bluuebunny/crossref_metadata_2025_split huggingface dataset:
Enhanced 4Pi single-molecule localization microscopy with coherent pupil based localization and light sheet illumination
Authors: Liu Sheng , Huang Fang 
Published: March 2019

AbstractOver the last decades, super-resolution techniques have revolutionized the field of fluorescence microscopy. Among them, interferometric or 4Pi microscopy methods exhibit supreme resolving power in the axial dimension. Combining with single-molecule detection/localization and adaptive optics, iPALM/4PiSMS/W-4PiSMSN allowed 10-15 nm isotropic 3D resolution throughout the whole cell. However, further improving the achieved 3D resolution poses significantly challenges which, in part, is blocked by the complexity of single-molecule emission pattern generated by these systems rendering a large portion of information carrying photons unusable. Here we introduce a localization algorithm that achieves the theoretical information limit for 4Pi based single-molecule switching nanoscopy (4Pi-SMSN), and demonstrates improvements in resolution, accuracy as well as applicability comparing with the state of art 4Pi-SMSN methods. Further, with a novel 4Pi-compatible light-sheet illumination reducing the fluorescence background by >5-fold, we demonstrated the new system enables further improvement in the achievable resolution of 4Pi/interferometric single-molecule imaging systems.
Cryo-EM Map of the CFA/I Pilus Rod
Authors: Zheng Weili , Andersson Magnus , Mortezaei Narges , Bullitt Esther , Egelman Edward H. 
Published: March 2019

AbstractEnterotoxigenicEscherichia coli(ETEC) are common agents of diarrhea for travelers and a major cause of mortality in children in developing countries. To attach to intestinal cells ETEC express colonization factors, among them CFA/I, which are the most prevalent factors and are the archetypical representative of Class 5 pili. Due to their helical quaternary structure that can be unwound and works as a damper of force, CFA/I pili help ETEC bacteria withstand intestinal fluid motion. We report in this work the CFA/I pilus structure at 4.0 Å resolution and report details of the donor strand complementation. The density map allows us to identify the buried surface area between subunits, and these regions are correlated to quaternary structural stability in class 5 and Chaperone-Usher pili. In addition, from the EM map we also predicted that residue 13 (proline) of the N-terminal β-strand could have a major impact on the filament’s structural stability. Therefore, we used optical tweezers to measure and compare the stability of the quaternary structure of wild type CFA/I and point-mutated CFA/I in which proline 13 was changed to a non-polar residue, phenylalanine. We found that pili with this mutated CFA/I require a lower force to unwind, supporting our hypothesis that Pro 13 is important for structural stability. The high-resolution CFA/I pilus structure presented in this work and the analysis of structural stability will be useful for the development of novel antimicrobial drugs that target the pilus structure to reduce its damping properties, which are needed for initial attachment and sustained adhesion of ETEC.SynopsisThe structure of a common virulence factor expressed on the surface of diarrheacausing bacteria, CFA/I pili, has been determined at 4.0 Å resolution. The role of proline 13 in stabilizing the pilus structure has been confirmed using force-measuring optical tweezers on wild type and point-mutated pili.
Coolpup.py:versatile pile-up analysis of Hi-C data
Authors: Flyamer Ilya M. , Illingworth Robert S. , Bickmore Wendy A. 
Published: March 2019

AbstractMotivationHi-C is currently the method of choice to investigate the global 3D organisation of the genome. A major limitation of Hi-C is the sequencing depth required to robustly detect loops in the data. A popular approach used to mitigate this issue, even in single-cell Hi-C data, is genome-wide averaging (piling-up) of peaks, or other features, annotated in high-resolution datasets, to measure their prominence in less deeply sequenced data. However current tools do not provide a computationally efficient and versatile implementation of this approach.ResultsHere we describecoolpup.py– a versatile tool to perform pile-up analysis on Hi-C data. We demonstrate its utility by replicating previously published findings regarding the role of cohesin and CTCF in 3D genome organization, as well as discovering novel details of Polycomb-driven interactions. We also present a novel variation of the pile-up approach that can aid the in statistical analysis of looping interactions. We anticipate thatcoolpup.pywill aid in Hi-C data analysis by allowing easy to use, versatile and efficient generation of pileups.Availability and implementationCoolpup.pyis cross-platform, open-source and free (MIT licensed) software. Source code is available fromhttps://github.com/Phlya/coolpuppyand it can be installed from the Python Packaging Index.ContactIlya.Flyamer@igmm.ed.ac.uk
Spatial structure governs the mode of tumour evolution
Authors: Noble Robert , Burri Dominik , Kather Jakob Nikolas , Beerenwinkel Niko 
Published: March 2019

