bluuebunny/crossref_metadata_2025_split huggingface dataset:
FLEXIBLE, PRODUCTION-SCALE, HUMAN WHOLE GENOME SEQUENCING ON A BENCHTOP SEQUENCER
Authors: Green Kevin , Krajacich Benjamin , Wiseman Kelly , Mains Peter T. , Robertson Samantha , Billings Sophie , Sudkamp Mitch , Lo Ching Shing , Lajoie Bryan R. , Kruglyak Semyon , Levy Shawn , Zhao Junhua 
Published: November 2024

ABSTRACTHuman whole-genome sequencing (hWGS) provides comprehensive genomic information that can potentially help guide research in disease prevention and treatment and ultimately improve human health. Recent advancements in sequencing technology have improved sequencing quality and further reduced sequencing costs on bench-top sized instruments, making whole-genome sequencing an accessible technology for broader use. Here, we demonstrate the feasibility of a large whole genome sequencing project using a benchtop sequencer in a small laboratory setting, on a scale previously reserved for production-scale factory-sized machines. In this project, 807 samples were prepared and sequenced across 313 flow cells, with high sequencing quality at a median %Q30 of 96.6% and a median %Q40 of 89.31%. To screen library quality and maximize sample yield, we utilized 48-plex sample pre-pool ‘QC’ runs to provide >1x coverage per sample prior to sample pooling and full-depth sequencing. With this strategy, we consistently achieved >30x human whole genome sequencing of three-plex sample trios with standard settings or up to 4 samples per run with a high-throughput run setting. Additionally, this low-pass data provided valuable sample-level insights, allowing for detection of chromosome copy number variations (CNV) prior to full-depth sequencing. With three instruments running concurrently, >2,800 30x human whole genomes could be sequenced per year. To further demonstrate additional flexibility present in the platform, we also explored two different use cases 1) large insert sizes (1kb+) library to achieve superior genome coverage; 2) a proof of concept for rapid WGS sequencing to minimize sample to answer turnaround time for time-critical sequencing applications. Sequencing of a 2×100 >30x human WGS can be achieved in <12 hours and subsequent generation of fastq, bam and vcf in <1 additional hour. This study provides a cost-effective and flexible real-world demonstration of achieving both high quality hWGS sequencing and instrument flexibility without the need for complex batching schemes or factory-sized sequencers.
Trophic Interactions ofAnopheles GambiaeMosquito Larvae in Aquatic Ecosystem: A Metagenomics Approach
Authors: Karikari Afia Serwaa , Akorli Jewelna , Gbogbo Francis , Ndong Ignatious Cheng , Karikari Akosua Bonsu , Karikari Akua Afriyie , Kyerematen Rosina , Mawuli Akwasi Agyenkwa , Gyimah Thomas , Aboagye-Antwi Fred 
Published: November 2024

AbstractBackgroundUnderstanding the trophic interactions ofAnopheles gambiaemosquito larvae in aquatic environments is essential for malaria prevention efforts such as in gene drive application. In this study, metagenomics method was employed to explore the feeding behavior and ecological associations ofAn. gambiaelarvae within their aquatic ecosystems.MethodsAnopheles gambiaelarvae and their co-existing fauna species were collected from December 2022 to June 2023. DNA from dissected midguts of the co-existing fauna was sequenced using Illumina NovaSeq for diet analysis through a shotgun sequencing approach.ResultsThe study revealed a complex network of trophic interactions in the freshwater habitat, with significant resource sharing betweenA. gambiaelarvae and other filter-feeding species, including various Diptera and non-Dipteran feeders. Contrary to assumptions, predator species did not exhibit exclusive predation onAnopheleslarvae, preferring other fauna instead.ConclusionThis study has demonstrated complex trophic interactions amongAnopheles gambiaelarvae and other organisms in freshwater ecosystems. It has offered essential insights for optimizing vector control strategies, such as in gene drive applications. Additionally, the study has provided valuable information on the aquatic fauna of the study area, which can serve as a baseline for developing a macroinvertebrate identification database for freshwater systems in Ghana. A complementary study that further explores the ecological role ofAn. gambiaelarvae in these freshwater habitats is presented in a subsequent publication.
CCR9 signal termination is governed by an arrestin-independent phosphorylation mechanism
Authors: Lamme Thomas D. , Smit Martine J. , Schafer Christopher T. 
Published: November 2024

