bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological Activation of NO-Sensitive Guanylyl Cyclase Ameliorates Obesity-Induced Arterial Stiffness
Authors: Budbazar Enkhjargal , Balmes Aylin , Elliott Danielle , Tintin Lisette Peres , Kopp Timo , Feil Susanne , Feil Robert , Schäffer Tilman E. , Seta Francesca
Published: February 2025

Objective

Arterial stiffness, or loss of elastic compliance in large arteries, is an independent precursor of cardiovascular disease (CVD)1and dementia2. Akin to anti-hypertensive and lipid-lowering drugs, arterial de-stiffening therapies could be beneficial at decreasing CVD risk. We previously discovered that enhanced cytoskeletal actin polymerization in vascular smooth muscle cells (VSMCs) contributes to increased arterial stiffness3. In aortas and VSMCs, we previously found that decreased NO-sensitive guanylyl cyclase (NO-GC), the NO receptor which synthesizes cGMP, caused downregulation of cGMP-dependent protein kinase I (cGKI) and of its target vasodilator-stimulated phosphoprotein (pVASPS239), leading to increased cytoskeletal actin polymerization3. In the current study, we tested whether activating NO-GC with an NO-GC activator (cinaciguat) modulates pVASPS239and cytoskeletal actin polymerization in VSMCs, thereby preventing obesity-induced arterial stiffness.

Approach & Results

Cinaciguat administration (5 mg/kg) to high fat, high sucrose diet (HFHS)-fed mice, our established model of arterial stiffness4, (1) decreased pulse wave velocity, thein vivoindex of arterial stiffness, without affecting blood pressure, (2) increased aortic pVASPS239levels, and (3) decreased the ratio of filamentous (F) to globular (G) actin, compared to vehicle administration. In cultured VSMCs, cinaciguat (10 μmol/L) increased pVASPS239levels and decreased the F/G actin ratio at baseline and after stimulation with the cytokine tumor necrosis factor α (TNFα), used to mimic the inflammatory milieu of HFHS aortas. These effects were abrogated in aortas and VSMCs from mice with smooth muscle-specific cGKI deletion (cGKISMKO), while being mimicked by a cell-permeable cGMP analog (8-Br-cGMP, 1 μmol/L), which also decreased VSMC stiffnessin vitro.

Conclusions

Collectively, our data strongly support the notion that pharmacological NO-GC activation would be beneficial in decreasing obesity-associated arterial stiffness by decreasing VSMC cytoskeletal actin hyper-polymerization. If translated to humans, NO-GC activators could become a viable approach to clinically treat arterial stiffness, which remains an unmet medical need.
The interactome of the Bakers’ yeast peroxiredoxin Tsa1 implicates it in the redox regulation of intermediary metabolism, glycolysis and zinc homeostasis
Authors: MacDiarmid Colin W. , Taggart Janet , Wang Yirong , Vashisht Ajay , Qing Xin , Wohlschlegel James A. , Eide David J.
Published: February 2025

Zinc (Zn) is an essential nutrient supporting a range of critical processes. In the yeastSaccharomyces cerevisiae, Zn deficiency induces a transcriptional response mediated by the Zap1 activator, which controls a regulon of ∼80 genes. A subset support zinc homeostasis by promoting zinc uptake and its distribution between compartments, while the remainder mediate an “adaptive response” to enhance fitness of zinc deficient cells. The peroxiredoxin Tsa1 is a Zap1-regulated adaptive factor essential for the growth of Zn deficient cells. Tsa1 can function as an antioxidant peroxidase, protein chaperone, or redox sensor: the latter activity oxidizes associated proteins via a redox relay mechanism. We previously reported that in Zn deficient cells, Tsa1 inhibits pyruvate kinase (Pyk1) to conserve phosphoenolpyruvate for aromatic amino acid synthesis. However, this regulation makes a relatively minor contribution to fitness in low zinc, suggesting that Tsa1 targets other pathways important to adaptation. Consistent with this model, the redox sensor function of Tsa1 was essential for growth of ZnD cells. Using an MBP-tagged version of Tsa1, we identified a redox-sensitive non-covalent interaction with Pyk1, and applied this system to identify multiple novel interacting partners. This interactome implicates Tsa1 in the regulation of critical processes including many Zn-dependent metabolic pathways. Interestingly, Zap1 was a preferred Tsa1 target, as Tsa1 strongly promoted the oxidation of Zap1 activation domain 2, and was essential for full Zap1 activity. Our findings reveal a novel posttranslational response to Zn deficiency, overlain on and interconnected with the Zap1-mediated transcriptional response.

Graphical abstract

In ZnD cells, Tsa1 mediates metabolic adaptation by regulation of glycolytic enzymes Pyk1 and Fba1, and supports activity of the Zap1 transcriptional activator via oxidation of the AD2 domain.
Disruption of the mRNA m6A writer complex triggers autoimmunity in Arabidopsis
Authors: Metheringham Carey L , Srivastava Anjil K , Thorpe Peter , Maji Ankita , Parker Matthew T , Barton Geoffrey J , Simpson Gordon G
Published: February 2025

