bluuebunny/crossref_metadata_2025 huggingface dataset:
The spliceosomal component GAMETOPHYTIC FACTOR 1 (GFA1) regulates a key photoperiodic switch
Authors: Tiedemann Sebastian , Sterck Lieven , Becker Hanna , Nielsen Nicola , Blohm Cordula , Blum Daniel , Lanz Christa , Maedler Kathrin , Weigel Detlef , Van de Peer Yves , Groß-Hardt Rita
Published: November 2024

Plants have evolved sophisticated mechanisms to perceive and interpret daytime in order to flower at an optimal time point. Here we show that the spliceosomal componentGAMETOPHYTIC FACTOR 1(GFA1) constitutes a previously unrecognized key photoperiodic switch, that is essential for flowering in long days. We show thatgfa1hypomorphic (gfa1hyp) plants fail to initiate flowering in long-days (LD), which correlates with ectopic activation of the short-day (SD) flowering repressorARABIDOPSIS THALIANA CENTRORADIALIS (ATC). Accordingly, flowering is restored upon inactivation ofATCingfa1hypmutants. A novel tissue-specificin-plantasplice assay and comprehensive RNAseq profiling ofgfa1hypmutants indicate that GFA1 mediated pre-mRNA splicing is substrate specific, as previously suggested for GFA1 orthologs. Furthermore, we show thatgfa1hypmutants accumulate nonsense transcripts of the photoreceptor componentsPHYBandRRC1, suggesting inappropriate photoreceptor signaling as a potential cause for the ectopic activation of the SD characteristic profile ingfa1hyp. In fact, known downstream targets of the phytochrome system such asRS31, SR34a, SRp30accumulate reduced amounts of light-dependent splice isoforms. Together, our data reveal a link between spliceosome composition and long-day flowering, based on complex transcriptional readouts in response to day length.
DNA replication fork stabilization and restart mechanisms revealed by biochemical reconstitution
Authors: Canal Berta , Bertolin Agostina P. , Lee Giselle C. , Minamino Masashi , Diffley John F.X.
Published: November 2024

Understanding how DNA replication forks stall and restart and how the DNA damage checkpoint prevents irreversible fork collapse in molecular detail are crucial for understanding how cells maintain stable genomes and how they prevent the genetic instability that drives cancer. Here we describe the reconstitution of fork stalling and restart with purified budding yeast proteins. After nucleotide depletion, leading strand DNA synthesis quickly stops but CMG helicase continues to unwind and Okazaki fragments continue to initiate on the lagging strand. Incomplete Okazaki fragments sequester PCNA, RFC and DNA polymerases δ and ε which prevents normal DNA synthesis restart and exposes nascent DNA to nuclease attack. The DNA damage checkpoint limits this sequestration by restraining fork progression, which protects stalled forks from collapse and ensures restart.
Tethering ferredoxin-NADP+reductase to photosystem I promotes photosynthetic cyclic electron transfer
Authors: Emrich-Mills Thomas Z. , Proctor Matthew S. , Degen Gustaf E. , Jackson Philip J. , Richardson Katherine H. , Hawkings Frederick R. , Buchert Felix , Hitchcock Andrew , Hunter C. Neil , Mackinder Luke C.M. , Hippler Michael , Johnson Matthew P.
Published: November 2024

Fixing CO2via photosynthesis requires ATP and NADPH. Linear electron transfer (LET) supplies both metabolites, yet depending on environmental conditions, additional ATP is required which can be generated by cyclic electron transfer (CET). How the balance between LET and CET is set remains largely unknown. Ferredoxin(FD)-NADP+reductase (FNR) has been suggested to act as the switch, channelling photosynthetic electrons to LET when it is bound to photosystem I (PSI) or CET when bound to cytochromeb6f. The essential role of FNR in LET precludes the use of a direct gene knock-out to test this hypothesis. We circumvented this problem by using CRISPR-Cas9 gene editing inChlamydomonas reinhardtiito create a chimeric form of FNR tethered to PSI via PSAF. Chimeric FNR mutants exhibited impaired photosynthetic growth relative to the wild type, and decreased LET and non-photochemical quenching (NPQ) under high CO2conditions. However, higher rates of CET and increased NPQ were found in the mutants under low CO2or low O2conditions where CET is normally up-regulated. Thus, rather than PSI-FNR interactions promoting LET, tethering FNR to PSI promotes CET at the expense of LET and CO2fixation.
Insights into the Translational Activation Mechanisms of theCOX1mRNA in Yeast Mitochondria
Authors: Zamudio-Ochoa Angelica , Camacho-Villasana Yolanda , García-Guerrero Aldo E. , Pérez-Martínez Xochitl
Published: November 2024

Mitochondrial translation is a critical regulatory step in mitochondrial genome expression. InSaccharomyces cerevisiae, translational activators are believed to bind to the 5’ UTRs of their target mRNAs to position the mitochondrial ribosome at the start codon. Pet309 and Mss51 are translational activators ofCOX1mRNA, which encodes subunit one of cytochromecoxidase. Pet309 physically interacts withCOX1mRNA, but no direct interaction of Mss51 with its target mRNA has been detected. Currently, the mechanisms underlying translational activation ofCOX1, or any other mitochondrial gene, remain poorly understood.

