bluuebunny/crossref_metadata_2025 huggingface dataset:
Deep Modeling of Gain-of-Function Mutations on Androgen Receptor
Authors: You Jiaying , Foo Jane , Lallous Nada , Cherkasov Artem
Published: January 2025

The efficiency of Androgen Receptor (AR) pathway inhibitors for prostate cancer (PCa) is on decline due to resistance mechanisms including the occurrence of gain-of-function mutations on human androgen receptor (AR). Hence, understanding and predicting such mutations is crucial for developing effective PCa treatment strategies. Leveraging accumulated data on clinically relevant AR mutants with recent advances in deep modeling techniques, this study aims to unveil and quantify critical AR mutation-drug relationships. By incorporating molecular descriptors for drugs and mutated genes sequences, this work represented these features as single vectors and demonstrates their effectiveness in modeling AR mutant responses to conventional antiandrogens. The developed approach achieves up to 80% accuracy in predicting the gain-of-function behavior of AR mutants and therefore can potentially uncover unknown agonist/antagonist relationships among mutant-drug pairs.
Shear stress inhibits adipocyte differentiation via downregulating lncRNA MALAT1
Authors: Caron Justin , Marino Madison , Wang Bo , Wang Shue
Published: January 2025

Adipocyte differentiation plays an important role in bone remodeling due to secretory factors that can directly modulate osteoblast and osteoclast, thus affecting overall bone mass and skeletal integrity. Excessive adipocyte differentiation within bone marrow microenvironment can lead to decreased bone mass, eventually causing osteoporosis. Mechanical microenvironment of bone marrow, including fluid shear, maintains the balance of adipocyte and osteoblast differentiation during bone remodeling. However, how mechanical cues interact with long noncoding RNA (lncRNA) and regulating adipocyte differentiation remains unexplored. In this study, we investigated the mechanosensitive role of lncRNA MALAT1 during mesenchymal stem cells (MSCs) adipocyte differentiation. By applying physiologically relevant shear stress, MSCs experienced morphological changes and adipocyte differentiation differences. Shear stress inhibits MSCs adipocyte differentiation with reduced oil-red-o-stained lipid droplets. Silencing MALATs results in reduced adipocyte differentiation. By leveraging a novel gapmer double stranded locked nuclei acid (dsLNA) nanobiosensor, we showed that shear stress inhibits MALAT1 expression with significantly reduced fluorescence intensity. Our findings suggest that shear stress influences adipocyte differentiation primarily through the downregulation of MALAT1, highlighting a significant interplay between biophysical cues and lncRNAs. This interaction is crucial for understanding the complexities of bone remodeling and the potential therapeutic targeting of lncRNAs to treat bone-related disorders.
Druggable genome CRISPRi screen in 3D hydrogels reveals regulators of cortactin-driven actin remodeling in invading glioblastoma cells
Authors: Hu Mufeng , Weldy Anna , Lovalvo Isabella , Akins Erin , Jain Saket , Chang Alexander , Sati Ankita , Lad Meeki , Lui Austin , Rajidi Akhil , Kothekar Ameya , Ding Erika , Kumar Sanjay , Aghi Manish K.
Published: January 2025

To identify new therapeutic targets that limit glioblastoma (GBM) invasion, we applied druggable-genome CRISPR screens to patient-derived GBM cells in micro-dissectible biomimetic 3D hydrogel platforms that permit separation and independent analysis of core vs. invasive fractions. We identified 12 targets whose suppression limited invasion, of which ACP1 (LMW-PTP) and Aurora Kinase B (AURKB) were validated in neurosphere assays. Proximity labeling analysis identified cortactin as an ACP1- AURKB link, as cortactin undergoes serine phosphorylation by AURKB and tyrosine dephosphorylation by ACP1. Suppression of ACP1 or AURKB in culture andin vivoshifted the balance of cortactin phosphorylation in GBM and reduced actin polymerization and actin-cortactin co-localization. Additional biophysical analysis implicated AURKB in GBM cell adhesion and cortical stiffness, and ACP1 in resistance to mechanical stress and shape plasticity needed for 3D migration. These findings reveal a novel targetable axis that balances kinase and phosphatase activities to regulate actin polymerization during GBM invasion.
Multiple, not single, recipient muscle tendon transfers produce well-directed thumb-tip forces in lateral pinch grasp: a simulation study with application to restoration of improved grasp after tetraplegia
Authors: Garcia Oliver , Towles Joseph D
Published: January 2025

Thumb tendon transfer surgical procedures in patients with cervical spinal injury engage the paralyzed flexor pollicis longus (FPL) muscle to enable lateral pinch grasp. However, functional outcomes are mixed, in part because the FPL cannot produce force at the thumb tip to promote a stable grasp. We used simulation to investigate whether a multi-site tendon transfer, targeting sets of paralyzed muscles driven by a single donor muscle, could outperform a single-site tendon transfer with the FPL alone and restore lateral pinch. We formed 36 groups of 2 muscles, 84 groups of 3 muscles, and 126 groups of 4 muscles. We used nonlinear optimization and in-situ measurements of endpoint forces in 3 lateral pinch postures. We found that 116 of the 246 muscle groups outperformed the FPL for wide and narrow lateral pinch postures and a posture in between. On average, the 116 muscle groups produced endpoint forces 8° to 40° better directed than the FPL, and groups of four muscles statistically significantly outperformed the FPL’s force production. Our findings highlight the possibility of using multi-insertion site tendon transfers to restore grasp following cervical spinal cord injury.
ResolVI - addressing noise and bias in spatial transcriptomics
Authors: Ergen Can , Yosef Nir
Published: January 2025

