bluuebunny/crossref_metadata_2025 huggingface dataset:
Dissociation of the nuclear basket triggers chromosome loss in aging yeast
Authors: Mirkovic Mihailo , McCarthy Jordan , Meinema Anne Cornelis , Parenteau Julie , Lee Sung Sik , Elela Sherif Abou , Barral Yves
Published: November 2024

In many organisms, aging is a clear risk factor for increased rates of chromosome mis-segregation, the main source of aneuploidy. Here, we report that old yeast mother cells lose chromosomes by partitioning them asymmetrically to their daughter cells together with the pre-existing (old) Sindle Pole Body (centrosome equivalent in yeast). Strikingly, remodelling of the NPC and the displacement of its nuclear basket triggered these asymmetric segregation events. Concomitantly, basket displacement also caused unspliced pre-mRNAs to leak to the cytoplasm. We show that removing the introns of three genes involved in chromosome segregation was sufficient to fully suppress chromosome loss in old cells. Furthermore, promoting pre-mRNA leakage in young cells also caused asymmetric chromosome partition and loss through the same three introns. Therefore, we propose that basket displacement from NPCs and its ensuing effects on pre-mRNA quality control are a key trigger of aging phenotypes such as aneuploidy.
Metabolic regulation of RNA methylation by the m6A-reader IGF2BP3
Authors: Sharma Gunjan , Gutierrez Martin , Jones Anthony E , Jaiswal Amit Kumar , Neeb Zachary T , Rios Amy , Thaxton Michelle L , Lin Tasha L , Tran Tiffany M , Kabbani Lyna E S , Ritter Alexander J , Stiles Linsey , ten Hoeve Johanna , Divakaruni Ajit S , Sanford Jeremy R , Rao Dinesh S
Published: November 2024

The interplay of RNA modifications - deposited by "writers", removed by "erasers" and identified by RNA binding proteins known as "readers" - forms the basis of the epitranscriptomic gene regulation hypothesis. Recent studies have identified the oncofetal RNA-binding protein IGF2BP3 as a "reader" of the N6-methyladenosine (m6A) modification and crucial for regulating gene expression. Yet, how its function as a reader overlaps with its critical oncogenic function in leukemia remains an open question. Here, we report the novel finding that the reader IGF2BP3 reprograms cellular metabolism, resulting in an altered ability of the "writers" to modify the epitranscriptome. In leukemia cells, IGF2BP3 supports increased glycolytic flux and one-carbon metabolism, leading to increased production of S-adenosyl methionine (SAM), a key substrate for methylation reactions within the cell. IGF2BP3 directly regulates the translation of MAT2B, the regulatory subunit of the methionine-adenosyltransferase complex, which is the final enzyme in a pathway leading to SAM production. This, in turn, results in increased m6A modifications on RNA, resulting in positive feedback regulation. This novel mechanism illustrates how metabolism mutually acts with epitranscriptomic modifications, underscoring the pervasive impact of IGF2BP3 in gene regulatory mechanisms governing a broad range of cancer-specific processes.
Subcellular plant carbohydrate metabolism under elevated temperature
Authors: Seydel Charlotte , Heß Martin , Schröder Laura , Klingl Andreas , Nägele Thomas
Published: November 2024

