bluuebunny/crossref_metadata_2025_split huggingface dataset:
Structural Basis for pre-tRNA Recognition and Processing by the Human tRNA Splicing Endonuclease Complex
Authors: Hayne Cassandra K. , Butay Kevin John U. , Stewart Zachary D. , Krahn Juno M. , Perera Lalith , Williams Jason G. , Petrovitch Robert M. , Deterding Leesa J. , Matera A. Gregory , Borgnia Mario J. , Stanley Robin E. 
Published: September 2022

AbstractAcross all walks of life, certain transfer RNA (tRNA) transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop (ASL). In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). The pathogenesis of PCH is poorly understood. Moreover, a lack of structures for any eukaryotic TSEN complex has hindered our understanding of tRNA recognition and processing. Here, we report Cryo-Electron Microscopy (cryo-EM) structures of the human TSEN•pre-tRNA complex, trapped in the pre-cleavage state, at near atomic resolution. These structures reveal the overall architecture of the complex, along with extensive tRNA binding interfaces within the complex. Although it shares structural homology with archaeal TSENs, the human TSEN complex contains additional features important for recognizing the acceptor stem and D-arm of the pre-tRNA. Our findings also establish the TSEN54 subunit as more than a simple molecular ruler; it functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits, TSEN2 and TSEN34. Finally, the human TSEN structures enable detailed visualization of the molecular environments of PCH-causing missense mutations, providing crucial insight into the mechanism of eukaryotic pre-tRNA splicing and neurodevelopmental disease.
FDA-Approved Drug Screening in Patient-Derived Organoids Demonstrates Potential of Drug Repurposing for Rare Cystic Fibrosis Genotypes
Authors: de Poel E. , Spelier S. , Hagemeijer M.C. , van Mourik P. , Suen S.W.F. , Vonk A.M. , Brunsveld J.E. , Ithakisiou G. N. , Kruisselbrink E. , Oppelaar H. , Berkers G. , de Winter-de Groot K.M. , Heida-Michel S. , Jans S.R. , van Panhuis H. , Bakker M. , van der Meer R. , Roukema J. , Dompeling E. , Weersink E.J.M. , Koppelman G.H. , Blaazer A.R. , Muijlwijk-Koezen J.E. , van der Ent C.K. , Beekman J.M. 
Published: September 2022

ABSTRACTBackgroundPreclinical cell-based assays that recapitulate human disease play an important role in drug repurposing. We previously developed a functional forskolin induced swelling (FIS) assay using patient-derived intestinal organoids (PDIOs), allowing functional characterization of CFTR, the gene mutated in people with cystic fibrosis (pwCF). CFTR function-increasing pharmacotherapies have revolutionized treatment for approximately 85% of people with CF, but a large unmet need remains to identify new treatments for all pwCF.MethodsWe used 76 non-homozygous F508del-CFTR PDIOs to test the efficacy of 1400 FDA-approved drugs on improving CFTR function, as measured in FIS assays.ResultsBased on the results of a secondary validation screen, we investigated CFTR elevating function of PDE4 inhibitors and currently existing CFTR modulators in further detail. We show that PDE4 inhibitors are potent CFTR function inducers in PDIOs and that CFTR modulator treatment rescues of CF genotypes that are currently not eligible for this therapy.ConclusionsThis study exemplifies the feasibility of high-throughput compound screening using PDIOs and we show the potential of repurposing drugs for pwCF that are currently not eligible for therapies.One-sentence SummaryWe screened 1400 FDA-approved drugs in CF patient-derived intestinal organoids using the previously established functional FIS assay, and show the potential of repurposing PDE4 inhibitors and CFTR modulators for rare CF genotypes.
A cryogenic, coincident fluorescence, electron and ion beam microscope
Authors: Boltje Daan B. , Hoogenboom Jacob P. , Jakobi Arjen J. , Jensen Grant J. , Jonker Caspar T.H. , Kaag Max J. , Koster Abraham J. , Last Mart G.F. , de Agrela Pinto Cecilia , Plitzko Jürgen M. , Raunser Stefan , Tacke Sebastian , Wang Zhexin , van der Wee Ernest B. , Wepf Roger , den Hoedt Sander 
Published: September 2022

