bluuebunny/crossref_metadata_2025_split huggingface dataset:
Neuronal Subcompartment Classification and Merge Error Correction
Authors: Li Hanyu , Januszewski Michał , Jain Viren , Li Peter H. 
Published: April 2020

AbstractRecent advances in 3d electron microscopy are yielding ever larger reconstructions of brain tissue, encompassing thousands of individual neurons interconnected by millions of synapses. Interpreting reconstructions at this scale demands advances in the automated analysis of neuronal morphologies, for example by identifying morphological and functional subcompartments within neurons. We present a method that for the first time uses full 3d input (voxels) to automatically classify reconstructed neuron fragments as axon, dendrite, or somal subcompartments. Based on 3d convolutional neural networks, this method achieves a mean f1-score of 0.972, exceeding the previous state of the art of 0.955. The resulting predictions can support multiple analysis and proofreading applications. In particular, we leverage finely localized subcompartment predictions for automated detection and correction of merge errors in the volume reconstruction, successfully detecting 90.6% of inter-class merge errors with a false positive rate of only 2.7%.
Expression of aMalasseziacodon optimized mCherry fluorescent protein in a bicistronic vector
Authors: Goh Joleen P.Z. , Ianiri Giuseppe , Heitman Joseph , Dawson Thomas L. 
Published: April 2020

AbstractThe use of fluorescent proteins allows a multitude of approaches from live imaging and fixed cells to labelling of whole organisms, making it a foundation of diverse experiments. Tagging a protein of interest or specific cell type allows visualization and studies of cell localization, cellular dynamics, physiology, and structural characteristics. In specific instances fluorescent fusion proteins may not be properly functional as a result of structural changes that hinder protein function, or when overexpressed may be cytotoxic and disrupt normal biological processes. In our study, we describe application of a bicistronic vector incorporating a Picornavirus 2A peptide sequence between a NAT antibiotic selection marker and mCherry. This allows expression of multiple genes from a single open reading frame and production of discrete protein products through a cleavage event within the 2A peptide. We demonstrate integration of this bicistronic vector into a modelMalasseziaspecies, the haploid strainM. furfurCBS 14141, with both active selection, high fluorescence, and proven proteolytic cleavage. Potential applications of this technology can include protein functional studies,Malasseziacellular localization, and co-expression of genes required for targeted mutagenesis.
Comparative RNA-Seq Analyses ofDrosophilaPlasmatocytes Reveal Gene Specific Signatures In Response To Clean Injury And Septic Injury
Authors: Ramond Elodie , Dudzic Jan Paul , Lemaitre Bruno 
Published: April 2020

AbstractDrosophila melanogaster’s blood cells (hemocytes) play essential roles in wound healing and are involved in clearing microbial infections. Here, we report the transcriptional changes of larval plasmatocytes after clean injury or infection with the Gram-negative bacteriumEscherichia colior the Gram-positive bacteriumStaphylococcus aureuscompared to hemocytes recovered from unchallenged larvae via RNA-Sequencing. This study reveals 676 differentially expressed genes (DEGs) in hemocytes from clean injury samples compared to unchallenged samples, and 235 and 184 DEGs inE. coliandS. aureussamples respectively compared to clean injury samples. The clean injury samples showed enriched DEGs for immunity, clotting, cytoskeleton, cell migration, hemocyte differentiation, and indicated a metabolic reprogramming to aerobic glycolysis, a well-defined metabolic adaptation observed in mammalian macrophages. Microbial infections trigger significant transcription of immune genes, with significant differences between theE. coliandS. aureussamples suggesting that hemocytes have the ability to engage various programs upon infection. Collectively, our data bring new insights onDrosophilahemocyte function and open the route to post-genomic functional analysis of the cellular immune response.
Benefits of Collisional Cross Section Assisted Precursor Selection (caps-PASEF) for Cross-linking Mass Spectrometry
Authors: Steigenberger Barbara , van den Toorn Henk W.P. , Bijl Emiel , Greisch Jean-François , Räther Oliver , Lubeck Markus , Pieters Roland J. , Heck Albert J.R. , Scheltema Richard A. 
Published: April 2020

