bluuebunny/crossref_metadata_2025 huggingface dataset:
Multi-species genome-wide CRISPR screens identify conserved suppressors of cold-induced cell death
Authors: Lam Breanna , Kajderowicz Kathrin M. , Keys Heather R. , Roessler Julian M. , Frenkel Evgeni M. , Kirkland Adina , Bisht Punam , El-Brolosy Mohamed A. , Jaenisch Rudolf , Bell George W. , Weissman Jonathan S. , Griffith Eric C. , Hrvatin Sinisa
Published: July 2024

Cells must adapt to environmental changes to maintain homeostasis. One of the most striking environmental adaptations is entry into hibernation during which core body temperature can decrease from 37°C to as low at 4°C. How mammalian cells, which evolved to optimally function within a narrow range of temperatures, adapt to this profound decrease in temperature remains poorly understood. In this study, we conducted the first genome-scale CRISPR-Cas9 screen in cells derived from Syrian hamster, a facultative hibernator, as well as human cells to investigate the genetic basis of cold tolerance in a hibernator and a non-hibernator in an unbiased manner. Both screens independently revealed glutathione peroxidase 4 (GPX4), a selenium-containing enzyme, and associated proteins as critical for cold tolerance. We utilized genetic and pharmacological approaches to demonstrate that GPX4 is active in the cold and its catalytic activity is required for cold tolerance. Furthermore, we show that the role of GPX4 as a suppressor of cold-induced cell death extends across hibernating species, including 13-lined ground squirrels and greater horseshoe bats, highlighting the evolutionary conservation of this mechanism of cold tolerance. This study identifies GPX4 as a central modulator of mammalian cold tolerance and advances our understanding of the evolved mechanisms by which cells mitigate cold-associated damage—one of the most common challenges faced by cells and organisms in nature.
The cold-inducible RNA-binding protein RBM3 stabilises viral mRNA at the cooler temperatures of the upper respiratory tract.
Authors: Coutts Hannah L , Turkington Hannah L , Bonazza Stefano , Getty Erin M P E , Sukumar Swathi , Power Ultan F , Courtney David G
Published: July 2024

Temperature regulation is a key aspect of homeostasis, and several systems are involved in orchestrating adjustments in gene expression at the cellular level. One such factor is RBM3, a cold-inducible RNA-binding protein implicated in several aspects of mRNA processing and regulation. The upper respiratory tract serves as a unique environment regarding temperature regulation. Physiologically, the lower tract is relatively stable at 37°C, while the upper tract fluctuates at or below 33°C. Adapting to this temperature differential, and the subsequent differences in transcriptome and proteome, is essential for respiratory viruses that infect and cause disease in the lower tract while simultaneously replicating and transmitting from the upper tract. At present, our understanding of the molecular mechanisms underlying influenza virus infection at cooler temperatures is lacking. Unsurprisingly, RBM3 protein levels are highest in nasopharyngeal tissue. Coupled with its known role in positively regulating bound RNA, it is an appealing candidate for manipulation by respiratory viruses. We found siRNA knockdown of RBM3 significantly decreased viral replication. To disentangle the direct effect of RMB3 from the shift in global gene expression at colder temperatures, we generated an A549 cell line constitutively overexpressing RBM3 at 37°C. Overexpression resulted in a significant increase in levels of viral replication. RBM3 was found to readily bind viral NP mRNA during infection and prolong the half-life of these transcripts. In contrast, an RBM3 RNA binding null mutant reverses this phenotype, validating that direct interaction of RBM3 has a stabilising effect on viral transcripts. The proviral nature of increased RBM3 levels was further validated in the more clinically relevant model of well-differentiated primary nasal epithelial cells. These data suggest a role for RBM3 in supporting influenza virus replication in the nasopharyngeal tract. Understanding IAV replication in this environment could prove fundamental to elucidating the mechanisms of influenza transmission and reassortment.
Major change in swine influenza virus diversity in France owing to emergence and widespread dissemination of a newly introduced H1N2 1C genotype in 2020
Authors: Richard Gautier , Hervé Séverine , Chastagner Amélie , Quéguiner Stéphane , Beven Véronique , Hirchaud Edouard , Barbier Nicolas , Gorin Stéphane , Blanchard Yannick , Simon Gaëlle
Published: July 2024