Distinguishing self from non-self is crucial to direct immune responses against pathogens. Unmodified RNAs stimulate human innate immunity, but RNA modifications suppress this response. mRNA m6A modification is essential forArabidopsis thalianaviability. However, the molecular basis of the impact of mRNA m6A depletion is poorly understood. Here, we show that disruption of the Arabidopsis mRNA m6A writer complex triggers autoimmunity. Most gene expression changes in m6A writer complexvir-1mutants grown at 17°C are explained by defence gene activation and are suppressed at 27°C, consistent with the established temperature sensitivity of Arabidopsis immunity. Accordingly, we found enhanced pathogen resistance and increased premature cell death invir-1mutants at 17°C but not 27°C. Global temperature-sensitive mRNA poly(A) tail length changes accompany these phenotypes. Our results demonstrate that autoimmunity is a major phenotype of mRNA m6A writer complex mutants, which has important implications for interpreting this modification’s role. Furthermore, we open the broader question of whether unmodified RNA triggers immune signalling in plants.
A genetic signature predicts aggressive paraganglioma sensitivity to dual PI3K-CDK4/6 inhibition therapy
Authors: Karna Bhargavi , Gentile Federica , Gulde Sebastian , Martino Daniela De , Maurer Julian , Ganesan Katyayni , Wang Katharina , Bechmann Nicole , Rapizzi Elena , Robledo Mercedes , Arroba Ester , Herzig Stephan , Nölting Svenja , Mohr Hermine , Pellegata Natalia S.
Published: February 2025

Effective medical therapies for metastatic paraganglioma (mPPGL) are currently lacking, leading to dismal prognosis. Building on our knowledge of molecular mechanisms driving PPGL progression, we assessed the therapeutic potential of targeting two critical processes: PI3K signaling and cell cycle regulation. The efficacy of buparlisib (PI3Ki) and ribociclib (CDK4/6i), individually and combined, was assessedin vitrousing PPGL cell lines, rat– and patient-derived primary cells, andin vivousing PPGL cells-derived mouse xenografts. The combination therapy demonstrated superior antitumor activity compared to single agents, particularlyin vivo. Mechanistically, the efficacy of the combination therapy was associated to the downregulation of FOXM1-controlled genes implicated in mitotic spindle assembly and chromosomal segregation, leading to mitotic catastrophe. Data mining and qRT-PCR showed this genetic signature to be upregulated in human mPPGLs. This suggests that aggressive PPGLs exhibit heightened vulnerability to dual PI3K and CDK4/6 inhibition, offering a promising therapeutic avenue for these challenging cancers.
Seek-a-Seq: Optical detection of target nucleic acids with attomolar sensitivity
Authors: Das Anand Kant , Saj Alam , Gan Hin Hark , Gunsalus Kristin C. , Shubeita George T.
Published: February 2025

We introduce Seek-a-Seq, an enzyme-free, PCR amplification-free, optical method to detect microbial nucleic acids based on fluorescent readout of hybridization between target sequences and complementary probes immobilized on the surface of customized sample chambers. We demonstrate the ability to detect ultralow concentration (~ 1 attomolar) of short target DNA and synthetic SARS-CoV-2 RNA with ease and specificity. Seek-a-Seq can enormously reduce costs, accelerate diagnostic workflows, and outperform existing enzyme-dependent methods.
Fabrication & Characterization of Hyaluronic Acid/Eucalyptus Hydrogels Loaded with PLGA Nanoparticles with Methotrexate as an Injectable Therapy for Rheumatoid Arthritis
Authors: Castilla-Bolanos Maria Alejandra , Duenas Mariana , Bustamante Laura , Castillo Samuel
Published: February 2025

Rheumatoid arthritis is an autoimmune disease that affects about 250,000 Colombians, 82% of whom are women. Current treatments include disease-modifying antirheumatic drugs (DMARDs), such as methotrexate (MTX), analgesics and physiotherapy. The most common DMARD is MTX, which binds to plasma proteins with low efficiency (50%) and has a half-life of 6 hours. Due to its limitations when administered orally, nanoparticles (NPs) have been proposed to overcome these limitations. NPs support the release of therapeutic molecules, minimizing side effects and increasing the bioavailability of the drug in a controlled administration. NPs synthesized from biodegradable polymers, such as polyglycolic lactic acid (PLGA), are convenient for drug delivery due to their high biocompatibility and ability to bind DMARDs such as MTX. PLGA NPs loaded with MTX (MTX-PLGA-NPs) have reduced the presence of proinflammatory factors such as IL-10 and TGF-β, suggesting their potential as anti-inflammatory therapies for arthritis. Therefore, this study aims to develop MTX-PLGA-NPs in bioactive and biocompatible hyaluronic acid-eucalyptus (GelHA-E) hydrogels to preserve their stability and delay their degradation. PLGA-NPs were synthesized with an average hydrodynamic diameter of 200 nm, the 1237 cm-1 band found in FITR indicated the successful covalent conjugation with MTX; the mass loss of only 1% in GelHA-E indicated the thermogravimetric stability of the biomaterial and the low hemolytic and platelet aggregation percentage confirmed the biocompatibility of the biomaterial as a potential localized, anti-inflammatory, and injectable treatment for rheumatoid arthritis.
Leishmania amazonensisinfection induces IL1β-dependent hyperalgesia, while dampening mechanical allodynia in C57BL/6 mice
Authors: Perez Gustavo G. , de Lima Emanuelle V. , Antonio Leticia S. , Vidal Eduardo Matheus M. , Christoff Raissa R. , Paiva Milla B. , Gonçalves Antônio José S. , Duarte Marcia Pereira O. , Bellio Maria , Silva Robson Coutinho , da Costa Robson , Figueiredo Rodrigo T. , Clarke Julia R. , Guedes Herbert L.
Published: February 2025