Technologies for estimating RNA expression at high throughput, in intact tissue slices, and with high spatial resolution (spatial transcriptomics; ST) shed new light on how cells communicate and tissues function. A fundamental step common to all ST protocols is quantification, namely segmenting the plane into regions, each approximating a cell, and then collating the molecules inside each region to estimate the cellular expression profile. Despite many advances in this area, a persisting problem is that of wrong assignment of molecules to cells, which limits most current applications to the level of a priori defined cell subsets and complicates the discovery of novel cell states. Here, we develop resolVI, a model that operates downstream of any segmentation algorithm to generate a probabilistic representation, correcting for misassignment of molecules, as well as for batch effects and other nuisance factors. We demonstrate that resolVI improves our ability to distinguish between cell states, to identify subtle expression changes in space, and to perform integrated analysis across datasets. ResolVI is available as open source software within scvi-tools.
Development of metagenomic methods for non-invasive health monitoring of endangered species: Unveiling hidden microbial threats in fecal samples
Authors: Sapino Román , Fernández-González Ángel , Castresana Jose
Published: January 2025

Metagenomic analysis of feces is emerging as a powerful tool for improving the monitoring of endangered species. A critical aspect in assessing the extinction risk of a species is the analysis of the burden of parasites and pathogens that can negatively affect the health of individuals. However, the identification of pathogens in non-model species using metagenomics is a major challenge due to the lack of reference genome sequences or data limited to distantly related species. In this study, we developed a pipeline for detecting potentially pathogenic bacteria from metagenomic sequences by mapping unassembled reads to available reference genomes. The approach uses the breadth of genome coverage rather than the number of mapped reads for species identification, thereby minimizing false positives due to conserved or repetitive genomic regions. We applied this method to fresh fecal samples of the Iberian desman (Galemys pyrenaicus), a critically endangered semi-aquatic mammal. Our analysis revealed the presence of 19 potentially pathogenic bacterial species, with prevalences ranging from a single individual to 30% of the samples. We also detected some desmans with elevated or altered pathogen loads, suggesting variations in individual health status or different environmental exposures. This work represents a novel application of metagenomic methods for species-level pathogen detection in wildlife using fecal samples. Application of this method across populations and over time for endangered species may provide essential health and epidemiological information to improve conservation strategies.
AlphaCross-XL: a seamless tool for automated and proteome-scale mapping of crosslinked peptides onto three-dimensional protein structures
Authors: Shenoy Sanjyot Vinayak , Biswas Deeptarup , Mandal Kamal , Zalevsky Arthur , Kishishita Audrey , Verma Ayushi , Upadhay Ishan , He Yi , Sali Andrej , Viner Rosa , Srivastava Sanjeeva , Wiita Arun P.
Published: January 2025

Crosslinking mass spectrometry (XL-MS) is an exciting proteomics technology to capture native protein conformations in real time within biological systems. Historically, however, implementation of this technology has typically been limited to single purified recombinant proteins orin vitroassembled protein complexes. These limitations are associated with inherent challenges in XL-MS analysis, including extremely low abundance of crosslinked (XL) peptides and complex deconvolution of XL peptide-derived spectral data. However, impressive recent developments in computation and instrumentation have now made it feasible to address biological questions using proteome-wide XL-MS analysis. Although some XL mapping software tools exist, these require manual input of specific Protein Data Bank (PDB) structures at the single protein level, and do not function at the high throughput scale required to analyze datasets derived from thousands of proteins. To address this need, we therefore sought to develop a strategy enabling automated mapping of XL peptides onto the three-dimensional (3D) structures of proteins, at a proteome-wide scale. Herein we describe AlphaCross-XL, a first-in-class seamless computational tool for automated mapping of XL peptides onto the protein structures for intra-protein crosslinks and loop-links. The AlphaCross-XL software first retrieves protein structures from the AlphaFold Protein Structure Database and maps all the identified crosslinks onto the 3D structure. It also calculates the Euclidian distance between the crosslinked residues and reports the violated and satisfied crosslink distances based on a user-defined distance threshold, which is visually discriminated by color in PyMOL. Lastly, the tool also supports further validation of user-submitted protein structures, that can include any computer-predicted protein structure and experimentally derived protein structures (i.e., from PDB). AlphaCross-XL is available athttps://github.com/sanjyotshenoy/alphacross-xl.
High cholesterol increases myeloma tumour burden and promotes resistance to bortezomib
Authors: Gámez Beatriz , Whipp Danielle , Rao Srinivasa , Morris Emma V. , Park Young Eun , Kaya Zeynep , Edwards Claire M.
Published: January 2025