AbstractCryogenic electron tomography (cryo-ET) combined with sub-tomogram averaging, allows in-situ visualisation and structure determination of macromolecular complexes at sub-nanometre resolution. Cryogenic focused ion beam (cryo-FIB) micromachining is used to prepare a thin lamella-shaped sample out of a frozen-hydrated cell for cryo-ET imaging, but standard cryo-FIB fabrication is blind to the precise location of the structure or proteins of interest. Fluorescence-guided focused ion beam (FIB) milling at target locations requires multiple sample transfers prone to contamination, and relocation and registration accuracy is often insufficient for 3D targeting. Here, we present in-situ fluoresence microscopy-guided FIB fabrication of a frozen-hydrated lamella to solve this problem: we built a coincident 3-beam cryogenic correlative microscope by retrofitting a compact cryogenic microcooler, custom positioning stage, and an inverted widefield fluorescence microscope (FM) on an existing focused ion-beam scanning electron microscope (FIB-SEM). We show FM controlled targeting at every milling step in the lamella fabrication process, validated with transmission electron microscope (TEM) tomogram reconstructions of the target regions. The ability to check the lamella during and after the milling process results in a higher success rate in the fabrication process and will increase the throughput of fabrication for lamellae suitable for high-resolution imaging.
PERK Kinase Activity is Regulated by Copper Binding-A New Regulatory Paradigm for Modulation of ER Stress Tolerance
Authors: Newton Sarah E. Bond , Beratan Noah R. , Akay-Espinoza Cagla , Bond Margaret K. , Shi Xinglong , Perhacs Julia , Gidalevitz Tali , Brady Donita C. , Jordan-Sciutto Kelly L. 
Published: September 2022

SummaryPERK is a kinase that, in response to ER stress, mediates duplicitous homeostatic and pro-apoptotic signaling. Thus, intricate regulation is required for physiologic function. Attempts to modulate PERK activity have shown that the determinants of adaptive vs. mal-adaptive signaling remain ambiguous. Here, with purified protein, we provide direct evidence that PERK binds copper, identify the residues that coordinate binding, and demonstrate that binding is necessary for kinase activity. Furthermore cellular PERK activity can be modulated via copper availability, and this regulatory relationship can be manipulated to dictate ER stress tolerance. Critically, these phenomenon translate to phenotypesin vivo, asC. elegansharboring a “copper-binding mutant” of PERK exhibit enhanced ER-stress sensitivity. The copper-PERK paradigm adds onto a new class of dynamic copper-binding enzymes and implicates that copper homeostasis, as a regulator of PERK, may constitute a previously unknown variable to resolve long-standing ambiguity in endeavors to therapeutically target PERK.
Chitosan-coated carboxylic acids show antimicrobial activity against antibiotic-resistant Gram-negative and positive pathogens
Authors: Cogan Tristan , James-Meyer Lynn 
Published: September 2022

AbstractAntibiotic resistance in bacteria is suggested to be the greatest risk to human health, but new agents are not being brought to market as the rapid evolution of resistance to them means that drug development costs cannot be recouped. Fatty acids have been proposed as a new generation of antibiotics, but toxicity and poor absorption has meant that their use has been impractical in the past. Chitosan has been used to encapsulate other agents as nanoparticles, but has not been used with fatty acids. Here we show that chitosan can be modified to direct fatty acids towards Gram-positive or negative bacteria so that they exert antimicrobial effects. We show that fatty acids work as effective antibiotics in vitro and in vivo, with activity against extremely drug resistant pathogens. Bacteria exposed to them do not develop resistance to these agents, and they are not toxic to mammalian cells. Activity was seen against salmonellosis andC. difficileinfection in animal models. Our results demonstrate that fatty acids formulated as chitosan nanoparticles are effective antibiotics, and can be used for a long period of time without resistance developing. This suggests that the usage of fatty acids coated in this manner could be sold in sufficient quantities to recoup its development costs, overcoming this barrier. These agents would form a new class of antibiotics, with the novel property of lack of bacterial resistance.
Environmental feedback drives oxidative stress response heterogeneity in bacterial populations
Authors: Choudhary Divya , Lagage Valentine , Foster Kevin , Uphoff Stephan 
Published: September 2022

