bluuebunny/crossref_metadata_2025 huggingface dataset:
Mycobacterial infection uncovers plasticity of Kupffer cells
Authors: Neuber Jana , Lohrmann Florens , Wald Samuel , Göçer Merve , Lösslein Anne Kathrin , Obwegs David , Gres Vitka , Goldmann Torsten , Rogg Manuel , Schell Christoph , Preißl Sebastian , Sagar  , Henneke Philipp
Published: February 2025

Bona fideKupffer cells (KCs) are prenatally seeded and show unique functional and immunophenotypic features among tissue macrophages. They are considered as terminally differentiated, and adaptability in disease is attributed to recruited, monocyte-derived KCs. Here, we investigated the extent of KC plasticity and the impact of origin in mycobacterial infections that target macrophages and can persist for months. Fate-mapping combined with high-resolution imaging revealed the emergence of a unique, infection specific KC subset which downregulated the signature markers CLEC4F and VSIG4 (“KClow”). KClowwere derived frombona fideKCs and located exclusively to granuloma cores. In contrast, monocyte-derived macrophages were contained at the granuloma borders and contributed to this tissue reaction. ATAC and single-cell RNA sequencing identified a specific signature of KClowwith high antimycobacterial activity and specialization to a hypoxic microenvironment. Despite their fundamental deviation from the classical KC phenotype, KClowshowed remarkable adaptability, and were capable to return to a homeostatic-like KC state. Accordingly, mycobacterial infections unmask KCs as highly plastic cells, capable of responding to extreme environmental changes.
Ångström-resolution imaging of cell-surface glycans
Authors: Masullo Luciano A. , Almahayni Karim , Pachmayr Isabelle , Honsa Monique , Heinze Larissa , Fritsche Sarah , Grabmayr Heinrich , Jungmann Ralf , Möckl Leonhard
Published: February 2025

Glycobiology is rooted in the study of monosaccharides, Ångström-sized molecules that are the building blocks of intricate glycosylation patterns. Glycosylated biomolecules form the glycocalyx, a dense coat encasing every human cell with central relevance – among others – in immunology, oncology, and virology. In order to understand glycosylation function, visualizing its molecular structure is fundamental. However, the ability to visualize the molecular architecture of the glycocalyx has remained elusive. Techniques like mass spectrometry, electron microscopy, and fluorescence microscopy lack the necessary cellular context, specificity, and resolution. Here, we address these limitations by combining metabolic labeling with Ångström-resolution fluorescence microscopy, enabling the first-ever visualization of individual sugars within glycans on the cell surface. Our work provides unprecedented insights into the molecular architecture of the glycocalyx and constitutes the foundation for future explorations of its function in health and disease.
Mechanical comparison ofEscherichia colibiofilms with altered matrix composition: a study combining shear-rheology and microindentation
Authors: Siri Macarena , Sarlet Adrien , Ziege Ricardo , Zorzetto Laura , Sotelo Guzman Carolina , Amini Shahrouz , Hengge Regine , Blank Kerstin G. , Bidan Cécile M.
Published: February 2025

The mechanical properties of bacterial biofilms depend on the composition and micro-structure of their extracellular matrix (ECM), which constitutes a network of extracellular proteins and polysaccharide fibers. In particular,E. colimacrocolony biofilms were suggested to present tissue-like elasticity due to a dense fiber network consisting of amyloid curli and phosphoethanolamine-modified cellulose (pEtN-cellulose). To understand the contribution of these two main ECM components to the emergent mechanical properties ofE. colibiofilms, we performed shear-rheology and microindentation experiments on biofilms grown fromE. colistrains that produce different ECM. We measured that biofilms containing curli fibers are stiffer in compression than curli-deficient biofilms. We further quantitatively demonstrate the crucial contribution of pEtN-cellulose, and especially of the pEtN modification, to the stiffness and structural stability of biofilms when associated with curli fibers. To compare the differences observed between the two methods, we also investigated how the structure and mechanical properties of biofilms with different ECM compositions are affected by the sample preparation method used for shear-rheology. We found that biofilm homogenization, used prior to shear-rheology, destroys the macroscale structure of the biofilm while the microscopic ECM architecture may remain intact. The resulting changes in biofilm mechanical properties highlight the respective advantages and limitations of the two complementary mechanical characterization techniques in the context of biofilm research. As such, our work does not only describe the role of the ECM on the mechanical properties ofE. colibiofilms. It also informs the biofilm community on considering sample preparation when interpreting mechanical data of biofilm-based materials.
Steering the chain-elongating microbiome to specific medium-chain carboxylates with ethanol and lactate as co-electron donors: maximizing C8 or C6
Authors: Wang Han , Jeon Byoung Seung , Ortiz-Ardila Andres E. , Angenent Largus T.
Published: February 2025