In many plant species, exposure to a changing environmental temperature regime induces an acclimation response which ultimately increases a plant’s thermotolerance. Under elevated temperature, membrane systems need remodelling to counteract de-stabilising thermodynamic effects. This also affects photosynthesis and carbohydrate metabolism due to heat affected protein functions, enzyme activities and transport processes across membrane systems. In the present study, a combination of electrolyte leakage assays and chlorophyll fluorescence measurements was applied to quantify heat tolerance before and after heat acclimation ofArabidopsis thalianaat different temperature regimes. Subcellular carbohydrate concentrations were determined in a combined approach of non-aqueous fractionation and 3D reconstruction of mesophyll cells and subcellular compartments using serial block-face scanning electron microscopy. Across temperature regimes between 32 °C and 38 °C, 7 days heat acclimation at 34 °C was found to most efficiently increase tissue heat tolerance. Under such conditions, cytosolic sucrose concentrations were stabilised by a shift of sucrose cleavage rates into the vacuolar compartment while invertase-driven cytosolic sucrose cleavage was found to be efficiently quenched by fructose and glucose acting as competitive and non-competitive inhibitors, respectively. Finally, this study provides strong evidence for a sucrose concentration gradient from the cytosol into the vacuole which might directly affect the physiological role and direction of proton gradient-driven sugar transport across the tonoplast.
Many transcription factor families have evolutionarily conserved binding motifs in plants
Authors: Zenker Sanja , Wulf Donat , Meierhenrich Anja , Viehöver Prisca , Becker Sarah , Eisenhut Marion , Stracke Ralf , Weisshaar Bernd , Bräutigam Andrea
Published: November 2024

Transcription factors control gene expression during development and in response to a broad range of internal and external stimuli. They regulate promoter activity by directly bindingcis- regulatory elements in DNA. The angiospermArabidopsis thalianacontains more than 1,500 annotated transcription factors, each containing a DNA-binding domain that is used to define transcription factor families.

Analyzing binding motifs of 686 and the binding sites of 335A. thalianatranscription factors as well as motifs of 92 transcription factors from other plants, we identified a constrained vocabulary of 74 conserved motifs spanning 50 families in plants. Among 21 transcription factor families, we found one core motif for all analyzed members and between 2 and 72% overlapping binding sites. Five families show conservation of the motif along phylogenetic clades. Five families including the C2H2 zinc finger family show high diversity among motifs in plants, suggesting potential for neofunctionalization of duplicated transcription factors based on the motif recognized. For conserved motifs we tested if they remained conserved since at least 450 million years ago by determining the binding motifs of 17 orthologous transcription factors from 11 families inM. polymorphausing amplified DNA affinity purification sequencing. We detected nearly identical binding motifs as predicted from the angiosperm data.

Taken together, the results show a large repertoire of overlapping binding sites within a TF family and species and a high degree of binding motif conservation for at least 450 million years. The results indicate more potential for evolution incis-rather thantrans-regulatory elements.
The microbiome diversifiesN-acyl lipid pools - including short-chain fatty acid-derived compounds
Authors: Mannochio-Russo Helena , Charron-Lamoureux Vincent , van Faassen Martijn , Lamichhane Santosh , Gonçalves Nunes Wilhan D. , Deleray Victoria , Patan Abubaker , Vittali Kyle , Rajkumar Prajit , Abiead Yasin El , Zhao Haoqi Nina , Portal Gomes Paulo Wender , Mohanty Ipsita , Lee Carlynda , Sund Aidan , Sharma Meera , Liu Yuanhao , Pattynama David , Walker Gregory T. , Norton Grant J. , Khatib Lora , Andalibi Mohammadsobhan S. , Wang Crystal X. , Ellis Ronald J. , Moore David J. , Iudicello Jennifer E. , Franklin Donald , Letendre Scott , Chin Loryn , Walker Corinn , Renwick Simone , Zemlin Jasmine , Meehan Michael J. , Song Xinyang , Kasper Dennis , Burcham Zachary , Kim Jane J. , Kadakia Sejal , Raffatellu Manuela , Bode Lars , Zengler Karsten , Wang Mingxun , Siegel Dionicio , Knight Rob , Dorrestein Pieter C.
Published: November 2024

N-acyl lipids are important mediators of several biological processes including immune function and stress response. To enhance the detection ofN-acyl lipids with untargeted mass spectrometry-based metabolomics, we created a reference spectral library retrievingN-acyl lipid patterns from 2,700 public datasets, identifying 851N-acyl lipids that were detected 356,542 times. 777 are not documented in lipid structural databases, with 18% of these derived from short-chain fatty acids and found in the digestive tract and other organs. Their levels varied with diet, microbial colonization, and in people living with diabetes. We used the library to link microbialN-acyl lipids, including histamine and polyamine conjugates, to HIV status and cognitive impairment. This resource will enhance the annotation of these compounds in future studies to further the understanding of their roles in health and disease and highlight the value of large-scale untargeted metabolomics data for metabolite discovery.
Histone variant H2BE controls activity-dependent gene expression and homeostatic scaling
Authors: Feierman Emily R. , Paranjapye Alekh , Su Steven , Qiu Qi , Wu Hao , Korb Erica
Published: November 2024

