bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of RNAlater™ as a field-compatible preservation method for metaproteomic analyses of bacteria-animal symbioses
Authors: Jensen Marlene , Wippler Juliane , Kleiner Manuel
Published: June 2021

Field studies are central to environmental microbiology and microbial ecology as they enable studies of natural microbial communities. Metaproteomics, the study of protein abundances in microbial communities, allows to study these communities ‘in situ’ which requires protein preservation directly in the field as protein abundance patterns can change rapidly after sampling. Ideally, a protein preservative for field deployment works rapidly and preserves the whole proteome, is stable in long-term storage, is non-hazardous and easy to transport, and is available at low cost. Although these requirements might be met by several protein preservatives, an assessment of their suitability in field conditions when targeted for metaproteomics is currently lacking. Here, we compared the protein preservation performance of flash freezing and the preservation solution RNAlater™ using the marine gutless oligochaete Olavius algarvensis and its symbiotic microbes as a test case. In addition, we evaluated long-term RNAlater™ storage after 1 day, 1 week and 4 weeks at room temperature (22-23 °C). We evaluated protein preservation using one dimensional liquid chromatography tandem mass spectrometry (1D-LC-MS/MS). We found that RNAlater™ and flash freezing preserved proteins equally well in terms of total number of identified proteins or relative abundances of individual proteins and none of the test time points were altered compared to t0. Moreover, we did not find biases against specific taxonomic groups or proteins with particular biochemical properties. Based on our metaproteomics data and the logistical requirements for field deployment we recommend RNAlater™ for protein preservation of field-collected samples when targeted for metaproteomcis.

Importance

Metaproteomics, the large-scale identification and quantification of proteins from microbial communities, provides direct insights into the phenotypes of microorganisms on the molecular level. To ensure the integrity of the metaproteomic data, samples need to be preserved immediately after sampling to avoid changes in protein abundance patterns. In laboratory set-ups samples for proteomic analyses are most commonly preserved by flash freezing; however, liquid nitrogen or dry ice is often unavailable at remote field locations due to its hazardous nature and transport restrictions. Our study shows that RNAlater™ can serve as a low hazard, easy to transport alternative to flash freezing for field preservation of samples for metaproteomics. We show that RNAlater™ preserves the metaproteome equally well as compared to flash freezing and protein abundance patterns remain stable during long-term storage for at least 4 weeks at room temperature.
Natian and Ryabhatta—graphical user interfaces to create, analyze and visualize single-cell transcriptomic datasets
Authors: Manivannan Sathiya N. , Garg Vidu
Published: June 2021

Single-cell transcriptomic analyses permit a high-resolution investigation of biological processes at the individual cell level. Single-cell transcriptomics technologies such as Drop-seq, Smart-seq, MARS-seq, sci-RNA-seq, and CELL-seq produce large volumes of data in the form of sequence reads. In general, the alignment of the reads to genomes and the enumeration of reads mapping to a specific gene results in a gene-count matrix. These gene-count matrix data require robust quality control and statistical analytical pipelines before data mining and interpretation. Among these post-alignment pipelines, the ‘Seurat’ package in ‘R’ is the most popular analytical pipeline for the analysis of single-cell data. This package provides quality control, normalization, principal component analysis, dimensional reduction, clustering, and marker identification among other functions needed to process and mine the single-cell transcriptomic data. While the Seurat package is continuously updated and includes a variety of functionalities, the user is still required to be proficient in the ‘R’ programming language and its data structures to be able to execute the Seurat functions. Hence, there is a demand for a graphical user interface (GUI) that takes in relevant input information and processes the single-cell data using the Seurat pipeline. A GUI will also highly improve the access to single-cell data for life sciences researchers who are not trained in the command-line operation of the ‘R’ platform. To meet this demand, we present R Shiny apps ‘Natian’ and ‘Ryabhatta’ to assist in the generation and analysis of Seurat files from a variety of different sources. The apps and example data can be downloaded from https://singlecelltranscriptomics.org. Natian allows users to create Seurat files from the output of multiple pipelines, integrate existing Seurat files, add metadata information, perform dimensional reduction analysis or upload dimensional reduction data, resume partially processed Seurat files and find cluster markers. Ryabhatta allows users to visualize gene expression using a variety of plotting options, analyze cluster markers, rename clusters, select cells from a graph or based on expression levels of markers, perform differential expression, count the number of cells in each condition, and perform pseudotime analysis using Monocle. We found that the use of these apps substantially improved the analytical and processing time and remove needless troubleshooting due to incompatible commands, typographical errors in scripts, and cluttering of the R environment with variables. We hope the use of these apps improves the use of single-cell data for life sciences research while also providing a tool to learn the functionalities of Seurat and R functions available for single-cell data analysis.
Novel LOTUS-domain proteins are organizational hubs that recruit C. elegans Vasa to germ granules
Authors: Cipriani P. Giselle , Bay Olivia , Zinno John , Gutwein Michelle , Gan Hin Hark , Mayya Vinay K. , Chung George , Chen Jia-Xuan , Fahs Hala , Guan Yu , Duchaine Thomas F. , Selbach Matthias , Piano Fabio , Gunsalus Kristin C.
Published: June 2021

