant to ADHD and found an inverse genetic correlation with educational attainment and a positive correlation with “ever smoker”, maternal smoking at birth, BMI, and schizophrenia. Overall, our findings highlight the power of TWAS to identify novel risk loci and prioritize putatively causal genes.
Pattern similarity analyses of frontoparietal task coding: Individual variation and genetic influences
Authors: Etzel Joset A. , Courtney Ya’el , Carey Caitlin E. , Gehred Maria Z. , Agrawal Arpana , Braver Todd S. 
Published: May 2019

AbstractPattern similarity analyses are increasingly used to characterize coding properties of brain regions, but relatively few have focused on cognitive control processes in FrontoParietal regions. Here, we use the Human Connectome Project (HCP) N-back task fMRI dataset to examine individual differences and genetic influences on the coding of working memory load (0-back, 2-back) and perceptual category (Face, Place). Participants were grouped into 105 MZ (monozygotic) twin, 78 DZ (dizygotic) twin, 99 non-twin sibling, and 100 unrelated pairs. Activation pattern similarity was used to test the hypothesis that FrontoParietal regions would have higher similarity for same load conditions, while Visual regions would have higher similarity in same perceptual category conditions. Results confirmed this highly robust regional double dissociation in neural coding, which also predicted individual differences in behavioral performance. In pair-based analyses, anatomically-selective genetic relatedness effects were observed: relatedness predicted greater activation pattern similarity in FrontoParietal only for load coding, and in Visual only for perceptual coding. Further, in related pairs, the similarity of load coding in FrontoParietal regions was uniquely associated with behavioral performance. Together, these results highlight the power of task fMRI pattern similarity analyses for detecting key coding and heritability features of brain regions.
A New Minimally Invasive Technique for Repairing Achilles Tendon Rupture:A Biomechanical Study
Authors: zhao Peng , Sun Dawei , Xiong Yaru , Zhuo Ribo 
Published: May 2019

AbstractIntroductionThe incidence of Achilles tendon rupture shows a gradually increasing trend, which is mainly managed by minimally invasive treatment due to its advantages, such as low wound infection rate. At present, the firmness of the commonly applied minimally invasive suture method for Achilles tendon remains controversial. Our research group has developed a novel suture method for Achilles tendon, which has achieved favorable clinical outcomes. Therefore, this experiment aimed to explore the optimal approach to repair Achilles tendon rupture through comparing the biomechanical strength of the commonly used Achilles tendon suture methods currently.Materials and methods6 fresh frozen human cadaveric Achilles tendon specimens were sutured by three kinds of technique, and were tested through the cyclical loading after repair.ResultsResults of cyclical loading showed that, the repair using the new technique was stronger after 10 cycles, 1000 cycles, and rupture. Moreover, the new technique had displayed superior anti-deformation strength to that of the Ma-Griffith technique.ConclusionsOur experimental results demonstrate that, the new technique proposed by our research group can attain comparable biomechanical properties to those of the Krachow technique. However, the sample size in this study is small, and further clinical trials are warranted.
The drift diffusion model as the choice rule in inter-temporal and risky choice: a case study in medial orbitofrontal cortex lesion patients and controls
Authors: Peters Jan , D’Esposito Mark 
Published: May 2019

AbstractSequential sampling models such as the drift diffusion model have a long tradition in research on perceptual decision-making, but mounting evidence suggests that these models can account for response time distributions that arise during reinforcement learning and value-based decision-making. Building on this previous work, we implemented the drift diffusion model as the choice rule in inter-temporal choice (temporal discounting) and risky choice (probability discounting) using a hierarchical Bayesian estimation scheme. We validated our approach in data from nine patients with focal lesions to the ventromedial prefrontal cortex / medial orbitofrontal cortex (vmPFC/mOFC) and nineteen age- and education-matched controls. Choice model parameters estimated via standard softmax action selection were reliably reproduced using the drift diffusion model as the choice rule, both for temporal discounting and risky choice. Model comparison revealed that, for both tasks, the data were best accounted for by a variant of the drift diffusion model including a non-linear mapping from value-differences to trial-wise drift rates. Posterior predictive checks of the winning models revealed a reasonably good fit to individual participants reaction time distributions. We then applied this modeling framework and 1) reproduced our previous results regarding temporal discounting in vmPFC/mOFC patients and 2) showed in a previously unpublished data set on risky choice that vmPFC/mOFC patients exhibit increased risk-taking relative to controls. Analyses of diffusion model parameters revealed that vmPFC/mOFC damage abolished neither value sensitivity nor asymptote of the drift rate. Rather, it substantially increased non-decision times and reduced response caution during risky choice. Our results highlight that novel insights can be gained from applying sequential sampling models in studies of inter-temporal and risky decision-making in cognitive neuroscience.
Benchmarking principal component analysis for large-scale single-cell RNA-sequencing
Authors: Tsuyuzaki Koki , Sato Hiroyuki , Sato Kenta , Nikaido Itoshi 
Published: May 2019

