bluuebunny/crossref_metadata_2025_split huggingface dataset:
A mechanistic model of the BLADE platform predicts performance characteristics of 256 different synthetic DNA recombination circuits
Authors: Bowyer Jack E. , Ding Chloe , Weinberg Benjamin H. , Wong Wilson W. , Bates Declan G. 
Published: April 2020

AbstractBoolean logic and arithmetic through DNA excision (BLADE) is a recently developed platform for implementing inducible and logical control over gene expression in mammalian cells, which has the potential to revolutionise cell engineering for therapeutic applications. This 2-input 2-output platform can implement 256 different logical circuits that exploit the specificity and stability of DNA recombination. Here, we develop the first mechanistic mathematical model of the 2-input BLADE platform based on Cre- and Flp-mediated DNA excision. After calibrating the model on experimental data from two circuits, we demonstrate close agreement between model outputs and data on the other 111 circuits that have so far been experimentally constructed using the 2-input BLADE platform. Model simulations of the remaining 143 circuits that have yet to be tested experimentally predict excellent performance of the 2-input BLADE platform across the range of possible circuits. Circuits from both the tested and untested subsets that perform less well consist of a disproportionally high number of STOP sequences. Model predictions suggested that circuit performance declines with a decrease in recombinase expression and new experimental data was generated that confirms this relationship.
Synaptic transmission induces site-specific changes in sialylation onN-linked glycoproteins in rat nerve terminals
Authors: Boll Inga , Jensen Pia , Schwämmle Veit , Larsen Martin R. 
Published: April 2020

AbstractSynaptic transmission leading to release of neurotransmitters in the nervous system is a fast and highly dynamic process. Previously, protein interaction and phosphorylation have been thought to be the main regulators of synaptic transmission. Here we show a novel potential modulator of synaptic transmission, sialylation ofN-linked glycosylation. The negatively charged sialic acids can be modulated, similarly to phosphorylation, by the action of sialyltransferases and sialidases thereby changing local structure and function of membrane glycoproteins. We characterized site-specific alteration in sialylation onN-linked glycoproteins in isolated rat nerve terminals after brief depolarization using quantitative sialiomics. We identified 1965 formerly sialylatedN-linked glycosites in synaptic proteins and found that the abundances of 430 glycosites changed after five seconds depolarization. We observed changes on essential synaptic proteins such as synaptic vesicle proteins, ion channels and transporters, neurotransmitter receptors and cell adhesion molecules. This study is to our knowledge the first to describe ultra-fast site-specific modulation of the sialiome after brief stimulation of a biological system.
The dynamic Nexus: Gap junctions control protein localization and mobility in distinct and surprising ways
Authors: McCutcheon Sean , Stout Randy F. , Spray David C. 
Published: April 2020

AbstractGap junction (GJ) channels permit molecules, such as ions, metabolites and second messengers, to transfer between cells. Their function is critical for numerous cellular interactions. GJ channels are composed of Connexin (Cx) hexamers paired across extracellular space and typically form large rafts of clustered channels, called plaques, at cell appositions. Cxs together with molecules that interact with GJ channels make up a supramolecular structure known as the GJ Nexus. While the stability of connexin localization in GJ plaques has been studied, mobility of other Nexus components has yet to be addressed. Colocalization analysis of several nexus components and other membrane proteins reveal that certain molecules are excluded from the GJ plaque (Aquaporin 4, EAAT2b), while others are quite penetrant (lipophilic molecules, Cx30, ZO-1, Occludin). Fluorescence recovery after photobleaching (FRAP) of tagged Nexus-associated proteins showed that mobility in plaque domains is affected by mobility of the Cx proteins. These novel findings indicate that the GJ Nexus is a dynamic membrane organelle, with cytoplasmic and membrane-embedded proteins binding and diffusing according to distinct parameters.Summary StatementGap junctions are clustered membrane channels in plasma membrane of astrocytes and other cells. We report new information on how gap junctions control location and mobility of other astrocyte proteins.
CryoEM Structure of CtBP2 Confirms Tetrameric Architecture
Authors: Jecrois Anne M. , Dcona M. Michael , Deng Xiaovan , Bandyopadhyay Dipankar , Grossman Steven R. , Schiffer Celia A. , Royer William E. 
Published: April 2020