AbstractIon mobility separates molecules in the gas-phase on their physico-chemical properties, providing information about their size as collisional cross-sections. The timsTOF Pro combines trapped ion mobility with a quadrupole, collision cell and a time-of-flight mass analyzer, to probe ions at high speeds with on-the-fly fragmentation. Here, we show that on this platform ion mobility is beneficial for cross-linking mass spectrometry (XL-MS). Cross-linking reagents covalently link amino acids in close proximity, resulting in peptide pairs after proteolytic digestion. These cross-linked peptides are typically present at low abundance in the background of normal peptides, which can partially be resolved by using enrichable cross-linking reagents. Even with a very efficient enrichable cross-linking reagent, like PhoX, the analysis of cross-linked peptides is still hampered by the co-enrichment of peptides connected to a partially hydrolyzed reagent – termed mono-linked peptides. For experiments aiming to uncover protein-protein interactions these are unwanted byproducts. Here, we demonstrate that gas-phase separation by ion mobility enables the separation of mono-linked peptides from cross-linked peptide pairs. A clear partition between these two classes is observed at a CCS of 500 Å2and a monoisotopic mass of 2 kDa, which can be used for targeted precursor selection. A total of 50 - 70% of the mono-linked peptides are prevented from sequencing, allowing the analysis to focus on sequencing the relevant cross-linked peptide pairs. In applications to both simple proteins and protein mixtures and a complete highly complex lysate this approach provides a substantial increase in detected cross-linked peptides.
The evaluation of miR-874 antagomiR and miR-146a toxicity in cardiomyocytes
Authors: Guo Linlin , Zhang Yue , Chen Zhiwei , Dou Wenjing , Wu Jiamei , Xu Heli , Tan Hongyou , Wang Xiyan 
Published: April 2020

AbstractMore and more studies have shown that microRNAs (miRNAs) play key roles in the treatment of heart failure. Studies have shown that miR-874 (miR-874) inhibitors can reduce H2O2-induced cardiomyocyte necrosis and may have a therapeutic effect on heart failure in terms of cardiomyocyte necrosis. Therefore, the purpose of this experiment is to study the other effects of miR-874 and miR-146a on the key pathways in cardiomyocytes and cardiac fibroblasts. In this study, the roles of miR-874 antagomiR and miR-146a in cardiomyocytes and cardiac fibroblasts were analyzed. we investigated the level of miR-874 expression in H9C2 cardiomyocytes after miR-874 antagomiR transfection and found that the miR-874 expression in the H9C2 cells transfected with miR-874 antagomiR group was significantly lower than that in the control group. The results showed that miR-874 was successfully knocked down by the transfection of miR-874 antagomiR. Our results demonstrated that miR-874 inhibition has no effect on the activity of Caspase-3/7 in cardiomyocytes. miR-874 antagomiR has no effect on the proliferation of cardiac fibroblasts, whereas it can inhibit the activity of caspase-8 in cardiac fibroblasts. In addition, the potential effect of miR-874 antagomiR on cardiac remodeling associated genes were examined. miR-874 antagomiR had no effect on SERCA2a mRNA level in H9C2 cells. In addition, miR-874 antagomiR were able to down-regulate the mRNA level of MMP9 and had no effect on MMP2 mRNA levels in H9C2 cells. Finally, The concentration of Ca2+was measured using Fluo-4 NW Calcium Assay Kits following transfection of miRNAs or negative control in primary cardiomyocytes. miR-874 antagomiR was found to have no effect on Ca2+concentration in cardiomyocytes. the concentration of Ca2+in the cardiomyocytes transfected with miR-146a mimics was significantly lower than the mimic negative control group. In summary, miR-874 antagomiR and miR-146a may have a therapeutic effect on heart failure, but may also have side effects on heart failure treatment. Therefore, miR-874 antagomiR should be further studied to provide a basis for the development of drugs for heart failure.
Elucidation and refinement of synthetic receptor mechanisms
Authors: Edelstein Hailey I. , Donahue Patrick S. , Muldoon Joseph J. , Kang Anthony K. , Dolberg Taylor B. , Battaglia Lauren M. , Allchin Everett R. , Hong Mihe , Leonard Joshua N. 
Published: April 2020