Swine influenza A viruses (swIAV) are a major cause of respiratory disease in pigs worldwide, presenting significant economic and health risks. These viruses can reassort, creating new strains with varying pathogenicity and cross-species transmissibility. This study aimed to monitor the genetic and antigenic evolution of swIAV in France from 2019 to 2022. Molecular subtyping revealed a marked increase in H1avN2 cases from 2020 onwards, altering the previously stable subtypes’ distribution. Whole-genome sequencing and phylogenetic analyses of H1av(1C) strains identified ten circulating genotypes, including five new genotypes, marked by a significant predominance of the H1avN2#E genotype. It was characterized by an HA-1C.2.4, an N2-Gent/84, and internal protein-encoding genes belonging to a newly defined genogroup within the Eurasian avian-like (EA) lineage, the EA-DK subclade. H1avN2#E emerged in Brittany, the country’s most pig-dense region, and rapidly became the most frequently detected swIAV genotype across France. This drastic change in the swIAV lineages proportions at a national scale was unprecedented, making H1avN2#E a unique case for understanding swIAV evolution and spreading patterns. Phylogenetic analyses suggested an introduction of the H1avN2#E genotype from a restricted source, likely originating from Denmark. It spread rapidly with low genetic diversity at the start of the epizootic in 2020, showing increasing diversification in 2021 and 2022, and exhibiting reassortments with other enzootic genotypes. Amino acid sequence alignments of H1avN2#E antigenic sites revealed major mutations and deletions compared to vaccine 1C strain (HA-1C.2.2) and previously predominant H1avN1 strains (HA-1C.2.1). Antigenic cartography confirmed significant antigenic distances between H1avN2#E and other 1C strains, suggesting the new genotype escaped from the swine population preexisting immunity. Epidemiologically, the H1avN2#E virus exhibited epizootic hallmarks with more severe clinical outcomes compared to H1avN1 viruses. These factors likely contributed to the spread of H1avN2#E within the pig population. The rapid rise of H1avN2#E highlighted the dynamic nature of swIAV genetic and antigenic diversity, underscoring the importance of adapted surveillance programs to support risk assessment in the event of new outbreaks. This also demonstrate the need to strengthen biosecurity measures when receiving pigs in a herd and to limit trading of swIAV-excreting live swine between European countries.
Spotted hyena gut cross-talks: Symbionts modulate mucosal immunity
Authors: Soares Susana P. V. , Jarquín-Díaz Victor Hugo , Veiga Miguel M. , Karl Stephan , Czirják Gábor Á. , Weyrich Alexandra , Metzger Sonja , East Marion L. , Hofer Heribert , Heitlinger Emanuel , Benhaiem Sarah , Ferreira Susana C. M.
Published: July 2024

The intestinal mucosa is at the front line of host-microbiome interactions, but little is known about these interactions within natural populations. Here, we non-invasively investigated associations between the gut microbiome and mucosal immune measures while controlling for host, social, and ecological factors in 199 samples of 158 wild spotted hyenas (Crocuta crocuta) in the Serengeti National Park, Tanzania. We profiled the microbiome composition, including bacteria, fungi and parasites, using a multi-amplicon approach, and measured faecal immunoglobulin A and mucin. Probabilistic models indicated that both immune measures predict microbiome similarity among individuals in an age-dependent manner. The strength of the association effect varied, being strongest within bacteria, intermediate within parasites, and weakest within fungi communities. Machine learning regression accurately predicted both measures and identified the taxa driving these associations: symbiotic bacteria reported in humans and laboratory mice, unclassified bacteria, a hookworm, host DNA likely reflecting inflammation, and diet. Our findings indicate a complex interplay between the host, its environment and symbionts. These findings increase our knowledge of the gut microbiome in natural populations, which harbour highly dynamic and diverse eukaryotes under the influence of unpredictable environmental factors and where selection is not artificially biased.
Environmental stiffness regulates neuronal maturation via Piezo1-mediated TTR activity
Authors: Kreysing Eva , Gautier Hélène , Humphrey Robert , Mooslehner Katrin , Muresan Leila , Haarhoff Daniel , Mukherjee Sudipta , Zhao Xiaohui , Winkel Alexander , Dimitracopoulos Andrea , Pillai Eva K. , Káradóttir Ragnhildur Thóra , Franze Kristian
Published: July 2024

During brain development, neurons extend axons to connect to their target cells while initiating a maturation process, during which neurons start expressing voltage-gated ion channels, form synapses, express synaptic transmitters and receptors, and start communicating via action potentials. Little is known about external factors regulating this process. Here, we identified environmental mechanics as an important regulator of neuronal maturation, and a molecular pathway linking tissue stiffness to this process. Using patch clamp electrophysiology, calcium imaging and immunofluorescence, we found that neurons cultured on stiffer substrates showed a delay in voltage-gated ion channel activity, spontaneous and evoked action potentials, and synapse formation. RNA sequencing and CRISPR/Cas9 knockdown strategies revealed that the mechanosensitive ion channel Piezo1 supresses transthyretin (TTR) expression on stiffer substrates, slowing down synaptic receptor expression and consequently electrical maturation. Stiffening of brain tissue inXenopus laevisembryos also resulted in a significant delay of synaptic activityin vivo. Our data indicate that environmental stiffness represents a fundamental regulator of neuronal maturation, which is important for the development of normal circuitry in the brain, and potentially for neurodevelopmental disorders.
Exploring the impacts of human breast milk functional lipidome on infant health and growth outcomes in early life using lipid bioinformatics
Authors: Ganeshalingam Moganatharsa , Cheema Sukhinder , Wagner Carol L. , Pham Thu Huong , Enstad Samantha , Andrews Chloe , Grapov Dmitry , Esposito Flavia , Sen Sarbattama , Thomas Raymond
Published: July 2024

