bluuebunny/crossref_metadata_2025 huggingface dataset:
Species- and strain-level assessment usingrrnlong-amplicons suggests donor’s influence on gut microbial transference via fecal transplants in metabolic syndrome subjects
Authors: Benítez-Páez Alfonso , Hartstra Annick V. , Nieuwdorp Max , Sanz Yolanda
Published: September 2020

Fecal microbiota transplantation (FMT) is currently used for treatingClostridium difficileinfection and explored for other clinical applications in experimental trials. However, the effectiveness of this therapy could vary, and partly depend on the donor’s bacterial species engraftment, whose evaluation is challenging because there are no cost-effective strategies for accurately tracking the microbe transference. In this regard, the precise identification of bacterial species inhabiting the human gut is essential to define their role in human health unambiguously. We used Nanopore-based device to sequence bacterialrrnoperons (16S-ITS-23S) and to reveal species-level abundance changes in the human gut microbiota of a FMT trial. By assessing the donor and recipient microbiota before and after FMT, we further evaluated whether this molecular approach reveals strain-level genetic variation to demonstrate microbe transfer and engraftment. Strict control over sequencing data quality and major microbiota covariates was critical for accurately estimating the changes in gut microbial species abundance in the recipients after FMT. We detected strain-level variation via single-nucleotide variants (SNVs) atrrnregions in a species-specific manner. We showed that it was possible to explore successfully the donor-bacterial strain (e.g.,Parabacteroides merdae)engraftment in recipients of the FMT by assessing the nucleotide frequencies at rrn-associated SNVs. Our findings indicate that the engraftment of donors’ microbiota is to some extent correlated with the improvement of metabolic health in recipients and that parameters such as the baseline gut microbiota configuration, sex, and age of donors should be considered to ensure the success of FMT in humans.

Trial registration

The study was prospectively registered at the Dutch Trial registry - NTR4488 (https://www.trialregister.nl/trial/4488).
Modelling illustrates that genomic selection provides new opportunities for intercrop breeding
Authors: Bančič Jon , Werner Christian , Gaynor Chris , Gorjanc Gregor , Odeny Damaris , Ojulong Henry F. , Dawson Ian K. , Hoad Steve , Hickey John M.
Published: September 2020

Intercrop breeding programs using genomic selection can produce faster genetic gain than intercrop breeding programs using phenotypic selection. Intercropping is an agricultural practice in which two or more component crops are grown together. It can lead to enhanced soil structure and fertility, improved weed suppression, and better control of pests and diseases. Especially in subsistence agriculture, intercropping has great potential to optimise farming and increase profitability. However, breeding for intercrop varieties is complex as it requires simultaneous improvement of two or more component crops that combine well in the field. We hypothesize that genomic selection can significantly simplify and accelerate the process of breeding crops for intercropping. Therefore, we used stochastic simulation to compare four different intercrop breeding programs implementing genomic selection and an intercrop breeding program entirely based on phenotypic selection. We assumed three different levels of genetic correlation between monocrop grain yield and intercrop grain yield to investigate how the different breeding strategies are impacted by this factor. We found that all four simulated breeding programs using genomic selection produced significantly more intercrop genetic gain than the phenotypic selection program regardless of the genetic correlation with monocrop yield. We suggest a genomic selection strategy which combines monocrop and intercrop trait information to predict general intercropping ability to increase selection accuracy in early stages of a breeding program and to minimize the generation interval.
Analysis of Fox genes inSchmidtea mediterraneareveals new families and a conserved role ofSmed-foxOin controlling cell death
Authors: Pascual-Carreras Eudald , Herrera-Úbeda Carlos , Rosselló Maria , Garcia-Fernandez Jordi , Saló Emili , Adell Teresa
Published: September 2020

The forkhead box (Fox) genes encode transcription factors that control several key aspects of development. Present in the ancestor of all eukaryotes, Fox genes underwent several duplications followed by loss and diversification events that gave rise to the current 25 families. However, few Fox members have been identified from the Lophotrochozoa clade, and specifically from planarians, which are a unique model for understanding development, due to the striking plasticity of the adult. The aim of this study was to identify and perform evolutionary and functional studies of the Fox genes of lophotrochozoan species and, specifically, of the planarianSchmidtea mediterranea. Generating a pipeline for identifying Forkhead domains and using phylogenetics allowed us the phylogenetic reconstruction of Fox genes. We corrected the annotation for misannotated genes and uncover a new family, the QD, present in all metazoans. According to the new phylogeny, the 27 Fox genes found inSchmidtea mediterraneawere classified into 12 families. In Platyhelminthes, family losses were accompanied by extensive gene diversification and the appearance of specific families, the A(P) and N(P). Among the newly identified planarian Fox genes, we found a single copy offoxO, which shows an evolutionary conserved role in controlling cell death.

