bluuebunny/crossref_metadata_2025_split huggingface dataset:
Therapeutic base and prime editing of COL7A1 mutations in recessive dystrophic epidermolysis bullosa
Authors: Hong Sung-Ah , Kim Song-Ee , Lee A-young , Hwang Gue-Ho , Kim Jong Hoon , Iwata Hiroaki , Kim Soo-Chan , Bae Sangsu , Lee Sang Eun 
Published: July 2021

ABSTRACTRecessive dystrophic epidermolysis bullosa (RDEB) is a severe skin fragility disorder caused by loss-of-function mutations in the COL7A1 gene, which encodes type VII collagen (C7), a protein that functions in skin adherence. From 36 Korean RDEB patients, we identified a total of 69 pathogenic mutations (40 variants without recurrence), including point mutations (72.5%) and insertion/deletion mutations (27.5%). We used base and prime editing to correct mutations in fibroblasts from two patients (Pat1, who carried a c.3631C>T mutation in one allele, and Pat2, who carried a c.2005C>T mutation in one allele). We applied adenine base editors (ABEs) to correct the pathogenic mutation or to bypass a premature stop codon in Pat1-derived primary fibroblasts. To expand the targeting scope, we also utilized prime editors (PEs) to correct the mutations in Pat1- and Pat2-derived fibroblasts. Ultimately, we found that both ABE- and PE-mediated correction of COL7A1 mutations restored full-length C7 expression, reversed the impaired adhesion and proliferation exhibited by the patient-derived fibroblasts, and, following transfer of edited patient-derived fibroblasts into the skin of immunodeficient mice, led to C7 deposition within the dermal-epidermal junction. These results suggest that base and prime editing could be feasible strategies for ex vivo gene editing to treat RDEB.
Arousal impacts distributed hubs modulating the integration of brain functional connectivity
Authors: Lee Kangjoo , Horien Corey , O’Connor David , Garand-Sheridan Bronwen , Tokoglu Fuyuze , Scheinost Dustin , Lake Evelyn M.R. , Constable R. Todd 
Published: July 2021

AbstractEven when subjects are at rest, it is thought that brain activity is organized into distinct brain states during which reproducible patterns are observable. Yet, it is unclear how to define or distinguish different brain states. A potential source of brain state variation is arousal, which may play a role in modulating functional interactions between brain regions. Here, we use simultaneous resting state functional magnetic resonance imaging and pupillometry to study the impact of arousal levels indexed by pupil area on the integration of large-scale brain networks. We employ a novel sparse dictionary learning-based method to identify hub regions participating in between-network integration stratified by arousal, by measuring k-hubness, the number (k) of functionally overlapping networks in each brain region. We show evidence of a brain-wide decrease in between-network integration and inter-subject variability at low relative to high arousal, with differences emerging across regions of the frontoparietal, default mode, motor, limbic, and cerebellum networks. State-dependent changes in k-hubness relate to the actual patterns of network integration within these hubs, suggesting a brain state transition from high to low arousal characterized by global synchronization and reduced network overlaps. We demonstrate that arousal is not limited to specific brain areas known to be directly associated with arousal regulation, but instead has a brain-wide impact that involves high-level between-network communications.
Scanning mutagenesis of RNA-binding protein ProQ reveals a quality control role for the Lon protease
Authors: Mouali Youssef El , Ponath Falk , Scharrer Vinzent , Wenner Nicolas , Hinton Jay C. D. , Vogel Jörg 
Published: July 2021