AbstractThe chemokine receptor CCR9 coordinates immune cell migration from the thymus to the small intestine along gradients of CCL25. Receptor dysregulation is associated with a variety of inflammatory bowel diseases such as Crohn’s and ulcerative colitis, while aberrant CCR9 overexpression correlates with tumor metastasis. Despite being an attractive therapeutic target, attempts to clinically antagonize CCR9 have been unsuccessful. This highlights the need for a deeper understanding of its specific regulatory mechanisms and signaling pathways. CCR9 is a G protein-coupled receptor (GPCR) and activates Giand Gqpathways. Unexpectedly, live-cell BRET assays reveal only limited G protein activation and signaling is rapidly terminated. Truncating the receptor C-terminus significantly enhanced G protein coupling, highlighting the regulatory role of this domain. Signal suppression was not due to canonical arrestin-coordinated desensitization. Rather, removal of GPCR kinase (GRK) phosphorylation led to sustained and robust G protein activation by CCR9. Using site-directed mutagenesis, we identified specific phosphorylation patterns that attenuate G protein coupling. Receptor internalization does not correlate with G protein activation capabilities. Instead, CCR9 phosphorylation appeared to directly destabilize the interaction of G protein heterotrimers with the receptor. This interference could lead to rapid loss of productive coupling and downstream signaling as phosphorylation would effectively render the receptor incapable of G protein coupling. An arrestin-independent, phosphorylation-driven deactivation mechanism could complement arrestin-dependent regulation of other GPCRs and have consequences for therapeutically targeting these receptors.
The mechanism of checkpoint-dependent DNA replication fork stabilization in human cells
Authors: Bertolin Agostina P. , Canal Berta , Yekezare Mona , Zeng Jingkun , Instrell Rachael , Howell Michael , Diffley John F.X. 
Published: November 2024

SummaryThe DNA damage checkpoint is crucial for maintaining genome stability after genotoxic stress; without it, excess DNA replication origins are activated, stalled DNA replication forks cannot restart normally, high levels of DNA damage and single-stranded DNA (ssDNA) accumulate and cells cannot complete S phase. Preventing excess origin firing suppresses all these effects. Here we show that when replication is not restrained by a functional checkpoint, excess DNA synthesis sequesters the processivity factor PCNA and its loader RFC, preventing normal fork restart. Nascent DNA ends unprotected by RFC/PCNA are attacked by the Helicase-Like Transcription Factor (HLTF), causing irreversible replication fork collapse and hyperaccumulation of single-stranded DNA. This explains how the checkpoint stabilizes stalled replication forks and has implications for how origin firing is normally coordinated with fork progression. Loss of HLTF suppresses fork collapse and cell lethality in checkpoint-deficient cells, which has implications for how resistance to anti-checkpoint therapies may arise.
A coordinated kinase and phosphatase network regulates Stu2 recruitment to yeast kinetochores
Authors: Stewart Michael G. , Carrier Joseph S. , Zahm Jacob A. , Harrison Stephen C. , Miller Matthew P. 
Published: November 2024

ABSTRACTCells coordinate diverse events at anaphase onset, including separase activation, cohesin cleavage, chromosome separation, and spindle reorganization. Regulation of the XMAP215 family member and microtubule polymerase, Stu2, at the metaphase-anaphase transition determines a specific redistribution from kinetochores to spindle microtubules. We show that cells modulate Stu2 kinetochore-microtubule localization by Polo-like kinase1/Cdc5-mediated phosphorylation of T866, near the Stu2 C-terminus, thereby promoting dissociation from the kinetochore Ndc80 complex. Cdk/Cdc28 likely primes Cdc5:Stu2 interaction. Cdc28 activity is also required for Stu2 nuclear import. PP2ACdc55actively opposes Cdc5 activity on Stu2T866during metaphase. This counter-regulation allows for switchlike redistribution of Stu2pT866at anaphase onset when separase inhibits PP2ACdc55. Blocking Stu2T866phosphorylation disrupts anaphase spindle progression, and we infer that PP2ACdc55regulates the mitotic spindle by dephosphorylating Stu2 and other MAPs. These data support a model in which increased phosphorylation at anaphase onset results from phosphatase inhibition and point to a larger regulatory network that facilitates rapid cytoskeletal modulation required for anaphase spindle maintenance.SUMMARYStu2 displays dynamic localization patterns in the cell cycle, with different kinetochore and microtubule distribution during distinct phases. Phosphorylation near Stu2’s C-terminus reduces its attachment to kinetochores to promote its microtubule activity in anaphase. Cdc5 and PP2ACdc55play counteracting roles in this pathway to promote proper timing of Stu2 phosphorylation.
Semisynthetic ferritin nanocages for flexible, site-specific targeting, cluster-formation and activation of membrane receptors
Authors: Neusch Andreas , Siepe Christina , Zitzke Liesa , Fux Alexandra C. , Monzel Cornelia 
Published: November 2024