Microbial chain elongation is a sustainable process to convert organic residues into valuable biochemicalsviaanaerobic fermentation. The operating conditions for the bioreactor and the ecological interactions among functional populations are crucial in controlling this process, but they have not been completely ascertained. Here, we unraveled two operating conditions (i.e., environmental factors):(1)the substrate ratio of ethanol and lactate as co-electron donors, and(2)the temperature, which affected both the product specificity (i.e., function) and microbial dynamics of chain-elongating microbiomes in a continuously fed bioreactor with product extraction. Specifically, we found that the increase in the substrate ratio of ethanol to lactate shifted the microbiomes towardn-caprylate (C8) production, while the slightly higher operating temperatures of 37°C or 42°C were advantageous ton-caproate (C6) production. We detected a core microbiome that was similar for all environmental conditions and the two bioreactors, consisting of populations fromSphaerochaetaspp.,Caproiciproducensspp., and Oscillospiraceae. Besides the core microbiome, we observed positive correlations between Erysipelaclostridiaceae UCG-004,Bacteroidesspp., Oscillospiraceae NK4A214, Rikenellaceae RC9, andPseudoclavibacterspp. withn-caprylate production. Similar populations compared to the core microbiome were positively correlated withn-caproate production. We showed that we can steer microbiomes toward a high specificity of certain medium-chain carboxylates.

Abstract Graphics

SYNOPSIS

Generating medium-chain carboxylates is a promising open-culture biotechnology production platform for converting organic waste streams into biofuels and chemicals.
An ensemble learning method for joint kernel association testing and principal component analysis on multiple kernels
Authors: Koh Hyunwook
Published: February 2025

In high-dimensional omics studies, researchers often conduct kernel association testing to power-fully detect the relationship of the genetic or microbial composition with human health or disease. Especially, in human microbiome studies, its dimension reduction analysis follows to visually represent complex microbiome data in a simple two- or three-dimensional coordinate space. However, various kernels exist, and they produce all different outcomes; hence, it is hard to interpret them all consistently. Then, omnibus testing has recently been a subject of intense investigation for a unified and powerful statistical inference. However, current omnibus tests are purely a test for significance producing only aP-value as their outcome with no related dimension reduction and visualization approach; hence, their utility is still limited. In this paper, I introduce an ensemble learning method, named as enKern, for joint kernel associating testing and principal component analysis on multiple kernels. enKern is based on a weight learning scheme that leverages complementary contributions from multiple kernels for powerful performance for various association patterns. I show that applying the weights to individual test statistics or individual kernels is equivalent, which in turn enables a visualization in a reduced dimensional coordinate space based on the weighted kernel to be matched with its original significance testing scheme. I demonstrate its use for human microbiomeβ-diversity analysis. I also demonstrate its outperformance in validity and power through simulation experiments. enKern is freely available in R computing environment athttps://github.com/hk1785/enkern.
Cocaine extended access gates compulsive-like seeking in rats
Authors: Tapia-Pacheco Mónica , de Peretti Jeanne-Laure , Vignal Lucie , Williams Maya , Baunez Christelle , Goaillard Jean-Marc , Degoulet Mickael
Published: February 2025

When does compulsive-like drug seeking emerge? Despite decades of research, and critical advances in our understanding of brain processes leading to addiction, this question remains widely unanswered. So far, behavioral models assessing compulsive-like cocaine seeking following an extended access to cocaine failed to capture the development of its compulsive seeking. In fact, compulsive-like animals immediately displayed pathological seeking when facing its negative consequences of drug seeking, usually materialized by an unescapable mild electric shock on the paws. Here, we designed a new task, ‘Punished Seeking during Extended access’ (PSE), by inserting punished seeking trials within the sessions of extended access to cocaine. We show that compulsive-like cocaine seeking progressively emerges after several PSE sessions, once cocaine intake has been escalated. We thus provide the addiction community a pertinent model to explore brain mechanisms sustaining the emergence of compulsive-like cocaine seeking.
Coralysis enables sensitive identification of imbalanced cell types and states in single-cell data via multi-level integration
Authors: Sousa António G.G. , Smolander Johannes , Junttila Sini , Elo Laura L.
Published: February 2025

Current state-of-the-art integration methods for single-cell transcriptomics often struggle with imbalanced cell types across heterogeneous datasets, particularly when the datasets include similar but unshared cell types. Here, we introduce Coralysis, an R package featuring a multi-level integration algorithm to overcome these challenges. Coralysis enables sensitive integration, reference-mapping, and cell state identification across single-cell datasets, demonstrating consistent performance across diverse single-cell RNA-seq integration tasks and outperforming state-of-the-art methods when similar cell types are unevenly distributed across batches or completely absent from some datasets. Beyond single-cell transcriptomics, Coralysis enables the integration of rare cell populations from single-cell proteomic assays, such as basophils (0.5%) from whole blood. It also accurately predicts cell type identities across various query-reference scenarios. For instance, it successfully reclassifies CD16+ monocytes and natural killer cells that were previously misclassified as CD14+ monocytes and cytotoxic T cells in peripheral blood mononuclear cells. Finally, a key feature of Coralysis is its ability to provide probability scores that enable identifying both transient and steady cell states along with their differential expression programs. Overall, Coralysis facilitates the study of subtle biological variation and its dynamics by improving the integration of imbalanced cell types and states, enabling a more faithful representation of the cellular landscape in complex single-cell experiments.
Cross-Species Biomechanical Determinants of Shape Diversity
Authors: Bailleul Richard , Cuny Nicolas , Khoromoskaia Diana , Basu Soham , Bergamini Giulia , Cucurachi Paolo , Rupp Sebastian , Guse Annika , Curantz Camille , Swinhoe Natalie , Cleves Phillip , Craggs Jamie , Fujita Sosuke , Nakajima Yuichiro , Steenbergen Petrus , Diz-Munoz Alba , Salbreux Guillaume , Ikmi Aissam
Published: February 2025