Human breast milk lipidome is complex, and how changes in the functional lipid metabolism converge systematically to alter infants’ health outcomes is poorly understood. We used human breast milk and infant-mother dyads as a test system to demonstrate how the application of improved lipid bioinformatics can be effective in discerning systematic changes in functional lipid metabolism providing novel discoveries of how lactational programming in early life can influence infant health and growth outcomes. The study consisted of 40 mother-infant dyads where breast milk, maternal diet, infant anthropometrics [fat mass index (FMI), length z score, BMI z score, fat-free mass index (FFMI)], and infant atopic disease outcome (ear infection, cold, wheezing, diarrhea, and eczema) were collected at one and four months postpartum. Integrated Lipid Bioinformatics analyses were conducted using XLSTAT, Metaboanalyst 5.0. R software, Lipid Search, Xcalibur, and Cytoscape software. The results showed breast milk lipidome ordinated into distinct clusters based on maternal BMI status, and differences in developmental and atopic disease outcomes following redundancy analysis. Specifically, lipids from obese mothers clustered with FMI and eczema, while lipids from non-obese mothers clustered with FFM and wheezing. Receiver operating analysis was effective in identifying potential lipid biomarkers that were significantly associated with infant FMI, FFMI, and eczema during early life. Sphingolipid and glycerophospholipid pathways were significantly associated with the altered breast milk lipidome impacting infant development and atopic disease outcome during the first year of life. The findings following the advanced lipid bioinformatics suggest that the breastmilk functional lipid metabolism appears to play a key role in lipid-mediated lactational programming influencing development and atopic disease outcome, and present opportunities for potential dietary intervention in early life.
Evolution of a central dopamine circuit underlies adaptation of light-evoked sensorimotor response in the blind cavefish,Astyanax mexicanus
Authors: Kozol RA , Canavan A , Tolentino B , Keene AC , Kowalko JE , Duboué ER
Published: July 2024

Adaptive behaviors emerge in novel environments through functional changes in neural circuits. While relationships between circuit function and behavior have been well studied, how evolution shapes those circuits and leads to behavioral adpation is poorly understood. The Mexican cavefish, Astyanax mexicanus, provides a unique genetically amendable model system, equipped with above ground eyed surface fish and multiple evolutionarily divergent populations of blind cavefish that have evolved in complete darkness. These differences in environment and vision provide an opprotunity to examine how a neural circuit is functionally influenced by the presence of light. Here, we examine differences in the detection, and behavioral response induced by non visual light reception. Both populations exhibit photokinetic behavior, with surface fish becoming hyperactive following sudden darkness and cavefish becoming hyperactive following sudden illumination. To define these photokinetic neural circuits, we integrated whole brain functional imaging with ourAstyanaxbrain atlas for surface and cavefish responding to light changes. We identified the caudal posterior tuberculum as the central modulator for both light or dark stimulated photokinesis. To unconver how spatiotemporal neuronal activity differed between surface fish and cavefish, we used stable pan-neuronal GCaMPAstyanaxtransgenics to show that a subpopulation of darkness sensitve neurons in surface fish are now light senstive in cavefish. Further functional analysis revealed that this integrative switch is dependent on dopmane signaling, suggesting a key role for dopamine and a highly conserved dopamine circuit in modulating the evolution of a circuit driving an essential behavior. Together, these data shed light into how neural circuits evolved to adapte to novel settings, and reveal the power ofAstyanaxas a model to elucidate mechanistic ingiths underlying sensory adaptation.

Abstract Figure
Evaluation of Methodologies in Anti-nephrin Autoantibody Detection
Authors: Liu Pan , Liu Shuping , Dalal Vidhi , Lane Jerome , Gessaroli Elisa , Forte Eleonora , Gallon Lorenzo , Jin Jing
Published: July 2024

Recent studies discovered the prominent presence of anti-nephrin autoantibodies in minimal change disease, steroid-sensitive nephrotic syndrome and/or post-transplant recurrent focal segmental glomerulosclerosis (FSGS). However, widely different, and often unconventional autoantibody detection methods were used among these studies, making it challenging to assess the pathogenic role for the antibodies. Here we examined methods of conventional ELISA, magnetic on-beads ELISA, immunoprecipitation-immunoblotting (IP-IB), and cell- and tissue-based antibody assays with 127 plasma samples of kidney and non-kidney diseases. On the antigen side, we compared commercially available recombinant human nephrin extracelluar domain (ECD) produced from human or mouse cell lines, as well as lab-made full length, ECD, and series of ECD truncates for measuring autoantibody reactivity and specificity. Surprisingly, different assay methods and different antigen preparations led to observation of assay-specific false-positive and false-negative results. In general, a set of tests that combines magnetic beads-enhanced ELISA, followed by IP-IB, and epitope mapping showed the most robust results for anti-nephrin autoantibodies, detected in two primary FSGS patients among all cases tested. It is interesting to note that cell/tissue-based results, also supported by antigen truncation studies, clearly suggest steric hindrance of reactive epitopes, as in full length nephrin that forms compact self-associated complexes. In conclusion, anti-nephrin positivity is rare among the tested patients (2/127), including those with FSGS (2/42), and autoantibody results can be affected by the choice of detection methods.
Coordinate control of the RNA polymerase II transcription cycle by CDK9-dependent, tripartite phosphorylation of SPT5
Authors: Sun Rui , Fisher Robert P.
Published: July 2024

