bluuebunny/crossref_metadata_2025_split huggingface dataset:
ORC1 binds tocis-transcribed RNAs for efficient activation of replication origins
Authors: Mas Aina Maria , Goñi Enrique , de los Mozos Igor Ruiz , Arcas Aida , Statello Luisa , Gonzalez Jovanna , Blázquez Lorea , Chelsea Lee Wei Ting , Gupta Dipika , Sejas Álvaro , Hoshina Shoko , Armaos Alexandros , Tartaglia Gian Gaetano , Waga Shou , Ule Jernej , Rothenberg Eli , Gómez María , Huarte Maite 
Published: March 2023

AbstractCells must coordinate the activation of thousands of replication origins dispersed throughout their genome. Active transcription is known to favor the formation of mammalian origins, although the role that RNA plays in this process remains unclear. We show that the ORC1 subunit of the human Origin Recognition Complex interacts with RNAs transcribed from genes with origins in their transcription start sites (TSSs), displaying a positive correlation between RNA binding and origin activity. RNA depletion, or the use of ORC1 RNA-binding mutant, result in inefficient activation of proximal origins, linked to impaired ORC1 chromatin release. ORC1 RNA binding activity resides in its intrinsically disordered region, involved in intra- and inter-molecular interactions, regulation by phosphorylation, and phase-separation. We show that RNA binding favors ORC1 chromatin release, by regulating its phosphorylation and subsequent degradation. We propose that fluctuating concentrations of RNA during the cell cycle may play a sequential role in controlling origins through interaction with this flexible region of ORC1. Our results unveil a novel non-coding function of RNA as a dynamic component of the chromatin, orchestrating the activation of replication origins.One sentence summaryThe human origin recognition complex subunit 1 ORC1, binds to RNAs transcribed from genes with origins of replication at the TSS, which is required for optimal origin activation.
Smart Lattice Light Sheet Microscopy for imaging rare and complex cellular events
Authors: Shi Yu , Tabet Jimmy S. , Milkie Daniel E. , Daugird Timothy A. , Yang Chelsea Q. , Giovannucci Andrea , Legant Wesley R. 
Published: March 2023

AbstractLight sheet microscopes enable rapid, high-resolution imaging of biological specimens; however, biological processes span a variety of spatiotemporal scales. Moreover, long-term phenotypes are often instigated by rare or fleeting biological events that are difficult to capture with a single imaging modality and constant imaging parameters. To overcome this limitation, we present smartLLSM, a microscope that incorporates AI-based instrument control to autonomously switch between epifluorescent inverted imaging and lattice light sheet microscopy. We apply this technology to two major scenarios. First, we demonstrate that the instrument provides population-level statistics of cell cycle states across thousands of cells on a coverslip. Second, we show that by using real-time image feedback to switch between imaging modes, the instrument autonomously captures multicolor 3D datasets or 4D time-lapse movies of dividing cells at rates that dramatically exceed human capabilities. Quantitative image analysis on high-content + high-throughput datasets reveal kinetochore and chromosome dynamics in dividing cells and determine the effects of drug perturbation on cells in specific mitotic stages. This new methodology enables efficient detection of rare events within a heterogeneous cell population and records these processes with high spatiotemporal 4D imaging over statistically significant replicates.
ROS-dependent palmitoylation is an obligate licensing modification for GSDMD pore formation
Authors: Du Gang , Healy Liam B. , David Liron , Walker Caitlin , Fontana Pietro , Dong Ying , Devant Pascal , Puthenveetil Robbins , Ficarro Scott B. , Banerjee Anirban , Kagan Jonathan C. , Lieberman Judy , Wu Hao 
Published: March 2023