How complex molecular mechanisms translate into diverse multicellular shapes remains unclear. By leveraging the bi-layered architecture of six cnidarian species that diverged 500 million years ago, we show that modularity in supracellular mechanics governs larval shape diversity. Using active surface theory, quantitative imaging, and an inducible genetic system, we identify species-specific variations in three biomechanical modules. Basally aligned stress fibers drive axial elongation, while oral geometry and aboral rigidity define shape polarity. Remarkably, manipulating these modules transforms one species' shape into another, demonstrating the causal relationship between module variation and shape diversity. Our analysis also uncovers instances of mechanical redundancies, where distinct module combinations generate similar shapes. These findings provide a general framework for how molecular complexity funnels into mesoscale mechanical determinants shaping morphological diversity.
High-throughput behavioural phenotyping of 25C. elegansdisease models including patient-specific mutations
Authors: O’Brien Thomas J. , Navarro Eneko P. , Barroso Consuelo , Menzies Lara , Martinez-Perez Enrique , Carling David , Brown André E. X.
Published: February 2025

Genetic diagnosis is fast and cheap, challenging our capacity to evaluate the functional impact of novel disease-causing variants or identify potential therapeutics. Model organisms includingC. eleganspresent the possibility of systematically modelling genetic diseases, yet robust, high-throughput methods have been lacking. Here we show that automated multi-dimensional behaviour tracking can detect phenotypes in 25 newC. elegansdisease models spanning homozygous loss-of-function alleles and patient-specific single-amino-acid substitutions. We find that homozygous loss-of-function (LoF) mutants across diverse genetic pathways (including BORC, FLCN, and FNIP-2) exhibit strong, readily detectable abnormalities in posture, locomotion, and stimulus responses compared to wild-type animals. Ansmc-3mutant strain—modelled by introducing a patient-identified missense change—causes developmental anomalies and distinct behavioural profiles even though complete loss of SMC-3 is lethal. In contrast, patient-derived missense mutations in another essential gene,tnpo-2, did not show a strong phenotype initially but it could be “sensitized” chemically (e.g., with aldicarb), potentially facilitating future drug screens. Our findings show that scalable behavioural phenotyping can capture a wide range of mutant effects—from strong to subtle—in patient-avatar worm lines. We anticipate that this standardized approach will enable systematic drug repurposing for rare genetic disorders as new disease variants are discovered.
Defining the limits of differential transpiration
Authors: Sinha Ranjita , Peláez-Vico María Ángeles , Fritschi Felix B. , Mittler Ron
Published: February 2025

Differential transpiration is a newly discovered acclimation strategy of annual plants to a combination of water deficit (WD) and heat stress (HS). Under these conditions (i.e.,WD+HS), transpiration of vegetative tissues is suppressed in plants such as soybean and tomato, while transpiration of reproductive tissues is not (termed ‘Differential Transpiration’; DT). This newly discovered acclimation process enables the cooling of reproductive organs under conditions of WD+HS, limiting HS-induced damage to plant reproduction. However, at what temperature and WD extremes will this process be active and functional at reducing the internal temperature of reproductive tissues, and at what developmental stages of the plant is it activated, remain unknown. Here, we report that DT occurs at most nodes (leaf developmental stages) of soybean plants subjected to WD+HS, and that it can function under extreme conditions of WD+HS (i.e.,18% of field water capacity and 42°C combined). Our findings reveal that DT is an effective acclimation strategy that protects reproductive processes from extreme conditions of WD+HS, at almost all developmental stages. In addition, our findings suggest that under field conditions DT could also be active in plants subjected to low or mild levels of WD during a heat wave.
The fluid genomic organisation of jingmenviruses
Authors: Valle Coralie , Parry Rhys H. , Coutard Bruno , Colmant Agathe M.G.
Published: February 2025

Jingmenviruses are a distinct group of flavi-like viruses characterized by a genome consisting of four to five segments. Here, we report the discovery of three novel putative jingmenviruses, identified by mining publicly available metagenomics data from mosquito and arachnid samples. Strikingly, these novel jingmenvirus sequences contain up to six genomic segments, with pairs of homologous segments coding for putative structural proteins. Following this discovery, we found an additional homologous segment for two other jingmenvirus genomes, which had gone unnoticed in the initial publications. The presence of a single version of the segments coding for non-structural proteins suggests we have indeed identified jingmenviruses with infectious units that contain up to six segments. We compared these novel jingmenvirus sequences to published sequences, in particular the segments with multiple open reading frames, and we propose that the putative translation initiation mechanisms involved for these segments are ribosomal frameshift resulting in the fusion of open reading frames and leaky scanning for overlapping ORFs. These putative mechanisms, conserved for all jingmenvirus sequences analysed, including in homologous segments, require biological confirmation. We also generated structural models of two putative structural proteins in the duplicated segments, and the corresponding alignments enabled us to confirm or identify the homologous relationship between sequences that shared limited nucleotide or amino acid identity. Altogether, these results highlight the fluid nature of jingmenviruses, which is a hallmark of multipartite viruses. Different combinations of segments packaged in different virus particles could facilitate the acquisition or loss of genomic segments and a segment duplication following genomic drift. Our data therefore contribute to the evidence of the multipartite nature of jingmenviruses and the evolutionary role this organisation may play.
VirMake: a flexible and user-friendly pipeline for viral taxonomic and functional characterisation from shotgun metagenomic sequencing data
Authors: Birkeland Einar , Istvan Paula , Avershina Ekaterina , Rognes Torbjørn , Rounge Trine B
Published: February 2025

