bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detailed metabolic phenotyping of four tissue specific Cas9 transgenic mouse lines
Authors: Bond Simon T. , Zhuang Aowen , Yang Christine , Gould Eleanor A.M. , Sikora Tim , Liu Yingying , Fu Ying , Watt Kevin I. , Tan Yanie , Kiriazis Helen , Lancaster Graeme I. , Gregorevic Paul , Henstridge Darren C. , McMullen Julie R. , Meikle Peter J. , Calkin Anna C. , Drew Brian G. 
Published: April 2021

AbstractCRISPR/Cas9 technology has revolutionized gene editing and fast tracked our capacity to manipulate genes of interest for the benefit of both research and therapeutic applications. Whilst many advances have, and continue to be made in this area, perhaps the most utilized technology to date has been the generation of knockout cells, tissues and animals by taking advantage of Cas9 function to promote indels in precise locations in the genome. Whilst the advantages of this technology are many fold, some questions still remain regarding the effects that long term expression of foreign proteins such as Cas9, have on mammalian cell function. Several studies have proposed that chronic overexpression of Cas9, with or without its accompanying guide RNAs, may have deleterious effects on cell function and health. This is of particular concern when applying this technology in vivo, where chronic expression of Cas9 in tissues of interest may promote disease-like phenotypes and thus confound the investigation of the effects of the gene of interest. Although these concerns remain valid, no study to our knowledge has yet to demonstrate this directly. Thus, in this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models with expression in the heart, liver, skeletal muscle and adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs. We demonstrated that Cas9 expression in these tissues had no detrimental effect on whole body health of the animals, nor did it induce any tissue-specific effects on energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass. Thus, our data suggests that these models are suitable for studying the tissue specific effects of gene deletion using the LSL-Cas9-Tg model, and that phenotypes observed utilizing these models can be confidently interpreted as being gene specific, and not confounded by the chronic overexpression of Cas9.
The nucleoporin Nup50 activates the Ran guanyl-nucleotide exchange factor RCC1 to promote mitotic NPC assembly
Authors: Holzer Guillaume , de Magistris Paola , Gramminger Cathrin , Sachdev Ruchika , Magalska Adriana , Schooley Allana , Anja Scheufen , Lennartz Birgitt , Tatarek-Nossol Marianna , Lue Hongqi , Linder Monika I. , Kutay Ulrike , Preisinger Christian , Moreno-Andres Daniel , Antonin Wolfram 
Published: April 2021

AbstractDuring mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. We show here that Nup50 plays a crucial role in NPC assembly that is independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of the protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α, β, the GTPase Ran or chromatin is crucial for its function in the assembly process. Instead, we discovered that an N-terminal fragment of Nup50 can stimulate the Ran guanine exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in mitotic NPC reformation. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild type protein in in vitro NPC assembly assays.
The Dendritic Ergic: Microtubule and Actin Cytoskeletons Mediate Stop-and-Go Movement of Mobile Carriers Between Stable Structures
Authors: de los Ángeles Juricic Urzúa María , Rojas Javiera Gallardo , Correa Andrés Couve , Cerda Mauricio , Gründler Steffen Härtel , González-Silva Carolina 
Published: April 2021

ABSTRACTThe ER-to-Golgi intermediate compartment (ERGIC) is a membranous organelle that mediates protein transport between the endoplasmic reticulum (ER) and Golgi apparatus. In neurons, clusters of these vesiculotubular structures are situated in throughout the cell in proximity to the ER, passing cargo to the cis-Golgi cisternae located mainly in the perinuclear region. Although ERGIC markers have been identified in neurons, the distribution and dynamics of neuronal ERGIC structures have not been characterized.Here, we argue that long-distance ERGIC transport occurs via an intermittent mechanism in neurons, with mobile elements moving between stationary structures. Using immunofluorescence microscopy, we detected discrete, irregular ERGIC structures in neural soma and dendrites. Slow live-cell imaging (2 frames/minute; 15 minutes) indicated that 8% of dendritic ERGIC structures were stable, remaining in place over long periods. On the other hand, fast live-cell imaging (2 frames/second; 180 seconds) captured mobile ERGIC structures advancing very short distances along dendrites. Importantly, these distances were consistent with the lengths between the stationary ERGIC structures. Kymography revealed ERGIC elements that moved intermittently, emerging from and fusing with stationary ERGIC structures. Surprisingly, this movement was apparently dependent not only on the integrity of the microtubule cytoskeleton, as has been previously reported, but on the actin cytoskeleton as well.Our results indicate that the dendritic ERGIC has a dual nature, with both stationary and mobile structures. The neural ERGIC network transports proteins via a stop-and-go movement that is mediated by the microtubule and actin cytoskeletons.
A Chromosome-level Assembly of a Wild Castor Genome Provides New Insights into the Adaptive Evolution in a Tropical Desert
Authors: Lu Jianjun , Pan Cheng , Fan Wei , Liu Wanfei , Zhao Huayan , Li Donghai , Wang Sen , Hu Lianlian , He Bing , Qian Kun , Qin Rui , Ruan Jue , Lin Qiang , Lü Shiyou , Cui Peng 
Published: April 2021