AbstractCharacterizing the mode – the way, manner, or pattern – of evolution in tumours is important for clinical forecasting and optimizing cancer treatment. DNA sequencing studies have inferred various modes, including branching, punctuated and neutral evolution, but it is unclear why a particular pattern predominates in any given tumour.1, 2Here we propose that differences in tumour architecture alone can explain the variety of observed patterns. We examine this hypothesis using spatially explicit population genetic models and demonstrate that, within biologically relevant parameter ranges, human tumours are expected to exhibit four distinct onco-evolutionary modes (oncoevotypes): rapid clonal expansion (predicted in leukaemia); progressive diversification (in colorectal adenomas and early-stage colorectal carcinomas); branching evolution (in invasive glandular tumours); and effectively almost neutral evolution (in certain non-glandular and poorly differentiated solid tumours). We thus provide a simple, mechanistic explanation for a wide range of empirical observations. Oncoevotypes are governed by the mode of cell dispersal and the range of cell-cell interaction, which we show are essential factors in accurately characterizing, forecasting and controlling tumour evolution.
3D microenvironment stiffness regulates tumor spheroid growth and mechanics via p21 and ROCK
Authors: Taubenberger Anna V. , Girardo Salvatore , Träber Nicole , Fischer-Friedrich Elisabeth , Kräter Martin , Wagner Katrin , Kurth Thomas , Richter Isabel , Haller Barbara , Binner Marcus , Hahn Dominik , Freudenberg Uwe , Werner Carsten , Guck Jochen 
Published: March 2019

AbstractMechanical properties of cancer cells and their microenvironment contribute to breast cancer progression. While mechanosensing has been extensively studied using two-dimensional (2D) substrates, much less is known about it in a physiologically more relevant 3D context. Here we demonstrate that breast cancer tumor spheroids, growing in 3D polyethylene glycol-heparin hydrogels, are sensitive to their environment stiffness. During tumor spheroid growth, compressive stresses of up to 2 kPa built up, as quantitated using elastic polymer beads as stress sensors. Atomic force microscopy (AFM) revealed that tumor spheroid stiffness increased with hydrogel stiffness. Also, constituent cell stiffness increased in a ROCK- and F-actin-dependent manner. Increased hydrogel stiffness correlated with attenuated tumor spheroid growth, a higher proportion of cells in G0/G1 phase and elevated levels of the cyclin-dependent kinase inhibitor p21. Drug-mediated ROCK inhibition reversed not only cell stiffening upon culture in stiff hydrogels but also increased tumor spheroid growth. Taken together, we reveal here a mechanism by which the growth of a tumor spheroid can be regulated via cytoskeleton rearrangements in response to its mechanoenvironment. Thus, our findings contribute to a better understanding of how cancer cells react to compressive stress when growing under confinement in stiff environments and provide the basis for a more in-depth exploration of the underlying mechanosensory response.
The polyploid genome of the mitotic parthenogenetic root-knot nematodeMeloidogyne enterolobii
Authors: Koutsovoulos Georgios D. , Poullet Marine , Ashry Abdelnaser El , Kozlowski Djampa K. , Sallet Erika , Rocha Martine Da , Martin-Jimenez Cristina , Perfus-Barbeoch Laetitia , Frey Juerg-Ernst , Ahrens Christian , Kiewnick Sebastian , Danchin Etienne G.J. 
Published: March 2019

