bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comprehensive Analysis of CRISPR Base Editing Outcomes for Multimeric Protein
Authors: Kabra Meha , Moosajee Mariya , Newby Gregory A. , Molugu Kaivalya , Saha Krishanu , Liu David R. , Pattnaik Bikash R. 
Published: June 2022

AbstractPoint mutations in the KCNJ13 gene cause an autosomal recessive, childhood blindness, Leber congenital amaurosis (LCA16) due to a loss-of-function Kir7.1 channel. In the present study, we investigated the etiology of LCA16 caused by a KCNJ13 missense mutation (c.431T>C, p.Leu144Pro) and explored the activity of two cytosine base editors mRNAs (CBEs, BE4max-WTCas9, and evoCDA-SpCas9-NG) as a proof-of-concept therapeutic option. We observed the KCNJ13-related retinopathy phenotype in patients harboring L144P mutation. Our in-silico prediction and in vitro validation demonstrated that L144P mutation affects the channel function. We observed high on-target efficiency in the CBEs treated L144P mutant gene expressing HEK-293 cells. Strikingly, our evaluation of base editing efficacy using electrophysiology showed negligible channel function. We found that the editing bystander ‘Cs’ in the protospacer region led to a missense change (L143F) in evoCDA edited cells and only silent changes in BE4max edited cells. Upon investigation of the effect of the synonymous codon, our extended analysis revealed distortion of mRNA structure, altered half-life, and/or low abundance of the cognate tRNA. We propose that KCNJ13-L144P mutation or other genes that share similar genetic complexity may be challenging to correct with the current generation of CRISPR base editors, and a combinational therapy using CRISPR base editors with a tighter editing window and requisite cognate-tRNA supplementation could be an alternative therapeutic approach to restore Kir7.1 channel function in LCA16 patients. Other options for hard-to-rescue alleles could employ homology-directed repair using CRISPR/Cas9 nucleases, Prime editing, and AAV-mediated gene augmentation.
Ex vivoExpansion Potential of Murine Hematopoietic Stem Cells: A Rare Property Only Partially Predicted by Phenotype
Authors: Zhang Qinyu , Olofzon Rasmus , Konturek-Ciesla Anna , Yuan Ouyang , Bryder David 
Published: June 2022

ABSTRACTThe scarcity of hematopoietic stem cells (HSCs) restricts their use in both clinical settings and experimental research. Here, we examined a recently developed method for expanding rigorously purified murine HSCsex vivo.After three weeks of culture, only 0.1% of cells exhibited the input HSC phenotype, but these accounted for almost all functional long-term HSC activity. Input HSCs displayed varying potential forex vivoself-renewal, with alternative outcomes revealed by single cell multimodal RNA- and ATAC-seq profiling. While most HSC progeny offered only transientin vivoreconstitution, these cells efficiently rescued mice from lethal myeloablation. The amplification of functional HSC activity allowed for long-term multilineage engraftment in unconditioned hosts that associated with a return of HSCs to quiescence. Thereby, our findings identify several key considerations forex vivoHSC expansion, with major implications also for assessment of normal HSC activity.Key point:Ex vivoself-renewal is an intrinsic property of rare candidate HSCs, with implications for assessments of HSC activity by transplantation.
Trim69 is a microtubule regulator that acts as a pantropic viral inhibitor
Authors: Song Yuxin , Nguyen Xuan-Nhi , Kumar Anuj , da Silva Claire , Picard Léa , Etienne Lucie , Cimarelli Andrea 
Published: June 2022

ABSTRACTTo identify novel cellular modulators of HIV-1 infection in IFN-stimulated myeloid cells, we have carried out a screen that combines functional and evolutionary analyses in THP-1-PMA cells that led us to the Tripartite Motif Protein 69 (Trim69), a poorly studied member of the Trim family of innate immunity regulators. Trim69 inhibits HIV-1, primate lentiviruses and the negative and positive-strand RNA viruses VSV and SARS-CoV2, overall indicating it is a broad-spectrum antiviral factor. Trim69 binds directly to microtubules and its antiviral activity is intimately linked to its ability to promote the accumulation of stable MTs, a specialized subset of microtubules. By analyzing the behavior of primary blood cells, we provide evidence that a program of MT stabilization is commonly observed in response to IFN-I in cells of the myeloid lineage and Trim69 is the key factor behind this program.Overall, our study identifies Trim69 as the first antiviral innate defense factor that regulates the properties of microtubules to limit viral spread, highlighting the possibility that the cytoskeleton may be a novel unappreciated fighting ground in the host-pathogen interactions that underlie viral infections.
Exercise preserves fitness capacity during aging through AMPK and mitochondrial dynamics
Authors: Campos Juliane Cruz , Marchesi Bozi Luiz Henrique , Traa Annika , van der Bliek Alexander M , Van Raamsdonk Jeremy M. , Blackwell T. Keith , Ferreira Julio Cesar Batista 
Published: June 2022

