bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of m6A residues at single-nucleotide resolution using eCLIP and an accessible custom analysis pipeline
Authors: Roberts Justin T. , Porman Allison M. , Johnson Aaron M. 
Published: March 2020

AbstractMethylation at the N6position of adenosine (m6A) is one of the most abundant RNA modifications found in eukaryotes, however accurate detection of specific m6A nucleotides within transcripts has been historically challenging due to m6A and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-Seq) have relied on m6A-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual m6A residues. More recently, modified cross-linking and immunoprecipitation (CLIP) based approaches that rely on inducing specific mutations during reverse transcription via UV crosslinking of the anti-m6A antibody to methylated RNA have been employed to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual m6A residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our m6A-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of m6A sites at true single nucleotide resolution. As the effort to accurately identify m6As in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the m6A epitranscriptome.
Autocrine IL11 cis-signaling in hepatocytes is an initiating nexus between lipotoxicity and non-alcoholic steatohepatitis
Authors: Dong Jinrui , Adami Eleonora , Chothani Sonia P. , Viswanathan Sivakumar , Ng Benjamin , Lim Wei Wen , Singh Brijesh K. , Zhou Jin , Ko Nicole SJ. , Shekeran Shamini G. , Tan Jessie , Lim Sze Yun , Wang Mao , Lio Pei Min , Yen Paul M. , Schafer Sebastian , Cook Stuart A. , Widjaja Anissa A. 
Published: March 2020

AbstractBackground and aimsIL11 signaling is important in non-alcoholic steatohepatitis (NASH) but how it contributes to NASH pathologies beyond fibrosis is not known. Here we investigate the role of IL11 signaling in hepatocyte lipotoxicity.MethodsHepatocytes were stimulated with IL6, IL11, HyperIL6, or HyperIL11 alone or in the presence of soluble gp130 (sgp130) or soluble IL11RA (sIL11RA), or loaded with palmitate in the presence of IgG or anti-IL11RA (X209) antibodies or sgp130. Effects were assessed using colorimetric ALT, GSH, or ELISA assays, immunoblots, and flow cytometry. The relative contributions of IL11cis-versus -transsignalingin vivowas assessed in two preclinical NASH models using a high fat methionine/choline deficient diet or a Western diet with liquid fructose in C57BL6/Ntac mice injected with AAV8-Alb-Cre, AAV8-Alb-sgp130, in mice with hepatocyte-specific deletion ofIl11ra(CKO), and in mice with global deletion ofIl11rainjected with AAV8-Alb-mIl11ra or AAV8-Alb-sIl11ra. Livers and serum were collected; serum samples were analyzed using biochemistry and liver tissues were analyzed by histology, qPCR, immunobloting, hydroxyproline, and GSH assays.ResultsWe show that lipid-laden hepatocytes secrete IL11, which acts via autocrinecis-signaling to cause lipoapoptosis. IL11 causes lipotoxic hepatocyte death through activation of non-canonical signaling pathways and increased NOX4-derived reactive oxygen species. In two preclinical models, hepatocyte-specific deletion ofIl11ra1protects mice from all aspects of NASH with beneficial effects on body weight. In accordance, restoration of IL11cis-signaling in hepatocytes only in mice globally deleted forIl11ra1reconstitutes steatosis and inflammation. Throughout, we found no evidence to support the existence of IL6 or IL11trans-signaling in the liver.ConclusionWe conclude that autocrine IL11-mediated cell death underlies hepatocyte lipotoxicity and that liver fibrosis and inflammation occur subsequently. These data highlight a new disease mechanism for the transition from compensated fatty liver disease to NASH.
PSP2, a gene encoding RGG-motif protein, is a novel suppressor of clathrin heavy chain deficiency
Authors: Roy Debadrita , Garg Mani , Rajyaguru Purusharth I 
Published: March 2020

