bluuebunny/crossref_metadata_2025 huggingface dataset:
Deconvolution of Expression for Nascent RNA Sequencing Data (DENR) Highlights Pre-RNA Isoform Diversity in Human Cells
Authors: Zhao Yixin , Dukler Noah , Barshad Gilad , Toneyan Shushan , Danko Charles G. , Siepel Adam
Published: March 2021

Quantification of mature-RNA isoform abundance from RNA-seq data has been extensively studied, but much less attention has been devoted to quantifying the abundance of distinct precursor RNAs based on nascent RNA sequencing data. Here we address this problem with a new computational method called Deconvolution of Expression for Nascent RNA sequencing data (DENR). DENR models the nascent RNA read counts at each locus as a mixture of user-provided isoforms. The performance of the baseline algorithm is enhanced by the use of machine-learning predictions of transcription start sites (TSSs) and an adjustment for the typical “shape profile” of read counts along a transcription unit. We show using simulated data that DENR clearly outperforms simple read-count-based methods for estimating the abundances of both whole genes and isoforms. By applying DENR to previously published PRO-seq data from K562 and CD4+ T cells, we find that transcription of multiple isoforms per gene is widespread, and the dominant isoform frequently makes use of an internal TSS. We also identify > 200 genes whose dominant isoforms make use of different TSSs in these two cell types. Finally, we apply DENR and StringTie to newly generated PRO-seq and RNA-seq data, respectively, for human CD4+ T cells and CD14+ monocytes, and show that entropy at the pre-RNA level makes a disproportionate contribution to overall isoform diversity, especially across cell types. Altogether, DENR is the first computational tool to enable abundance quantification of pre-RNA isoforms based on nascent RNA sequencing data, and it reveals high levels of pre-RNA isoform diversity in human cells.
A novel heteromeric pantothenate kinase complex in apicomplexan parasites
Authors: Tjhin Erick T. , Howieson Vanessa M. , Spry Christina , van Dooren Giel G. , Saliba Kevin J.
Published: March 2021

Coenzyme A is synthesised from pantothenate via five enzyme-mediated steps. The first step is catalysed by pantothenate kinase (PanK). All PanKs characterised to date form homodimers. Many organisms express multiple PanKs. In some cases, these PanKs are not functionally redundant, and some appear to be non-functional. Here, we investigate the PanKs in two pathogenic apicomplexan parasites,Plasmodium falciparumandToxoplasma gondii. Each of these organisms express two PanK homologues (PanK1 and PanK2). We demonstrate thatPfPanK1 andPfPanK2 associate, forming a single, functional PanK complex that includes the multi-functional protein,Pf14-3-3I. Similarly, we demonstrate thatTgPanK1 andTgPanK2 form a single complex that possesses PanK activity. BothTgPanK1 andTgPanK2 are essential forT. gondiiproliferation, specifically due to their PanK activity. Our study constitutes the first examples of heteromeric PanK complexes in nature and provides an explanation for the presence of multiple PanKs within certain organisms.
A chemogenetic platform for controlling plasma membrane signaling and synthetic signal oscillation
Authors: Hatano Yuka , Suzuki Sachio , Nakamura Akinobu , Yoshii Tatsuyuki , Atsuta-Tsunoda Kyoko , Aoki Kazuhiro , Tsukiji Shinya
Published: March 2021

Chemogenetic methods that enable the rapid translocation of specific signaling proteins in living cells using small molecules are powerful tools for manipulating and interrogating intracellular signaling networks. However, existing techniques rely on chemically induced dimerization of two protein components and have certain limitations, such as a lack of reversibility, bioorthogonality, and usability. Here, by expanding our self-localizing ligand-induced protein translocation (SLIPT) approach, we have developed a versatile chemogenetic system for plasma membrane (PM)-targeted protein translocation. In this system, a novel engineeredEscherichia colidihydrofolate reductase in which a hexalysine (K6) sequence is inserted in a loop region (iK6DHFR) is used as a universal protein tag for PM-targeted SLIPT. Proteins of interest that are fused to theiK6DHFR tag can be specifically recruited from the cytoplasm to the PM within minutes by addition of a myristoyl-d-Cys-tethered trimethoprim ligand (mDcTMP). We demonstrated the broad applicability and robustness of this engineered protein–synthetic ligand pair as a tool for the conditional activation of various types of signaling molecules, including protein and lipid kinases, small GTPases, heterotrimeric G proteins, and second messengers. In combination with a competitor ligand and a culture-medium flow chamber, we further demonstrated the application of the system for chemically manipulating protein localization in a reversible and repeatable manner to generate synthetic signal oscillations in living cells. The present bioorthogonaliK6DHFR/mDcTMP-based SLIPT system affords rapid, reversible, and repeatable control of the PM recruitment of target proteins, offering a versatile and easy-to-use chemogenetic platform for chemical and synthetic biology applications.
Cost efficiency of institutional incentives in finite populations
Authors: Duong Manh Hong , Anh Han The
Published: March 2021