Background

Bacteriophages (phages) are recognized as key regulators of microbial communities, and it is pivotal to understand viral ecology, diversity, and evolution. However, identification and characterization of phages from shotgun metagenome sequencing pose unique bioinformatic challenges owing to the inherent complexity and scale of the data.

Results

Here, we introduce VirMake, a comprehensive, flexible, and scalable pipeline developed for viral taxonomic and functional analysis of shotgun metagenome data. It enables high-throughput end-to-end processing, assembly, and annotation of metagenomic data, including viral sequence identification, taxonomic and functional annotation, and using a modular framework, allows users to integrate viral analysis at different stages of metagenome data analysis. VirMake can be installed on Linux operating systems that support Conda and is freely available athttps://github.com/Rounge-lab/VirMake. Here, we showcase VirMake capabilities using three metagenome datasets of varying sizes (up to 2.9 Tbp), generated from various environments (human gut and water) and using different sample preparation protocols (VLP enrichment and bulk DNA extraction).

Conclusions

VirMake enabled robust processing of all datasets, showing viral genome richness, taxonomy and integration status to vary according to sampling environments and laboratory protocols.
Integrated multi-omics profiling reveals the role of the DNA methylation landscape in shaping biological heterogeneity and clinical behaviour of metastatic melanoma
Authors: Anichini Andrea , Caruso Francesca P. , Lagano Vincenzo , Noviello Teresa M.R. , Tufano Rossella , Nicolini Gabriella , Molla Alessandra , Bersani Ilaria , Sgambelluri Francesco , Covre Alessia , Lofiego Maria F. , Coral Sandra , Di Giacomo Anna Maria , Simonetti Elena , Valeri Barbara , Cossa Mara , Ugolini Filippo , Simi Sara , Massi Daniela , Milione Massimo , Maurichi Andrea , Patuzzo Roberto , Santinami Mario , Maio Michele , Ceccarelli Michele , Mortarini Roberta
Published: February 2025

The biological and clinical relevance of the DNA methylation landscape in metastatic melanoma (MM) remains underexplored. In a retrospective cohort of 191 MM lesions from 165 AJCC Stage III and IV patients (EPICA cohort) we identified four tumor subsets (i.e. DEMethylated, LOW, INTermediate and CIMP) with progressively increasing levels of DNA methylation. These findings were validated in the TCGA MM. In EPICA, patients with LOW methylation tumors exhibited a significantly longer survival and a lower progression rate to more advanced AJCC stages, compared to patients with CIMP tumors. Furthermore, in an independent adjuvant immune checkpoint blockade MM cohort, patients with DEM/LOW pre-therapy lesions showed significantly longer relapse-free survival compared to those with INT/CIMP lesions. RNA-seq data analysis revealed that LOW and CIMP EPICA tumors showed opposite activation of master molecules influencing prognostic target genes, and differential expression of immunotherapy response and melanoma differentiation signatures. Compared to CIMP tumors, LOW lesions showed enrichment for pre-exhausted and exhausted T cells and more frequent retention of HLA Class I antigens. The differentiation and immune-related transcriptional features associated with LOW vs CIMP lesions were tumor-intrinsic programs retained in-vitro by melanoma cell lines. Consistently, treatment of differentiated melanoma cell lines with a DNMT inhibitor induced global DNA de-methylation, promoted de-differentiation and upregulated viral mimicry and IFNG predictive signatures of immunotherapy response. These findings underscore the role of DNA methylation in driving MM biological and clinical heterogeneity and support exploration of methylome targeting strategies for precision immunotherapy in melanoma.
Long-read detection of transposable element mobilization in the soma of hypomethylatedArabidopsis thalianaindividuals
Authors: Movilli Andrea , Sushko Svitlana , Rabanal Fernando , Weigel Detlef
Published: February 2025

Because transposable elements (TEs) can cause heritable genetic changes, past work on TE mobility inArabidopsis thalianahas mostly focused on new TE insertions in the germline of hypomethylated plants. It is, however, well-known that TEs can also be active in the soma, although the high-confidence detection of somatic events has been challenging. Here, we leveraged the high accuracy of PacBio HiFi long reads to evaluate the somatic mobility of TEs in individuals of anA. thaliananon-reference strain lacking activity of METHYLTRANSFERASE1 (MET1), a major component of the DNA methylation maintenance machinery. Most somatically mobile families coincided with those found in germline studies of hypomethylated genotypes, although the exact TE copies differed. We also discovered mobile elements that had been missed by standard TE annotation methods. Somatic TE activity was variable among individual plants, but also within TE families. Finally, our approach pointed to the possible involvement of alternative transposition as a cause for somatic hypermutability in a region that contains two closely spaced VANDAL21 elements. We conclude that long-read sequencing can reveal widespread TE transposition in the soma ofA. thalianahypomethylated mutants. Assessing somatic instead of germline mobilization is a fast and reliable method to investigate different aspects of TE mobility at the single plant level.
Computing pathogenicity of mutations in human cytochrome P450 superfamily
Authors: Mondal Somnath , Shrivastava Pranchal , Mehra Rukmankesh
Published: February 2025