The RNA polymerase II (RNAPII) transcription cycle is regulated throughout its duration by reversible protein phosphorylation. The elongation factor SPT5 contains two regions targeted by cyclin-dependent kinase 9 (CDK9) and previously implicated in promoter-proximal pausing and termination: the linker between KOWx-4 and KOW5 domains and carboxy-terminal repeat (CTR) 1, respectively. Here we show that phosphorylations in the KOWx-4/5 linker, CTR1 and a third region, CTR2, coordinately control pause release, elongation speed and RNA processing. Pausing was increased by mutations preventing CTR1 or CTR2 phosphorylation, but attenuated when both CTRs were mutated. Whereas mutating CTR1 alone slowed elongation and repressed nascent transcription, simultaneous mutation of CTR2 partially reversed both effects. Nevertheless, mutating both CTRs led to aberrant splicing, dysregulated termination and diminished steady-state mRNA levels, and impaired cell proliferation more severely than did either single-CTR mutation. Therefore, tripartite SPT5 phosphorylation times pause release and regulates RNAPII elongation rates positively and negatively to ensure productive transcription and cell viability.
Climatic determinants of plant phenology in vernal pool habitats
Authors: Hendrickson Brandon Thomas , Heckel Aubrie , Martin Robert , Sexton Jason
Published: July 2024

Vernal pool plants are small, colorful, and specialized to both desiccated and inundated conditions that distinguish the ephemeral wetlands in which they grow. These species germinate rapidly in response to the first rain and grow quickly to take advantage of available water supplies. The floral phenology of vernal pool plant species is little understood despite being a crucial developmental stage for producing seeds and determining population growth rates. The current study focuses on two vernal pool plants,Limnanthes douglasiissp.rosea(meadowfoam), a vernal pool specialist, andTrifolium variegatum(whitetip clover), a generalist vernal pool associate, and characterizes their phenology in response to interannual climate variation. We recorded phenology and climate data over seven years during a period of highly variable precipitation and temperature patterns, which serve as a robust dataset for quantifying the relationship of floral phenology with various climatic factors. We found that warmer and drier environmental conditions occurring during early growth periods were strongly associated with advanced floral phenology later in the life cycle for both species. Over the seven-year dataset, which was increasingly warm and dry, phenology advanced by 4.7 days per year for meadowfoam and 5.6 days per year for whitetip clover, respectively. The floral duration of the habitat specialist was influenced by microtopographic features of vernal pools, whereas no such patterns were observed for the habitat generalist. Finally, warmer and drier conditions were associated with reduced occupancy rates of both focal species within vernal pools. To our knowledge, this is the first study quantifying the relationship between vernal pool floral phenology and climate, offering insights into how phenology may shift in response to modern climate change.
Prediction of protein subcellular localization in single cells
Authors: Zhang Xinyi , Tseo Yitong , Bai Yunhao , Chen Fei , Uhler Caroline
Published: July 2024

The subcellular localization of a protein is important for its function and interaction with other molecules, and its mislocalization is linked to numerous diseases. While atlas-scale efforts have been made to profile protein localization across various cell lines, existing datasets only contain limited pairs of proteins and cell lines which do not cover all human proteins. We present a method that uses both protein sequences and cellular landmark images to performPredictions ofUnseenProteins’Subcellular localization (PUPS), which can generalize to both proteins and cell lines not used for model training. PUPS combines a protein language model and an image inpainting model to utilize both protein sequence and cellular images for protein localization prediction. The protein sequence input enables generalization to unseen proteins and the cellular image input enables cell type specific prediction that captures single-cell variability. PUPS’ ability to generalize to unseen proteins and cell lines enables us to assess the variability in protein localization across cell lines as well as across single cells within a cell line and to identify the biological processes associated with the proteins that have variable localization. Experimental validation shows that PUPS can be used to predict protein localization in newly performed experiments outside of the Human Protein Atlas used for training. Collectively, PUPS utilizes both protein sequences and cellular images to predict protein localization in unseen proteins and cell lines with the ability to capture single-cell variability.
Evolutionarily divergentMycobacterium tuberculosisCTP synthase filaments are under selective pressure
Authors: Lynch Eric M. , Lu Yao , Park Jin Ho , Shao Lin , Kollman Justin , Rego E. Hesper
Published: July 2024

The final and rate-limiting enzyme in pyrimidine biosynthesis, CTP synthase (CTPS), is essential for the viability ofMycobacterium tuberculosisand other mycobacteria. Its product, CTP, is critical for RNA, DNA, lipid and cell wall synthesis, and is involved in chromosome segregation. In various organisms across the tree of life, CTPS assembles into higher-order filaments, leading us to hypothesize thatM. tuberculosisCTPS (mtCTPS) also forms higher-order structures. Here, we show that mtCTPS does assemble into filaments but with an unusual architecture not seen in other organisms. Through a combination of structural, biochemical, and cellular techniques, we show that polymerization stabilizes the active conformation of the enzyme and resists product inhibition, potentially allowing for the highly localized production of CTP within the cell. Indeed, CTPS filaments localize near the CTP-dependent complex needed for chromosome segregation, and cells expressing mutant enzymes unable to polymerize are altered in their ability to robustly form this complex. Intriguingly, mutants that alter filament formation are under positive selection in clinical isolates ofM. tuberculosis, pointing to a critical role needed to withstand pressures imposed by the host and/or antibiotics. Taken together, our data reveal an unexpected mechanism for the spatially organized production of a critical nucleotide inM. tuberculosis, which may represent a vulnerability of the pathogen that can be exploited with chemotherapy.
Alleviation of salt stress in strawberries by hydrogen-rich water: physiological, transcriptomic and metabolomic responses
Authors: Wang Renyuan , Chu Shaohua , Zhang Dan , Zhang Xia , Chi Yaowei , Ma Xianzhong , Chen Xunfeng , Yang Haiyan , Ding Wenjiang , Zhao Ting , Ren Yongfeng , Yang Xijia , Zhou Pei
Published: July 2024