AbstractExercise is a nonpharmacological intervention that improves health during aging, and a valuable tool in the diagnostics of aging-related diseases. In muscle, exercise transiently alters mitochondrial functionality and metabolism. Mitochondrial fission and fusion are critical effectors of mitochondrial plasticity, which allows a fine-tuned regulation of organelle connectiveness, size and function. Here we have investigated the role of mitochondrial dynamics during exercise in the genetically tractable modelCaenorhabditis elegans. We show that in body wall muscle a single exercise session induces a cycle of mitochondrial fragmentation followed by fusion after a recovery period, and that daily exercise sessions delay the mitochondrial fragmentation and fitness capacity decline that occur with aging. The plasticity of this mitochondrial dynamics cycle is essential for fitness capacity and its enhancement by exercise training. Surprisingly, among longevity-promoting mechanisms we analyzed, constitutive activation of AMPK uniquely preserves fitness capacity during aging. As with exercise training, this benefit of AMPK is abolished by impairment of mitochondrial fission or fusion. AMPK is also required for fitness capacity to be enhanced by exercise, with our findings together suggesting that exercise enhances muscle function through AMPK regulation of mitochondrial dynamics. Our results indicate that mitochondrial connectivity and the mitochondrial dynamics cycle are essential for maintaining fitness capacity and exercise responsiveness during aging, and suggest that AMPK activation may recapitulate some exercise benefits. Targeting mechanisms to optimize mitochondrial fission and fusion balance, as well as AMPK activation, may represent promising strategies for promoting muscle function during aging.Significance StatementExercise is a powerful anti-aging intervention. In muscle exercise remodels mitochondrial metabolism and connectiveness, but the role of mitochondrial dynamics in exercise responsiveness has remained poorly understood. Working inCaenorhabditis elegans, we find that the mitochondrial dynamics cycle of fission and fusion is critical for fitness capacity, that exercise delays an aging-associated decline in mitochondrial connectiveness and fitness capacity, and that the mitochondrial dynamics cycle is required for the latter benefit. AMPK, which regulates mitochondrial dynamics, is needed for exercise to maintain fitness capacity with age and can recapitulate this exercise benefit. Our data identify the mitochondrial dynamics cycle as an essential mediator of exercise responsiveness, and an entry point for interventions to maintain muscle function during aging.
Regeneration and Developmental Enhancers Are Differentially Compatible with Minimal Promoters
Authors: Begeman Ian J. , Emery Benjamin , Kurth Andrew , Kang Junsu 
Published: June 2022

ABSTRACTEnhancers and promoters are cis-regulatory elements that control gene expression. Enhancers are activated in a cell type-, tissue-, and condition-specific manner to stimulate promoter function and transcription. Zebrafish have emerged as a powerful animal model for examining the activities of enhancers derived from various species through transgenic enhancer assays, in which an enhancer is coupled with a minimal promoter. However, the efficiency of minimal promoters and their compatibility with multiple developmental and regeneration enhancers have not been systematically tested in zebrafish. Thus, we assessed the efficiency of six minimal promoters and comprehensively interrogated the compatibility of the promoters with developmental and regeneration enhancers. We found that the fos minimal promoter and Drosophila synthetic core promoter (DSCP) yielded high rates of leaky expression that may complicate the interpretation of enhancer assays. Notably, the adenovirus E1b promoter, the zebrafish lepb 0.8-kb (P0.8) and lepb 2-kb (P2) promoters, and a new zebrafish synthetic promoter (ZSP) that combines elements of the E1b and P0.8 promoters drove little or no ectopic expression, making them suitable for transgenic assays. We also found significant differences in compatibility among specific combinations of promoters and enhancers, indicating the importance of promoters as key regulatory elements determining the specificity of gene expression. Our study provides guidelines for transgenic enhancer assays in zebrafish to aid in the discovery of functional enhancers regulating development and regeneration.
Adenosine Reduces Sinoatrial Node Cell AP Firing Rate by uncoupling its Membrane and Calcium Clocks
Authors: Wirth Ashley N. , Tsutsui Kenta , Maltsev Victor A. , Lakatta Edward G. 
Published: June 2022