Cytochrome P450 (CYP) are heme-containing enzymes involved in the metabolism of drugs and endogenous compounds. Humans have 57 known CYPs, with >200 mutations linked to severe disorders, highlighting their role in disease etiology. To investigate their pathogenicity, we performed an in-depth computational study, comparing pathogenic mutations with non-pathogenic ones. First, we evaluated the effects of all possible mutations across 26 known CYP’s structures using five structure-and five sequence-based methods, aiming to reproduce pathogenesis. We computed 23,46,410 mutations, categorized into: all possible, non-pathogenic, and pathogenic. Comparisons consistently revealed a meaningful stability pattern: non-pathogenic > all > pathogenic. Second, we found that a significantly higher number of pathogenic mutations were buried in CYP structures, relating to their higher pathogenesis potential. Third, we analyzed mutation allele frequency relative to solvent accessibility. Fourth, we computed change in 48 amino acid properties upon mutation and identified three distinguishing features: Gibbs free energy, isoelectric point and volume. Fifth, the positive residue content was reduced significantly in diseased mutations, with arginine mutations being the main culprit, directly linked to isoelectric point change. Sixth, we found a higher propensity for pathogenic mutations in conserved sites, suggesting disruption of CYP function. Finally, analysis of heme versus substrate sites showed a higher frequency of pathogenic mutations in heme site, with arginine being the main mutating residue, possibly disrupting arginine-heme interactions. We provide the first comprehensive analysis of mutation effects across multiple CYPs. We model the chemical basis of CYP-related pathogenicity, paving way for a semi-quantitative model to predict diseases.
Computation-through-Dynamics Benchmark: Simulated datasets and quality metrics for dynamical models of neural activity
Authors: Versteeg Christopher , McCart Jonathan D. , Ostrow Mitchell , Zoltowski David M. , Washington Clayton B. , Driscoll Laura , Codol Olivier , Michaels Jonathan A. , Linderman Scott W. , Sussillo David , Pandarinath Chethan
Published: February 2025

A primary goal of systems neuroscience is to discover how ensembles of neurons transform inputs into goal-directed behavior, a process known as neural computation. A powerful framework for understanding neural computation uses neural dynamics – the rules that describe the temporal evolution of neural activity – to explain how goal-directed input-output transformations occur. As dynamical rules are not directly observable, we need computational models that can infer neural dynamics from recorded neural activity. We typically validate such models using synthetic datasets with known ground-truth dynamics, but unfortunately existing synthetic datasets don’t reflect fundamental features of neural computation and are thus poor proxies for neural systems. Further, the field lacks validated metrics for quantifying the accuracy of the dynamics inferred by models. The Computation-through-Dynamics Benchmark (CtDB) fills these critical gaps by providing: 1) synthetic datasets that reflect computational properties of biological neural circuits, 2) interpretable metrics for quantifying model performance, and 3) a standardized pipeline for training and evaluating models with or without known external inputs. In this manuscript, we demonstrate how CtDB can help guide the development, tuning, and troubleshooting of neural dynamics models. In summary, CtDB provides a critical platform for model developers to better understand and characterize neural computation through the lens of dynamics.
Pannagram: unbiased pangenome alignment and the Mobilome calling
Authors: Igolkina Anna A. , Bezlepsky Alexander D. , Nordborg Magnus
Published: February 2025

Pannagram is a toolkit for unbiased pangenome alignment and Mobilome detection based on full-genome assemblies. It consists of two components: a command-line interface for performing the multiple genome alignment in different modes, extracting features such as structural variants (SVs), and searching sequences between datasets; and an R library that provides tools for analyzing alignments and SVs, as well as visualizing every step of the Mobilome analysis pipeline. As a proof of concept, we applied Pannagram to 12Cucumis sativusgenomes, identified SVs, and constructed the graph of nestedness for these variants. Within this graph, we identified families of actually mobile elements belonging to known transposable element families (LINEs, LTRs, and TIRs) and uncovered candidates for a novel type of mobile elements. Our results highlight the power of Pannagram in Mobilome discovery while ensuring an unbiased approach to pangenome analysis.
Exploring ID4 as a Driver of Aggression and a Therapeutic Target in Triple-Negative Breast Cancer
Authors: Toro C , Real S , Laurito S , Branham MT
Published: February 2025

Basal-like breast cancer (BLBC) is characterized by an aggressive clinical course, high genomic instability, and limited therapeutic options. The Inhibitor of Differentiation 4 (ID4) protein has been identified as a critical regulator of BLBC, where its overexpression correlates with poor prognosis. However, the mechanistic contributions of ID4 to BLBC tumorigenesis remain incompletely understood. In this study, we employed an integrative approach combining CRISPR-Cas9-mediated ID4 knockout, small-molecule inhibition, in vivo tumor modeling, and in silico transcriptional analyses to investigate the functional role of ID4 in BLBC.