ABSTRACTThe FinO-domain protein ProQ belongs to a widespread family of RNA-binding proteins (RBPs) involved in gene regulation in bacterial chromosomes and mobile elements. Whilst the cellular RNA targets of ProQ have been established in diverse bacteria, the functionally crucial ProQ residues remain to be identified under physiological conditions. Following our discovery that ProQ deficiency alleviates growth suppression of Salmonella with succinate as the sole carbon source, an experimental evolution approach was devised to exploit this phenotype. By coupling mutational scanning with loss-of-function selection, we identified multiple ProQ residues in both the N-terminal FinO domain and the variable C-terminal region required for ProQ activity. Two C-terminal mutations abrogated ProQ function and mildly impaired binding of a model RNA target. By contrast, several mutations in the FinO domain rendered ProQ both functionally inactive and unable to interact with target RNA in vivo. Alteration of the FinO domain stimulated the rapid turnover of ProQ by Lon-mediated proteolysis, suggesting a quality control mechanism that prevents the accumulation of non-functional ProQ molecules. We extend this observation to Hfq, the other major sRNA chaperone of enteric bacteria. The Hfq Y55A mutant protein, defective in RNA-binding and oligomerization, proved to be labile and susceptible to degradation by Lon. Taken together, our findings connect the major AAA+ family protease Lon with RNA-dependent quality control of Hfq and ProQ, the two major sRNA chaperones of Gram-negative bacteria.SIGNIFICANCEProteins that interact with RNA play a vital role in controlling key functions in pathogenic bacteria. RNA-binding proteins regulate how, when and where bacteria feed, swim or interact with a host, and it is critical that we understand how RNAs associate with these proteins. ProQ is one of the three major RNA-binding proteins (RBPs) in Gram-negative bacteria. In this study, we mapped the amino acid residues of ProQ that are essential for function. We successfully identified residue substitutions that rendered the ProQ RBP both non-functional and unable to interact with RNA. Our findings raise the possibility that the Lon protease mediates a quality control mechanism of ProQ that targets this RBP in the absence of RNA. A posttranslational quality control mechanism of this type could prevent the accumulation of nonfunctional RBPs in the bacterial cytoplasm.
Substrate recognition determinants of human eIF2α phosphatases
Authors: Hodgson George , Andreeva Antonina , Bertolotti Anne 
Published: July 2021

AbstractPhosphorylation of the translation initiation factor eIF2α is a rapid and vital cellular defence against many forms of stress. In mammals, the levels of eIF2α phosphorylation are set through the antagonistic action of four protein kinases and two heterodimeric protein phosphatases. The phosphatases are composed of the catalytic subunit PP1 and one of two related non-catalytic subunits, PPP1R15A or PPP1R15B (R15A or R15B). Attempts at reconstituting recombinant holophosphatases have generated two models, one proposing that substrate recruitment requires the addition of actin, whilst the second proposes that this function is encoded by R15s. The biological relevance of actin in substrate recruitment has not been evaluated. Here we generated a series of truncation mutants and tested their properties in mammalian cells. We show that substrate recruitment is encoded by an evolutionary conserved region in R15s, R15A325-554 and R15B340-639. Actin does not bind these regions establishing that it is not required for substrate recruitment. Activity assays in cells showed that R15A325-674 and R15B340-713, encompassing the substrate-binding region and the PP1 binding-region, exhibit wild-type activity. This study identifies essential regions of R15s and demonstrates they function as substrate receptors. This work will guide the design of future structural studies with biological significance.
The use of high-resolution computed tomography to explore age-related trabecular change in human ribs
Authors: Mavroudas Sophia R. , Dominguez Victoria M. 
Published: July 2021

ABSTRACTHigh-resolution computed tomography was used to explore age-related trabecular change in male human ribs ranging in age from 20–95 years (Mean=55 years, SD=21.634 years) from the Texas State Donated Skeletal Collection (TXSTDSC). Two regions of interest (ROIs), midshaft (50%) and anterior (75%) were extracted from each scan to analyze age-related trabecular change. Dragonfly V4.1 was used to isolate cortical bone volumes of interest (VOIs) and three trabecular VOIs for each ROI; one each along the cutaneous cortex, the center of the medullary cavity, and the pleural cortex. Each trabecular VOI was analyzed for bone volume fraction (BV/TV), trabecular thickness (TbTh), trabecular spacing (TbSp), connectivity density (Conn.D), and degree of anisotropy (DA), within and between the 50 and 75% ROIs. Overall, the cutaneous VOIs at both the 50% and 75% ROIs exhibited greater BV/TV, TbTh, and Conn.D when compared to the center and pleural VOIs. All results are consistent with expected biomechanical strain on human ribs. Both trabecular variables and cortical bone volume are only weakly associated with age. These results show that 3D analysis of trabecular bone volume does not improve visualization or understanding of trabecular bone changes with age over traditional 2D methods. Future research should incorporate female samples to explore sex-related trabecular change variation.HIGHLIGHTS3D analysis of change in trabecular structure along the rib length and with ageTrabecular spacing at midshaft shows highest correlations with age-at-deathRelative cortical area is more strongly correlated with age in anterior ribs than at midshaft
The genomic basis of copper tolerance inDrosophilais shaped by a complex interplay of regulatory and environmental factors
Authors: Green Llewellyn , Coronado-Zamora Marta , Radio Santiago , Rech Gabriel E. , Salces-Ortiz Judit , González Josefa 
Published: July 2021

