bluuebunny/crossref_metadata_2025_split huggingface dataset:
Determining the pathogenicity of variants of uncertain significance and identification of a founder variant in the epilepsy-associated gene,SZT2
Authors: Calhoun Jeffrey D , Aziz Miriam C , Happ Hannah C , Gunti Jonathan , Gleason Colleen , Mohamed Najma , Zeng Kristy , Hiller Meredith , Bryant Emily , Mithal Divakar , Bellinski Irena , Kinsley Lisa , Grimmel Mona , Schwaibold Eva MC , Smith-Hicks Constance , Chassevent Anna , Scala Marcello , Accogli Andrea , Torella Annalaura , Striano Pasquale , Capra Valeria , Bird Lynne M. , Ben-Sahra Issam , Ekhilevich Nina , Hershkovitz Tova , Weiss Karin , Millichap John , Gerard Elizabeth E , Carvill Gemma L 
Published: January 2021

AbstractBiallelic pathogenic variants inSZT2result in a neurodevelopmental disorder with shared features, including early-onset epilepsy, developmental delay, macrocephaly, and corpus callosum abnormalities. SZT2 is as a critical scaffolding protein in the amino acid sensing arm of the mTOR signaling pathway. Due to its large size (3432 amino acids), lack of crystal structure, and absence of functional domains, it is difficult to determine the pathogenicity ofSZT2missense and in-frame deletions. We report a cohort of twelve individuals with biallelicSZT2variants and phenotypes consistent withSZT2-related neurodevelopmental disorder. The majority of this cohort contained one or moreSZT2variants of uncertain significance (VUS). We developed a novel individualized platform to functionally characterizeSZT2VUSs. We identified a recurrent in-frame deletion (SZT2 p.Val1984del) which was determined to be a loss-of-function variant and therefore likely pathogenic. Haplotype analysis determined this single in-frame deletion is a founder variant in those of Ashkenazi Jewish ancestry. Overall, we present a FACS-based rapid assay to distinguish pathogenic variants from VUSs inSZT2, using an approach that is widely applicable to other mTORopathies including the most common causes of the focal genetic epilepsies,DEPDC5, TSC1/2, MTORandNPRL2/3.
Neuropilin-1 Assists SARS-CoV-2 Infection by Stimulating the Separation of Spike Protein Domains S1 and S2
Authors: Li Zhen-lu , Buck Matthias 
Published: January 2021

AbstractThe cell surface receptor Neuropilin-1 (Nrp1) was recently identified as a host factor for SARS-CoV-2 entry. As the Spike protein of SARS-CoV-2 is cleaved into the S1 and the S2 domain by furin protease, Nrp1 binds to the newly created C-terminal RRAR amino acid sequence of the S1 domain. In this study, we model the association of a Nrp1 (a2-b1-b2) protein with the Spike protein computationally and analyze the topological constraints in the SARS-CoV-2 Spike protein for binding with Nrp1 and ACE2. Importantly, we study the exit mechanism of S2 from the S1 domain with the assistance of ACE2 as well as Nrp1 by molecular dynamics pulling simulations. In the presence of Nrp1, by binding the S1 more strongly to the host membrane, there is a high probability of S2 being pulled out, rather than S1 being stretched. Thus, Nrp1 binding could stimulate the exit of S2 from the S1 domain, which will likely increase virus infectivity as the liberated S2 domain mediates the fusion of virus and host membranes. Understanding of such a Nrp1-assisted viral infection opens the gate for the generation of protein-protein inhibitors, such as antibodies, which could attenuate the infection mechanism and protect certain cells in a future combination therapy.
Hypertrophied human adipocyte spheroids asin vitromodel of weight gain and adipose tissue dysfunction
Authors: Ioannidou Anna , Alatar Shemim , Åhlander Matilda , Hornell Amanda , Fisher Rachel M. , Hagberg Carolina E 
Published: January 2021