CRISPR-Cas9-mediated knockout of ID4 in MDA-MB-231 cells resulted in significant reductions in proliferation, colony formation, and Ki67 expression, indicating a loss of aggressive phenotypic traits. In vivo xenograft studies further revealed that ID4-silenced cells exhibited markedly delayed tumor formation and a significant reduction in metastatic potential compared to controls. Kaplan-Meier survival analysis of basal-like tumors from The Cancer Genome Atlas (TCGA) dataset demonstrated that patients with low ID4 expression had improved relapse-free survival.

Gene set enrichment analysis (GSEA) of BLBC tumors stratified by ID4 expression revealed a shift toward luminal-like transcriptional programs in the ID4-low subgroup, including increased estrogen response and inflammatory signaling pathways. Furthermore, transcription factor activity analysis identified the activation of MYC, JUN, and STAT in ID4-low tumors, suggesting a transition toward a more differentiated phenotype. Finally, pharmacological inhibition of ID4 using the small-molecule degrader AGX51 effectively reduced proliferation in TNBC cells, highlighting ID4 as a potential therapeutic target.

Together, these findings establish ID4 as a key driver of BLBC aggressiveness and suggest that its inhibition may represent a viable therapeutic strategy. This study provides compelling evidence supporting the development of ID4-targeted therapies for TNBC patients, with the potential to improve clinical outcomes in this challenging disease subset.
Opposing Functions of Distinct Regulatory T Cell Subsets in Colorectal Cancer
Authors: Huang Xiao , Feng Dan , Mitra Sneha , Andretta Emma S. , Hooshdaran Nima B. , Ghelani Aazam P. , Wang Eric Y. , Frost Joe N. , Lawless Victoria , Vancheswaran Aparna , Jiang Qingwen , Leslie Christina S. , Rudensky Alexander Y.
Published: February 2025

Regulatory T (Treg) cells contribute to solid organ cancer progression, except in colorectal cancer (CRC) despite being abundantly present. Here, we demonstrate that two distinct tumoral IL-10⁺ and IL-10⁻ Treg cell subsets exert opposing functions by counteracting and promoting CRC tumor growth, respectively. The tumor restraining activity of IL-10⁺ Treg cells was mediated by their suppression of effector CD4 T cell production of IL-17, which directly stimulates CRC tumor cell proliferation. Consistently, IL-10⁻ Treg cells were more abundant in both mouse and human CRC tumors than in tumor-adjacent normal tissues, whereas IL-10+Treg cells exhibited the opposite distribution. Furthermore, relative abundance of IL-10⁺ and IL-10⁻ Treg cells correlated with better and worse disease prognoses in human CRC, respectively. This functional dichotomy between Treg cell subsets provides a rationale for therapeutic strategies to selectively target pro-tumoral Treg cells while preserving their anti-tumoral counterparts across barrier tissue cancers that harbor both subsets.
Genomic insights reveal community structure and phylogenetic associations of endohyphal bacteria and viruses in fungal endophytes
Authors: Escudero-Leyva E. , Belle M. , Dadkhahtehrani A. , Culver J.N. , Araya-Salas M. , Kutza J.P. , Goldson N. , Chavarría M. , Chaverri P.
Published: February 2025

Background

Endohyphal microbiomes, comprising bacteria and viruses, significantly influence fungal phenotypes, host fitness, and ecological interactions. Endohyphal bacterial symbionts are known to affect fungal pathogenicity, secondary metabolite production, and adaptability, yet many aspects of their diversity and interactions remain uncertain. In contrast, endohyphal viruses, despite their widespread presence, are poorly understood in terms of their diversity, ecological roles, and evolutionary relationships with fungal hosts. These gaps highlight the need for integrative studies to explore the composition, diversity, host associations, and functional roles of both bacterial and viral communities in fungi. This study aimed to (1) characterize the diversity of endohyphal bacterial and viral communities within selected fungal endophytes using genomic and transcriptomic approaches, and (2) test for host specialization through phylogenetic signals and core microbial taxa.

Results

Endohyphal microbial communities from 19 fungal isolates from eight fungal orders (Amphisphaeriales, Botryosphaeriales, Diaporthales, Glomerellales, Mucorales, Pleosporales, Sordariales, andXylariales) obtained fromFagus grandifolialeaves were characterized. Bacterial communities exhibited high diversity and significant phylogenetic signals, with core taxa such asBacillales, Burkholderiales, Enterobacterales, Hyphomicrobiales, andPseudomonadales, shared across all fungal samples. Specific bacterial taxa displayed potential host specialization, includingMoraxellales, Sphingomonadales, andStreptosporangiaceaeforAmphisphaerialesfungal samples;Enterobacterales(e.g.,Enterobacteraceae),Hyphomicrobiales(e.g.,Rhizobiaceae), andMicrococcalesforGlomerellales; andCytophagalesforDiaporthales. Viral communities were less diverse, withBamfordviraeandHeunggongvirae(double-stranded DNA viruses) identified as core taxa in metagenomic data, while metatranscriptomic data revealed no core taxa. Surprisingly, only a few reads of double-stranded RNA mycoviruses were detected.