Institutions can provide incentives to increase cooperation behaviour in a population where this behaviour is infrequent. This process is costly, and it is thus important to optimize the overall spending. This problem can be mathematically formulated as a multi-objective optimization problem where one wishes to minimize the cost of providing incentives while ensuring a desired level of cooperation within the population. In this paper, we provide a rigorous analysis for this problem. We study cooperation dilemmas in both the pairwise (the Donation game) and multi-player (the Public Goods game) settings. We prove the regularity of the (total incentive) cost function, characterize its asymptotic limits (infinite population, weak selection and large selection) and show exactly when reward or punishment is more efficient. We prove that the cost function exhibits a phase transition phenomena when the intensity of selection varies. We calculate the critical threshold in regards to the phase transition and study the optimization problem when the intensity of selection is under and above the critical value. It allows us to provide an exact calculation for the optimal cost of incentive, for a given intensity of selection. Finally, we provide numerical simulations to demonstrate the analytical results. Overall, our analysis provides for the first time a selection-dependent calculation of the optimal cost of institutional incentives (for both reward and punishment) that guarantees a minimum amount of cooperation. It is of crucial importance for real-world applications of institutional incentives since intensity of selection is specific to a given population and the underlying game payoff structure.

In this Supplementary Information, we provide detailed calculations and proofs as well as illustrative figures for the analytical results in the main text of the present paper.
Chronic neuronal excitation leads to homeostatic suppression of structural long-term potentiation
Authors: Ueda Hiromi H. , Sato Aiko , Onda Maki , Murakoshi Hideji
Published: March 2021

Synaptic plasticity is long-lasting changes in synaptic currents and structure. When neurons are exposed to signals that induce aberrant neuronal excitation, they increase the threshold for the induction of synaptic plasticity, called homeostatic plasticity. To further understand the homeostatic regulation of synaptic plasticity and its molecular mechanisms, we investigated glutamate uncaging/photoactivatable (pa)CaMKII-dependent sLTP induction in hippocampal CA1 neurons after chronic neuronal excitation by GABAAreceptor antagonists. The neuronal excitation suppressed the glutamate uncaging-evoked Ca2+influx and failed to induce sLTP. Single-spine optogenetic stimulation using paCaMKII also failed to induce sLTP, suggesting that CaMKII downstream signaling is impaired in response to chronic neuronal excitation. Furthermore, while the inhibition of Ca2+influx was protein synthesis-independent, paCaMKII-induced sLTP depended on it. Our findings demonstrate that chronic neuronal excitation suppresses sLTP in two independent ways (i.e., the inhibitions of Ca2+influx and CaMKII downstream signaling), which may contribute to the robust neuronal protection in excitable environments.
Integrated single-cell transcriptomics and epigenomics reveals strong germinal center-associated etiology of autoimmune risk loci
Authors: King Hamish W , Wells Kristen L , Shipony Zohar , Kathiria Arwa S , Wagar Lisa E , Lareau Caleb , Orban Nara , Capasso Robson , Davis Mark M , Steinmetz Lars M , James Louisa K , Greenleaf William J
Published: March 2021

The germinal center (GC) response is critical for both effective adaptive immunity and establishing peripheral tolerance by limiting auto-reactive B cells. Dysfunction in these processes can lead to defects in immune response to pathogens or contribute to autoimmune disease. To understand the gene regulatory principles underlying the GC response, we generated a single-cell transcriptomic and epigenomic atlas of the human tonsil, a widely studied and representative lymphoid tissue. We characterize diverse immune cell subsets and build a trajectory of dynamic gene expression and transcription factor activity during B cell activation, GC formation, and plasma cell differentiation. We subsequently leverage cell type-specific transcriptomic and epigenomic maps to interpret potential regulatory impact of genetic variants implicated in autoimmunity, revealing that many exhibit their greatest regulatory potential in GC cell populations. Together, these analyses provide a powerful new cell type-resolved resource for the interpretation of cellular and genetic causes underpinning autoimmune disease.

One sentence summary

Single-cell chromatin accessibility landscapes of immune cell subsets reveal regulatory potential of autoimmune-associated genetic variants during the germinal center response.
Inhibition of the ubiquitin-proteasome system by a bioactivatable compound
Authors: Giovannucci Tatiana A. , Salomons Florian A. , Haraldsson Martin , Elfman Lotta H. M. , Wickström Malin , Young Patrick , Lundbäck Thomas , Eirich Jürgen , Altun Mikael , Jafari Rozbeh , Gustavsson Anna-Lena , Johnsen John Inge , Dantuma Nico P.
Published: March 2021

