bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genome editing in animals with minimal PAM CRISPR-Cas9 enzymes
Authors: Vicencio Jeremy , Sánchez-Bolaños Carlos , Moreno-Sánchez Ismael , Brena David , Kukhtar Dmytro , Ruiz-López Miguel , Cots-Ponjoan Mariona , Vejnar Charles E. , Rubio Alejandro , Melero Natalia Rodrigo , Carolis Carlo , Pérez-Pulido Antonio J. , Giráldez Antonio J. , Kleinstiver Benjamin P. , Cerón Julián , Moreno-Mateos Miguel A. 
Published: June 2021

ABSTRACTThe requirement for Cas nucleases to recognize a specific PAM is a major restriction for genome editing. SpCas9 variants SpG and SpRY, recognizing NGN and NRN PAM, respectively, have contributed to increase the number of editable genomic sites in cell cultures and plants. However, their use has not been demonstrated in animals.We have characterized and optimized the activity of SpG and SpRY in zebrafish and C. elegans. Delivered as mRNA-gRNA or ribonucleoprotein (RNP) complexes, SpG and SpRY were able to induce mutations in vivo, albeit at a lower rate than SpCas9 in equivalent formulations. This lower activity was overcome by optimizing mRNA-gRNA or RNP concentration, leading to efficient mutagenesis at regions inaccessible to SpCas9. We also found that the CRISPRscan algorithm can predict SpG and SpRY activity in vivo. Finally, we applied SpG and SpRY to generate knock-ins by homology-directed repair. Altogether, our results expand the CRISPR-Cas targeting genomic landscape in animals.
Comparative Analysis of Sorghum EMS Mutants and Natural Populations
Authors: Wang Liya , Lipzen Anna , Lu Zhenyuan , Chen Junping , Wang Xiaofei , Tello-Ruiz Marcela K. , Barry Kerrie , Mortimer Jenny , Ware Doreen , Xin Zhanguo 
Published: June 2021

AbstractTo build a large-scale genomic resource for functional validation of sorghum genes through EMS-mutagenized BTx623 seeds, we deep sequenced (30-60X) an additional 445 phenotyped EMS mutant lines. 4.2 million EMS mutations are called with nearly 36,800 mutations that could have a disruptive effect on functions of over 15,500 genes. Combining variants carried by both the natural population and previous EMS efforts, over 69% of sorghum coding genes (23644) are now presented with one or more mutations that are, or are predicted to be, disruptive to their functions. Our results show that the EMS population carries more significant mutations but less in each sample than the natural population, which makes it more powerful in elucidating sorghum gene functions on a large scale and requiring less work in validation of candidate causal genes. We have made the data available through two ways, one is the integration with the BSAseq workflow that supports retrieving independent EMS samples carrying the same genes with significant mutation for complementary testing, and the other is a web application for directly querying genes with significant mutations onSciApps.org.
Glyoxal as alternative fixative for single cell RNA sequencing
Authors: Bageritz Josephine , Krausse Niklas , Yousefian Schayan , Leible Svenja , Valentini Erica , Boutros Michael 
Published: June 2021

AbstractSingle cell RNA sequencing (scRNA-seq) has become an important method to identify cell types, delineate the trajectories of cell differentiation in whole organisms and understand the heterogeneity in cellular responses. Nevertheless, sample collection and processing remain a severe bottleneck for scRNA-seq experiments. Cell isolation protocols often lead to significant changes in the transcriptomes of cells, requiring novel methods to preserve cell states. Here, we developed and benchmarked protocols using glyoxal as a fixative for scRNA-seq application. Using Drop-seq methodology, we detected high numbers of transcripts and genes from glyoxal-fixed Drosophila cells after scRNA-seq. The effective glyoxal fixation of transcriptomes in Drosophila and human cells was further supported by a high correlation of gene expression data between glyoxal-fixed and unfixed samples. Accordingly, we also found highly expressed genes overlapping to a large extent between experimental conditions. These results indicated that our fixation protocol did not induce considerable changes in gene expression and conserved the transcriptome for subsequent single cell isolation procedures. In conclusion, we present glyoxal as a suitable fixative for Drosophila cells and potentially cells of other species that allows high-quality scRNA-seq applications.
Cross-reactive TCR with alloreactivity for immunodominant HIV-1 epitope Gag TL9 with enhanced control of viral infection
Authors: Liu Yang , San Dan , Yin Lei 
Published: June 2021