Conclusions

The findings suggest a level of host specialization in bacterial communities and a more complex, limited association for viral communities, with dsDNA dominating the endohyphal virome. This study enhances our understanding of fungal-microbe interactions and highlights the ecological and evolutionary dynamics of endohyphal microbiota. Future efforts to expand reference databases and investigate the functional roles of these microbial taxa will further elucidate their contributions to fungal biology, impacts on their plant hosts, and ecosystem processes.
Divergent Hox cluster collinearity in horned beetles reveals adult head patterning function of labial
Authors: Nadolski Erica M , Manley Isabel G , Gill Sukhmani , Moczek Armin P
Published: February 2025

Hox genes play critical roles in specifying the regionalization of the body axis across metazoa, with the exception of the anterior dorsal head of bilaterian animals, which instead is instructed by a deeply conserved set of non-Hox regulators. The anterior dorsal head is also a hot spot of evolutionary diversification, raising the question as to the developmental-genetic underpinnings of such innovation. Onthophagine dung beetles develop evolutionarily novel and highly diversified horns on the dorsal head used as weapons during intrasexual conflicts. Preliminary RNAseq data unexpectedly documented Hox gene expression in the dorsal head of premetamorphic onthophagine larvae. Motivated by this observation, we aimed to (i) investigate the genomic content and arrangement of the Hox cluster across three onthophagine species, and (ii) assess expression patterns and (iii) potential functions of the anterior Hox genes labial, proboscipedia, and Deformed in patterning the adult beetle head and cephalic horns. We document an unexpected, derived Hox cluster configuration in the Onthophagus sagittarius genome, a species with apomorphic cephalic horn morphology. Yet despite this genomic rearrangement, embryonic expression patterns of labial and proboscipedia as well as the adult segment patterning functions of proboscipedia and Deformed were found to be conserved. In contrast, labial RNAi revealed an adult head patterning function outside horn-forming regions previously undescribed for any insect. Lastly, we show that electrosurgical ablation of the presumptive larval labial-expressing head region phenocopies this conspicuous adult labial RNAi defect. We discuss the implications of these data for current models of insect head development and diversification.
Xylazine potentiates the interoceptive effects of fentanyl in male and female rats
Authors: Bender Brooke N. , Carew Joseph M. , Bedard Madigan L. , McElligott Zoe A. , Besheer Joyce
Published: February 2025

Rationale

Xylazine, a sedative typically used in veterinary medicine, has been increasingly detected as an adulterant in the unregulated opioid supply and present in opioid overdose deaths. Therefore, xylazine-adulterated fentanyl is a growing public health concern. People who use drugs have reported that xylazine changes and prolongs the effects of fentanyl

Objectives

We used standard operant drug discrimination procedures to better understand how xylazine impacts the discriminative stimulus/interoceptive effects of fentanyl.

Methods

Male and female Long-Evans rats (n=23) were trained to discriminate fentanyl (0.032 mg/kg intraperitoneal) such that one lever was reinforced with sucrose on days when fentanyl was administered, and the other lever was reinforced when vehicle was administered. Once rats met testing criteria, we tested a dose range of fentanyl to confirm discriminative stimulus control, then we tested if xylazine alone produced fentanyl-like effects and if the addition of xylazine to fentanyl impacted fentanyl interoceptive effects.

Results

Stimulus control was confirmed, as rats showed increased percent responses on the fentanyl-appropriate lever as well as decreased response rates for increasing doses of fentanyl. Xylazine alone did not substitute for the stimulus effects of fentanyl but produced similar response rate reductions as fentanyl alone. Xylazine co-administered with fentanyl potentiated the stimulus effects of lower doses of fentanyl in both males and females and potentiated response rate reductions.

Conclusions

These results indicate that xylazine enhances the interoceptive effects of fentanyl, which may inform clinical research about xylazine-adulterated fentanyl.
A piezoelectric electroporator (Piezopen) for enhanced “naked” RNA vaccine delivery
Authors: Michalaki Eleftheria , Van Zanten Ananya , Najjar Jade , Byagathvalli Gaurav
Published: February 2025

Despite the success of COVID-19 mRNA vaccines, they still face challenges with high costs, complex manufacturing, off-target biodistribution, and systemic reactogenicity stemming from their inflammatory carriers: lipid nanoparticles (LNPs). While “naked” RNA delivery could in principle solve these issues, studies have suggested that it is infeasible due to rapid degradation by RNases and poor cellular entry, thereby necessitating formulations that enhance intracellular delivery and RNA stability. Now, we challenge this paradigm by showing that a simple and inexpensive (<$1), lighter-derived electroporator with microneedle electrodes (Piezopen) can augment gene expression and immunogenicity to naked mRNA leading to comparable responses to LNPs at low doses. We achieve robust responses in the absence of systemic inflammation and reactogenicity using skin-targeted delivery, administer diverse construct types (i.e., mRNA, self-amplifying RNA (saRNA), circular RNA (circRNA)), and demonstrate cross-species validation in live human skin to derisk subsequent clinical application. Our results introduce Piezopen as an inexpensive, well-tolerated, and efficacious alternative to LNPs for mRNA vaccine delivery, designed to facilitate routine vaccinations and pandemic response.
Somatic mutations distinguish melanocyte subpopulations in human skin
Authors: Tandukar Bishal , Deivendran Delahny , Chen Limin , Bahrani Neda , Weier Beatrice , Sharma Harsh , Cruz-Pacheco Noel , Hu Min , Marks Kayla , Zitnay Rebecca G , Bandari Aravind K. , Nekoonam Rojina , Yeh Iwei , Judson-Torres Robert , Shain A. Hunter
Published: February 2025