Malignant cells display an increased sensitivity towards drugs that reduce the function of the ubiquitin-proteasome system (UPS), which is the primary proteolytic system for destruction of aberrant proteins. Here, we report on the discovery of the bioactivatable compound CBK77, which causes an irreversible collapse of the UPS, accompanied by a general accumulation of ubiquitylated proteins and caspase-dependent cell death. CBK77 caused accumulation of ubiquitin-dependent, but not ubiquitin-independent, reporter substrates of the UPS, suggesting a selective effect on ubiquitin-dependent proteolysis. In a genome-wide CRISPR interference screen, we identified the redox enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) as a critical mediator of CBK77 activity, and further demonstrated its role as the compound bioactivator. Through affinity-based proteomics, we found that CBK77 covalently interacts with ubiquitin. In vitro experiments showed that CBK77-treated ubiquitin conjugates were less susceptible to disassembly by deubiquitylating enzymes.In vivoefficacy of CBK77 was validated by reduced growth of NQO1-proficient human adenocarcinoma cells in nude mice treated with CBK77. This first-in-class NQO1-activatable UPS inhibitor suggests that it may be possible to exploit the intracellular environment in malignant cells for leveraging the impact of compounds that impair the UPS.
Source imaging of high-density visual evoked potentials with multi-scale brain parcellations and connectomes
Authors: Pascucci David , Tourbier Sebastien , Rué-Queralt Joan , Carboni Margherita , Hagmann Patric , Plomp Gijs
Published: March 2021

We describe the multimodal neuroimaging dataset VEPCON (OpenNeuro Dataset ds003505). It includes raw data and derivatives of high-density EEG, structural MRI, diffusion weighted images (DWI) and single-trial behavior (accuracy, reaction time). Visual evoked potentials (VEPs) were recorded while participants (n=20) discriminated briefly presented faces from scrambled faces, or coherently moving stimuli from incoherent ones. EEG and MRI were recorded separately from the same participants. The dataset contains pre-processed EEG of single trials in each condition, behavioral measures, structural MRIs, individual brain parcellations at 5 spatial resolutions (83 to 1015 regions), and the corresponding structural connectomes computed from fiber count, fiber density, average fractional anisotropy and mean diffusivity maps. For source imaging, VEPCON provides EEG inverse solutions based on individual anatomy, with Python and Matlab scripts to derive activity time-series in each brain region, for each parcellation level. The BIDS-compatible dataset can contribute to multimodal methods development, studying structure-function relations, and to unimodal optimization of source imaging and graph analyses, among many other possibilities.
Cluster Analysis of SARS-CoV-2 Gene using Deep Learning Autoencoder: Gene Profiling for Mutations and Transitions
Authors: Miyake Jun , Sato Takaaki , Baba Shunsuke , Nakamura Hayao , Niioka Hirohiko , Nakazawa Yoshihisa
Published: March 2021

We report on a method for analyzing the variant of coronavirus genes using autoencoder. Since coronaviruses have mutated rapidly and generated a large number of genotypes, an appropriate method for understanding the entire population is required. The method using autoencoder meets this requirement and is suitable for understanding how and when the variants emarge and disappear. For the over 30,000 SARS-CoV-2 ORF1ab gene sequences sampled globally from December 2019 to February 2021, we were able to represent a summary of their characteristics in a 3D plot and show the expansion, decline, and transformation of the virus types over time and by region. Based on ORF1ab genes, the SARS-CoV-2 viruses were classified into five major types (A, B, C, D, and E in the order of appearance): the virus type that originated in China at the end of 2019 (type A) practically disappeared in June 2020; two virus types (types B and C) have emerged in the United States and Europe since February 2020, and type B has become a global phenomenon. Type C is only prevalent in the U.S. and is suspected to be associated with high mortality, but this type also disappeared at the end of June. Type D is only found in Australia. Currently, the epidemic is dominated by types B and E.
Alignment and Integration of Spatial Transcriptomics Data
Authors: Zeira Ron , Land Max , Raphael Benjamin J.
Published: March 2021

Spatial transcriptomics (ST) is a new technology that measures mRNA expression across thousands of spots on a tissue slice, while preserving information about the spatial location of spots. ST is typically applied to several replicates from adjacent slices of a tissue. However, existing methods to analyze ST data do not take full advantage of the similarity in both gene expression and spatial organization across these replicates. We introduce a new methodPASTE(Probabilistic Alignment of ST Experiments) to align and integrate ST data across adjacent tissue slices leveraging both transcriptional similarity and spatial distances between spots. First, we formalize and solve the problem of pairwise alignment of ST data from adjacent tissue slices, or layers, using Fused Gromov-Wasserstein Optimal Transport (FGW-OT), which accounts for variability in the composition and spatial location of the spots on each layer. From these pairwise alignments, we construct a 3D representation of the tissue. Next, we introduce the problem of simultaneous alignment and integration of multiple ST layers into a single layer with a low rank gene expression matrix. We derive an algorithm to solve the problem by alternating between solving FGW-OT instances and solving a Non-negative Matrix Factorization (NMF) of a weighted expression matrix. We show on both simulated and real ST datasets that PASTE accurately aligns spots across adjacent layers and accurately estimates a consensus expression matrix from multiple ST layers. PASTE outperforms integration methods that rely solely on either transcriptional similarity or spatial similarity, demonstrating the advantages of combining both types of information.