Leishmaniases are neglected diseases causing significant deaths and disabilities. In Brazil, the most prevalent form is cutaneous leishmaniasis, characterized by painless lesions despite intense inflammation and ulceration. While BALB/c mice models exhibit hypersensitization to inflammatory stimuli, C57BL/6 mice better mimic human-like lesion progression and nociceptive responses. This study aimed to investigate the mechanisms underlying nociceptive changes in cutaneous leishmaniasis using the C57BL/6 model. Following infection withL. amazonensis, behavioral and nociceptive tests revealed unaltered mechanical nociception and motor capacity, though thermal hypersensitivity emerged during the chronic phase. Elevated IL-1β production in the lesions and upregulation of TRPV1 in dorsal root ganglia (DRG) neurons were detected via ELISA and qPCR. Mice deficient in IL-1β-related proteins or receptors exhibited higher thermal nociception thresholds, highlighting IL-1β’s role in heat hypersensitization during late infection stages. Microscopy of chronic lesions revealed tissue deformities, indicating desensitization to mechanical and inflammatory stimuli due to nerve terminal alterations and fibroplasia from regenerative processes. Conversely, thermal hypersensitivity in chronic phases was driven by IL-1β effects on thermal nociceptive neurons in the DRG. These findings suggest that IL-1β and TRPV1 contribute to thermal hypersensitivity, while structural changes in lesions underlie mechanical desensitization. This model provides insights into the complex nociceptive mechanisms of cutaneous leishmaniasis.
Structural Insights into the Dynamics of Water in SOD1 Catalysis and Drug Interactions
Authors: Yapici Ilkin , Tokur Arda Gorkem , Sever Belgin , Ciftci Halilibrahim , Basak Ayse Nazli , DeMirci Hasan
Published: February 2025

Superoxide dismutase 1 (SOD1) is a crucial enzyme that protects cells from oxidative damage by converting superoxide radicals into H2O2 and O2. This detoxification process, essential for cellular homeostasis, relies on a precisely orchestrated catalytic mechanism involving the copper cation, while the zinc cation contributes to the structural integrity of the enzyme. This study presents the 2.3 Å crystal structure of human SOD1 (PDB ID: 9IYK), revealing an assembly of six homodimers and twelve distinct active sites. The water molecules form a complex hydrogen-bonding network that drives proton transfer and sustains active site dynamics. Our structure also uncovers subtle conformational changes that highlight the intrinsic flexibility of SOD1, which is essential for its function. Additionally, we observe how these dynamic structural features may be linked to pathological mutations associated with amyotrophic lateral sclerosis (ALS). By advancing our understanding of hSOD1's mechanistic intricacies and the influence of water coordination, this study offers valuable insights for developing therapeutic strategies targeting ALS. Our structure's unique conformations and active site interactions illuminates new facets of hSOD1 function, underscoring the critical role of structural dynamics in enzyme catalysis. Besides, we conducted molecular docking analysis using SOD1 for potential radical scavengers and Abl1 inhibitors targeting misfolded SOD1 aggregation along with oxidative stress and apoptosis, respectively. The results showed that CHEMBL1075867, a free radical scavenger derivative, showed the most promising docking results and interactions in the binding site of hSOD1, highlighting its promising role for further studies against SOD1-mediated ALS.
GATA2 Mediates Macrophage Proliferation During Atherosclerosis
Authors: Aktar Amena , Vrieze Angela M , Telesnicki Kiera , Mun Minhyuk , Wodz Kasia , Yin Charles , Cox-Duvall Paisley , Nagpal David , Heit Bryan
Published: February 2025

Atherosclerosis is fueled by the buildup of lipid-laden macrophages within the vascular intima. These macrophages are derived from monocytes that are recruited from the circulation into the developing lesion, where they proliferate and differentiate into macrophages, with proliferation producing most of the macrophages in the resulting lesions. However, the signals and transcriptional events driving the proliferation of atherosclerotic plaque macrophages remain poorly understood. Analysis of human plaque spanning a range of disease severity identified a subpopulation of macrophages that expressed the hematopoietic transcription factor GATA2. These GATA2-expressing macrophages had a transcriptional profile that was intermediary between monocytes and mature macrophages, and selectively upregulated genes associated with proliferation and apoptosis. The expression of GATA2 was concomitant with plaque macrophage proliferation at all stages of disease, with over 90% of proliferating macrophages expressing GATA2. GATA2 was upregulated in macrophages following exposure to oxLDL, with GATA2 expression being necessary and sufficient for the proliferation of these macrophages. In these cells, GATA2 mediates proliferation by upregulating expression of the proto-oncogene MYB, while simultaneously decreasing sensitivity to apoptosis induced by the unfolded protein response. Together, these data identify GATA2 as a transcription factor upregulated by atherogenic stimuli that functions as the primary mediator of macrophage proliferation in atherosclerotic plaque.
BENDER DB: a database of protein binding sites across neglected disease proteomes
Authors: Paiva Vinícius A. , Pires Douglas E. V. , Bressan Gustavo C. , Izidoro Sandro C. , Silveira Sabrina A.
Published: February 2025