AbstractClathrin, made up of the heavy- and light-chains, constitutes one of the most abundant protein in vesicles involved in intracellular protein trafficking and endocytosis.YPR129W, which encodes RGG-motif containing translation repressor was identified as a part of multi-gene construct (SCD6) that suppressed clathrin deficiency. However, the contribution ofYPR129Walone in suppressing clathrin deficiency has not been documented. In this study we identifyYPR129Was a necessary and sufficient gene in a multigene construct SCD6 that suppresses clathrin deficiency. Importantly, we identify cytoplasmic RGG-motif protein encoding genePSP2as a novel suppressor of clathrin deficiency. Three other RGG-motif protein encoding genesSBP1, DED1andGBP2do not suppress clathrin deficiency.DHH1, a DEAD-box RNA helicase with translation repression activity also fails to rescue clathrin deficiency. α-factor secretion assay suggests that suppression of clathrin deficiency bySCD6andPSP2is not mediated by the rescue of the trans-Golgi network (TGN) protein sorting defect observed in the absence ofCHC1. Detailed domain analysis of the two suppressors reveals that the RGG-motif of both Scd6 and Psp2 is important for suppressing clathrin deficiency. Additionally, the Lsm domain deletion as well as the arginine to alanine mutation in the arginine methylation defective (AMD) mutant render Scd6 defective in suppressing clathrin deficiency. Overall based on our results usingSCD6andPSP2proteins, we identify a novel role of RGG-motif in suppressing clathrin deficiency. Since both the suppressors are RNA-binding granule-resident proteins, this study opens an exciting avenue for exploring the connection between clathrin function and cytoplasmic RNA metabolism.
Revealing the polarity of actin filaments by cryo-electron tomography
Authors: Martins Bruno , Sorrentino Simona , Chung Wen-Lu , Tatli Meltem , Medalia Ohad , Eibauer Matthias 
Published: March 2020

SummaryThe actin cytoskeleton plays a fundamental role in numerous cellular processes, such as cell motility, cytokinesis, and adhesion to the extracellular matrix. Revealing the polarity of individual actin filaments in cells, would foster an unprecedented understanding of cytoskeletal processes and their associated mechanical forces. Cryo-electron tomography provides the means for high-resolution structural imaging of cells. However, the low signal-to-noise ratio of cryo-tomograms obscures the high frequencies and therefore the polarity of actin filaments cannot be directly measured. Here, we developed an approach that enables to determine the polarity of actin filaments in cellular cryo-tomograms. We applied it to reveal the actin polarity distribution in focal adhesions, and show a linear relation between actin polarity and distance from the apical boundary of the adhesion site.
Extracellular Ca2+-sensitive fluorescent protein biosensor based on a collagen-binding domain
Authors: Okkelman Irina A. , McGarrigle Ryan , O’Carroll Shane , Berrio Daniel Carvajal , Schenke-Layland Katja , Hynes James , Dmitriev Ruslan I. 
Published: March 2020

AbstractThe importance of extracellular gradients of biomolecules becomes increasingly appreciated in the processes of tissue development and regeneration, in health and disease. In particular, dynamics of extracellular calcium concentration is rarely studied. Here, we present low affinity Ca2+biosensor based on Twitch-2B fluorescent protein fused with the cellulose- and collagen-binding peptides. These recombinant chimeric proteins can bind cellulose and collagen scaffolds and enable for scaffold-based biosensing of Ca2+in proximity of live 3D tissue models. We found that the Twitch-2B mutant is compatible with intensity-based ratiometric and fluorescence lifetime imaging microscopy (FLIM) measurement formats, under one- and two-photon excitation modes. Furthermore, the donor fluorescence lifetime of ColBD-Twitch displays response to [Ca2+] over a range of ∼2-2.5 ns, making it attractive biosensor for multiplexed FLIM microscopy assays. To evaluate performance of this biosensor in physiological measurements, we applied ColBD-Twitch to the live Lgr5-GFP mouse intestinal organoid culture and measured its responses to the changes in extracellular Ca2+upon chelation with EGTA. When we combined it with spectrally resolved FLIM of lipid droplets using Nile Red dye, we observed changes in cytoplasmic and basal membrane-associated lipid droplet composition in response to the extracellular Ca2+depletion, suggesting that intestinal epithelium can respond to and compensate such treatment. Altogether, our results demonstrate ColBD-Twitch as a prospective Ca2+sensor for multiplexed FLIM analysis in a complex 3D tissue environment.
Plasticity as an Underlying Mechanism of Tumor Heterogeneity in Breast Cancer
Authors: Akbar Muhammad Waqas , Isbilen Murat , Kucukkaraduman Baris , Canli Secil Demirkol , Dedeoglu Ege , Azizolli Shila , Cela Isli , Akcay Abbas Guven , Hakanoglu Hasim , Gure Ali Osmay 
Published: March 2020