ABSTRACTInduction of phenotypic heterogeneity is a ubiquitous consequence of bacterial stress responses. It is commonly postulated that isogenic cells exploit stochastic molecular fluctuations to generate phenotypic heterogeneity as a population survival strategy (termed bet-hedging). However, it is also possible that each cell attempts to maximise its own chances of survival. In that case, the apparent heterogeneity could either be caused by inevitable molecular noise or by underlying deterministic mechanisms which have escaped observation. Here, we investigated the sources and consequences of gene expression heterogeneity in the response of Escherichia coli cells to hydrogen peroxide (H2O2) stress. A machine-learning model accurately predicted the variable responses of individual cells growing in structured populations, indicating that the phenotypic heterogeneity has a deterministic origin. The model further showed that spatio-temporal dynamics in H2O2 concentration were dictated by the H2O2 scavenging capacity and morphology of cells in the local environment. Hence, oxidative stress response fluctuations were in fact the result of a precise and rapid feedback between each cell and its immediate environment. Although single cells optimise their individual responses, the formation of short-range H2O2 gradients by their scavenging activities increases stress tolerance at the population level and leads to spatial variation in mutation rates.
Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke,Schistosoma mansoni
Authors: Ittiprasert Wannaporn , Moescheid Max F. , Chaparro Cristian , Mann Victoria H. , Quack Thomas , Rodpai Rutchanee , Miller André , Wistiphongpun Prapakorn , Buakaew Watunyoo , Mentink-Kane Margaret , Schmid Sarah , Popratiloff Anastas , Grevelding Christoph G. , Grunau Christoph , Brindley Paul J. 
Published: September 2022

SummaryThe identification and characterization of genomic safe harbor sites (GSH) aims to facilitate consistent transgene activity without disruption to the host cell genome. We combined genome annotation and chromatin structure analysis by computational approach to predict the location of four GSHs in the human blood fluke,Schistosoma mansoni, a major infectious pathogen of the tropics. Introduction of a transgene into the egg of the parasite was accomplished using CRISPR/Cas-assisted homology-directed repair and overlapping guide RNAs. Gene editing efficiencies of 24% and transgene-encoded fluorescence of 75% of gene-edited schistosome eggs were observed. These outcomes advance functional genomics for schistosomes by providing a tractable path towards transgenic helminths using homology directed repair-catalyzed transgene insertion. This approach should be adaptable to helminths generally.MotivationFunctional genomics methods are needed in the arena of the Neglected Tropical Diseases, especially for helminth parasites, to facilitate basic and transformational studies to improve global public health. Gain-of-function phenotypes would be valuable in this context. Hence, the motivation for this investigation was to identify a genome safe harbor (GSH) site in the chromosomes of the human blood fluke,Schistosoma mansoniand, in a pilot approach, and to develop methods for transgene insertion and methods to characterize transgene performance following homology directed insertion at the schistosome GSH. The progress reported here can be expected to advance functional genomics for species of the Platyhelminthes – including model, free living species of planarians, and should be adaptable to helminths generally.
The spatiotemporal dynamics of semantic integration in the human brain
Authors: Murphy Elliot , Forseth Kiefer J. , Donos Cristian , Rollo Patrick S. , Tandon Nitin 
Published: September 2022