Identifying binding sites is crucial for expanding our knowledge of various biological processes, supporting drug discovery, and repositioning strategies, particularly in the early research phases of target hopping. This is especially important for neglected diseases, which predominantly impact vulnerable populations in developing regions. To address this, we created BENDER DB, a database designed to map predicted protein binding sites within the proteomes of pathogens associated with neglected diseases. Utilizing AlphaFold-predicted structures, BENDER DB integrates results from five leading binding site prediction tools, resulting in over one million binding sites across over 100,000 proteins from 10 different proteomes. BENDER DB offers unique features, such as integrating multiple predictors, interactive visualization tools, and comprehensive graphical representations, allowing for a detailed visual comparative analysis of different prediction methods and the predicted binding sites. We combined the computational approaches to offer a consensus of binding site predictions, leveraging the strengths of multiple techniques. Additionally, we introduce BENDER AI, a meta-predictor that combines the outputs of the predictors to provide a unified binding site classification. BENDER AI outperformed all individual methods in 4 out of 5 evaluated metrics. Specifically, it achieved an MCC of 0.64 and an AUC of 0.89, surpassing the highest MCC of 0.63 and the highest AUC of 0.84 obtained by individual predictors, demonstrating strong performance and effectiveness in supporting binding site prediction. By consolidating these results, BENDER DB enables detailed comparative analyses of prediction methods and integrates interactive visualization tools to provide users with an intuitive platform for binding site exploration. The database aims to accelerate drug discovery efforts, particularly in underdeveloped regions, by providing a robust and detailed resource for studying protein binding sites. BENDER DB is available athttps://benderdb.ufv.br.
Gonococcal polarization dynamics during adaptation to low oxygen levels
Authors: Schiefer Paul , Maier Berenike
Published: February 2025

Neisseria gonorrhoeaecan switch between aerobic and microaerophilic lifestyles, but little is known about the dynamics of this transition. In particular, switching from respiration to partial denitrification is likely to affect the electrophysiology of these bacteria. Here, we use a Nernstian dye to measure the membrane potential of single planktonic cells while they continuously consume the oxygen in the growth medium. We show that cells undergo a characteristic pattern of transient depolarization followed by transient hyperpolarization at a critical oxygen concentration, reminiscent of the response to sudden changes in membrane voltage driven by gated ion-channels. Subsequently, the cells depolarize strongly to a near-constant membrane potential. In the presence of nitrite, the cells repolarize after a delay and the repolarization depends on the oxygen-dependent regulatorfnrrequired for denitrification. The temporal behaviour of planktonic cells explains the complex spatio-temporal polarization pattern of the colonies formed byN. gonorrhoeae. This sexually transmitted human pathogen experiences different growth environments during transmission. Our results are an important step towards understanding how electrophysiology adapts to changing environments.
Long-Term Functional Rescue of Trauma-Induced Vision Loss by a Novel, Small Molecule TrkB Activator
Authors: Modgil Shweta , Walker Christopher L. , Chrenek Micah A. , Grossniklaus Hans E. , McDonald Frank E. , Iuvone P. Michael
Published: February 2025

Brain-derived neurotrophic factor (BDNF) signaling through the tropomyosin-related kinase B (TrkB) receptor promotes neuronal growth and survival following an injury. However, its short half-life and pleiotropic effects limit the clinical use of BDNF as a therapy in neurodegenerative disorders. Identification of novel and selective TrkB activators may ameliorate the damage caused to retinal neurons during eye-related injuries, and may reduce adverse visual outcomes associated with visual trauma. We previously described a selective TrkB agonist, N-[2-(5-hydroxy-1H-indol-3-yl) ethyl]-2-oxopiperidine-3-carboxamide (HIOC), that reduces the decline in visual function in a mouse model of ocular trauma (1). Using the lead optimization approach, we subsequently synthesized a fluoropyridine analog of HIOC, 2-fluoro-N-(2-(5-hydroxy-1H-indol-3-yl) ethyl) nicotinamide (HIFN), which also successfully activates TrkB. HIFN is a more potent TrkB activator than the parent compound, HIOC. Further, treatment with HIFN demonstrated neuroprotection in an animal model of overpressure ocular blast injury, ameliorating blast-related visual functional decline. Mice treated with HIFN had better visual acuity, contrast sensitivity, and retinal function supported by enhanced survival of retinal ganglion cells compared to vehicle-treated animals. Moreover, HIFN exhibited better protective effects than HIOC. The therapeutic effects of HIFN were attributed to TrkB activation, as blocking the receptor with a selective receptor antagonist (ANA-12) abrogated the neuroprotection. Together, our results identify HIFN, a novel TrkB receptor activator, as a strategy for decreasing retinal degeneration and progressive vision loss associated with traumatic ocular injury. In addition, this compound may have broader applications treating other diseases with altered TrkB activity.
Machine Learning-Based Model for Behavioral Analysis in Rodents: Application to the Forced Swim Test
Authors: Della Valle Andrea , De Carlo Sara , Sonsini Gregorio , Pilati Sebastiano , Perali Andrea , Ubaldi Massimo , Ciccocioppo Roberto
Published: February 2025