AbstractThe rise in obesity prevalence has created an urgent need for new and improved methods to study human adipocytes and the pathogenic effects of weight gainin vitro. Despite numerous studies showing the advantages of culturing adipocyte progenitors as 3D structures, the majority continue using traditional 2D cultures which result in small, multilocular adipocytes with poor representability. We hypothesized that providing differentiating pre-adipocytes with a vascular growth niche would mimicin vivoadipogenesis and improve the differentiation process. Here we present a simple, easily applicable culture protocol that allows for the differentiation and culturing of human adipocytes with a more unilocular morphology and larger lipid droplets than previous protocols. We moreover offer a protocol for inducing adipocyte enlargementin vitro, resulting in larger lipid droplets and development of several key features of adipocyte dysfunction, including altered adipokine secretions and impaired lipolysis. Taken together, our hypertrophied human adipocyte spheroids offer an improved culture system for studying the cellular and molecular mechanisms causing metabolic dysfunction and inflammation during weight gain.
Noncanonical Electromechanical Coupling Mechanism of an HCN Channel
Authors: Dai Gucan , Aman Teresa K. , DiMaio Frank , Zagotta William N. 
Published: January 2021

ABSTRACTPacemaker hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels exhibit a reversed voltage-dependent gating, activating by membrane hyperpolarization instead of depolarization. Sea urchin HCN (spHCN) channels also undergo an inactivation with hyperpolarization which occurs only in the absence of cyclic nucleotide. Here we applied transition metal ion FRET and Rosetta modeling to measure differences in the structural rearrangements between activation and inactivation of spHCN channels. We found that removal of cAMP produced a largely rigid-body rotation of the C-linker relative to transmembrane domain, bringing the A’ helix in close proximity to the voltage-sensing S4 helix. In addition, rotation of the C-linker was elicited by hyperpolarization in the absence but not in the presence of cAMP. These results suggest the A’ helix functions like a mechanical clutch to engage inactivation. cAMP binding disengages the clutch, permitting the hyperpolarization-dependent activation mediated by the S5 helix. Furthermore, rearrangement of A’ helix during recovery from inactivation of spHCN channels was similar to that for the activation of KCNH channels, suggesting a conserved mechanism for this noncanonical electromechanical coupling in the CNBD channel family.
Evidence for a hydrogen sulfide-sensing E3 ligase in yeast
Authors: Johnson Zane M. , Wang Yun , Sutter Benjamin M. , Tu Benjamin P. 
Published: January 2021

ABSTRACTIn yeast, control of sulfur amino acid metabolism relies upon Met4, a transcription factor which activates the expression of a network of enzymes responsible for the biosynthesis of cysteine and methionine. In times of sulfur abundance, the activity of Met4 is repressed via ubiquitination by the SCFMet30E3 ubiquitin ligase, but the mechanism by which the F-box protein Met30 senses sulfur status to tune its E3 ligase activity remains unresolved. Herein, we show that Met30 responds to flux through the transsulfuration pathway to regulate theMETgene transcriptional program. In particular, Met30 is responsive to the biological gas hydrogen sulfide, which is sufficient to induce ubiquitination of Met4in vivo. Additionally, we identify important cysteine residues in Met30’s WD-40 repeat region that sense the availability of sulfur in the cell. Our findings reveal how SCFMet30dynamically senses the flow of sulfur metabolites through the transsulfuration pathway to regulate synthesis of these special amino acids.
Compensating complete loss of signal recognition particle during co-translational protein targeting by the translation speed and accuracy
Authors: Zhao Liuqun , Fu Gang , Cui Yanyan , Xu Zixiang , Cai Tao , Zhang Dawei 
Published: January 2021

AbstractSignal recognition particle (SRP) is critical for delivering co-translational proteins to the bacterial inner membrane. Previously, we identified SRP suppressors inEscherichia colithat inhibit translation initiation and elongation, which provided insights into the mechanism of bypassing the requirement of SRP. Suppressor mutations tended to be located in regions that govern protein translation under evolutionary pressure. To test this hypothesis, we re-executed the suppressor screening of SRP. Here we isolated a novel SRP suppressor mutation located in the Shine-Dalgarno sequence of the S10 operon, which partially offset the targeting defects of SRP-dependent proteins. We found that the suppressor mutation decreased the protein translation rate, which extended the time window of protein targeting. This increased the possibility of the correct localization of inner membrane proteins. Furthermore, the fidelity of translation was decreased in suppressor cells, suggesting that the quality control of translation was inactivated to provide an advantage in tolerating toxicity caused by the loss of SRP. Our results demonstrated that the inefficient protein targeting due to SRP deletion can be rescued through modulating translational speed and accuracy.
The selenophosphate synthetase,selD, is important forClostridioides difficilephysiology
Authors: McAllister Kathleen N. , Aguirre Andrea Martinez , Sorg Joseph A. 
Published: January 2021