AbstractGasdermin D (GSDMD) is the common effector for cytokine secretion and pyroptosis downstream of inflammasome activation by forming large transmembrane pores upon cleavage by inflammatory caspases. Here we report the surprising finding that GSDMD cleavage is not sufficient for its pore formation. Instead, GSDMD is lipidated by S-palmitoylation at Cys191 upon inflammasome activation, and only palmitoylated GSDMD N-terminal domain (GSDMD-NT) is capable of membrane translocation and pore formation, suggesting that palmitoylation licenses GSDMD activation. Treatment by the palmitoylation inhibitor 2-bromopalmitate and alanine mutation of Cys191 abrogate GSDMD membrane localization, cytokine secretion, and cell death, without affecting GSDMD cleavage. Because palmitoylation is formed by a reversible thioester bond sensitive to free thiols, we tested if GSDMD palmitoylation is regulated by cellular redox state. Lipopolysaccharide (LPS) mildly and LPS plus the NLRP3 inflammasome activator nigericin markedly elevate reactive oxygen species (ROS) and GSDMD palmitoylation, suggesting that these two processes are coupled. Manipulation of cellular ROS by its activators and quenchers augment and abolish, respectively, GSDMD palmitoylation, GSDMD pore formation and cell death. We discover that zDHHC5 and zDHHC9 are the major palmitoyl transferases that mediate GSDMD palmitoylation, and when cleaved, recombinant and partly palmitoylated GSDMD is 10-fold more active in pore formation than bacterially expressed, unpalmitoylated GSDMD, evidenced by liposome leakage assay. Finally, other GSDM family members are also palmitoylated, suggesting that ROS stress and palmitoylation may be a general switch for the activation of this pore-forming family.One-Sentence SummaryGSDMD palmitoylation is induced by ROS and required for pore formation
Interpreting Generative Adversarial Networks to Infer Natural Selection from Genetic Data
Authors: Riley Rebecca , Mathieson Iain , Mathieson Sara 
Published: March 2023

AbstractUnderstanding natural selection in humans and other species is a major focus for the use of machine learning in population genetics. Existing methods rely on computationally intensive simulated training data. Unlike efficient neutral coalescent simulations for demographic inference, realistic simulations of selection typically requires slow forward simulations. Because there are many possible modes of selection, a high dimensional parameter space must be explored, with no guarantee that the simulated models are close to the real processes. Mismatches between simulated training data and real test data can lead to incorrect inference. Finally, it is difficult to interpret trained neural networks, leading to a lack of understanding about what features contribute to classification.Here we develop a new approach to detect selection that requires relatively few selection simulations during training. We use a Generative Adversarial Network (GAN) trained to simulate realistic neutral data. The resulting GAN consists of a generator (fitted demographic model) and a discriminator (convolutional neural network). For a genomic region, the discriminator predicts whether it is “real” or “fake” in the sense that it could have been simulated by the generator. As the “real” training data includes regions that experienced selection and the generator cannot produce such regions, regions with a high probability of being real are likely to have experienced selection. To further incentivize this behavior, we “fine-tune” the discriminator with a small number of selection simulations. We show that this approach has high power to detect selection in simulations, and that it finds regions under selection identified by state-of-the art population genetic methods in three human populations. Finally, we show how to interpret the trained networks by clustering hidden units of the discriminator based on their correlation patterns with known summary statistics. In summary, our approach is a novel, efficient, and powerful way to use machine learning to detect natural selection.
Elastin Recoil is Driven by the Hydrophobic Effect
Authors: Jamhawi Nour M. , Koder Ronald L. , Wittebort Richard J. 
Published: March 2023

AbstractElastin is an extracellular matrix material found in all vertebrates. Its reversible elasticity, robustness and low stiffness are essential for the function of arteries, lungs, and skin. It is among the most resilient elastic materials known: During a human lifetime, arterial elastin undergoes in excess of 2x109stretching/contracting cycles without replacement and slow oxidative hardening has been identified as a limiting factor on human lifespan. For over fifty years, the mechanism of entropic recoil has been controversial. Herein, we report a combined NMR and thermomechanical study that establishes the hydrophobic effect as the primary driver of elastin function. Water ordering at the solvent:protein interface was observed as a function of stretch using double quantum2H NMR and the most extensive thermodynamic analysis performed to date was obtained by measuring elastin length and volume as a function of force and temperature in normal water, heavy water and with co-solvents. When stretched, elastin’s heat capacity increases, water is ordered proportional to the degree of stretching, the internal energy decreases, and heat is released in excess of the work performed. These properties show that recoil in elastin under physiological conditions is primarily driven by the hydrophobic effect rather than by configurational entropy as is the case for rubber. Consistent with this conclusion are decreases in the thermodynamic signatures when co-solvents that alter the hydrophobic effect are introduced. We propose that hydrophobic effect-driven recoil, as opposed to a configurational entropy mechanism, where hardening from crystallization can occur, is the origin of elastin’s unusual resilience.SignificanceElastin, found in tissues that require reversible elasticity, has low stiffness and great resiliency. It is a self-assembled material that has been a target for regenerative medicine. However, the basis for its elasticity has been controversial for more than 50 years. Formed from a hydrophobic protein with an equivalent mass of water, the controversy is whether recoil is driven by entropy gain of the protein and/or the water. We demonstrate that matrix water is progressively ordered upon stretching and that the thermodynamics of elastin recoil are those of the hydrophobic effect and different from those of rubber. We conclude that recoil is primarily driven by the hydrophobic effect and suggest that this accounts for elastin’s low stiffness and high resilience.
Inactivation of phosphodiesterase-4B gene in rat nucleus accumbens shell by CRISPR/Cas9 modulates the motivation to chronically self-administer nicotine
Authors: Sharp Burt M , Jiang Qin , Kim Panjun , Chen Hao 
Published: March 2023