We describe MIP-1 and MIP-2, novel paralogous C. elegans germ granule components that interact with the intrinsically disordered MEG-3 protein. These proteins promote P granule condensation, form granules independently of MEG-3 in the postembryonic germ line, and balance each other in regulating P granule growth and localization. MIP-1 and MIP-2 each contain two LOTUS domains and intrinsically disordered regions and form homo- and heterodimers. They bind and anchor the Vasa homolog GLH-1 within P granules and are jointly required for coalescence of MEG-3, GLH-1, and PGL proteins. Animals lacking MIP-1 and MIP-2 show temperature-sensitive embryonic lethality, sterility, and mortal germ lines. Germline phenotypes include defects in stem cell self-renewal, meiotic progression, and gamete differentiation. We propose that these proteins serve as scaffolds and organizing centers for ribonucleoprotein networks within P granules that help recruit and balance essential RNA processing machinery to regulate key developmental transitions in the germ line.
Teaching Python for Data Science: Collaborative development of a modular & interactive curriculum
Authors: Duda Marlena , Sovacool Kelly L. , Farzaneh Negar , Nguyen Vy Kim , Haynes Sarah E. , Falk Hayley , Furman Katherine L. , Walker Logan A. , Diao Rucheng , Oneka Morgan , Drotos Audrey C. , Woloshin Alana , Dotson Gabrielle A. , Kriebel April , Meng Lucy , Thiede Stephanie N. , Lapp Zena , Wolford Brooke N.
Published: June 2021

We are bioinformatics trainees at the University of Michigan who started a local chapter of Girls Who Code to provide a fun and supportive environment for high school women to learn the power of coding. Our goal was to cover basic coding topics and data science concepts through live coding and hands-on practice. However, we could not find a resource that exactly met our needs. Therefore, over the past three years, we have developed a curriculum and instructional format using Jupyter notebooks to effectively teach introductory Python for data science. This method, inspired by The Carpentries organization, uses bite-sized lessons followed by independent practice time to reinforce coding concepts, and culminates in a data science capstone project using real-world data. We believe our open curriculum is a valuable resource to the wider education community and hope that educators will use and improve our lessons, practice problems, and teaching best practices. Anyone can contribute to our educational materials on GitHub.
Incorporation of data from multiple hypervariable regions when analyzing bacterial 16S rRNA sequencing data
Authors: Jones Carli B. , White James R. , Ernst Sarah E. , Sfanos Karen S. , Peiffer Lauren B.
Published: June 2021

Short read 16S rRNA amplicon sequencing is a common technique used in microbiome research. However, inaccuracies in estimated bacterial community composition can occur due to amplification bias of the targeted hypervariable region. A potential solution is to sequence and assess multiple hypervariable regions in tandem, yet there is currently no consensus as to the appropriate method for analyzing this data. Additionally, there are many sequence analysis resources for data produced from the Illumina platform, but fewer open-source options available for data from the Ion Torrent platform. Herein, we present an analysis pipeline using an open-source analysis platform that integrates data from multiple hypervariable regions and is compatible with data produced from the Ion Torrent platform. We used the ThermoFisher Ion 16S™ Metagenomics Kit and a mock community of 20 bacterial strains to assess taxonomic classification of amplicons from 6 separate hypervariable regions (V2, V3, V4, V6-7, V8, V9) using our analysis pipeline. We report that different hypervariable regions have different specificities for taxonomic classification, which also had implications for global level analyses such as alpha and beta diversity. Finally, we utilize a generalized linear modeling approach to statistically integrate the results from multiple hypervariable regions and apply this methodology to data from a small clinical cohort. We conclude that scrutinizing sequencing results separately by hypervariable region provides a more granular view of the taxonomic classification achieved by each primer set as well as the concordance of results across hypervariable regions. However, the data across all hypervariable regions can be combined using generalized linear models to statistically evaluate overall differences in community structure and relatedness among sample groups.
Presynaptic Mitochondrial Volume and Density Scale with Presynaptic Power Demand
Authors: Justs Karlis A. , Lu Zhongmin , Chouhan Amit K. , Borycz Jolanta A. , Lu Zhiyuan , Meinertzhagen Ian A. , Macleod Gregory T.
Published: June 2021

Stable neural function requires an energy supply that can meet the intense episodic power demands of neuronal activity. The bioenergetic machinery of glycolysis and oxidative phosphorylation is highly responsive to such demands, but it must occupy a minimum volume if it is to accommodate these demands. We examined the trade-off between presynaptic power demands and the volume available to the bioenergetic machinery. We quantified the energy demands of sixDrosophilamotor nerve terminals through direct measurements of neurotransmitter release and Ca2+entry, and via theoretical estimates of Na+entry and power demands at rest. Electron microscopy revealed that terminals with the highest power demands contained the greatest volume of mitochondria, indicating that mitochondria are allocated according to presynaptic power demands. In addition, terminals with the greatest power demand-to-volume ratio (∼66 nmol·min-1·μL-1) harbor the largest mitochondria packed at the greatest density. If we assume sequential and complete oxidation of glucose by glycolysis and oxidative phosphorylation, then these mitochondria are required to produce ATP at a rate of 52 nmol·min-1·μL-1at rest, rising to 963 during activity. Glycolysis would contribute ATP at 0.24 nmol·min-1·μL-1of cytosol at rest, rising to 4.36. These data provide a quantitative framework for presynaptic bioenergeticsin situ, and reveal that, beyond an immediate capacity to accelerate ATP output from glycolysis and oxidative phosphorylation, over longer time periods presynaptic terminals optimize mitochondrial volume and density to meet power demand.