AbstractRoot-knot nematodes (genusMeloidogyne) are plant parasitic species that cause huge economic loss in the agricultural industry and affect the prosperity of communities in developing countries. Control methods against these plant pests are sparse and the current preferred method is deployment of plant cultivars bearing resistance genes againstMeloidogynespecies. However, some species such asM. enterolobiiare not controlled by the resistance genes deployed in the most important crop plants cultivated in Europe. The recent identification of this species in Europe is thus a major concern. Like the other most damaging Meloidogyne species (e.g.M. incognita,M. arenariaandM. javanica),M. enterolobiireproduces by obligatory mitotic parthenogenesis. Genomic singularities such as a duplicated genome structure and a relatively high proportion of transposable elements have previously been described in the above mentioned mitotic parthenogenetic Meloidogyne.To gain a better understanding of the genomic and evolutionary background we sequenced the genome ofM. enterolobiiusing high coverage short and long read technologies. The information contained in the long reads helped produce a highly contiguous genome assembly ofM. enterolobii, thus enabling us to perform high quality annotations of coding and non-coding genes, and transposable elements.The genome assembly and annotation reveals a genome structure similar to the ones described in the other mitotic parthenogenetic Meloidogyne, described as recent hybrids. Most of the genome is present in 3 different copies that show high divergence. Because most of the genes belong to these duplicated regions only few gene losses took place, which suggest a recent polyploidization. The most likely hypothesis to reconcile high divergence between genome copies despite few gene losses and translocations is also a recent hybrid origin. Consistent with this hypothesis, we found an abundance of transposable elements at least as high as the one observed in the mitotic parthenogenetic nematodesM. incognitaandM. javanica.
FSHR-1/GPCR activates the mitochondrial unfolded protein response in Caenorhabditis elegans
Authors: Kim Sungjin , Sieburth Derek 
Published: March 2019

AbstractThe mitochondrial unfolded protein response (UPRmt) is an evolutionarily conserved adaptive response that functions to maintain mitochondrial homeostasis following mitochondrial damage. In C. elegans, the nervous system plays a central role in responding to mitochondrial stress by releasing endocrine signals that act upon distal tissues to activate the UPRmt. The mechanisms by which mitochondrial stress is sensed by neurons and transmitted to distal tissues is not fully understood. Here, we identify a role for the conserved follicle-stimulating hormone G protein coupled receptor (GPCR), FSHR-1, in promoting UPRmt activation. Genetic deficiency of fshr-1 severely attenuates UPRmt activation and organism-wide survival in response to mitochondrial stress. FSHR-1 functions in a common genetic pathway with SPHK-1/sphingosine kinase to promote UPRmt activation, and FSHR-1 regulates the mitochondrial association of SPHK-1 in the intestine. Through tissue-specific rescue assays, we show that FSHR-1 functions in neurons to activate the UPRmt, to promote mitochondrial association of SPHK-1 in the intestine, and to promote organism-wide survival in response to mitochondrial stress. We propose that FSHR-1 functions cell non-autonomously in neurons to activate UPRmt upstream of SPHK-1 signaling in the intestine.
Bipartite anchoring of SCREAM enforces stomatal initiation by coupling MAP Kinases to SPEECHLESS
Authors: Putarjunan Aarthi , Ruble Jim , Srivastava Ashutosh , Zhao Chunzhao , Rychel Amanda L. , Hofstetter Alex K. , Tang Xiaobo , Zhu Jian-Kang , Tama Florence , Zheng Ning , Torii Keiko U. 
Published: March 2019

AbstractCell-fate in eukaryotes is regulated by MAP Kinases (MAPKs) that translate external cues to cellular responses. In plants, two MAPKs, MPK3/6, regulate diverse processes of development, environmental response, and immunity. Yet, the mechanism bridging these shared signaling components with a specific target remains unresolved. Focusing on the development of stomata, epidermal valves for gas exchange and transpiration, we report here that the bHLH protein SCREAM functions as a scaffold by recruiting MPK3/6 to downregulate SPEECHLESS, a transcription factor initiating stomatal cell lineages. SCREAM directly binds with MPK3/6 through an evolutionarily-conserved yet unconventional bipartite motif. Mutations in this motif abrogate association, phosphorylation and degradation of SCREAM, unmask hidden non-redundancies between MPK3 and MPK6, and result in uncontrolled stomatal differentiation. Structural analyses of MPK6 at the 2.75Å resolution unraveled bipartite binding of SCREAM with MPK6, that is distinct from an upstream MAPKK. Our findings elucidate, at the atomic resolution, the mechanism directly linking extrinsic signals to transcriptional reprogramming during the establishment of stomatal cell-fate, and highlight a unique substrate-binding mode adopted by plant MAPKs.
Constitutive expression of a fluorescent protein reports the size of live human cells
Authors: Berenson Daniel F. , Zatulovskiy Evgeny , Xie Shicong , Skotheim Jan M. 
Published: March 2019

