bluuebunny/crossref_metadata_2025_split huggingface dataset:
A framework for evaluating edited cell libraries created by massively parallel genome engineering
Authors: Cawley Simon , Abbate Eric , Abraham Christopher G. , Alvarez Steven , Barber Mathew , Bolte Scott , Bruand Jocelyne , Church Deanna M. , Davis Clint , Estes Matthew , Federowicz Stephen , Fox Richard , Gander Miles W. , Garst Andrew D. , Gencer Gozde , Halweg-Edwards Andrea L. , Hardenbol Paul , Hraha Thomas , Jain Surbhi , Johnson Charlie , Juneau Kara , Krishnamurthy Nandini , Lambert Shea , Leland Bryan , Pearson Francesca , Ray J. Christian J. , Sanada Chad D. , Shaver Timothy M. , Shepherd Tyson R. , Shorenstein Joshua , Spindler Eileen C. , Struble Craig A. , Swat Maciej H. , Tanner Stephen , Tian Tian , Wishart Ken , Graige Michael S. 
Published: September 2021

AbstractGenome engineering methodologies are transforming biological research and discovery. Approaches based on CRISPR technology have been broadly adopted and there is growing interest in the generation of massively parallel edited cell libraries. Comparing the libraries generated by these varying approaches is challenging and researchers lack a common framework for defining and assessing the characteristics of these libraries. Here we describe a framework for evaluating massively parallel libraries of edited genomes based on established methods for sampling complex populations. We define specific attributes and metrics that are informative for describing a complex cell library and provide examples for estimating these values. We also connect this analysis to generic phenotyping approaches, using either pooled (typically via a selection assay) or isolate (often referred to as screening) phenotyping approaches. We approach this from the context of creating massively parallel, precisely edited libraries with one edit per cell, though the approach holds for other types of modifications, including libraries containing multiple edits per cell (combinatorial editing). This framework is a critical component for evaluating and comparing new technologies as well as understanding how a massively parallel edited cell library will perform in a given phenotyping approach.
PcGCE is a potent elicitor of defense responses in aspen
Authors: Donev Evgeniy N. , Derba-Maceluch Marta , Liu Xiao-Kun , Bwanika Henri Colyn , Dobrowolska Izabela , Thapa Mohit , Leśniewska Joanna , Šimura Jan , Tsai Alex Yi-Lin , Boström Dan , Kleczkowski Leszek A. , Eriksson Maria E. , Ljung Karin , Master Emma R. , Mellerowicz Ewa J. 
Published: September 2021

AbstractUsing microbial enzymes in transgenesis is a powerful means to introduce new functionalities in plants. Glucuronoyl esterase (GCE) is a microbial enzyme hydrolyzing the ester bond between lignin and 4-O-methyl-α-D-glucuronic acid present as a side chain of glucuronoxylan. This bond mediates lignin-carbohydrate complex (LCC) formation, considered as crucial factor of lignocellulose recalcitrance to saccharification. Previous studies showed that hybrid aspen (Populus tremula L. x tremuloides Michx.) constitutively expressing Phanerochaete carnosa Burt GCE (PcGCE) had better efficiency of cellulose-to-glucose conversion but were stunned and had lower cellulose content indicating that more studies are needed to design strategy for deployment of this enzyme in planta. Here we report that the transgenic plants exhibit premature leaf senescence, increased accumulation of calcium oxalate crystals, tyloses and necrotic lesions and have strongly activated immune defense reactions as revealed by their altered profiles of transcriptomes, metabolomes and hormones in the leaves. To elucidate if these effects are triggered by damage-associated molecular patterns (DAMPs) or by PcGCE protein perceived as a pathogen-associated molecular pattern (PAMP), we ectopically expressed in aspen an enzymatically inactive PcGCES217A. The mutated PcGCE induced similar growth retardation, leaf necrosis and premature senescence as the active one, providing evidence that PcGCE protein is recognized as PAMP. Transcriptomics analysis of young expanding leaves of 35S:PcGCE plants identified several candidates for receptors of PcGCE, which were not expressed in developing wood tissues. Grafting experiments showed that PcGCE transcripts are not cell-to-cell mobile and that PcGCE expressing leaves augment systemic responses. In agreement, expressing PcGCE in developing wood by using the wood-specific promoter (WP), avoided all off-target effects. Moreover, WP:PcGCE lines grew better than control plants providing evidence that this strategy can be used in transgenic crops dedicated for biorefinery.
Inhibition of Bruton’s tyrosine kinase activity attenuates trauma-induced multiple organ dysfunction in rats
Authors: Patel Nikita M , Oliveira Filipe RMB , Ramos Hanna Pillmann , Aimaretti Eleonora , Alves Gustavo Ferreira , Coldewey Sina M , Collino Massimo , Sordi Regina , Thiemermann Christoph 
Published: September 2021