To explore in depth the mechanism ofCOX1mRNA translational activation, we studied the association of Pet309 and Mss51 with the mitochondrial ribosome. Both Pet309 and Mss51 interact with the mitoribosome regardless of the presence ofCOX1mRNA or of each other. Pet309’s association with the ribosome and withCOX1mRNA depends on its N-terminal domain. These findings indicate that Pet309 and Mss51 stably interact with the mitoribosome independently of an active translation. By integrating our data with previously published research, we propose a new mechanism ofCOX1mRNA translation activation.

SUMMARY STATEMENT

Yeast mitochondrial mRNAs require translational activators by an almost unknown mechanism. Based on our findings on Mss51 and Pet309 function, we present a new model for translation of theCOX1mRNA
Long-read nanopore shotgun eDNA sequencing for river biodiversity, pollution and environmental health monitoring
Authors: Nousias Orestis , Duffy Fiona G. , Duffy Isabelle J. , Whilde Jenny , Duffy David J.
Published: November 2024

As global temperatures rise, species populations and biodiversity decline, and infectious diseases emerge all at unprecedented rates, it is more vital than ever to accurately understand the current state of natural habitats. While traditional methods including direct sampling and observation have their merits, newer technologies offer additional sampling capabilities.

Here, we assess the feasibility of a single assay: shotgun long-read sequencing, to monitor species eDNA from across the tree of life, from viruses to complex multicellular organisms, across a river system (mountain tributary to sea). We conducted eDNA sampling and shotgun long-read sampling from water taken from the Avoca River watercourse, Co. Wicklow, Ireland, from a mountain tributary through to the sea, and comparative nearby beach sand sampling. We report that shotgun long-read sequencing and metagenomic analysis have utility for the detection and quantification of organismal DNA present in eDNA samples, from across the tree of life, from microbes (including DNA viruses) to mammals. With this single assay we were able to simultaneously quantify differences in eDNA abundance for a broad range of biodiversity and pathogens across sites and sample types. This included human, wildlife, plant and microbial pathogens and parasites with health, agricultural and economic importance. Additionally, the generated eDNA genomic data enabled population genetic applications even from natural complex community settings, as demonstrated here for blue mussels (Mytilus edulis). The results demonstrate that Oxford Nanopore sequencing provides a quantitative approach for river biodiversity, pollution and environmental health monitoring.

This method is more cost-effective and requires less laboratory preparation time or molecular expertise (barcode/primer design) than alternative biodiversity eDNA approaches (e.g. metabarcoding or qPCR). Shotgun analysis approaches will also benefit from the continual expansion of reference genome databases, for environmental, evolutionary and medical reasons, among others. Computation, cloud and artificial intelligence (AI) tools (such as the cloud-based analyses utilized here) can analyze shotgun sequencing eDNA data in a matter of hours and can be used by conservation practitioners, environmentalists and public health personnel without the need for coding or in-depth bioinformatics skills. The proven citizen/community scientist applicability and ease of eDNA sampling may further revolutionize and democratize biodiversity research, conservation surveillance, and environmental health monitoring. Long-read shotgun sequencing of eDNA offers the means to assess whole ecosystems, and the ecological, trophic, and host-pathogen interactions occurring within them.
CELF2 promotes tau exon 10 inclusion via hinge domain-mediated nuclear condensation
Authors: Li Xin , Syed Ishana , Zhang Zhao , Adhikari Rashmi , Tang Dan , Ko SuHyuk , Liu Zhijie , Chen Lizhen
Published: November 2024

Alternative splicing is a fundamental process that contributes to the functional diversity and complexity of proteins. The regulation of each alternative splicing event involves the coordinated action of multiple RNA-binding proteins, creating a diverse array of alternatively spliced products. Dysregulation of alternative splicing is associated with various diseases, including neurodegeneration. Here we demonstrate that CELF2, a splicing regulator and a GWAS-identified risk factor for Alzheimer’s disease, binds to mRNAs associated with neurodegenerative diseases, with a specific interaction observed in the intron adjacent to exon 10 on Tau mRNA. Loss of CELF2 in the mouse brain results in a decreased inclusion of Tau exon 10, leading to a reduced 4R:3R ratio. Further exploration shows that the hinge domain of CELF2 possesses an intrinsically disordered region (IDR), which mediates CELF2 condensation and function. The functionality of IDR in regulating CELF2 function is underscored by its substitutability with IDRs from FUS and TAF15. Using TurboID we identified proteins that interact with CELF2 through its IDR. We revealed that CELF2 co-condensate with NOVA2 and SFPQ, which coordinate with CELF2 to regulate the alternative splicing of Tau exon 10. A negatively charged residue within the IDR (D388), which is conserved among CELF proteins, is critical for CELF2 condensate formation, interactions with NOVA2 and SFPQ, and function in regulating tau exon 10 splicing. Our data allow us to propose that CELF2 regulates Tau alternative splicing by forming condensates through its IDR with other splicing factors, and that the composition of the proteins within the condensates determines the outcomes of alternative splicing events.
SloR-SRE binding to theS. mutans mntHpromoter is cooperative
Authors: Ziogas Myrto , Drummond India , Todorovic Igor , Kraczkowsky Katie , Zhang Hua , Wu Hui , Spatafora Grace
Published: November 2024