ABSTRACTAlthough both HLA B*81:01 and HLA B*42:01 are members of the B7 supertype and can present many of the same HIV-1 epitopes, the identification of a dual-reactive T-cell phenotype was unexpected, since structural data suggested that TL9 peptide binds to each allele in a distinct conformation. How the dual-reactive TCR recognizes these radically distinct p-MHC surfaces is revealed by our structural study, that the introduction of TCR T18A induces a molecular switch of the TL9 peptide in B4201 to approach its conformation in B8101. Most importantly, unique docking of CDR3β towards MHC but not peptide ligand strengthens the peptide tolerance of T18A, extends the ability of TCR to adapt mutations. Moreover, the high affinity of dual-reactive TCR for WT and escape mutant TL9 highlights the functional advantage of the alloreactive phenotype.
Magnetic Bead-Based Separation (MBS) of Pneumococcal Serotypes
Authors: York Anna , Huynh Emily T. , Mbodj Sidiya , Yolda-Carr Devyn , Hislop Maikel S. , Echlin Haley , Rosch Jason W. , Weinberger Daniel M. , Wyllie Anne L. 
Published: June 2021

SUMMARYThe separation of pneumococcal serotypes from a complex polymicrobial mixture may be required for different applications. For instance, a minority strain could be present at a low frequency in a clinical sample, making it difficult to identify and isolate by traditional culture-based methods. We therefore developed an assay to separate mixed pneumococcal samples using serotype-specific antiserum and a magnetic bead-based separation method. Using qPCR and colony counting methods, we first show that serotypes (12F, 23F, 3, 14, 19A and 15A) present at ∼0.1% of a dual serotype mixture can be enriched to between 10% and 90% of the final sample. We demonstrate two applications for this method: extraction of a known pneumococcal serotype from saliva samples and efficient purification of capsule switch variants from experimental transformation experiments. Moreover, this method may have further laboratory or clinical applications when the selection of specific serotypes is required.
Plasmodium falciparum Calcium Dependent Protein Kinase 4 is critical for male gametogenesis and transmission to the mosquito vector
Authors: Kumar Sudhir , Haile Meseret T. , Hoopmann Michael R. , Tran Linh T. , Michaels Samantha A. , Morrone Seamus R. , Ojo Kayode K. , Reynolds Laura M. , Kusebauch Ulrike , Vaughan Ashley M. , Moritz Robert L. , Kappe Stefan H.I. , Swearingen Kristian E. 
Published: June 2021