ABSTRACTObjectiveThe aim of this study was to investigate (a) the potential of the Bruton’s tyrosine kinase (BTK) inhibitors (BTKi) acalabrutinib and fenebrutinib to reduce multiple organ dysfunction syndrome (MODS) in acute and chronic hemorrhagic shock (HS) rat models and (b) whether treatment with either acalabrutinib or fenebrutinib attenuates BTK, NF-κB and NLRP3 activation in HS.BackgroundThe MODS caused by an excessive systemic inflammatory response following trauma is associated with a high morbidity and mortality. The protein BTK is known to play a role in the activation of the NLRP3 inflammasome, which is a key component of the innate inflammatory response. However, its role in trauma-hemorrhage is unknown.MethodsAcute and chronic HS rat models were performed to determine the influence of acalabrutinib or fenebrutinib on MODS. The activation of BTK, NF-κB and NLRP3 pathways were analyzed by western blot in the kidney.ResultsWe demonstrated that (a) HS caused organ injury and/or dysfunction and hypotension (post resuscitation) in rats, while (b) treatment of HS-rats with either acalabrutinib or fenebrutinib attenuated the organ injury and dysfunction in acute and chronic HS models and (c) reduced the activation of BTK, NF-κB and NLRP3 pathways in the kidney.ConclusionOur results point to a role of BTK in the pathophysiology of organ injury and dysfunction caused by trauma/hemorrhage and indicate that BTK inhibitors may be repurposed as a potential therapeutic approach for MODS after trauma and/or hemorrhage.MINI-ABSTRACTThis study evaluated the role of Bruton’s tyrosine kinase (BTK) in trauma/hemorrhage. Patients with trauma had elevated gene expression of BTK. The BTK inhibitors acalabrutinib (irreversible) and fenebrutinib (reversible) attenuated the trauma-induced multiple organ dysfunction in rats with hemorrhagic shock, indicating that BTK could be a potential therapeutic target.
Gene regulation by DNA methylation is contingent on chromatin accessibility during transgenerational plasticity in the purple sea urchin
Authors: Bogan Samuel N , Strader Marie E , Hofmann Gretchen E 
Published: September 2021

AbstractEpigenetic processes are proposed to contribute to phenotypic plasticity. In invertebrates, DNA methylation commonly varies across environments and can correlate or causally associate with phenotype, but its role in transcriptional responses to the environment remains unclear. Maternal environments experienced by the sea urchin Strongylocentrotus purpuratus induce 3 – 6x greater differential CpG methylation in offspring larvae relative to larval developmental environments, suggesting a role for DNA methylation in transgenerational plasticity (TGP). However, a negligible association has been observed between differentially methylated and differentially expressed genes. What gene regulatory roles does invertebrate DNA methylation possess under environmental change, if any? We quantified DNA methylation and gene expression in S. purpuratus larvae exposed to different ecologically relevant conditions during gametogenesis (maternal conditioning) or embryogenesis (developmental conditioning). We modeled differential gene expression and differential splicing under maternal conditioning as functions of DNA methylation, incorporating variables for genomic feature and chromatin accessibility. We detected significant interactions between differential methylation, chromatin accessibility, and genic architecture associated with differential expression and splicing. Observed transcriptional responses to maternal conditioning were also 4 – 13x more likely when accounting for interactions between methylation and chromatin accessibility. Our results provide evidence that DNA methylation possesses multiple functional roles during TGP in S. purpuratus, but its effects are contingent upon other genomic and epigenomic states. Singularly unpredictive of transcription, DNA methylation is likely one cog in the epigenomic machinery contributing to environmental responses and phenotypic plasticity in S. purpuratus and other invertebrates.
Modulation of sensory perception by hydrogen peroxide enables Caenorhabditis elegans to find a niche that provides both food and protection from hydrogen peroxide
Authors: Schiffer Jodie A. , Stumbur Stephanie V. , Seyedolmohadesin Maedeh , Xu Yuyan , Serkin William T. , McGowan Natalie G , Banjo Oluwatosin , Torkashvand Mahdi , Lin Albert , Hosea Ciara N. , Assié Adrien , Samuel Buck S. , O’Donnell Michael P. , Venkatachalam Vivek , Apfeld Javier 
Published: September 2021