Multiple myeloma (MM) is an incurable hematologic cancer where malignant plasma cells accumulate in the bone marrow (BM), with disease progression highly dependent on cellular interactions. Obesity is a major risk factor for myeloma, however the underlying mechanisms are unclear. Here we used a murine myeloma model to show that a high cholesterol diet increases local and circulating levels of low density lipoprotein cholesterol (LDL), increasing bone marrow tumour burden in vivo. Exogenous LDL induced bortezomib-specific drug resistance in metabolically stressed myeloma cells in vitro and ex vivo. RNA-seq analysis revealed bortezomib-induced changes in the cholesterol biosynthesis/homeostasis pathway that were completely reverted with LDL pretreatment. In silico analysis of patient data supported a role for cholesterol in response to bortezomib-based therapies. Targeted proteome profiling revealed changes in expression of the adipokine resistin in the bone marrow of cholesterol-treated or myeloma-bearing mice, with elevated resistin expression observed in MGUS patients. Bone marrow adipocytes were found to be a major source of resistin, which was further increased in response to LDL. In summary, we demonstrate that high cholesterol promotes both myeloma development and bortezomib resistance, identifying resistin as a potential mediator so revealing new mechanisms underlying myeloma pathogenesis.
Epigenetic clocks and inflammaging: pitfalls caused by ignoring cell-type heterogeneity
Authors: Guo Xiaolong , Teschendorff Andrew E.
Published: January 2025

Epigenetic clocks are machine learning predictors of chronological age that have consistently been shown to be also informative of biological age measures, such as all-cause mortality. A recent study has argued against the use of machine learning methods for building epigenetic clocks, on grounds that these predictors do not enrich for features that correlate with disease. In particular, it argues that standard epigenetic clocks can’t measure inflammaging, an age-related trait, proposing an ad-hoc ‘feature rectification’ strategy and an ‘inflammation clock’ that predicts age-acceleration in inflammatory diseases. Here we demonstrate that this inflammation clock only captures an increase in the neutrophil to lymphocyte ratio associated with inflammatory diseases like rheumatoid arthritis, and that it fails to capture the shift from naïve to mature lymphocytes, which is a key feature of inflammaging. As such, their clock does not measure inflammaging, and is subsumed and outperformed by an ordinary cell-type deconvolution algorithm. Our analysis underscores the critical importance for epigenetic clock and epigenome-wide association studies to always estimate cell-type fractions in the tissue of consideration, and to adjust for its variation, in order to disentangle effects due to changing cell-type composition from DNA methylation changes that happen in specific cell-types.
Mechanical efficiency during sub-maximal cycling is underestimated because negative muscular power is ignored
Authors: Vos Roeland T. , Bobbert Maarten F. , Kistemaker Dinant A.
Published: January 2025

Thein vivomechanical efficiency of muscles has often been estimated during sub-maximal cycling. In this approach, it has implicitly been assumed that the average amount of positive mechanical muscle power equals the average mechanical power output, i.e., that no power is dissipated by muscles. Here, we investigated the validity of this assumption using an optimal control musculoskeletal model. We identified optimal muscle stimulation patterns for 4 cadences (60, 80, 100 and 120RPM) and 5 levels of average mechanical power output (50, 100, 150, 200 and 250W). We found that the amount of negative mechanical muscular power was substantial, with the average across all conditions being -84,6W (56,4%). The amount of negative mechanical muscular power was found to increase with increasing cadence and was independent of the average mechanical power output. To investigate the effect of negative muscular power onin vivoestimates of the muscular efficiency, we used our simulation results to correct gross efficiencies measured during sub-maximal cycling. The resulting increases in the gross efficiency were substantial, with the average increasing from 16.9% to 27.5%. These results suggest that current estimates of the muscular efficiency during sub-maximal cycling underestimate the true muscular efficiency.
Mitochondrial ATP-Sensitive K+Channels (MitoKATP) Regulate Brown Adipocyte Differentiation and Metabolism
Authors: Pereira Osvaldo R. , Serna Julian D.C. , Caldeira da Silva Camille C. , Camara Henrique , Kodani Sean D. , Festuccia William T. , Tseng Yu-Hua , Kowaltowski Alicia J.
Published: January 2025

Brown adipose tissue (BAT) plays a central role in mammalian non-shivering thermogenesis, dissipating mitochondrial membrane potentials through the activity of uncoupling protein UCP1 to release heat. Inner membranes of mitochondria are known to be permeable to potassium ions (K+), which enter the matrix either through ATP-sensitive channels (MitoKATP) or leakage across the bilayer driven by inner membrane potentials. Mitochondrial K+influx is associated with increased osmotic pressure, promoting water influx and increasing matrix volume. Since BAT mitochondria have lower inner membrane potentials due to uncoupling protein 1 (UCP1) activity, we hypothesized this could involve compensatory changes in MitoKATP activity, and thus tested MitoKATP involvement in brown adipocyte activities under basal and stimulated conditions. We find that cold exposure and adrenergic stimulation in mice modulate BAT MitoK levels, the channel portion of MitoKATP. Genetic ablation of the gene that codes for the pore-forming subunit of MitoKATP in human pre-adipocytes decreased cellular respiration and proliferation, compromising differentiation into mature adipocytes. In mouse cell lines, the absence of the protein limited cellular oxygen consumption in the precursor stage, but not in mature adipocytes. Interestingly, inhibition of MitoKATP in mature adipocytes increased adrenergic-stimulated oxygen consumption, indicating that shutdown of this pathway is important for full BAT thermogenesis. Similarly, MitoKATP inhibition increased oxygen consumption in BAT mitochondria isolated from mice treated with beta 3 adrenergic receptor agonist CL316,243. Overall, our results suggest that the activity of MitoKATP regulates differentiation and metabolism of brown adipocytes, impacting on thermogenesis.
Unveiling G-Protein-Coupled Receptor Conformational Dynamics via Metadynamics Simulations and Markov State Models
Authors: Roessner Rita A. , Floquet Nicolas , Louet Maxime
Published: January 2025