Streptococcus mutansis a commensal member of the plaque microbiome. It is especially prevalent when dietary sugars are available forS. mutansfermentation, generating acid byproducts that lower plaque pH and foster tooth decay.S. mutanscan survive in the transient conditions of the mouth, in part because it can regulate the uptake of manganese and iron during periods of feast when metal ions are available, and famine when they are limiting.S. mutansdepends on a 25kDa metalloregulatory protein, called SloR, to modulate uptake of these cations across the bacterial cell surface. When bound to manganese, SloR, binds to palindromic recognition elements in the promoter of thesloABCgenes that encode the major manganese transporter inS. mutans. Reports in the literature describ MntH, an ancillary manganese transporter inS. mutans, that is also subject to SloR control. In the present study, we performed expression profiling experiments that reveal coordinate regulation of thesloABCandmntHgenes at the level of transcription. In addition, we describe a role for themntHgene product that is redundant with that of thesloABC-encoded metal ion uptake machinery. The results of DNA binding studies support direct SloR binding to themntHpromoter region which, like that at thesloABCpromoter, harbors three palindromic recognition elements to which SloR binds cooperatively to repress downstream transcription. These findings expand our understanding of the SloR metalloregulome and elucidate SloR-DNA binding that is essential forS. mutansmetal ion homeostasis and fitness in the oral cavity.
The nephronophthisis proteinGLIS2/NPHP7is required for the DNA damage response in kidney tubular epithelial cells
Authors: Ebert Lena K. , Schloesser Lukas , Frech Laura E. , Jain Manaswita , Dafinger Claudia , Liebau Max C. , Benzing Thomas , Schermer Bernhard , Slaats Gisela G.
Published: November 2024

Nephronophthisis (NPH) is an autosomal-recessive cystic kidney disease representing the most frequent genetic cause of end-stage kidney failure in children and adolescents. NPH is caused by genetic variants in >20 NPHP genes. While nearly all NPHP genes encode ciliary proteins, classifying NPH as a renal ciliopathy, there is evidence for a pathogenic role of a compromised DNA damage response (DDR). Here, we present a novelNphp7/Glis2-deficient mouse model with an early stop codon using CRISPR/Cas9-mediated genome editing (Glis2Y122X). Homozygous mice develop cystic kidney disease with significant fibrosis at a higher age. Interestingly, the kidneys of these animals exhibit an accumulation of DNA damage (DD) early on, even before any functional impairment of the kidneys becomes apparent. Interactome analysis for GLIS2 revealed an array of DDR-related proteins within the GLIS2 protein complex. Consistent with thein vivodata, the knockdown ofGlis2in kidney epithelial cells led to increased DNA damage. Moreover, supporting the role of GLIS2 in the DDR, we demonstrate that a substantial proportion of GLIS2 is present within the chromatin fraction of cells which is further increased upon UV-induced DD. Live-cell imaging revealed the rapid recruitment of GFP-tagged GLIS2 to sites of laser-induced DD, a response diminished in Glis2Y122Xand a variant of Glis2 resembling a known patient mutation. Overall, our data provide compelling evidence for the direct involvement of GLIS2 in the DNA damage response, highlighting the loss of genome stability as an important factor contributing to the pathogenesis of renal ciliopathies.

Author Summary

Nephronophthisis (NPH) is a rare inherited kidney disease characterized by cyst formation, fibrosis, and kidney failure at a young age. It is typically caused by mutations in genes essential for the proper function of cilia, small sensory, antenna-like cell protrusions. However, emerging evidence suggests that genome instability and impaired DNA repair may also contribute to NPH. Thus, in some cases, ciliopathies may result from defects in nuclear proteins rather than ciliary proteins. To investigate this, we generated a mouse model with a defectiveNPHP7/Glis2gene using genome-editing tools. The encoded protein GLIS2 has been described to be in the cilium and the nucleus, while its detailed function remained unclear. As these mice aged, they exhibited signs of DNA damage and later developed cystofibrotic kidney disease. Consistently, we demonstrated that the GLIS2 protein interacts with key DNA repair proteins and is rapidly recruited to sites of DNA damage and repair. Thus, inefficient DNA damage repair appears to contribute to kidney disease in this mouse model. These findings underscore the crucial role of genome stability in preventing kidney disease, providing new insights into the underlying causes of NPH.
Nitrate Promoting Hair Growth Through Enhancing Wnt/β-catenin and Downregulating TGF-β/Samd/BMP signaling pathway in C57BL/6 Mice
Authors: Lan Yanjie , Li Shenglan , Wang Jiachen , Duan Ling , Huang Mengqian , Yang Xin , Zhang Chunmei , Wang Songling , Li Wenbin
Published: November 2024

Nitrate found widely distributed in vegetables and fruits. It has been proved to have many physiological functions through nitrate-nitrite-NO pathway, such as attenuate oxidative stress, enhance the skeletal muscle and tone skin vascular. But the function of nitrate in regulating hair growth is unclear. Here, we evaluated potential roles of nitrate on hair growth and its mechanisms by in vivo models. Mice treated with 10mM nitrate supplementation significantly promoting hair growth, along with an increase in the number of hair follicles and thickness of dermis. We further demonstrated that Wnt3a and β-catenin were highly expressed in the nitrate-treated groups, while the expression of TGF-β was attenuated. Moreover, nitrate also increased the expression levels of growth factors (Fgf, Igf-1, and Vegf). Our data indicated that nitrate supplementation could effectively accelerate hair regrowth through activating the Wnt/β-catenin and inhibiting TGF-β1/Smad/BMP signaling pathway. Therefore, these findings provide support for a potential therapeutic role of nitrate in preventing and treating alopecia.
Membrane mimetic thermal proteome profiling (MM-TPP) towards mapping membrane protein-ligand dynamics
Authors: Jandu Rupinder Singh , Al-Seragi Mohammed , Aoki Hiroyuki , Babu Mohan , van Hoa Franck Duong
Published: November 2024