ABSTRACTSynthetic receptors are powerful tools for engineering mammalian cell-based devices. These biosensors enable cell-based therapies to perform complex tasks such as regulating therapeutic gene expression in response to sensing physiological cues. Although multiple synthetic receptor systems now exist, many aspects of receptor performance are poorly understood. In general, it would be useful to understand how receptor design choices influence performance characteristics. In this study, we examined the modular extracellular sensor architecture (MESA) and systematically evaluated previously unexamined design choices, yielding substantially improved receptors. A key finding that might extend to other receptor systems is that the choice of transmembrane domain (TMD) is important for generating high-performing receptors. To provide mechanistic insights, we adopted and employed a Förster resonance energy transfer (FRET)-based assay to elucidate how TMDs affect receptor complex formation and connected these observations to functional performance. To build further insight into these phenomena, we developed a library of new MESA receptors that sense an expanded set of ligands. Based upon these explorations, we conclude that TMDs affect signaling primarily by modulating intracellular domain geometry. Finally, to guide the design of future receptors, we propose general principles for linking design choices to biophysical mechanisms and performance characteristics.
Immune modulation to improve survival of respiratory virus infections in mice
Authors: Wali Shradha , Flores Jose R. , Jaramillo Ana Maria , Goldblatt David L. , Pantaleón García Jezreel , Tuvim Michael J. , Dickey Burton F. , Evans Scott E. 
Published: April 2020

AbstractViral pneumonia remains a global health threat requiring novel treatment strategies, as strikingly exemplified in the SARS-CoV-2 pandemic of 2019-2020. We have reported that mice treated with a combination of inhaled Toll-like receptor (TLR) 2/6 and TLR 9 agonists (Pam2-ODN) to stimulate innate immunity are broadly protected against respiratory pathogens, but the mechanisms underlying this protection remain incompletely elucidated. Here, we show in a lethal paramyxovirus model that Pam2-ODN-enhanced survival is associated with robust virus inactivation by reactive oxygen species (ROS), which occurs prior to internalization by lung epithelial cells. However, we also found that mortality in sham-treated mice temporally corresponded with CD8+T cell-enriched lung inflammation that peaks on days 11-12 after viral challenge, when the viral burden has waned to a scarcely detectable level. Pam2-ODN treatment blocked this injurious inflammation by reducing the viral burden, and alternatively, depleting CD8+T cells 8 days after viral challenge also decreased mortality. These findings reveal opportunities for targeted immunomodulation to protect susceptible individuals against the morbidity and mortality of respiratory viral infections.
CRISPR-Cas9 cytidine and adenosine base editing of splice-sites mediates highly-efficient disruption of proteins in primary cells
Authors: Kluesner Mitchell G. , Lahr Walker S. , Lonetree Cara-Lin , Smeester Branden A. , Claudio-Vázquez Patricia N. , Pitzen Samuel P. , Vignes Madison J. , Lee Samantha C. , Bingea Samuel P. , Andrews Aneesha A. , Webber Beau R. , Moriarity Branden S. 
Published: April 2020

ABSTRACTBase editors allow for precise nucleotide editing without the need for genotoxic double-stranded breaks. Prior work has used base editors to knockout genes by introducing premature stop codons or by disrupting conserved splice-sites, but no direct comparison exists between these methods. Additionally, while base editor mediated disruption of splice sites has been used to shift the functional isoform pool, its utility for gene knockout requires further validation. To address these needs, we developed the program SpliceR (z.umn.edu/spliceR) to design cytidine-deaminase base editor (CBE) and adenosine-deaminase base editor (ABE) splice-site targeting guides. We compared the splice-site targeting and premature stop codon introduction in a knockout screen against the TCR-CD3 immune synapse in primary human T-cells. Our data suggests that 1) the CBE, BE4 is more reliable than the ABE, ABE7.10 for splice-site targeting knockout and 2) for both CBEs and ABEs, splice-donor targeting is the most reliable approach for base editing induced knockout.
Nucleotide excision repair hotspots and coldspots of UV-induced DNA damage in the human genome
Authors: Jiang Yuchao , Li Wentao , Lindsey-Boltz Laura A , Yang Yuchen , Li Yun , Sancar Aziz 
Published: April 2020