Significance Statement

The remarkable energy demands of the brain are supported by the complete oxidation of its fuel but debate continues regarding a division of labor between glycolysis and oxidative phosphorylation across different cell types. Here we leverage the neuromuscular synapse, a model for studying neurophysiology, to elucidate fundamental aspects of neuronal energy metabolism that ultimately constrain rates of neural processing. We quantified energy production rates required to sustain activity at individual nerve terminals and compared these data with the volume capable of oxidative phosphorylation (mitochondria) and glycolysis (cytosol). We find strong support for oxidative phosphorylation playing a primary role in presynaptic terminals and provide the firstin vivoestimates of energy production rates per unit volume of presynaptic mitochondria and cytosol.
Dynamic interplay between reward and voluntary attention determines stimulus processing in visual cortex
Authors: Grahek Ivan , Schettino Antonio , Koster Ernst H.W. , Andersen Søren K.
Published: June 2021

Reward enhances stimulus processing in the visual cortex, but the mechanisms through which this effect occurs remain unclear. Reward prospect can both increase the deployment of voluntary attention and increase the salience of previously neutral stimuli. In this study we orthogonally manipulated reward and voluntary attention while human participants performed a global motion detection task. We recorded steady-state visual evoked potentials (SSVEPs) to simultaneously measure the processing of attended and unattended stimuli linked to different reward probabilities, as they compete for attentional resources. The processing of the high rewarded feature was enhanced independently of voluntary attention, but this gain diminished once rewards were no longer available. Neither the voluntary attention nor the salience account alone can fully explain these results. Instead, we propose how these two accounts can be integrated to allow for the flexible balance between reward-driven increase in salience and voluntary attention.
Task-Dependent Warping of Semantic Representations During Search for Visual Action Categories
Authors: Shahdloo Mo , Çelik Emin , Ürgen Burcu A. , Gallant Jack L. , Çukur Tolga
Published: June 2021

Object and action perception in cluttered dynamic natural scenes relies on efficient allocation of limited brain resources to prioritize the attended targets over distractors. It has been suggested that during visual search for objects, distributed semantic representation of hundreds of object categories is warped to expand the representation of targets. Yet, little is known about whether and where in the brain visual search for action categories modulates semantic representations. To address this fundamental question, we studied human brain activity recorded via functional magnetic resonance imaging while subjects viewed natural movies and searched for either communication or locomotion actions. We find that attention directed to action categories elicits tuning shifts that warp semantic representations broadly across neocortex, and that these shifts interact with intrinsic selectivity of cortical voxels for target actions. These results suggest that attention serves to facilitate task performance during social interactions by dynamically shifting semantic selectivity towards target actions, and that tuning shifts are a general feature of conceptual representations in the brain.
PLEKHA5, PLEKHA6 and PLEKHA7 bind to PDZD11 to target the Menkes ATPase ATP7A to the cell periphery and regulate copper homeostasis
Authors: Sluysmans Sophie , Méan Isabelle , Xiao Tong , Boukhatemi Amina , Ferreira Flavio , Jond Lionel , Chang Christopher J. , Citi Sandra
Published: June 2021

Copper homeostasis is crucial for cellular physiology and development, and its dysregulation leads to disease. The Menkes ATPase ATP7A plays a key role in copper efflux, by trafficking from the Golgi to the plasma membrane upon cell exposure to elevated copper, but the mechanisms that target ATP7A to the cell periphery are poorly understood. PDZD11 interacts with the C-terminus of ATP7A, which contains sequences involved in ATP7A trafficking, but the role of PDZD11 in ATP7A localization is unknown. Here we identify PLEKHA5 and PLEKHA6 as new interactors of PDZD11, which similarly to the junctional protein PLEKHA7 bind to PDZD11 N-terminus through their WW domains. Using CRISPR-KO kidney epithelial cells, we show by immunofluorescence that WW-PLEKHAs (PLEKHA5, PLEKHA6, PLEKHA7) recruit PDZD11 to distinct plasma membrane localizations, and that they are required for the efficient anterograde targeting of ATP7A to the cell periphery in elevated copper. Pulldown experiments show that WW-PLEKHAs promote PDZD11 interaction with the C-terminus of ATP7A. However, WW-PLEKHAs and PDZD11 are not necessary for ATP7A Golgi localization in basal copper, ATP7A copper-induced exit from the Golgi, and ATP7A retrograde trafficking to the Golgi. Finally, measuring bioavailable copper with the labile copper probe CF4 shows that WW-PLEKHAs and PDZD11 are required to maintain low intracellular copper levels when cells are exposed to elevated copper. These data indicate that WW-PLEKHAs-PDZD11 complexes regulate the localization and function of ATP7A to modulate cellular copper homeostasis.
TMEM120A contains a specific coenzyme A-binding site and might not mediate poking- or stretch-induced channel activities in cells
Authors: Rong Yao , Jiang Jinghui , Gao Yiwei , Guo Jianli , Song Danfeng , Liu Wenhao , Zhao Yan , Xiao Bailong , Liu Zhenfeng
Published: June 2021

