bluuebunny/crossref_metadata_2025_split huggingface dataset:
Helical transition of the bridge helix of Cas12a is an allosteric regulator of R-loop formation and RuvC activation
Authors: Ganguly Chhandosee , Martin Lindsie , Aribam Swarmistha , Thomas Leonard M. , Rajan Rakhi 
Published: January 2025

ABSTRACTCRISPR-Cas12a is widely used for genome editing and biomarker detection since it can create targeted double-stranded DNA breaks and promote non-specific DNA cleavage after identifying specific DNA. To mitigate the off-target DNA cleavage of Cas12a, we previously developed aFrancisella novicidaCas12a variant (FnoCas12aKD2P) by introducing double proline substitutions (K969P/D970P) in a conserved helix called the bridge helix (BH). In this work, we used cryogenic electron microscopy (cryoEM) to understand the molecular mechanisms of BH-mediated activation of Cas12a. We captured five structures of FnoCas12aKD2Pat different states of conformational activation. Comparison with wild-type (FnoCas12aWT) structures unravels a mechanism where BH acts as a trigger that allosterically activates REC lobe movements by tracking the number of base pairs in the growing RNA-DNA hybrid to undergo a loop-to-helical transition and bending to latch onto the hybrid. The transition of the BH is coupled to the previously reported loop-to-helix transition of the “lid”, essential for opening RuvC endonuclease, through direct interactions of residues of the BH and the lid. We also observe structural details of cooperativity of BH and “helix-1” of RuvC for activation, a previously proposed interaction. Overall, our study enables development of high-fidelity Cas12a and Cas9 variants by BH-modifications.
Guidestar: a spike-in approach to improve RNA detection accuracy in imaging-based spatial transcriptomics
Authors: Tan Jazlynn Xiu Min , Wang Lurong , Seow Wan Yi , Boonruangkan Jeeranan , Huang Mike , Toh Jiamin , Chen Kok Hao , Chou Nigel 
Published: January 2025

SummaryImaging-based spatial transcriptomics technologies, such as MERFISH and seq-FISH, use combinatorial barcoding and imaging to simultaneously detect individual RNA molecules from 10s to 10,000s of genes. These technologies require the decoding of individual RNA molecules’ location and gene identity from stacks of images. However, beyond using ‘blank’ code-words as negative controls, there is a lack of ground truth information embedded within the assay to experimentally measure the accuracy and sensitivity of the decoding algorithm. We introduce Guidestar, a system of spike-in controls integrated within a combinatorial FISH assay, that labels a subset of RNA transcripts with additional probes. These probes are imaged separately as ‘guide bits’, which serve as ground-truth data to assess decoding accuracy at the level of individual RNA molecules. Using Guidestar to evaluate accuracy of an existing decoding method suggested alternative parameter settings that increased sensitivity with minimal impact on accuracy. We also used the Guidestar dataset to train a machine-learning based classifier to distinguish true from false RNA calls, yielding 9% and 40% higher F1 scores across cell line and tissue samples, respectively.
Molecular Insights into Cross-Species Spillover of Coronavirus HKU5 via ACE2 Receptor Recognition
Authors: Xia Lingyun , Zhang Yuanyuan , Li Yuya , Li Dexin , Zhou Qiang 
Published: January 2025

AbstractCoronaviruses represent a significant zoonotic threat, with host adaptation serving as a pivotal determinant of cross-species transmission. The bat-derived β-coronavirus HKU5 utilizes its spike (S) protein for receptor recognition and viral entry. Here, we report the cryo-electron microscopy (cryo-EM) structure of the HKU5 S protein in a closed conformation. Two fatty acids are bound to the HKU5 S protein, stabilizing the S protein in the closed conformation. Furthermore, we solve the structure of the HKU5 receptor-binding domain (RBD) in complex with the peptidase domain (PD) ofPipistrellus abramusangiotensin-converting enzyme 2 (ACE2), uncovering a unique binding mode distinct from other coronaviruses that use ACE2 as their receptor. Evolutionary and functional analyses indicate that mutations on RBD can modulate receptor binding, while conservation and structural modeling suggest that HKU5 has the potential to cross species barrier. Notably, we identify ACE2 orthologs in avian species, such asPitta sordida, that support stable HKU5 RBD binding and interaction. These findings provide molecular insights into the host adaptation of HKU5 and underscore the importance of surveillance for this virus and its zoonotic risk.
Timed chromatin invasion during mitosis governs prototype foamy virus integration site selection and infectivity
Authors: Lagadec F. , Singh P. K. , Calmels C. , Lapaillerie D. , Lindemann D. , Parissi V. , Cherepanov P. , Engelman A. N. , Lesbats P. 
Published: January 2025