AbstractC-terminal binding proteins 1 and 2 (CtBP1 and CtBP2) are transcriptional regulators that activate or repress many genes involved in cellular development, apoptosis and metastasis. CtBP proteins are activated under hypoxic conditions where NAD(H) levels tend to be higher. NADH-dependent activation of CtBP2 has direct implication in multiple types of cancers and poor patient prognosis. Previous studies have proposed dimeric CtBP as the relevant oligomeric state, however our studies with multi-angle light scattering have shown that the primary effect of NADH binding is to promote the assembly of two CtBP dimers into tetramers. Here, we present the cryoEM structures of two different constructs of CtBP2 corroborating that the native state of CtBP2 in the presence of NADH is indeed tetrameric. The physiological relevance of tetrameric CtBP2 was tested in HCT116; CtBP2 −/− cells transfected with tetramer destabilizing_mutants. Mutants that inhibit tetramer formation show a decrease in expression of the CtBP transcriptional target TIAM1 and exhibit a decrease in the ability to promote cell migration. Together with our cryoEM studies, these results highlight the tetramer as the functional oligomeric form of CtBP2.
Analysis of stress responses inAstyanaxlarvae reveals heterogeneity among different populations
Authors: Chin Jacqueline SR , Loomis Cody L. , Albert Lydia T. , Medina-Trenche Shirley , Kowalko Johanna , Keene Alex C. , Duboué Erik R. 
Published: April 2020

AbstractStress responses are conserved physiological and behavioral outcomes as a result of facing potentially harmful stimuli, yet in pathological states, stress becomes debilitating. Stress responses vary considerably throughout the animal kingdom, but how these responses are shaped evolutionarily is unknown. The Mexican cavefish has emerged as a powerful system for examining genetic principles underlying behavioral evolution. Here, we demonstrate that caveAstyanaxhave reduced behavioral and physiological measures of stress when examined at larval stages. We also find increased expression of the glucocorticoid receptor, a repressible element of the neuroendocrine stress pathway. Additionally, we examine stress in three different cave populations, and find that some, but not all, show reduced stress measures. Together, these results reveal a mechanistic system by which cave-dwelling fish reduced stress, presumably to compensate for a predator poor environment.Research HighlightCavefish populations ofA. mexicanushave reduced stress relative to surface conspecific at larval stagesWe show that a glucocorticoid receptor, a negative regulator of the neuroendocrine stress axis, is upregulated in stress-resistant cavefishThere exists much ontological heterogeneity between different cavefish populations.
Deep learning models for lipid-nanoparticle-based drug delivery
Authors: Harrison Philip J , Wieslander Håkan , Sabirsh Alan , Karlsson Johan , Malmsjö Victor , Hellander Andreas , Wählby Carolina , Spjuth Ola 
Published: April 2020

AbstractLarge-scale time-lapse microscopy experiments are useful to understand delivery and expression in RNA-based therapeutics. The resulting data has high dimensionality and high (but sparse) information content, making it challenging and costly to store and process. Early prediction of experimental outcome enables intelligent data management and decision making. We start from time-lapse data of HepG2 cells exposed to lipid-nanoparticles loaded with mRNA for expression of green fluorescent protein (GFP). We hypothesize that it is possible to predict if a cell will express GFP or not based on cell morphology at time-points prior to GFP expression. Here we present results on per-cell classification (GFP expression/no GFP expression) and regression (level of GFP expression) using three different approaches. In the first approach we use a convolutional neural network extracting per-cell features at each time point. We then utilize the same features combined with: a long-short-term memory (LSTM) network encoding temporal dynamics (approach 2); and time-series feature extraction using the python package tsfresh followed by principal component analysis and gradient boosting machines (approach 3), to reach a final classification or regression result. Application of the three approaches to a previously unanalyzed test set of cells showed good predictive performance of all three approaches but that accounting for the temporal dynamics via LSTMs or tsfresh led to significantly improved performance. The predictions made by the LSTM and tsfresh applications were not significantly different. The results highlight the benefit of accounting for temporal dynamics when studying drug delivery using high content imaging.
Insights into calcium signaling and gene expression in astrocytes uncovered with 129S4 Slc1a3-2A-CreERT2 knock-in mice
Authors: Kaczmarczyk Lech , Reichenbach Nicole , Blank Nelli , Jonson Maria , Dittrich Lars , Petzold Gabor C. , Jackson Walker 
Published: April 2020

