bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACTThe 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

AbstractPlasmids 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

SummaryNative 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

ABSTRACTBreast 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.SUMMARYThere 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

AbstractBackgroundEpigenetic 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.MethodsWe 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.ResultsFollowing 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.ConclusionsDNA 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

AbstractC. 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

AbstractHeterochromatin 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

AbstractThe 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

AbstractThe 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.SignificanceThis 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

AbstractPurposeTo evaluate the performance of LLMs in extracting data from stroke CT reports in the presence and absence of an annotation guideline.MethodsIn 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.ResultsGPT-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).ConclusionOur 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

AbstractHumans 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.