ABSTRACTCell size is intimately related to cell physiology because it sets the geometric scale of organelles and biosynthesis. A number of methods exist to measure different aspects of size of individual cells, but each has significant drawbacks. Here, we present an alternative method to measure the size of single human cells using a nuclear localized fluorescent protein expressed from a constitutive promoter. We validate this method by comparing it to several established cell size measurement strategies, including flow cytometry optical scatter, total protein dyes, and quantitative phase microscopy. We directly compare our fluorescent protein measurement to the commonly used measurement of nuclear volume and show that our measurements are more robust and less dependent on image segmentation. We apply our method to examine how cell size impacts the cell division cycle, which reaffirms the importance of G1/S size control. Importantly, combining our size reporter with fluorescent labeling of a different protein in a different color channel allows measurement of concentration dynamics using simple widefield fluorescence imaging. Thus, we expect our method will be of use to other researchers interested in the topics of cell size control and, more broadly, how dynamically changing protein concentrations control cell fates.
Impact of Genome Architecture Upon the Functional Activation and Repression of Hox Regulatory Landscapes
Authors: Rodríguez-Carballo Eddie , Lopez-Delisle Lucille , Yakushiji-Kaminatsui Nayuta , Ullate-Agote Asier , Duboule Denis 
Published: March 2019

BackgroundThe spatial organization of the mammalian genome relies upon the formation of chromatin domains of various scales. At the level of gene regulation in cis, collections of enhancer sequences define large regulatory landscapes that usually match with the presence of topologically associating domains (TADs). These domains are largely determined by bound CTCF molecules and often contain ranges of enhancers displaying similar or related tissue specificity, suggesting that in some cases such domains may act as coherent regulatory units, with a global on or off state.ResultsBy using the HoxD gene cluster as a paradigm, we investigated the effect of large genomic rearrangements affecting the two TADs flanking this locus, including their fusion into a single chromatin domain. We show that, within a single hybrid TAD, the activation of both proximal and distal limb enhancers initially positioned in either TADs globally occurred as when both TADs are intact. We also show that the timely implementation of distal limb enhancers depends on whether or not target genes had previously responded to proximal enhancers, due to the presence or absence of H3K27me3 marks.ConclusionsFrom this work, we conclude that antagonistic limb proximal and distal enhancers can exert their specificities when positioned into the same TAD and in the absence of their genuine target genes. We also conclude that removing these target genes reduced the coverage of a regulatory landscape by chromatin marks associated with silencing and thus prolonged its activity in time. Since Polycomb group proteins are mainly recruited at the Hox gene cluster, our results suggest that Polycomb Repressive Complex 2 (PRC2) can extend its coverage to far-cis regulatory sequences as long as confined to the neighboring TAD structure.
High-throughput Multimodal Automated Phenotyping (MAP) with Application to PheWAS
Authors: Liao Katherine P. , Sun Jiehuan , Cai Tianrun A. , Link Nicholas , Hong Chuan , Huang Jie , Huffman Jennifer E. , Gronsbell Jessica , Zhang Yichi , Ho Yuk-Lam , Castro Victor , Gainer Vivian , Murphy Shawn N. , O’Donnell Christopher J. , Gaziano J. Michael , Cho Kelly , Szolovits Peter , Kohane Isaac S. , Yu Sheng , Cai Tianxi ,   with the VA Million Veteran Program
Published: March 2019

AbstractObjectiveElectronic health records (EHR) linked with biorepositories are a powerful platform for translational studies. A major bottleneck exists in the ability to phenotype patients accurately and efficiently. The objective of this study was to develop an automated high-throughput phenotyping method integrating International Classification of Diseases (ICD) codes and narrative data extracted using natural language processing (NLP).MethodWe developed a mapping method for automatically identifying relevant ICD and NLP concepts for a specific phenotype leveraging the UMLS. Aggregated ICD and NLP counts along with healthcare utilization were jointly analyzed by fitting an ensemble of latent mixture models. The MAP algorithm yields a predicted probability of phenotype for each patient and a threshold for classifying subjects with phenotype yes/no. The algorithm was validated using labeled data for 16 phenotypes from a biorepository and further tested in an independent cohort PheWAS for two SNPs with known associations.ResultsThe MAP algorithm achieved higher or similar AUC and F-scores compared to the ICD code across all 16 phenotypes. The features assembled via the automated approach had comparable accuracy to those assembled via manual curation (AUCMAP 0.943, AUCmanual 0.941). The PheWAS results suggest that the MAP approach detected previously validated associations with higher power when compared to the standard PheWAS method based on ICD codes.ConclusionThe MAP approach increased the accuracy of phenotype definition while maintaining scalability, facilitating use in studies requiring large scale phenotyping, such as PheWAS.
Lipid membranes trigger misfolding and self-assembly of amyloid β 42 protein into aggregates
Authors: Banerjee Siddhartha , Hashemi Mohtadin , Zagorski Karen , Lyubchenko Yuri L. 
Published: March 2019