AbstractGenetic variation is a primary determinant of phenotypic diversity within populations. In laboratory mice, genetic variation has often been regarded as a serious experimental confounder, and thus minimized through inbreeding. However, generalizations of results obtained with inbred strains need to be made with caution. Effects of genetic background on traits need to be controlled, especially when working with complex phenotypes and disease models. Here we compared behavioral parameters of C57Bl/6 – the mouse strain most widely used for biomedical research - with those of 129S4. Our data demonstrate high within-strain and intra-litter behavioral hyperactivity in C57Bl/6. In contrast, 129S4 had relatively consistent activity levels throughout life. This consistency would be advantageous for studying neurodegeneration and aging, when mice need to be analyzed for long periods. However, the majority of mouse models and transgenic tools are on a C57Bl/6 background. We recently established six popular Cre driver lines and two Cre effector lines in 129S4. To augment this collection, we genetically engineered a Cre mouse line to study astrocytes directly in 129S4, which we describe here. For functional validation, it was crossed with two Cre effector lines, each in a different genomic locus, and showed in both cases that it was functional and astrocyte-specific. Calcium currents studied with gCaMP5g-tdTomato were more heterogenous, lasted longer and had a higher amplitude in cortical compared to hippocampal astrocytes. Translatomes studied with RiboTag revealed that some genes thought to mark neurons are also expressed in astrocytes, that genes linked to a single neurodegenerative disease have highly divergent expression patterns, and that ribosome proteins are non-uniformly expressed across brain regions and cell types.
Larger active site in an ancestral hydroxynitrile lyase increases catalytically promiscuous esterase activity
Authors: Jones Bryan J. , Evans Robert L. , Mylrea Nathan J. , Chaudhury Debayan , Luo Christine , Guan Bo , Pierce Colin T. , Gordon Wendy R. , Wilmot Carrie M. , Kazlauskas Romas J. 
Published: April 2020

AbstractHydroxynitrile lyases (HNL’s) belonging to the α/β-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the α/β-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared toHbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 °C higher than forHbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-Å resolution shows the expected α/β-hydrolase fold, but a 60% larger active site as compared toHbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site thanHbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis ofHbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely thanHbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/ mol·M forHbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.
Mitochondrial Aurora kinase A induces mitophagy by interacting with MAP1LC3 and Prohibitin 2
Authors: Bertolin Giulia , Alves-Guerra Marie-Clotilde , Cheron Angélique , Burel Agnès , Prigent Claude , Borgne Roland Le , Tramier Marc 
Published: April 2020

AbstractEpithelial and haematologic tumours often show the overexpression of the serine/threonine kinase AURKA. Recently, AURKA was shown to localise at mitochondria, where it regulates mitochondrial dynamics and ATP production. Here we define the molecular mechanisms of AURKA in regulating mitochondrial turnover by mitophagy. When overexpressed, AURKA induces the disappearance of Outer Mitochondrial Membrane proteins by the proteasome. Then, AURKA triggers the degradation of Inner Mitochondrial Membrane (IMM)/matrix proteins by interacting with core components of the autophagy pathway. On the IMM, the kinase forms a tripartite complex with MAP1LC3 and the mitophagy receptor PHB2. This complex is necessary to trigger mitophagy in a PARK2/Parkin-independent manner. The formation of the tripartite complex is induced by the phosphorylation of PHB2 on Ser39, which is required for MAP1LC3 to interact with PHB2. Last, treatment with the PHB2 ligand Xanthohumol blocks AURKA-induced mitophagy by destabilising the tripartite complex. This treatment also restores normal ATP production levels. Altogether, these data provide evidence for a previously undetected role of AURKA in promoting mitophagy through the interaction with PHB2 and MAP1LC3. This work paves the way to the use of function-specific pharmacological inhibitors to counteract the effects of the overexpression of AURKA in cancer.
Broad geographical and ecological diversity from similar genomic toolkits in the ascomycete genusTetracladium
Authors: Anderson Jennifer L. , Marvanová Ludmila 
Published: April 2020

ABSTRACTThe ascomycete genusTetracladiumis best known for containing aquatic hyphomycetes, which are important decomposers in stream food webs. However, some species ofTetracladiumare thought to be multifunctional and are also endobionts in plants. Suprisingly,Tetracladiumsequences are increasingly being reported from metagenomics and metabarcoding studies of both plants and soils world-wide. It is not clear how these sequences are related to the described species and little is known about the non-aquatic biology of these fungi. Here, the genomes of 24Tetracladiumstrains, including all described species, were sequenced and used to resolve relationships among taxa and to improve our understanding of ecological and genomic diversity in this group. All genome-sequencedTetracladiumfungi form a monophyletic group. Conspecific strains ofT. furcatumfrom both aquatic saprotrophic and endobiont lifestyles and a putative cold-adapted clade are identified. Analysis of ITS sequences from water, soil, and plants from around the world reveals that multifunctionality may be widespread through the genus. Further, frequent reports of these fungi from extreme environments suggest they may have important but unknown roles in those ecosystems. Patterns of predicted carbohydrate active enzymes (CAZyme) and secondary metabolites in theTetracladiumgenomes are more similar to each other than to other ascomycetes, regardless of ecology, suggesting a strong role for phylogeny shaping genome content in the genus.Tetracladiumgenomes are enriched for pectate lyase domains (including PL3-2), GH71α-1,3-glucanase domains and CBM24α-1,3-glucan/mutan binding modules, and both GH32 and CBM38, inulinase and inulin binding modules. These results indicate that these fungi are well-suited to digesting pectate and pectin in leaves when living as aquatic hyphomycetes, and inulin when living as root endobionts. Enrichment forα-1,3-glucanase domains may be associated with interactions with biofilm forming microorganisms in root and submerged leaf environments.
Distance-based metrics for comparing conformational ensembles of intrinsically disordered proteins
Authors: Lazar Tamas , Guharoy Mainak , Vranken Wim , Rauscher Sarah , Wodak Shoshana J. , Tompa Peter 
Published: April 2020