The dynamic character of G-protein-coupled receptors (GPCRs) is essential in their functionality as signal transducers. However, the molecular details of how ligands affect this conformational repertoire to steer intracellular signaling pathways remain elusive. Here, we address this question by modeling the conformational landscape of the growth hormone secretagogue receptor (GHSR-1a), a prototypical peptide-activated class A GPCR, in its apo state and bound to pharmacologically distinct ligands. We present a generally applicable protocol to efficiently explore the conformational space of GHSR-1a that is sensitive to the bound ligand. Combining metadynamics simulations and Markov state modeling, we computed the free energy landscape of GHSR-1a in its apo state and bound to an agonist, antagonist, or inverse agonist, respectively. Consistent with the current multi-state model of GPCR activity, we found that GHSR-1a populates multiple metastable states whose energies and transition probabilities change depending on the bound ligand. Furthermore, our Markov state models (MSM) have revealed two intermediate states that have not yet been described by experimental structures and which we assume to facilitate the binding of extracellular ligands and intracellular protein partners, respectively. Lastly, our MSMs allowed us to shed light on the molecular differences between basal and agonistinduced GHSR-1a activation. Our results are not only compatible with previously reported experimental data, but they capture the equilibria governing GHSR-1a activation in unprecedented detail. Due to its applicability to all class A GPCRs, our protocol is a valuable tool for the development of pharmaceuticals targeting this protein family.
Chromosomal capture of beneficial genes drives plasmids towards ecological redundancy
Authors: MacLean R. Craig , Lood Cédric , Wheatley Rachel M.
Published: January 2025

Plasmids are a ubiquitous feature of bacterial genomes, but the evolutionary forces driving genes to become associated with plasmids are poorly understood. To address this problem, we compared the fitness effects of chromosomal and plasmid genes in the plant symbiontRhizobium leguminosarum. Here we show that plasmids are depleted in beneficial genes compared to the chromosome, and this effect is stronger for ancient plasmids compared to recently acquired plasmids. These findings support the hypothesis that evolution drives beneficial genes to become localized to the bacterial chromosome, resulting in a gradual decay in the ecological value of plasmids. These findings question the ecological importance of plasmids and highlight the challenge of understanding how plasmids persist over the long term.
Lethal effects of phyllosphere fungi associated with local plants on invasive plantAgeratina adenophoraseedlings
Authors: Zeng Zhao-Ying , Liu Zi-Qing , Yang Ai-Ling , Li Yu-Xuan , Wang Yong-Lan , Siemann Evan , Li Bo , Zhang Han-Bo
Published: January 2025

Native community resistance to plant invasion may reflect microbial effects on invader establishment. Here, we compared the leaf and soil effects of 25 local native plant species on the germination and survival of invaderAgeratina adenophora.The foliar physical and chemical traits vary among 26 plant species and shape the microbial community. Leaves had more adverse effects on the germination and seedling survival ofA. adenophorathan did soil. Foliar physicochemical properties negatively affect seed germination, but foliar microbes mainly explained survival ofA. adenophora. Moreover, foliar effects of native plants more closely related to the invader more strongly lengthened its germination time and decreased its seedling survival. Interestingly, the most of the predictive bacteria were positively related to seedling survival, whereas most of the predictive fungi were negatively related to seedling survival and were specialist pathogens. We also obtained phyllosphere fungi belonging toAlternaria,FusariumandStagonosporopsis, which killed more than 60% ofA. adenophoraseedlings, which have potential as biocontrol agents for invaders. This is the first study to provide evidence for the effects of phylogenetic relatedness and leaf traits (both functional and microbial) on local community resistance to invaders through the regulation of germination and seedling survival.
Hetero-multicellular stromal cells incorporate into scaffold-free 3D cultures of epithelial cancer cells to drive invasion
Authors: Ortiz Elizabeth , Thway Kyaw Hsu , Ortiz-Soto Gabriela , Yao Paulina , Kelber Jonathan A
Published: January 2025

Breast cancer (BC) is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids (PDOs) and patient-derived xenograft organoids (PDXOs) are pathophysiologically relevant, they are costly to propagate, difficult to manipulate and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. Advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these hetero-multicellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells(THP-1) and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent of the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, hetero-multicellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in hetero-multicellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture hetero-cellular crosstalk to study cancer metastasis. Our study presents the generation of hetero-multicellular stromal-epithelial in a scaffold and scaffold free environment that can be used to study invasion and cellular spatial distributions.
DNA methylation patterns in breast cancer, paired benign tissue from ipsilateral and contralateral breast, and healthy controls
Authors: Dennis Saya R. , Tsukioki Takahiro , Cottone Gannon , Zhou Wanding , Ganz Patricia A. , Sehl Mary E. , Luo Yuan , Khan Seema A. , Clare Susan
Published: January 2025