Author summary

Transcription factors are the key elements that regulate gene expression in the nucleus. The forkhead box (Fox) transcription factors are one of the most numerous and they control key aspects of development. Fox genes were already present in the ancestor of all eukaryotes, and then underwent several duplications followed by loss and diversification events that gave rise to the current Fox families in the different species. The available data classifies Fox genes in 25 families, but they include few members corresponding to Lophotrocozoa, one of the two invertebrate phyla that includes annelids, molluscs or platyhelmintes. In this study we identify and perform evolutionary studies of the Fox genes of several lophotrochozoan species and, specifically, of the planarianSchmidtea mediterranea. The result is the correction of the annotation of Fox genes from many species, proposing a new nomenclature, and the identification of new families; the QD family, present in all metazoans, and the A(P) and N(P) families, specific of Platyhelminthes. We also study the function ofSchmidtea mediterranea foxO, a gene involved in aging and cancer in other species, showing its evolutionary conserved role in controlling cell death according to cell metabolism.
Rapid Development of Improved Data-dependent Acquisition Strategies
Authors: Davies Vinny , Wandy Joe , Weidt Stefan , van der Hooft Justin J. J. , Miller Alice , Daly Rónán , Rogers Simon
Published: September 2020

Tandem mass spectrometry (LC-MS/MS) is widely used to identify unknown ions in untargeted metabolomics. Data Dependent Acquisition (DDA) chooses which ions to fragment based upon intensity observed in MS1 survey scans and typically only fragment a small subset of the ions present. Despite this inefficiency, relatively little work has addressed the development of new DDA methods, partly due to the high overhead associated with running the many extracts necessary to optimise approaches in busy MS facilities. In this work, we firstly provide theoretical results that show how much improvement is possible over current DDA strategies. We then describe anin silicoframework for fast and cost efficient development of new DDA acquisition strategies using a previously developed Virtual Metabolomics Mass Spectrometer (ViMMS). Additional functionality is added to ViMMS to allow methods to be used both in simulation and on real samples via an instrument application programming interface (API).

We demonstrate this framework through the development and optimisation of two new DDA methods which introduce new advanced ion prioritisation strategies. Upon application of the here developed methods to two complex metabolite mixtures, our results show that they are able to fragment more unique ions than standard DDA acquisition strategies.
Phylogeny of docks and sorrels (Rumex, Polygonaceae) reveals plasticity of reproductive systems
Authors: Grant Kirstie D. , Koenemann Daniel , Mansaray Janet , Ahmed Aisha , Khamar Hamid , Oualidi Jalal El , Burke Janelle M.
Published: September 2020

The genusRumexis a unique member of the Polygonaceae (Buckwheat) family of plants. A source of intrigue forRumexlies in the diversity of the reproductive systems associated with the subgenera, species, and subspecies within this genus. Four previously circumscribed subgenera, some 200 species, and a number of subspecies comprise the collectiveRumexgenus. These species exhibit monoecious, dioecious, synoecious (hermaphroditic), and polygamous reproductive systems. Moreover, some of the dioecious species contain sex chromosomes, a phenomenon that is very rare in angiosperms. Apart from the confirmed morphological and phytogeographical distinctions, two of the four described subgenera,AcetosaandAcetosella, are distinctive in their exhibited sex chromosome systems. For this study, we used three chloroplast markers,rbcL, trnH-psbA, trnL-F, and dense taxon sampling, to reconstruct a molecular phylogeny forRumex.The reconstructed phylogeny for this work resolves six major clades and one large grade inRumex. In addition, the species with known dioecious reproductive systems derived from unique sex chromosome systems are resolved in two different clades nested within “the dioecious clade”. These results suggest that the species with divergent sexual systems are more closely related to each other than to other species comprising the rest of theRumexgenus. Furthermore, some species with known synoecious reproductive systems are resolved in a single clade which is also nested within “the dioecious clade”. These results imply a possible reversal occurring over time which suggests the highly plastic nature of reproductive systems amongRumexspecies.
Reorganization of the neurobiology of language after sentence overlearning
Authors: Skipper Jeremy I , Aliko Sarah , Brown Stephen , Jo Yoon Ju , Lo Serena , Molimpakis Emilia , Lametti Daniel R
Published: September 2020

There is a widespread assumption that there are a static set of ‘language regions’ in the brain. Yet, people still regularly produce familiar ‘formulaic’ expressions when those regions are severely damaged. This suggests that the neurobiology of language varies with the extent of word sequence learning and might not be fixed. We test the hypothesis that perceiving sentences is mostly supported by sensorimotor regions involved in speech production and not ‘language regions’ after overlearning. Twelve participants underwent two sessions of behavioural testing and functional magnetic resonance imaging (fMRI), separated by 15 days. During this period, they repeated two sentences 30 times each, twice a day. In both fMRI sessions, participants ‘passively’ listened to those two sentences and novel sentences. Lastly, they spoke novel sentences. Behavioural results confirm that participants overlearned sentences. Correspondingly, there was an increase or recruitment of sensorimotor regions involved in sentence production and a reduction in activity or inactivity for overlearned sentences in regions involved in listening to novel sentences. The global network organization of the brain changed by ∼45%, mostly through lost connectivity. Thus, there was a profound reorganization of the neurobiology of speech perception after overlearning towards sensorimotor regions not considered in most contemporary models and away from the ‘language regions’ posited by those models. These same sensorimotor regions are generally preserved in aphasia and Alzheimer’s disease, perhaps explaining residual abilities with formulaic language. These and other results warrant reconsidering static neurobiological models of language.
Molecular Characterization, Phylogenetic and Variation Analyzes of SARS-CoV-2 strains in Turkey
Authors: Murat Karamese , Didem Ozgur , Ediz Tutuncu Emin
Published: September 2020

Introduction

We present the sequence analysis for 47 complete genomes for SARS-CoV-2 isolates on Turkish patients. To identify their genetic similarity, phylogenetic analysis was performed by comparing the worldwide SARS-CoV-2 sequences, selected from GISAID, to the complete genomes from Turkish isolates. In addition, we focused on the variation analysis to show the mutations on SARS-CoV-2 genomes.

