bluuebunny/crossref_metadata_2025_split huggingface dataset:
Personalized structural biology reveals the molecular mechanisms underlying heterogeneous epileptic phenotypes caused by de novo KCNC2 variants
Authors: Mukherjee Souhrid , Cassini Thomas A. , Hu Ningning , Yang Tao , Li Bian , Shen Wangzhen , Moth Christopher W. , Rinker David C. , Sheehan Jonathan H. , Cogan Joy D. ,   Undiagnosed Diseases Network, Newman John H. , Hamid Rizwan , Macdonald Robert L. , Roden Dan M. , Meiler Jens , Kuenze Georg , Phillips John A. , Capra John A. 
Published: February 2022

ABSTRACTBackgroundNext-generation whole exome sequencing (WES) is ubiquitous as an early step in the diagnosis of rare diseases and the interpretation of variants of unknown significance (VUS). Developmental and epileptic encephalopathies (DEE) are a group of rare devastating epilepsies, many of which have unknown causes. Increasing WES in the clinic has identified several rare monogenic DEEs caused by ion channel variants. However, WES often fails to provide actionable insight, due to the challenges of proposing functional hypotheses for candidate variants. Here, we describe a “personalized structural biology” (PSB) approach that addresses this challenge by leveraging recent innovations in the determination and analysis of protein 3D structures.ResultsWe illustrate the power of the PSB approach in an individual from the Undiagnosed Diseases Network (UDN) with DEE symptoms who has a novel de novo VUS in KCNC2 (p.V469L), the gene that encodes the Kv3.2 voltage-gated potassium channel. A nearby KCNC2 variant (p.V471L) was recently suggested to cause DEE-like phenotypes. We find that both variants are located in the conserved hinge region of the S6 helix and likely to affect protein function. However, despite their proximity, computational structural modeling suggests that the V469L variant is likely to sterically block the channel pore, while the V471L variant is likely to stabilize the open state. Biochemical and electrophysiological analyses demonstrate heterogeneous loss-of-function and gain-of-function effects, respectively, as well as differential inhibition in response to 4-aminopyridine (4-AP) treatment. Using computational structural modeling and molecular dynamics simulations, we illustrate that the pore of the V469L variant is more constricted increasing the energetic barrier for K+ permeation, whereas the V471L variant stabilizes the open conformationConclusionsOur results implicate KCNC2 as a causative gene for DEE and guided the interpretation of a UDN case. They further delineate the molecular basis for the heterogeneous clinical phenotypes resulting from two proximal pathogenic variants. This demonstrates how the PSB approach can provide an analytical framework for individualized hypothesis-driven interpretation of protein-coding VUS suspected to contribute to disease.
Hospitalisation for COVID-19 predicts long lasting cerebrovascular impairment: A prospective observational cohort study
Authors: Tsvetanov Kamen A , Spindler Lennart R B , Stamatakis Emmanuel A , Newcombe Virginia FJ , Lupson Victoria C , Chatfield Doris A , Manktelow Anne E , Outtrim Joanne G , Elmer Anne , Kingston Nathalie , Bradley John R , Bullmore Edward T , Rowe James B , Menon David K ,   The Cambridge NeuroCOVID Group,   The NIHR COVID-19 BioResource,   The Cambridge NIHR Clinical Research Facility,   The CITIID-NIHR BioResource COVID-19 Collaboration
Published: February 2022