Background

Epigenetic changes, particularly DNA methylation, are crucial to breast cancer development. Tumor-adjacent normal (AN) tissue frequently serves as a reference for characterizing genomic alterations but is reported to share some characteristics with tumors. However, it is unclear whether AN’s epigenetic profiles reflect a predisposition to cancer or a response to the presence of the tumor. We address this gap by systematically comparing methylation profiles of tumor, AN, and matched-benign tissues from both breasts, as well as to healthy donated breast tissue.

Methods

We studied four different sample categories from 69 cancer cases: tumor (TU), AN, ipsilateral opposite quadrant (OQ), and contralateral unaffected breast (CUB); and healthy donated breast (HDB) tissue from 182 cancer-unaffected donors. These constitute a “tumor proximity axis” (TPxA): HDB→CUB→OQ→AN→TU. Methylation profiles were assayed using Illumina’s Infinium Methylation EPICv1.0 BeadChip. Differential methylation (DM) analysis was conducted, and the significantly DM CpGs were analyzed for enrichment of transcription factor binding sites (TFBS) and other features.

Results

Following data processing and quality control, there were 69 TU, 60 AN, 67 OQ, 68 CUB, and 182 HDB samples for analysis. DM analysis showed distinct methylation profiles of TU relative to benign tissues, whereas case-benign tissues were similar to each other but distinct from HDB. Hypomethylated sites in case-benign versus HDB were enriched for TF binding sites of TP63, GATA3, ESR1, PR, AR, NR3C1, and GREB1. TU hypermethylation events were enriched for Polycomb-repressive complex 2 (PRC2) binding, including EZH2, SUZ12, and JARID2, with hypermethylation enrichment for PRC2-related binding motifs in both ER+ and ER- tumors. TU methylation profiles were otherwise highly distinct by ER status: TFBS enrichment of hypomethylation events for hormone receptor-related pathways in ER+ tumors and for hematopoiesis/immune-related pathways in ER- tumors. We found no differential methylation between benign tissues from patients with ER+ vs. ER- tumors.

Conclusions

DNA methylation profiles differ profoundly at two points: tumor to case-benign and case-benign to HDB, with clear distinction between ER+ and ER- tumors. Case-benign tissues are not epigenetically “normal”, are similar across both breasts, and do not differ by ER status of paired tumors.
Molecular Requirements forC. elegansTransgenerational Epigenetic Inheritance of Pathogen Avoidance
Authors: Kaletsky Rachel , Moore Rebecca , Sengupta Titas , Seto Renee , Ceballos-Llera Borja , Murphy Coleen T.
Published: January 2025

C. elegans’major food source is bacteria, and worms are naturally attracted to many bacterial species, including pathogenicPseudomonas; in fact, worms prefer PA14 as well as wild bacteria over the labE. colistrain (OP50) standardly used in the laboratory setting. Many labs have shown that despite this natural attraction to PA14, prior exposure to PA14 causes the worms to instead avoid PA14. This behavioral switch can happen on a relatively fast time scale, even within the duration of the choice assay. Here we show that accurate assessment of the animals’ true first choice requires the use of a paralytic (azide) to trap the worms at their initial choice, and to prevent the switch from attraction to avoidance of PA14 within the assay period. We previously discovered that exposure ofC. elegansto 25°C plate-grown PA14 at 20°C for 24hrs not only leads to these animals switching from attraction to avoidance of PA14, but also to their progeny avoiding PA14 in the naïve state, and this avoidance persists through the F4 generation. Other types of PA14 training can also cause P0 and/or F1 avoidance, but do not induce transgenerational (F2 and beyond) inheritance. We also previously showed that the transgenerational (P0-F4) learned avoidance is mediated by P11, a small RNA produced by PA14. P11 is both necessary and sufficient for transgenerational epigenetic inheritance of avoidance behavior. P11 is highly expressed in our standard growth conditions (25°C on surfaces), but not in other conditions, suggesting that reported failure to observe F2-F4 avoidance is most likely due to the absence of P11 expression in PA14 in the experimenters’ growth conditions. Through mutant analyses, we have tested many genes – including germline regulators, small RNA uptake, RNA interference/processing, chromatin modifiers, and neuronal genes - for their involvement in transgenerational inheritance of learned pathogen avoidance, allowing us to better understand the molecular requirements for this process. We previously found that wildC. elegansstrains also show TEI of learned pathogen avoidance, and that at least two other wild bacteria,P. vranovensisandP. fluorescens 15,induce this transgenerational avoidance. The avoidance induced by eachPseudomonasspecies functions through a specific, distinct small RNA (Pv1 inP. vranovensisand Pfs1 inP. fluorescens 15, respectively) that either directly or indirectly reduce the levels of the genemaco-1, which in turn regulatesdaf-7expression in the ASI neuron and subsequent avoidance behavior. The conservation of multiple components of this small RNA TEI mechanism acrossC. elegansstrains and in multiplePseudomonasspecies suggests that this transgenerational learned avoidance behavior is likely to be functional and physiologically important in wild conditions.
Auto-methylation of the histone methyltransferase SetDB1 at its histone-mimic motifs ensures the spreading and maintenance of heterochromatin
Authors: Tang Qing , Zhang An , Sullivan Michael , Fejes Toth Katalin , Aravin Alexei A.
Published: January 2025