AbstractSelection of a suitable chromatin environment during retroviral integration is a tightly regulated and multilayered process that involves interplay between viral and host factors. However, whether intrinsic chromatin dynamics during mitosis modulate retroviral genome invasion is currently poorly described. Direct interaction between the spumaretrovirus prototype foamy virus (PFV) Gag protein and cellular chromatin has been described as a major determinant for integration site selection. A previous Gag chromatin-binding site (CBS)–nucleosome co-crystal structure revealed an interaction with the histone H2A-H2B acidic patch via a highly conserved arginine anchor residue. Yet, the molecular mechanisms regulating Gag-chromatin capture during PFV infection remain obscure. Here, we investigated the kinetics of Gag-chromatin interactions during mitosis and proviral integration of PFV-infected synchronized cells. Using Gag CBS variant viruses, we showed that alteration of Gag affinity for nucleosome binding induced untimely chromatin tethering during mitosis, decreased infectivity and redistributed viral integration sites to markers associated with late replication timing of host chromosomes. Mutant Gag proteins were moreover defective in their ability to displace the histone H4 tail from the nucleosome acidic patch of highly condensed mitotic chromatin. These data indicate that the mitotic chromatin landscape during Gag–nucleosome interactions hosts PFV integration site selection determinants and that spumaretroviruses evolved high-affinity chromatin binding to overcome early mitosis chromatin condensation for optimal viral DNA tethering, integration and infection.
MiRNA Locker: A Modularized DNA Assembly As miRNA Inhibitors
Authors: Zhu Lingyun , Min Lu , Zhu Chu-shu , Wu Xiaomin , Li Wenying , Ma Jiaxin , Lei Yilin , Gao Changsong , Qiu Xinyuan , Liu Chuanyang 
Published: January 2025

AbstractMicroRNAs (miRNAs) are vital post-transcriptional regulators that govern key cellular processes such as proliferation, migration, and apoptosis. Current loss-of-function approaches, including chemically modified antisense oligonucleotides (ASOs), face significant challenges, including high costs, limited scalability, and off-target effects. To overcome these limitations, we developed “miRNA Locker”, a novel miRNA inhibition platform created using the Overlapped Oligo Assembly (OOA) method. This innovative platform constructs highly stable dumbbell-shaped single-stranded DNA structures, offering improved target specificity, scalability, and cost-effectiveness. Using miR-214 as a proof-of-concept target, we demonstrated that miRNA Lockers effectively bind Argonaute-miRNA complexes, reduce miRNA levels, and induce downstream changes in gene expression and cellular phenotypes, surpassing the performance of commercial antagomirs. Furthermore, applying miRNA Lockers to miR-654 validated the regulatory role of miR-654 in modulating RNF8 expression and promoting epithelial-mesenchymal transition (EMT) in lung cancer cells. Our results highlight the potential of miRNA Lockers as a versatile tool for studying miRNA function and advancing miRNA-based therapies.Graphical Abstract
A cytoskeletal scaffold promotes motile cilia assembly by regulating transition-zone integrity
Authors: Park Hyunji , Choi Minjun , Cheung Helen Oi-Lam , Zhang Yu , Makino Shigeru , Yoshikawa Yoshiaki , Qi Haoran , Liu Zheng , Lan Guocheng , Fu Guoling , Wang Qian , Guo Shiny Shengzhen , Liu Pentao , Liu Zhen , Ti Shih-Chieh , Wang Won-Jing , Li Xiang David Xiang , Ni Tao , Hui Chi Chung , He Mu 
Published: January 2025