ABSTRACTWe recently developed high-throughput sequencing approaches, eXcision Repair sequencing (XR-seq) and Damage-seq, to generate genome-wide mapping of DNA excision repair and damage formation, respectively, with single-nucleotide resolution. Here, we used time-course XR-seq data to profile UV-induced excision repair dynamics, paired with Damage-seq data to quantify the overall induced DNA damage. We identified genome-wide repair hotspots exhibiting high-level nucleotide excision repair immediately after UV irradiation. We show that such repair hotspots do not result from hypersensitivity to DNA damage, and are thus not damage hotspots. We find that the earliest repair occurs preferentially in promoters and enhancers from open-chromatin regions. The repair hotspots are also significantly enriched for frequently interacting regions and super-enhancers, both of which are themselves hotspots for local chromatin interactions. Further interrogation of chromatin organization to include DNA replication timing allows us to conclude that early-repair hotspots are enriched for early-replication domains. Collectively, we report genome-wide early-repair hotspots of UV-induced damage, in association with chromatin states and epigenetic compartmentalization of the human genome.
Detecting Genes Associated with Pathogenicity and Antimicrobial Resistance in Three New Zealand Waterways
Authors: Davis Meredith T. , Midwinter Anne C. , Death Russell G. , Cosgrove Richard , Winkworth Richard C. 
Published: April 2020

BackgroundMore than 100 zoonoses may be transmitted via water, among them enteric diseases are leading causes of human mortality. Traditional monitoring for zoonoses relies on culturing of indicator species, but environmental DNA (eDNA) provides an alternative, allowing direct testing for genetic loci associated with pathogenicity and/or antimicrobial resistance in zoonotic bacteria.ObjectivesTo evaluate whether genes associated with Shiga toxin producingEscherichia coli(STEC) and antimicrobial resistance can be monitored in waterways using culture-free sampling of eDNA combined with PCR-based testing.MethodsWater and sediment samples were collected from two sites on each of three rivers in Canterbury, New Zealand; sample sites were situated above and below reaches bordered by intensive dairy farming. Samples from each site were tested for genes typically associated withEscherichia coli, STEC serogroups O26 and O157, human pathogenic strains of STEC, and resistance to a broad range of antibiotics.ResultsBoth culturing and genetic testing confirmed the presence ofE. coliin all samples. In contrast, presence of genes associated with STEC and antibiotic resistance varied by season and substrate. The O157 serogroup was identified at more than twice as many sites as O26, with the latter more common in autumn samples. In autumn, genes associated with pathogenic STEC were detected in one Ashley and both Rangitata River samples but were present in all spring samples, except one Ashley and one Selwyn River collection. The antibiotic resistance gene was only identified in spring, predominantly at sites downstream of intensive dairying.DiscussionWhile our sample is small this study indicates that genetic testing of eDNA can be a useful tool for monitoring the presence and persistence of zoonoses in waterways. How the presence of these genetic elements is related to that of pathogenic STEC and incidence of disease in humans now needs to be examined.
Employing NaChBac for cryo-EM analysis of toxin action on voltage-gated Na+channels in nanodisc
Authors: Gao Shuai , Valinsky William C , On Nguyen Cam , Houlihan Patrick R , Qu Qian , Liu Lei , Pan Xiaojing , Clapham David E. , Yan Nieng 
Published: April 2020

AbstractNaChBac, the first bacterial voltage-gated Na+(Nav) channel to be characterized, has been the prokaryotic prototype for studying the structure-function relationship of Navchannels. Discovered nearly two decades ago, the structure of NaChBac has not been determined. Here we present the cryo-EM analysis of NaChBac in both detergent micelles and nanodiscs. Under both conditions, the conformation of NaChBac is nearly identical to that of the potentially inactivated NavAb. Determining the structure of NaChBac in nanodiscs enabled us to examine gating modifier toxins (GMTs) of Navchannels in lipid bilayers. To study GMTs in mammalian Navs, we generated a chimera in which the extracellular fragment of the S3 and S4 segments in the second voltage-sensing domain from Nav1.7 replaces the corresponding sequence in NaChBac. Cryo-EM structures of the nanodisc-embedded chimera alone and in complex with HuwenToxin IV (HWTX-IV) were determined to 3.5 Å and 3.2 Å resolutions, respectively. Compared to the structure of HWTX-IV-bound human Nav1.7, which was obtained at an overall resolution of 3.2 Å, the local resolution of the toxin has been improved from ~ 6 Å to ~ 4 Å. This resolution enabled visualization of toxin docking. NaChBac can thus serve as a convenient surrogate for structural studies of the interactions between GMTs and Navchannels in a membrane environment.
Responses to Visual Speech in Human Posterior Superior Temporal Gyrus Examined with iEEG Deconvolution
Authors: Metzger Brian A. , Magnotti John F. , Wang Zhengjia , Nesbitt Elizabeth , Karas Patrick J. , Yoshor Daniel , Beauchamp Michael S. 
Published: April 2020