A cell’s ability to respond and adapt to environmental stimuli relies in part on transcriptional programs controlled by histone proteins. Histones affect transcription through numerous mechanisms including through replacement with variant forms that carry out specific functions. We recently identified the first widely expressed H2B histone variant, H2BE and found that it promotes transcription and is critical for neuronal function and long-term memory. However, how H2BE is regulated by extracellular stimuli and whether it controls activity-dependent transcription and cellular plasticity remain unknown. We used CUT&Tag and RNA-sequencing of primary neurons, single-nucleus sequencing of cortical tissue, and multielectrode array recordings to interrogate the expression of H2BE in response to stimuli and the role of H2BE in activity-dependent gene expression and plasticity. We find that unlike Further, we show that neurons lacking H2BE are unable to mount proper long-term activity-dependent transcriptional responses both in cultured neurons and in animal models. Lastly, we demonstrate that H2BE knockout neurons fail to undergo the electrophysiological changes associated with homeostatic plasticity in neurons after long-term stimulation. In summary, these data demonstrate that H2BE expression is inversely correlated to activity and necessary for long-term activity-dependent responses, revealing the first instance of a histone variant involved in the homeostatic plasticity response in neurons.
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

Human 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

Background

Understanding 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.

Methods

Anopheles 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.

Results

The 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.

Conclusion

This 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.
Dominant-negative TP53 mutations potentiated by the HSF1-regulated proteostasis network
Authors: Sebastian Rebecca M. , Patrick Jessica E. , Hui Tiffani , Amici David R. , Giacomelli Andrew O. , Butty Vincent L. , Hahn William C. , Mendillo Marc L. , Lin Yu-Shan , Shoulders Matthew D.
Published: November 2024

Protein mutational landscapes are sculpted by the impacts of the resulting amino acid substitutions on the proteins stability and folding or aggregation kinetics. These properties can, in turn, be modulated by the composition and activities of the cellular proteostasis network. Heat shock factor 1 (HSF1) is the master regulator of the cytosolic and nuclear proteostasis networks, dynamically tuning the expression of cytosolic and nuclear chaperones and quality control factors to meet demand. Chronic increases in HSF1 levels and activity are prominent hallmarks of cancer cells. One plausible explanation for this observation is that the consequent upregulation of proteostasis factors could biophysically facilitate the acquisition of oncogenic mutations. Here, we experimentally evaluate the impacts of chronic HSF1 activation on the mutational landscape accessible to the quintessential oncoprotein p53. Specifically, we apply quantitative deep mutational scanning of p53 to assess how HSF1 activation shapes the mutational pathways by which p53 can escape cytotoxic pressure conferred by the small molecule nutlin-3, which is a potent antagonist of the p53 negative regulator MDM2. We find that activation of HSF1 broadly increases the fitness of dominant-negative substitutions within p53. This effect of HSF1 activation was particularly notable for non-conservative, biophysically unfavorable amino acid substitutions within buried regions of the p53 DNA-binding domain. These results indicate that chronic HSF1 activation profoundly shapes the oncogenic mutational landscape, preferentially supporting the acquisition of cancer-associated substitutions that are biophysically destabilizing. Along with providing the first experimental and quantitative insights into how HSF1 influences oncoprotein mutational spectra, these findings also implicate HSF1 inhibition as a strategy to reduce the accessibility of mutations that drive chemo-therapeutic resistance and metastasis.
CCR9 signal termination is governed by an arrestin-independent phosphorylation mechanism
Authors: Lamme Thomas D. , Smit Martine J. , Schafer Christopher T.
Published: November 2024