AbstractMotile cilia are eukaryotic organelles with essential chemo- and mechano-sensing functions across evolution, from single cell organisms to humans. Motile cilia of the mammalian nervous, respiratory and reproductive systems are characterized by unique motility proteins to generate fluid flow essential for transporting metabolites and removing mucus. The molecular mechanism of motile cilia biogenesis remains unknown. Here, we use mouse genetics, single-molecule motility assays, proteomics, high-resolution imaging, andin situcryo-tomography to identify mammalian KIF27, a motor protein of the Kinesin-4 family and homologue of the Hedgehog pathway regulator COS2/KIF7, as a key regulator of motile cilia assembly. We show that KIF27 promotes the integrity of the transition zone, a diffusion barrier situated at the cilium base. Loss of KIF27 causes specific and profound defects in axonemal structure and disrupts cilia beating, which collectively lead to organismal phenotypes that recapitulate primary ciliary dyskinesia. We show that the motile properties of KIF27 are dispensable for its function in motile cilia biogenesis. Instead, KIF27 acts as a microtubule scaffold to regulate the transition zone architecture and enable correct ciliary incorporation of motility-generating proteins. Given that KIF27 homologues exist in different evolutionarily lineages, we propose that the ancestral activities of KIF27/KIF7 kinesins were to form a microtubule-associated scaffold for protein-protein interactions pertinent to cilia formation and signaling. The transition-zone associated KIF27 activities may represent a general building principle for motile cilia assembly in diverse species and cell types.
High-field fMRI at 7 Tesla reveals topographic responses tuned to number in the developing human brain
Authors: Jeong Ga-Ram , Soch Joram , Trampel Robert , Nieder Andreas , Skeide Michael A. 
Published: January 2025

AbstractIn the adult brain, hemodynamic responses to visually presented numerosities reveal receptive field-like tuning curves in topographically organized maps across association cortices. It is currently unknown whether such tuned topographic responses to numerosities can also be detected in the developing brain. Here we conducted a 7 Tesla fMRI experiment in which we presented a large set of visual dot displays to 11-12-year-old children and adults. We found that developing hemodynamic responses indeed revealed logarithmic Gaussian tuning to quantitative information. Tuning models explained comparable amounts of variance in children and adults. In most subjects, six bilateral cortical maps consistently exhibited topographic responses to numerosities. The present study goes beyond previous work by uncovering a population code for quantity detection in individual developing human brains. Our work lays a foundation for a model-based neuroimaging approach to individual cognitive differences in the context of developmental dyscalculia and mathematical giftedness.
Layer 1 NDNF interneurons form distinct subpopulations with opposite activation patterns during sleep in freely behaving mice
Authors: Aurélie Brécier , Gaëlle Mailhos , Przemyslaw Jarzebowski , Yuqi Li , Ole Paulsen , Audrey Hay Y. 
Published: January 2025

ABSTRACTNon-rapid eye movement (NREM) sleep facilitates memory consolidation by transferring information from the hippocampus to the neocortex. Recent evidence suggests that this transfer occurs primarily when hippocampal sharp-wave ripples (SWRs) and thalamocortical spindles are synchronized. In this study, we asked what role cortical layer 1 NDNF-expressing (L1 NDNF) interneurons play during NREM sleep in gating information transfer during SWR-spindle synchronization. Using simultaneous cell-specific calcium imaging and local field potential recordings in freely moving mice, we discovered that L1 NDNF neurons form cell assemblies tuned to specific sleep stages, exhibiting differential responses to spindle synchronization. L1 NDNF neurons mediate slow inhibition through GABABreceptors. Systemic application of a GABABreceptor antagonist increased pyramidal neuron excitability during NREM sleep, enhanced inhibitory responses during SWRs, and disrupted SWR-spindle coupling. Overall, these findings suggest an important contribution of L1 NDNF neuron-mediated slow inhibition to the synchronization of sleep oscillations with potential implications for memory consolidation processes.
Processing of Genomic RNAs by Dicer in Bat Cells Limits SARS-CoV-2 Replication
Authors: Owolabi Iyanuoluwani J. , Karim Shazeed-UI , Khanal Sweta , Valdivia Sergio , Frenzel Christopher , Bai Fengwei , Flynt Alex S. 
Published: January 2025