AbstractPrincipal component analysis (PCA) is an essential method for analyzing single-cell RNA-seq (scRNA-seq) datasets, but large-scale scRNA-seq datasets require long computational times and a large memory capacity.In this work, we review 21 fast and memory-efficient PCA implementations (10 algorithms) and evaluate their application using 4 real and 18 synthetic datasets. Our benchmarking showed that some PCA algorithms are faster, more memory efficient, and more accurate than others. In consideration of the differences in the computational environments of users and developers, we have also developed guidelines to assist with selection of appropriate PCA implementations.
Reconstructing wells from high density regions extracted from super-resolution single particle trajectories
Authors: Parutto P. , Heck J. , Heine M. , Holcman D. 
Published: May 2019

AbstractLarge amount of super-resolution single particle trajectories has revealed that the cellular environment is enriched in heterogenous regions of high density, which remain unexplained. The biophysical properties of these regions are characterized by a drift and their extension (a basin of attraction) that can be estimated from an ensemble of trajectories. We develop here two statistical methods to recover the dynamics and local potential wells (field of force and boundary) using as a model a truncated Ornstein-Ulhenbeck process. The first method uses the empirical distribution of points, which differs inside and outside the potential well, while the second focuses on recovering the drift field. Finally, we apply these two methods to voltage-gated calcium channels and phospholipids moving on the surface of neuronal cells and recover the energy and size of these high-density regions with nanometer precision.
Robinson-Foulds Reticulation Networks
Authors: Markin Alexey , Anderson Tavis K. , Vadali Venkata SKT , Eulenstein Oliver 
Published: May 2019

AbstractPhylogenetic (hybridization) networks allow investigation of evolutionary species histories that involve complex phylogenetic events other than speciation, such as reassortment in virus evolution or introgressive hybridization in invertebrates and mammals. Reticulation networks can be inferred by solving thereticulation network problem, typically known as thehybridization network problem. Given a collection of phylogenetic input trees, this problem seeks aminimum reticulation networkwith the smallest number of reticulation vertices into which the input trees can be embedded exactly. Unfortunately, this problem is limited in practice, since minimum reticulation networks can be easily obfuscated by even small topological errors that typically occur in input trees inferred from biological data. We adapt the reticulation network problem to address erroneous input trees using the classic Robinson-Foulds distance. TheRF embedding costallows trees to be embedded into reticulation networksinexactly, but up to a measurable error. The adapted problem, called theRobinson-Foulds reticulation network (RF-Network) problemis, as we show and like many other problems applied in molecular biology, NP-hard. To address this, we employ local search strategies that have been successfully applied in other NP-hard phylogenetic problems. Our local search method benefits from recent theoretical advancements in this area. Further, we introduce inpractice effective algorithms for the computational challenges involved in our local search approach. Using simulations we experimentally validate the ability of our method,RF-Net, to reconstruct correct phylogenetic networks in the presence of error in input data. Finally, we demonstrate how RF-networks can help identify reassortment in influenza A viruses, and provide insight into the evolutionary history of these viruses. RF-Net was able to estimate a large and credible reassortment network with 164 taxa.
CD8+T cell immunity is compromised by anti-CD20 treatment and rescued by IL-17A
Authors: Vernengo Facundo Fiocca , Beccaria Cristian G. , Furlan Cintia L. Araujo , Boari Jimena Tosello , Almada Laura , Serrán Melisa Gorosito , Gazzoni Yamila , Montes Carolina L. , Rodríguez Eva V. Acosta , Gruppi Adriana 
Published: May 2019