Integral membrane proteins (IMPs) remain the principal target of small-molecule therapeutics, and yet modalities towards probing on and off-target hits against this protein class in a robust, unbiased, and detergent-free manner remain starkly underdeveloped. Previously, we introduced the Peptidisc membrane mimetic (MM) for the water-soluble stabilization of theEscherichia colimembrane proteome and interactome (Carlson et al., 2019). Herein, we implement the Peptidisc into thermal proteome profiling (TPP), enabling for the first time a broad-scale level characterization of membrane protein-ligand interactions while completely circumventing structural perturbations invoked by detergents. Using a library prepared from the whole mouse liver, we determine the influence of ATP and orthovanadate on the thermal stability of IMPs, including pharmaceutically relevant ATP-binding cassette ABC transporters and G-protein coupled receptors. MM-TPP also detects thermal stability changes driven by ATP by-products, where non-canonical ATP binders can be validated with next-generation computational tools. MM-TPP thus offers a robust platform for identifying on- and off-target ligand effects, providing insights into the druggable membrane proteome and its stability as a consequence of changing and often dynamic small molecules.
Methuselah Proteins in Longevity: Unraveling Their Impact Through Mathematical Genomics
Authors: Hassan Sk. Sarif , Nawn Debaleena , Ghosh Ankita , Sil Moumita , Goswami Arunava , Basu Pallab , Lundstrom Kenneth , Uversky Vladimir N.
Published: November 2024

This study provides a quantitative and comprehensive analysis of 18 Methuselah (mth) protein variants from fruit flies, focusing on their evolutionary relationships, structural features, and functional roles in aging and longevity. Phylogenetic analysis identified two major clades of mth proteins, with the first clade indicating conserved functions across Drosophila species and the second clade reflecting gene duplication and diversification. The study found five distinct functional subclasses of mth proteins through amino acid frequency and poly-string analyses, linked to their structural diversity and role in longevity. Structural topology and post-translational modifications reveal similarities with G-protein-coupled receptors (GPCRs), suggesting that mth proteins are crucial for signal transduction and cellular health. Variability in propeptide cleavage sites and intrinsic protein disorder further highlight adaptive roles in signaling. The findings underscore the importance of a quantitative and comprehensive approach to studying Methuselah genes, offering insights into their functional versatility and evolutionary dynamics. This enhanced quantitative understanding contributes to advancing research on aging and longevity.
Platelet Membrane-Coated SPION for Targeted Paclitaxel Delivery and Magnetic Hyperthermia in Cancer Therapy
Authors: Tavakoli Mohamadreza , Maghsoudian Samane , Rezaei-Aderiani Amir , Hajiramezanali Maliheh , Amani Mahdiyar , Sharifikolouei Elham , Ghahremani Mohammad Hossein , Raoufi Mohammad , Motasadizadeh Hamidreza , Dinarvand Rassoul
Published: November 2024

Due to the limited therapeutic efficacy and side effects associated with conventional chemotherapy, researchers have turned their attention to developing targeted drug delivery systems using advanced nanotechnology. Coating nanoparticles (NPs) with cell membranes is a promising strategy because it extends their circulation times and allows them to selectively adhere to damaged vessel sites through the platelet membrane surface, thereby enhancing tumor uptake. Herein, we have developed a biomimetic drug delivery system consisting of superparamagnetic iron oxide nanoparticles (SPIONs) coated by platelet membranes (PM) for carrying Paclitaxel (PTX) to exploit the synergism effect of chemotherapy and magnetic hyperthermia. Controlled-release PTX nanoparticles exhibited consistent behavior over time, indicating no significant difference in release between SPION/PTX and SPION/PTX/PM at pH 7.4. However, at pH 5.5, improved release was observed, specifically a 1.4-fold increase for SPION/PTX/PM. The confocal and flow cytometry results show an enhancement in the cellular uptake of SPION/PTX/PM nanoparticles, with an average fluorescence intensity of 142 ±12.5. MTT results indicated that SPION/PTX/PM demonstrates superior cytotoxic effects compared to SPION/PTX and PTX alone, showing an IC50 value of 5 μg/mL after 48 h of treatment. Furthermore, the IC50 decreased to 1 μg/mL when an alternating magnetic field was applied. Hence, the in vivo results and histopathological staining showed that the SPION/PTX/PM-AFM treatment group exhibited the highest rate of tumor growth inhibition, reaching nearly 92.14 %. These findings highlight the potential of using platelet membrane-coated nanoparticles for targeted delivery, combining magnetic hyperthermia and chemotherapy to minimize chemotherapy’s undesirable effects while maximizing therapeutic outcomes.
Phylogeography of the electric rays of the family Narcinidae (Gill, 1862): systematic review and evidence of the dispersal routes to America.
Authors: Rodrigues-Filho Luis Fernando Silva , Silva Richard Klein Castro , Lima Eduardo Lopes , Siccha-Ramirez Raquel , Rincon Getulio , Nunes Jorge Luiz Silva , Sampaio Iracilda , Sales Joao Braullio Luna
Published: November 2024