Abstract Figure

With the continuous advancement of climate change and human activities, cultivable land is becoming increasingly susceptible to the influence of salt ions. Therefore, it is crucial to mitigate the salt stress on plants through various approaches. In this study, strawberry seedlings were selected as the experimental plants, and a comprehensive transcriptomic and metabolomic analysis was conducted to elucidate the effects of hydrogen-rich water (HRW) on strawberries under salt stress. The results indicated that HRW significantly promoted plant growth, particularly increasing root biomass by 49.50%. Additionally, HRW regulated the levels of soluble sugars, malondialdehyde (MDA), and antioxidant enzymes, enhancing the cellular uptake of potassium ions and the expulsion of sodium ions. The levels of Ca2+and Mg2+in organelles increased by 2.06 and 2.45-fold, respectively. Transcriptomic analysis revealed that HRW substantially altered gene expression in strawberry roots; under salt stress, HRW up-regulated beneficial biological processes. Furthermore, genes related to ion absorption and transport, antioxidant enzymes, and cell wall biosynthesis were screened. Meanwhile, key common pathways were identified in differentially expressed metabolites (DEMs) and differentially expressed genes (DEGs) related to phenylpropane biosynthesis, alanine, aspartate, and glutamate metabolism, amino and nucleotide sugar metabolism, and galactose metabolism. A molecular mechanism for mitigating salt stress in strawberry seedlings by HRW was provided by the integrated approaches in this research, reflecting the potential applications of hydrogen gas in agriculture.
Spatially variable competition contributes to mismatched responses of plant fitness and occurrence to environmental gradients
Authors: Hayashi Kenji T. , Kraft Nathan J. B.
Published: July 2024

Species distributions have long been understood to depend on the complex interplay between the abiotic environment and biotic interactions. Empirical work in ecological communities has increasingly revealed how competition can mediate species’ demographic responses to environmental variation, but understanding how the demographic consequences of spatially variable competition manifest in observed distribution patterns remains an important challenge.

Here, we describe a conceptual framework for characterizing competition-driven mismatches between responses of fitness and occurrence to environmental gradients. We then explore these mismatches for eight annual plant species in an edaphically variable California grassland landscape.

We experimentally quantified how species’ demographic rates (germination rate, fecundity) and fitness respond to spatial variation in the soil environment, either in the presence or absence of naturally occurring neighbors. We also surveyed species’ occurrence across the study landscape. Combining these demographic and occurrence data, we asked whether observed occurrence patterns are congruent with responses of intrinsic fitness (i.e., fitness in the absence of competitors) to soil gradients.

We found that competition altered responses of fitness to the primary soil gradient (soil texture) for many (4/8) species. In turn, occurrence patterns were often poorly or even inversely related to responses of intrinsic fitness to this environmental axis. In contrast, we found that competition had relatively little effect on responses of fitness and occurrence to a secondary soil gradient (soil Ca:Mg).

Synthesis. We demonstrate that spatially variable competition can contribute to mismatched responses of fitness and occurrence to environmental variation. Importantly, these quantitative mismatches depend on the species and environmental gradient in question. Our results caution against assuming that variation in occurrence implies variation in intrinsic fitness (or vice versa) without first disentangling how the abiotic environment and competition impact the demographic processes that underlie species distributions.
Genetic variance in the murine defensin locus modulates glucose homeostasis
Authors: Masson Stewart W.C. , Simpson Rebecca C. , Cutler Harry B. , Carlos Patrick W. , Marian Oana C. , Potter Meg , Madsen Søren , Cooke Kristen C. , Craw Niamh R. , Fuller Oliver K. , Harney Dylan J. , Larance Mark , Cooney Gregory J. , Morahan Grant , Shanahan Erin R. , Hodgkins Christopher , Payne Richard J. , Stöckli Jacqueline , James David E.
Published: July 2024

Insulin resistance is a heritable risk factor for many chronic diseases; however, the genetic drivers remain elusive. In seeking these, we performed genetic mapping of insulin sensitivity in 670 chow-fed Diversity Outbred in Australia (DOz) mice and identified a genome-wide significant locus (QTL) on chromosome 8 encompassing 17 defensin genes. By taking a systems genetics approach, we ultimately identified alpha-defensin 26 (Defa26) as the causal gene in this region. To validate these findings, we synthesized Defa26 and performed diet supplementation experiments in two mouse strains with distinct endogenous Defa26 expression levels. In the strain with relatively lower endogenous expression (C57BL/6J) supplementation improved insulin sensitivity and reduced gut permeability, while in the strain with higher endogenous expression (A/J) it caused hypoinsulinemia, glucose intolerance and muscle wasting. Based on gut microbiome and plasma bile acid profiling this appeared to be the result of disrupted microbial bile acid metabolism. These data illustrate the danger of single strain over-reliance and provide the first evidence of a link between host-genetics and insulin sensitivity which is mediated by the microbiome.
Decoding RNA Metabolism by RNA-linked CRISPR Screening in Human Cells
Authors: Nugent Patrick J. , Park Heungwon , Wladyka Cynthia L. , Chen Katharine Y. , Bynum Christine , Quarterman Grace , Hsieh Andrew C. , Subramaniam Arvind Rasi
Published: July 2024