ABSTRACTBats are reservoirs for numerous viruses that cause serious diseases in other animals and humans. Several mechanisms are proposed to contribute to the tolerance of bats to these pathogens. This study investigates the response of bat cells to double-stranded RNA generated by SARS-CoV-2 replication. Here, we found the involvement of Dicer in the processing of viral genomic RNAs during SARS-CoV-2 infection. Examining RNA sequencing of infected cells, small-interfering RNA (siRNA)-like fragments were found derived from viral RNAs. Depletion of Dicer showed a reduction in these RNAs and an increase in viral loads suggesting unlike other mammals, bats may use Dicer to limit viral replication. This prompted the exploration of key dsRNA sensors in bat cells. Our analysis showed significant upregulation of OAS1 and MX1 in response to dsRNA, while PKR levels remained low, suggesting alternative dsRNA-response mechanisms are present that eschew the common PKR-based system. These results further show how bats employ distinct strategies for antiviral defense that may contribute to tolerating viral infections. They suggest the involvement of Dicer in antiviral mechanisms in bats, a function not observed in other mammals. This highlights a mechanism for bat originating viruses to evolve features that in other animals could cause extreme antiviral responses such as is seen with SARS-CoV-2.GRAPHICAL SUMMARY
Kinetic analysis of strand invasion duringC. elegansmeiosis reveals similar rates of sister- and homolog-directed repair
Authors: Hamrick Antonia , Cope Henry D. , Forbis Divya , Rog Ofer 
Published: January 2025

AbstractMeiotic chromosome segregation requires reciprocal exchanges between the parental chromosomes (homologs). Exchanges are formed via tightly-regulated repair of double-strand DNA breaks (DSBs). However, since repair intermediates are mostly quantified in fixed images, our understanding of the mechanisms that control the progression of repair remains limited. Here, we study meiotic repair kinetics inCaenorhabditis elegansby extinguishing new DSBs and following the disappearance of a crucial intermediate - strand invasion mediated by the conserved RecA-family recombinase RAD-51. We find that RAD-51 foci have a half-life of 42-132 minutes for both endogenous and exogenous DSBs. Surprisingly, we find that repair templated by the sister chromatid is not slower than repair templated by the homolog. This suggests that differential kinetics are unlikely to underlie ’homolog bias’: the preferential use of the homolog as a repair template. We also use our kinetic information to revisit the total number of DSBs per nucleus - the ‘substrate’ for the formation of exchanges - and find an average of 40 DSBs in wild-type meiosis and >50 DSBs when homolog pairing is perturbed. Our work opens the door for analysis of the interplay between meiotic repair kinetics and the fidelity of genome inheritance.Key pointsBy extinguishing new meiotic DSBs, we define the lifetime of a key repair intermediate.We find similar rates of meiotic DNA repair templated by the sister and the homolog.Kinetic information allows calculation of the total number of meiotic DSBs inC. elegans.
Site-specific DNA demethylation during spermatogenesis presets the sites of nucleosome retention in mouse sperm
Authors: Maezawa So , Yukawa Masashi , Sakashita Akihiko , Barski Artem , Namekawa Satoshi H. 
Published: January 2025

AbstractDNA methylation patterns are inherited from the parental germline to the embryo. In mature sperm, the sites of unmethylated DNA are tightly coupled to sites of histone retention at gene regulatory elements that are implicated in paternal epigenetic inheritance. The timing and mechanism of site-specific DNA demethylation in the male germline currently remains unknown. Here, we perform genome-wide profiling of DNA methylation during spermatogenesis by capturing methylated DNA through interaction with a methyl-DNA binding protein domain (MBD). Our data demonstrate that there is a site-specific change in DNA methylation during the mitosis-to-meiosis transition. Importantly, the genomic sites that are demethylated during this transition predetermine nucleosome retention sites in spermatozoa. These results suggest that site-specific DNA demethylation during the mitosis-to-meiosis transition of spermatogenesis prepares embryonic gene expression after fertilization. We therefore propose DNA demethylation during spermatogenesis as a novel phase of epigenetic reprogramming that contributes to embryonic gene regulation.
Green advertising: a hybrid literature review using citation and TCM analysis
Authors: Goyal Sakshi , Ramaswamy Venkat Narasimhan , Goyal Praveen , Chanda Udayan 
Published: January 2025

Purpose
Given the multifaceted nature of research in green advertising (GA), it is critical to holistically review the extant literature to understand their contributions. This study aims to perform a hybrid literature review by combining citation analysis and a theory-context-method (TCM) framework to map the conceptual development of GA in the past three decades.