TMEM120A, a member of the Transmembrane protein 120 (TMEM120) family, has pivotal function in adipocyte differentiation and metabolism, and may also contribute to sensing mechanical pain by functioning as an ion channel named TACAN. Here we report that expression of TMEM120A is not sufficient in mediating poking- or stretch-induced currents in cells, and have solved cryo-EM structures of human TMEM120A (HsTMEM120A) in complex with an endogenous metabolic cofactor (coenzyme A, CoASH) and in the apo form. HsTMEM120A forms a symmetrical homodimer with each monomer containing an amino-terminal coiled-coil motif followed by a transmembrane domain with six membrane-spanning helices. Within the transmembrane domain, a CoASH molecule is hosted in a deep cavity and forms specific interactions with nearby amino acid residues. Mutation of a central tryptophan residue involved in binding CoASH dramatically reduced the binding affinity of HsTMEM120A with CoASH. HsTMEM120A exhibits distinct conformations at the states with or without CoASH bound. Our results suggest that TMEM120A may have alternative functional roles potentially involved in CoASH transport, sensing or metabolism.
Interpreting ciliopathy-associated missense variants of uncertain significance (VUS) in an animal model
Authors: Lange Karen I. , Best Sunayna , Tsiropoulou Sofia , Berry Ian , Johnson Colin A. , Blacque Oliver E.
Published: June 2021

Purpose

Better methods are required to interpret the pathogenicity of disease-associated variants of uncertain significance (VUS), which cannot be actioned clinically. In this study, we explore the use of a tractable animal model (Caenorhabditis elegans) for in vivo interpretation of missense VUS alleles of TMEM67, a cilia gene associated with ciliopathies.

Methods

CRISPR/Cas9 gene editing was used to generate homozygous knock-in C. elegans worm strains carrying TMEM67 patient variants engineered into the orthologous gene (mks-3). Quantitative phenotypic assays of sensory cilia structure and function measured if the variants affect mks-3 gene function. Results from worms were validated by a genetic complementation assay in a human TMEM67 knock-out hTERT-RPE1 cell line that tests a TMEM67 signaling function.

Results

Assays in C. elegans accurately distinguished between known benign (Asp359Glu, Thr360Ala) and known pathogenic (Glu361Ter, Gln376Pro) variants. Analysis of eight missense VUS generated evidence that three are benign (Cys173Arg, Thr176Ile, Gly979Arg) and five are pathogenic (Cys170Tyr, His782Arg, Gly786Glu, His790Arg, Ser961Tyr).

Conclusion

Efficient genome editing and quantitative functional assays in C. elegans make it a tractable in vivo animal model that allows rapid, cost-effective interpretation of ciliopathy-associated missense VUS alleles.
Zebrafish pigment cells develop directly from persistent highly multipotent progenitors
Authors: Nikaido Masataka , Subkhankulova Tatiana , Uroshlev Leonid A. , Kasianov Artem J. , Sosa Karen Camargo , Bavister Gemma , Yang Xueyan , Rodrigues Frederico S. L. M. , Carney Thomas J. , Schwetlick Hartmut , Dawes Jonathan H.P. , Rocco Andrea , Makeev Vsevelod , Kelsh Robert N.
Published: June 2021

Neural crest cells (NCCs) are highly multipotent stem cells. A long-standing controversy exists over the mechanism of NCC fate specification, specifically regarding the presence and potency of intermediate progenitors. The direct fate restriction (DFR) model, based on early in vivo clonal studies, hypothesised that intermediates are absent and that migrating cells maintain full multipotency1–6. However, most authors favour progressive fate restriction (PFR) models, with fully multipotent early NCCs (ENCCs) transitioning to partially-restricted intermediates before committing to individual fates7–12. Here, single cell transcriptional profiling of zebrafish pigment cell development leads to us proposing a Cyclical Fate Restriction mechanism of NCC development that reconciles the DFR and PFR models. Our clustering of single NCC Nanostring transcriptional profiles identifies only broadly multipotent intermediate states between ENCCs and differentiated melanocytes and iridophores. Leukocyte tyrosine kinase (Ltk) marks the multipotent progenitor and iridophores, consistent with biphasic ltk expression13–15. Ltk inhibitor and constitutive activation studies support expression at an early multipotent stage, whilst lineage-tracing of ltk-expressing cells reveals their multipotency extends beyond pigment cell-types to neural fates. We conclude that pigment cell development does not involve a conventional PFR mechanism, but instead occurs directly and more dynamically from a broadly multipotent intermediate state.
splatPop: simulating population scale single-cell RNA sequencing data
Authors: Azodi Christina B. , Zappia Luke , Oshlack Alicia , McCarthy Davis J.
Published: June 2021

With improving technology and decreasing costs, single-cell RNA sequencing (scRNA-seq) at the population scale has become more viable, opening up the doors to study functional genomics at the single-cell level. This development has lead to a rush to adapt bulk methods and develop new single-cell-specific methods and tools for computational analysis of these studies. Many single-cell methods have been tested, developed, and benchmarked using simulated data. However, current scRNA-seq simulation frameworks do not allow for the simulation of population-scale scRNA-seq data. Here, we present splatPop, a new Splatter model, for flexible, reproducible, and well documented simulation of population-scale scRNA-seq data with known expression quantitative trait loci (eQTL) effects. The splatPop model also allows for the simulation of complex batch effects, cell group effects, and conditional effects between individuals from different cohorts.
Transcription factor-like 5 is a potential DNA/RNA-binding protein essential for maintaining male fertility in mice
Authors: Xu Weiya , Zhang Yiyun , Qin Dongdong , Gui Yiqian , Wang Shu , Du Guihua , Yang Fan , Li Lufan , Yuan Shuiqiao , Wang Mei , Wu Xin
Published: June 2021