Methods

Illumina MiSeq platform was used for sequencing the libraries. The raw reads were aligned to the known SARS-CoV-2 genome (GenBank:MN908947.3) using the Burrows-Wheeler aligner (v.0.7.1). The phylogenetic tree was constructer using Phylip v.3.6 with Neighbor-Joining and composite likelihood method. The variants were detected by using Genome Analysis Toolkit-HaplotypeCaller v.3.8.0 and were inspected on GenomeBrowse v2.1.2.

Results

All viral genome sequences of our isolates was located in lineage B under the different clusters such as B.1 (n=3), B.1.1 (n=28), and B.1.9 (n=16). According to the GISAID nomenclature, all our complete genomes were placed in G, GR and GH clades. Five hundred forty-nine total and 53 unique variants were detected. All 47 genomes exhibited different kinds of variants. The distinct variants consist of 274 missense, 225 synonymous, and 50 non-coding alleles.

Conclusion

The results indicated that the SARS-CoV-2 sequences of our isolates have great similarity with all Turkish and European sequences. Further studies should be performed for better comparison of strains, after more complete genome sequences will be released. We also believe that collecting and sharing any data about SARS-CoV-2 virus and COVID-19 will be effective and may help the related studies.
Functional binding dynamics relevant to the evolution of zoonotic spillovers in endemic and emergentBetacoronavirusstrains
Authors: Rynkiewicz Patrick , Babbitt Gregory A. , Cui Feng , Hudson André O. , Lynch Miranda L.
Published: September 2020

Comparative functional analysis of the dynamic interactions between variousBetacoronavirusmutant strains and broadly utilized target proteins such as ACE2 and CD26, is crucial for a more complete understanding of zoonotic spillovers of viruses that cause diseases such as COVID-19. Here, we employ machine learning to replicated sets of nanosecond scale GPU accelerated molecular dynamics simulations to statistically compare and classify atom motions of these target proteins in both the presence and absence of different endemic and emergent strains of the viral receptor binding domain (RBD) of the S spike glycoprotein. Machine learning was used to identify functional binding dynamics that are evolutionarily conserved from bat CoV-HKU4 to human endemic/emergent strains. Conserved dynamics regions of ACE2 involve both the N-terminal helices, as well as a region of more transient dynamics encompassing K353, Q325 and a novel motif AAQPFLL 386-92 that appears to coordinate their dynamic interactions with the viral RBD at N501. We also demonstrate that the functional evolution ofBetacoronaviruszoonotic spillovers involving ACE2 interaction dynamics are likely pre-adapted from two precise and stable binding sites involving the viral bat progenitor strain’s interaction with CD26 at SAMLI 291-5 and SS 333-334. Our analyses further indicate that the human endemic strains hCoV-HKU1 and hCoV-OC43 have evolved more stable N-terminal helix interactions through enhancement of an interfacing loop region on the viral RBD, whereas the highly transmissible SARS-CoV-2 variants (B.1.1.7, B.1.351 and P.1) have evolved more stable viral binding via more focused interactions between the viral N501 and ACE2 K353 alone.
Itk promotes the integration of TCR and CD28 costimulation, through its direct substrates, SLP-76 and Gads
Authors: Hallumi Enas , Shalah Rose , Lo Wan-Lin , Corso Jasmin , Oz Ilana , Beach Dvora , Wittman Samuel , Isenberg Amy , Sela Meirav , Urlaub Henning , Weiss Arthur , Yablonski Deborah
Published: September 2020

The costimulatory receptor, CD28, synergizes with the T cell antigen receptor (TCR) to promote IL-2 production, cell survival and proliferation. Despite their profound synergy, the obligatory interdependence of the signaling pathways initiated by these two receptors is not well understood. Upon TCR stimulation, Gads, a Grb2-family adaptor, bridges the interaction of two additional adaptors, LAT and SLP-76, to form a TCR-induced effector signaling complex. SLP-76 binds the Tec-family tyrosine kinase, Itk, which phosphorylates SLP-76 at Y173 and PLC-γ1 at Y783. Here we identified Gads Y45 as an additional TCR-inducible, Itk-mediated phosphorylation site. Y45 is found within the N-terminal SH3 domain of Gads, an evolutionarily conserved domain with no known binding partners or signaling function. Gads Y45 phosphorylation depended on the interaction of Gads with SLP-76 and on the preferentially-paired binding of Gads to phospho-LAT. Three Itk-related features, Gads Y45, SLP-76 Y173, and a proline-rich Itk SH3-binding motif on SLP-76, were selectively required for activation of the CD28 RE/AP transcriptional element from the IL-2 promoter, but were not required to activate NFAT. This study illuminates a new regulatory module, in which Itk-targeted phosphorylation sites on two adaptor proteins, SLP-76 and Gads, control the transcriptional response to TCR/CD28 costimulation, thus enforcing the obligatory interdependence of the TCR and CD28 signaling pathways.
Site-specific lipidation enhances IFITM3 membrane interactions and antiviral activity
Authors: Garst Emma , Lee Hwayoung , Das Tandrila , Bhattacharya Shibani , Percher Avital , Wiewiora Rafal , Witte Isaac P. , Li Yumeng , Goger Michael , Peng Tao , Im Wonpil , Hang Howard C.
Published: September 2020