AbstractExperimentalists studying multisensory integration compare neural responses to multisensory stimuli with responses to the component modalities presented in isolation. This procedure is problematic for multisensory speech perception since audiovisual speech and auditory-only speech are easily intelligible but visual-only speech is not. To overcome this confound, we developed intracranial encephalography (iEEG) deconvolution. Individual stimuli always contained both auditory and visual speech but jittering the onset asynchrony between modalities allowed for the time course of the unisensory responses and the interaction between them to be independently estimated. We applied this procedure to electrodes implanted in human epilepsy patients (both male and female) over the posterior superior temporal gyrus (pSTG), a brain area known to be important for speech perception. iEEG deconvolution revealed sustained, positive responses to visual-only speech and larger, phasic responses to auditory-only speech. Confirming results from scalp EEG, responses to audiovisual speech were weaker than responses to auditory- only speech, demonstrating a subadditive multisensory neural computation. Leveraging the spatial resolution of iEEG, we extended these results to show that subadditivity is most pronounced in more posterior aspects of the pSTG. Across electrodes, subadditivity correlated with visual responsiveness, supporting a model in visual speech enhances the efficiency of auditory speech processing in pSTG. The ability to separate neural processes may make iEEG deconvolution useful for studying a variety of complex cognitive and perceptual tasks.Significance statementUnderstanding speech is one of the most important human abilities. Speech perception uses information from both the auditory and visual modalities. It has been difficult to study neural responses to visual speech because visual-only speech is difficult or impossible to comprehend, unlike auditory-only and audiovisual speech. We used intracranial encephalography (iEEG) deconvolution to overcome this obstacle. We found that visual speech evokes a positive response in the human posterior superior temporal gyrus, enhancing the efficiency of auditory speech processing.
Fungi of the Fortuna Forest Reserve: Taxonomy and ecology with emphasis on ectomycorrhizal communities
Authors: Corrales Adriana , Ovrebo Clark L. 
Published: April 2020

ABSTRACTPanamanian montane forests harbor a high diversity of fungi, particularly of ectomycorrhizal (ECM) fungi, however their taxonomy and diversity patterns remain for the most part unexplored. Here we present state of the art fungal taxonomy and diversity patterns at Fortuna Forest Reserve based on morphological and molecular identification of over 1,000 fruiting body collections of macromycetes made over a period of five years. We compare these new results with previously published work based on environmental sampling ofOreomunnea mexicanaroot tips. We compiled a preliminary list of species and report 22 new genera and 29 new fungal species for Panama. Based on fruiting body collection data we compare the species composition of ECM fungal communities associated withOreomunneastands across sites differing in soil fertility and amount of rainfall. We also examine the effect of a long-term nitrogen addition treatment on the fruiting body production of ECM fungi. Finally, we discuss the biogeographic importance of Panama collections which fill in the knowledge gap of ECM fungal records between Costa Rica and Colombia. Given that the isthmus of Panama was an important migration route of ECM tree and fungal species from northern temperate areas to South America, the ECM fungal communities of Panama might show a high degree of isolation and therefore a high level of endemism. We expect that the forests at Fortuna will continue to yield new ECM macromycete data as we continue to study the collected specimens and describe new species.
Optimal sampling design for spatial capture-recapture
Authors: Dupont Gates , Royle J. Andrew , Nawaz Muhammad Ali , Sutherland Chris 
Published: April 2020

AbstractSpatial capture-recapture (SCR) has emerged as the industry standard for estimating population density by leveraging information from spatial locations of repeat encounters of individuals. The precision of density estimates depends fundamentally on the number and spatial configuration of traps. Despite this knowledge, existing sampling design recommendations are heuristic and their performance remains untested for most practical applications. To address this issue, we propose a genetic algorithm that minimizes any sensible, criteria-based objective function to produce near-optimal sampling designs. To motivate the idea of optimality, we compare the performance of designs optimized using three model-based criteria related to the probability of capture. We use simulation to show that these designs out-perform those based on existing recommendations in terms of bias, precision, and accuracy in the estimation of population size. Our approach allows conservation practitioners and researchers to generate customized and improved sampling designs for wildlife monitoring.