RNAs undergo a complex choreography of metabolic processes in human cells that are regulated by thousands of RNA-associated proteins. While the effects of individual RNA-associated proteins on RNA metabolism have been extensively characterized, the full complement of regulators for most RNA metabolic events remain unknown. Here we present a massively parallel RNA-linked CRISPR (ReLiC) screening approach to measure the responses of diverse RNA metabolic events to knockout of 2,092 human genes encoding all known RNA-associated proteins. ReLiC screens highlight modular interactions between gene networks regulating splicing, translation, and decay of mRNAs. When combined with biochemical fractionation of polysomes, ReLiC reveals striking pathway-specific coupling between growth fitness and mRNA translation. Perturbing different components of the translation and proteostasis machineries have distinct effects on ribosome occupancy, while perturbing mRNA transcription leaves ribosome occupancy largely intact. Isoform-selective ReLiC screens capture differential regulation of intron retention and exon skipping by SF3b complex subunits. Chemogenomic screens using ReLiC decipher translational regulators upstream of mRNA decay and uncover a role for the ribosome collision sensor GCN1 during treatment with the anti-leukemic drug homoharringtonine. Our work demonstrates ReLiC as a versatile platform for discovering and dissecting regulatory principles of human RNA metabolism.
Transfer RNA acetylation regulates in vivo mammalian stress signaling
Authors: Thalalla Gamage Supuni , Khoogar Roxane , Howpay Manage Shereen , Crawford McKenna C. , Georgeson Joe , Polevoda Bogdan V. , Sanders Chelsea , Lee Kendall A. , Nance Kellie D. , Iyer Vinithra , Kustanovich Anatoly , Perez Minervo , Thu Chu T. , Nance Sam R. , Amin Ruhul , Miller Christine N. , Holewinski Ronald J. , Meyer Thomas , Koparde Vishal , Yang Acong , Jailwala Parthav , Nguyen Joe T. , Andresson Thorkell , Hunter Kent , Gu Shuo , Mock Beverly A. , Edmondson Elijah F. , Difilippantonio Simone , Chari Raj , Schwartz Schraga , O’Connell Mitchell R. , Wu Colin Chih-Chien , Meier Jordan L.
Published: July 2024

Transfer RNA (tRNA) modifications are crucial for protein synthesis, but their position-specific physiological roles remain poorly understood. Here we investigate the impact of N4-acetylcytidine (ac4C), a highly conserved tRNA modification, using a Thumpd1 knockout mouse model. We find that loss of Thumpd1-dependent tRNA acetylation leads to reduced levels of tRNALeu, increased ribosome stalling, and activation of eIF2α phosphorylation. Thumpd1 knockout mice exhibit growth defects and sterility. Remarkably, concurrent knockout of Thumpd1 and the stress-sensing kinase Gcn2 causes penetrant postnatal lethality, indicating a critical genetic interaction. Our findings demonstrate that a modification restricted to a single position within type II cytosolic tRNAs can regulate ribosome-mediated stress signaling in mammalian organisms, with implications for our understanding of translation control as well as therapeutic interventions.
On the use of kinship and familiarity associated social information in mediatingDrosophila melanogasteroviposition decisions
Authors: Rakosy Emily , Talagala Sanduni , Long Tristan A.F.
Published: July 2024

Decisions where an individual lays their eggs are important, as the choice may affect their offspring's survival and lifetime reproductive success. Information produced by conspecifics can potentially be useful in decision-making as this "social information" may provide an energetically cheaper means of assessing oviposition site suitability rather than acquiring it personally. However, as not all public information may be equally beneficial, cues produced by kin may be especially valuable as they might signal suitable microenvironments, and are associated with other fitness advantages resulting from improved foraging success and/or a decreased risk of competition/cannibalism compared to sites where unrelated conspecifics are located. Using the fruit fly,Drosophila melanogaster, we explored whether public information use is associated with kin-based egg-laying decisions. Kinship is potentially recognized in several ways, including environmentally-associated proxy cues, so we explored whether there were biases in how focal females interacted with cues from conspecifics that differed in both genetic relatedness, and environmental "familiarity". In a series of inter-connected assays, we examined the behaviour of focal females that interacted with a choice of potential egg-laying substrates that differed in the manner of their prior conspecific exposure, and counted the offspring that eclosed from these different substrates. Sites that had exhibited cues produced by conspecific demonstrators were visited more, and yielded more focal offspring compared to unexposed substrates. Furthermore, patterns of bias in offspring production were consistent with ovipositing females exhibiting sensitivity to the kinship status of the prior substrate's occupants. The basis of the kinship categorization by ovipositing females appears to be based on phenotypes that reflect true genetic relatedness, but the nature of the social information can be affected by other factors. These results further highlight the potential usefulness ofD. melanogasteras a model to understand the evolution of social behaviour in the expression of decision-making.
A new regulator of sporulation sheds light on spore morphogenesis and ballistospory in mushroom-forming fungi
Authors: Hou Zhihao , Merényi Zsolt , Yang Yashu , Zhang Yan , Csernetics Árpád , Bálint Balázs , Hegedüs Botond , Földi Csenge , Wu Hongli , Kristóffy Zsolt , Ábrahám Edit , Miklovics Nikolett , Virágh Máté , Liu Xiao-Bin , Zsibrita Nikolett , Lipinszki Zoltán , Karcagi Ildiko , Gao Wei , Nagy László G.
Published: July 2024