Interferon-induced transmembrane proteins (IFITMs) areS-palmitoylated proteins in vertebrates that restrict a diverse range of viruses.S-palmitoylated IFITM3 in particular directly engages incoming virus particles, prevents their cytoplasmic entry, and accelerates their lysosomal clearance by host cells. However, the precise molecular mechanisms of action for IFITM-mediated viral restriction are still unclear. To investigate how site-specificS-palmitoylation controls IFITM3 antiviral activity, here we employed computational, chemical, and biophysical approaches to demonstrate that site-specific lipidation of IFITM3 at highly conserved cysteine 72 modulates its conformation and interaction with lipid membranes leading to enhanced antiviral activity of IFITM3 in mammalian cells. Collectively, our results demonstrate that site-specificS-palmitoylation of IFITM3 directly alters its biophysical properties and activity in cells to prevent virus infection.
NuMorph: tools for cellular phenotyping in tissue cleared whole brain images
Authors: Krupa Oleh , Fragola Giulia , Hadden-Ford Ellie , Mory Jessica T. , Liu Tianyi , Humphrey Zachary , Rees Benjamin W. , Krishnamurthy Ashok , Snider William D. , Zylka Mark J. , Wu Guorong , Xing Lei , Stein Jason L.
Published: September 2020

Tissue clearing methods allow every cell in the mouse brain to be imaged without physical sectioning. However, the computational tools currently available for cell quantification in cleared tissue images have been limited to counting sparse cell populations in stereotypical mice. Here we introduce NuMorph, a group of image analysis tools to quantify all nuclei and nuclear markers within the mouse cortex after tissue clearing and imaging by a conventional light-sheet microscope. We applied NuMorph to investigate two distinct mouse models: aTopoisomerase 1(Top1) conditional knockout model with severe neurodegenerative deficits and aNeurofibromin 1(Nf1) conditional knockout model with a more subtle brain overgrowth phenotype. In each case, we identified differential effects of gene deletion on individual cell-type counts and distribution across cortical regions that manifest as alterations of gross brain morphology. These results underline the value of 3D whole brain imaging approaches and the tools are widely applicable for studying 3D structural deficits of the brain at cellular resolution in animal models of neuropsychiatric disorders.
Identifying zoonotic origin of SARS-CoV-2 by modeling the binding affinity between Spike receptor-binding domain and host ACE2
Authors: Huang Xiaoqiang , Zhang Chengxin , Pearce Robin , Omenn Gilbert S. , Zhang Yang
Published: September 2020

Despite considerable research progress on SARS-CoV-2, the direct zoonotic origin (intermediate host) of the virus remains ambiguous. The most definitive approach to identify the intermediate host would be the detection of SARS-CoV-2-like coronaviruses in wild animals. However, due to the high number of animal species, it is not feasible to screen all the species in the laboratory. Given that the recognition of the binding ACE2 proteins is the first step for the coronaviruses to invade host cells, we proposed a computational pipeline to identify potential intermediate hosts of SARS-CoV-2 by modeling the binding affinity between the Spike receptor-binding domain (RBD) and host ACE2. Using this pipeline, we systematically examined 285 ACE2 variants from mammals, birds, fish, reptiles, and amphibians, and found that the binding energies calculated on the modeled Spike-RBD/ACE2 complex structures correlate closely with the effectiveness of animal infections as determined by multiple experimental datasets. Built on the optimized binding affinity cutoff, we suggested a set of 96 mammals, including 48 experimentally investigated ones, which are permissive to SARS-CoV-2, with candidates from primates, rodents, and carnivores at the highest risk of infection. Overall, this work not only suggested a limited range of potential intermediate SARS-CoV-2 hosts for further experimental investigation; but more importantly, it proposed a new structure-based approach to general zoonotic origin and susceptibility analyses that are critical for human infectious disease control and wildlife protection.
PanACoTA: A modular tool for massive microbial comparative genomics
Authors: Perrin Amandine , Rocha Eduardo P.C.
Published: September 2020

The study of the gene repertoires of microbial species, their pangenomes, has become a key topic of study in microbial evolution and genomics. Yet, the increasing number of genomes available complicates the establishment of the basic building blocks of comparative genomics. Here, we present PanACoTA (https://github.com/gem-pasteur/PanACoTA), a tool that allows to download all genomes of a species, build a database with those passing quality and redundancy controls, uniformly annotate, and then build their pangenome, several variants of core genomes, their alignments, and a rapid but accurate phylogenetic tree. While many programs building pangenomes have become available in the last few years, we have focused on a modular method, that tackles all the key steps of the process, from download to phylogenetic inference. While all steps are integrated, they can also be run separately and multiple times to allow rapid and extensive exploration of the parameters of interest. The software is built in Python3 and includes features to facilitate its future development. We believe PanACoTa is an interesting addition to the current set of comparative genomics tools, since it will accelerate and standardize the more routine parts of the work, allowing microbial genomicists to more quickly tackle their specific questions.
Principles of resilient coding for plant ecophysiologists
Authors: Stinziano Joseph R , Roback Cassaundra , Gamble Demi , Murphy Bridget K , Hudson Patrick J , Muir Christopher D
Published: September 2020