To better understand the homeostatic mechanisms governing melanocytes, we performed deep phenotyping of clonal expansions of single melanocytes from human skin. In total, we interrogated the mutational landscapes, gene expression profiles, and morphological features of 297 melanocytes from 31 donors. To our surprise, a population of melanocytes with low mutation burden was maintained in sun damaged skin. These melanocytes were more stem-like, smaller, less dendritic and displayed distinct gene expression profiles compared to their counterparts with high mutation burdens. We used single-cell spatial transcriptomics (10X Xenium) to reveal the spatial distribution of melanocytes inferred to have low and high mutation burdens (LowMut and HighMut cells), based on their gene expression profiles. LowMut melanocytes were found in hair follicles as well as in the interfollicular epidermis, whereas HighMut melanocytes resided almost exclusively in the interfollicular epidermis. We propose that melanocytes in the hair follicle occupy a privileged niche, protected from UV radiation, but periodically migrate out of the hair follicle to replenish the interfollicular epidermis after waves of photodamage. More broadly, our study illustrates the advantages of a cell atlas that includes mutational information, as cells can change their cellular states and positional coordinates over time, but mutations are like scars, providing a historical record of the homeostatic processes that were operative on each cell.
Optogenetic interrogation of the lateral-line sensory system reveals mechanisms of pattern separation in the zebrafish brain
Authors: Velez-Angel Nicolas , Lu Sihao , Fabella Brian , Reagor Caleb C. , Brown Holland R. , Vázquez Yuriria , Jacobo Adrian , Hudspeth A. J.
Published: February 2025

The ability of animals to interact with their environment hinges on the brain’s capacity to distinguish between patterns of sensory information and accurately attribute them to specific sensory organs. The mechanisms by which neuronal circuits discriminate and encode the source of sensory signals remain elusive. To address this, we utilized as a model the posterior lateral line system of larval zebrafish, which is used to detect water currents. This system comprises a series of mechanosensory organs called neuromasts, which are innervated by neurons from the posterior lateral line ganglion. By combining single-neuromast optogenetic stimulation with whole-brain calcium imaging, we developed a novel approach to investigate how inputs from neuromasts are processed. Upon stimulating individual neuromasts, we observed that neurons in the brain of the zebrafish show diverse selectivity properties despite a lack of topographic organization in second-order circuits. We further demonstrated that complex combinations of neuromast stimulation are represented by sparse ensembles of neurons within the medial octavolateralis nucleus (MON) and found that neuromast input can be integrated nonlinearly. Our approach offers an innovative method for spatiotemporally interrogating the zebrafish lateral line system and presents a valuable model for studying whole-brain sensory encoding.
Hexokinase detachment from mitochondria drives the Warburg effect to support compartmentalized ATP production
Authors: Huggler Kimberly S. , Mellado Fritz Carlos A. , Flickinger Kyle M. , Chang Gavin R. , McGuire Meghan F. , Cantor Jason R.
Published: February 2025

Hexokinase (HK) catalyzes the synthesis of glucose-6-phosphate, marking the first committed step of glucose metabolism. Most cancer cells express two homologous isoforms (HK1 and HK2) that can each bind to the outer mitochondrial membrane (OMM). CRISPR screens across hundreds of cancer cell lines indicate that both are dispensable for cell growth in traditional culture media. By contrast,HK2deletion impairs cell growth in Human Plasma-Like Medium (HPLM). Here, we find that HK2 is required to maintain sufficient cytosolic (OMM-detached) HK activity under conditions that enhance HK1 binding to the OMM. Notably, OMM-detached rather than OMM-docked HK promotes “aerobic glycolysis” (Warburg effect), an enigmatic phenotype displayed by most proliferating cells. We show that several proposed theories for this phenotype cannot explain theHK2dependence and instead find thatHK2deletion severely impairs glycolytic ATP production with little impact on total ATP yield for cells in HPLM. Our results reveal a basis for conditionalHK2essentiality and suggest that demand for compartmentalized ATP synthesis underlies the Warburg effect.
Overexpression of the NEK8 kinase inhibits homologous recombination
Authors: Turner Joshua L. , Moore Georgia , McCraw Tyler J. , Mason Jennifer M.
Published: February 2025

Homologous recombination maintains genome stability by repairing double strand breaks and protecting replication fork stability. Defects in homologous recombination results in cancer predisposition but can be exploited due to increased sensitivity to certain chemotherapeutics such as PARP inhibitors. The NEK8 kinase has roles in the replication response and homologous recombination. NEK8 is overexpressed in breast cancer, but the impact of NEK8 overexpression on homologous recombination has not been determined. Here, we demonstrate NEK8 overexpression inhibits RAD51 focus formation resulting in a defect in homologous recombination and degradation of stalled replication forks. Importantly, NEK8 overexpression sensitizes cells to the PARP inhibitor, Olaparib. Together, our results suggest NEK8 overexpressing tumors may be recombination-deficient and respond to chemotherapeutics that target defects in recombination such as Olaparib.