ABSTRACTGametocytes of the malaria parasite Plasmodium are taken up by the mosquito vector with an infectious blood meal, representing a critical stage for parasite transmission. Calcium dependent protein kinases (CDPKs) play key roles in calcium-mediated signaling across the complex life cycle of the parasite. We sought to understand their role in human parasite transmission from the host to the mosquito vector and thus investigated the role of the human-infective parasite Plasmodium falciparum CDPK4 in the parasite life cycle. P. falciparum cdpk4− parasites created by targeted gene deletion showed no effect in blood stage development or gametocyte development. However, cdpk4− parasites showed a severe defect in male gametogenesis and the emergence of flagellated male gametes. To understand the molecular underpinnings of this defect, we performed mass spectrometry-based phosphoproteomic analyses of wild type and Plasmodium falciparum cdpk4− late gametocyte stages, to identify key CDPK4-mediated phosphorylation events that may be important for the regulation of male gametogenesis. We further employed in vitro assays to identify these putative substrates of Plasmodium falciparum CDPK4. This indicated that CDPK4 regulates male gametogenesis by directly or indirectly controlling key essential events such as DNA replication, mRNA translation and cell motility. Taken together, our work demonstrates that PfCDPK4 is a central kinase that regulates exflagellation, and thereby is critical for parasite transmission to the mosquito vector.IMPORTANCETransmission of the malaria parasite to the mosquito vector is critical for the completion of the sexual stage of the parasite life cycle and is dependent on the release of male gametes from the gametocyte body inside the mosquito midgut. In the present study, we demonstrate that PfCDPK4 is critical for male gametogenesis and is involved in phosphorylation of proteins essential for male gamete emergence. Targeting PfCDPK4 and its substrates may provide insights into achieving effective malaria transmission-blocking strategies.
Combining Multi-Dimensional Molecular Fingerprints to Predict hERG Cardiotoxicity of Compounds
Authors: Ding Weizhe , Zhang Li , Nan Yang , Wu Juanshu , Xin Xiangxin , Han Chenyang , Li Siyuan , Liu Hongsheng 
Published: June 2021

AbstractAt present, drug toxicity has become a critical problem with heavy medical and economic burdens. acLQTS (acquired Long QT Syndrome) is acquired cardiac ion channel disease caused by drugs blocking the hERG channel. Therefore, it is necessary to avoid cardiotoxicity in the drug design and computer models have been widely used to fix this plight. In this study, we present a molecular fingerprint based on the molecular dynamic simulation and uses it combined with other molecular fingerprints (multi-dimensional molecular fingerprints) to predict hERG cardiotoxicity of compounds. 203 compounds with hERG inhibitory activity (pIC50) were retrieved from a previous study and predicting models were established using four machine learning algorithms based on the single and multi-dimensional molecular fingerprints. Results showed that MDFP has the potential to be an alternative to traditional molecular fingerprints and the combination of MDFP and traditional molecular fingerprints can achieve higher prediction accuracy. Meanwhile, the accuracy of the best model, which was generated by consensus of four algorithms with multi-dimensional molecular fingerprints, was 0.694 (RMSE) in the test dataset. Besides, the number of hydrogen bonds from MDFP has been determined as a critical factor in the predicting models, followed by rgyr and sasa. Our findings provide a new sight of MDFP and multi-dimensional molecular fingerprints in building models of hERG cardiotoxicity prediction.
SE(3)-Equivariant Energy-based Models for End-to-End Protein Folding
Authors: Wu Jiaxiang , Shen Tao , Lan Haidong , Bian Yatao , Huang Junzhou 
Published: June 2021

AbstractAccurate prediction of protein structures is critical for understanding the biological function of proteins. Nevertheless, most structure optimization methods are built upon pre-defined statistical energy functions, which may be sub-optimal in formulating the conformation space. In this paper, we propose an end-to-end approach for protein structure optimization, powered by SE(3)-equivariant energy-based models. The conformation space is characterized by a SE(3)-equivariant graph neural network, with substantial modifications to embed the protein-specific domain knowledge. Furthermore, we introduce continuously-annealed Langevin dynamics as a novel sampling algorithm, and demonstrate that such process converges to native protein structures with theoretical guarantees. Extensive experiments indicate that SE(3)-Fold achieves comparable structure optimization accuracy, compared against state-of-the-art baselines, with over 1-2 orders of magnitude speed-up.
Insulin is expressed by enteroendocrine cells during human fetal development
Authors: Egozi Adi , Llivichuzhca-Loja Dhana , McCourt Blake , Farack Lydia , An Xiaojing , Wang Fujing , Chen Kong , Konnikova Liza , Itzkovitz Shalev 
Published: June 2021