ABSTRACTIncreases in industrialization and anthropogenic activity have resulted in an increase of pollutants released into the environment. Of these pollutants, heavy metals such as copper are particularly concerning due to their bio-accumulative nature. Due to its highly heterogeneous distribution and its dual nature as an essential micronutrient and toxic element, the genetic basis of copper tolerance is likely shaped by a complex interplay of genetic and environmental factors.In this study, we utilized the natural variation present in multiple populations ofDrosophila melanogastercollected across Europe to screen for variation in copper tolerance. We found that latitude and the degree of urbanization at the collection sites, rather than any other combination of environmental factors, were linked to copper tolerance. While previously identified copper-related genes were not differentially expressed in tolerantvs. sensitive strains, genes involved in metabolism, reproduction, and protease induction contributed to the differential stress response. Additionally, the greatest transcriptomic and physiological responses to copper toxicity were seen in the midgut; where we found that preservation of gut acidity is strongly linked to greater tolerance. Finally, we identified transposable element insertions likely to play a role in copper stress response.Overall, by combining genome-wide approaches with environmental association analysis, and functional analysis of candidate genes, our study provides a unique perspective on the genetic and environmental factors that shape copper tolerance in naturalD. melanogasterpopulations, and identifies new genes, transposable elements and physiological traits involved in this complex phenotype.
Cell adhesion factors during adolescence support amygdalo-cortical connections and flexible action later in life
Authors: Kietzman Henry W. , Shapiro Lauren P. , Trinoskey-Rice Gracy , Guo Jidong , Gourley Shannon L. 
Published: July 2021

AbstractAdolescent brain development is characterized by dramatic neuronal remodeling in the prefrontal cortex. This plasticity is presumed to act in part to “set the stage” for prefrontal cortical function in adulthood, but causal relationships have largely not been verified. Integrins are cell adhesion factors that provide a link between the extracellular matrix and the intracellular actin cytoskeleton. We find that β1-integrin presence in the prelimbic subregion of the prefrontal cortex (PL) during adolescence, but not adulthood, is necessary for adult mice to select actions based on reward likelihood and value. These behaviors require coordinated limbic-frontal-striatal circuits. We identified projections from the basolateral amygdala (BLA) to PL as being necessary for mice to express learned response strategies. We then visualized adolescent PL neurons receiving input from the BLA and projecting to the dorsomedial striatum (DMS), a primary striatal output by which the PL controls reward-related behavior. These projection-defined neurons had a more “adult-like” morphology relative to a general population of layer V PL neurons. β1-integrin loss caused the overexpression of stubby-type dendritic spines at the expense of more mature spines, a phenotype not observed when β1-integrins were silenced before or after adolescence. Together, these experiments localize β1-integrin-mediated cell adhesion activity within a developing di-synaptic circuit that coordinates flexible action.
A complete reference genome improves analysis of human genetic variation
Authors: Aganezov Sergey , Yan Stephanie M. , Soto Daniela C. , Kirsche Melanie , Zarate Samantha , Avdeyev Pavel , Taylor Dylan J. , Shafin Kishwar , Shumate Alaina , Xiao Chunlin , Wagner Justin , McDaniel Jennifer , Olson Nathan D. , Sauria Michael E.G. , Vollger Mitchell R. , Rhie Arang , Meredith Melissa , Martin Skylar , Lee Joyce , Koren Sergey , Rosenfeld Jeffrey A. , Paten Benedict , Layer Ryan , Chin Chen-Shan , Sedlazeck Fritz J. , Hansen Nancy F. , Miller Danny E. , Phillippy Adam M. , Miga Karen H. , McCoy Rajiv C. , Dennis Megan Y. , Zook Justin M. , Schatz Michael C. 
Published: July 2021