Tissue-specific transcription factors often play key roles in the development of specific cell lineages. Transcription factor-like 5 (TCFL5) is a testis-specific protein that contains the basic helix-loop-helix domain, although the in vivo functions of TCFL5 remain unknown. Herein, we generated CRISPR/Cas9-mediated knockout mice to dissect the function of TCFL5 in mouse testes. Surprisingly, we found that it was difficult to generate homozygous mice with the Tcfl5 deletion since the heterozygous males (Tcfl5+/−) were infertile. We did, however, observe markedly abnormal phenotypes of spermatids and spermatozoa in the testes and epididymides of Tcfl5+/− mice. Mechanistically, we demonstrated that TCFL5 transcriptionally regulated a set of genes participating in male germ cell development, which we uncovered via RNA-sequencing and TCFL5 ChIP-sequencing. We also found that TCFL5 interacted with RNA-binding proteins (RBPs) that regulated RNA processing, and further identified the fragile X mental retardation gene 1, autosomal homolog (FXR1, a known RBP) as an interacting partner of TCFL5 that may coordinate the transition and localization of TCFL5 in the nucleus. Collectively, we herein report for the first time that Tcfl5 is haploinsufficient in vivo and hypothesize that TCFL5 may be a dual-function protein that mediates DNA and RNA to regulate spermatogenesis.
The endoplasmic reticulum membrane protein complex localizes to the mitochondrial - endoplasmic reticulum interface and its subunits modulate phospholipid biosynthesis in Trypanosoma brucei
Authors: Iyer Advaitha , Niemann Moritz , Serricchio Mauro , Dewar Caroline E. , Oeljeklaus Silke , Farine Luce , Warscheid Bettina , Schneider André , Bütikofer Peter
Published: June 2021

The endoplasmic reticulum membrane complex (EMC) is a versatile complex that plays a key role in membrane protein biogenesis in the ER. Deletion of the complex has wide-ranging consequences including ER stress, disturbance in lipid transport and organelle tethering, among others. Here we report the function and organization of the evolutionarily conserved EMC (TbEMC) in the highly diverged eukaryote, Trypanosoma brucei. Using (co-) immunoprecipitation experiments in combination with mass spectrometry and whole cell proteomic analyses of parasites after depletion of select TbEMC subunits, we demonstrate that the TbEMC is composed of 9 subunits that are present in a high molecular mass complex localizing to the mitochondrial-endoplasmic reticulum interface. Knocking out or knocking down of single TbEMC subunits led to growth defects of T. brucei procyclic forms in culture. Interestingly, we found that depletion of individual TbEMC subunits lead to disruption of de novo synthesis of phosphatidylcholine (PC) or phosphatidylethanolamine (PE), the two most abundant phospholipid classes in T. brucei. Downregulation of TbEMC1 or TbEMC3 inhibited formation of PC while depletion of TbEMC8 inhibited PE synthesis, pointing to a role of the TbEMC in phospholipid synthesis. In addition, we found that in TbEMC7 knock-out parasites, TbEMC3 is released from the complex, implying that TbEMC7 is essential for the formation or the maintenance of the TbEMC.
MatrixQCvis: shiny-based interactive data quality exploration for omics data
Authors: Naake Thomas , Huber Wolfgang
Published: June 2021

Motivation

First-line data quality assessment and exploratory data analysis are integral parts of any data analysis workflow. In high-throughput quantitative omics experiments (e.g. transcriptomics, proteomics, metabolomics), after initial processing, the data are typically presented as a matrix of numbers (feature IDs × samples). Efficient and standardized data-quality metrics calculation and visualization are key to track the within-experiment quality of these rectangular data types and to guarantee for high-quality data sets and subsequent biological question-driven inference.

Results

We present MatrixQCvis, which provides interactive visualization of data quality metrics at the per-sample and per-feature level using R’s shiny framework. It provides efficient and standardized ways to analyze data quality of quantitative omics data types that come in a matrix-like format (features IDs × samples). MatrixQCvis builds upon the Bioconductor SummarizedExperiment S4 class and thus facilitates the integration into existing workflows.

Availability

MatrixQCVis is implemented in R. It is available via Bioconductor and released under the GPL v3.0 license.

Contact

thomas.naake@embl.de

Supplementary information

Supplementary Information is available at bioRxiv online.
Dual Dysfunction of Kir2.1 Underlies Conduction and Excitation-Contraction Coupling Defects Promoting Arrhythmias in a Mouse Model of Andersen-Tawil Syndrome Type 1
Authors: Macías Álvaro , González-Guerra Andrés , Moreno-Manuel Ana I. , Cruz Francisco M. , García-Quintáns Nieves , Gutiérrez Lilian K. , Roche-Molina Marta , Bermúdez-Jiménez Francisco , Andrés Vicente , Vera-Pedrosa María Linarejos , Martínez-Carrascoso Isabel , Bernal Juan A. , Jalife José
Published: June 2021