AbstractThe spontaneous action potential (AP) firing rate of sinoatrial nodal cells (SANC) is regulated by a system of intracellular Ca2+ and membrane ion current clocks driven by Ca2+-calmodulin-activated adenylyl cyclase-protein kinase A (PKA) signaling. The mean AP cycle length (APCL) and APCL variability inform on the effectiveness of clock coupling. Endogenous ATP metabolite adenosine (ado) binds to adenosine receptors that couple to Gi protein-coupled receptors, reducing spontaneous AP firing rate via Gβγ signaling that activates an membrane-clock outward current, IKACh. Ado also inhibits adenylyl cyclase activity via Giα signaling, impacting cAMP-mediated PKA-dependent protein phosphorylation and intracellular Ca2+ cycling. We hypothesize that in addition to IKAdo activation, ado signaling impacts Ca2+ via Giα signaling and that both effects reduce AP firing rate by reducing the effectiveness of the Ca2+ and membrane clock coupling. To this end, we measured Ca2+ and membrane potential characteristics in enzymatically isolated single rabbit SANC. 10 µM ado substantially increased both the mean APCL (on average by 43%, n=10) and AP beat-to-beat variability from 5.1±1.7% to 7.2±2.0% (n=10) measured via membrane potential and 5.0±2.2 to 10.6±5.9 (n=40) measured via Ca2+ (assessed as the coefficient of variability, CV=SD/mean). These effects were mediated by hyperpolarization of the maximum diastolic membrane potential (membrane clock effect) and suppression of diastolic spontaneous, local Ca2+ releases (LCRs) (Ca2+ clock effect): as LCR size distributions shifted from larger to smaller values, the time of LCR occurrence during diastolic depolarization (LCR period) became prolonged, and the ensemble LCR Ca2+ signal became reduced. The tight linear relationship of coupling between LCR period to the APCL in the presence of ado “drifted” upward and leftward, i.e. for a given LCR period, APCL was prolonged, becoming non-linear indicating clock uncoupling. An extreme case of uncoupling occurred at higher ado concentrations (>100 µM): small stochastic LCRs of the Ca2+ clock failed to self-organize and synchronize to the membrane clock, thus creating a failed attempt to generate an AP resulting in arrhythmia and cessation of AP firing. Thus, the effects of ado to activate Gβγ and IKACh, Ado and to activate Giα, suppressing adenylyl cyclase activity, both contribute to the ado-induced increase in the mean APCL and APCL variability by reducing the fidelity of clock coupling and AP firing rate.
Direct Force Measurement and Loading on Developing Tissues in Intact Avian Embryos
Authors: Chan Chon U , Xiong Fengzhu , Michaut Arthur , Vidigueira Joana M. N. , Pourquie Olivier , Mahadevan L. 
Published: June 2022

AbstractDevelopmental morphogenesis is driven by tissue stresses acting on tissue rheology. Direct measurements of forces in small tissues (100μm-1mm)in situsuch as in early embryos require high spatial precision and minimal invasiveness. Here we report tissue force microscopy (TiFM) integrating a vertical cantilever probe and live imaging to enable close-loop control of mechanical loading in early chicken embryos. By testing previously qualitatively characterized force-producing tissues in the elongating body axis, we show that TiFM quantitatively captures stress dynamics with high sensitivity. TiFM also provides the capacity of applying a stable, minimally-invasive and physiologically relevant load to drive tissue deformation, which alters morphogenetic progression and cell movements. Together, TiFM addresses a key technological gap in tissue force measurement and manipulation in small developing embryos, and promises to contribute to the quantitative understanding of complex multi-tissue mechanics during development.
Probing the biophysical constraints of SARS-CoV-2 spike N-terminal domain using deep mutational scanning
Authors: Ouyang Wenhao O. , Tan Timothy J.C. , Lei Ruipeng , Song Ge , Kieffer Collin , Andrabi Raiees , Matreyek Kenneth A. , Wu Nicholas C. 
Published: June 2022

