bluuebunny/crossref_metadata_2025_split huggingface dataset:
Geographic pair-matching in large-scale cluster randomized trials
Authors: Arnold Benjamin F. , Rerolle Francois , Tedijanto Christine , Njenga Sammy M. , Rahman Mahbubur , Ercumen Ayse , Mertens Andrew , Pickering Amy , Lin Audrie , Arnold Charles D. , Das Kishor , Stewart Christine P. , Null Clair , Luby Stephen P. , Colford John M. , Hubbard Alan E. , Benjamin-Chung Jade 
Published: May 2023

AbstractCuster randomized trials are often used to study large-scale public health interventions. In large trials, even small improvements in statistical efficiency can have profound impacts on the required sample size and cost. Pair matched randomization is one strategy with potential to increase trial efficiency, but to our knowledge there have been no empirical evaluations of pair-matching in large-scale, epidemiologic field trials. Location integrates many socio-demographic and environmental characteristics into a single feature. Here, we show that geographic pair-matching leads to substantial gains in statistical efficiency for 14 child health outcomes that span growth, development, and infectious disease through a re-analysis of two large-scale trials of nutritional and environmental interventions in Bangladesh and Kenya. We estimate relative efficiencies ≥ 1.1 for all outcomes assessed and relative efficiencies regularly exceed 2.0, meaning an unmatched trial would have needed to enroll at least twice as many clusters to achieve the same level of precision as the geographically pair-matched design. We also show that geographically pair-matched designs enable estimation of fine-scale, spatially varying effect heterogeneity under minimal assumptions. Our results demonstrate broad, substantial benefits of geographic pair-matching in large-scale, cluster randomized trials.
Repeated Omicron exposures override ancestral SARS-CoV-2 immune imprinting
Authors: Yisimayi Ayijiang , Song Weiliang , Wang Jing , Jian Fanchong , Yu Yuanling , Chen Xiaosu , Xu Yanli , Yang Sijie , Niu Xiao , Xiao Tianhe , Wang Jing , Zhao Lijuan , Sun Haiyan , An Ran , Zhang Na , Wang Yao , Wang Peng , Yu Lingling , Lv Zhe , Gu Qingqing , Shao Fei , Jin Ronghua , Shen Zhongyang , Xie Xiaoliang Sunney , Wang Youchun , Cao Yunlong 
Published: May 2023

AbstractThe continuous emergence of highly immune evasive SARS-CoV-2 variants, like XBB.1.51,2and XBB.1.163,4, highlights the need to update COVID-19 vaccine compositions. However, immune imprinting induced by wildtype (WT)-based vaccination would compromise the antibody response to Omicron-based boosters5-9. Vaccination strategies that can counter immune imprinting are critically needed. In this study, we investigated the degree and dynamics of immune imprinting in mouse models and human cohorts, especially focusing on the role of repeated Omicron stimulation. Our results show that in mice, the efficacy of single Omicron-boosting is heavily limited by immune imprinting, especially when using variants antigenically distinct from WT, like XBB, while the concerning situation could be largely mitigated by a second Omicron booster. Similarly, in humans, we found that repeated Omicron infections could also alleviate WT-vaccination-induced immune imprinting and generate high neutralizing titers against XBB.1.5 and XBB.1.16 in both plasma and nasal mucosa. By isolating 781 RBD-targeting mAbs from repeated Omicron infection cohorts, we revealed that double Omicron exposure alleviates immune imprinting by generating a large proportion of highly matured and potent Omicron-specific antibodies. Importantly, epitope characterization using deep mutational scanning (DMS) showed that these Omicron-specific antibodies target distinct RBD epitopes compared to WT-induced antibodies, and the bias towards non-neutralizing epitopes observed in single Omicron exposures due to imprinting was largely restored after repeated Omicron stimulation, together leading to a substantial neutralizing epitope shift. Based on the DMS profiles, we identified evolution hotspots of XBB.1.5 RBD and demonstrated the combinations of these mutations could further boost XBB.1.5’s immune-evasion capability while maintaining high ACE2 binding affinity. Our findings suggest the WT component should be abandoned when updating COVID-19 vaccine antigen compositions to XBB lineages, and those who haven’t been exposed to Omicron yet should receive two updated vaccine boosters.
An expanded palette of ATP sensors for subcellular and multiplexed imaging
Authors: Sahan Ayse Z. , Youngblood Eventine , Das Siddharth , Schmitt Danielle L. , Zhang Jin 
Published: May 2023