Andersen-Tawil Syndrome (ATS) is associated with life threatening arrhythmias of unknown mechanism. We report on a mouse model carrying the trafficking-deficient mutant Kir2.1Δ314-315. The mouse recapitulates the electrophysiological phenotype of type 1 (ATS1), with slower conduction velocities in response to flecainide, QT prolongation exacerbated by isoproterenol, and increased vulnerability to calcium-mediated arrhythmias resembling catecholaminergic polymorphic ventricular tachycardia (CPVT). Kir2.1Δ314-315 expression significantly reduced inward rectifier K+ and Na+ inward currents, depolarized resting membrane potential and prolonged action potential duration. Immunolocalization in wildtype cardiomyocytes and skeletal muscle cells revealed a novel sarcoplasmic reticulum (SR) microdomain of functional Kir2.1 channels contributing to intracellular Ca2+ homeostasis. Kir2.1Δ314-315 cardiomyocytes showed defects in SR Kir2.1 localization and function, which contributed to abnormal spontaneous Ca2+ release events. This is the first in-vivo demonstration of a dual arrhythmogenic mechanism of ATS1 defects in Kir2.1 channel function at the sarcolemma and the SR, with overlap between ATS1 and CPVT.
The predicted bZIP transcription factor ZIP-1 promotes resistance to intracellular infection in Caenorhabditis elegans
Authors: Lažetić Vladimir , Wu Fengting , Cohen Lianne B. , Reddy Kirthi C. , Chang Ya-Ting , Gang Spencer S. , Bhabha Gira , Troemel Emily R.
Published: June 2021

Defense against intracellular infection has been extensively studied in vertebrate hosts, but less is known about invertebrate hosts. For example, almost nothing is known about the transcription factors that induce defense against intracellular intestinal infection in the model nematode Caenorhabditis elegans. Two types of intracellular pathogens that naturally infect the C. elegans intestine are the Orsay virus, which is a positive-sense RNA virus, and microsporidia, which are fungal pathogens. Surprisingly, these molecularly distinct pathogens induce a common host transcriptional response called the Intracellular Pathogen Response (IPR). Here we describe zip-1 as an IPR regulator that functions downstream of all known IPR activating and regulatory pathways. zip-1 encodes a putative bZIP transcription factor of previously unknown function, and we show how zip-1 controls induction of a subset of genes upon IPR activation. ZIP-1 protein is expressed in the nuclei of intestinal cells, and is required in the intestine to upregulate IPR gene expression. Importantly, zip-1 promotes resistance to infection by the Orsay virus and by microsporidia in intestinal cells. Altogether, our results indicate that zip-1 represents a central hub for all triggers of the IPR, and that this transcription factor plays a protective role against intracellular pathogen infection in C. elegans.
Heparan Sulfate-dependent RAGE oligomerization is indispensable for pathophysiological functions of RAGE
Authors: Li Miaomiao , Ong Chih Yean , Langouët-Astrié Christophe J , Tan Lisi , Verma Ashwni , Yang Yimu , Zhang Xiaoxiao , Shah Dhaval K. , Schmidt Eric P. , Xu Ding
Published: June 2021

RAGE, a druggable inflammatory receptor, is known to function as an oligomer but the exact oligomerization mechanism remains poorly understood. Previously we have shown that heparan sulfate (HS) plays an active role in RAGE oligomerization. To understand the physiological significance of HS-induced RAGE oligomerization in vivo, we generated RAGE knock-in mice (RageAHA/AHA) by introducing point mutations to specifically disrupt HS–RAGE interaction. The RAGE mutant demonstrated normal ligand-binding but impaired capacity of HS-binding and oligomerization. Remarkably, RageAHA/AHA mice phenocopied Rage−/− mice in two different pathophysiological processes, namely bone remodeling and neutrophil-mediated liver injury, which demonstrates that HS-induced RAGE oligomerization is essential for RAGE signaling. Our findings suggest that it should be possible to block RAGE signaling by inhibiting HS-RAGE interaction. To test this, we generated a monoclonal antibody that targets the HS-binding site of RAGE. This antibody blocks RAGE signaling in vitro and in vivo, recapitulating the phenotype of RageAHA/AHA mice. By inhibiting HS-RAGE interaction genetically and pharmacologically, our work validated an alternative strategy to antagonize RAGE. Finally, we have performed RNA-seq analysis of neutrophils and lungs and found that while Rage−/− mice had a broad alteration of transcriptome in both tissues compared to wild-type mice, the changes of transcriptome in RageAHA/AHA mice were much more restricted. This unexpected finding suggests that by preserving the expression of RAGE protein (in a dominant-negative form), RageAHA/AHA mouse might represent a cleaner genetic model to study physiological roles of RAGE in vivo compared to Rage−/− mice.
Acentrosomal spindle assembly and stability in C. elegans oocytes requires a kinesin-12 non-motor microtubule interaction domain
Authors: Wolff Ian D. , Hollis Jeremy A. , Wignall Sarah M.
Published: June 2021