The 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

The 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.
Site-specific DNA insertion into the human genome with engineered recombinases
Authors: Fanton Alison , Bartie Liam J. , Martins Juliana Q. , Tran Vincent Q. , Goudy Laine , Durrant Matthew G. , Wei Jingyi , Pawluk April , Konermann Silvana , Marson Alexander , Gilbert Luke A. , Hsu Patrick D.
Published: November 2024

Technologies for precisely inserting large DNA sequences into the genome are critical for diverse research and therapeutic applications. Large serine recombinases (LSRs) can mediate direct, site-specific genomic integration of multi-kilobase DNA sequences without a pre-installed landing pad, but current approaches suffer from low insertion rates and high off-target activity. Here, we present a comprehensive engineering roadmap for the joint optimization of DNA recombination efficiency and specificity. We combined directed evolution, structural analysis, and computational models to rapidly identify additive mutational combinations. We further enhanced performance through donor DNA optimization and dCas9 fusions, enabling simultaneous target and donor recruitment. Top engineered LSR variants achieved up to 53% integration efficiency and 97% genome-wide specificity at an endogenous human locus, and effectively integrated large DNA cargoes (up to 12 kb tested) for stable expression in challenging cell types, including non-dividing cells, human embryonic stem cells, and primary human T cells. This blueprint for rational engineering of DNA recombinases enables precise genome engineering without the generation of double-stranded breaks.
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

Cells 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.

Stu2 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

Homopolymerization 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.
Genomic Architecture of the Resistance to Phytophthora Cactorum 2 (RPc2) Locus in Strawberry (Fragaria × ananassa)
Authors: Han Hyeondae , Oh Youngjae , Jang Yoon Jeong , Chandra Saket , Gill Upinder , Verma Sujeet , Noh Young-Hee , Isobe Sachiko , Pincot Dominique D. A. , cole Glenn S , Famula Randi , Feldmann Mitchell J , Knapp Steve J. , Whitaker Vance , Lee Seonghee
Published: November 2024

Phytophthora crown rot (PhCR), caused by Phytophthora cactorum, is a soilborne disease with broad impacts to cultivated strawberries in the United States and worldwide. While a resistance locus, RPc2, has been identified in octoploid strawberries, the underlying genomic architecture and mechanism remain unclear. Here, we fine-mapped the RPc2 region to 546 kb, containing 92 genes, and constructed a chromosome-scale haplotype-phased genome of resistant breeding selection FL 16.33-8. Comparative genome analyses with high-quality octoploid reference genomes, 'Florida Brilliance' (FaFB1) and 'Royal Royce' (FaRR1), identified two candidate genes, Wall Associated Kinase 1 (WAK1) and Cyclic Nucleotide Gated Channel 1 (CNGC1). Gene functions were validated using an Agrobacterium-mediated transient expression assay. Furthermore, by leveraging the population structure of the RPc2 locus among genetically diverse breeding populations, we unveiled the complexity of genetic architecture and recent selective sweeps associated with RPc2. Notably, the predominant resistant haplotype (RPc2-H3) is prevalent in most commercial strawberry varieties in the US. The findings from this study are facilitating the advancement of genome-assisted breeding strategies for resistance to PhCR in strawberries.
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

We 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

Aortic 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

Kinetoplastid 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

The 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

The 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

Chitons 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

In 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

In 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.
Intrinsic bioenergetic adaptations compensate for reduced mitochondrial content in HER2-driven mammary tumors
Authors: Frangos Sara M , Brunetta Henver S , Wang Dongdong , Jabile Maria Joy Therese , Ma David W L , Muller William J , Khursigara Cezar M , Fisher-Wellman Kelsey H , Steinberg Gregory R , Holloway Graham P
Published: November 2024