The electric rays belonging family Narcinidae are a cosmopolitan group, which has come under increasing scrutiny in relation to the taxonomic composition of the family and its phylogenetic position within the order Torpediniformes. Phylogeographic inferences can provide important insights into the present-day distribution of the narcinid species, as well as the systematics of the group. In this context, the present study evaluates the evolutionary and biogeographic history of the family Narcinidae, inferring distribution patterns and the molecular systematics of these organisms based on the DNA barcode. The results of the analysis indicate that the electric rays originated in the Central Indo-Pacific realm, with all other dispersal events beginning from this region. Two different dispersal routes were observed for the American narcinids. The first route was from the Central Indo-Pacific realm to the Pacific Ocean and then the Caribbean Sea, prior to the closure of the Isthmus of Panama. The second route start on Central Indo-Pacific realm passed South Africa continent before crossing the Atlantic. The principal torpediniform lineages diversified between the end of the Cretaceous and the early Paleocene, with the present-day narcinid taxa arising and diversifying between 25 and 20 Mya. The present study confirms the lack of monophyly in the Narcinidae, validates the genus Narcinops, and restricts the occurrence of the genus Narcine to the coastal environments of the Americas, which emphasizes the need for the review and revalidation of the synonym Syrraxis for the narcinids of the Central/Western Indo-Pacific realm. This study also describes additional lineages for Narcine entemedor and Narcine maculata and validates the occurrence of three species (Narcinesp., Narcine brasiliensis, and Narcine bancroftii) in the Atlantic Ocean, based on the published data, and emphasizes the need for a definitive description of Narcine sp.
Augmenting Transcriptome Annotations through the Lens of Splicing Evolution
Authors: Zang Xiaofei Carl , Chen Ke , Khan Irtesam Mahmud , Shao Mingfu
Published: November 2024

Alternative splicing (AS) is a ubiquitous mechanism in eukaryotes. It is estimated that 90% of human genes are alternatively spliced. Despite enormous efforts, transcriptome annotations remain, nevertheless, incomplete. Conventional means of annotation were largely driven by experimental data such as RNA-seq and protein sequences, while little insight was shed on understanding transcriptomes and alternative splicings from the perspective of evolution. This study addresses this critical gap by presenting TENNIS (Transcript EvolutioN for New Isoform Splicing), an evolution-based model to predict unannotated isoforms and refine existing annotations without requiring additional data. The model of TENNIS is based on two minimal premises–AS isoforms evolve sequentially from existing isoforms, and each evolutionary step involves a single AS event. We formulate the identification of missing transcripts as an optimization problem and parsimoniously find the minimal number of novel transcripts. Our analysis showed approximately 80% of multi-transcript groups from six transcriptome annotations satisfy our evolutionary model. At a high confidence level, 40% of isoforms predicted by TENNIS were validated by deep long-read RNA-seq. In a simulated incomplete annotation scenario, TENNIS dramatically outperforms two randomized baseline approaches by a 2.25-3 fold-change in precision or a 3.5-3.9 fold-change in recall, after controlling the same level of recall or precision of the baseline methods. These results demonstrate that TENNIS effectively identifies missing transcripts by complying with minimal propositions, offering a powerful approach for transcriptome augmentations through the lens of alternative splicing evolutions. TENNIS is freely available athttps://github.com/Shao-Group/tennis.
Microtubule dynamics are defined by conformations and stability of clustered protofilaments
Authors: Kalutskii Maksim , Grubmüller Helmut , Volkov Vladimir A , Igaev Maxim
Published: November 2024

Microtubules are dynamic cytoskeletal polymers that add and lose tubulin dimers at their ends. Microtubule growth, shortening and transitions between them are linked to GTP hydrolysis. Recent evidence suggests that flexible tubulin protofilaments at microtubule ends adopt a variety of shapes, complicating structural analysis using conventional techniques. Therefore, the link between GTP hydrolysis, protofilament structure and microtubule polymerization state is poorly understood. Here, we investigate the conformational dynamics of microtubule ends using coarse-grained modeling supported by atomistic simulations and cryo-electron tomography. We show that individual bent protofilaments organize in clusters, transient precursors to a straight microtubule lattice, with GTP-bound ends showing elevated and more persistent cluster formation. Differences in the mechanical properties of GTP- and GDP-protofilaments result in differences in intra-cluster tension, determining both clustering propensity and protofilament length. We propose that conformational selection at microtubule ends favors long-lived clusters of short GTP-protofilaments that are more prone to form a straight microtubule lattice and accommodate new tubulin dimers. Conversely, microtubule ends trapped in states with unevenly long and stiff GDP-protofilaments are more prone to shortening. We conclude that protofilament clustering is the key phenomenon that links the hydrolysis state of single tubulins to the polymerization state of the entire microtubule.
Moisture and competition constrain ephemeral resource quality for burying beetle reproduction
Authors: Hayes Tracie E. , Lassérès Léo , Yang Louie H.
Published: November 2024