AbstractLarge scale human genome wide association studies (GWAS) have identified a growing pool of genes associated with cigarette smoking. One of the most prominent, phosphodiesterase-4B (PDE4B), has been associated with multiple smoking phenotypes. Although PDE4B modulates the half-life of neuronal cAMP, its precise role in smoking behaviors is unknown. To address this knowledge gap, we used a reverse translational approach. We inactivatedPDE4Bin bilateral medial nucleus accumbens shell (NAcs) neurons by injecting AAV containing a specific gRNA in female transgenic Cas9+ Long Evans rats. These rats then were given 23-hour chronic access to nicotine intravenous self-administration (IVSA) under a schedule of increasing fixed ratios (FR). With the increased effort required at FR7, nicotine SA (i.e. active presses and drug infusions) declined significantly in controls, whereas it was maintained in the mutagenized group. A progressive ratio (PR) study also showed significantly greater cumulative nicotine infusions in the mutant group. Hence, we hypothesized that enhanced PDE4B protein activity would reduce nicotine IVSA. A positive allosteric modulator,2-(3-(4-chloro-3-fluorophenyl)-5-ethyl-1H-1,2,4-triazol-1-yl)-N-(3,5-dichlorobenzyl)acetamide (MR-L2), was microinfused into NAcs bilaterally at FR3 or FR5; in both cohorts, MR-L2 acutely reduced nicotine IVSA. In summary, these studies show that the activity of PDE4B regulates the capacity of NAcs to maintain nicotine IVSA in face of the cost of increasing work. This finding and the results of the PR study indicate that PDE4B affects the motivation to obtain nicotine. These reverse translational studies in rats provide insight into the motivational effects of NAcs PDE4B that advance our understanding of the smoking behaviors mapped in human GWAS.
Temporal Coordination of the Transcription Factor Response to H2O2stress
Authors: Jose Elizabeth , March-Steinman Woody , Wilson Bryce A. , Shanks Lisa , Paek Andrew L. 
Published: March 2023

AbstractThe p53 and FOXO transcription factors (TFs) share many similarities despite their distinct evolutionary origins. Both TFs are activated by a variety of cellular stresses and upregulate genes in similar pathways including cell-cycle arrest and apoptosis. Oxidative stress from excess H2O2activates both FOXO1 and p53, yet whether they are activated at the same time is unclear. Here we found that cells respond to high H2O2levels in two temporal phases. In the first phase FOXO1 rapidly shuttles to the nucleus while p53 levels remain low. In the second phase FOXO1 exits the nucleus and p53 levels rise. We found that other oxidative stress induced TFs are activated in the first phase with FOXO1 (NF-kB, NFAT1), or the second phase with p53 (NRF2, JUN) but not both following H2O2 stress. The two TF phases result in large differences in gene expression patterns. Finally, we provide evidence that 2-Cys peroxiredoxins control the timing of the TF phases in response to H2O2.
POTENTIATION OF CORTICO-SPINAL OUTPUT VIA TARGETED ELECTRICAL STIMULATION OF THE MOTOR THALAMUS
Authors: Ho Jonathan C. , Grigsby Erinn M. , Damiani Arianna , Liang Lucy , Balaguer Josep-Maria , Kallakuri Sridula , Barrios-Martinez Jessica , Karapetyan Vahagn , Fields Daryl , Gerszten Peter C. , Hitchens T. Kevin , Constantine Theodora , Adams Gregory M. , Crammond Donald J. , Capogrosso Marco , Gonzalez-Martinez Jorge A. , Pirondini Elvira 
Published: March 2023