AbstractThe endospore-forming pathogen,Clostridioides difficile, is the leading cause of antibiotic-associated diarrhea and is a significant burden on the community and healthcare.C. difficile, like all forms of life, incorporates selenium into proteins through a selenocysteine synthesis pathway. The known selenoproteins inC. difficileare involved in a metabolic process that uses amino acids as the sole carbon and nitrogen source (Stickland metabolism). The Stickland metabolic pathway requires the use of two selenium-containing reductases. In this study, we built upon our initial characterization of the CRISPR-Cas9-generatedselDmutant by creating a CRISPR-Cas9-mediated restoration of theselDgene at the native locus. Here, we use these CRISPR-generated strains to analyze the importance of selenium-containing proteins onC. difficilephysiology. SelD is the first enzyme in the pathway for selenoprotein synthesis and we found that multiple aspects ofC. difficilephysiology were affected (e.g., growth, sporulation, and outgrowth of a vegetative cell post-spore germination). Using RNAseq, we identified multiple candidate genes which likely aid the cell in overcoming the global loss of selenoproteins to grow in medium which is favorable for using Stickland metabolism. Our results suggest that the absence of selenophosphate (i.e., selenoprotein synthesis) leads to alterations toC. difficilephysiology so that NAD+can be regenerated by other pathways.ImportanceC. difficileis a Gram-positive, anaerobic gut pathogen which infects thousands of individuals each year. In order to stop theC. difficilelifecycle, other non-antibiotic treatment options are in urgent need of development. Towards this goal, we find that a metabolic process used by only a small fraction of the microbiota is important forC. difficilephysiology – Stickland metabolism. Here, we use our CRISPR-Cas9 system to ‘knock in’ a copy of theselDgene into the deletion strain to restoreselDat its native locus. Our findings support the hypothesis that selenium-containing proteins are important for several aspects ofC. difficilephysiology – from vegetative growth to spore formation and outgrowth post-germination.
Functional phenotyping of small multidrug resistance proteins fromStaphylococcus aureusandFrancisella tularensisreveals functional homology to EmrE
Authors: Spreacker Peyton J. , Beeninga Will F. , Young Brooke L. , Porter Colin J. , Henzler-Wildman Katherine A. 
Published: January 2021

ABSTRACTSmall multidrug resistance (SMR) transporters efflux toxic substrates from bacterial cells. These transporters were recently divided into two subfamilies: the GdX-like and EmrE-like SMRs. The EmrE-like subfamily of SMRs is predicted to contain transporters that are highly promiscuous in both substrate specificity and mechanism based on extensive characterization of the founding member of this subfamily, EmrE. However, there is only limited functional analysis of other members of this family from pathogenic strains such asStaphylococcus aureusandFrancisella tularensis. Here, we use a small compound screen to explore the substrate specificity and diversity of EmrE-subfamily SMRs from these two bacterial species and confirm that they are functionally more like EmrE than the GdX-like subfamily of toxic-metabolite transporters. The results of these experiments lay the foundation for understanding the complex substrate specificity profiles of SMR family transporters and assess the potential for targeting these transporters for future antibiotic development, either broadly or in a species-specific manner.
To make or take: bacterial lipid homeostasis during infection
Authors: Adams Felise G. , Trappetti Claudia , Waters Jack K. , Zang Maoge , Brazel Erin B. , Paton James C. , Snel Marten F. , Eijkelkamp Bart A. 
Published: January 2021