ABSTRACTIncreasing the expression level of the SARS-CoV-2 spike (S) protein has been critical for COVID-19 vaccine development. While previous efforts largely focused on engineering the receptor-binding domain (RBD) and the S2 subunit, the N-terminal domain (NTD) has been long overlooked due to the limited understanding of its biophysical constraints. In this study, the effects of thousands of NTD single mutations on S protein expression were quantified by deep mutational scanning. Our results revealed that in terms of S protein expression, the mutational tolerability of NTD residues was inversely correlated with their proximity to the RBD and S2. We also identified NTD mutations at the interdomain interface that increased S protein expression without altering its antigenicity. Overall, this study not only advances the understanding of the biophysical constraints of the NTD, but also provides invaluable insights into S-based immunogen design.
Structure of the GOLD-domain seven-transmembrane helix protein family member TMEM87A
Authors: Hoel Christopher M. , Zhang Lin , Brohawn Stephen G. 
Published: June 2022

AbstractTMEM87s are eukaryotic transmembrane proteins with two members (TMEM87A and TMEM87B) in humans. TMEM87s have proposed roles in protein transport to and from the Golgi, as mechanosensitive ion channels, and in developmental signaling. TMEM87 disruption has been implicated in cancers and developmental disorders. To better understand TMEM87 structure and function, we determined a cryo-EM structure of human TMEM87A in lipid nanodiscs. TMEM87A consists of a Golgi-dynamics (GOLD) domain atop a membrane spanning seven-transmembrane helix domain with a large cavity open to solution and the membrane outer leaflet. Structural and functional analyses suggest TMEM87A may not function as an ion channel or G-protein coupled receptor. We find TMEM87A shares its characteristic domain arrangement with seven other proteins in humans; three that had been identified as evolutionary related (TMEM87B, GPR107, and GPR108) and four previously unrecognized homologs (GPR180, TMEM145, TMEM181, and WLS)). Among these structurally related GOLD domain seven-transmembrane helix (GOST) proteins, WLS is best characterized as a membrane trafficking and secretion chaperone for lipidated Wnt signaling proteins. We find key structural determinants for WLS function are conserved in TMEM87A. We propose TMEM87A and structurally homologous GOST proteins could serve a common role in trafficking membrane-associated cargo.
Humanized mice bearing CRISPR/Cas9 Disruption of Signal Transducer and Activator of Transcription 1 (STAT1) to Model Primary Immunodeficiency
Authors: Aron Jennifer L. , Thauland Timothy , Khan Humza , Butte Manish J. 
Published: June 2022

AbstractBackgroundThe search for a single, pathogenic genetic variant in a patient suspected to have a monogenic inborn error of immunity (IEI) often reveals a multitude of rare variants of unknown significance (VUS). Distinguishing which VUS is disease-causing versus the irrelevant, rare variants from the genetic background is slow and difficult. Advances in gene editing technology, particularly CRISPR/Cas9, promise to accelerate the timeline for the development of single-variant animal models, thus affording an experimental system for validating new genes and their variants.ObjectiveWe sought to demonstrate a proof-of-concept of using CRISPR/Cas9 in human hematopoietic stem cells (hHSC) to develop of humanized mice bearing a hematopoietic deficiency in signal transducer and activator 1 (STAT1).MethodsUsing CRISPR/Cas9, we introduced indels into the STAT1 gene of hHSCs and implanted them into immunodeficient mice. The reconstituted immune systems were assessed by flow cytometry.ResultsMice transplanted with cells edited to eliminate STAT1 developed human immune systems with diverse cell phenotypes. Lymphocytes from these reconstituted mice showed low expression of STAT1 protein and diminished phosphorylation of STAT1 in response to interferon stimulation. These data mirror the impaired, but not abolished, response to interferons seen in human partial STAT1 deficiency. CRISPR/Cas9 genome editing techniques can be used to rapidly and inexpensively create functional, humanized models of primary immune deficiencies.
Corner flows induced by surfactant-producing bacteria Bacillus subtilis and Pseudomonas fluorescens
Authors: Li Yuan , Sanfilippo Joe , Kearns Daniel , Yang Judy Q. 
Published: June 2022