Plant ecophysiology is founded on a rich body of physical and chemical theory, but it is challenging to connect theory with data in unambiguous, analytically rigorous, and reproducible ways. Custom scripts written in computer programming languages (coding) enable plant ecophysiologists to model plant processes and fit models to data reproducibly using advanced statistical techniques. Since many ecophysiologists lack formal programming education, we have yet to adopt a unified set of coding principles and standards that could make coding easier to learn, use, and modify. We identify eight principles to help in plant ecophysiologists without much programming experience to write resilient code: 1) standardized nomenclature, 2) consistency in style, 3) increased modularity/extensibility for easier editing and understanding, 4) code scalability for application to large datasets, 5) documented contingencies for code maintenance, 6) documentation to facilitate user understanding; 7) extensive tutorials, and 8) unit testing. We illustrate these principles using a new R package, {photosynthesis}, which provides a set of analytical and simulation tools for plant ecophysiology. Our goal with these principles is to advance scientific discovery in plant ecophysiology by making it easier to use code for simulation and data analysis, reproduce results, and rapidly incorporate new biological understanding and analytical tools.
Correction of frameshift mutations in theatpBgene by translational recoding in chloroplasts ofOenotheraand tobacco
Authors: Malinova Irina , Zupok Arkadiusz , Massouh Amid , Schöttler Mark Aurel , Meyer Etienne H. , Yaneva-Roder Liliya , Szymanski Witold , Rößner Margit , Ruf Stephanie , Bock Ralph , Greiner Stephan
Published: September 2020

Translational recoding, also known as ribosomal frameshifting, is a process that causes ribosome slippage along the messenger RNA, thereby changing the amino acid sequence of the synthesized protein. Whether the chloroplast employs recoding, is unknown. I-iota, a plastome mutant ofOenothera(evening primrose), carries a single adenine insertion in an oligoA stretch ofatpB(encoding a β-subunit of the ATP synthase). The mutation is expected to cause synthesis of a truncated, non-functional protein. We report that a full-length AtpB protein is detectable in I-iota leaves, suggesting operation of a recoding mechanism. To characterize the phenomenon, transplastomic tobacco lines were generated, in which theatpBreading frame was altered by insertions or deletions in the oligoA motif. We found that insertion of two adenines was more efficiently compensated than insertion of a single adenine, or deletion of one or two adenines. We further show that homopolymeric composition of the oligoA stretch is essential for recoding. Plants carrying a disrupted oligoA stretch have an albino-phenotype, indicating absence of indel correction. Our work provides evidence for the operation of translational recoding in chloroplasts. Recoding enables correction of frameshift mutations and can restore photoautotrophic growth in mutants that otherwise would be lethal.
Human trans-editing enzyme displays tRNA acceptor stem specificity and relaxed amino acid selectivity
Authors: Vargas-Rodriguez Oscar , Bakhtina Marina , McGowan Daniel , Abid Jawad , Goto Yuki , Suga Hiroaki , Musier-Forsyth Karin
Published: September 2020

Accurate translation of genetic information into proteins is vital for cell sustainability. ProXp-ala prevents proteome-wide Pro-to-Ala mutations by hydrolyzing misacylated Ala-tRNAPro, which is synthesized by prolyl-tRNA synthetase (ProRS). Bacterial ProXp-ala was previously shown to combine a size-based exclusion mechanism with conformational and chemical selection for the recognition of the alanyl moiety, while tRNAPro is selected via recognition of tRNA acceptor stem elements G72 and A73. The identity of these critical bases changed during evolution with eukaryotic cytosolic tRNAPro possessing a cytosine at the corresponding positions. The mechanism by which eukaryotic ProXp-ala adapted to these changes remains unknown. In this work, recognition of the aminoacyl moiety and tRNA acceptor stem by human (Hs) ProXp-ala was examined. Enzymatic assays revealed that Hs ProXp-ala requires C72 and C73 in the context of Hs cytosolic tRNAPro for efficient deacylation of mischarged Ala-tRNAPro. The strong dependence on these bases prevents cross-species deacylation of bacterial Ala-tRNAPro or of Hs mitochondrial Ala-tRNAPro by the human enzyme. Similar to the bacterial enzyme, Hs ProXp-ala showed strong tRNA acceptor-stem recognition but differed in its amino acid specificity profile relative to bacterial ProXp-ala. Changes at conserved residues in both the Hs and bacterial ProXp-ala substrate binding pockets modulated this specificity. These results illustrate how the mechanism of substrate selection diverged during the evolution of the ProXp-ala family and provides the first example of a trans-editing domain whose specificity evolved to adapt to changes in its tRNA substrate.
Proximity-dependent proteomics reveals extensive interactions of Protocadherin-19 with regulators of Rho GTPases and the microtubule cytoskeleton
Authors: Emond Michelle R. , Biswas Sayantanee , Morrow Matthew L. , Jontes James D.
Published: September 2020

Protocadherin-19 belongs to the cadherin family of cell surface receptors and has been shown to play essential roles in the development of the vertebrate nervous system. Mutations in human Protocadherin-19 (PCDH19) lead toPCDH19Female-limited epilepsy (PCDH19FLE) in humans, characterized by the early onset of epileptic seizures in children and a range of cognitive and behavioral problems in adults. Despite being considered the second most prevalent gene in epilepsy, very little is known about the intercellular pathways in which it participates. In order to characterize the protein complexes within which Pcdh19 functions, we generated Pcdh19-BioID fusion proteins and utilized proximity-dependent biotinylation to identify neighboring proteins. Proteomic identification and analysis revealed that the Pcdh19 interactome is enriched in proteins that regulate Rho family GTPases, microtubule binding proteins and proteins that regulate cell divisions. We cloned the centrosomal protein Nedd1 and the RacGEF Dock7 and verified their interactions with Pcdh19in vitro. Our findings provide the first comprehensive insights into the interactome of Pcdh19, and provide a platform for future investigations into the cellular and molecular biology of this protein critical to the proper development of the nervous system.
Vital Insights into Prokaryotic Genome Compaction by Nucleoid-Associated Protein (NAP) and Illustration of DNA Flexure Angles at Single Molecule Resolution
Authors: Purkait Debayan , Bandyopadhyay Debolina , Mishra Padmaja P.
Published: September 2020