AbstractGenetically encoded fluorescent biosensors that detect changes in ATP levels in live cells have enabled the discovery of novel roles for ATP in cellular processes and signaling. Many of these available ATP biosensors have a limited dynamic range, or have ATP affinities that are not suitable for sensing the physiological concentrations of ATP in mammalian cells. To address these limitations, we developed a FRET-based ATP biosensor with enhanced dynamic range and signal to noise ratio, eATeam. Using eATeam, we uncovered distinct spatiotemporal dynamics of ATP changes upon inhibition of cellular energy production. We also developed dimerization-dependent GFP and RFP-based ATP biosensors with enhanced dynamic ranges compared to the current standard in the field. Using the single-color ATP biosensors, we visualized the complex interplay between AMPK activity, ATP, lactate, and calcium by multiplexed imaging in single cells. This palette of ATP sensors expands the toolbox for interrogating subcellular ATP regulation and metabolic signaling in living cells.
Regulation of lipolysis by 14-3-3 proteins on human adipocyte lipid droplets
Authors: Yang Qin , Loureiro Zinger Yang , Desai Anand , DeSouza Tiffany , Li Kaida , Wang Hui , Nicoloro Sarah M , Solivan-Rivera Javier , Corvera Silvia 
Published: May 2023

ABSTRACTAdipocyte lipid droplets (LDs) play a crucial role in systemic lipid metabolism by storing and releasing lipids to meet the organism’s energy needs. Hormonal signals such as catecholamines and insulin act on adipocyte LDs, and impaired responsiveness to these signals can lead to uncontrolled lipolysis, lipotoxicity, and a higher risk of metabolic diseases. To investigate the mechanisms that control LD function in human adipocytes, we employed techniques to obtain mesenchymal progenitor cells on a large scale and applied proximity labeling mediated by enhanced ascorbate peroxidase (APEX2) to identify the interactome of PLIN1 in differentiated adipocytes. We identified 70 proteins that interact specifically with PLIN1, including PNPLA2 and LIPE, which are the primary effectors of regulated triglyceride hydrolysis, and four members of the 14-3-3 protein family (YWHAB, YWHAE, YWHAZ, and YWHAG), which are known to regulate diverse signaling pathways. Functional studies showed that YWHAB is required for maximum cAMP-stimulated lipolysis and helps to mitigate the anti-lipolytic effects of insulin. These findings reveal new regulatory mechanisms that control lipolysis in human metabolism.SIGNIFICANCE STATEMENTLipid droplets are ubiquitous cytoplasmic organelles that store metabolic energy and play a key role in cellular lipid metabolism (1). Adipocyte LDs play an additional, crucial role, as they supply the energy needs of the whole body through hormonally regulated triglyceride synthesis, storage, and release. The mechanisms by which adipocyte lipid droplets release lipids for systemic use has been mostly studied in mouse models and cell lines. To understand how lipid mobilization is controlled in human adipocytes, we used proximity labeling to identify proteins that interact with PLIN1, a major component of the lipid droplet, in adipocytes generated from primary human progenitor cells. Our study catalogues the interactome of human PLIN1 and identifies previously unrecognized potential mechanism for control of human adipocyte lipolysis through 14-3-3 proteins.
Glycogen phosphorylase inhibition alongside taxol chemotherapy synergistically elicits ferroptotic cell death in clear cell ovarian and kidney cancers
Authors: Khan Tashbib , Kryza Thomas , He Yaowu , Gunter Jennifer H , Gough Madeline , Snell Cameron , Hooper John D. 
Published: May 2023

ABSTRACTBackgroundClear cell carcinomas (CCCs) are a distinct histopathological subtype defined by a clear cytoplasm comprised of glycogen and lipids and characterised by poor prognosis and widespread chemoresistance. In the present work we investigate glycogen metabolism as a targetable modality for these cancers.Methods and ResultsAdopting the indole carboxamide site pan-glycogen phosphorylase inhibitor CP91149 against clear cell ovarian and renal cancer cell line models, we note antiproliferative and antimigratory effects, as well as energetic stress reflected by reduced ATP pools and increased superoxide-derived reactive oxygen species. Following this, using the agent alongside standard of care chemotherapies for clear cell ovarian (ccOC) and renal cell carcinoma (ccRCC), we note specific synergy with microtubule disrupting chemotherapy paclitaxel, a phenomenon retained in ccOC lines made stably resistant to paclitaxel. Rescue experiments, as well as phenotypic assays suggest that combination-treated cells undergo ferroptotic cell death. We postulate this synergistic efficacy to arise from subjecting the already hypersensitive clear cell cancers to the mitochondrial stress elicited by taxol chemotherapy alongside the oxidative stress augured by glycogen phosphorylase inhibition.ConclusionsGiven that CCCs are widely chemoresistant, the present work potentially presents a novel therapeutic avenue for this shared histotype.
KairoSight-3.0: A Validated Optical Mapping Software to Characterize Cardiac Electrophysiology, Excitation-Contraction Coupling, and Alternans
Authors: Haq Kazi T , Roberts Anysja , Berk Fiona , Allen Samuel , Swift Luther M , Posnack Nikki Gillum 
Published: May 2023