AbstractThe assembly of polypeptides and proteins into nanoscale aggregates is a phenomenon observed in a vast majority of proteins. Importantly, aggregation of amyloid β (Aβ) proteins is considered as a major cause for the development of Alzheimer’s disease. The process depends on various conditions and typical test-tube experiments require high protein concentration that complicates the translation of results obtained in vitro to understanding the aggregation process in vivo. Here we demonstrate that Aβ42 monomers at the membrane bilayer are capable of self-assembling into aggregates at physiologically low concentrations, and the membrane in this aggregation process plays a role of a catalyst. We applied all-atom molecular dynamics to demonstrate that the interaction with the membrane surface dramatically changes the conformation of Aβ42 protein. As a result, the misfolded Aβ42 rapidly assembles into dimers, trimers and tetramers, so the on-surface aggregation is the mechanism by which amyloid oligomers are produced and spread.
Human DNA-PK activates a STING-independent DNA sensing pathway
Authors: Burleigh Katelyn , Maltbaek Joanna H. , Cambier Stephanie , Green Richard , Gale Michael , James Richard C. , Stetson Daniel B. 
Published: March 2019

SummaryDetection of intracellular DNA by the cGAS-STING pathway activates a type I interferon-mediated innate immune response that protects from virus infection and can be harnessed to promote anti-tumor immunity. Whether there are additional DNA sensing pathways, and how such pathways might function, remains controversial. We show here that humans – but not mice – have a second, potent, STING-independent DNA sensing pathway that is blocked by the E1A viral oncogene of human adenovirus 5. We identify human DNA-PK as the sensor of this pathway and demonstrate that DNA-PK kinase activity drives a robust and broad antiviral response. We discover that the heat shock protein HSPA8/HSC70 is a unique target of DNA-PK. Finally, we demonstrate that detection of foreign DNA and DNA damage trigger distinct modalities of DNA-PK activity. These findings reveal the existence, sensor, unique target, and viral antagonists of a STING-independent DNA sensing pathway (SIDSP) in human cells.
Class A GPCRs use the membrane potential to increase their sensitivity and selectivity
Authors: Shalaeva Daria N. , Cherepanov Dmitry A. , Galperin Michael Y. , Vriend Gert , Mulkidjanian Armen Y. 
Published: March 2019

AbstractThe human genome contains about 700 genes of G protein-coupled receptors (GPCRs) of class A; these seven-helical membrane proteins are the targets of almost half of all known drugs. In the middle of the helix bundle, crystal structures revealed a highly conserved sodium-binding site, which is connected with the extracellular side by a water-filled tunnel. Sodium ions are observed in GPCRs crystallized in their inactive conformations, but not in GPCRs that were trapped in agonist-bound active conformations. The escape route of the sodium ion upon the inactive-to-active transition and its very direction, either into the cytoplasm or back outside the cell, hitherto remained obscure. We modeled sodium-binding GPCRs as electrogenic carriers of sodium ions. In this model the sodium gradient over the cell membrane would increase the sensitivity of GPCRs if their activation is thermodynamically coupled to the translocation of the sodium ion into the cytoplasm, while decreasing it if the sodium ion retreats into the extracellular space upon receptor activation. The model quantitatively describes the available data on both activation and suppression of distinct GPCRs by membrane voltage. The model also predicts selective amplification of the signal from (endogenous) agonists if only they, but not their (partial) analogs, could induce sodium translocation. Comparative structure and sequence analyses of sodium-binding GPCRs indicate a key role for the conserved leucine residue in the second transmembrane helix (Leu2.46) in coupling sodium translocation to receptor activation. Hence, class A GPCRs appear to utilize the energy of the transmembrane sodium potential to increase their sensitivity and selectivity.