ABSTRACTHomopolymerization and cluster formation of cellular membrane receptors (MR) is closely related to their signaling activity. However, underlying mechanisms and effects of clustering are often hardly understood. This lack of knowledge is due to the lack of suitable tools which enable to specifically target and activate distinct MRs, without causing side-effects. In this study, we designed a fluorescent semisynthetic nanoparticle (NP) based on the iron-storage protein ferritin andS. aureusProtein A, that is readily equipped with a variety of antibodies withKDvalues below 5 nM. Specificity of the NP antigen recognition was evaluated in cell experiments with cells expressing Transferrin Receptor 1 or the death receptor CD95, both of which displayed rapid cluster formation upon contact with the NP. Lastly, it was possible to induce apoptosis solely by induced clustering of CD95 via our engineered NP.
Failing to account for RNA quantity inflates background and leads to the misleading appearance that PRC2 and GFP bind to RNAin vivo
Authors: Guo Jimmy K. , Blanco Mario R. , Guttman Mitchell 
Published: November 2024

ABSTRACTWe recently published biochemical and quantitative evidence that challenges the widespread claims that PRC2 binds directly to many RNAsin vivo. A recent preprint performs a re-analysis of some of our data using a different metric and claims to identify contradictory results – that PRC2 binds to more RNAs than the well-characterized PTBP1 and SAF-A RNA-binding proteins. The preprint also reports other counterintuitive observations, including that PRC2, and all proteins examined, bind to RNAs that donotexist within the same cell and therefore could not interactin vivonor could they be UV-crosslinked. Because these observations are incongruent with orthogonal, non-PCR based, measurements as well as fundamental RNA biology, we explored why their analysis would lead to these unexpected observations. After carefully examining the methods used and regenerating the reported results, we found a very simple explanation for these discrepancies: the authors effectively ignore the vast majority of RNAs in the sample. Here we show that this failure to properly account for the total RNA in each sample leads to inaccurate estimates of the true proportions of each RNA in the sample and therefore the enrichment calculated. We find that the number of RNA reads discarded is dependent on the RNA binding properties of the protein and therefore dramatically inflates background signals (e.g., RNA detected with GFP) while deflating specific signals (e.g., RNA detected with PTBP1). Importantly, we show that this issue alone explains the discrepancy between their observations and our previously reported results. The fact that PRC2 binding can only be observed when analyzed in this way further reinforces the fundamental point of our original paper: the existing evidence in support of direct PRC2-RNA binding requires critical re-evaluation.
Colchicine Inhibits Smooth Muscle Cell Phenotypic Switch and Aortic Dissection in Mice
Authors: Jiang Hui , Zhao Yaping , Jin Mei , Zhang Zhidan , Wang Li , Liu Zhiping , Xu Suowen , Weng Jianping 
Published: November 2024

AbstractAortic dissection is a serious cardiovascular disease characterized by intimal tearing and vascular delamination. Smooth muscle cell phenotypic transformation, extracellular matrix degradation and vascular inflammation are the main pathogenic mechanisms of aortic dissection. Colchicine is an alkaloid derived from the plant Colchicum autumnale L. and was the first FDA-approved anti-inflammatory drug in the cardiovascular field. In this study, we investigated the protective effects of colchicine in aortic dissection in mice and found that colchicine resisted aortic dissection by inhibiting vascular inflammation and smooth muscle cell phenotypic transformation, and that colchicine reversed smooth muscle cell phenotypic transformation by targeting the transcriptional activator myocardin. This study provides new directions for the development of drug therapies for aortic dissection.
A high-throughput protein tagging toolkit that retains endogenous UTRs for studying gene regulation in Kinetoplastids
Authors: Carbajo Carla Gilabert , Han Xiaoyang , Savur Bhairavi , Upadhyaya Arushi , Taha Fatima , Tinti Michele , Wheeler Richard J , Yates Phillip A. , Tiengwe Calvin 
Published: November 2024