AbstractTreatment with anti-CD20, used in many diseases in which B cells play a pathogenic role, has been associated with susceptibility to intracellular infections. Here, we studied the effect of anti-CD20 injection on CD8+T cell immunity using an experimental model ofTrypanosoma cruziinfection, in which CD8+T cells play a pivotal role. C57BL/6 mice were treated with anti-CD20 for B cell depletion prior toT. cruziinfection. Infected anti-CD20-treated mice exhibited a CD8+T cell response with a conserved expansion phase followed by an early contraction, resulting in a strong reduction in total and parasite-specific CD8+T cells at 20 days postinfection. Anti-CD20 injection decreased the number of effector and memory CD8+T cells and reduced the frequency of proliferating and cytokine producing CD8+T cells. Accordingly, infected anti-CD20-treated mice presented a lower cytotoxicity ofT. cruzipeptide-pulsed target cellsin vivo. All of these alterations in CD8+T cell immunity were associated with increased tissue parasitism. Anti-CD20 injection also dampened an established CD8+T cell response, indicating that B cells were involved in the maintenance rather than the induction of CD8+T cell immunity. Anti-CD20 injection also resulted in a marked reduction in the frequency of IL-6- and IL-17A-producing cells, and only rIL-17A injection partially restored the CD8+T cell response in infected anti-CD20-treated mice. Thus, anti-CD20 reduced CD8+T cell immunity, and IL-17A is a candidate for rescuing deficient responses either directly or indirectly.ImportanceMonoclonal antibody targeting the CD20 antigen on B cells is used to treat the majority of Non-Hodgkin lymphoma patients and some autoimmune disorders. This therapy generates adverse effects, notably opportunistic infections and activation of viruses from latency. Here, using the infection murine model with the intracellular parasiteTrypanosoma cruzi, we report that anti-CD20 treatment not only affects B cell response but also CD8+T cells, the most important immune effectors involved in control of intracellular pathogens. Anti-CD20 treatment, directly or indirectly, affects cytotoxic T cell number and function and this deficient response was rescued by the cytokine IL-17A. The identification of IL-17A as the cytokine capable of reversing the poor response of CD8+T cells provide information about a potential therapeutic treatment aimed at enhancing defective immunity induced by B cell depletion.
Benchmarking algorithms for gene regulatory network inference from single-cell transcriptomic data
Authors: Pratapa Aditya , Jalihal Amogh P. , Law Jeffrey N. , Bharadwaj Aditya , Murali T. M. 
Published: May 2019

AbstractWe present a comprehensive evaluation of state-of-the-art algorithms for inferring gene regulatory networks (GRNs) from single-cell gene expression data. We develop a systematic framework called BEELINE for this purpose. We use synthetic networks with predictable cellular trajectories as well as curated Boolean models to serve as the ground truth for evaluating the accuracy of GRN inference algorithms. We develop a strategy to simulate single-cell gene expression data from these two types of networks that avoids the pitfalls of previously-used methods. We selected 12 representative GRN inference algorithms. We found that the accuracy of these methods (measured in terms of AUROC and AUPRC) was moderate, by and large, although the methods were better in recovering interactions in the synthetic networks than the Boolean models. Techniques that did not require pseudotime-ordered cells were more accurate, in general. The observation that the endpoints of many false positive edges were connected by paths of length two in the Boolean models suggested that indirect effects may be predominant in the outputs of the algorithms we tested. The predicted networks were considerably inconsistent with each other, indicating that combining GRN inference algorithms using ensembles is likely to be challenging. Based on the results, we present some recommendations to users of GRN inference algorithms, including suggestions on how to create simulated gene expression datasets for testing them. BEELINE, which is available athttp://github.com/murali-group/BEELINEunder an open-source license, will aid in the future development of GRN inference algorithms for single-cell transcriptomic data.
Unique and redundant roles of SOX2 and SOX17 in regulating the germ cell tumour fate
Authors: Jostes Sina , Fellermeyer Martin , Merges Gina , Kristiansen Glen , Nettersheim Daniel , Schorle Hubert 
Published: May 2019

ABSTRACTEmbryonal carcinomas (ECs) and seminomas are testicular germ cell tumours. ECs display expression of SOX2, while seminomas display expression of SOX17. In somatic differentiation, SOX17 drives endodermal cell fate. However, seminomas lack expression of endoderm markers, but show features of pluripotency. Here, we use ChIP-sequencing to report and compare the binding pattern of SOX17 in seminoma-like TCam-2 cells to SOX2 in EC-like 2102EP cells and SOX17 in somatic cells. In seminoma-like cells, SOX17 was detected at canonical (SOX2/OCT4), compressed (SOX17/OCT4) and other SOX family member motifs. SOX17 directly regulatesTFAP2C,PRDM1andPRDM14, thereby maintaining latent pluripotency and supressing somatic differentiation. In contrast, in somatic cells canonical motifs are not bound by SOX17. In sum only 11% of SOX17 binding sites overlap in seminoma-like and somatic cells. This illustrates that binding site choice is highly dynamic and cell type specific. Deletion of SOX17 in seminoma-like cells resulted in loss of pluripotency, marked by a reduction of OCT4 protein level and loss of alkaline phosphatase activity. Further, we found that in EC-like cells SOX2 regulates pluripotency-associated genes, predominantly by partnering with OCT4. In conclusion, SOX17 (in seminomas) functionally replaces SOX2 (in ECs) to maintain expression of the pluripotency cluster.
Single-cell transcriptomics reveals expansion of cytotoxic CD4 T-cells in supercentenarians
Authors: Hashimoto Kosuke , Kouno Tsukasa , Ikawa Tomokatsu , Hayatsu Norihito , Miyajima Yurina , Yabukami Haruka , Terooatea Tommy , Sasaki Takashi , Suzuki Takahiro , Valentine Matthew , Pascarella Giovanni , Okazaki Yasushi , Suzuki Harukazu , Shin Jay W. , Minoda Aki , Taniuchi Ichiro , Okano Hideyuki , Arai Yasumichi , Hirose Nobuyoshi , Carninci Piero 
Published: May 2019