Integration Host Factor (IHF) is a heterodimeric site-specific nucleoid-associated protein (NAP) well known for its DNA bending ability. The binding is mediated through the narrow minor grooves of the consensus sequence, involving van der-Waals interaction and hydrogen bonding. Although the DNA bend state of IHF has been captured by both X-ray Crystallography and Atomic Force Microscopy (AFM), the range of flexibility and degree of heterogeneity in terms of quantitative analysis of the nucleoprotein complex has largely remained unexplored. Here we have monitored and compared the trajectories of the conformational dynamics of a dsDNA upon binding of wild-type (wt) and single-chain (sc) IHF at millisecond resolution through single-molecule FRET (smFRET). Our findings reveal that the nucleoprotein complex exists in a ‘Slacked-Dynamic’ state throughout the observation window where many of them have switched between multiple ‘Wobbling States’ in the course of attainment of packaged form. A range of DNA ‘Flexure Angles’ has been calculated that give us vital insights regarding the nucleoid organization and transcriptional regulation in prokaryotes. This study opens up an opportunity to improve the understanding of the functions of other nucleoid-associated proteins (NAPs) by complementing the previous detailed atomic-level structural analysis, which eventually will allow accessibility towards a better hypothesis.
Filaggrin deficient mice have a lower threshold for cutaneous allergen sensitization but do not develop spontaneous skin inflammation or atopy
Authors: Muhandes Lina , Pippel Martin , Chapsa Maria , Behrendt Rayk , Ge Yan , Dahl Andreas , Yi Buqing , Dalpke Alexander , Winkler Sylke , Hiller Michael , Beissert Stefan , Jessberger Rolf , Fallon Padraic G. , Roers Axel
Published: September 2020

Defects of filaggrin (FLG) compromise epidermal barrier function and represent an important known genetic risk factor for atopic dermatitis (AD), but also for systemic atopy, including allergic sensitization and asthma. The flaky tail mouse model, widely used to address mechanisms of atopy induction by barrier-defective skin, harbors two mutations that affect the skin barrier, the mutationFlgft, resulting in near-complete loss of FLG expression, and the matted mutation inactivating transmembrane protein 79 (Tmem79). Upon separation of the two mutant loci, which are closely linked on chromosome 3, mice defective only for Tmem79 featured pronounced dermatitis and systemic atopy. Upon extensive backcross to BALB/c, alsoFlgft/ftmice (assumed to be wild type forTmem79), developed AD-like dermatitis and reproduced the human ‘atopic march’, with high IgE levels and spontaneous asthma, suggesting a key role for functional Flg in protection from atopy also in mice. In contrast, BALB/c mice congenic for a targetedFlgknock out mutation did not develop skin inflammation or atopy. To resolve this discrepancy, we generated Flg-deficient mice on a pure BALB/c background by inactivating theFlggene in BALB/c embryos. These animals feature an ichthyosis phenotype, but do not develop spontaneous dermatitis or systemic atopy. We sequenced the genome of the atopicFlgftBALB/c congenics and discovered that they were unexpectedly homozygous for the atopy-causingTmem79mattedmutation. In summary, we show that Flg-deficiency does not cause atopy in mice. This finding is in line with lack of atopic disease in a fraction of Ichthyosis vulgaris patients carrying two FLG null alleles. However, absence of FLG may promote and modulate dermatitis caused by other genetic barrier defects, as skin inflammation inTmem79ma/maFlgft/ftBALB/c congenics is qualitatively different compared toTmem79ma/mamice.
Diurnal Dynamics of the Arabidopsis Rosette Proteome and Phosphoproteome
Authors: Uhrig R. Glen , Echevarría-Zomeño Sira , Schlapfer Pascal , Grossmann Jonas , Roschitzki Bernd , Koerber Niklas , Fiorani Fabio , Gruissem Wilhelm
Published: September 2020

Plant growth depends on the diurnal regulation of cellular processes, but it is not well understood if and how transcriptional regulation controls diurnal fluctuations at the protein-level. Here we report a high-resolutionArabidopsis thaliana(Arabidopsis) leaf rosette proteome acquired over a 12 h light : 12 h dark diurnal cycle and the phosphoproteome immediately before and after the light-to-dark and dark-to-light transitions. We quantified nearly 5000 proteins and 800 phosphoproteins, of which 288 fluctuated in their abundance and 226 fluctuated in their phosphorylation status. Of the phosphoproteins, 60% were quantified for changes in protein abundance. This revealed six proteins involved in nitrogen and hormone metabolism that had concurrent changes in both protein abundance and phosphorylation status. The diurnal proteome and phosphoproteome changes involve proteins in key cellular processes, including protein translation, light perception, photosynthesis, metabolism and transport. The phosphoproteome at the light-dark transitions revealed the dynamics at phosphorylation sites in either anticipation of or response to a change in light regime. Phosphorylation site motif analyses implicate casein kinase II and calcium/calmodulin dependent kinases among the primary light-dark transition kinases. The comparative analysis of the diurnal proteome and diurnal and circadian transcriptome established how mRNA and protein accumulation intersect in leaves during the diurnal cycle of the plant.

