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Results for “molecular biology”

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bioRxiv2025

Active Gaussian Network Model: a non-equilibrium description of protein fluctuations and allosteric behavior

Understanding the link between structure and function in proteins is fundamental in molecular biology and proteomics. A central question in this context is whether allostery -- where the binding of a molecule at one site affects the activity of a distant site -- emerges as a further manifestation of the intricate interplay between structure, function, and intrinsic dynamics. This study explores how allosteric regulation is modified when intrinsic protein dynamics operate under out-of-equilibrium conditions. To this purpose, we introduce a simple nonequilibrium model of protein dynamics, inspired by active matter systems, by generalizing the widely employed Gaussian Network Model (GNM) to incorporate non-thermal effects. Our approach underscores the advantage of framing allostery as a causal process by using, as a benchmark system, the second PDZ domain of the human phosphatase hPTP1E that mediates protein-protein interactions. We employ causal indicators, such as response functions and transfer entropy, to identify the network of PDZ2 residues through which the allosteric signal propagates across the protein structure. These indicators reveal specific regions that align well with experimental observations. Furthermore, our results suggest that deviations from purely thermal fluctuations can significantly influence allosteric communication by introducing distinct timescales and memory effects. This influence is particularly relevant when the allosteric response unfolds on timescales incompatible with relaxation to equilibrium. Accordingly, non-thermal fluctuations may become essential for accurately describing protein responses to ligand binding and developing a comprehensive understanding of allosteric regulation.

biophysics↗

bioRxiv2025

The Filterprep: A Simple and Efficient Approach for High-Yield, High-Purity Plasmid DNA Purification

Plasmid DNA purification remains a fundamental yet resource-intensive step in molecular biology and biotechnology. Conventional protocols often yield plasmids with suboptimal purity, while commercial kits, though efficient, are costly and use proprietary formulations that limit transparency and customization. Here, we present Filterprep, a hybrid method combining classical ethanol precipitation with a single spin-column cleanup step, enabling rapid recovery of high-purity plasmid DNA with yields notably higher than those obtained using commercial kits, in approximately 40 minutes. Filterprep offers a transparent, cost-effective, and scalable alternative, simplifying workflows and reducing hands-on time without compromising DNA quality. The purified plasmids are compatible with a variety of downstream applications, including mammalian cell transfection, protein expression, and reporter assays. With its balance of efficiency and practicality, Filterprep is well suited for high-throughput laboratories and resource-limited settings.

biochemistry↗

bioRxiv2025

A miniaturized, high-throughput buffer-centric method for protein solubility screening

Efficient access to soluble recombinant proteins remains a major bottleneck in biochemical and structural studies. We describe an aqueous, solvent-centric, fully miniaturized 96-well workflow to screen extraction conditions that preserve soluble recombinant protein during lysis and clarification in a single working day. Liquid-nitrogen-frozen E. coli pellets are cryogenically bead-milled with stainless-steel beads, retaining the native intracellular milieu while ensuring uniform disruption. The resulting wet, frozen cell powder can therefore be extracted with user-defined solvent, enabling systematic exploration of pH, ionic strength, detergents, and chaotropes. Protein solubility is assessed by a 1 {micro}L chromogenic anti-His dot-blot. We demonstrate the use of the protocol by solubilizing a set of highly challenging de novo-generated proteins and show that dot-blot intensity provides a practical semi-quantitative proxy for successful extraction of soluble proteins. We also provide experimentally supported guidelines on the influence of solvent reagents on subsequent steps of protein production, SDS-PAGE, and Ni-NTA purification. This workflow is compatible with upstream genetic solubility-enhancement, chassis- and cultivation-based strategies and enables direct transition from screening hits to scale-up. Because the workflow uses standard molecular biology equipment and inexpensive consumables, it can be readily adopted or automated in most laboratories.

biochemistry↗

bioRxiv2025

DCX Enhances Glioblastoma Metabolism Through Synergistic Regulation of Glutamine Synthesis and Metabolism-Related Genes for Cellular Homeostasis