AbstractWild castor grows in the high-altitude tropical desert of the African Plateau, a region known for high ultraviolet radiations, strong lights and extremely dry conditions. To investigate the potential genetic basis of adaptation to both highland and tropical deserts, we generated a chromosome-level genome sequence of the wild castor accession WT05, with genome size of 316 Mb and scaffold and contig N50 sizes of 31.93 Mb and 8.96 Mb, respectively. Compared with cultivated castor and other Euphorbiaceae species, the wild castor exhibits positive selection and gene family expansion for genes involved in DNA repair, photosynthesis and abiotic stress responses. Genetic variations associated with positive selection were identified in several key genes, such asLIG1, DDB2, andRECG1, involved in nucleotide excision repair. Moreover, a study of genomic diversity among wild and cultivated accessions revealed genomic regions containing selection signatures associated with the adaptation to extreme environments. The identification of the genes and alleles with selection signatures provides insights into the genetic mechanisms underlying the adaptation of wild castor to the high-altitude tropical desert and would facilitate direct improvement of modern castor varieties.
Sequencing of methylase-accessible regions in integral circular extrachromosomal DNA reveals differences in chromatin structure
Authors: Chen Weitian , Weng Zhe , Xie Zhe , Xie Yeming , Zhang Chen , Chen Zhichao , Ruan Fengying , Wang Juan , Sun Yuxin , Fang Yitong , Guo Mei , Tong Yiqin , Li Yaning , Tang Chong 
Published: April 2021

AbstractAlthough extrachromosomal DNA (ecDNA) has been intensively studied for several decades, the mechanisms underlying its tumorigenic effects have been revealed only recently. In the majority of conventional sequencing studies, the high-throughput short-read sequencing largely ignores the epigenetic status of most ecDNA regions except for the junctional areas. Here, we developed the sequencing of enzyme-accessible chromatin in circular DNA (CCDA-seq) method, which uses methylase to label open chromatin without fragmentation and exonuclease to enrich the ecDNA sequencing depth, followed by long-read nanopore sequencing. Using CCDA-seq, we observed significantly different patterns in nucleosome/regulator binding in ecDNA at a single-molecule resolution. These results deepen the understanding of ecDNA regulatory mechanisms.
Chromatin interaction aware gene regulatory modeling with graph attention networks
Authors: Karbalayghareh Alireza , Sahin Merve , Leslie Christina S. 
Published: April 2021

AbstractLinking distal enhancers to genes and modeling their impact on target gene expression are longstanding unresolved problems in regulatory genomics and critical for interpreting non-coding genetic variation. Here we present a new deep learning approach called GraphReg that exploits 3D interactions from chromosome conformation capture assays in order to predict gene expression from 1D epigenomic data or genomic DNA sequence. By using graph attention networks to exploit the connectivity of distal elements up to 2Mb away in the genome, GraphReg more faithfully models gene regulation and more accurately predicts gene expression levels than state-of-the-art deep learning methods for this task. Feature attribution used with GraphReg accurately identifies functional enhancers of genes, as validated by CRISPRi-FlowFISH and TAP-seq assays, outperforming both CNNs and the recently proposed Activity-by-Contact model. Sequence-based GraphReg also accurately predicts direct transcription factor (TF) targets as validated by CRISPRi TF knockout experiments via in silico ablation of TF binding motifs. GraphReg therefore represents an important advance in modeling the regulatory impact of epigenomic and sequence elements.
Re-profiling of natural inhibitor via combinatorial drug screening: Brefeldin A variant design as an effective antagonist leading to EPAC2 structure modification and antibody design for identification
Authors: Uttarkar Akshay , Niranjan Vidya 
Published: April 2021