It is now recognized that mitochondria play a crucial role in tumorigenesis, however, it has become clear that tumor metabolism varies significantly between cancer types. The failure of recent clinical trials attempting to directly target tumor respiration with inhibitors of oxidative phosphorylation has highlighted the critical need for additional studies comprehensively assessing mitochondrial bioenergetics. Therefore, we systematically assessed the bulk tumor and mitochondrial metabolic phenotype between murine HER2-driven mammary cancer tumors and paired benign mammary tissue. Transcriptomic and proteomic profiling revealed that HER2-driven mammary tumors are characterized by a downregulation of mitochondrial genes/proteins compared to benign mammary tissue, including a general downregulation of OXPHOS subunits comprising Complexes I-IV. Despite this observation, mitochondrial respiration supported by both carbohydrate-derived substrates (pyruvate) and lipids (palmitoyl-carnitine) was several-fold higher in HER2-driven tumors which persisted regardless of normalization method (i.e. wet weight, total protein content and when corrected for mitochondrial content). This upregulated respiratory capacity could not be explained by OXPHOS uncoupling; however, several subunits/regulators of Complex V function were not downregulated in the tumors, suggesting possible compensatory effects may contribute to high respiratory rates. Furthermore, tumor mitochondria displayed a smaller and more punctate morphology, aligning with a general reduction in mitochondrial fusion and increase in mitochondrial fission markers, which could contribute to improved OXPHOS efficiency. Together, this data highlights that the typical correlation of mitochondrial content and respiratory capacity may not apply to all tumor types and implicates the activation of mitochondrial respiration supporting tumorigenesis in this model.
Emergence of a Novel Reassortant Clade 2.3.2.1c Avian Influenza A/H5N1 Virus Associated with Human Cases in Cambodia
Authors: Siegers Jurre Y. , Xie Ruopeng , Byrne Alexander M.P. , Edwards Kimberly M. , Hu Shu , Yann Sokhoun , Sin Sarath , Tok Songha , Chea Kimlay , Horm Sreyviseth , Rith Chenthearath , Keo Seangmai , Pum Leakhena , Duong Veasna , Auerswald Heidi , Phou Yisuong , Kol Sonita , Spiegel Andre , Harvey Ruth , Tum Sothyra , Sorn San , Seng Bunnary , Sengdoeurn Yi , Darapheak Chau , Savuth Chin , Hak Makara , Ieng Vanra , Patel Sarika , Thielen Peter , Claes Filip F. , Lewis Nicola S. , Sovann Ly , Dhanasekaran Vijaykrishna , Karlsson Erik A.
Published: November 2024

I.

After nearly a decade without reported human A/H5N1 infections, Cambodia faced a sudden resurgence with 16 cases between February 2023 and August 2024, all caused by A/H5 clade 2.3.2.1c viruses. Fourteen cases involved a novel reassortant A/H5N1 virus with gene segments from both clade 2.3.2.1c and clade 2.3.4.4b viruses. The emergence of this novel genotype underscores the persistent and ongoing threat of avian influenza in Southeast Asia. This study details the timeline and genomic epidemiology of these infections and related poultry outbreaks in Cambodia.
Decoding Liver Cancer Prognosis: From Multi-omics Subtypes, Prognostic Models to Single Cell Validation
Authors: Wang Yanbin , Wu Yuqi , Zhang Hong , Liu Xinyue , Ling Jing , Zhou Xiao , Song Anping , Sun Li , Qiu Hong , Yuan Xianglin , Xiong Hua , Zou Yanmei
Published: November 2024

Purpose

Hepatocellular carcinoma (HCC) is a highly aggressive tumor characterized by significant heterogeneity and invasiveness, leading to a lack of precise individualized treatment strategies and poor patient outcomes. This necessitates the urgent development of accurate patient stratification methods and targeted therapies based on distinct tumor characteristics.

Experimental Design

By integrating gene expression data from The Cancer Genome Atlas (TCGA), International Cancer Genome Consortium (ICGC), and Gene Expression Omnibus (GEO), we identified subtypes through a multi-omics consensus clustering approach amalgamated from 10 clustering techniques. Subsequently, we developed a prognostic model, employing machine learning algorithms, based on subtype classification features. Finally, by analyzing single cell sequencing data, we investigated the mechanisms driving prognostic variations among distinct subtypes.