Code availability

Software is available athttps://github.com/raphael-group/paste
The spatial network structure of intertidal meiofaunal communities derived from environmental DNA metabarcoding surveys in Northwest Iberia
Authors: Bellisario Bruno , Fais Maria , Duarte Sofia , Vieira Pedro E. , Canchaya Carlos , Costa Filipe O.
Published: March 2021

The identification of the patterns and mechanisms behind species’ distribution is one of the major challenges in ecology, having also important outcomes for the conservation and management of ecosystems. This is especially true for those components of biodiversity providing essential ecosystem functions and for which standard surveys may underestimate their real taxonomic diversity due to their high degree of cryptic diversity and inherent diagnosis difficulties, such as meiofaunal communities. Environmental DNA (eDNA) metabarcoding may provide a fast and reliable way to refine and scale-up the characterization of biological diversity in complex environmental samples, allowing to bypass such drawbacks and increase the resolution of biodiversity estimates. Moreover, the possibility of integrating eDNA metabarcoding-derived data with tools and methods rooted in network theory would deepen the knowledge of the structuring processes of ecological communities in ways that cannot be predicted from studying individual species/communities in isolation. Here, a sediment eDNA metabarcoding of mitochondrial cytochrome c oxidase I (COI) and the nuclear hypervariable V4 region of the 18S rDNA (18S) was used to reconstruct the spatial networks of intertidal meiofaunal OTUs from three estuaries of North-Western Iberian Peninsula. Null models were used to identify the role of environmental and spatial constraints on the structure of COI- and 18S-derived spatial networks and to characterize the macroecological features of surveyed phyla. Our results show the feasibility of eDNA metabarcoding, not only to capture a fair amount of diversity hard to detect with standard surveys procedures, but also to identify hierarchical spatial structures in intertidal meiofaunal assemblages. This suggests that exclusivity of occurrence rather than pervasiveness appears to be the norm in meiofaunal organisms and that niche-based processes predominantly drive the spatial aggregation and contemporary distribution of meiofaunal phyla within the system.
Accurate reconstruction of dynamic gene expression and growth rate profiles from noisy measurements
Authors: Vidal Gonzalo , Vidal-Céspedes Carlos , Rudge Timothy J.
Published: March 2021

Cells face changing environments to which they sense and respond in complex ways, changing their rates of gene expression and growth. Measuring these dynamics is therefore essential to understanding natural and synthetic regulatory networks that give rise to functional phenotypes. However, reconstruction of gene expression and growth rate profiles from typically noisy measurements of cell populations is difficult due to the effects of noise at low cell densities among other factors. We present here a method for estimation of dynamic gene expression rates and biomass growth rates from noisy measurement data, and show that it is several times more accurate than current approaches. We applied our method to multiple promoter-reporter fusion genes. Gene expression rates of such promoter-reporter fusions are typically used as a proxy for transcription rates. However, using our method we show that fusion gene expression rate dynamics are determined at least by the promoter of interest and the downstream reporter.
L-Thyroxine improves vestibular compensation in a rat model of acute peripheral vestibulopathy
Authors: Rastoldo Guillaume , Marouane Emna , El Mahmoudi Nada , Pericat David , Watabe Isabelle , Lapotre Agnes , Tonetto Alain , López-Juárez Alejandra , Caron Philippe , Fraysse Marie-José Esteve , Zwergal Andreas , Tighilet Brahim
Published: March 2021

Unilateral vestibular lesions induce a vestibular syndrome, which recovers over time due to vestibular compensation. The therapeutic effect of L-Thyroxine (L-T4) on vestibular compensation was investigated by behavioral testing and immunohistochemical analysis in a rat model of unilateral vestibular neurectomy (UVN). We demonstrated that an acute L-T4 treatment reduced the vestibular syndrome and significantly promoted vestibular compensation. Thyroid hormone receptors (TRα and TRβ) and type II iodothyronine deiodinase (DIO2) were present in the vestibular nuclei (VN), supporting a local action of L-T4. We confirmed the T4-induced metabolic effects by demonstrating an increase in the number of cytochrome oxidase-labelled neurons in the VN three days after the lesion. L-T4 treatment modulated glial reaction by decreasing both microglia and oligodendrocytes in the deafferented VN three days after UVN and increased cell proliferation. The survival of newly generated cells was not affected, but neuronal differentiation was altered by the L-T4 treatment.
No-U-Turn sampling for phylogenetic trees
Authors: Wahle Johannes
Published: March 2021

The inference of phylogenetic trees from sequence data has become a staple in evolutionary research. Bayesian inference of such trees is predominantly based on the Metropolis-Hastings algorithm. For high dimensional and correlated data this algorithm is known to be inefficient. There are gradient based algorithms to speed up such inference. Building on recent research which uses gradient based approaches for the inference of phylogenetic trees in a Bayesian framework, I present an algorithm which is capable of performing No-U-Turn sampling for phylogenetic trees. As an extension to Hamiltonian Monte Carlo methods, No-U-Turn sampling comes with the same benefits, such as proposing distant new states with a high acceptance probability, but eliminates the need to manually tune hyper parameters. Evaluated on real data sets, the new sampler shows that it converges faster to the target distribution. The results also indicate that a higher number of topologies are traversed during sampling by the new algorithm in comparison to traditional Markov Chain Monte Carlo approaches. This new algorithm leads to a more efficient exploration of the posterior distribution of phylogenetic tree topologies.