Shifts in abiotic factors such as temperature and moisture can change the availability of resources, especially under climate change. Both abiotic and biotic drivers can have profound, rapid effects on species distribution, survival, and reproduction. Little is known about how abiotic factors affect the availability of ephemeral resources. Burying beetles (Nicrophorusspp.) are specialist users of ephemeral resources, as their reproduction requires locating, defending, and burying a small carcass. Environmental moisture, such as coastal fog, could change how quickly carcasses dry out. We tested the role of carcass moisture and interspecific competition with a generalist scavenger,Heterosilphaspp., on reproduction by placing pairs ofNicrophorus guttulain field chambers with fresh and experimentally dehydrated mouse carcasses. Pairs that were given fresh mouse carcasses were more likely to carry out reproductive behaviors and produce viable offspring than pairs that were given a partially dehydrated mouse. For those pairs that reproduced, competition limited the number of offspring. These results indicate that shifts in abiotic factors under climate change, along with biotic factors like competition, can reduce the availability of ephemeral resource patches for consumers.
The maize recombination landscape evolved during domestication
Authors: Epstein Ruth , Wheeler JJ , Hubisz Melissa , Sun Qi , Bukowski Robert , Zhai Jingjing , Lai Wei-Yun , Buckler Edward , Pawlowski Wojtek P.
Published: November 2024

Meiotic recombination is an important evolutionary process because it can increase the amount of genetic variation within populations through the breakage of unfavorable linkages and creation of novel allelic combinations. Despite the plethora of knowledge about population-level benefits of recombination and numerous theoretical studies examining how recombination rates can evolve over time, there is a lack of empirical evidence for any hypotheses that have been put forward. To alleviate this gap in knowledge, we characterized the evolution of the recombination landscape inZea maysssp.mays(maize) during its domestication fromZea maysssp.parviglumis(teosinte), explored hypotheses that permitted the evolution of the maize recombination landscape and tied these alterations to changes in the genetic basis of recombination. Using experimental populations and the population genomics approach of ancestral recombination graph (ARG) inference, our data demonstrated that maize had a 12% increase in its genome-wide recombination rate during domestication. Although the maize and teosinte recombination landscapes are highly correlated, r = 0.85 at 1Mb resolution, maize has evolved to have higher recombining regions in interstitial chromosome regions, compared to teosinte which only harbors high recombining regions sub-telomerically. Our data show that the re-patterning of COs towards interstitial chromosome regions came from reduced CO interference levels within maize. Supporting the idea that CO interference is reduced within maize, we found evidence for selection acting on trans-acting recombination-modifiers that participate in the class I CO pathway or CO interference directly. Lastly, we showed that the re-patterning of COs was beneficial to maize evolution because regions that significantly increased in recombination were targeted to gene-rich regions harboring domestication related loci. Because we found regions with significant increases in recombination had a lower deleterious mutation load, compared to regions with decreases in recombination, we concluded that the domestication-related variation in these regions, in which selection acted upon during domestication, was shielded from the Hill-Robertson effect. In conclusion, the re-patterning of CO events during domestication allowed maize to adapt and evolve at a faster rate than previously understood.
Acidic Conditions Promote Clustering of Cancer Cell Derived Extracellular Vesicles and Enhance their Fusion with Synthetic Liposomes
Authors: Fisher William S. , Douglas Jessica , Roshan Sherwin , Perez Ramon , Wei Sophia , Roberts Logan , Ewert Kai K. , Safinya Cyrus R.
Published: November 2024

Extracellular vesicles (EVs) are endogenous vesicles secreted by cells. Exosomes, a subset of EVs ranging from 30 to 150 nanometers in diameter, contain cytosolic proteins, gene-silencing miRNA, and gene-encoding mRNA and have roles in intercellular communication. Exosomes show promise as cancer chemotherapeutic drug delivery vehicles given their low immunogenicity and cell-specific delivery of luminal contents to the cytosol of target cells. However, loading exosomes with cancer chemotherapeutic drugs is inefficient, which limits their therapeutic application. To overcome this barrier, methods have been developed which allow fusion of EVs with synthetic liposomes preloaded with therapeutic drug. While these methods show more efficient fusion than passive incubation of EVs with liposomes, they risk either damage to the membrane proteins of the EVs or contamination of the final EV-liposome hybrid with residual depletant molecules, which can cause side effects or hinder content delivery. Here, we present a new, weakly perturbative method which uses acidic conditions (pH 5) to significantly enhance the fusion of EVs and synthetic, neutral liposomes (NLs) compared to passive incubation in pH 7.4 at 37 °C. An adapted Forster resonance energy transfer (FRET) based lipid mixing assay confirms that fusion is enhanced with this method. This significant finding implies that lipid-only synthetic liposomes are able to fuse with EVs, creating EV-liposome hybrids under relevant temperature and pH conditions, with no other non-lipidic component, such as fusogenic amphipathic peptides, added to the synthetic liposomes. Remarkably, differential interference contrast (DIC) and fluorescence microscopy show that this enhancement of fusion corresponds with the onset of clustering of mixtures of EVs and NLs, or EVs alone, in acidicbut not neutralpH conditions. The findings support a hypothesis that content release from EVs in early to late endocytic environment may be a combination of protein-protein clustering interactions and a lipidic component. Further, this study provides a novel method for enhanced fusion of EVs and liposomes which is expected to preserve EV membrane proteins and functionality towards the development of therapeutic hybrid drug delivery vehicles in nanomedicine applications.
A journey towards developing a new cleavable crosslinker reagent for in-cell crosslinking
Authors: Mueller Fraenze , Brutiu Bogdan Razvan , Saridakis Iakovos , Leischner Thomas , Birklbauer Micha Johannes , Matzinger Manuel , Madalinski Mathias , Lendl Thomas , Shaaban Saad , Dorfer Viktoria , Maulide Nuno , Mechtler Karl
Published: November 2024