AbstractHuman coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has multiple neurological consequences, but its long-term effect on brain health is still uncertain. The cerebrovascular consequences of COVID-19 may also affect brain health. Here we assess cerebrovascular health in 45 hospitalised patients using the resting state fluctuation amplitudes (RSFA) from functional magnetic resonance imaging, in relation to disease severity and in contrast with 42 controls. Widespread changes in frontoparietal RSFA were related to the severity of the acute COVID-19 episode, as indexed by COVID-19 WHO Progression Scale, inflammatory and coagulatory biomarkers. This relationship was not explained by chronic cardiorespiratory dysfunction, age, or sex. Exploratory analysis suggests that the level of cerebrovascular dysfunction is associated with cognitive, mental, and physical health at follow-up. The principal findings were consistent across univariate and multivariate approaches. The results indicate chronic cerebrovascular impairment following severe acute COVID-19, with the potential for long-term consequences on cognitive function and mental wellbeing.
Core and Flanking bHLH-PAS:DNA interactions mediate specificity and drive obesity
Authors: Bersten DC , Sullivan AE , McDougal D , Breen J , Fitzsimmons RL , Muscat GEO , Pederson S , Bruning JB , Fan CM , Thomas PQ , Russell DL , Peet DJ , Whitelaw ML 
Published: February 2022

AbstractThe basic-Helix-Loop-Helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical biological sensors of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet derived metabolites and environmental pollutants) stimuli to regulate genes involved in signal adaptation and homeostasis1. bHLH TFs bind DNA as homo or heterodimers via E-box (CANNTG) response elements, however the DNA binding specificity of the PAS domain-containing bHLH subfamily remains unresolved1. We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1α, HIF2α, AhR, NPAS4, SIM1) and demonstrate distinct core (NNCGTG) specificities for different heterodimer classes. The results also show that bHLH-PAS TFs bind over a large footprint 12-15bp and recognise preferential DNA sequences flanking the core. For example, specificity beyond otherwise identical core binding by SIM1 and the HIFs is mediated through N-terminal HIFα-DNA interactions. We also reveal an intimate relationship between DNA shape and both core and flanking TF binding allowing motif sequence flexibility and underpinning TF binding specificity. Furthermore, DNA-shape affinity relationships revealed that novel downstream PAS-A-loop DNA interactions are associated with AT-rich sequences that lead to high-affinity binding, and that loss of this function underpins a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model.Importantly, models of protein-DNA binding accurately predict in vivo occupancy, while response element methylation blocks DNA binding and predicts cell type specific chromatin occupancy. These data provide a definitive and accurate map of bHLH-PAS TF specificity and target selectivity through novel flanking protein-DNA interactions that are crucial for in vivo biological function.
Augmented glucose dependency of autoreactive B cells provides a treatment target for lupus
Authors: Wilson John J. , Wei Jian , Daamen Andrea R. , Sears John D. , Bechtel Elaine , Mayberry Colleen L. , Stafford Grace A. , Bechtold Lesley , Grammer Amrie C. , Lipsky Peter E. , Roopenian Derry C. , Chang Chih-Hao 
Published: February 2022

AbstractHeightened glycolysis is inherent to immune/inflammatory disorders, but little is known of its role in the pathogenesis of systemic lupus erythematosus (lupus). Here, we profile key autoimmune populations in acute and chronic lupus-prone models and their response to glycolytic inhibition. We demonstrate that glycolysis is specifically required for autoreactive germinal center B cells (GCB), but not for T follicular helper cells (Tfh) to survive. This augmented reliance on glucose oxidation to maintain ATP production in pathogenic GCB renders them highly susceptible to oxidative stress-induced apoptosis triggered by glycolysis blockade via 2-deoxyglucose (2DG). We show that 2DG can preferentially reduce GCB in lupus-prone mice, while sparing other autoreactive populations, including Tfh, but still significantly improving lifespan and kidney function. Furthermore, the subset of GCB expressing B-cell maturation antigen (BCMA) exhibits an exaggerated dependence on glycolysis to sustain their growth. Depletion of these cells with a proliferation-inducing ligand-based CAR T-cells leads to greatly prolonged lifespan of mice with severe autoimmune activation. These results reveal that glycolysis dependent GCB, especially those expressing BCMA, are key lupus mediators and highlight that they can be selectively targeted to improve disease outcomes for lupus patients.
Investigating RNA-RNA interactions through computational and biophysical analysis
Authors: Mrozowich Tyler , Park Sean M. , Waldl Maria , Henrickson Amy , Tersteeg Scott , Nelson Corey R. , Deklerk Anneke , Demeler Borries , Hofacker Ivo L. , Wolfinger Michael T. , Patel Trushar R. 
Published: February 2022