Author summary

Phylogentic trees are important for our understanding of evolutionary relationships. But even for only a small number of entities the number of possible trees is immense. In order to efficiently search through this large space and analyze the distributions of these trees, different algorithmic solutions have been proposed. A phylogenetic tree is not only defined by the way it groups the entities but also by the length of the branches. This nature of the phylogentic trees complicates the exploration of these distributions. Building on research and algorithmic ideas for non tree like data and recent developments on the mathematics of trees, I propose a new method which is able to traverse the space of possible trees more rapidly. By using a search strategy which is guided by the data and the underlying evolutionary model, the algorithm is able to better sample from the desired distribution. I am able to show that the new algorithm can indeed analyze the correct distribution but can do so with a higher efficiency than other algorithms.
Vegfr3-tdTomato, a reporter mouse for microscopic visualization of lymphatic vessel by multiple modalities
Authors: Redder Esther , Kirschnick Nils , Hägerling René , Hansmeier Nils , Kiefer Friedemann
Published: March 2021

Lymphatic vessels are indispensable for tissue fluid homeostasis, transport of solutes and dietary lipids and immune cell trafficking. In contrast to blood vessels, which are easily visible by their erythrocyte cargo, lymphatic vessels are not readily detected in the tissue context. Their invisibility interferes with the analysis of the three-dimensional lymph vessel structure in large tissue volumes and hampers dynamic intravital studies on lymphatic function and pathofunction. An approach to overcome these limitations are mouse models, which express transgenic fluorescent proteins under the control of tissue-specific promotor elements.

We introduce here the BAC-transgenic mouse reporter strainVegfr3-tdTomato that expresses a membrane-tagged version of tdTomato under control of Flt4 regulatory elements.Vegfr3-tdTomato mice inherited the reporter in a mendelian fashion and showed selective and stable fluorescence in the lymphatic vessels of multiple organs tested, including lung, kidney, heart, diaphragm, intestine, mesentery and dermis. In this model, tdTomato expression was sufficient for direct visualisation of lymphatic vessels by epifluorescence microscopy. Furthermore, lymph vessels were readily visualized using a number of microscopic modalities including confocal laser scanning, light sheet fluorescence and two-photon microscopy. Due to the early onset of VEGFR-3 expression in venous embryonic vessels and the short maturation time of tdTomato, this reporter offers an interesting alternative to Prox1-promoter driven lymphatic reporter mice for instance to study the developmental differentiation of venous to lymphatic endothelial cells.
A graph-based cell tracking algorithm with few manually tunable parameters and automated segmentation error correction
Authors: Löffler Katharina , Scherr Tim , Mikut Ralf
Published: March 2021

Automatic cell segmentation and tracking enables to gain quantitative insights into the processes driving cell migration. To investigate new data with minimal manual effort, cell tracking algorithms should be easy to apply and reduce manual curation time by providing automatic correction of segmentation errors. Current cell tracking algorithms, however, are either easy to apply to new data sets but lack automatic segmentation error correction, or have a vast set of parameters that needs either manual tuning or annotated data for parameter tuning. In this work, we propose a tracking algorithm with only few manually tunable parameters and automatic segmentation error correction. Moreover, no training data is needed. We compare the performance of our approach to three well-performing tracking algorithms from the Cell Tracking Challenge on data sets with simulated, degraded segmentation – including false negatives, over- and under-segmentation errors. Our tracking algorithm can correct false negatives, over- and under-segmentation errors as well as a mixture of the aforementioned segmentation errors. On data sets with under-segmentation errors or a mixture of segmentation errors our approach performs best. Moreover, without requiring additional manual tuning, our approach ranks several times in the top 3 on the 6thedition of the Cell Tracking Challenge.
Growth-Rate Dependent And Nutrient-Specific Gene Expression Resource Allocation In Fission Yeast
Authors: Kleijn Istvan T. , Martínez-Segura Amalia , Bertaux François , Saint Malika , Kramer Holger , Shahrezaei Vahid , Marguerat Samuel
Published: March 2021

Cellular resources are limited and their relative allocation to gene expression programmes determines physiological states and global properties such as the growth rate. Quantitative studies using various growth conditions have singled out growth rate as a major physiological variable explaining relative protein abundances. Here, we used the simple eukaryoteSchizosaccharomyces pombeto determine the importance of growth rate in explaining relative changes in protein and mRNA levels during growth on a series of non-limiting nitrogen sources. Although half of fission yeast genes were significantly correlated with the growth rate, this came alongside wide-spread nutrient-specific regulation. Proteome and transcriptome often showed coordinated regulation but with notable exceptions, such as metabolic enzymes. Genes positively correlated with growth rate participated in every level of protein production with the notable exception of RNA polymerase II, whereas those negatively correlated mainly belonged to the environmental stress response programme. Critically, metabolic enzymes, which represent ∼55-70% of the proteome by mass, showed mainly condition-specific regulation. Specifically, many enzymes involved in glycolysis and NAD-dependent metabolism as well as the fermentative and respiratory pathways were condition-dependent and not consistently correlated with growth. In summary, we provide a rich account of resource allocation to gene expression in a simple eukaryote, advancing our basic understanding of the interplay between growth-rate dependent and nutrient-specific gene expression.
OCRbayes: A Bayesian hierarchical modeling framework for Seahorse extracellular flux oxygen consumption rate data analysis
Authors: Zhang Xiang , Yuan Taolin , Keijer Jaap , de Boer Vincent C. J.
Published: March 2021