Gliomas are the most common primary intracranial tumors, comprising 81% of malignant brain tumors, and currently lack effective therapies. Recent advances in molecular biology have shown that cancer cells exploit microtubule-associated proteins (MAPs) under stress to activate various signaling pathways. This study investigates the role of Doublecortin (DCX) in glioma metabolism and its impact on tumor proliferation. In this study, CRISPR-engineered glioma models with DCX overexpression or knockdown were analyzed using integrated genomic, transcriptomic, and metabolomic approaches. Metabolic activity was assessed via RNA sequencing, Seahorse assays, and targeted mass spectrometry. Pharmacological inhibition of key pathways validated functional dependencies. We demonstrate that gliomas enriched with DCX exhibit elevated glycolytic activity while also relying on cellular respiration and oxidative phosphorylation (OXPHOS) for energy to support the abnormal proliferation of glioma cells. Upon integrative analysis of enriched genes and proteins, we observed genetic and metabolome-level signatures associated with differences in central carbon and energy metabolism in CRISPR-modified glioma cells expressing high DCX. Whole-genome transcriptome analysis revealed enriched metabolic entities promoting hydrolysis of glutamine and glutaminolysis in glioma cells and inhibition of selected differentially enriched genes with small molecule inhibitors abrogated metabolic enrichment and resulted in reduced energy levels and protein translation required for aberrant growth. Finally, we establish that DCX stimulates glutaminolysis to regulate homeostasis for energy supplements in glioma cells. Targeting DCX-mediated metabolic pathways may provide a novel therapeutic approach for glioblastoma, highlighting the potential for innovative treatments in this challenging disease.

cancer biology↗

bioRxiv2025

Post-translational modifications of microtubules are crucial for malaria parasite transmission

Microtubules, composed of - and {beta}-tubulin polymers, are essential components of the eukaryotic cytoskeleton. They maintain cellular shape and structural integrity and play critical roles in cell division and in intracellular vesicular transport. In Plasmodium, the parasite that causes malaria, nuclear replication during the liver stage is among the fastest known in eukaryotic cells and relies heavily on microtubules for DNA segregation and cytoskeletal organization. Despite their importance, the role of microtubules in liver stage development remains largely unexplored. Here, we investigated microtubule dynamics during liver stage development using a combination of cell and molecular biology techniques, expansion microscopy, and live-cell imaging. By employing antibodies specific for -tubulin post-translational modifications (PTMs), we found that the Plasmodium sporozoites subpellicular microtubules (SSPM) persist during liver infection, giving rise to liver stage parasite microtubule bundles (LSPMB). These LSPMB form multimeric tubulin structures within hepatocytes and are redistributed to the hemi-spindle poles of parasite nuclei during schizogony. Deletion of the C-terminal region encompassing all known Plasmodium -tubulin PTM sites prevented sporozoite migration from the mosquito midgut to the salivary glands, effectively blocking parasite transmission. Using Plasmodium microtubule-specific depolymerisation drugs, we found that while LSPMB are stable in sporozoites, they exhibit dynamic behavior during hepatocyte infection. Given the regulatory role of PTMs in microtubule dynamics, we generated parasite mutants by substituting and deleting key -tubulin C-terminal residues involved in PTMs. Substitution of the polyglutamylation site with alanine and deletion of the C-terminal tyrosination/detyrosination motifs impaired parasite growth during liver infection. Together, our findings reveal extensive microtubule remodeling during liver stage development and establish -tubulin C-terminal modifications as critical regulators of both intracellular development and parasite transmission of Plasmodium parasites.

cell biology↗

bioRxiv2025

RtmR is a membrane-embedded RRM-family RNA-binding protein that regulates biofilm formation

The animal symbiont Vibrio fischeri has served as a model organism for molecular processes underlying bacterial group behaviors, including quorum sensing and biofilm development. Here, using a genetic approach to identify negative regulators of biofilm formation in V. fischeri, we identified a membrane-bound RNA-binding protein, RtmR (VF_2432), that acts as an inhibitor of the symbiosis polysaccharide (SYP) biofilm. Membrane localization of the protein seems to be required for protein stability or expression, as truncation of the transmembrane helices led to an inability to detect the protein. The conserved RNP1 and RNP2 motifs in RtmRs cytoplasmic RNA recognition motif (RRM) domain are required for function, and we demonstrate binding to RNA substrates. Identification of RtmR RNA ligands was conducted with a iiCLIP-seq approach that revealed a large interactome. One transcript identified was that of the biofilm regulatory histidine kinase RscS. We found that RtmR biofilm inhibition depends on RscS activity and that RtmR negatively regulates levels of RscS. Overall, this work characterizes a novel type of bacterial RNA-binding protein. IMPORTANCEBacterial RNA-binding proteins (RBPs) perform key functions to regulate stress responses and development. Bacterial RBPs including the RNA chaperones Hfq and ProQ, the global regulator CsrA, and the cold shock proteins (Csps) have been extensively studied, although additional classes of RBPs have been predicted by bioinformatic methods including those carrying an RRM domain. This work expands on recent studies of RRM domain proteins in bacteria to characterize a membrane-bound RRM protein that regulates bacterial biofilm development. Given our rapidly-expanding knowledge regarding the role for RNA-binding proteins in bacterial molecular biology, this work contributes a new class of membrane-bound regulators with homologs in human pathogens and marine symbionts.