Crosslinking mass spectrometry (XL-MS) is a powerful technology that recently emerged as an essential complementary tool for elucidating protein structures and mapping interactions within a protein network. Crosslinkers which are amenable to post-linking backbone cleavage simplify peptide identification, aid in 3D structure determination and enable system-wide studies of protein-protein interactions (PPIs) in cellular environments. However, state-of-the-art cleavable linkers are fraught with practical limitations, including extensive evaluation of fragmentation energies and fragmentation behaviour of the crosslinker backbone. We herein introduce DiSPASO as a lysine-selective, MS-cleavable cross-linker with an alkyne handle for affinity enrichment. DiSPASO was designed and developed for efficient cell membrane permeability and crosslinking while securing low cellular perturbation. We tested DiSPASO employing three different copper-based enrichment strategies using model systems with increasing complexity (Cas9-Halo, purified ribosomes, live cells). Fluorescence microscopy in-cell crosslinking experiments revealed a rapid uptake of DiSPASO into HEK 293 cells within 5 minutes. While DiSPASO represents progress in cellular PPI analysis, its limitations and low crosslinking yield in cellular environments require careful optimisation of the crosslinker design, highlighting the complexity of developing effective XL-MS tools and the importance of continuous innovation in accurately mapping PPI networks within dynamic cellular environments.
Evolutionary Dynamics of G-Quadruplexes in Human and Other Great Ape Telomere-to-Telomere Genomes
Authors: Mohanty Saswat K. , Chiaromonte Francesca , Makova Kateryna D.
Published: November 2024

G-quadruplexes (G4s) are non-canonical DNA structures that can form at approximately 1% of the human genome. G4s contribute to point mutations and structural variation and thus facilitate genomic instability. They play important roles in regulating replication, transcription, and telomere maintenance, and some of them evolve under purifying selection. Nevertheless, the evolutionary dynamics of G4s has remained underexplored. Here we conducted a comprehensive analysis of predicted G4s (pG4s) in the recently released, telomere-to-telomere (T2T) genomes of human and other great apes—bonobo, chimpanzee, gorilla, Bornean orangutan, and Sumatran orangutan. We annotated tens of thousands of new pG4s in T2T compared to previous ape genome assemblies, including 41,236 in the human genome. Analyzing species alignments, we found approximately one-third of pG4s shared by all apes studied and identified thousands of species– and genus-specific pG4s. pG4s accumulated and diverged at rates consistent with divergence times between the studied species. We observed a significant enrichment and hypomethylation of pG4 shared across species at regulatory regions, including promoters, 5’ and 3’UTRs, and origins of replication, strongly suggesting their formation and functional role in these regions. pG4s shared among great apes displayed lower methylation levels compared to species-specific pG4s, suggesting evolutionary conservation of functional roles of the former. Many species-specific pG4s were located in the repetitive and satellite regions deciphered in the T2T genomes. Our findings illuminate the evolutionary dynamics of G4s, their role in gene regulation, and their potential contribution to species-specific adaptations in great apes, emphasizing the utility of high-resolution T2T genomes in uncovering previously elusive genomic features.
Manipulation of alternative splicing ofIKZF1elicits distinct gene regulatory responses in T cells
Authors: Pastor Lucia , Newman Jeremy R.B. , Callahan Colin M. , Pickin Rebecca R. , Atkinson Mark A. , Onengut-Gumuscu Suna , Concannon Patrick
Published: November 2024

Genome-wide studies have identified significant allelic associations between genetic variants in or near theIKZF1gene and multiple autoimmune disorders.IKZF1, encoding the transcription factor IKAROS, produces at least 10 distinct transcripts. To explore the impact of alternative splicing ofIKZF1on the function of mature T cells, we generated a panel of human T-cell clones with truncating mutations inIKZF1exons 4, 6 or both. Differences in gene expression, chromatin accessibility, and protein abundance among clones were assessed by RNA-seq, ATAC-seq and immunoblotting. Clones with single targeting events clustered separately from double-targeted clones on multiple parameters, but overall, clone responses were highly heterogeneous. Perturbation ofIKZF1splicing resulted in significant differences in expression and chromatin accessibility of other autoimmunity-associated genes and elicited compensatory expression changes in other IKAROS family members. Our results suggest that even modest alterations ofIKZF1splicing can have significant effects on gene expression and function in mature T cells, potentially contributing to autoimmunity in susceptible individuals.

Author Summary

RNA sequencing has revealed an unexpectedly large population of alternative transcripts produced by most human genes, but the functional significance of such transcripts has been debated, with some authors arguing that they represent non-functional “noise” and others arguing that they are largely functional and greatly expand the potential human proteome. Here, we explore these issues for the transcription factorIKZF1, which produces numerous alternative transcripts in human T cells and is significantly associated with risk for type 1 diabetes and other autoimmune disorders. We introduce stop codons at multiple sites in alternatively spliced exons and evaluate transcript levels and chromatin accessibility in individually targeted clones, allowing us to assay the broad impact of alternative splicing ofIKZF1in human T cells. Our studies reveal that perturbation ofIKZF1splicing results in significant differences in gene expression, chromatin accessibility and protein production of other autoimmunity-associated genes and elicits compensatory expression changes in other IKAROS family members. Even modest alterations inIKZF1splicing had significant effects on gene expression and function in mature T cells, potentially contributing to autoimmunity in susceptible individuals and suggesting that transcript isoforms produced by alternative splicing can and do have functional impacts.
Efficient mRNA DeliveryIn VitroandIn VivoUsing a Polycharged Biodegradable Nanomaterial
Authors: Yang Xuejin , Xiao Jingya , Staveness Daryl , Zang Xiaoyu
Published: November 2024