SummaryHydrogen peroxide (H2O2) is the most common chemical threat that organisms face. Here, we show that H2O2 alters the bacterial food preference of Caenorhabditis elegans, enabling the nematodes to find a safe environment with food. H2O2 induces the nematodes to leave food patches of laboratory and microbiome bacteria when those bacterial communities have insufficient H2O2-degrading capacity. The nematode’s behavior is directed by H2O2-sensing neurons that promote escape from H2O2 and by bacteria-sensing neurons that promote attraction to bacteria. However, the input for H2O2-sensing neurons is removed by bacterial H2O2-degrading enzymes and the bacteria-sensing neurons’ perception of bacteria is prevented by H2O2. The resulting cross-attenuation provides a general mechanism that ensures the nematode’s behavior is faithful to the lethal threat of hydrogen peroxide, increasing the nematode’s chances of finding a niche that provides both food and protection from hydrogen peroxide.
Ratiometric RNA labeling allows dynamic multiplexed analysis of gene circuits in single cells
Authors: Xu Shuhui , Li Kai , Ma Liang , Zhang Jianhan , Yoon Shinae , Elowitz Michael B. , Lin Yihan 
Published: September 2021

ABSTRACTBiological processes are highly dynamic and are regulated by genes that connect with one and another, forming regulatory circuits and networks. Understanding how gene regulatory circuits operate dynamically requires monitoring the expression of multiple genes in the same cell. However, it is limited by the relatively few distinguishable fluorescent proteins. Here, we developed a multiplexed real-time transcriptional imaging method based on two RNA stem-loop binding proteins, and employed it to analyze the temporal dynamics of synthetic gene circuits. By incorporating different ratios of MS2 and PP7 stem-loops, we were able to monitor the real-time nascent transcriptional activities of up to five genes in the same cell using only two fluorescent proteins. Applying this multiplexing capability to synthetic linear or branched gene regulatory cascades revealed that propagation of transcriptional dynamics is enhanced by non-stationary dynamics and is dictated by the slowest regulatory branch in the presence of combinatorial regulation. Mathematical modeling provided further insight into temporal multi-gene interactions and helped to understand potential challenges in regulatory inference using snapshot single-cell data. Ratiometric multiplexing should scale exponentially with additional labelling channels, providing a way to track the dynamics of larger circuits.
Multiple molecular events underlie stochastic switching between two heritable cell states in a eukaryotic system
Authors: Ziv Naomi , Brenes Lucas R. , Johnson Alexander 
Published: September 2021