AbstractGeneration of beta cells via transdifferentiation of other cell types is a promising avenue for the treatment of diabetes. Here, we reconstruct a single cell atlas of enteroendocrine cells in the human fetal and neonatal small intestine. We identify a subset of fetal enteroendocrine K/L cells that express high levels of insulin and other beta cell genes. Our findings highlight a potential extra-pancreatic source of beta cells and exposes its molecular blueprint.
Chromosome specific telomere lengths and the minimal functional telomere revealed by nanopore sequencing
Authors: Sholes Samantha L. , Karimian Kayarash , Gershman Ariel , Kelly Thomas J. , Timp Winston , Greider Carol W. 
Published: June 2021

ABSTRACTWe developed a method to tag telomeres and measure telomere length by nanopore sequencing in the yeast S. cerevisiae. Nanopore allows long read sequencing through the telomere, subtelomere and into unique chromosomal sequence, enabling assignment of telomere length to a specific chromosome end. We observed chromosome end specific telomere lengths that were stable over 120 cell divisions. These stable chromosome specific telomere lengths may be explained by stochastic clonal variation or may represent a new biological mechanism that maintains equilibrium unique to each chromosomes end. We examined the role of RIF1 and TEL1 in telomere length regulation and found that TEL1 is epistatic to RIF1 at most telomeres, consistent with the literature. However, at telomeres that lack subtelomeric Y’ sequences, tel1Δ rif1Δ double mutants had a very small, but significant, increase in telomere length compared to the tel1Δ single mutant, suggesting an influence of Y’ elements on telomere length regulation. We sequenced telomeres in a telomerase-null mutant (est2Δ) and found the minimal telomere length to be around 75bp. In these est2Δ mutants there were many apparent telomere recombination events at individual telomeres before the generation of survivors, and these events were significantly reduced in est2Δ rad52Δ double mutants. The rate of telomere shortening in the absence of telomerase was similar across all chromosome ends at about 5 bp per generation. This new method gives quantitative, high resolution telomere length measurement at each individual chromosome end, suggests possible new biological mechanisms regulating telomere length, and provides capability to test new hypotheses.
Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus
Authors: Kick L.M. , von Wrisberg M.-K. , Runtsch L. S. , Schneider S. 
Published: June 2021

AbstractCas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key-residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression.
spicyR: Spatial analysis of in situ cytometry data in R
Authors: Canete Nicolas P. , Iyengar Sourish S. , Wilmott James S. , Ormerod John T. , Harman Andrew N. , Patrick Ellis 
Published: June 2021

AbstractMotivationHigh parameter histological techniques have allowed for the identification of a variety of distinct cell types within an image, providing a comprehensive overview of the tissue environment. This allows the complex cellular architecture and environment of diseased tissue to be explored. While spatial analysis techniques have revealed how cell-cell interactions are important within the disease pathology, there remains a gap in exploring changes in these interactions within the disease process. Specifically, there are currently no established methods for performing inference on cell localisation changes across images, hindering an understanding of how cellular environments change with a disease pathology.ResultsWe have developed the spicyR R package to perform inference on changes in the spatial localisation of cell types across groups of images. Application to simulated data demonstrates a high sensitivity and specificity. We demonstrate the utility of spicyR by applying it to a type 1 diabetes imaging mass cytometry dataset, revealing changes in cellular associations that were relevant to the disease progression. Ultimately, spicyR allows changes in cellular environments to be explored under different pathologies or disease states.Availability and ImplementationR package freely available at http://bioconductor.org/packages/release/bioc/html/spicyR.html and shiny app implementation at http://shiny.maths.usyd.edu.au/spicyR/Contactellis.patrick@sydney.edu.auSupplementary informationCode for reproducing key figures available at https://github.com/nickcee/spicyRPaper.
drda: An R package for dose-response data analysis
Authors: Malyutina Alina , Tang Jing , Pessia Alberto 
Published: June 2021