AbstractKinetoplastid parasites cause diseases that threaten human and animal health. To survive transitions between vertebrate hosts and insect vectors, these parasites rely on precise regulation of gene expression to adapt to environmental changes. Since gene regulation in Kinetoplastids is primarily post-transcriptional, developing efficient genetic tools for modifying genes at their endogenous loci while preserving regulatory mRNA elements is crucial for studying their complex biology.We present a CRISPR/Cas9-based tagging system that preserves untranslated regulatory elements and uses a viral 2A peptide fromThosea asignato generate two separate proteins from a single transcript: a drug-selectable marker and a tagged protein of interest. This dual-function design maintains native control elements, allowing discrimination between regulation of transcript abundance, translational efficiency, and post-translational events.We validate the system by tagging sixTrypanosoma bruceiproteins and demonstrate: (i) high-efficiency positive selection and separation of drug-selectable marker and target protein, (ii) preservation of regulatory responses to environmental cues like heat shock and iron availability, and (iii) maintenance of stage-specific regulation during developmental transitions. This versatile toolkit is applicable to all kinetoplastids amenable to CRISPR/Cas9 editing, providing a powerful reverse genetic tool for studying post-transcriptional regulation and protein function in organisms where post-transcriptional control is dominant.
Pre-rRNA spatial distribution and functional organization of the nucleolus
Authors: Pan Yu-Hang , Shan Lin , Yang Zheng-Hu , Zhang Yu-Yao , Zhang Yuan , Cao Shi-Meng , Zhang Jun , Yang Li , Chen Ling-Ling 
Published: November 2024

AbstractThe self-organized nucleolus is where the pre-ribosomal RNA (pre-rRNA) processing and pre-ribosomal ribonucleoprotein (RNP) assembly take place. Here we present the spatiotemporal distribution of pre-rRNA intermediates in the human nucleolus, revealing a segregated spatial distribution of small subunit (SSU) and large subunit (LSU) precursors. Notably, the 5’ external transcribed spacer (5’ ETS)-containing SSU pre-rRNAs are retained across FC-DFC-PDFC regions, while the internal transcribed spacer 2 (ITS2)-containing LSU pre-rRNAs move to PDFC-GC regions for processing. Inhibiting 5’ ETS processing disrupts the SSU pre-rRNA distribution and the nested FC-DFC sub-nucleolar structure. Cells in amniotes possess a multi-layered nucleolus, whereas anamniotes have only a bipartite structure with a merged FC-DFC. Kinetic labeling of pre-rRNA outflow shows a 7-fold higher pace in tetrapartite nucleoli over that in bipartite nucleoli, indicating that the emergence of the nested FC-DFC may facilitate an efficient SSU pre-rRNA processing over the course of evolution. Collectively, depicting the spatiotemporal distribution of pre-rRNAs reveals a key role of processing steps in organizing the multi-layered nucleolus and suggests a possible evolutionary advantage of the multi-layered structure in amniotes.
Self-Authenticating Genomic Materials with Advanced Genome Signatures
Authors: Wang Zhaoguan , Cui Jingsong , Liu Qian , Li Jiawei , Guo Chi , Liu Yiyang , Jiang Changyue , Xue Hui , Li Jiaojiao , Ke Yonggang , Qi Hao 
Published: November 2024