Summary Statement

The manuscript provides quantitative information of diurnal changes in the accumulation and phosphorylation of proteins inArabidopsis thalianarosettes grown in a 12 h photoperiod. The highly resolved time-scale of the datasets offer new proteome-level insights for future targeted studies.
Extracellular Vesicle-Mediated Purinergic Signaling Contributes to Host Microenvironment Plasticity and Metastasis in Triple Negative Breast Cancer
Authors: Duan Suzann , Nordmeier Senny , Byrnes Aidan E. , Buxton Iain L. O.
Published: September 2020

Metastasis accounts for over 90% of cancer-related deaths. The mechanisms guiding this process remain unclear. Secreted nucleoside diphosphate kinase A and B (NDPK) support breast cancer metastasis. Proteomic evidence confirms their presence in breast cancer-derived extracellular vesicles (EVs). We investigated the role of EV-associated NDPK in modulating the host microenvironment in favor of pre-metastatic niche formation. We measured NDPK expression and activity in EVs isolated from triple-negative breast cancer (MDA-MB-231) and non-tumorigenic mammary epithelial (HME1) cells using flow cytometry, western blot, and ATP assay. We evaluated the effects of EV-associated NDPK on endothelial cell migration, vascular remodeling, and metastasis. We further assessed MDA-MB-231 EV induced-proteomic changes in support of pre-metastatic lung niche formation. NDPK-B expression and phosphotransferase activity were enriched in MDA-MB-231 EVs that promote vascular endothelial cell migration and disrupt monolayer integrity. MDA-MB-231 EV-treated mice demonstrate pulmonary vascular leakage and enhanced experimental lung metastasis, whereas treatment with an NDPK inhibitor or a P2Y1 purinoreceptor antagonist blunts these effects. We identified perturbations to the purinergic signaling pathway in experimental lungs, lending evidence to support a role for EV-associated NDPK-B in lung pre-metastatic niche formation and metastatic outgrowth.
Decellularization enables functional analysis of ECM remodeling in planarian regeneration
Authors: Sonpho Ekasit , Mann Frederick G. , Levy Michaella , Ross Eric J. , Guerrero-Hernández Carlos , Florens Laurence , Saraf Anita , Doddihal Viraj , Ounjai Puey , Alvarado Alejandro Sánchez
Published: September 2020

The extracellular matrix (ECM) is a three-dimensional network of macromolecules that provides a microenvironment capable of supporting and regulating cell functions. However, only a few research organisms are available for the systematic dissection of the composition and functions of the ECM, particularly during regeneration. We utilized a free-living flatwormSchmidtea mediterraneato develop an integrative approach consisting of decellularization, proteomics, and RNA-interference (RNAi) to characterize and investigate ECM functions during tissue homeostasis and regeneration. High-quality ECM was isolated from planarians, and its matrisome profile was characterized by LC-MS/MS. The functions of identified ECM components were interrogated using RNAi. Using this approach, we discovered that heparan sulfate proteoglycan and kyphoscoliosis peptidase are essential for both tissue homeostasis and regeneration. Our strategy provides a robust experimental approach for identifying novel ECM components involved in regeneration that might not be discovered bioinformatically.
Real-time structural motif searching in proteins using an inverted index strategy
Authors: Bittrich Sebastian , Burley Stephen K. , Rose Alexander S.
Published: September 2020

Biochemical and biological functions of proteins are the product of both the overall fold of the polypeptide chain, and, typically, structural motifs made up of smaller numbers of amino acids constituting a catalytic center or a binding site. Detection of such structural motifs can provide valuable insights into the function(s) of previously uncharacterized proteins. Technically, this remains an extremely challenging problem because of the size of the Protein Data Bank (PDB) archive. Existing methods depend on a clustering by sequence similarity and can be computationally slow. We have developed a new approach that uses an inverted index strategy capable of analyzing>160,000 PDB structures with unmatched speed. The efficiency of the inverted index method depends critically on identifying the small number of structures containing the query motif and ignoring most of the structures that are irrelevant. Our approach (implemented atmotif.rcsb.org) enables real-time retrieval and superposition of structural motifs, either extracted from a reference structure or uploaded by the user. Herein, we describe the method and present five case studies that exemplify its efficacy and speed for analyzing 3D structures of both proteins and nucleic acids.

Author summary

The Protein Data Bank (PDB) provides open access to more than 160,000 three-dimensional structures of proteins, nucleic acids, and biological complexes. Similarities between PDB structures give valuable functional and evolutionary insights but such resemblance may not be evident at sequence or global structure level. Throughout the database, there are recurring structural motifs – groups of modest numbers of residues in proximity that, for example, support catalytic activity. Identification of common structural motifs can unveil subtle similarities between proteins and serve as fingerprints for configurations such as the His-Asp-Ser catalytic triad found in serine proteases or the zinc coordination site found in Zinc Finger DNA-binding domains. We present a highly efficient yet flexible strategy that allows users for the first time to search for arbitrary structural motifs across the entire PDB archive in real-time. Our approach scales favorably with the increasing number and complexity of deposited structures, and, also, has the potential to be adapted for other applications in a macromolecular context.
Loss of the Pioneer Factor FOXA1 Results in Genome-wide Epigenetic Reprogramming and activation of Interferon-Response Genes includingCD274/PD-L1
Authors: Hu Wenhuo , Yamashita Hironobu , Craig Jenna , Walter Vonn , Warrick Joshua I. , Al-Ahmadie Hikmat , DeGraff David J.
Published: September 2020