Background

Mitochondrial dysfunction is involved in many complex diseases. Efficient and accurate evaluation of mitochondrial functionality is crucial for understanding pathology as well as facilitating novel therapeutic developments. As a popular platform, Seahorse extracellular flux (XF) analyzer is widely used for measuring mitochondrial oxygen consumption rate (OCR) in living cells. A hidden feature of Seahorse XF OCR data is that it has a complex data structure, caused by nesting and crossing between measurement cycles, wells and plates. Surprisingly, statistical analysis of Seahorse XF data has not received sufficient attention, and current methods completely ignore the complex data structure, impairing the robustness of statistical inference.

Results

To rigorously incorporate the complex structure into data analysis, here we developed a Bayesian hierarchical modeling framework, OCRbayes, and demonstrated its applicability based on analysis of published data sets.

Conclusions

We showed that OCRbayes can analyze Seahorse XF OCR experimental data derived from either single or multiple plates. Moreover, OCRbayes has potential to be used for diagnosing patients with mitochondrial diseases.
Timing of increased temperature sensitivity coincides with nervous system development in winter moth embryos
Authors: van Dis Natalie E. , van der Zee Maurijn , Hut Roelof A. , Wertheim Bregje , Visser Marcel E.
Published: March 2021

Climate change is rapidly altering the environment and many species will need to genetically adapt their seasonal timing to keep up with these changes. Insect development rate is largely influenced by temperature, but we know little about the mechanisms underlying temperature sensitivity of development. Here we investigate seasonal timing of egg hatching in the winter moth, one of the few species which has been found to genetically adapt to climate change, likely through selection on temperature sensitivity of egg development rate. To study when during development winter moth embryos are most sensitive to changes in ambient temperature, we gave eggs an increase or decrease in temperature at different moments during their development. We measured their developmental progression and timing of egg hatching, and used fluorescence microscopy to construct a timeline of embryonic development for the winter moth. We found that egg development rate responded more strongly to temperature once embryos were in the fully extended germband stage. This is the phylotypic stage at which all insect embryos have developed a rudimentary nervous system. Furthermore, at this stage timing of ecdysone signaling determines developmental progression, which could act as an environment dependent gateway. Intriguingly, this may suggest that, from the phylotypic stage onward, insect embryos can start to integrate internal and environmental stimuli to actively regulate important developmental processes. As we found evidence that there is genetic variation for temperature sensitivity of egg development rate in our study population, such regulation could be a target of selection imposed by climate change.
Metagenomic analysis reveals the signature of gut microbiota associated with human chronotypes
Authors: Carasso Shaqed , Fishman Bettina , Lask Liel Stelmach , Shochat Tamar , Geva-Zatorsky Naama , Tauber Eran
Published: March 2021

Patterns of diurnal activity differ substantially between individuals, with early risers and late sleepers being examples of opposite chronotypes. Growing evidence suggests that the late chronotype significantly impacts the risk of developing mood disorders, obesity, diabetes, and other chronic diseases. Despite the vast potential of utilizing chronotype information for precision medicine, those factors that shape chronotypes remain poorly understood. Here, we assessed whether the various chronotypes are associated with different gut microbiome compositions. Using metagenomic sequencing analysis, we established a distinct signature associated with chronotype based on two bacterial genera,Alistipes(elevated in “larks”) andLachnospira(elevated in “owls”). We identified three metabolic pathways associated with the early chronotype, and linked distinct dietary patterns with different chronotypes. Our work demonstrates an association between the gut microbiome and chronotype and may represent the first step towards developing dietary interventions aimed at ameliorating the deleterious health correlates of the late chronotype.
DNA spike-ins enable confident interpretation of SARS-CoV-2 genomic data from amplicon-based sequencing
Authors: Lagerborg Kim A. , Normandin Erica , Bauer Matthew R. , Adams Gordon , Figueroa Katherine , Loreth Christine , Gladden-Young Adrianne , Shaw Bennett , Pearlman Leah , Shenoy Erica S. , Hooper David , Pierce Virginia M. , Zachary Kimon C. , Park Daniel J. , MacInnis Bronwyn L. , Lemieux Jacob E. , Sabeti Pardis C. , Reilly Steven K , Siddle Katherine J.
Published: March 2021

The rapid global spread and continued evolution of SARS-CoV-2 has highlighted an unprecedented need for viral genomic surveillance and clinical viral sequencing. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine lab processes and results. This challenge will only increase with expanding global production of sequences by diverse research groups for epidemiological and clinical interpretation. We present an approach which uses synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination through a sequencing workflow. Applying this approach to the ARTIC Consortium’s amplicon design, we define a series of best practices for Illumina-based sequencing and provide a detailed characterization of approaches to increase sensitivity for low-viral load samples incorporating the SDSIs. We demonstrate the utility and efficiency of the SDSI method amidst a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases.
Persistent ventricle partitioning in the adult zebrafish heart
Authors: Pfefferli Catherine , Moran Hannah R. , Felker Anastasia , Mosimann Christian , Jaźwińska Anna
Published: March 2021