AbstractCerebral white matter lesions prevent cortico-spinal descending inputs from effectively activating spinal motoneurons, leading to loss of motor control. However, in most cases, the damage to cortico-spinal axons is incomplete offering a potential target for new therapies aimed at improving volitional muscle activation. Here we hypothesized that, by engaging direct excitatory connections to cortico-spinal motoneurons, stimulation of the motor thalamus could facilitate activation of surviving cortico-spinal fibers thereby potentiating motor output. To test this hypothesis, we identified optimal thalamic targets and stimulation parameters that enhanced upper-limb motor evoked potentials and grip forces in anesthetized monkeys. This potentiation persisted after white matter lesions. We replicated these results in humans during intra-operative testing. We then designed a stimulation protocol that immediately improved voluntary grip force control in a patient with a chronic white matter lesion. Our results show that electrical stimulation targeting surviving neural pathways can improve motor control after white matter lesions.
Prevalence of IgG and IgM to SARS-CoV-2 and other human coronaviruses in The Democratic Republic of Congo, Sierra Leone and Uganda: A Longitudinal Study
Authors: Lawal Bolarinde J. , Gallagher Katherine E , Kitonsa Jonathan , Tindanbil Daniel , Kasonia Kambale , Drammeh Abdoulie , Lowe Brett , Mukadi-Bamuleka Daniel , Patterson Catriona , Greenwood Brian , Samai Mohamed , Leigh Bailah , Tetteh Kevin K. A. , Ruzagira Eugene , Watson-Jones Deborah , Kavunga-Membo Hugo 
Published: March 2023

AbstractObjectivesWe assessed the prevalence of immunoglobulin G (IgG) and immunoglobulin M (IgM) against four endemic human coronaviruses (HCoVs) and two SARS-CoV-2 antigens, among vaccinated and unvaccinated staff at health care centres in Uganda, Sierra Leone, and the Democratic Republic of Congo (DRC).MethodsGovernment health facility staff who had patient contact in Goma (DRC), Kambia District (Sierra Leone), and Masaka District (Uganda) were enrolled. Questionnaires and blood samples were collected at three timepoints over four months. Blood samples were analysed with the Luminex MAGPIX®.ResultsAmong unvaccinated participants, the prevalence of IgG/IgM antibodies against SARS-CoV-2 RBD or N-protein at enrolment was 70% in Goma (138/196), 89% in Kambia (112/126) and 89% in Masaka (190/213). IgG responses against endemic HCoVs at baseline were not associated with SAR-CoV-2 sero-acquisition during follow-up. Among vaccinated participants, those who had evidence of SARS-CoV-2 IgG/IgM at baseline tended to have higher IgG responses to vaccination compared to those SARS-CoV-2 seronegative at baseline, controlling for the time of sample collection since vaccination.ConclusionsThe high levels of natural immunity and hybrid immunity should be incorporated into both vaccination policy and prediction models of the impact of subsequent waves of infection in these settings.
A complete digital karyotype of the B-cell leukemia REH cell line resolved by long-read sequencing
Authors: Wiklander Mariya Lysenkova , Arvidsson Gustav , Bunikis Ignas , Lundmark Anders , Raine Amanda , Marincevic-Zuniga Yanara , Gezelius Henrik , Bremer Anna , Feuk Lars , Ameur Adam , Nordlund Jessica 
Published: March 2023

ABSTRACTThe B-cell acute lymphoblastic leukemia (ALL) cell line REH, with the t(12;21)ETV6-RUNX1translocation, is known to have a complex karyotype defined by a series of large-scale chromosomal rearrangements. Taken from a 15-year-old at relapse, the cell line offers a practical model for the study of high-risk pediatric B-ALL patients. In recent years, short-read DNA and RNA sequencing have emerged as a complement to analog karyotyping techniques in the resolution of structural variants in an oncological context. However, it is challenging to create a comprehensive digital karyotype of a genome with these techniques alone. Here, we explore the integration of long-read PacBio and Oxford Nanopore whole genome sequencing (WGS), IsoSeq RNA-sequencing, and short-read sequencing to create a detailed digital karyotype of the REH cell line. WGS refined the breakpoints of known aberrations and clarified the molecular traits of disrupted ALL-associated genesBTG1andTBL1XR1, as well as the glucocorticoid receptorNR3C1. Several previously underreported structural variants were also uncovered, including deletions affecting the ALL-associated genesVPREB1andNFATC1. Meanwhile, transcriptome sequencing identified seven fusion genes within the genomic breakpoints. Together, our extensive whole-genome investigation makes high-quality open-source data available to the leukemia genomics community.KEY POINTSA complete digital karyotype of the REH cell line was produced with short- and long-read DNA and RNA sequencing technologies.The study enabled precise identification of structural variants, and the fusion genes expressed as the result of these variants.
A toxic ankyrin cysteine protease effector RipBH of brown rot triggered autophagy-associated cell death
Authors: Zheng Xueao , Huang Mengshu , Tan Xiaodan , Wang Bingsen , Li Yanping , Xue Hao , Cheng Dong , Qiu Huishan , Li Wenhao , Song Botao , Chen Huilan 
Published: March 2023