AbstractLanguage depends critically on the integration of lexical information to derive semantic concepts. The neurobiology of this fundamental aspect of human cognition remains mostly unknown. Limitations of spatiotemporal resolution have previously rendered it difficult to disentangle processes involved in semantic integration from independent computations such as tracking word meaning and establishing referents. We utilized intracranial recordings (n = 58) during the presentation of orthographic descriptions that were either referential or non-referential to a common object. Referential contexts enabled high frequency broadband gamma activation (70–150 Hz) of a distributed network including the inferior frontal sulcus (IFS), medial parietal cortex (MPC) and medial temporal lobe (MTL) in the left, language-dominant hemisphere. Components of this network (IFS, MPC), alongside posterior superior temporal sulcus, showed greater engagement for trials that did not progressively isolate a limited set of referents, relative to trials that did. IFS and posterior middle temporal gyrus activity was modulated by semantic coherence, regardless of whether the sentence picked out a common object. Centered around IFS and spreading out dorsally towards middle frontal gyrus and ventrally towards anterior/posterior inferior frontal gyrus, we uncovered a cortical mosaic of functional specialization for reference, coherence and semantic narrowing. Early processing windows implicated IFS in all aspects of semantics, and in later windows sub-portions of IFS exposed a clearer functional tessellation with some remaining overlap. This work unveils how lateral fronto-temporal regions make distinctly rich semantic contributions and are involved jointly in semantic integration and conflict resolution, depending on the sub-region and processing stage.
Immuno-targeting of Gram-positive Pathogens via a Cell Wall Binding Tick Antifreeze Protein
Authors: Dalesandro Brianna E. , Pires Marcos M. 
Published: September 2022

AbstractThe human immune system employs several mechanisms to defend against pathogenic bacteria. However, pathogenic bacterial cells have evolved means to counter these responses, rendering our immune system less effective. Immunological agents that supplement or modulate the host immune response have proven to have powerful therapeutic potential, although this modality is less explored against bacterial pathogens. We describe the application of a bacterial binding protein to re-engage the immune system towards pathogenic bacteria. More specifically, a hapten was conjugated to a protein expressed by Ixodes scapularis ticks, called Ixodes scapularis antifreeze glycoprotein (IAFGP), that has high affinity for the D-alanine residue on the peptidoglycan of the bacterial cell wall. We showed that a fragment of this protein retained high surface binding affinity. Moreover, when conjugated to a hapten this conjugate led to the display of haptens on the cell surface of vancomycin-resistant Enterococcus faecalis. Hapten display then induced the recruitment of antibodies and promoted immune-cell mediated uptake of bacterial pathogens. These results demonstrate the feasibility in using cell wall binding agents as the basis of a class of bacterial immunotherapies against bacterial pathogens.
Diversity and ecology of Caudoviricetes phages with genome terminal repeats in fecal metagenomes from four Dutch cohorts
Authors: Gulyaeva Anastasia , Garmaeva Sanzhima , Kurilshikov Alexander , Vich Vila Arnau , Riksen Niels P. , Netea Mihai G. , Weersma Rinse K. , Fu Jingyuan , Zhernakova Alexandra 
Published: September 2022

AbstractThe human gut harbors numerous viruses infecting the human host, microbes and other inhabitants of the gastrointestinal tract. Most of these viruses remain undiscovered, and their influence on human health is unknown. Here we characterize viral genomes in gut metagenomic data from 1,950 individuals from four population and patient cohorts. We focus on a subset of viruses that is highly abundant in the gut, remains largely uncharacterized, and allows confident complete genome identification – phages that belong to the class Caudoviricetes and possess genome terminal repeats. We detect 1,899 species-level units belonging to this subset, 19% of which do not have complete representative genomes in major public gut virome databases. These units display diverse genomic features, are predicted to infect a wide range of microbial hosts, and on average account for < 1% of metagenomic reads. Analysis of longitudinal data from 338 individuals shows that the composition of this fraction of the virome remained relatively stable over a period of 4 years. We also demonstrate that 54 species-level units are highly prevalent (detected in > 5% of individuals in a cohort). Finally, we find 34 associations between highly prevalent phages and human phenotypes, 24 of which can be explained by the relative abundance of potential hosts.
The mitochondrial ferredoxin-like is essential for the formation of complex I-containing respiratory supercomplexes in Arabidopsis thaliana
Authors: Röhricht Helene , Przybyla-Toscano Jonathan , Forner Joachim , Boussardon Clément , Keech Olivier , Rouhier Nicolas , Meyer Etienne H. 
Published: September 2022