Design/methodology/approach
Using the SPAR-4-SLR technique, the current study identified the literature contributions of 133 articles published between 1993 and 2024 in the Scopus and Web of Science databases.

Findings
The study’s findings identified that certain theories, such as attribution theory and signalling theory have been used frequently in investigating the influence of GA on consumer behavior. The context analysis identified emerging themes like green trolling and green demarketing. This study also proposes substantial avenues with alternative theories for further advancement of GA research.

Research limitations/implications
By conducting a hybrid literature review, this study identifies the highly cited articles and sources and provides a comprehensive synthesis of GA research.

Practical implications
Analysis of 133 articles from eight subject areas identified various themes in GA, aiding future researchers in identifying potential research opportunities and for marketers to develop appropriate strategies for their GA campaigns.

Originality/value
This study is the initial attempt to perform a hybrid literature review on GA using citation analysis and TCM framework.
A person-centered perspective on entrepreneurial success: combining proactive behavioral strategies across various life domains
Authors: Boesten Renée M. , Demerouti Evangelia , Le Blanc Pascale M. , Pekaar Keri A. 
Published: January 2025

PurposeThis study adopts a person-centered perspective to explore how entrepreneurs combine multiple proactive behavioral strategies across the business, personal and business-environment domains. We research whether certain combinations of proactive behavioral strategies (i.e. seeking resources, optimizing demands, seeking challenges, idea generation, environmental exploration, network crafting, self-insight and boundary management) relate to well-being and business performance. Moreover, we investigate whether entrepreneurs’ daily use of these strategies aligns with their general profiles.Design/methodology/approachA sample of 286 Dutch entrepreneurs participated in a general survey study. Moreover, 49 of these entrepreneurs also participated in a six-day diary study (N = 255). Latent profile analysis was used to find specific profiles and multilevel regression to understand the daily patterns.FindingsWe uncovered four meaningful profiles: the minimum business effort entrepreneurs, the solid and self-caring entrepreneurs, the needy and self-ignorant entrepreneurs and the proactive business entrepreneurs, each with different outcomes in business performance and well-being. Daily proactive behavior showed stability, suggesting a foundation deeply rooted in their general proactive behavior.Originality/valueThe study highlights the value of proactive strategies across all life domains, as profiles encompassing this combination show greater entrepreneurial success.
Gotta take my avatar shopping: impacts of interactive virtual shopping in esports
Authors: Hwang Yongjin , Gupta Keshav , Ock Deokkyung 
Published: January 2025

PurposeEsports game developers are incorporating interactive virtual shopping (IVS) experiences into their products to facilitate consumer–brand interactions. Despite the potential influence of IVS, existing research lacks an understanding of its impact on consumers’ overall affect– particularly game enjoyment, a critical factor in sustaining engagement with esports products – and on attitudinal change toward brands in the real world.Design/methodology/approachAn experiment was designed to examine the impact of shopping in the virtually interactive esports marketplace on game enjoyment. Mediating effects of positive change in brand attitude, perceived realism and character attachment and the moderating effect of self-avatar gender congruity were investigated using SPSS PROCESS Macro. Additionally, the impact of brand popularity on the relationship between IVS and brand attitude was examined using analysis of covariance (ANCOVA).FindingsThere was a positive impact of IVS on game enjoyment, mediated by a positive change in brand attitude, perceived realism and character attachment. Self-avatar gender congruity moderated the mediation effect of perceived realism. Notably, less popular brands exhibited a greater change in brand attitude compared to more popular brands.Originality/valueThe findings add to the body of literature on esports, particularly in investigating the role of IVS within the esports virtual world and advancing research on cognitive appraisal theory. This study informs practitioners in the esports industry about the value of creating interactive virtual marketplaces, as they serve to benefit sponsoring brands and enhance game enjoyment for players.