AbstractBacterial fatty acids are critical components of the cellular membrane. A shift in environmental conditions or in the bacterium’s lifestyle may result in the requirement for a distinct pool of fatty acids with unique biophysical properties. This can be achieved by the modification of existing fatty acids or viade novosynthesis. Furthermore, bacteria have evolved efficient means to acquire these energy-rich molecules from their environment. However, the balance betweende novofatty acid synthesis and exogenous acquisition during pathogenesis is poorly understood. Here we studied the mouse fatty acid landscape prior and post infection withAcinetobacter baumannii, a Gram-negative, opportunistic human pathogen. The lipid fluxes observed following infection revealed fatty acid- and niche-specific changes. Lipidomic profiling ofA. baumanniiisolated from the pleural cavity of mice identified novelA. baumanniimembrane phospholipid species and an overall increased abundance of unsaturated fatty acid species. Importantly, we found thatA. baumanniirelies largely upon fatty acid acquisition in all but one of the studied niches, the blood, where the pathogen biosynthesises its own fatty acids. This work is the first to reveal the significance of balancing the making and taking of fatty acids in a Gram-negative bacterium during infection, which provides new insights into the validity of targeting fatty acid synthesis as a treatment strategy.ImportanceAcinetobacter baumanniiis one of the world’s most problematic superbugs, and is associated with significance morbidity and mortally in the hospital environment. The critical need for new antimicrobial strategies is recognised, but our understanding of its behaviour and adaptation to a changing environment during infection is limited. Here, we investigated the role of fatty acids at the host-pathogen interface using a mouse model of disease. We provide comprehensive insights into the bacterial membrane composition when they colonise the pleural cavity. Further, we show thatA. baumanniiheavily relies upon making its fatty acids when residing in the blood, whereas the bacterium favours fatty acid acquisition in most other host niches. Our new knowledge aids in understanding the importance of host fatty acids in infectious diseases. Further, fatty acid synthesis is an attractive target for the development of new antimicrobial strategies, but our work emphasizes the critical need to understand the microbial lipid homeostasis before this can be deemed suitable.
Multi-organ complement deposition in COVID-19 patients
Authors: Macor Paolo , Durigutto Paolo , Mangogna Alessandro , Bussani Rossana , D’Errico Stefano , Zanon Martina , Pozzi Nicola , Meroni PierLuigi , Tedesco Francesco 
Published: January 2021

AbstractBackgroundIncreased levels of circulating complement activation products have been reported in COVID-19 patients, but only limited information is available on complement involvement at tissue level. The mechanisms and pathways of local complement activation remain unclear.MethodsWe performed immunofluorescence analyses of autopsy specimens of lungs, kidney and liver from nine COVID-19 patients who died of acute respiratory failure. Snap-frozen samples embedded in OCT were stained with antibodies against complement components and activation products, IgG and spike protein of SARS-CoV-2.FindingsLung deposits of C1q, C4, C3 and C5b-9 were localized in the capillaries of the interalveolar septa and on alveolar cells. IgG displayed a similar even distribution, suggesting classical pathway activation. The spike protein is a potential target of IgG, but its uneven distribution suggests that other viral and tissue molecules may be targeted by IgG. Factor B deposits were also seen in COVID-19 lungs and are consistent with activation of the alternative pathway, whereas MBL and MASP-2 were hardly detectable. Analysis of kidney and liver specimens mirrored findings observed in the lung. Complement deposits were seen on tubules and vessels of the kidney with only mild C5b-9 staining in glomeruli, and on hepatic artery and portal vein of the liver.InterpretationComplement deposits in different organs of deceased COVID-19 patients caused by activation of the classical and alternative pathways support the multi-organ nature of the disease.FundingGrants from the Italian Ministry of Health (COVID-2020-12371808) to PLM and National Institutes of Health HL150146 to NP are gratefully acknowledged.
Quarantine fatigue thins fat-tailed coronavirus impacts in U.S. cities by making epidemics inevitable
Authors: Conte Marc N. , Gordon Matthew , Sims Charles 
Published: January 2021

AbstractWe use detailed location data to show that contacts between individuals in most U.S. cities and counties are fat tailed, suggesting that the fat tails documented in a small number of superspreading clusters are widespread. We integrate these results into a stochastic compartmental model to show that COVID-19 cases were also fat tailed for many U.S. cities for several weeks in the spring and summer. Due to epidemiological thresholds, fat-tailed cases would have been more prevalent if not for the gradual increase in contact rates throughout the summer that made outbreaks more certain.
Parental feeding and childhood genetic risk for obesity: Exploring hypothetical interventions with causal inference methods
Authors: Herle Moritz , Pickles Andrew , Micali Nadia , Abdulkadir Mohamed , De Stavola Bianca 
Published: January 2021