AbstractNumerous viruses utilize essential long-range RNA-RNA genome interactions, specifically flaviviruses. Using Japanese encephalitis virus (JEV) as a model system, we computationally predicted and then biophysically validated and characterized its long-range RNA-RNA genomic interaction. Using multiple RNA computation assessment programs, we determine the primary RNA-RNA interacting site among JEV isolates and numerous related viruses. Followingin vitrotranscription of RNA, we provide, for the first time, characterization of an RNA-RNA interaction using multi-angle light scattering (SEC-MALS) and analytical ultra-centrifugation (AUC). Next, we report the first RNA-RNA interaction study quantified by microscale thermophoresis (MST), demonstrating that the 5’ and 3’ TR of JEV interact with nM affinity, which is significantly reduced when the conserved cyclization sequence is not present. Furthermore, we perform computational kinetic analyses validating the cyclization sequence as the primary driver of this RNA-RNA interaction. Finally, we examined the 3-dimensional structure of the interaction using small-angle X-ray scattering, revealing a flexible yet stable interaction. This pathway can be adapted and utilized to study various viral and human long-non-coding RNA-RNA interactions, and determine their binding affinities, a critical pharmacological property of designing potential therapeutics.Graphical Abstract
USP22 controls type III interferon signaling and SARS-CoV-2 infection through activation of STING
Authors: Karlowitz Rebekka , Stanifer Megan L. , Roedig Jens , Andrieux Geoffroy , Bojkova Denisa , Smith Sonja , Kowald Lisa , Schubert Ralf , Boerries Melanie , Cinatl Jindrich , Boulant Steeve , van Wijk Sjoerd J. L. 
Published: February 2022

AbstractPattern recognition receptors (PRRs) and interferons (IFNs) serve as essential antiviral defense against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Type III IFN (IFN-λ) exhibit cell-type specific and long-lasting functions in autoinflammation, tumorigenesis and antiviral defense. Here, we identify the deubiquitinating enzyme USP22 as central regulator of basal IFN-λ secretion and SARS-CoV-2 infections in native human intestinal epithelial cells (hIECs). USP22-deficient hIECs strongly upregulate genes involved in IFN signaling and viral defense, including numerous IFN-stimulated genes (ISGs), with increased secretion of IFN-λ and enhanced STAT1 signaling, even in the absence of exogenous IFNs or viral infection. Interestingly, USP22 controls basal and 2’3’-cGAMP-induced STING activation and loss of STING reversed STAT activation and ISG and IFN-λ expression. Intriguingly, USP22-deficient hIECs are protected against SARS-CoV-2 infection, viral replication and the formation of de novo infectious particles, in a STING-dependent manner. These findings reveal USP22 as central host regulator of STING and type III IFN signaling, with important implications for SARS-CoV-2 infection and antiviral defense.
Pyruvate supports RET-dependent mitochondrial ROS production necessary to control Mycobacterium avium infection in human primary macrophages
Authors: Røst Lisa Marie , Louet Claire , Bruheim Per , Flo Trude Helen , Gidon Alexandre 
Published: February 2022

ABSTRACTMacrophages deploy a variety of antimicrobial programs to contain mycobacterial infection. Upon activation, they undergo extensive metabolic reprogramming to meet an increase in energy demand, but also to support immune effector functions such as secretion of cytokines and antimicrobial activities. Here, we report that mitochondrial import of pyruvate is linked to production of mitochondrial ROS and control of Mycobacterium avium infection in human primary macrophages. Using chemical inhibition, targeted mass spectrometry and single cell image analysis, we show that macrophages infected with M. avium switched to aerobic glycolysis without any major imbalances in the tricarboxylic acid cycle or changes in the energy charge. Instead, we found that pyruvate import contributed to hyperpolarization of mitochondria in infected cells and increased production of mitochondrial reactive oxygen species by the complex I via reverse electron transport, which reduced the macrophage burden of M. avium. While mycobacterial infections are extremely difficult to treat and notoriously resistant to antibiotics, this work stresses out that compounds specifically inducing mitochondrial reactive oxygen species could present themself as valuable adjunct treatments.
Interphase chromosomes of the Aedes aegypti mosquito are liquid crystalline and can sense mechanical cues
Authors: Contessoto Vinícius G. , Dudchenko Olga , Aiden Erez Lieberman , Wolynes Peter G. , Onuchic José N. , Di Pierro Michele 
Published: February 2022