ABSTRACTBackgroundCardiac optical mapping is an imaging technique that measures fluorescent signals across a cardiac preparation. Dual optical mapping of voltage-sensitive and calcium-sensitive probes allow for simultaneous recordings of cardiac action potentials and intracellular calcium transients with high spatiotemporal resolution. The analysis of these complex optical datasets is both time intensive and technically challenging; as such, we have developed a software package for semi-automated image processing and analysis. Herein, we report an updated version of our software package (KairoSight-3.0) with features to enhance characterization of cardiac parameters using optical signals.MethodsTo test software validity and applicability, we used Langendorff-perfused heart preparations to record transmembrane voltage and intracellular calcium signals from the epicardial surface. Isolated hearts from guinea pigs and rats were loaded with a potentiometric dye (RH237) and/or calcium indicator dye (Rhod-2AM) and fluorescent signals were acquired. We used Python 3.8.5 programming language to develop theKairoSight-3.0software. Cardiac maps were validated with a user-specified manual mapping approach.ResultsManual maps of action potential duration (30 or 80% repolarization), calcium transient duration (30 or 80% reuptake), action potential and calcium transient alternans were constituted to validate the accuracy of software-generated maps. Manual and software maps had high accuracy, with >97% of manual and software values falling within 10 ms of each other and >75% within 5 ms for action potential duration and calcium transient duration measurements (n=1000-2000 pixels). Further, our software package includes additional cardiac metric measurement tools to analyze signal-to-noise ratio, conduction velocity, action potential and calcium transient alternans, and action potential-calcium transient coupling time to produce physiologically meaningful optical maps.ConclusionsKairoSight-3.0has enhanced capabilities to perform measurements of cardiac electrophysiology, calcium handling, and the excitation-contraction coupling with satisfactory accuracy.Graphical Abstract Demonstrating Experimental and Data Analysis WorkflowCreated withBiorender.com
Unveiling the Catalytic Mechanism of GTP Hydrolysis in Microtubules
Authors: Beckett Daniel , Voth Gregory A. 
Published: May 2023

AbstractMicrotubules (MTs) are large cytoskeletal polymers, composed of αβ-tubulin heterodimers, capable of stochastically converting from polymerizing to depolymerizing states and vice-versa. Depolymerization is coupled with hydrolysis of GTP within β-tubulin. Hydrolysis is favored in the MT lattice compared to free heterodimer with an experimentally observed rate increase of 500 to 700 fold, corresponding to an energetic barrier lowering of 3.8 to 4.0 kcal/mol. Mutagenesis studies have implicated α-tubulin residues, α:E254 and α:D251, as catalytic residues completing the β-tubulin active site of the lower heterodimer in the MT lattice. The mechanism for GTP hydrolysis in the free heterodimer, however, is not understood. Additionally, there has been debate concerning whether the GTP-state lattice is expanded or compacted relative to the GDP-state and whether a “compacted” GDP-state lattice is required for hydrolysis. In this work, extensive QM/MM simulations with transition-tempered metadynamics free energy sampling of compacted and expanded inter-dimer complexes, as well as free heterodimer, have been carried out to provide clear insight into the GTP hydrolysis mechanism. α:E254 was found to be the catalytic residue in a compacted lattice, while in the expanded lattice disruption of a key salt bridge interaction renders α:E254 less effective. The simulations reveal a barrier decrease of 3.8 ± 0.5 kcal/mol for the compacted lattice compared to free heterodimer, in good agreement with experimental kinetic measurements. Additionally, the expanded lattice barrier was found to be 6.3 ± 0.5 kcal/mol higher than compacted, demonstrating that GTP hydrolysis is variable with lattice state and slower at the MT tip.Significance StatementMicrotubules (MTs) are large and dynamic components of the eukaryotic cytoskeleton with the ability to stochastically convert from a polymerizing to a depolymerizing state and vice-versa. Depolymerization is coupled to the hydrolysis of guanosine-5’-triphosphate (GTP), which is orders of magnitude faster in the MT lattice than in free tubulin heterodimers. Our results computationally ascertain the catalytic residue contacts in the MT lattice that accelerate GTP hydrolysis compared to the free heterodimer as well as confirm that a compacted MT lattice is necessary for hydrolysis while a more expanded lattice is unable to form the necessary contacts and thereby hydrolyze GTP.
Improving AlphaFold2-based Protein Tertiary Structure Prediction with MULTICOM in CASP15
Authors: Liu Jian , Guo Zhiye , Wu Tianqi , Roy Raj S. , Chen Chen , Cheng Jianlin 
Published: May 2023