ABSTRACTPotato brown rot, caused byRalstonia solanacearum, is one of the most destructive diseases of potatoes. The pathogen could hide in the tuber, leading to the rotting tubers. However, few mechanisms of pathogenesis in tubers caused by brown rot were reported. Here, we identified a highly virulent type III effector RipBH, which is not only required for the pathogenesis of potato brown rot but also displays strong cell toxicity in yeast and tobacco. We found RipBH is a novel structural cysteine protease with a large ankyrin repeat domain that contains 10 ankyrin repeats, we named it as an ankyrin cysteine protease. Biochemical analysis showed that all the ankyrin repeats are required for virulence, and the first five ankyrin repeats are indispensable for auto-cleavage site recognition. Further analysis showed that RipBH triggered autophagy-associated cell death. The ankyrin cysteine protease effector existed extensively in plant and animal pathogens suggesting the ankyrin cysteine protease effectors are functionally essential for pathogen pathogenesis. Our study enhances our understanding of this type of cysteine protease and illustrates the pathogenesis of cysteine protease in potato brown rot.
Structures of wild-type and selected CMT1X mutant connexin 32 gap junction channels and hemichannels
Authors: Qi Chao , Lavriha Pia , Bayraktar Erva , Vaithia Anand , Schuster Dina , Pannella Micaela , Sala Valentina , Picotti Paola , Bortolozzi Mario , Korkhov Volodymyr M. 
Published: March 2023

AbstractIn myelinating Schwann cells, communication between myelin layers is mediated by gap junction channels (GJC) formed by docked connexin 32 hemichannels (HCs). Mutations in Cx32 cause the X-linked Charcot–Marie–Tooth disease (CMT1X), a degenerative neuropathy with no cure. A molecular link between Cx32 dysfunction and CMT1X pathogenesis is still missing. Here, we describe the high resolution cryo-EM structures of the Cx32 GJC and HC, along with two CMT1X-linked mutants, W3S and R22G. While the structures of wild-type and mutant GJCs are virtually identical, the HCs show a major difference: in the W3S and R22G mutant HCs, the N-terminal helix partially occludes the pore, consistent with an impaired HC activity. Our results suggest that HC dysfunction may be involved in the pathogenesis of CMT1X.One-Sentence SummaryConnexin 32 channel structures reveal a gating helix defect in CMT1X disease-associated mutant hemichannels
cell-analysis-tools: an open-source library for single-cell analysis of multi-dimensional microscopy images
Authors: Guzman Emmanuel Contreras , Rehani Peter R. , Skala Melissa C. 
Published: March 2023

ABSTRACTSingle cell analysis of multi-dimensional microscopy images is repetitive, time consuming, and arduous. Numerous analysis steps are required to quantify and visualize cell heterogeneity and trends between experimental groups. The open-source community has created tools to facilitate this process. To further simplify analysis, we created a library of functions called cell-analysis-tools. This library includes functions that can streamline single-cell analysis for faster quality checking and automation. This library also includes example code with randomly generated data for dimensionality reduction [t-distributed stochastic neighbor embedding (t-SNE), principal component analysis (PCA), Uniform Manifold Approximation and Projection (UMAP)] and machine learning models [random forest, support vector machine (SVM), linear regression] that scientists can swap with their own data to visualize trends. Lastly, this library includes template scripts for feature extraction that can help identify differences between experimental groups and cell heterogeneity within a group. This library can significantly decrease user time while increasing robustness and reproducibility of results.