As RNA rises as one of the most significant modalities for clinical applications and life science research, efficient tools for delivering and integrating RNA molecules into biological systems become essential. We hereby report a formulation using a polycharged biodegradable nano-carrier, N1-501, which demonstrates superior efficiency and versatility in mRNA encapsulation and delivery in both cell and animal models. N1-501 is a polymeric material designed to function through a one-step facile formulation process suitable for various research settings. Its capability for mRNA transfection is investigated across a wide range of mRNA doses and in different biological models, including 18 tested cell lines and mouse models. This study also comprehensively analyzes N1-501’s application for mRNA transfection by examining factors such as buffer composition and pH, incubation condition, and media type. Additionally, N1-501’s superiorin vivomRNA transfection capability ensures its potential as an efficient and consistent tool for advancing mRNA-based therapies and genetic research.
Potential spillover effects on diagnostic delay for cancer during the NHS-Galleri trial: a quasi-experimental difference-in-differences study
Authors: Mann Sean , Eagan Joshua , Griffin Beth Ann
Published: November 2024

Objective

To examine whether regions participating in the NHS-Galleri trial of a multi-cancer early detection test experienced changes in cancer diagnostic delay rates that could indicate the presence of spillover due to constraints on care delivery.

Design

Quasi-experimental difference-in-differences analysis.

Setting

NHS England, April 2021-September 2023.

Participants

All 21 cancer alliance regions in NHS England, 8 of which participated in the trial and 13 of which did not.

Main outcomes

Rates of cancer diagnostic delays, defined as the percentage of patient referrals for investigation of possible cancer that resulted in diagnostic resolution within 28 days of initial referral. A difference-in-differences analysis was used to compare diagnostic delay rates in regions that participated in the NHS-Galleri trial to regions that did not participate, before and after the start of the trial. Three primary analyses were conducted: for all types of suspected cancer; for the most commonly referred cancers (breast, skin, lower gastrointestinal); and for less commonly referred cancers (all other types).

Results

We observed no significant differences in rates of diagnostic delay for all cancers or for the most commonly referred cancers. For less commonly referred cancers, the percentage of patients experiencing diagnostic delays increased from 32.6% to 35.1% in regions participating in the NHS-Galleri trial from the 6 months before to the 6 months after trial start, compared with a small reduction from 32.7% to 31.7% in regions that did not participate (adjusted difference 3.4 percentage points, 95% confidence interval 1.2 to 5.7). These differences persisted from 6 to 12 months after trial start and receded the following year.

Conclusions

Regional participation in the NHS-Galleri trial was associated with a statistically significant increase in diagnostic delay rates for less commonly referred cancers in the first year of the trial, but not for cancer overall or the most commonly referred cancers. Rollout of the trial intervention may have increased demand for downstream diagnostic services and, in a capacity constrained environment, led to region-wide increases in delayed diagnosis for less commonly referred cancers. If patients in the control group faced delayed diagnosis as a result, this would represent spillover due to constraints on care delivery during the first year and could affect measurement of the trial’s primary outcome, incidence of later stage cancer at time of diagnosis. This study underscores the importance of addressing spillover in clinical trials of interventions that affect availability of healthcare services.
“Around the clock”. Exploring health care professionals’ experience of discharge of older people during out of hours from the emergency department: a qualitative study
Authors: Dunnion Mary , Goode Debbie , Ryan Assumpta , McIlfatrick Sonja
Published: November 2024

Background

Older adults constitute a considerable number of attendances at emergency departments (EDs). Whilst many require hospital admission, a greater focus now is on admission avoidance with older adults being subsequently discharged from EDs. Little is known, however, about the experiences out of hours (OOH) when specialist older person support services are unavailable.

Aim

To explore senior health care professionals’ experiences of discharging older people during out of hours from EDs.

Method

A qualitative study involving individual semi-structured interviews was used to explore the experiences of healthcare professionals. Fourteen participants in total responded from a wide geographical spread. Data analysis was undertaken using Braun and Clarke’s (2022) six-step framework for Thematic Analysis.

Results

One overarching theme was identified focusing on risks and safety for the older person whilst being discharged OOH from the ED. Three sub-themes included “Should they stay, or should they go?”, “Bright lights and noise”, and “New ways of working”, which included risks in discharging an older person OOH from the ED, risks in delaying discharge, and recommendations for future practice. Significant differences were reported between office hours and OOH discharge of older adults from the ED. Diversity in practice assessments, and resources available was also evident across EDs. Significant adverse consequences were identified for older adults having to stay for prolonged periods in the ED.

Conclusions

There have been many welcome developments in healthcare services for older people who attend and are subsequently discharged from the ED. However, further innovative practice and collaboration with senior decision makers is needed to meet the healthcare needs of a rapidly ageing population. Safe, equitable and effective discharge practice 24/7 should be a norm for all older people in every ED regardless of location. Gaps in care identified must be addressed taking cognisance of the relevant recommendations for future practice.