AbstractCompared to its predecessors, the Telomere-to-Telomere CHM13 genome adds nearly 200 Mbp of sequence, corrects thousands of structural errors, and unlocks the most complex regions of the human genome to clinical and functional study. Here we demonstrate how the new reference universally improves read mapping and variant calling for 3,202 and 17 globally diverse samples sequenced with short and long reads, respectively. We identify hundreds of thousands of novel variants per sample—a new frontier for evolutionary and biomedical discovery. Simultaneously, the new reference eliminates tens of thousands of spurious variants per sample, including up to 12-fold reduction of false positives in 269 medically relevant genes. The vast improvement in variant discovery coupled with population and functional genomic resources position T2T-CHM13 to replace GRCh38 as the prevailing reference for human genetics.One Sentence SummaryThe T2T-CHM13 reference genome universally improves the analysis of human genetic variation.
Development of photo-lenalidomide for cellular target identification
Authors: Lin Zhi , Amako Yuka , Kabir Farah , Flaxman Hope A. , Budnik Bogdan , Woo Christina M. 
Published: July 2021

ABSTRACTThe thalidomide analog lenalidomide is a clinical therapeutic that alters the substrate engagement of cereblon (CRBN), a substrate receptor for the CRL4 E3 ubiquitin ligase. Here, we report the development of photo-lenalidomide, a lenalidomide probe with a photo-affinity label and enrichment handle, for target identification by chemical proteomics. After evaluating a series of lenalidomide analogs, we identified a specific amide linkage to lenalidomide that allowed for installation of the desired functionality, while preserving the substrate degradation profile, phenotypic anti-proliferative and immunomodulatory properties of lenalidomide. Photo-lenalidomide maintains these properties by enhancing binding interactions with the thalidomide-binding domain of CRBN, as revealed by binding site mapping and molecular modeling. Using photo-lenalidomide, we captured the known targets IKZF1 and CRBN from multiple myeloma MM.1S cells, and further identified a new target, eukaryotic translation initiation factor 3 subunit i (eIF3i), from HEK293T cells. eIF3i is directly labeled by photolenalidomide and forms a complex with CRBN in the presence of lenalidomide, but is itself not ubiquitylated or degraded. These data point to the potentially broader array of substrates induced by ligands to CRBN that may or may not be degraded, which can be revealed by the highly translatable application of photo-lenalidomide and chemical proteomics in additional biological settings.Abstract Figure
VCP protects neurons from proteopathic seeding
Authors: Zhu Jiang , Pittman Sara , Dhavale Dhruva , French Rachel , Patterson Jessica N. , Kaleelurrrahuman Salman , Sun Yuanzi , Buscher William J. , Bieschke Jan , Kotzbauer Paul , Ayala Yuna , Davis Albert A. , Weihl Conrad 
Published: July 2021

SUMMARYUptake and spread of proteopathic seeds, such as αS, Tau, and TDP-43, contribute to neurodegeneration. The cellular machinery necessary for this process is poorly understood. Using a genome-wide CRISPR-Cas9 screen, we identified Valosin Containing Protein (VCP) as a suppressor of αS seeding. Dominant mutations in VCP cause multisystem proteinopathy (MSP) with muscle and neuronal degeneration. VCP inhibition or disease mutations increase αS seeding in cells and neurons. This is not associated with an increase in seed uptake and is similar to treatment with the lysosomal damaging agent, LLoME. Intrastriatal injection of αS seeds into VCP disease mice enhances seeding efficiency compared with controls. This is not specific to αS since VCP inhibition or disease mutations increased TDP-43 seeding in neurons. These data support that VCP protects against proteopathic spread of pathogenic aggregates. The spread of distinct aggregate species may dictate pleiotropic phenotypes and pathologies in VCP associated MSP.
Lack of blocking activity in anti-CTLA-4 antibodies reduces toxicity, but not anti-tumor efficacy
Authors: Stone Erica L. , Carter Kyle P. , Wagner Ellen K. , Asensio Michael A. , Benzie Emily , Chiang Yao Y. , Coles Garry L. , Edgar Chelsea , Gautam Bishal K. , Gras Ashley , Leong Jackson , Leong Renee , Manickam Vishal A. , Mizrahi Rena A. , Niedecken Ariel R. , Saini Jasmeen , Sandhu Savreet K. , Simons Jan Fredrick , Stadtmiller Kacy , Tinsley Brendan , Tracy LaRee , Wayham Nicholas P. , Lim Yoong Wearn , Adler Adam S. , Johnson David S. 
Published: July 2021