AbstractIntrinsically disordered proteins (IDPs) are proteins whose native functional states represent ensembles of highly diverse conformations. Such ensembles are a challenge for quantitative structure comparisons as their conformational diversity precludes optimal superimposition of the atomic coordinates, necessary for deriving common similarity measures such as the root-mean-square deviation (RMSD) of these coordinates. Here we introduce superimposition-free metrics, which are based on computing matrices of Cα-Cα distance distributions within ensembles and comparing these matrices between ensembles. Differences between two matrices yield information on the similarity between specific regions of the polypeptide, whereas the global structural similarity is captured by the ens_dRMS, defined as the root-mean-square difference between the medians of the Cα-Cαdistance distributions of two ensembles. Together, our metrics enable rigorous investigations of structure-function relationships in conformational ensembles of IDPs derived using experimental restraints or by molecular simulations, and for proteins containing both structured and disordered regions.Statement of SignificanceImportant biological insight is obtained from comparing the high-resolution structures of proteins. Such comparisons commonly involve superimposing two protein structures and computing the residual root-mean-square deviation of the atomic positions. This approach cannot be applied to intrinsically disordered proteins (IDPs) because IDPs do not adopt well-defined 3D structures, rather, their native functional state is defined by ensembles of heterogeneous conformations that cannot be meaningfully superimposed. We report new measures that quantify the local and global similarity between different conformational ensembles by evaluating differences between the distributions of residue-residue distances and their statistical significance. Applying these measures to IDP ensembles and to a protein containing both structured and intrinsically disordered domains provides deeper insights into how structural features relate to function.
Testing the Effect of the Toba Volcanic Eruption on Population Sizes in Worldwide Mammal Species
Authors: Torosin Nicole S. , Raff Jennifer A. , Hayes M. Geoffrey 
Published: April 2020

AbstractThe volcanic eruption of Toba in northern Sumatra at 71 kyBP (±5 kyBP) emitted sulfur gas and deposited thick layers of dust throughout the surrounding region. It is thought to have had a significant and dramatic cooling impact on the paleoclimate worldwide. Ambrose [1] conjectured this to be the cause of the contemporaneous (50-100 kyBP) population bottleneck observed in humans. We hypothesize that a volcanic winter of sufficient magnitude to cause a population bottleneck in humans would similarly affect other mammals. To test this hypothesis, we estimated pairwise mismatch distributions using mtDNA control region sequences of 28 mammal species archived on NCBI to assess whether each species underwent a population bottleneck. For any species fitting the sudden expansion model, we estimated the timing of the bottleneck and compared it to the date range of the Toba eruption. Only 3 of the 28 species show evidence of rapid population expansion overlapping in time with the Toba eruption. Therefore, the hypothesis that the volcanic winter triggered by the Toba eruption caused a significant bottleneck impacting mammal species worldwide is not supported by mitochondrial evidence. Our results question the hypothesis that the Toba eruption contributed to the bottleneck observed in humans at this time.
A Non-Destructive Method for Quantifying Tissue Vascularity Using Quantitative Deep Learning Image Processing
Authors: Veith Austin , Baker Aaron B. 
Published: April 2020

AbstractThe quantitative analysis of blood vessel networks is an important component in the understanding and analysis of vascular disease, ischemia, cancer and many other disease states. However, many imaging techniques used for imaging vascular networks are time consuming, prevent further analyses or are technically challenging. Here, we describe a nondestructive technique for imaging the vessels in harvested tissue samples that relatively rapid and effective visualization ofin situvasculature. Furthermore, this technique allows for further analysis of the sample using histochemical staining, immunostaining and other techniques that can be performed on fixed tissue. The method uses ex vivo permeation of the tissue with an iodine solution and micro CT imaging to visualize the vascular network. Using a deep learning algorithm, we trained a convolutional neural network to automatically segment blood vessels from the surrounding tissue for analysis and create a map of the vasculature. While this method cannot achieve the resolution obtainable through destructive techniques, it is a simple and quick method for visualizing and quantifying vascular networks in three dimensions prior to analysis with conventional histological techniques. The global visualization of the three-dimensional vascular network provided by this method gives additional insights into complex changes in the vascular structure and can guide further histological analyses to specific regions of the vascular network. Overall, this method is a simple technique that can be added to most conventional tissue analyses to enhance the quantification and information derived from animal models with vascular network remodeling.