AbstractSince CASP14, AlphaFold2 has become the standard method for protein tertiary structure prediction. One remaining challenge in the field is to further improve the accuracy of AlphaFold2-based protein structure prediction. To address this challenge, we developed a new version of the MULTICOM system to sample diverse multiple sequence alignments (MSAs) and structural templates to improve the input for AlphaFold2 to generate structural models. The models are then ranked by both the pairwise model similarity and AlphaFold2 self-reported model quality score. The top ranked models are further refined by a novel structure alignment-based refinement method powered by Foldseek. Moreover, for a monomer target that is a subunit of a protein assembly (complex), MULTICOM integrates tertiary and quaternary structure prediction together to account for tertiary structural changes induced by protein-protein interaction in the assembly. The MULTICOM system participated in the tertiary structure prediction in the 15thCritical Assessment of Techniques for Protein Structure Prediction (CASP15) in 2022 as server and human predictors. Our best server predictor (MULTICOM_refine) ranked 3rdamong 47 CASP15 server predictors and our best human predictor (MULTICOM) ranked 7thamong all 132 human and server predictors. The average GDT-TS score and TM-score of the first structural models that MULTICOM_refine predicted for 94 CASP15 domains are ∼0.80 and ∼0.92, 9.6% and 8.2% and higher than ∼0.73 and 0.85 of the standard AlphaFold2 predictor respectively. The results demonstrate that our approach can significantly improve the accuracy of the AlphaFold2-based protein tertiary structure prediction. The source code of MULTICOM is available at:https://github.com/BioinfoMachineLearning/MULTICOM3.
Lysine-independent ubiquitination and degradation of REV-ERBα involves a bi-functional degradation control sequence at its N-terminus
Authors: Suen Ting-Chung , DeBruyne Jason P. 
Published: May 2023

AbstractREV-ERBα and REV-ERBβ proteins play crucial roles in linking the circadian system to overt daily rhythms in mammalian physiology and behavior. In most tissues, REV-ERBα protein robustly cycles such that it is detected only within a tight interval of 4-6 hours each day, suggesting both its synthesis and degradation are tightly controlled. Several ubiquitin ligases are known to drive REV-ERBα degradation, but how they interact with REV-ERBα and which lysine residues they ubiquitinate to promote degradation are unknown. In this study, we attempted to identify both ubiquitin-ligase-binding and ubiquitination sites within REV-ERBα required for its degradation. Surprisingly, mutating all lysine residues, the common sites for ubiquitin conjugation, in REV-ERBα to arginines (K20R), did very little to impair its degradation in cells. K20R were degraded much faster by co-expression of two E3 ligases, SIAH2 or SPSB4, suggesting possible N-terminal ubiquitination. To explore this, we examined if small deletions at the N-terminus of REV-ERBα would alter its degradation. Interestingly, deletion of amino acid (AA) residues 2 to 9 (delAA2-9) clearly resulted in a less stable REV-ERBα. We found that it was the length (i.e. 8 AA), and not the specific sequence, that confers stability in this region. Simultaneously, we also mapped the interaction site of the E3 ligase SPSB4 to this same region, specifically requiring AA4-9 of REV-ERBα. Thus, the first 9 AA of REV-ERBα has two opposing roles in regulating REV-ERBα turnover. Further, deleting eight additional AAs (delAA2-17) from the N-terminus strongly prevents REV-ERBα degradation. Combined, these results suggest that complex interactions within the first 25AAs potentially act as an endogenous ‘switch’ that allows REV-ERBα to exist in a stabilized conformation in order to accumulate at one time of day, but then rapidly shifts to a destabilized form, to enhance its removal at the end of its daily cycle.
Functional genomic analysis of non-canonical DNA regulatory elements of the aryl hydrocarbon receptor
Authors: Patel Tajhal D , Nakka Manjula , Grimm Sandra L , Coarfa Cristian , Gorelick Daniel A 
Published: May 2023