AbstractParental feeding behaviors are common intervention targets for childhood obesity, but often only deliver small changes. Childhood BMI is partly driven by genetic effects, and the extent to which parental feeding interventions can mediate child genetic liability is not known. Here we aim to examine how potential interventions on parental feeding behaviors can mitigate some of the association between child genetic liability and BMI in early adolescence, using causal inference based methods. Data were from the Avon Longitudinal Study of Parents and Children and we quantified the interventional disparity measure of child genetic risk for BMI (PRS-BMI) on objectively BMI at 12 years, if we were to intervene on parental feeding styles measured when children were 10-11 years (n=4,248). Results are presented as Adjusted Total Association (Adj-Ta) between genetic liability and BMI at 12 years, versus the Interventional Disparity Measure Direct Effect (IDM-DE), which represents the association, that would remain, had we intervened on the parental feeding. For children with the top quintile of genetic liability, an intervention shifting parental feeding to the levels of children with lowest genetic risk, resulted in a difference of 0.81 kg/m2in BMI at 12y (Adj-Ta= 3.27, 95%CI: 3.04, 3.49; versus IDM-DE=2.46, 95%CI: 2.24, 2.67). Findings suggest that parental feeding interventions have the potential to buffer some of the genetic liability for childhood obesity. Further, we highlight a novel way to analyze potential interventions for health conditions only using secondary data analyses, by combining methodology from statistical genetics and social epidemiology.
Online learning for orientation estimation during translation in an insect ring attractor network
Authors: Robinson Brian S. , Norman-Tenazas Raphael , Cervantes Martha , Symonette Danilo , Johnson Erik C. , Joyce Justin , Rivlin Patricia K. , Hwang Grace , Zhang Kechen , Gray-Roncal William 
Published: January 2021

AbstractInsect neural systems are a promising source of inspiration for new algorithms for navigation, especially on low size, weight, and power platforms. There have been unprecedented recent neuroscience breakthroughs withDrosophilain behavioral and neural imaging experiments as well as the mapping of detailed connectivity of neural structures. General mechanisms for learning orientation in the central complex (CX) ofDrosophilahave been investigated previously; however, it is unclear how these underlying mechanisms extend to cases where there is translation through an environment (beyond only rotation), which is critical for navigation in robotic systems. Here, we develop a CX neural connectivity-constrained model that performs sensor fusion, as well as unsupervised learning of visual features for path integration; we demonstrate the viability of this circuit for use in robotic systems in simulated and physical environments. Furthermore, we propose a theoretical understanding of how distributed online unsupervised network weight modification can be leveraged for learning in a trajectory through an environment by minimizing of orientation estimation error. Overall, our results here may enable a new class of CX-derived low power robotic navigation algorithms and lead to testable predictions to inform future neuroscience experiments.SummaryAn insect neural connectivity-constrained model performs sensor fusion and online learning for orientation estimation.
Restoration of deficient DNA Repair Genes Mitigates Genome Instability and Increases Productivity of Chinese Hamster Ovary Cells
Authors: Spahn Philipp N. , Zhang Xiaolin , Hu Qing , Hamaker Nathaniel K. , Hefzi Hooman , Li Shangzhong , Kuo Chih-Chung , Huang Yingxiang , Lee Jamie C. , Ly Peter , Lee Kelvin H. , Lewis Nathan E. 
Published: January 2021

AbstractChinese Hamster Ovary (CHO) cells are the primary host used for manufacturing of therapeutic proteins. However, production instability of high-titer cell lines is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein titer. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide polymorphisms (SNPs) in DNA repair genes. Comparison with other mammalian cells confirmed DNA repair is compromised in CHO. Restoration of key DNA repair genes by SNP reversal or expression of intact cDNAs improved DNA repair and genome stability. Moreover, the restoration of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show for the first time that correction of key DNA repair genes yields considerable improvements in stability and protein expression in CHO, and provide new opportunities for cell line development and a more efficient and sustainable production of therapeutic proteins.