During the meiotic divisions in oocytes, microtubules are sorted and organized by motor proteins to generate a bipolar spindle in the absence of centrosomes [1]. In most organisms, kinesin-5 family members crosslink and slide microtubules to generate outward force that promotes acentrosomal spindle bipolarity [2–7]. However, the mechanistic basis for how other kinesin families act on acentrosomal spindles has not been explored. We investigated this question in C. elegans oocytes, where kinesin-5 is not required to generate outward force [8]. Instead, the kinesin-12 family motor KLP-18 performs this function [9–12]. KLP-18 acts with adaptor protein MESP-1 (meiotic spindle 1) to sort microtubule minus ends to the periphery of a microtubule array, where they coalesce into spindle poles [12]. If either of these proteins is depleted, outward sorting of microtubules is lost and minus ends converge to form a monoaster. Here we use a combination of in vitro biochemical assays and in vivo mutant analysis to provide insight into the mechanism by which these proteins collaborate to promote acentrosomal spindle assembly. We identify a microtubule binding site on the C-terminal stalk of KLP-18 and demonstrate that a direct interaction between the KLP-18 stalk and MESP-1 activates non-motor microtubule binding. We also provide evidence that this C-terminal domain is required for KLP-18 activity during spindle assembly and show that KLP-18 is continuously required to maintain spindle bipolarity. This study thus provides new insight into the construction and maintenance of the oocyte acentrosomal spindle as well as into kinesin-12 mechanism and regulation.
Exosomal TAR DNA binding protein 43 profile in canine model of amyotrophic lateral sclerosis: A preliminary study in developing blood-based biomarker for neurodegenerative diseases
Authors: Pfeiffer Penelope , Coates Joan R. , Esqueda Yajaira M. , Kennedy Andrew , Getchell Kyleigh , McLenon Myra , Kosa Edina , Agbas Abdulbaki
Published: June 2021

Objective

Blood-based biomarkers provide a crucial information in progress of neurodegenerative diseases with minimally invasive sampling method. Validated blood-based biomarker application in people with amyotrophic lateral sclerosis would derive numerous benefits. Canine degenerative myelopathy is a naturally occurring animal disease model to study the biology of human amyotrophic lateral sclerosis. Serum derived exosomes are potential carriers for cell-specific cargoes making them ideal venue to study biomarkers for a variety of diseases and biological processes. This study assessed the exosomal proteins that may be assigned as surrogate biomarker that may reflect biochemical changes in central nervous system.

Methods

Exosomes were isolated from canine serum using commercial exosome isolation reagents. Exosomes target proteins contents were analysed by Western blotting method.

Results

The profiles of potential biomarker candidates in spinal cord homogenate and that of serum-derived exosomes were found elevated in dogs with degenerative myelopathy as compare to control subjects.

Conclusions

Serum-derived exosomal biomolecules can serve as surrogate biomarkers in neuro degenerative diseases.

Key Messages

A canine with degenerative myelopathy can serve as a model animal to study human amyotrophic lateral sclerosis.

Serum-derived exosomes contains Transactive Response DNA Binding Protein 43 (TDP-43), potential biomarker candidate.

The levels of spinal cord TDP-43 proteins and that of serum-derived exosomes exhibited a similar profiling. Therefore, serum derived exosomes may be used as a venue for establishing blood-based biomarkers for neurodegenerative diseases.
Predicting microbiome compositions from species assemblages through deep learning
Authors: Michel-Mata Sebastian , Wang Xu-Wen , Liu Yang-Yu , Angulo Marco Tulio
Published: June 2021

Microbes can form complex communities that perform critical functions in maintaining the integrity of their environment or their hosts’ well-being. Rationally managing these microbial communities requires improving our ability to predict how different species assemblages affect the final species composition of the community. However, making such a prediction remains challenging because of our limited knowledge of the diverse physical, biochemical, and ecological processes governing microbial dynamics. To overcome this challenge, here we present a deep learning framework that automatically learns the map between species assemblages and community compositions from training data only, without knowledge of any of the above processes. First, we systematically validate our framework using synthetic data generated by classical population dynamics models. Then, we apply it to experimental data of both in vitro and in vivo communities, including ocean and soil microbial communities, Drosophila melanogaster gut microbiota, and human gut and oral microbiota. In particular, we show how our framework learns to perform accurate out-of-sample predictions of complex community compositions from a small number of training samples. Our results demonstrate how deep learning can enable us to understand better and potentially manage complex microbial communities.
Light-induction of endocannabinoids and activation of Drosophila TRPC channels
Authors: Sokabe Takaaki , Bradshaw Heather B. , Tominaga Makoto , Leishman Emma , Montell Craig
Published: June 2021

Drosophila phototransduction represents a classical model for signaling cascades that culminate with activation of TRP channels. TRP and TRPL are the canonical TRP (TRPC) channels, which are gated by light stimulation of rhodopsin and engagement of Gq and phospholipase Cβ (PLC). Despite decades of investigation, the mechanism of TRP activation in photoreceptor cells is unresolved. Here, using a combination of genetics, lipidomics and Ca2+ imaging, we found that light increased the levels of an abundant endocannabinoid, 2-linoleoyl glycerol (2-LG) in vivo. The elevation in 2-LG strictly depended on the PLC encoded by norpA. Moreover, this endocannabinoid upregulated TRPC-dependent Ca2+ influx in a heterologous expression system and in dissociated ommatidia from compound eyes. We propose that 2-LG is a physiologically relevant endocannabinoid that activates TRPC channels in photoreceptor cells.
Structural basis for the context-specific action of classic peptidyl transferase inhibitors
Authors: Syroegin Egor A. , Flemmich Laurin , Klepacki Dorota , Vazquez-Laslop Nora , Micura Ronald , Polikanov Yury S.
Published: June 2021