Forkhead Box A1 (FOXA1) is a pioneer transcription factor critical in epigenetic regulation of chromatin and cell fate determination. Reduced FOXA1 expression is an independent predictor of poor overall survival in bladder cancer patients. However, the impact ofFOXA1loss on chromatin epigenetics in bladder cancer is unknown. Therefore, we determined the impact ofFOXA1knock out (KO) on epigenetic modification of chromatin and associated gene expression. We identified 8,230 differentially expressed genes followingFOXA1KO. Surprisingly, Gene Set Enrichment Analysis (GSEA) identified IFNɑ/ɣ gene expression signatures as enriched followingFOXA1KO.FOXA1KO induced both increased and decreased numbers of histone 3 lysine 27 acetylation (H3K27ac) sites throughout the genome. As expected, the majority of differences in H3K27ac across genomic areas inFOXA1KO cells is mapped to intergenic and intronic regions where enhancers reside. In addition, a subset of differential H3K27ac levels were also mapped to proximal promoters and within gene bodies. Integrated analysis of RNA/ChIP-seq data shows changes in gene expression that are mirrored by differences in H3K27ac. Motif analysis of DNA sequence enriched for H3K27ac identified significant increases in transcription factor binding motifs including the interferon sensitive response element (ISRE) and interferon response factors such as IRF1. Moreover, we identified increased H3K27ac of regulatory elements as being associated with several upregulated interferon sensitive genes (ISGs) inFOXA1KO cells, includingCD274/PD-L1. Western blotting and Q-RT-PCR confirmed upregulation ofCD274/PD-L1 followingFOXA1KO. Analysis of TCGA data confirmed an inverse relationship betweenFOXA1andCD274in bladder cancer, as well as in other cancers. In summary, we provide evidence of widespread epigenetic reprogramming afterFOXA1KO in bladder cancer cells. Additionally, we provide evidence thatFOXA1KO-induced epigenetic changes contribute to activation of a global interferon-dominant expression signature, including the immune checkpoint targetCD274/PD-L1 in a cancer cell-intrinsic manner.
RNA sequence and structure determinants of Pol III transcriptional termination in human cells
Authors: Verosloff Matthew S. , Corcoran William K. , Dolberg Taylor B. , Leonard Joshua N. , Lucks Julius B.
Published: September 2020

The precise mechanism of transcription termination of the eukaryotic RNA polymerase III (Pol III) has been a subject of considerable debate. Although previous studies have clearly shown that at the end of RNA transcripts, tracts comprised of multiple uracils are required for Pol III termination, whether upstream RNA secondary structure in the nascent transcript is necessary for robust transcriptional termination is still subject to debate. We sought to address this directly through the development of anin celluloPol III transcription termination assay using a synthetic biology approach. Specifically, we utilized the recently developed Tornado expression system and a stabilized Corn RNA aptamer to create a Pol III-transcribed RNA that produces a detectable fluorescent signal when transcribed in human cells. To study the effects of RNA sequence and structure on Pol III termination, we systematically varied the sequence context upstream of the aptamer and identified sequence characteristics that enhance or diminish termination. We found that in the absence of predicted secondary structure, only poly-U tracts longer than then the average length found in the human genome (4–5 nucleotides), efficiently terminate Pol III transcription. We found that shorter poly-U tracts could induce termination when placed in proximity to secondary structural elements, while secondary structure by itself was not sufficient to induce termination. These findings demonstrate a key role for sequence and structural elements within Pol III-transcribed nascent RNA for efficient transcription termination, and demonstrate a generalizable assay for characterizing Pol III transcription in human cells.
A long-range chromatin interaction regulates SATB homeobox 1 gene expression in trophoblast stem cells
Authors: Yu Wei , Chakravarthi V. Praveen , Borosha Shaon , Ratri Anamika , Dalal Khyati , Wolfe Michael W. , Starks Rebekah R. , Tuteja Geetu , Rumi M.A. Karim
Published: September 2020

SATB homeobox proteins are important regulators of developmental gene expression. Among the stem cell lineages determined during early embryonic development, trophoblast stem (TS) cells exhibit robust SATB expression. Both SATB1 and SATB2 act to maintain trophoblast stem-state. However, the molecular mechanisms that regulate TS-specificSatbexpression are not yet known. We identifiedSatb1variant 2 as the predominant transcript in trophoblasts. Histone marks, and RNA polymerase II occupancy in TS cells indicated active state of the promoter. A novel cis-regulatory region with active histone marks was identified ∼21kbp upstream of variant 2 promoter. CRISPR/Cas9 mediated disruption of this sequence decreasedSatb1expression in TS cells and chromatin conformation capture confirmed looping of this regulatory region into the promoter. Scanning position weight matrices across the enhancer predicted two ELF5 binding sites in close vicinity of SATB1 sites, which were confirmed by chromatin immunoprecipitation. Knockdown of ELF5 downregulatedSatb1expression in TS cells and overexpression of ELF5 increased the enhancer-reporter activity. Interestingly, ELF5 interacts with SATB1 in TS cells, and the enhancer activity was upregulated following SATB overexpression. Our findings indicate that trophoblast-specificSatb1expression is regulated by long-range chromatin looping of an enhancer that interacts with ELF5 and SATB proteins.