AbstractEukaryotic transcriptional networks are often large and contain several levels of feedback regulation. Many of these networks have the ability to generate and maintain several distinct transcriptional states across multiple cell divisions and to switch between them. In certain instances, switching between cell states is stochastic, occurring in a small subset of cells of an isogenic population in a seemingly homogenous environment. Given the scarcity and unpredictability of switching in these cases, investigating the determining molecular events is challenging. White-opaque switching in the fungal species Candida albicans is an example of stably inherited cell states that are determined by a complex transcriptional network and can serve as an experimentally accessible model system to study characteristics important for stochastic cell fate switching in eukaryotes. In standard lab media, genetically identical cells maintain their cellular identity (either “white” or “opaque”) through thousands of cell divisions and switching between the states is rare. By isolating populations of white or opaque cells, previous studies have elucidated the many differences between the two stable cell states and identified a set of transcriptional regulators needed for cell type switching. Yet little is known about the molecular events that determine the rare, stochastic switching events that occur in single cells. We use microfluidics combined with fluorescent reporters to directly observe rare switching events between the white and opaque states. We investigate the stochastic nature of switching by beginning with white cells and monitoring the activation of Wor1, a master regulator and marker for the opaque state, in single cells and throughout cell pedigrees. Our results indicate that switching requires two stochastic steps; first an event occurs that predisposes a lineage of cells to switch. In the second step, some but not all, of those predisposed cells rapidly express high levels of Wor1 and commit to the opaque state. To further understand the rapid rise in Wor1, we used a synthetic inducible system in Saccharomyces cerevisiae into which a controllable C. albicans Wor1 and a reporter for its transcriptional control region have been introduced. We document that Wor1 positive autoregulation is highly cooperative (Hill coefficient > 3), leading to rapid activation and producing an “all or none” rather than a graded response. Taken together, our results suggest that reaching a threshold level of a master regulator is sufficient to drive cell type switching in single cells and that an earlier molecular event increases the probability of reaching that threshold in certain small lineages of cells. Quantitative molecular analysis of the white-opaque circuit can serve as a model for the general understanding of complex circuits.
Prevalence of viral frequency-dependent infection in coastal marine prokaryotes revealed using monthly time series virome analysis
Authors: Tominaga Kento , Ogawa-Haruki Nana , Nishimura Yosuke , Watai Hiroyasu , Yamamoto Keigo , Ogata Hiroyuki , Yoshida Takashi 
Published: September 2021

AbstractViruses infecting marine prokaryotes have large impacts on the diversity and dynamics of their hosts. Model systems suggest viral infection is frequency-dependent and constrained by the virus-host encounter rate. However, it is unclear whether the frequency-dependent infection is pervasive among the abundant prokaryotic populations with different growth strategies (i.e. r-strategy and K-strategy). To address this question, we performed a comparison of prokaryotic and viral communities using 16S rRNA amplicon and virome sequencing based on samples collected monthly for two years at a Japanese coastal site, Osaka Bay. Concurrent seasonal shifts observed in prokaryotic and viral community dynamics indicated that abundances of viruses correlated with that of their predicted host phyla (or classes). Co-occurrence network analysis between abundant prokaryotes and viruses revealed 6 423 co-occurring pairs, suggesting a tight coupling of host and viral abundances and their “one to many” correspondence. Although dominant K-strategist like species, such as SAR11, showed few co-occurring viruses, a fast succession of their viruses suggests viruses infecting these populations changed continuously. Our results suggest the frequency-dependent viral infection prevailed in coastal marine prokaryotes regardless of host taxa and growth strategy.
A population based expression atlas provides insights into disease resistance and other physiological traits in cassava (Manihot esculentaCrantz)
Authors: Ogbonna Alex C. , Ramu Punna , Williams Esuma , Nandudu Leah , Morales Nicolas , Powell Adrian , Kawuki Robert , Bauchet Guillaume , Jannink Jean-Luc , Mueller Lukas A. 
Published: September 2021

AbstractCassava, a food security crop in Africa, is grown throughout the tropics and subtropics. Although cassava can provide high productivity in suboptimal conditions, the yield in Africa is substantially lower than in other geographies. The yield gap is attributable to many challenges faced by cassava in Africa, including susceptibility to diseases and poor soil conditions. In this study, we carried out 3’RNA sequencing on 150 accessions from the National Crops Resources Research Institute, Ugandan for 5 tissue types, providing population-based transcriptomics resources to the research community in a web-based queryable cassava expression atlas. Differential expression and weighted gene co-expression network analysis were performed to detect 8,820 significantly differentially expressed genes (DEGs), revealing similarity in expression patterns between tissue types and the clustering of detected DEGs into 18 gene modules. As a confirmation of data quality, differential expression and pathway analysis targeting cassava mosaic disease (CMD) identified 27 genes observed in the plant-pathogen interaction pathway, several previously identified CMD resistance genes and two peroxidase family proteins different from theCMD2gene. Present research work represents a novel resource towards understanding complex traits at expression and molecular levels for the development of resistant and high-yielding cassava varieties, as exemplified with CMD.
Personalizing the control law of an upper-limb exoskeleton using EMG signal
Authors: Treussart Benjamin , Caron Remi , Geffard Franck , Marin Frederic , Vignais Nicolas 
Published: September 2021