Heterochromatin plays a critical role in nuclear organization and the regulation of gene expression by directing 3D genome organization, regulating lineage-specific gene expression, and ensuring the repression of transposable elements and endogenous retroviruses. Functionally and structurally distinct chromatin domains are defined by the so-called histone code, which consists of combinations of post-translational histone modifications deposited by “code writers” and recognized by “code readers.” The primary mark of heterochromatin, trimethylation of histone H3 at lysine 9 (H3K9me3), is deposited by histone methyltransferases, such as SetDB1, and serves as a binding platform for readers, most notably HP1 family proteins.

Using a reporter system to monitor the dynamics of heterochromatin establishment and maintenance, we demonstrated that transient tethering of HP1 triggers the SetDB1-dependent establishment of stable heterochromatin. This finding indicates the presence of a feedback mechanism wherein the reader of the H3K9me3 mark recruits the writer. We further discovered that the genetic interaction between SetDB1 and HP1 is mirrored by a direct physical interaction. This interaction requires the auto-methylation of two conserved histone mimic motifs located in unstructured regions of SetDB1. HP1 binds these SetDB1 motifs using the same molecular interface it employs to recognize the modified histone tail.

Our findings show that SetDB1 auto-methylation is essential for the spreading and stable maintenance of heterochromatin. This includes its roles in processes such as X-chromosome inactivation and the negative feedback regulation of a large gene family encoding KRAB-ZNF transcriptional repressors. Thus, the primary heterochromatin mark is not limited to nucleosomes but is also deployed on the mark’s writer itself. This fosters a direct physical interaction between the writer and the reader, ensuring key features of heterochromatin: its spreading to establish extended domains and its stable maintenance through cell divisions.
Alfalfa transcriptomic responses to the field pathobiome
Authors: Nemchinov Lev G. , Irish Brian M. , Grinstead Sam , Postnikova Olga A.
Published: January 2025

The pathobiome is a comprehensive biotic environment that includes a community of all disease-causing organisms within the plant, defining their mutual interactions and the resultant effect on plant health. We have previously reported on the diverse composition of the alfalfa pathobiome in the field production environment. We have also briefly addressed host reactions to a consortium of pathogenic microorganisms by profiling gene expression patterns shaped by the plant pathobiome in the field. In this study, we expand on the ‘field host genomics’ approach by surveying gene expression changes in visually healthy and diseased plants collected from different commercial alfalfa fields. In addition to offering insights into the genetic basis of host resistance to multi-pathogenic infections in natural ecosystems, this strategy can also help to identify plants with tolerant genotypes adapted to field pathobiome that could be used in breeding programs.
In situNMR reveals a pH sensor motif in an outer membrane protein that drives bacterial vesicle production
Authors: Wood Nicholas A , Gopinath Tata , Shin Kyungsoo , Marassi Francesca M.
Published: January 2025

The outer membrane vesicles (OMVs) produced by diderm bacteria have important roles in cell envelope homeostasis, secretion, interbacterial communication, and pathogenesis. The facultative intracellular pathogenSalmonella entericaTyphimurium (STm) activates OMV biogenesis inside the acidic vacuoles of host cells by upregulating the expression of the outer membrane (OM) protein PagC, one of the most robustly activated genes in a host environment. Here, we used solid-state nuclear magnetic resonance (NMR) and electron microscopy (EM), with native bacterial OMVs, to demonstrate that three histidines, essential for the OMV biogenic function of PagC, constitute a key pH-sensing motif. The NMR spectra of PagC in OMVs show that they become protonated around pH 6, and His protonation is associated with specific perturbations of select regions of PagC. The use of bacterial OMVs is an essential aspect of this work enabling NMR structural studies in the context of the physiological environment. PagC expression upregulates OMV production inE. coli, replicating its function in STm. Moreover, the presence of PagC drives a striking aggregation of OMVs and increases bacterial cell pellicle formation at acidic pH, pointing to a potential role as an adhesin active in biofilm formation. The data provide experimental evidence for a pH-dependent mechanism of OMV biogenesis and aggregation driven by an outer membrane protein.