AbstractWe use data-driven physical simulations to study the three-dimensional architecture of the Aedes aegypti genome. Hi-C maps exhibit both a broad diagonal and compart-mentalization with telomeres and centromeres clustering together. Physical modeling reveals that these observations correspond to an ensemble of 3D chromosomal structures that are folded over and partially condensed. Clustering of the centromeres and telomeres near the nuclear lamina appears to be a necessary condition for the formation of the observed structures. Further analysis of the mechanical properties of the genome reveals that the chromosomes of Aedes aegypti, by virtue of their atypical structural organization, are highly sensitive to the deformation of the nuclei. This last finding provides a possible physical mechanism linking mechanical cues to gene regulation.
Modeling of Alpha-1 Antitrypsin Deficiency with Syngeneic Human iPSC-Hepatocytes Reveals Metabolic Dysregulation and Cellular Heterogeneity in PiMZ and PiZZ Hepatocytes
Authors: Kaserman Joseph E , Werder Rhiannon B. , Wang Feiya , Matte Taylor , Higgins Michelle I. , Dodge Mark , Lindstrom-Vautrin Jonathan , Hinds Anne , Bullitt Esther , Caballero Ignacio S. , Shi Xu , Gerszten Robert E. , Brunetti-Pierri Nicola , Liesa Marc , Villacorta-Martin Carlos , Hollenberg Anthony N. , Kotton Darrell N. , Wilson Andrew A. 
Published: February 2022

AbstractIndividuals homozygous for the pathogenic “Z” mutation in alpha-1 antitrypsin deficiency (AATD) are known to be at increased risk for chronic liver disease. That some degree of risk is similarly conferred by the heterozygous state, estimated to affect 2% of the US population, has also become clear. A lack of model systems that recapitulate heterozygosity in human hepatocytes has limited the ability to study the impact of expressing a single ZAAT allele on hepatocyte biology. Here, through the application of CRISPR-Cas9 editing, we describe the derivation of syngeneic induced pluripotent stem cells (iPSCs) engineered to determine the effects of ZAAT heterozygosity in iPSC-derived hepatocytes (iHeps) relative to homozygous mutant (ZZ) or corrected (MM) cells. We find that heterozygous MZ iHeps exhibit an intermediate disease phenotype and share with ZZ iHeps alterations in AAT protein processing and downstream perturbations in hepatic metabolic function including ER and mitochondrial morphology, reduced mitochondrial respiration, and branch-specific activation of the unfolded protein response in subpopulations of cells. Our cellular model of MZ heterozygosity thus provides evidence that expression of a single Z allele is sufficient to disrupt hepatocyte homeostatic function and suggest a mechanism underlying the increased risk of liver disease observed among MZ individuals.
IZUMO1 is a sperm fusogen
Authors: Brukman Nicolas G. , Nakajima Kohdai P. , Valansi Clari , Flyak Kateryna , Li Xiaohui , Higashiyama Tetsuya , Podbilewicz Benjamin 
Published: February 2022