Sporulation is the most widespread means of reproduction and dispersal in fungi. In the Basidiomycota, sexual spores are produced on specialised cells known as basidia, from which they are discharged forcibly by a powered process called ballistospory, the highest known acceleration in nature. However, the genetics of sporulation, in particular postmeiotic events related to spore morphogenesis and ballistospory, remain poorly known. Here, we explore the genetics of these processes, based on a new, highly conserved transcription factor, Sporulation-Related Regulator 1 (SRR1), and its putative downstream regulatory network. Reverse genetics ofSrr1in the model mushroomCoprinopsis cinereaand commercially produced oyster mushroom indicated a conserved role ofSrr1in sporulation across Agaricomycetes. RNA-Seq analysis and motif-based inference of a hypothetical SRR1 gene regulatory network allowed delimiting putative targets regulated by SRR1 in a direct and indirect manner. Using this network and comparative genomics, we identified genes associated with ballistospory, including a putative SRR1-target chitinase, which was found to be required for normal spore production and morphology. Overall, our study offers new insights into the genetic mechanisms governing postmeiotic spore morphogenesis and ballistospory in the Agaricomycetes.
A finite set of content-free pointers in visual working memory: MEG evidence
Authors: Yu Xinchi , Lau Ellen
Published: July 2024

Human visual working memory (VWM) is known to be capacity-limited, but the nature of this limit continues to be debated. Recent work has proposed that VWM is supported by a finite (∼ 3) set of content-free pointers, acting as stand-ins for individual objects and binding features together. Here, based on two visual working memory experiments (N=20 each) examining memory for simple and complex objects, we report a sustained MEG response over right posterior cortex whose magnitude tracks the core hypothesized properties of this system: load-dependent, capacity-limited and content-free. These results provide novel evidence for a finite set of content-free pointers underlying VWM.
PLK-1 regulates MEX-1 polarization in theC. eleganszygote
Authors: Kim Amelia J. , Miller Stephanie I. , Greiner Elora C. , Kettenbach Arminja N. , Griffin Erik E.
Published: July 2024

The one-cellC. elegansembryo undergoes an asymmetric cell division during which germline factors such as the RNA-binding proteins POS-1 and MEX-1 segregate to the posterior cytoplasm, leading to their asymmetric inheritance to the posterior germline daughter cell. Previous studies found that the RNA-binding protein MEX-5 recruits polo-like kinase PLK-1 to the anterior cytoplasm where PLK-1 inhibits the retention of its substrate POS-1, leading to POS-1 segregation to the posterior. In this study, we tested whether PLK-1 similarly regulates MEX-1 polarization. We find that both the retention of MEX-1 in the anterior and the segregation of MEX-1 to the posterior depend on PLK kinase activity and on the interaction between MEX-5 and PLK-1. Human PLK1 directly phosphorylates recombinant MEX-1 on 9 predicted PLK-1 sites in vitro, four of which were identified in previous phosphoproteomic analysis ofC. elegansembryos. The introduction of alanine substitutions at these four PLK-1 phosphorylation sites (MEX-1(4A)) significantly weakened the inhibition of MEX-1 retention in the anterior, thereby weakening MEX-1 segregation to the posterior. In contrast, mutation of a predicted CDK1 phosphorylation site had no effect on MEX-1 retention or on MEX-1 segregation. MEX-1(4A) mutants are viable and fertile but display significant sterility and fecundity defects at elevated temperatures. Taken together with our previous findings, these findings suggest PLK-1 phosphorylation drives both MEX-1 and POS-1 polarization during the asymmetric division of the zygote.
Adaptive resistance to FLT3 inhibitors is potentiated by ROS-driven DNA repair signalling
Authors: Staudt Dilana E. , Germon Zacary P. , Mannan Abdul , McLachlan Tabitha , Murray Heather C. , Duchatel Ryan J. , Thomas Bryce C. , Beitaki Tyrone , McEwen Holly P. , Persson Mika L. , Calvert Leah , Findlay Izac J. , Jackson Evangeline R. , Smith Nathan D. , Skerrett-Byrne David A. , Mossman David , Nixon Brett , De Iullis Geoffry , Douglas Alicia M. , Enjeti Anoop K. , Sillar Jonathan R. , Chamberlain Janis , Alvaro Frank , Wei Andrew H. , Connerty Patrick , Verrills Nicole , Dun Matthew D.
Published: July 2024