Significance

This work sheds light on the mechanism for extracellular vesicle biogenesis by Gram negative bacteria. It shows that theSalmonellasurface protein PagC, a major driver of extracellular vesicle formation, harbors a set of pH-sensitive histidines that become protonated at acidic pH, increasing vesicle production, and promoting bacterial cell aggregation. NMR analysis of PagC in natively secreted bacterial vesicles is introduced as a new important tool forin situstructural analysis of bacterial membrane proteins. The results have important implications for understanding the molecular factors that drive the formation of bacterial extracellular vesicles, their functions in human infection, as well as their roles as vaccine, drug delivery and nanotechnology platforms.
Data Extraction from Free-Text Stroke CT Reports Using GPT-4o and Llama-3.3-70B: The Impact of Annotation Guidelines
Authors: Wihl Jonas , Rosenkranz Enrike , Schramm Severin , Berberich Cornelius , Griessmair Michael , Woźnicki Piotr , Pinto Francisco , Ziegelmayer Sebastian , Adams Lisa C. , Bressem Keno K. , Kirschke Jan S. , Zimmer Claus , Wiestler Benedikt , Hedderich Dennis , Kim Su Hwan
Published: January 2025

Purpose

To evaluate the performance of LLMs in extracting data from stroke CT reports in the presence and absence of an annotation guideline.

Methods

In this study, performance of GPT-4o and Llama-3.3-70B in extracting ten imaging findings from stroke CT reports was assessed in two datasets from a single academic stroke center. Dataset A (n = 200) was a stratified cohort including various pathological findings, whereas Dataset B (n = 100) was a consecutive cohort. Initially, an annotation guideline providing clear data extraction instructions was designed based on a review of cases with inter-annotator disagreements in dataset A. For each LLM, data extraction was performed under two conditions – with the annotation guideline included in the prompt and without it. Queries for both LLMs were run with a temperature setting of 0. For GPT-4o, additional queries with a temperature of 1 were performed.

Results

GPT-4o consistently demonstrated superior performance over Llama-3.3-70B under identical conditions, with micro-averaged precision ranging from 0.83 to 0.95 for GPT-4o and from 0.65 to 0.86 for Llama-3.3-70B. Across both models and both datasets, incorporating the annotation guideline into the LLM input resulted in higher precision rates, while recall rates largely remained stable. In dataset B, precision of GPT-4o and Llama-3-70B improved from 0.83 to 0.95 and from 0.87 to 0.94, respectively. The greatest increase in precision on a variable-level was seen in infarct demarcation (0.59 to 1.00) and subdural hematoma (0.67 to 1.00). Overall classification performance with and without annotation guideline was significantly different in five out of six conditions (e.g. dataset B/Llama-3.3/temp=0: p = 0.001).

Conclusion

Our results demonstrate the potential of GPT-4o and Llama-3.3-70B in extracting imaging findings from stroke CT reports, with GPT-4o steadily exceeding the performance of Llama-3-70B. We further provide evidence that well-defined annotation guidelines can enhance LLM data extraction accuracy.
Sex-specific transcriptome similarity networks elucidate comorbidity relationships
Authors: Sánchez-Valle Jon , Flores-Rodero María , Xavier Costa Felipe , Carbonell-Caballero Jose , Núñez-Carpintero Iker , Tabarés-Seisdedos Rafael , Rocha Luis Mateus , Cirillo Davide , Valencia Alfonso
Published: January 2025

Humans present sex-driven biological differences. Consequently, the prevalence of analyzing specific diseases and comorbidities differs between the sexes, directly impacting patients’ management and treatment. Despite its relevance and the growing evidence of said differences across numerous diseases (with 4,370 PubMed results published within the past year), knowledge at the comorbidity level remains limited. In fact, to date, no study has attempted to identify the biological processes altered differently in women and men, promoting differences in comorbidities. To shed light on this problem, we analyze expression data for more than 100 diseases from public repositories, analyzing each sex independently. We calculate similarities between differential expression profiles by disease pairs and find that 13-16% of transcriptomically similar disease pairs are sex-specific. By comparing these results with epidemiological evidence, we recapitulate 53-60% of known comorbidities distinctly described for men and women, finding sex-specific transcriptomic similarities between sex-specific comorbid diseases. The analysis of shared underlying pathways shows that diseases can co-occur in men and women by altering alternative biological processes. Finally, we identify different drugs differentially associated with comorbid diseases depending on patients’ sex, highlighting the need to consider this relevant variable in the administration of drugs due to their possible influence on comorbidities.
Clonal interference and changing selective pressures shape the escape of SARS-CoV-2 from hundreds of antibodies
Authors: Haddox Hugh K , Abdel Aziz Omar , Galloway Jared G , Kent Javen , Cooper Cameron R , Bloom Jesse D , Matsen Frederick A
Published: January 2025

SARS-CoV-2 rapidly evolves to evade human immunity. While the virus's overall resistance to human polyclonal antibody responses has steadily increased over time, the dynamics by which it escaped individual monoclonal antibodies within these responses have not been thoroughly explored. Recently, a series of studies by Cao et al. used deep mutational scanning (DMS) to identify which mutations allow the Wuhan-Hu-1 receptor-binding domain to escape binding by individual antibodies, doing so for thousands of antibodies. Here, we sought to use these data to retrospectively examine the evolutionary dynamics of escape from a set of 1,603 antibodies. For each antibody, we used the DMS data to predict an antibody-escape score for each of thousands of globally circulating viral sequences from the first 3.5 years of the pandemic, and then computed an escape trajectory that quantifies how the population's average escape score changed over time. We use pseudovirus neutralization data from Cao et al. and Wang et al. to validate common patterns in escape trajectories. While some trajectories increase monotonically over time, others show large fluctuations as a result of clade-displacement events that reduce the frequency of antibody-escape mutations in the viral population. Fitness effects of mutations estimated from natural sequences suggest that the mutations are displaced due to clonal interference. Further, these estimates suggest that the order in which escape mutations arose is shaped by changing selective pressures. Overall, this work helps describe how SARS-CoV-2 evaded the individual components of a polyclonal immune response in nature, and suggests that evasion occurred via complex evolutionary dynamics.
Multifaceted Actions of Neurosteroids
Authors: Kumar Ajeet , Qian Mingxing , Xu Yuanjian , Benz Ann , Covey Douglas F. , Zorumski Charles F. , Mennerick Steven
Published: January 2025