The vertebrate heart integrates cells from the early-differentiating first heart field (FHF) and the later-differentiating second heart field (SHF) emerging from the lateral plate mesoderm. In mammals, this process forms the basis for the development of the left and right ventricle chambers and subsequent chamber septation. The single ventricle-forming zebrafish heart also integrates FHF and SHF lineages during embryogenesis, yet the contributions of these two myocardial lineages to the adult zebrafish heart remain incompletely understood. Here, we characterize the myocardial labeling of FHF descendants in both the developing and adult zebrafish ventricle. Expanding previous findings, late gastrulation-stage labeling usingdrl-driven CreERT2 recombinase with a myocardium-specific,myl7-controlledloxPreporter results in predominant labeling of FHF-derived outer curvature and the right side of the embryonic ventricle. Raised to adulthood, such lineage-labeled hearts retain broad areas of FHF cardiomyocytes in a region of the ventricle that is positioned at the opposite side to the atrium and encompasses the apex. Our data add to the increasing evidence for a persisting cell-based compartmentalization of the adult zebrafish ventricle even in the absence of any physical boundary.
Binary interactome models of inner- versus outer-complexome organisation
Authors: Lambourne Luke , Yadav Anupama , Wang Yang , Desbuleux Alice , Kim Dae-Kyum , Cafarelli Tiziana , Pons Carles , Kovács István A. , Jailkhani Noor , Schlabach Sadie , Ridder David De , Luck Katja , Bian Wenting , Shen Yun , Yang Zhipeng , Mee Miles W. , Helmy Mohamed , Jacob Yves , Lemmens Irma , Rolland Thomas , Coté Atina G. , Gebbia Marinella , Kishore Nishka , Knapp Jennifer J. , Mellor Joseph C. , Reimand Jüri , Tavernier Jan , Cusick Michael E. , Falter-Braun Pascal , Spirohn Kerstin , Zhong Quan , Aloy Patrick , Hao Tong , Charloteaux Benoit , Roth Frederick P. , Hill David E. , Calderwood Michael A. , Twizere Jean-Claude , Vidal Marc
Published: March 2021

Hundreds of different protein complexes that perform important functions across all cellular processes, collectively comprising the “complexome” of an organism, have been identified1. However, less is known about the fraction of the interactome that exists outside the complexome, in the “outer-complexome”. To investigate features of “inner”- versus outer-complexome organisation in yeast, we generated a high-quality atlas of binary protein-protein interactions (PPIs), combining three previous maps2–4and a new reference all-by-all binary interactome map. A greater proportion of interactions in our map are in the outer-complexome, in comparison to those found by affinity purification followed by mass spectrometry5–7or in literature curated datasets8–11. In addition, recent advances in deep learning predictions of PPI structures12mirror the existing experimentally resolved structures in being largely focused on the inner complexome and missing most interactions in the outer-complexome. Our new PPI network suggests that the outer-complexome contains considerably more PPIs than the inner-complexome, and integration with functional similarity networks13–15reveals that interactions in the inner-complexome are highly detectable and correspond to pairs of proteins with high functional similarity, while proteins connected by more transient, harder-to-detect interactions in the outer-complexome, exhibit higher functional heterogeneity.
Cis-regulatory variants affect gene expression dynamics in yeast
Authors: Shih Ching-Hua , Fay Justin C.
Published: March 2021

Evolution ofcis-regulatory sequences depends on how they effect gene expression and motivates both the identification and prediction ofcis-regulatory variants responsible for expression differences within and between species. While much progress has been made in relatingcis-regulatory variants to expression levels, the timing of gene activation and repression may also be important to the evolution ofcis-regulatory sequences. We investigated allele-specific expression (ASE) dynamics within and betweenSaccharomycesspecies during the diauxic shift and found appreciablecis-acting variation in gene expression dynamics. Within species ASE is associated with intergenic variants, but ASE dynamics are more strongly associated with insertions and deletions than ASE levels. To refine these associations we used a high-throughput reporter assay to test promoter regions and individual variants. Within the subset of regions that recapitulated endogenous expression we identified and characterizedcis-regulatory variants that affect expression dynamics. Between species, chimeric promoter regions generate novel patterns and indicate constraints on the evolution of gene expression dynamics. We conclude that changes incis-regulatory sequences can tune gene expression dynamics and that the interplay between expression dynamics and other aspects expression are relevant to the evolution ofcis-regulatory sequences.
Complementary resource preferences spontaneously emerge in diauxic microbial communities
Authors: Wang Zihan , Goyal Akshit , Dubinkina Veronika , George Ashish B. , Wang Tong , Fridman Yulia , Maslov Sergei
Published: March 2021