Ribosome-targeting antibiotics serve both as powerful antimicrobials and as tools for studying the ribosome. The ribosomal catalytic site, the peptidyl transferase center (PTC), is targeted by a large number of various drugs. The classical and best-studied PTC-acting antibiotic chloramphenicol, as well as the newest clinically significant linezolid, were considered indiscriminate inhibitors of every round of peptide bond formation, presumably inhibiting protein synthesis by stalling ribosomes at every codon of every gene being translated. However, it was recently discovered that chloramphenicol or linezolid, and many other PTC-targeting drugs, preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms and structural bases that underlie this phenomenon of context-specific action of even the most basic ribosomal antibiotics, such as chloramphenicol, are unknown. Here we present high-resolution structures of ribosomal complexes, with or without chloramphenicol, carrying specific nascent peptides that support or negate the drug action. Our data suggest that specific amino acids in the nascent chains directly modulate the antibiotic affinity to the ribosome by either establishing specific interactions with the drug molecule or obstructing its placement in the binding site. The model that emerged from our studies rationalizes the critical importance of the penultimate residue of a growing peptide for the ability of the drug to stall translation and provides the first atomic-level understanding of context specificity of antibiotics that inhibit protein synthesis by acting upon the PTC.
Electrodetection of small molecules by conformation-mediated signal enhancement
Authors: Murugappan Krishnan , Sundaramoorthy Uthayasuriya , Damry Adam M. , Nisbet David R. , Jackson Colin J. , Tricoli Antonio
Published: June 2021

Electrochemical biosensors allow the rapid, selective, and sensitive transduction of critical biological parameters into measurable signals. However, current electrochemical biosensors often fail to selectively and sensitively detect small molecules due to their small size and low molecular complexity. We have developed an electrochemical biosensing platform that harnesses the analyte-dependent conformational change of highly selective solute-binding proteins to amplify the signal generated by analyte binding. Using this platform, we constructed and characterized two biosensors that can sense leucine and glycine, respectively. We show that these biosensors can selectively and sensitively detect their targets over a wide range of concentrations – up to seven orders of magnitude – and that the selectivity of these sensors can be readily altered by switching the bioreceptor’s binding domain. Our work represents a new paradigm for the design of a family of modular electrochemical biosensors, where access to electrode surfaces can be controlled by protein conformational change.
Generation and transmission of inter-lineage recombinants in the SARS-CoV-2 pandemic
Authors: Jackson Ben , Boni Maciej F , Bull Matthew J , Colleran Amy , Colquhoun Rachel M , Darby Alistair , Haldenby Sam , Hill Verity , Lucaci Anita , McCrone John T , Nicholls Samuel , O’Toole Áine , Pacchiarini Nicole , Poplawski Radoslaw , Scher Emily , Todd Flora , Webster Hermione , Whitehead Mark , Wierzbicki Claudia ,   The COVID-19 Genomics UK (COG-UK) consortium, Loman Nicholas J , Connor Thomas R , Robertson David L , Pybus Oliver G , Rambaut Andrew
Published: June 2021

We present evidence for multiple independent origins of recombinant SARS-CoV-2 viruses sampled from late 2020 and early 2021 in the United Kingdom. Their genomes carry single nucleotide polymorphisms and deletions that are characteristic of the B.1.1.7 variant of concern, but lack the full complement of lineage-defining mutations. Instead, the remainder of their genomes share contiguous genetic variation with non-B.1.1.7 viruses circulating in the same geographic area at the same time as the recombinants. In four instances there was evidence for onward transmission of a recombinant-origin virus, including one transmission cluster of 45 sequenced cases over the course of two months. The inferred genomic locations of recombination breakpoints suggest that every community-transmitted recombinant virus inherited its spike region from a B.1.1.7 parental virus, consistent with a transmission advantage for B.1.1.7’s set of mutations.
Proteasome complexes experience profound structural and functional rearrangements throughout mammalian spermatogenesis
Authors: Živković Dušan , Sanchez Dafun Angelique , Menneteau Thomas , Schahl Adrien , Lise Sandrine , Kervarrec Christine , Toste Rêgo Ana , da Fonseca Paula C. A. , Chavent Matthieu , Pineau Charles , Burlet-Schiltz Odile , Marcoux Julien , Bousquet Marie-Pierre
Published: June 2021

During spermatogenesis, spermatogonia undergo a series of mitotic and meiotic divisions on their path to spermatozoa. To achieve this, a succession of processes requiring high proteolytic activity are in part orchestrated by the proteasome. The spermatoproteasome (s20S) is specific to the developing gametes, in which the gamete-specific α4s subunit replaces the α4 isoform found in the constitutive proteasome (c20S). Although the s20S is conserved across species and was shown to be crucial for germ cell development, its mechanism, function and structure remain incompletely characterized. Here, we used advanced mass spectrometry (MS) methods to map the composition of proteasome complexes and their interactomes throughout spermatogenesis. We observed that the s20S becomes highly activated as germ cells enter meiosis, mainly through a particularly extensive 19S activation and, to a lesser extent, PA200 binding. Additionally, the proteasome population shifts from c20S (98%) to s20S (>82-92%) during differentiation, presumably due to the shift from α4 to α4s expression. We demonstrated that s20S, but not c20S, interacts with components of the meiotic synaptonemal complex, where it may localize via association with the PI31 adaptor protein. In vitro, s20S preferentially binds to 19S, and displays higher trypsin- and chymotrypsin-like activities, both with and without PA200 activation. Moreover, using MS methods to monitor protein dynamics, we identified significant differences in domain flexibility between α4 and α4s. We propose that these differences induced by α4s incorporation result in significant changes in the way the s20S interacts with its partners, and dictate its role in germ cell differentiation.