ABSTRACTThe aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that binds DNA and regulates genes in response to halogenated and polycyclic aromatic hydrocarbons. AHR also regulates the development and function of the liver and the immune system. In the canonical pathway, AHR binds a consensus DNA sequence, termed the xenobiotic response element (XRE), recruits protein coregulators, and regulates target gene expression. Emerging evidence suggests that AHR may regulate gene expression via an additional pathway, by binding to a non-consensus DNA sequence termed the non-consensus XRE (NC-XRE). The prevalence of NC-XRE motifs in the genome is not known. Studies using chromatin immunoprecipitation and reporter genes provide indirect evidence of AHR-NC-XRE interactions, but direct evidence for an AHR-NCXRE interaction that regulates transcription in a natural genomic context is lacking. Here, we analyzed AHR binding to NC-XRE DNA on a genome-wide scale in mouse liver. We integrated ChIP-seq and RNA-seq data and identified putative AHR target genes with NC-XRE motifs in regulatory regions. We also performed functional genomics at a single locus, the mouseSerpine1gene. Deleting NC-XRE motifs from theSerpine1promoter reduced the upregulation ofSerpine1by TCDD, an AHR ligand. We conclude that AHR upregulatesSerpine1via NC-XRE DNA. NC-XRE motifs are prevalent throughout regions of the genome where AHR binds. Taken together, our results suggest that AHR regulates genes via NC-XRE motifs. Our results will also improve our ability to identify AHR target genes and their physiologic relevance.
GPCR binding and JNK3 activation by arrestin-3 have different structural requirements
Authors: Zheng Chen , Weinstein Liana D. , Nguyen Kevin K. , Grewal Abhijeet , Gurevich Eugenia V. , Gurevich Vsevolod V. 
Published: May 2023

AbstractArrestins bind active phosphorylated G protein-coupled receptors (GPCRs). Among the four mammalian subtypes, only arrestin-3 facilitates the activation of JNK3 in cells. In available structures, Lys-295 in the lariat loop of arrestin-3 and its homologue Lys-294 in arrestin-2 directly interact with the activator-attached phosphates. We compared the role of arrestin-3 conformational equilibrium and of Lys-295 in GPCR binding and JNK3 activation. Several mutants with enhanced ability to bind GPCRs showed much lower activity towards JNK3, whereas a mutant that does not bind GPCRs was more active. Subcellular distribution of mutants did not correlate with GPCR recruitment or JNK3 activation. Charge neutralization and reversal mutations of Lys-295 differentially affected receptor binding on different backgrounds, but had virtually no effect on JNK3 activation. Thus, GPCR binding and arrestin-3-assisted JNK3 activation have distinct structural requirements, suggesting that facilitation of JNK3 activation is the function of arrestin-3 that is not bound to a GPCR.
The genetic code assembles via division and fusion, basic cellular events
Authors: Yarus Michael 
Published: May 2023

AbstractStandard Genetic Code (SGC) evolution is quantitatively modeled in computed ‘worlds’ containing up to 2000 independent coding ‘environments’. Environments can host multiple codes that may fuse or divide, with division yielding identical descendants. Code division may be selected - sophisticated gene products could be required for orderly separation. Several unforeseen results emerge: more rapid evolution requires unselective code division, rather than its selective form. Combining selective and unselective code division, with/without code fusion, with/without independent environmental coding tables and with/without wobble defines 25= 32 possible pathways for SGC evolution. These 32 possible histories are compared, particularly, for speed and accuracy. Pathways differ greatly; for example, ≈ 300-fold different in time to evolve SGC-like codes. Eight of 32 pathways, employing code division, are quickest. Four of these eight, that combine fusion and division, also unite speed and accuracy. The two precise, swiftest paths, thus the most likely routes to the SGC, are similar, differing only in fusion with independent environmental codes. Code division instead of fusion with unrelated codes implies that independent codes can be dispensable. Instead, a single ancestral code that divides and fuses can initiate fully encoded peptide biosynthesis. Division and fusion create a ‘crescendo of competent coding’, facilitating search for the SGC, and also assist advent of otherwise disfavored wobble coding. Code fusion readily unites multiple codon assignment mechanisms. But via code division and fusion, the SGC is shown to emerge from a single primary origin, via familiar cellular events.