Many microbes grow diauxically, utilizing the available resources one at a time rather than simultaneously. The properties of communities of microbes growing diauxically remain poorly understood, largely due to a lack of theory and models of such communities. Here, we develop and study a minimal model of diauxic microbial communities assembling in a serially diluted culture. We find that unlike co-utilizing communities, diauxic community assembly repeatably and spontaneously leads to communities with complementary resource preferences, i.e., communities where species prefer different resources as their top choice. Simulations and theory explain that the emergence of complementarity is driven by the disproportionate contribution of the top choice resource to the growth of a diauxic species. Additionally, we develop a geometric approach for analyzing serially diluted communities, with or without diauxie, which intuitively explains several additional emergent community properties, such as the apparent lack of species which grow fastest on a resource other than their most preferred resource. Overall, our work provides testable predictions for the assembly of natural as well as synthetic communities of diauxically shifting microbes.
An egg-derived sulfated N-Acetyllactosamine glycan is an antigenic decoy of influenza virus vaccines
Authors: Guthmiller Jenna J. , Utset Henry A. , Henry Carole , Li Lei , Zheng Nai-Ying , Sun Weina , Vieira Marcos Costa , Zost Seth , Huang Min , Hensley Scott E. , Cobey Sarah , Palese Peter , Wilson Patrick C.
Published: March 2021

Influenza viruses grown in eggs for the purposes of vaccine generation often acquire mutations during egg adaptation or possess differential glycosylation patterns than viruses circulating amongst humans. Here, we report that seasonal influenza virus vaccines possess an egg-derived sulfated N-acetyllactosamine (LacNAc) that is an antigenic decoy. Half of subjects that received an egg-grown vaccine mounted an antibody response against this egg-derived antigen. Egg-binding monoclonal antibodies specifically bind viruses grown in eggs, but not viruses grown in other chicken derived cells, suggesting only egg-grown vaccines can induce anti-LacNAc antibodies. Notably, antibodies against the sulfated LacNAc utilized a restricted antibody repertoire and possessed features of natural antibodies, as most antibodies were IgM and have simple heavy chain complementarity determining region 3. By analyzing a public dataset of influenza virus vaccine induced plasmablasts, we discovered egg-binding public clonotypes that were shared across studies. Together, this study shows that egg-grown vaccines can induce antibodies against an egg-associated glycan, which may divert the host immune response away from protective epitopes.
Understanding C4photosynthesis in Setaria by a proteomic and kinetic approach
Authors: Calace Paula , Tonetti Tomás , Margarit Ezequiel , Figueroa Carlos M. , Lobertti Carlos , Andreo Carlos S. , Wheeler Mariel C. Gerrard , Saigo Mariana
Published: March 2021

Plants performing C4photosynthesis have a higher productivity per crop area related to an optimized use of water and nutrients. This is achieved through a series of anatomical and biochemical features that allow the concentration of CO2around RuBisCO. In C4plants the photosynthetic reactions are distributed between two cell types, they initially fix the carbon to C4acids within the mesophyll cells (M) and then transport these compounds to the bundle sheath cells (BS), where they are decarboxylated so that the resulting CO2is incorporated into the Calvin cycle (CC).

This work is focused on the comparative analysis of the proteins present in M and BS ofSetaria viridis, a C4model close relative of several major feed, fuel, and bioenergy grasses. The integration of kinetic and proteomic approaches agrees that the C4compound malate is mainly decarboxylated in the chloroplasts of BS cells by NADP-malic enzyme (NADP-ME). Besides, NAD-malic enzyme (NAD-ME) located in the mitochondria could also contribute to the C4carbon shuttle. We presented evidence of metabolic strategies that involve chloroplastic, mitochondrial and peroxisomal proteins to avoid the leakage of C4intermediates in order to sustain an efficient photosynthetic performance.

Highlight

Proteomic and kinetic analyses show metabolic strategies involving chloroplastic, mitochondrial and peroxisomal proteins to maintain the C4cycle performance in parallel to other metabolic pathways.
A novel, open source, low-cost bioreactor for load-controlled cyclic loading of tendon explants
Authors: Pedaprolu Krishna , Szczesny Spencer E
Published: March 2021

A major risk factor for tendinopathy is tendon overuse (i.e., fatigue loading). Fatigue loading of tendon damages the extracellular matrix and induces tissue degeneration. However, the specific mechanisms linking tendon fatigue damage with tissue degeneration are unclear. While explant models of tendon fatigue loading have been used to address this knowledge gap, they predominantly employ bioreactors that apply cyclic displacements/strains rather than loads/stresses, which are more physiologically relevant. This is because of the technical complexity and cost of building a load-controlled bioreactor, which requires multiple motors, load cells, and computationally intensive feedback loops. Here, we present a novel, low-cost, load-controlled bioreactor that applies cyclic loading to multiple tendon explants by offloading weights from a single motorized stage. Using an optional load cell, we validated that the bioreactor can effectively provide load-controlled fatigue testing of mouse and rat tendon explants while maintaining tissue viability. Furthermore, all the design files, bill of materials, and operating software are available “open source” (https://github.com/Szczesnytendon/Bioreactor) so that anyone can easily manufacture and use the bioreactor for their own research. Therefore, this novel load-controlled bioreactor will enable researchers to study the mechanisms driving fatigue-induced tendon degeneration in a more physiologically relevant and cost-effective manner.