SummaryMammalian sperm-egg adhesion depends on the trans-interaction between the sperm-specific type I glycoprotein IZUMO1 and its oocyte-specific GPI-anchored receptor JUNO. However, the mechanisms and proteins (fusogens) which mediate the following step of gamete fusion remain unknown. Using live imaging and content mixing assays in a heterologous system and structure-guided mutagenesis, we unveil an unexpected function for IZUMO1 in cell-to-cell fusion. We show that IZUMO1 alone is sufficient to induce fusion, and that this ability is retained in a mutant unable to bind JUNO. On the other hand, a triple mutation in exposed aromatic residues prevents this fusogenic activity without impairing JUNO interaction. Our findings suggest a second, crucial function for IZUMO1 as a unilateral mouse gamete fusogen.HighlightsIZUMO1 expression in somatic cells in culture induces cell-to-cell fusionThe fusogenic activity of IZUMO1 is unilateralCell fusion is independent of the binding of IZUMO1 to JUNOIZUMO1-mediated cell merger depends on its transmembrane domain, and three solvent-exposed aromatic residuesGraphical abstract
Trpc6 gain-of-function disease mutation enhances phosphatidylserine exposure in murine platelets
Authors: Boekell Kimber L. , Brown Brittney J. , Talbot Brianna E. , Schlondorff Johannes S. 
Published: February 2022

AbstractPlatelets enhance coagulation by exposing phosphatidylserine (PS) on their cell surface in response to strong agonist activation. Transient receptor potential channels, including TRPC6, have been implicated in the calcium influx central to this process. Here, we characterize the effect of a Trpc6 gain-of-function (GOF) disease-associated, and a dominant negative (DN), mutation on murine platelet activation. Platelets from mice harboring Trpc6E896K/E896K (GOF) and Trpc6DN/DN mutations were subject to in vitro analysis. Trpc6E896K/E896K and Trpc6DN/DN mutant platelets show enhanced and absent calcium influx, respectively, upon addition of the TRPC3/6 agonist GSK1702934A (GSK). GSK was sufficient to induce integrin αIIbβ3 activation, P-selection and PS exposure, talin cleavage, and MLC2 phosphorylation in Trpc6E896K/E896K, but not in wild-type, platelets. Thrombin-induced calcium influx and PS exposure were enhanced, and clot retraction delayed, by GOF TRPC6, while no differences were noted between wild-type and Trpc6DN/DN platelets. In contrast, Erk activation upon GSK treatment was absent in Trpc6DN/DN, and enhanced in Trpc6E896K/E896K, platelets, compared to wild-type. The positive allosteric modulator, TRPC6-PAM-C20, and fluoxetine maintained their ability to enhance and inhibit, respectively, GSK-mediated calcium influx in Trpc6E896K/E896K platelets. The data demonstrate that gain-of-function mutant TRPC6 channel can enhance platelet activation, including PS exposure, while confirming that TRPC6 is not necessary for this process. Furthermore, they suggest that Trpc6 GOF disease mutants do not simply increase wild-type TRPC6 responses, but can affect pathways not usually modulated by TRPC6 channel activity, displaying a true gain-of-function phenotype.
Scale of analysis drives the observed ratio of spatial to non-spatial variance in microbial water quality: insights from two decades of citizen science data
Authors: Weller Daniel L. , Weller Donald E. , Strawn Laura K. , Love Tanzy M. T. 
Published: February 2022

AbstractWhile fecal indicator bacteria (FIB) testing is used to monitor surface waters for potential health hazards, recent studies report substantial variation in FIB levels and that observed variation appeared dependent on scale of analysis (SOA). Citizen science data and random effects models were used to quantify variance in FIB levels attributable to spatial versus temporal factors. Separately, Bayesian models were used to quantify the ratio of spatial to nonspatial variance in FIB levels, and identify associations between environmental factors and FIB levels. Separate analyses were performed for each stream, lake, and watershed as well as at the state-level (three SOAs). As SOA increased (from waterway to watershed to statewide models), variance attributable to spatial sources generally increased and variance attributable to temporal sources generally decreased. While relationships between FIB levels and environmental factors, such as flow conditions, were constant across SOA, the effect of land cover was both highly dependent on SOA and consistently smaller than the effect of outfalls. This study demonstrates the importance of scale when designing monitoring programs or using FIB monitoring to inform management. Moreover, these data represent a comprehensive survey of water quality in Upstate NY, and the study highlights a valuable publicly available database.