AbstractBreast cancer shows plasticity in terms of classification. Upon drug treatment and metastasis some tumors switch to another subtype leading to loss of response to therapy. In this study, we ask the question which molecular subclasses of breast cancer are more switchable upon drug therapy and metastasis. We used in silico data to classify breast cancer tumors in PAM50 molecular classes before treatment and after treatment using gene expression data. Similar analysis was performed for primary tumors and their metastatic growth. Our analysis showed that in both scenarios some breast tumors shift from one class to another. This suggests that patients who underwent chemotherapy but resulted in relapse or/and metastasis should be retyped for molecular subclass so that treatment protocol should be adopted according to those subtypes. Additionally, 20 genes were identified as biomarkers for metastasis in breast cancer.
Molecular evolution and developmental expression of melanin pathway genes in Lepidoptera
Authors: Kuwalekar Muktai , Deshmukh Riddhi , Padvi Ajay , Kunte Krushnamegh 
Published: March 2020

ABSTRACTPigmentation is involved in a wide array of biological functions across insect orders, including body patterning, thermoregulation, and immunity. The melanin pathway, in particular, has been characterized in several species. However, molecular evolution of the genes involved in this pathway is poorly characterized, and their roles in pigmentation of early developmental stages are just beginning to be explored in non-model organisms. We traced the molecular evolution of six melanin pathway genes in 53 species of Lepidoptera covering butterflies and moths, and representing over 100 million years of diversification. We compared the rates of synonymous and nonsynonymous substitutions within and between these genes to study signatures of selection at the level of individual sites, genes, and branches of the gene tree. We found that molecular evolution of all six genes was governed by strong purifying selection. Yet, a number of sites showed signs of being under positive selection, including in the highly conserved domain regions of three genes. Further, we traced the expression of these genes across developmental stages, tissues, and sexes in thePapilio polytesbutterfly using a developmental transcriptome dataset. We observed that the expression patterns of the genes inP. polyteslargely reflected their known tissue-specific function in other species. The expression of sequentially acting genes in the melanin pathway was correlated. Interestingly, four out of six melanin pathway genes (ebony, pale, aaNAT, andDDC) showed a sexually dimorphic pattern of developmental heterochrony; i.e., females showed peak activity much earlier in pupal development compared to that of males. Our evolutionary and developmental analyses suggest that the vast diversity of wing patterning and pigmentation in Lepidoptera may have been aided largely by differential developmental regulation of genes in a highly conserved pathway, in which the sequence evolution of individual genes is highly constrained.
Disruption of oncogenic targeting by ISWI via phosphorylation of a prion-like domain
Authors: Chen Mark , Foster Joseph P. , Lock Ian C. , Leisenring Nathan H. , Daniel Andrea R. , Floyd Warren , Xu Eric S. , Davis Ian J. , Kirsch David G. 
Published: March 2020