AbstractThe authenticity and integrity of synthetic genomic materials containing valuable intellectual property are essential for advancing scientific knowledge and enhancing biosafety. Existing DNA tags and watermarks, however, have limited efficacy due to low mutation tolerance and inadequate digital encoding capacity. Here, we present “Genome Signature”, a biochemically stable and tamper-resistant DNA labeling system that enables the creation of self-authenticating genomes. Central to this system is a novel Golomb-ruler-derived Genome-Comb, which efficiently maps extensive nucleotide sequences onto limited codons within endogenous genes, significantly improving error correction and data encoding across millions of nucleotides. Our method successfully recorded a 4.5 million-nucleotide genome in livingE. coli. The Genome Signature autonomously identifies and corrects mutations and effectively encodes data within codon orders, ensuring genome integrity and authenticity. Furthermore, it allows precise tracking of sequences across different cells, potentially revolutionizing the development of reliable genomic materials in synthetic biology.
Radular teeth matrix protein 1 directs iron oxide deposition in chiton teeth
Authors: Nemoto Michiko , Okada Koki , Akamine Haruka , Odagaki Yuki , Narahara Yuka , Okoshi Kenji , Obuse Kiori , Kisailus David , Moriya Hisao , Satoh Akira 
Published: November 2024

AbstractChitons deposit magnetite, a type of iron oxide, on the cusps of their radular teeth. The molecules that control the deposition of iron oxide on chiton teeth have not been identified. Using immunofluorescence staining, we found that chiton-specific radular teeth matrix protein 1 (RTMP1) is highly expressed in tooth epithelial cells prior to iron oxide deposition and preexists in the cusp at the site of iron oxide deposition. RTMP1 was found to be secreted from the elongated epithelial cells covering teeth and transported into teeth through microvilli. Moreover, recombinant RTMP1 exhibited the ability to form iron oxide. These results indicate that RTMP1 is a key molecule for iron oxide deposition in chiton teeth.
Mouse Pachytene piRNAs Cleave Hundreds of Transcripts, But Alter the Steady-State Abundance of Only a Minority of Targets
Authors: Cecchini Katharine , Ajaykumar Nandagopal , Bagci Ayca , Zamore Phillip D. , Gainetdinov Ildar 
Published: November 2024

SUMMARYIn animals, 18–35-nt piRNAs guide PIWI proteins to regulate complementary RNAs. During male meiosis, mammals produce an exceptionally abundant class of piRNAs called pachytene piRNAs. Pachytene piRNAs are required for spermatogenesis and have been proposed to control gene expression by various mechanisms. Here, we show that pachytene piRNAs regulate targets predominantly, if not exclusively, by endonucleolytic cleavage. Remarkably, pachytene piRNAs slice hundreds of RNAs, yet a change in steady-state level is detectable for a small fraction of transcripts. Our data suggest that cleavage of the few targets whose abundance is reduced significantly by piRNAs is essential for male fertility. Other pachytene piRNA targets are enriched for highly transcribed genes, which may explain why piRNA cleavage is often inconsequential for the steady-state abundance of targets. We propose that the retention of pachytene piRNAs throughout mammalian evolution is driven by the selective advantage conferred by a tiny minority of piRNAs.
Marker gene fishing for single-cell data with complex heterogeneity
Authors: Shao Yiren , Gao Qi , Wang Liuyang , Li Dongmei , Nixon Andrew B. , Chan Cliburn , Li Qi-Jing , Xie Jichun 
Published: November 2024

AbstractIn single-cell studies, cells can be characterized with multiple sources of heterogeneity such as cell type, developmental stage, cell cycle phase, activation state, and so on. In some studies, many nuisance sources of heterogeneity (SOH) are of no interest, but may confound the identification of the SOH of interest, and thus affect the accurate annotate the corresponding cell subpopulations. In this paper, we develop B-Lightning, a novel and robust method designed to identify marker genes and cell subpopulations correponding to a SOH (e.g., cell activation status), isolating it from other sources of heterogeneity (e.g., cell type, cell cycle phase). B-Lightning uses an iterative approach to enrich a small set of trustworthy marker genes to more reliable marker genes and boost the signals of the SOH of interest. Multiple numerical and experimental studies showed that B-Lightning outperforms existing methods in terms of sensitivity and robustness in identifying marker genes. Moreover, it increases the power to differentiate cell subpopulations of interest from other heterogeneous cohorts. B-Lightning successfully identified new senescence markers in ciliated cells from human idiopathic pulmonary fibrosis (IPF) lung tissues, new T cell memory and effector markers in the context of SARS-COV-2 infections, and their synchronized patterns which were previously neglected. This paper highlights B-Lightning’s potential as a powerful tool for single-cell data analysis, particularly in complex data sets where sources of heterogeneity of interest are entangled with numerous nuisance factors.