AbstractDrug discovery can be impactful with re-purposing and combinatorial strategies leading to potential pharmaceutical outputs. Epac2 has been a target of interest for various physiological conditions where suppression is inevitable to achieve desired therapeutic effect. Epac isoforms inhibition is crucial in vascular functions to prevent chronic inflammation leading to hypertension and myocardial infarction. An attempt utilizing brefeldin A, a natural inhibitor was subjected to substitution of 3 side chain groups with 43 fragments via combinatorial strategy. This resulted in generating a library of 79507 brefeldin A variants. High throughput virtual screening yielded 68,043 variants followed by precision docking providing 117 lead like brefeldin A variants. The best docked variant (3-((1R,2E,6R,10E,11aS,13S,14A)-6-(methylsulfonamido)-13-(3-methylureido)-4-oxo-4,6,7,8,9,11a,12,13,14,14a-decahydro-1H-cyclopenta[f][1]oxacyclotridecin-1-yl)-2,3-dihydro-1Himidazol-1-ium) has an increased binding efficiency of −10.841 kcal/mol. Simulation studies up to 200ns of complex lead to re-orientation of target tertiary structure resulted in RMSD change of 30.221 Å, suggesting the epac2 structure modification leading to unavailability of RAS-GEF domain and its interaction with Rap1b. A single domain antibody was designed to bind specifically to re-structured epac2 for potential identification over the native target structure. The resulting Brefeldin variant can be potentially labelled as a most effective antagonist against epac2 which induces theoretically irreversible structural re-conformation. This study also provides a robustin-silicoworkflow for searching of chemical space, generating and screening of combination libraries and the efficient utilization of known inhibitor.
An improved molecular crowding sensor CRONOS for detection of crowding changes in membrane-less organelles under pathological conditions
Authors: Miyagi Tamami , Yamanaka Yoshiaki , Harada Yuichiro , Narumi Satoshi , Hayamizu Yuhei , Kuroda Masahiko , Kanekura Kohsuke 
Published: April 2021

AbstractMembrane-less organelles (MLOs) formed by liquid-liquid phase separation (LLPS) play pivotal roles in biological processes. During LLPS, proteins and nucleotides are extremely condensed, resulting in changes of their conformation and biological functions. Disturbed LLPS homeostasis in MLOs cause fatal diseases such as amyotrophic lateral sclerosis. Therefore, it is important to detect changes of the degree of crowding in MLOs. However, it has not been investigated well due to lack of an appropriate method. To address this, we developed a genetically-encoded molecular crowding sensor CRONOS that senses the degree of macromolecular crowding in MLOs using fluorescence resonance energy transfer (FRET) system. CRONOS is a very bright biosensor with wider dynamic range and detect changes in the macromolecular volume fraction better than the previously reported mCer-mCit sensor in solution. By fusing to scaffold protein of each MLO, we successfully delivered CRONOS to MLO of interest and detected previously undescribed difference of the degree of crowding in each MLO. If not tagged, CRONOS localized to interstitial space of MLOs, giving us the crowding information of inspace. CRONOS also detected changes of degree of macromolecular crowding in nucleolus induced by environmental stress or inhibition of transcription. These findings suggest that CRONOS can be a useful tool for determination of molecular crowding and detection of pathological changes in MLOs in live cells.
ReFeaFi: Genome-wide prediction of regulatory elements driving transcription initiation
Authors: Umarov Ramzan , Li Yu , Arakawa Takahiro , Takizawa Satoshi , Gao Xin , Arner Erik 
Published: April 2021

AbstractRegulatory elements control gene expression through transcription initiation (promoters) and by enhancing transcription at distant regions (enhancers). Accurate identification of regulatory elements is fundamental for annotating genomes and understanding gene expression patterns. While there are many attempts to develop computational promoter and enhancer identification methods, reliable tools to analyze long genomic sequences are still lacking. Prediction methods often perform poorly on the genome-wide scale because the number of negatives is much higher than that in the training sets. To address this issue, we propose a dynamic negative set updating scheme with a two-model approach, using one model for scanning the genome and the other one for testing candidate positions. The developed method achieves good genome-level performance and maintains robust performance when applied to other species, without re-training. Moreover, the unannotated predicted regulatory regions made on the human genome are enriched for disease-associated variants, suggesting them to be potentially true regulatory elements rather than false positives. We validated high scoring “false positive” predictions using reporter assay and all tested candidates were successfully validated, demonstrating the ability of our method to discover novel human regulatory regions.
A new contact killing toxin permeabilizes cells and belongs to a large protein family
Authors: Crisan Cristian V. , Chandrashekar Harshini , Everly Catherine , Steinbach Gabi , Hill Shannon E. , Yunker Peter J. , Lieberman Raquel R. , Hammer Brian K. 
Published: April 2021