Alterations in the FMS-like tyrosine kinase 3 (FLT3) gene are the most frequent driver mutations in acute myeloid leukaemia (AML), linked to a high risk of relapse in patients with internal tandem duplications (FLT3-ITD). Tyrosine kinase inhibitors (TKIs) targeting the FLT3 protein are approved for clinical use, yet resistance often emerges. This resistance is mainly seen following the acquisition of additional point mutations in the tyrosine kinase domain (TKD), resulting in a double mutant FLT3-ITD/TKD, which sustains cell signalling and survival despite the presence of FLT3 inhibitors. Here, we developed a FLT3-mutant AML model with adaptive resistance to type II TKIs, sorafenib, and quizartinib byin vitrodrug selection. Through global multiomic profiling, we identified upregulation of proteins involved in reactive oxygen species (ROS) production, particularly NADPH-oxidases, driving cellular ’ROS-addiction’, with resistant cells relying on ROS for survival, and genome fidelity preserved by ATM-driven DNA repair. Transcriptomic analysis of adult and paediatric AML (pAML) patients identified high ATM expression as a biomarker for shorter median overall survival in both the de novo and relapsed settings. Inhibition of ATM with clinically relevant therapy WSD-0628 effectively killed TKI- and chemotherapy-resistant AML cellsin vitroand significantly extended the survival of mice with sorafenib- and quizartinib-resistant FLT3-ITD AMLin vivo. We propose a new treatment strategy to improve survival of patients who develop resistance to sorafenib and quizartinib, as well as relapsed and refractory pAML, exploiting resistance mechanisms to precision therapies and cell-intrinsic features of high-risk cases, highlighting a clinically relevant salvage strategy.
Optimized reporters for multiplexed detection of transcription factor activity
Authors: Trauernicht Max , Filipovska Teodora , Rastogi Chaitanya , van Steensel Bas
Published: July 2024

In any given cell type, dozens of transcription factors (TFs) act in concert to control the activity of the genome by binding to specific DNA sequences in regulatory elements. Despite their considerable importance in determining cell identity and their pivotal role in numerous disorders, we currently lack simple tools to directly measure the activity of many TFs in parallel. Massively parallel reporter assays (MPRAs) allow the detection of TF activities in a multiplexed fashion; however, we lack basic understanding to rationally design sensitive reporters for many TFs. Here, we use an MPRA to systematically optimize transcriptional reporters for 86 TFs and evaluate the specificity of all reporters across a wide array of TF perturbation conditions. We thus identified critical TF reporter design features and obtained highly sensitive and specific reporters for 60 TFs, many of which outperform available reporters. The resulting collection of “prime” TF reporters can be used to uncover TF regulatory networks and to illuminate signaling pathways.

HIGHLIGHTS

Systematic design and optimization of transcriptional reporters for 86 TFs

Characterization of TF-specific reporter design optimization rules

Evaluation of reporter TF-specificity across a wide array of TF perturbations

Identification of a collection of 60 “prime” TF reporters with optimized performance
Learning cell fate landscapes from spatial transcriptomics using Fused Gromov-Wasserstein
Authors: Huizing Geert-Jan , Peyré Gabriel , Cantini Laura
Published: July 2024

In dynamic biological processes such as development, spatial transcriptomics is revolutionizing the study of the mechanisms underlying spatial organization within tissues. Inferring cell fate trajectories from spatial transcriptomics profiled at several time points has thus emerged as a critical goal, requiring novel computational methods. Wasserstein gradient flow learning is a promising framework for analyzing sequencing data across time, built around a neural network representing the differentiation potential. However, existing gradient flow learning methods cannot analyze spatially resolved transcriptomic data.

Here, we propose STORIES, a method that employs an extension of Optimal Transport to learn a spatially informed potential. We benchmark our approach using three large Stereo-seq spatiotemporal atlases and demonstrate superior spatial coherence compared to existing approaches. Finally, we provide an in-depth analysis of axolotl neural regeneration and mouse gliogenesis, recovering gene trends for known markers asNptx1in neuron regeneration andAldh1l1in gliogenesis and additional putative drivers.
Global coordination of protrusive forces in migrating immune cells
Authors: Reis-Rodrigues Patricia , Canigova Nikola , Avellaneda Mario J. , Gaertner Florian , Vaahtomeri Kari , Riedl Michael , Merrin Jack , Hauschild Robert , Fukui Yoshinori , Garcia Alba Juanes , Sixt Michael
Published: July 2024

Efficient immune responses rely on the capacity of leukocytes to traverse diverse and complex tissues. To meet such changing environmental conditions, leukocytes usually adopt an amoeboid configuration, utilizing their forward-positioned nucleus as a probe to identify and follow the path of least resistance among pre-existing pores. We show that in dense environments, where even the largest pores preclude free passage, leukocytes switch polarity and position their nucleus behind centrosome and organelles. In this mesenchymal configuration, local compression of the cell body triggers assembly of a central F-actin pool, located between cell front and nucleus. Central actin pushes outward to transiently dilate a path for organelles and nucleus. Pools of central and front actin are tightly coupled and experimental depletion of the central pool enhances actin accumulation and protrusion formation at the cell front. Although this shifted balance speeds up cells in permissive environments, migration in restrictive environments is impaired, as the unleashed leading edge dissociates from the trapped cell body. Our findings establish an actin regulatory loop that balances path dilation with advancement of the leading edge to maintain cellular coherence.