The Forced Swim Test (FST) is a widely used preclinical model for assessing antidepressant efficacy, studying stress response, and evaluating depressive-like behaviours in rodents. Over the last 10 years, more than 5,500 scientific articles reporting the use of the FST have been published. Despite its widespread use, the FST behaviours are still manually scored, resulting in a labor-intensive and time-consuming process that is prone to human bias and variability. Despite eliminating some biases, existing automated systems are costly and typically only able to distinguish between immobility and active behaviours. Therefore, they are often unable to accurately differentiate the major subtypes of movement patterns, such as swimming and climbing. To address these limitations, we propose a novel approach based on machine learning (ML) using a three-dimensional residual convolutional neural network (3D RCNN) that processes video pixels directly, capturing the spatiotemporal dynamics of rodent behaviour. Our ML model was validated against manual scoring in rats treated with fluoxetine and desipramine, two antidepressants known to induce distinct behavioural patterns. The ML model successfully differentiated among swimming, climbing, and immobility behaviours, demonstrating its potential as a standardized and unbiased tool for automatized behavioural analysis in the FST. Subsequently, we successfully validated our model by testing its ability to distinguish between drugs that predominantly evoke climbing (i.e., amitriptyline), those that preferentially facilitate swimming (i.e., paroxetine), and those that evoke both in a more balanced manner (i.e., venlafaxine). This approach represents a significant advancement in preclinical research, providing a more accurate and efficient method to analyze forced swimming data in rodents. We anticipate that due to its adaptability, in addition to the FST, the model could be applied to various behavioural tests in laboratory animals.
BRCA1-A and LIG4 complexes mediate ecDNA biogenesis and cancer drug resistance
Authors: Chung Oliver W. , Yao Shun , Yang Fu , Wang Ling , Cerda-Smith Christian , Hutchinson Haley M. , Wood Kris C. , Su Weijia , Khasraw Mustafa , Zou Lee , Ramsden Dale A. , Zhang ZZ Zhao
Published: February 2025

Extrachromosomal circular DNA (ecDNA) are commonly produced within the nucleus to drive genome dynamics and heterogeneity, enabling cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes mediate ecDNA production. Following DNA fragmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively catalyzes ecDNA production inDrosophilaand mammals, with patient tumor ecDNA harboring junctions marked by LIG4 activity. Notably, disrupting LIG4 or BRCA1-A in cancer cells impairs ecDNA-mediated adaptation, hindering resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers new therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.
Tumor Treating Fields inhibit cell proliferation and promote immunogenic cell death in biliary tract cancer cells
Authors: Yue Ying , Wang Yingying , Yao Min , Suo Yuanzhen
Published: February 2025

Tumor Treating Fields (TTFields) are low-intensity, intermediate-frequency alternating electric fields that exert antimitotic effects on cancer cells. This study evaluated the in vitro efficacy of TTFields on biliary tract cancer (BTC) cell lines HCCC-9810 and RBE, investigating their sensitivity to TTFields across varying frequencies and electric field intensities. The results demonstrated that a frequency of 150 kHz induced the most pronounced cytotoxic effects, significantly impairing clonogenicity and migratory capacity while inducing abnormal mitotic processes. Furthermore, TTFields treatment triggered a marked upregulation of immunogenic cell death (ICD) biomarkers, including enhanced surface exposure of calreticulin (CRT) and increased extracellular release of high mobility group box 1 (HMGB1) and ATP. These findings indicate that TTFields represent a promising therapeutic approach for BTC, not only by suppressing tumor proliferation but also by enhancing ICD.
Epigenetic regulation of visual system remodeling during flatfish metamorphosis: DNA methylation dynamics in ocular migration and visual adaptation
Authors: Guerrero-Peña Laura , Suarez-Bregua Paula , Sánchez-Baizán Nuria , Piferrer Francesc , Tena Juan J. , Rotllant Josep
Published: February 2025

Flatfish metamorphosis is characterized by extensive tissue remodeling, associated with a transition from pelagic to benthic lifestyle, being the migration of one eye the most dramatic change. Epigenetic mechanisms exert a pivotal role in developmental programs. This study investigates the DNA methylation profiles of migrating and non-migrating eyes using reduced-representation bisulfite sequencing (RRBS) during three developmental stages of turbot: pre-metamorphosis, climax and post-metamorphosis. Over 31% of all identified regions were hypermethylated during climax stage in both eyes, coinciding with elevated expression of thednmt3agene, responsible for de novo methylation. Additionally, transcription factors crucial for retinal ganglion cells (RGCs) development, including theeomesaandtbr1b, exhibited differential methylation and expression between the migrating and non-migrating eye during the climax phase. These findings underscore the significance of DNA methylation in the intricate remodeling of the visual system during turbot metamorphosis, particularly regarding RGC-mediated ocular migration and the transmission of visual signals.
C.La.P.: Enhancing transformer-based genomic signal modeling by integrating DNA sequences and chromatin accessibility data
Authors: Nisantzis Panos Firbas , Gonçalves Carolina , de Polavieja Gonzalo
Published: February 2025

Transformers have shown promise in chromatin modeling but have primarily relied solely on reference DNA sequences, limiting their utility across multiple biological contexts. In this work we enhance transformer models by integrating reference sequences with ATAC-seq, a chromatin accessibility assay. Our Chromatin LAnguage Processing (CLaP) model combines a convolutional tokenizer, a transformer encoder, and task-specific components to predict multiple genomic signals from a single input. After pre-training with masked nucleotide prediction, CLaP achieved per-token F1 scores exceeding 0.8 for three target ChIP-seq assays in fine-tuning. Attention mechanism analysis revealed that CLaP detects CTCF binding sites with nucleotide-level precision by learning the sequence preference of the CTCF factor and the characteristic ATAC-seq patterns that are caused by protein binding events. Additionally, CLaP predicts protein-DNA binding events not captured by the ChIP-seq ground truth. These findings highlight CLaP’s potential to expand chromatin modeling by incorporating molecular assay data alongside sequence information.
Graph-based Contrastive Learning Enables Unified Integration and Niche Transfer Across Single-Cell and Spatial Multi-Omics
Authors: Zhou Weige , Fan Xueying , Li Lanxiang , Zheng Jianrong , Liu Xiaodong , Jin Wenfei , Tian Luyi
Published: February 2025