AbstractIn eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of five multiprotein complexes (complexes I to V). Analyses of the OXPHOS system by native gel electrophoresis revealed an organization of OXPHOS complexes in supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mFDX-like). This protein was previously found associated with complex I, being part of the bridge domain linking the matrix and membrane arms of the complex. A phylogenetic analysis suggests that the Arabidopsis thaliana mFDX-like evolved from classical mitochondrial ferredoxin, but it lost one of the cysteines required for the coordination of the iron-sulfur (Fe-S) cluster essential for the electron transfer function of ferredoxins. Accordingly, our biochemical study shows that AtmFDX-like does not bind an Fe-S cluster, and is therefore unlikely to be involved in electron transfer reactions. To study the function of mFDX-like, we created deletion lines in Arabidopsis using a CRISPR/Cas9 approach. These lines do not show any growth phenotype under standard growth condition. However, the characterization of the OXPHOS system demonstrates that mFDX-like is important for the assembly of complex I, and essential for the formation of complex I-containing supercomplexes. We propose that mFDX-like and the bridge domain are required for the correct conformation of the membrane arm of complex I that is essential for the association of complex I with complex III to form supercomplexes.
Global mapping of RNA-chromatin contacts reveals a proximity-dominated connectivity model for ncRNA-gene interactions
Authors: Limouse Charles , Smith Owen K. , Jukam David , Fryer Kelsey A. , Greenleaf William J. , Straight Aaron F. 
Published: September 2022

AbstractNon-coding RNAs (ncRNAs) are transcribed throughout the genome and provide regulatory inputs to gene expression through their interaction with chromatin. Yet, the genomic targets and functions of most ncRNAs are unknown. Here we use chromatin-associated RNA sequencing (ChAR-seq) to map the global network of ncRNA interactions with chromatin in human embryonic stem cells, and the dynamic changes in interactions during differentiation into definitive endoderm. We uncover general principles governing the organization of the RNA- chromatin interactome, demonstrating that nearly all ncRNAs exclusively interact with genes in close three-dimensional proximity to their locus, and provide a model predicting the interactome. We uncover RNAs that interact with many loci across the genome, and unveil thousands of unannotated RNAs that dynamically interact with chromatin. By relating the dynamics of the interactome to changes in gene expression, we demonstrate that activation or repression of individual genes is unlikely to be controlled by a single ncRNA.
Distinguishing Signal from Noise: Understanding Patterns of Non-Detections to Inform Accurate Quantitative Metabarcoding
Authors: Gold Zachary , Shelton Andrew Olaf , Casendino Helen R. , Duprey Joe , Gallego Ramón , Van Cise Amy , Fisher Mary , Jensen Alexander J. , D’Agnese Erin , Allan Elizabeth Andruszkiewicz , Ramón-Laca Ana , Garber-Yonts Maya , Labare Michaela , Parsons Kim M. , Kelly Ryan P. 
Published: September 2022

AbstractCorrecting for amplification biases in genetic metabarcoding data can yield quantitative estimates of template DNA concentrations. However, a major source of uncertainty in metabarcoding data is the presence of non-detections, where a technical PCR replicate fails to detect a species observed in other replicates. Such non-detections are an important special case of variability among technical replicates in metabarcoding data, particularly in environmental samples. While many sampling and amplification processes underlie observed variation in metabarcoding data, understanding the causes of non-detections is an important step in distinguishing signal from noise in metabarcoding studies. Here, we use both simulated and empirical data to 1) develop a qualitative understanding of how non-detections arise in metabarcoding data, 2) outline steps to recognize uninformative data in practice, and 3) identify the conditions under which amplicon sequence data can reliably detect underlying biological signals. We show in both simulations and empirical data that, for a given species, the rate of non-detections among technical replicates is a function of both the template DNA concentration and species-specific amplification efficiency. Consequently, we conclude metabarcoding datasets are strongly affected by (1) deterministic amplification biases during PCR and (2) stochastic sampling of amplicons during sequencing — both of which we can model — but also by (3) stochastic sampling of rare molecules prior to PCR, which remains a frontier for quantitative metabarcoding. Our results highlight the importance of estimating species-specific amplification efficiencies and critically evaluating patterns of non-detection in metabarcoding datasets to better distinguish environmental signal from the noise inherent in molecular detections of rare targets.