AbstractImplementing an intuitive control law for an upper-limb exoskeleton dedicated to force augmentation is a challenging issue in the field of human-robot collaboration. The goal of this study is to adapt an EMG-based control system to a user based on individual caracteristics. To this aim, a method has been designed to tune the parameters of control using objective criteria, improving user’s feedback. The user’s response time is used as an objective value to adapt the gain of the controller. The proposed approach was tested on 10 participants during a lifting task. Two different conditions have been used to control the exoskeleton: with a generic gain and with a personalized gain. EMG signals was captured on five muscles to evaluate the efficiency of the conditions and the user’s adaptation. Results showed a statistically significant reduction of mean muscle activity of the deltoid between the beginning and the end of each situation (28.6 ± 13.5% to 17.2 ± 7.3% of Relative Maximal Contraction for the generic gain and from 24.9 ± 8.5% to 18.0 ± 6.8% of Relative Maximal Contraction for the personalized gain). When focusing on the first assisted movements, the personalized gain induced a mean activity of the deltoïd significantly lower (29.0 ± 8.0% of Relative Maximal Contraction and 37.4 ± 9.5% of Relative Maximal Contraction, respectively). Subjective evaluation showed that the system with a personalized gain was perceived as more intuitive, and required less concentration when compared to the system with a generic gain.
Engineering CAR-NK cells to secrete IL15 sustains their anti-AML functionality, but is associated with systemic toxicities
Authors: Christodoulou Ilias , Ho Won Jin , Marple Andrew , Ravich Jonas W. , Tam Ada , Rahnama Ruyan , Fearnow Adam , Rietberg Cambrynne , Yanik Sean , Solomou Elena E. , Varadhan Ravi , Koldobskiy Michael A. , Bonifant Challice L. 
Published: September 2021

AbstractBackgroundThe prognosis of patients with recurrent/refractory acute myelogenous leukemia (AML) remains poor and cell-based immunotherapies hold promise to improve outcomes. NK cells can elicit an anti-leukemic response via a repertoire of activating receptors that bind AML surface ligands. NK cell adoptive transfer is safe but thus far has shown limited anti-AML efficacy. Here, we aimed to overcome this limitation by engineering NK cells to express chimeric antigen receptors (CARs) to boost their anti-AML activity, and interleukin-15 (IL15) to enhance their persistence.MethodsWe characterized in detail NK cell populations expressing a panel of AML (CD123)-specific CARs and/or IL15 in vitro and in AML xenograft models.ResultsCARs with 2B4.ζ or 4-1BB.ζ signaling domains demonstrated greater cell surface expression and endowed NK cells with improved anti-AML activity in vitro. Initial in vivo testing revealed that only 2B4.ζ CAR-NK cells had improved anti-AML activity in comparison to untransduced (UTD) and 4-1BB.ζ CAR-NK cells. However, the benefit was transient due to limited CAR-NK cell persistence. Transgenic expression of secretory (s)IL15 in 2B4.ζ CAR and UTD NK cells improved their effector function in the setting of chronic antigen simulation in vitro. Multiparameter flow analysis after chronic antigen exposure identified the expansion of unique NK cell subsets. 2B4.ζ/sIL15 CAR and sIL15 NK cells maintained an overall activated NK cell phenotype. This was confirmed by transcriptomic analysis, which revealed a highly proliferative and activated signature in these NK cell groups. In vivo, 2B4.ζ/sIL15 CAR-NK cells had potent anti-AML activity in one model, while 2B4.ζ/sIL15 CAR and sIL15 NK cells induced lethal toxicity in a second model.ConclusionTransgenic expression of CD123-CARs and sIL15 enabled NK cells to function in the setting of chronic antigen exposure but was associated with systemic toxicities. Thus, our study provides the impetus to explore inducible and controllable expression systems to provide cytokine signals to AML-specific CAR-NK cells before embarking on early phase clinical testing.Abstract FigureIn BriefSecretion of IL15 from anti-CD123.2B4.CD3ζ CAR-NK cells enhances their activation and killing potential against Acute Myelogenous Leukemia, but carries the risk of severe systemic toxicity.