The rapid growth of single-cell and spatial omics has outpaced computational methods capable of unifying these data into a cohesive framework for tissue atlas construction and cross-sample analysis. A critical bottleneck lies in the inability of existing tools to co-embed cells from diverse technologies-spanning transcriptomics, epigenomics, and proteomics-into a shared reference space while preserving spatial architecture and molecular specificity. Here, we present Garfield (Graph-based Contrastive Learning Enables Fast Single-Cell Embedding), a geometric deep-learning framework that addresses these challenges through spatially or molecularly aware cell embedding. Leveraging a graph contrastive learning framework, Garfield learns a shared embedding space for data generated by diverse technologies, enabling seamless construction and querying of spatial reference atlases. Our results show that Garfield consistently outperforms state-of-the-art benchmark models in identifying spatial niches across multiple datasets. We further demonstrate Garfield's versatility by applying it to multimodal spatial data, including gene expression and chromatin accessibility, where it successfully identifies distinct niches in the mouse brain. Notably, Garfield reveals tumor microenvironment heterogeneity in non-small cell lung cancer and breast cancer, uncovered conserved, barrier-like immune niches at tumor margins orchestrating CD80-mediated T cell-B cell-dendritic cell interactions and IFN-y/B cell activation pathways, forming spatially coordinated immune surveillance hubs. These findings underscore Garfield's potential to advance spatial omics research by offering a robust, scalable solution for integrating and interpreting complex spatial data across diverse tissue types and modalities.
Target-Specificity and Repeatability in Neuro-Cardiac-Guided TMS for Heart-Brain Coupling
Authors: Feng Zi-Jian , Martin Sandra , Numssen Ole , Weise Konstantin , Jing Ying , Gerardos Gerasimos , Martin Carla , Hartwigsen Gesa , Knösche Thomas R.
Published: February 2025

The dorsolateral prefrontal cortex (DLPFC) is a principal target for repetitive transcranial magnetic stimulation (rTMS) in the treatment of major depressive disorder, with therapeutic effects hypothesized to be mediated by the connectivity between the DLPFC and the subgenual anterior cingulate cortex (sgACC). Interestingly, these depression-related hubs are also part of the heart-brain axis, thus potentially rendering the stratification of individual depression targets possible by tapping into short-term heart rate modulation after DLPFC stimulation. Recently, a set of stimulation protocols has been proposed to objectively quantify these downstream effects. While these neuro-cardiac-guided TMS protocols (NCG-TMS) are promising to improve clinical responses, rigorous, third-party assessments of these approaches including replicability, robustness, and the impact of stimulation side-effects are critically missing. Here, we employed a 10 Hz TMS protocol (NCG-TMS 2.0) across three sessions to evaluate the effects of stimulation intensity and DLPFC target specificity on heart–brain coupling (HBC) in a cohort of healthy participants. Our results demonstrate a significant nonlinear increase in HBC with higher stimulation intensity, with the F3 lateral and F3 posterior targets eliciting more robust effects than the sham condition. For the first time, we are able to demonstrate reliable target- and intensity-specific HBC modulation across multiple NCG-TMS 2.0 sessions. Although the repeatability within subjects was limited when including the initial session, subsequent sessions yielded consistent results for the F3 anterior and F3 lateral targets at higher intensities. Although pain and other side effects influenced HBC, they did not fully account for the observed modulation of HBC. These findings underscore the critical role of spatial specificity and stimulation intensity in modulating heart–brain interactions and offer a potential framework for optimizing individualized rTMS treatment protocols for depression.
Acylation of the RTX Toxin MbxA stimulates host membrane disruption through a specific interaction with cholesterol
Authors: Chacko Feby M , Ganz Sarah M , Pfitzer-Bilsing Anne , Haensch Sebastian , Westhoff Philipp , Weidtkamp-Peters Stefanie , Reichert Sander HJ , Exterkate Marten , Schmitt Lutz
Published: February 2025

RTX toxins, characterized by their calcium-binding glycine-rich repeat regions, are known for their ability to disrupt host cell membranes. Although the acylation of specific lysine residues in these toxins is established as crucial for their hemolytic activity, the precise mechanisms underlying this enhancement remain unclear. In this study, we explore the role of acylation in the pore-forming behaviour of the RTX toxin MbxA by comparing the lytic activities of acylated MbxA and its non-acylated form, proMbxA, onin vitromembrane systems, as well as living cells. Our findings demonstrate that a cholesterol specific interaction promotes MbxA-induced membrane disruption in an acylation-dependent manner. Further analysis revealed that acylation is not necessary for initial membrane binding but markedly enhances pore formation. Overall, our results offer valuable insights into the molecular determinants that regulate RTX toxin activity, highlighting a complex interplay between lipid composition (sterols), acylation, and membrane disruption, thereby advancing our understanding of RTX toxin pathogenesis
In vitroandin vivoAntiviral Activity of the Acyclic Nucleoside Phosphonate Prodrug LAVR-289 against Poxvirus and African Swine Fever Virus Replication
Authors: Marcheteau Elie , Mosca Estelle , Frenois-Veyrat Gaelle , Kappler-Gratias Sandrine , Boutin Laetitia , Top Sokunthea , Mathieu Thomas , Colas Cyril , Favetta Patrick , Garnier Tony , Barbe Peggy , Keck Mathilde , Gillet Daniel , Mas Alicia , Alejo Ali , Yu Yinyi , Toth Karoly , Abate Getahun , Roy Vincent , Skerry Janet , Wetzel Kelly S , Joshua Shamblin D , Joseph Golden W , Panchal Rekha G , Mucker Eric M , Mudhasani Rajini , Agrofoglio Luigi A. , Iseni Frederic , Gallardo Franck
Published: February 2025