Results

First, we developed a novel consensus clustering method that categorizes liver cancer patients into two subtypes, CS1 and CS2. Second, we constructed a prognostic prediction model, which demonstrated superior predictive accuracy compared to several models published in the past five years. Finally, we observed differences between CS1 and CS2 in various metabolic pathways, biological processes, and signaling pathways, such as fatty acid metabolism, hypoxia levels, PI3K-AKT and MIF signaling pathway.
UCF-MultiOrgan-Path:A Benchmark Dataset of Histopathologic Images for Deep Learning-Based Organ Classification
Authors: Hossain Md Sanzid Bin , Piazza Yelena , Braun Jacob , Bilic Anthony , Hsieh Michael , Fouissi Samir , Borowsky Alexander , Kaseb Hatem , Renduchintala Chaithanya , Fraser Amoy , Wray Britney-Ann , Chen Chen , Wang Liqiang , Husain Mujtaba , Hadley Dexter
Published: November 2024

A pathologist typically diagnoses tissue samples by examining glass slides under a light microscope. The entire tissue specimen can be stored digitally as a Whole Slide Image (WSI) for further analysis. However, managing and diagnosing large numbers of images manually is time-consuming and requires specialized expertise. Consequently, computer-aided diagnosis of these pathology images is an active research area, with deep learning showing promise in disease classification and cancer cell segmentation. Robust deep learning models need many annotated images, but public datasets are limited, often constrained to specific organs, cancer types, or binary classifications, which limits generalizability. To address this, we introduce the UCF multi-organ histopathologic (UCF-MultiOrgan-Path) dataset, containing 977 WSIs from cadaver tissues across 15 organ classes, including lung, kidney, liver, and pancreas. This dataset includes ∼2.38 million patches of 512×512 pixels. For technical validation, we provide patch-based and slide-based approaches for patch- and slide-level classification. Our dataset, containing millions of patches, can serve as a benchmark for training and validating deep learning models in multi-organ classification.
Analysis of Glomerular Transcriptomes from Nephrotic Patients SuggestAPOL1Risk Variants Impact Parietal Epithelial Cells
Authors: Gonzalez-Vicente Agustin , Crawford Dana C. , Bush William S. , Wu Zhenzhen , Bruggeman Leslie A. , Nair Viji , Eichinger Felix , Wessely Oliver , Kretzler Matthias , O’Toole John F. , Sedor John R. ,   Kidney Precision Medicine Project,   Nephrotic Syndrome Study Network
Published: November 2024

The disproportionate risk for idiopathic proteinuric podocytopathies in Black people is explained, in part, by the presence of two risk alleles (G1 or G2) in theAPOL1gene. The pathogenic mechanisms responsible for this genetic association remain incompletely understood. We analyzed glomerular RNASeq transcriptomes from patients with idiopathic nephrotic syndrome of which 72 had inferred African ancestry (AA) and 152 did not (noAA). Using gene coexpression networks we found a significant association betweenAPOL1risk allele number and the coexpression metamodule 2 (MM2), even after adjustment for eGFR and proteinuria at biopsy. Unadjusted Kaplan-Meier curves showed that unlike noAA, AA with the highest tertile of MM2 gene activation scores were less likely to achieve complete remission (p≤0.014). Characteristic direction (ChDir) identified a signature of 1481 genes, which separated patients withAPOL1risk alleles from those homozygous for referenceAPOL1. Only in AA, the tertile with the highest activation scores of these 1481 genes was less likely to achieve complete remission (p≤0.022) and showed a trend to faster progression to the composite event of kidney failure or loss of 40% eGFR (p≤0.099). The MM2 and ChDir genes significantly overlapped and were both enriched for Epithelial Mesenchymal Transition and inflammation terms. Finally, MM2 significantly overlapped with a parietal epithelial cell (PEC)-identity gene signature but not with a podocyte identity signature. Podocytes expressing variant APOL1s may generate inflammatory signals that activate PECs by paracrine mechanisms contributing toAPOL1nephropathy.