AbstractAnalysis of dose-response data is an important step in many scientific disciplines, including but not limited to pharmacology, toxicology, and epidemiology. The R package drda is designed to facilitate the analysis of dose-response data by implementing efficient and accurate functions with a familiar interface. With drda, it is possible to fit models by the method of least squares, perform goodness of fit tests, and conduct model selection. Compared to other similar packages, drda provides, in general, more accurate estimates in the least-squares sense. This result is achieved by a smart choice of the starting point in the optimization algorithm and by implementing the Newton method with a trust region with analytical gradients and Hessian matrices. In this article, drda is presented through the description of its methodological components and examples of its user-friendly functions. Performance is finally evaluated using a real, large-scale drug sensitivity screening dataset.
Insulin signaling couples growth and early maturation to cholesterol intake
Authors: Texada Michael J. , Lassen Mette , Pedersen Lisa H. , Malita Alina , Rewitz Kim 
Published: June 2021

ABSTRACTNutrition is one of the most important influences on growth and the timing of developmental maturation transitions including mammalian puberty and insect metamorphosis. Childhood obesity is associated with precocious puberty, but the assessment mechanism that links body fat to early maturation is unknown. During development, intake of nutrients promotes signaling through insulin-like systems that govern the growth of cells and tissues and also regulates the timely production of the steroid hormones that initiate the juvenile-adult transition. We show here that the dietary lipid cholesterol, required as a component of cell membranes and as a substrate for steroid biosynthesis, also governs body growth and maturation in Drosophila via promoting the expression and release of insulin-like peptides. This nutritional input acts via the Niemann-Pick-type-C (Npc) cholesterol sensors/transporters in the glia of the blood-brain barrier and cells of the adipose tissue to remotely drive systemic insulin signaling and body growth. Furthermore, increasing intracellular cholesterol levels in the steroid-producing prothoracic gland strongly promotes endoreduplication, leading to accelerated attainment of a nutritional checkpoint that ensures that animals do not initiate maturation prematurely. These findings couple sensing of the lipid cholesterol to growth control and maturational timing, which may help explain both the link between cholesterol and cancer as well as the critical connection between body fat (obesity) and early puberty.HIGHLIGHTSDietary cholesterol promotes developmental growth and leads to early maturationInsulin signaling couples cholesterol intake with systemic growthCholesterol promotes insulin signaling and growth via glial and fat-tissue relaysCholesterol sensing affects a nutritional checkpoint that prevents early maturationGRAPHICAL ABSTRACT
Education as a key to provide the growth of entrepreneurial intentions
Authors: Pinto Borges Ana , Lopes João M. , Carvalho Catarina , Vieira Bruno Miguel , Lopes Jorge 
Published: June 2021

PurposeThe purpose of this study is to analyze the entrepreneurial intentions of higher education students in a private higher education institution (HEI), enrolled in the following courses: Business Management, Hospitality Management, Tourism and Business Relations.Design/methodology/approachA quantitative methodology was used through the application of a questionnaire in a private HEI located in the northern region of Portugal and 228 valid responses were collected.FindingsWe have found out that (1) the elder the students are, the higher is the probability that they will consider entrepreneurship as the first option to enter the job market; (2) the courses of Business Management and Hotel Management are those that influence the entrepreneurial intentions most positively; (3) the more prepared a student feels in order to start a business, the more likely he will become an entrepreneur; (4) to consider entrepreneurship as the first option to enter the job market is positively influenced by the dimensions of the Attitude Toward Enterprise (ATE), namely, leadership, creativity, achievement and personal control; (5) to have a business idea could be motivated by the course in which the students are enrolled in, if they feel they are prepared to start a business, and by the dimensions of ATE.Practical implicationsHEIs and/or regional governments may have an entrepreneurship support office, where students can get assistance in the process of creating their companies and develop innovative entrepreneurial models adapted to older students. The creation of “senior entrepreneurial ecosystems” can be an interesting path to be explored. HEIs can make extracurricular activities available to students. Policymakers must introduce entrepreneurship to primary and secondary education.Originality/valueThis study increases the understanding of the individual characteristics of the students in a private HEI, as well as the courses that positively influence entrepreneurial intentions.