Background and purpose

Neurosteroids modulate neuronal function and are promising therapeutic agents for neuropsychiatric disorders. Neurosteroid analogues are approved for treating postpartum depression and are of interest in other disorders. GABA-A receptors are well characterized targets of natural neurosteroids, but other biological pathways are likely relevant to therapeutic mechanisms and/or to off-target effects. We performed hypothesis-generatingin silicoanalyses and broadin vitrobiological screens to assess the range of actions of neurosteroids analogues of varying structural attributes.

Key Results

We employedin silicomolecular similarity analysis and network pharmacology to elucidate likely targets. This analysis confirmed likely targets beyond GABA-A receptors. We then functionally screened 19 distinct neurosteroid structures across 78 targets representing interconnected signaling pathways, complemented with a limited screen of kinase activation. Results revealed unanticipated modulation of targets by neurosteroids with some structural selectivity. Many compounds-initiated androgen receptor translocation with little or no enantioselectivity. Modulation of multiple G-protein receptors was also unexpected.

Conclusions and implications

Neurosteroids are ascendant treatments in neuropsychiatry, but their full spectrum of actions remains unclear. This virtual and biological screening discovery approach opens new vistas for exploring mechanism of neurosteroids analogues. The multifaceted approach provides an unbiased, holistic exploration of the potential effects of neurosteroids across various molecular targets and provides a platform for future validation studies to aid drug discovery.
DUX4-stimulated genes define the antiviral response to herpesviruses in human trophoblasts
Authors: Hatterschide Joshua , Yang Liheng , Coyne Carolyn B.
Published: January 2025

The placenta serves as a barrier against the vertical transmission of viruses, due to the protective functions of fetal-derived trophoblasts. Although some antiviral programs of trophoblasts are well-documented, our understanding of how trophoblasts respond to teratogenic viruses remains incomplete. To address this, we profiled the transcriptional responses of human trophoblast organoids to seven teratogenic viruses. We discovered that herpesviruses including HSV-1, HSV-2, and HCMV did not trigger an interferon (IFN) response but instead induced the expression ofDUX4and downstream target genes, termed DUX4-stimulated genes (DSGs). This program was uniquely, highly expressed in trophoblasts. Through single-cell RNA sequencing, we defined the trophoblast response toDUX4and demonstrated that this program defines cells with low viral transcripts following HSV-1 infection. Similar to the IFN-stimulated genes, we observed that many DSGs with diverse predicted functions exhibited anti-herpesvirus activity. These findings establish DUX4 and DSGs as a critical antiviral defense mechanism in trophoblasts.
Macrophage depletion restores the DRG microenvironment and prevents axon degeneration in bortezomib-induced neuropathy
Authors: Thomsen Michael B. , Singh Abhishek , Thebeau Christina N. , Gao Vivian D. , Schulze Nicholas F. , Avraham Oshri , Yang Sarah X. , Koneru Shriya , Geier Sami S. , Landon Shannon M. , Pelea Aidan , Cavalli Valeria , Geisler Stefanie
Published: January 2025

Peripheral neuropathy is a common and debilitating side effect of the chemotherapeutic bortezomib (BTZ). To explore the mechanisms underlying BTZ-induced neuropathy (BIPN), we developed a mouse model that replicates the route of administration and approximates the prolonged BTZ exposure experienced by patients. We find that male mice treated with BTZ experience more severe sensorimotor dysfunction and axon loss compared to females and observed similar results when analyzing human data. Using single cell RNA-sequencing, we reveal that BTZ significantly alters the dorsal root ganglia (DRG) microenvironment in mice, producing pronounced sex-specific changes in satellite glial cells (SGCs) in males and females and dysregulation of the extracellular matrix (ECM), particularly in males. These changes are accompanied by expansion of macrophages, which is more pronounced in males. We identify four macrophage subtypes in the DRG, including a pro-fibrotic population that is exclusively associated with BIPN. Depletion of macrophages via anti-CSF1R treatment in male mice prevents BTZ-induced SGC activation and aberrant collagen deposition in DRGs, potently preserves peripheral axons, and improves functional outcomes. These findings highlight SGCs, neuroinflammation and dysregulation of the ECM as drivers of sex-specific differences in BIPN and suggest that targeting neuroinflammation is a promising therapeutic strategy to treat this disease.

One Sentence Summary

Inflammation and neurofibrosis in DRGs underlie sex-specific differences in bortezomib-induced neuropathy and are promising treatment targets.