ABSTRACTAnti-CTLA-4 antibodies such as ipilimumab were among the first immune-oncology agents to show significantly improved outcomes for patients. However, existing anti-CTLA-4 therapies fail to induce a response in a majority of patients and can induce severe, immune-related adverse events. It has been assumed that checkpoint inhibition, i.e., blocking the interaction between CTLA-4 and its ligands, is the primary mechanism of action for ipilimumab. In this study we present evidence that checkpoint inhibition is not a primary mechanism of action for efficacy of anti-CTLA-4 antibodies. Instead, the primary mechanism for efficacy is FcR-mediated Treg depletion in the tumor microenvironment. First, we identified a monoclonal antibody (mAb) that binds to CTLA-4 at an epitope that differs from ipilimumab’s by only a few amino acids, yet has limited checkpoint inhibitor activity. Surprisingly, the weak checkpoint inhibitor has superior anti-tumor activity compared to ipilimumab in a murine model. The weak checkpoint inhibitor also induces less Treg proliferation and has increased ability to inducein vitroFcR signaling andin vivodepletion of intratumoral Tregs. Further experiments showed that the enhanced FcR activity of the weak checkpoint inhibitor likely contributes to its enhanced anti-tumor activity. Importantly, we also showed that weak checkpoint inhibition was associated with lower toxicity in murine models. Our work suggests that new anti-CTLA-4 drugs should be optimized for Treg depletion rather than checkpoint inhibition.
Pharmacokinetics of caffeine: A systematic analysis of reported data for application in metabolic phenotyping and liver function testing
Authors: Grzegorzewski Jan , Bartsch Florian , Köller Adrian , König Matthias 
Published: July 2021

ABSTRACTCaffeine is by far the most ubiquitous psychostimulant worldwide found in tea, coffee, cocoa, energy drinks, and many other beverages and food. Caffeine is almost exclusively metabolized in the liver by the cytochrome P-450 enzyme system to the main product paraxanthine and the additional products theobromine and theophylline. Besides its stimulating properties, two important applications of caffeine are metabolic phenotyping of cytochrome P450 1A2 (CYP1A2) and liver function testing. An open challenge in this context is to identify underlying causes of the large inter-individual variability in caffeine pharmacokinetics. Data is urgently needed to understand and quantify confounding factors such as lifestyle (e.g. smoking), the effects of drug-caffeine interactions (e.g. medication metabolized via CYP1A2), and the effect of disease. Here we report the first integrative and systematic analysis of data on caffeine pharmacokinetics from 148 publications and provide a comprehensive high-quality data set on the pharmacokinetics of caffeine, caffeine metabolites, and their metabolic ratios in human adults. The data set is enriched by meta-data on the characteristics of studied patient cohorts and subjects (e.g. age, body weight, smoking status, health status), the applied interventions (e.g. dosing, substance, route of application), measured pharmacokinetic time-courses, and pharmacokinetic parameters (e.g. clearance, half-life, area under the curve). We demonstrate via multiple applications how the data set can be used to solidify existing knowledge and gain new insights relevant for metabolic phenotyping and liver function testing based on caffeine. Specifically, we analyzed (i) the alteration of caffeine pharmacokinetics with smoking and use of oral contraceptives; (ii) drug-drug interactions with caffeine as possible confounding factors of caffeine pharmacokinetics or source of adverse effects; (iii) alteration of caffeine pharmacokinetics in disease; and (iv) the applicability of caffeine as a salivary test substance by comparison of plasma and saliva data. In conclusion, our data set and analyses provide important resources which could enable more accurate caffeine-based metabolic phenotyping and liver function testing.