AbstractSupercentenarians, people who have reached 110 years of age, are a great model of healthy aging. Their characteristics of delayed onset of age-related diseases and compression of morbidity imply that their immune system remains functional. Here we performed single-cell transcriptome analysis of 61,202 peripheral blood mononuclear cells (PBMCs), derived from seven supercentenarians and five younger controls. We identified a marked increase of cytotoxic CD4 T-cells (CD4 CTLs) coupled with a substantial reduction of B-cells as a novel signature of supercentenarians. Furthermore, single-cell T-cell receptor sequencing of two supercentenarians revealed that CD4 CTLs had accumulated through massive clonal expansion, with the most frequent clonotypes accounting for 15% to 35% of the entire CD4 T-cell population. The CD4 CTLs exhibited substantial heterogeneity in their degree of cytotoxicity as well as a nearly identical transcriptome to that of CD8 CTLs. This indicates that CD4 CTLs utilize the transcriptional program of the CD8 lineage while retaining CD4 expression. Our study reveals that supercentenarians have unique characteristics in their circulating lymphocytes, which may represent an essential adaptation to achieve exceptional longevity by sustaining immune responses to infections and diseases.SignificanceExceptionally long-lived people such as supercentenarians tend to spend their entire lives in good health, implying that their immune system remains active to protect against infections and tumors. However, their immunological condition has been largely unexplored. We profiled thousands of circulating immune cells from supercentenarians at single-cell resolution, and identified a large number of CD4 T-cells that have cytotoxic features. This characteristic is very unique to supercentenarians, because generally CD4 T-cells have helper, but not cytotoxic, functions under physiological conditions. We further profiled their T-cell receptors, and revealed that the cytotoxic CD4 T-cells were accumulated through clonal expansion. The conversion of helper CD4 T-cells to a cytotoxic variety might be an adaptation to the late stage of aging.
Thymically-derived Foxp3+ regulatory T cells are the primary regulators of type 1 diabetes in the non-obese diabetic mouse model
Authors: Holohan Daniel R. , Van Gool Frédéric , Bluestone Jeffrey A. 
Published: May 2019

AbstractRegulatory T cells (Tregs) are an immunosuppressive population that are identified based on the stable expression of the fate-determining transcription factor forkhead box P3 (Foxp3). Tregs can be divided into distinct subsets based on whether they developed in the thymus (tTregs) or in the periphery (pTregs). Whether there are unique functional roles that distinguish pTregs and tTregs remains largely unclear. To elucidate these functions, efforts have been made to specifically identify and modify individual Treg subsets. Deletion of the conserved non-coding sequence (CNS)1 in the Foxp3 locus leads to selective impairment of pTreg generation without disrupting tTreg generation in the C57BL/6J background. Using CRISPR-Cas9 genome editing technology, we removed the Foxp3 CNS1 region in the non-obese diabetic (NOD) mouse model of spontaneous type 1 diabetes mellitus (T1D) to determine if pTregs contribute to autoimmune regulation. Deletion of CNS1 impaired in vitro induction of Foxp3 in naïve NOD CD4+ T cells, but it did not alter Tregs in most lymphoid and non-lymphoid tissues analyzed except for the large intestine lamina propria, where a small but significant decrease in RORγt+ Tregs and corresponding increase in Helios+ Tregs was observed in NOD CNS1−/− mice. CNS1 deletion also did not alter the development of T1D or glucose tolerance despite increased pancreatic insulitis in pre-diabetic female NOD CNS1−/− mice. CNS1 Furthermore, the proportions of autoreactive Tregs and conventional T cells (Tconvs) within pancreatic islets were unchanged. These results suggest that pTregs dependent on the Foxp3 CNS1 region are not the dominant regulatory population controlling T1D in the NOD mouse model.