Poxviruses are double-stranded DNA viruses including relevant zoonotic pathogens with high morbidity. Although African swine fever virus (ASFV) belongs to theAsfarviridaefamily and is not strictly classified as a member of thePoxviridae, both fall within the same class ofPokkesviricetesthat replicate in the cytoplasm, and some poxviruses pose potential biological warfare threats. Among compounds targeting these viruses, acyclic nucleoside phosphonate prodrugs are nucleoside analogues inhibitors of viral DNA polymerases that have been identified as promising agents. However, some limitations related to their toxicity and the rapid emergence of resistance highlight the need for new antiviral molecules. In this study, the new nucleoside analogue LAVR-289 was shown to effectively inhibit the viral replication by intervening early in the viral replication step, targeting a specific domain of the poxvirus DNA polymerase. Using monkeypox virus models, the subcutaneous or oral administration of LAVR-289 demonstrates protective efficacy in infected animal models without toxicity or behavioral modification. The stabilityin vivo, long shelf-life and efficacy make LAVR-289 a promising candidate for further development and stockpiling as a medical countermeasure against dsDNA virus outbreaks. Its broad-spectrum efficacy is a real asset in a context of recurrent viral epidemics, risk of bioterrorism and emergence of resistance strains in the population.

Highlights

LAVR-289 is a unique acyclic nucleoside phosphonate prodrug targeting viral DNA polymerases.

LAVR-289 displays antiviral activity against dsDNA viruses, ASFV and poxviruses.

First report ofin vivoevaluation of LAVR-289 against MPXV by subcutaneous and oral administration.

LAVR-289 reduces clinical signs and increase survival in animal models.
Discovery of non-opioid receptor protein targets of fentanyl and remifentanil by affinity-based protein profiling in diverse animal model and human tissues
Authors: Lin Vivian S. , Suazo Kiall F. , Kim Doo Nam , Leach Damon T. , Sveistyte Agne , Walker Jennifer , Gorham Leo J. , Schultz Katherine J. , Mo Kai-For , Callister Stephen J. , Stratton Kelly G. , Lomas Gerard X. , Nelson William C. , Colburn Heather A. , Paurus Vanessa L. , Lalli Priscila M. , Moore Ronald J. , Powell Samantha M. , Rodriguez Oscar , Cort John R. , Wright Aaron T.
Published: February 2025

Synthetic opioids such as fentanyl and related analogs have been widely used for pain management. However, their negative side effects, including respiratory depression and high potential for addiction, underscore the need for a deeper understanding of fentanyl’s interactions with proteins throughout the human body. Fentanyl analogs bind and activate opioid receptors in the central and peripheral nervous systems, triggering numerous downstream signaling pathways. Increasingly, fentanyl has been shown to interact with non-opioid receptors, and elucidation of these non-canonical fentanyl-protein interactions may provide insights into the mechanisms contributing to fentanyl’s adverse effects and illuminate novel countermeasure strategies. To identify proteins in mammalian tissues that may interact with fentanyl, we designed and synthesized three affinity-based probes (AfBPs) that include the fentanyl core and feature a diazirine photoaffinity group and alkyne handle for click chemistry at different positions. Molecular docking simulations predicted that these AfBPs bind the mu opioid receptor similarly to fentanyl.

Affinity-based protein profiling using the FA-T1 probein vitroin tissues from six animal species identified histamine N-methyltransferase (HNMT), endophilin-B1 (SH3GLB1), fructosamine-3-kinase (FN3K), cutA divalent cation tolerance analog (CUTA), and monoamine oxidase B (MAOB) among the top proteins that bind fentanyl in multiple species and tissue types. Molecular docking of fentanyl and remifentanil with these protein structures identified putative binding sites. The interaction of fentanyl with specific proteins was empirically assessed through protein structural analyses. These findings highlight potential fentanyl-protein interactions that may contribute to the acute and long-term impacts of fentanyl exposures.
Chemical inhibition of a bacterial immune system
Authors: Zang Zhiyu , Duncan Olivia K. , Sabonis Dziugas , Shi Yun , Miraj Gause , Fedorova Iana , Le Shuai , Deng Jun , Zhu Yuhao , Cai Yanyao , Zhang Chengqian , Arya Garima , Duerkop Breck A. , Liang Haihua , Bondy-Denomy Joseph , Ve Thomas , Tamulaitiene Giedre , Gerdt Joseph P.
Published: February 2025

The rise of antibiotic resistance motivates a revived interest in phage therapy. However, bacteria possess dozens of anti-bacteriophage immune systems that confer resistance to therapeutic phages. Chemical inhibitors of these anti-phage immune systems could be employed as adjuvants to overcome resistance in phage-based therapies. Here, we report that anti-phage systems can be selectively inhibited by small molecules, thereby sensitizing phage-resistant bacteria to phages. We discovered a class of chemical inhibitors that inhibit the type II Thoeris anti-phage immune system. These inhibitors block the biosynthesis of a histidine-ADPR intracellular ‘alarm’ signal by ThsB and prevent ThsA from arresting phage replication. These inhibitors promiscuously inhibit type II Thoeris systems from diverse bacteria—including antibiotic-resistant pathogens. Chemical inhibition of the Thoeris defense improved the efficacy of a model phage therapy against a phage-resistant strain ofP. aeruginosain a mouse infection, suggesting a therapeutic potential. Furthermore, these inhibitors may be employed as chemical tools to dissect the importance of the Thoeris system for phage defense in natural microbial communities.
RNA-guided nucleases enable a gene drive of insertion sequences in plasmids
Authors: Mears Kepler S. , Rossine Fernando W. , Quinones-Olvera Natalia , Souque Célia , Baym Michael
Published: February 2025