SummaryChromosomal translocations generate oncogenic fusion proteins in approximately one-third of sarcomas, but how these proteins promote tumorigenesis and the effect of cancer therapies on their function are not well understood. Here, we reveal a molecular mechanism by which the fusion oncoprotein FUS-CHOP promotes tumor maintenance that also explains the remarkable radiation sensitivity of myxoid liposarcomas. We identified novel interactions between FUS-CHOP and chromatin remodeling complexes that regulate sarcoma cell proliferation. One of these chromatin remodelers, SNF2H, co-localizes with FUS-CHOP genome-wide at active enhancers. Following ionizing radiation, DNA damage response kinases phosphorylate the prion-like domain of FUS-CHOP to impede these protein-protein interactions, which are required for transformation. Therefore, the DNA damage response after irradiation disrupts oncogenic targeting of chromatin remodelers required for FUS-CHOP-driven sarcomagenesis.SignificancePrion-like domains translocated in cancer have been shown to drive global epigenetic changes that are oncogenic. However, some translocation-driven cancers exhibit dramatic clinical responses to therapy, though the mechanism for these responses are not well-understood. Here we show that ionizing radiation can disrupt oncogenic interactions between a fusion oncoprotein and a chromatin remodeling complex, ISWI. This mechanism of disruption links phosphorylation of the prion-like domain in an oncogenic fusion protein to DNA damage after ionizing radiation and reveals that a dependence on oncogenic chromatin remodeling underlies sensitivity to radiation therapy in myxoid liposarcoma.
A bottom-up approach for thede novodesign of functional proteins
Authors: Yang Che , Sesterhenn Fabian , Bonet Jaume , van Aalen Eva , Scheller Leo , Abriata Luciano A , Cramer Johannes T , Wen Xiaolin , Rosset Stéphane , Georgeon Sandrine , Jardetzky Theodore , Krey Thomas , Fussenegger Martin , Merkx Maarten , Correia Bruno E 
Published: March 2020

AbstractDe novoprotein design has enabled the creation of novel protein structures. To design novel functional proteins, state-of-the-art approaches use natural proteins or first design protein scaffolds that subsequently serve as templates for the transplantation of functional motifs. In these approaches, the templates are function-agnostic and motifs have been limited to those with regular secondary structure. Here, we present a bottom-up approach to buildde novoproteins tailored to structurally complex functional motifs. We applied a bottom-up strategy to design scaffolds for four different binding motifs, including one bi-functionalized protein with two motifs. Thede novoproteins were functional as biosensors to quantify epitope-specific antibody responses and as orthogonal ligands to activate a signaling pathway in engineered mammalian cells. Altogether, we present a versatile strategy for the bottom-up design of functional proteins, applicable to a wide range of functional protein design challenges.
Powerful detection of polygenic selection and evidence of environmental adaptation in US beef cattle
Authors: Rowan Troy N. , Durbin Harly J. , Seabury Christopher M. , Schnabel Robert D. , Decker Jared E. 
Published: March 2020

AbstractSelection on complex traits can rapidly drive evolution, especially in stressful environments. This polygenic selection does not leave intense sweep signatures on the genome, rather many loci experience small allele frequency shifts, resulting in large cumulative phenotypic changes. Directional selection and local adaptation are actively changing populations; but, identifying loci underlying polygenic or environmental selection has been difficult. We use genomic data on tens of thousands of cattle from three populations, distributed over time and landscapes, in linear mixed models with novel dependent variables to map signatures of selection on complex traits and local adaptation. We identify 207 genomic loci associated with an animal’s birth date, representing ongoing selection for monogenic and polygenic traits. Additionally, hundreds of additional loci are associated with continuous and discrete environments, providing evidence for local adaptation. These candidate loci highlight the nervous system’s possible role in local adaptation. While advanced technologies have increased the rate of directional selection in cattle, it has likely been at the expense of historically generated local adaptation, which is especially problematic in changing climates. When applied to large, diverse cattle datasets, these selection mapping methods provide an insight into how selection on complex traits continually shapes the genome. Further, by understanding the genomic loci implicated in adaptation, may help us breed more adapted and efficient cattle and begin understanding the basis for mammalian adaptation, especially in changing climates. These selection mapping approaches help clarify selective forces and loci in evolutionary, model, and agricultural contexts.Author SummaryInterest in mapping the impacts of selection and local adaptation on the genome is increasing due to the novel stressors presented by climate change. Until now, approaches have largely focused on mapping “sweeps” on large-effect loci. Highly powered datasets that are both temporally and geographically distributed have not existed. Recently, large numbers of beef cattle have been genotyped across the United States, including influential individuals with cryopreserved semen. This has created multiple powerful datasets distributed over time and landscapes. Here, we map the recent effects of selection and local adaptation in three cattle populations. The results provide insight into the biology of mammalian adaptation and generate useful tools for selecting and breeding better-adapted cattle for a changing environment.
Structural basis for capsid recruitment and coat formation during HSV-1 nuclear egress
Authors: Draganova Elizabeth B. , Zhang Jiayan , Zhou Z. Hong , Heldwein Ekaterina E. 
Published: March 2020