AbstractMechanistic understanding of bacterial spreading in soil is critical to control pathogenic contamination of groundwater and soil as well as design bioremediation projects. However, our understanding is currently limited by the lack of direct bacterial imaging in soil conditions. Here, we overcome this limitation by directly observing the spread of bacterial solution in a transparent chamber with varying corner angles designed to replicate soil-like conditions. We show that two common soil bacteria, Bacillus subtilis and Pseudomonas fluorescens, generate flows along sharp corners (< 60°) by producing surfactants that turn nonwetting solid surfaces into wetting surfaces. We further show that a surfactant-deficient mutant of B. subtilis cannot generate corner flows along sharp corners, confirming that the bacteria-generated corner flows require the production of bacterial surfactants. The speed of biosurfactant-induced corner flow at the sharp corner is about several millimeters per hour, similar to that of bacterial swarming, the fastest mode of known bacterial surface translocation. We further demonstrate that the bacteria-generated corner flow only occurs when the corner angle is less than a critical value, which can be predicted from the contact angle of the bacterial solution. Furthermore, we show that the corner flow has a maximum height due to the roundness or cutoff of corners. The mechanistic understanding and mathematical theories of bacterial spreading presented in this study will help improve predictions of bacterial spreading in soil, where corners are ubiquitous, and facilitate future designs of soil contamination mitigation and other bioremediation projects.ImportanceThe spread of bacterial cells in soil regulates soil biogeochemical cycles, increases the possibility of soil and groundwater contamination, and controls the efficiency of many bacteria-based bioremediation projects. However, mechanistic understanding of bacterial spreading in soil remains incomplete due to a lack of direct or in-situ observations. Here, we simulate confined spaces of soil using a transparent material with similar hydrophobicity as hydrocarbon-covered soil and directly visualize the spread of two common soil bacteria, Bacillus subtilis and Pseudomonas fluorescens. We show that both bacteria can generate vertical flows along sharp corners of the transparent chamber. The velocity of the bacterial corner flow is several millimeters per hour. We further demonstrate that the corner flow was generated by bacteria-produced bio-surfactants, which are soap-like chemicals and turn nonwetting solid surfaces into wetting surfaces. Our results will help improve predictions of bacterial spreading in soil and facilitate designs of soil-related bioremediation projects.
How knowledge sharing encourages innovative work behavior through occupational self-efficacy? The moderating role of entrepreneurial leadership
Authors: Islam Talat , Zahra Iram , Rehman Saif Ur , Jamil Saqib 
Published: June 2022

Purpose
Innovation has become a necessity for the information technology (IT) sector, especially during COVID-19 pandemic. Therefore, this study aims to investigate how knowledge sharing affects employees’ innovative work behavior (IWB). Specifically, the study examined occupational self-efficacy (as mediating mechanism) and entrepreneurial leadership (as boundary condition) to encourage IWB.

Design/methodology/approach
The study used social media platforms to collect data from 270 employees working in the IT sector through “google forms” on convenience basis between March and August, 2021. The study applied structural equation modeling in two stages to examine the measurement model (for uni-dimensionality) and the structural model (for hypotheses testing).

Findings
The study noted that knowledge sharing positively affects employees’ IWB and occupational self-efficacy positively explains this association. In addition, employees’ perception of entrepreneurial leadership strengthens the association between knowledge sharing and IWB.

Research limitations/implications
The study collected data from a developing country during COVID-19 by using a cross-sectional design that may restrict causality. However, the findings suggest the management not only encourages knowledge sharing environment but also engages employees in various training that motivate them to experiment with new ideas and techniques.

Originality/value
This study extends the existing literature on knowledge sharing and IWB by exploring occupational self-efficacy as mediating mechanism and entrepreneurial leadership as a boundary condition.
Parameter estimation for grey system models: gradient matching versus integral matching
Authors: Wei Baolei , Xie Naiming , Yang L.U. 
Published: June 2022

PurposeThe cumulative sum (Cusum) operator, also referred to as accumulating generation operator, is the fundamental of grey system models and proves to be successful in various real-world applications. This paper aims to uncover the advantages of the Cusum operator from a parameter estimation perspective, i.e. comparing integral matching with classical gradient matching.Design/methodology/approachGrey system models are represented as a state space form to investigate the effect of measurement errors on estimation performance; subsequently, gradient matching and integral matching are respectively formulated to estimate parameters from noisy observations and, then, their quantitative relationships are established by using matrix computation tricks.FindingsExtensive simulations, which are conducted on both linear and non-linear models under different sample size and noise level combinations, show that integral matching is superior to gradient matching, and, also the former is less sensitive to measurement error.Originality/valueThis paper explains why the Cusum operator is widely utilized in grey system models, thereby further solidifying the mathematical fundamentals of grey system models.