Mobile genetic elements (MGEs) and the interactions between them are a major source of evolutionary innovation. Insertion sequences, the simplest MGEs usually encoding only the necessary genes for transposition and maintenance, are widespread in bacterial genomes, and are particularly common in plasmids. Plasmids, self-replicating extrachromosomal DNA elements, often exist in multiple copies imparting a stochastic barrier to the fixation of an insertion sequence by limiting the proportion of the plasmid population harboring the IS. In this work we demonstrate that to overcome this, the IS200/605 family of insertion sequences utilizes programmable RNA guided nucleases as gene drive to spread the IS through the plasmid population. TnpB, the likely ancestor of Cas12, records the specific insertion site of the IS in its RNA guide to prevent loss of the IS during transposition. When introduced to a plasmid TnpB will be reprogrammed to target and cleave IS-plasmids, resulting in biased replication of IS+ plasmids. Furthermore, the gene drive activity is critical for the IS to invade high copy plasmid populations. Because TnpB can only be mobilized between microbes on other mobile genetic elements, this advantage to fixing in plasmids may help explain the prevalence of TnpB across the tree of life. More generally, the unique pressures arising from movement between genetic contexts with different multiplicities shapes the evolution of strategies for MGE spread.
AnNlrp5-null mutation leads to attenuatedde novomethylation in oocytes, accompanied by a significant reduction in DNMT3L
Authors: Nic Aodha Leah , Pokhilko Alexandra , Rosen Leah U , Galatidou Styliani , Walewska Edyta , Belton Christian , Galvao Antonio , Okkenhaug Hanneke , Yu Lu , Nakhuda Asif , Mansfield Bill , Khan Soumen , Oxley David , Barragán Montserrat , Kelsey Gavin
Published: February 2025

Nlrp5encodes a core component of the subcortical maternal complex (SCMC) a cytoplasmic protein structure unique to the mammalian oocyte and cleavage-stage embryo.NLRP5mutations have been identified in patients presenting with early embryo arrest, recurrent molar pregnancies and imprinting disorders. Correct patterning of DNA methylation over imprinted domains during oogenesis is necessary for faithful imprinting of genes. It was previously shown that oocytes with mutation in the human SCMC geneKHDC3Lhad globally impaired methylation, indicating that integrity of the SCMC is essential for correct establishment of DNA methylation at imprinted regions. Here, we present a multi-omic analysis of anNlrp5- null mouse model, which in GV oocytes displays a misregulation of a broad range of maternal proteins, including proteins involved in several key developmental processes. This misregulation likely underlies impaired oocyte developmental competence. Amongst impacted proteins are several epigenetic modifiers, including a substantial reduction in DNMT3L; we show thatde novoDNA methylation is attenuated inNlrp5-null oocytes. This provides evidence for mechanisms leading to downstream misregulation of imprinted genes, which in turn, may result in imprinting syndromes, multi-locus imprinting disturbances (MLID) and hydatidiform moles.
Cell type-specific epigenetic regulatory circuitry of coronary artery disease loci
Authors: Hecker Dennis , Song Xiaoning , Baumgarten Nina , Diagel Anastasiia , Katsaouni Nikoletta , Li Ling , Li Shuangyue , Kumar Maji Ranjan , Behjati Ardakani Fatemeh , Ma Lijiang , Tews Daniel , Wabitsch Martin , Björkegren Johan L.M. , Schunkert Heribert , Chen Zhifen , Schulz Marcel H.
Published: February 2025

Coronary artery disease (CAD) is the leading cause of death worldwide. Recently, hundreds of genomic loci have been shown to increase CAD risk, however, the molecular mechanisms underlying signals from CAD risk loci remain largely unclear. We sought to pinpoint the candidate causal coding and non-coding genes of CAD risk loci in a cell type-specific fashion. We integrated the latest statistics of CAD genetics from over one million individuals with epigenetic data from 45 relevant cell types to identify genes whose regulation is affected by CAD-associated single nucleotide variants (SNVs) via epigenetic mechanisms. Applying two statistical approaches, we identified 1,580 genes likely involved in CAD, about half of which have not been associated with the disease so far. Enrichment analysis and phenome-wide association studies linked the novel candidate genes to disease-specific pathways and CAD risk factors, corroborating their disease relevance. We showed that CAD-SNVs are enriched to regulate gene expression by affecting the binding of transcription factors (TFs) with cellular specificity. Of all the candidate genes, 23.5% represented non-coding RNAs (ncRNA), which likewise showed strong cell type specificity. We conducted a proof-of-concept biological validation for the novel CAD ncRNA geneIQCH-AS1. CRISPR/Cas9-based gene knockout ofIQCH-AS1, in a human preadipocyte strain, resulted in reduced preadipocyte proliferation, less adipocyte lipid accumulation, and atherogenic cytokine profile. The cellular data are in line with the reduction ofIQCH-AS1in adipose tissues of CAD patients and the negative impact of risk alleles on its expression, suggestingIQCH-AS1to be protective for CAD. Our study not only pinpoints CAD candidate genes in a cell type-specific fashion but also spotlights the roles of the understudied ncRNA genes in CAD genetics.