ABSTRACTVibrio choleraeis an aquatic Gram-negative bacterium that causes severe diarrheal cholera disease when ingested by humans. To eliminate competitor cells in both the external environment and inside hosts,V. choleraeuses the Type VI Secretion System (T6SS). The T6SS is a macromolecular weapon employed by many Gram-negative bacteria to deliver cytotoxic proteins into adjacent cells. In addition to canonical T6SS gene clusters encoded by all sequencedV. choleraeisolates, strain BGT49 encodes an additional locus, which we named auxiliary cluster 4 (Aux 4). The Aux 4 cluster is located on a mobile genetic element and can be used by killer cells to eliminate bothV. choleraeandEscherichia colicells in a T6SS-dependent manner. A putative toxin encoded in the cluster, which we name TpeV (Type VIPermeabilizingEffectorVibrio), shares no homology to known proteins and does not contain motifs or domains indicative of function. Ectopic expression of TpeV in the periplasm ofE. colipermeabilizes cells and disrupts the membrane potential. Using confocal microscopy, we confirm that susceptible target cells become permeabilized when competed with killer cells harboring the Aux 4 cluster. We also determine thattpiV, the gene located immediately downstream oftpeV, encodes an immunity protein that neutralizes the toxicity of TpeV. Finally, we show that TpeV homologs are broadly distributed across important animal and plant pathogens and are localized in proximity to other T6SS genes. Our results suggest that TpeV is a toxin that belongs to a large family of T6SS proteins.IMPORTANCEBacteria live in polymicrobial communities where competition for resources and space is essential for survival. Proteobacteria use the T6SS to eliminate neighboring cells and cause disease. However, the mechanisms by which many T6SS toxins kill or inhibit susceptible target cells are poorly understood. The sequence of the TpeV toxin we describe here is unlike any previously described protein. We demonstrate that it has antimicrobial activity by permeabilizing cells, eliminating membrane potentials and causing severe cytotoxicity. TpeV homologs are found near known T6SS genes in human, animal and plant bacterial pathogens, indicating that the toxin is a representative member of a broadly distributed protein family. We propose that TpeV-like toxins contribute to the fitness and pathogenicity of many bacteria. Finally, since antibiotic resistance is a critical global health threat, the discovery of new antimicrobial mechanisms could lead to the development of new treatments against resistant strains.
Light-dependent inhibition of clathrin-mediated endocytosis in yeast
Authors: Prischich Davia , del Dedo Javier Encinar , Cambra Maria , Prat Judit , Camarero Nuria , Nevola Laura , Martín-Quirós Andrés , Rebollo Elena , Giralt Ernest , Isabel Geli María , Gorostiza Pau 
Published: April 2021

AbstractClathrin-mediated endocytosis (CME) is an essential cellular process, which is evolutionarily conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of pharmacological agents that could complement genetics in selectively and reversibly interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor that targets the AP2/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP and demonstrate that TL2 retains its inhibitory activity inS. cerevisiaespheroplasts, thus providing a unique tool for acute and reversible CME modulation in yeast.
Species and strain cultivation of skin, oral, and gut microbiota
Authors: Fleming Elizabeth , Pabst Victor , Hoyt Amelia , Zhou Wei , Hardy Rachel , Peterson Anna , Beach Ryan , Ondouah-Nzutchi Yvette , Dong Jinhong , Oh Julia 
Published: April 2021

ABSTRACTGenomics-driven discovery of microbial species have provided extraordinary insights into the biodiversity of human microbiota. High resolution genomics to investigate species- and strain-level diversity and mechanistic studies, however, rely on the availability of individual microbes from a complex microbial consortia. Here, we describe and validate a streamlined workflow for cultivating microbes from the skin, oral, and gut microbiota, informed by metagenomic sequencing, mass spectrometry, and strain profiling.
Callosal fiber length scales with brain size according to functional lateralization, evolution, and development
Authors: Yang Liyuan , Zhao Chenxi , Xiong Yirong , Zhong Suyu , Wu Di , Peng Shaoling , Thiebaut de Schotten Michel , Gong Gaolang 
Published: April 2021

AbstractBrain size significantly impacts the organization of white matter fibers. Fiber length scaling – the degree to which fiber length varies according to brain size – was overlooked. We investigated how fiber lengths within the corpus callosum, the most prominent white matter tract, vary according to brain size. The results showed substantial variation in length scaling among callosal fibers, replicated in two large healthy cohorts (∼2000 individuals). The underscaled callosal fibers mainly connected the precentral gyrus and parietal cortices, whereas the overscaled callosal fibers mainly connected the prefrontal cortices. The variation in such length scaling was biologically meaningful: larger scaling corresponded to larger neurite density index but smaller fractional anisotropy values; cortical regions connected by the callosal fibers with larger scaling were more lateralized functionally as well as phylogenetically and ontogenetically more recent than their counterparts. These findings highlight an interaction between interhemispheric communication and organizational and adaptive principles underlying brain development and evolution.Significance StatementBrain size varies across evolution, development, and individuals. Relative to small brains, the neural fiber length in large brains is inevitably increased, but the degree of such increase may differ between fiber tracts. Such a difference, if it exists, is valuable for understanding adaptive neural principles in large versus small brains during evolution and development. The present study showed a substantial difference in the length increase between the callosal fibers that connect the two hemispheres, replicated in two large healthy cohorts. Altogether, our study demonstrates that reorganization of interhemispheric fibers length according to brain size is intrinsically related to fiber composition, functional lateralization, cortical myelin content, evolutionary and developmental expansion.