AbstractDuring herpesvirus infection, egress of nascent viral capsids from the nucleus is mediated by the viral nuclear egress complex (NEC). NEC deforms the inner nuclear membrane (INM) around the capsid by forming a hexagonal array. However, how the NEC coat interacts with the capsid and how curved coats are generated to enable budding is yet unclear. Here, by structure-guided truncations, confocal microscopy, and cryoelectron tomography, we show that binding of the capsid protein UL25 promotes the formation of NEC pentagons rather than hexagons. We hypothesize that during nuclear budding, binding of UL25 situated at the pentagonal capsid vertices to the NEC at the INM promotes formation of NEC pentagons that would anchor the NEC coat to the capsid. Incorporation of NEC pentagons at the points of contact with the vertices would also promote assembly of the curved hexagonal NEC coat around the capsid, leading to productive egress of UL25-decorated capsids.
Three-dimensional genome structure and chromatin accessibility reorganization duringin vivoinduction of human T cell tolerance
Authors: Li Ruifeng , Guo Huidong , Hou Yingping , Liu Shuoshuo , Wang Ming , Peng Ting , Zhao Xiang-Yu , Lu Liming , Han Yali , Shao Yiming , Chang Ying-Jun , Li Cheng , Huang Xiao-Jun 
Published: March 2020

SUMMARYAchieving T cell tolerance is a pivotal goal for the field of transplantation and autoimmune diseases. Here, we characterized the gene expression profiles, 3D genome architecture and chromatin accessibility in human steady-state and tolerant T cells, which had been induced in healthy donors by granulocyte-colony-stimulating factorin vivo. We provided the first high-resolution 3D genomic landscape of human tolerant T cellsin vivoand identified highly expressed suppressor of cytokine signaling 1 (SOCS1), which is essential for maintaining T cell tolerance and was validated byex vivoexperiments. Mechanistically, SOCS1 is activated by STAT3, which mediates a new interaction between theSOCS1locus and downstream super-enhancers and is accompanied by the disruption of the CTCF loop between theSOCS1locus and upstream heterochromatin. This competitive regulation pattern between STAT3 and CTCF is present in the whole genome. Our study defines a regulatory model of transcription factors and provides insight into the induction of immune tolerance.
ProNetView-ccRCC: A web-based portal to interactively explore clear cell renal cell carcinoma proteogenomics networks
Authors: Kalayci Selim , Petralia Francesca , Wang Pei , Gümüş Zeynep H. 
Published: March 2020

ABSTRACTTo better understand the molecular basis of cancer, the NCI’s Clinical Proteomics Tumor Analysis Consortium (CPTAC) has been performing comprehensive large-scale proteogenomic characterizations of multiple cancer types. Gene/protein regulatory networks has subsequently been derived based on these proteogenomic profiles, which serve as useful tools to gain system-level understanding of the molecular regulatory factories underlying the diseases. On the other hand, it remains a challenge to effectively visualize and navigate the resulting network models, which capture higher order structures in the proteogenomic profiles. There is a pressing need to have a new open community resource tool for intuitive visual exploration, interpretation and communication of these gene/protein regulatory networks by the cancer research community. In this work, we introduce ProNetView-ccRCC (http://ccrcc.cptac-network-view.org/), an interactive web-based network exploration portal for investigating phosphopeptide co-expression network inferred based on the CPTAC clear cell renal cell carcinoma (ccRCC) phosphoproteomics data. ProNetView-ccRCC enables quick, user-intuitive visual interactions with the ccRCC tumor phosphoprotein co-expression network comprised of 3,614 genes, as well as 30 functional pathway-enriched network modules. Users can interact with the network portal and can conveniently query for association between abundance of each phosphopeptide in the network and clinical variables such as tumor grade.