microbiology↗

bioRxiv2025

An Automated End-to-End Workflow for Production of Secreted Proteins in Transfected Mammalian Cells

The advancement of automation technologies has helped to enable a surge in large-scale screening efforts across fields such as molecular biology, protein biochemistry, cell biology, and structural biology. In the context of this "omics"-driven research, there is a need to generate automation platforms that are more flexible and less expensive, so that they can be utilized for basic research conducted by small groups. A key challenge in automation lies in developing methods that can replicate fine motor techniques that are normally performed manually by researchers at the bench. We are engaged in a large-scale project to map interactions among human cell-surface and secreted proteins and assess their effects on cells. This project involves production of a library of more than 2000 recombinant His-tagged fusion proteins secreted from transfected Expi293 cells. To execute such a project with a small group at an academic institution required construction of an affordable automated system that could also be used by other investigators. This led us to develop a high-throughput, 96-well format automation platform for end-to-end protein production. The workflow includes transformation of E. coli, plasmid DNA preparation, transient transfection, protein purification, desalting and buffer exchange, protein quantification, and normalization of protein concentrations, resulting in assay-ready proteins. The system is built around an in-house engineered modular robotic platform that integrates liquid handling with a suite of interchangeable plug-and-play mobile enclosed device modules. Housed within a BSL-2 sterile environment, the platform enables flexible, fully automated workflows and can be readily customized for diverse user-defined protocols.

biochemistry↗

bioRxiv2025

Morphological and MALDI-TOF MS identification of freshwater snails, including intermediate hosts of schistosomes in Gabon.

A thorough knowledge of intermediate snail hosts and their geographical distribution is essential to understanding the transmission of schistosomiasis and to inform interventions in Gabon. Accurate identification of both snails species and Schistosoma species is therefore essential. The aim of this study was to identify the intermediate hosts of Schistosoma spp. (snails) using MALDI-TOF MS. MethodsSnails were collected from various sites across Gabon and were morphologically identified to species or genus level. Snail species were randomly selected, dissected and subjected to MALDI-TOF MS and molecular analysis. Parasites were detected by cercarial emission, followed by qPCR analysis and sequencing. ResultsA total of 3,722 snails were collected and morphologically identified as Bulinus truncatus (n=1845), Bulinus forskalii (n=381), Gyraulus costulatus (n=680), Bellamya sp. (n=67) and Melania sp. (n=749). Of the 3,722 snails collected, 21.2% (n = 791) were selected for further analysis, including Bu. truncatus (n = 301), Bu. forskalii (n = 158), and Gy. costulatus (n=147). A subset of 65 snails was subjected to molecular identification to confirm morphological classification, using COI, ITS, and 18S gene. BLAST analysis of the resulting sequences showed 97-100% identity with reference sequences of Bu. truncatus, Bu. forskalii, and Gy. costulatus available in GenBank. Analysis of the MS spectra from the 791 snails revealed that 78.2% (n = 618) were of high quality, with signal intensities exceeding 3,000 arbitrary units (a.u.). The MALDI-TOF MS reference database was subsequently updated with 12 high-quality spectra, comprising four representative spectra for each snail species. A blind test of 606 remaining spectra against the database. A blind test of the remaining 606 spectra, using the updated MALDI-TOF MS reference database, demonstrated reliable identification of 100% (Bulinus forskalii, 147/147; Gyraulus costulatus, 158/158) and 96% (Bulinus truncatus, 289/301) of the specimens. These results were consistent with morphological identification, with log score values (LSVs) ranging from 1.7 to 2.8. Principal component analysis (PCA) showed that Bulinus species clustered according to their geographical origin. Overall, 17,66% of the snails analysed were found to be positive for Schistosoma sp. Further analysis, including qPCR and sequencing, revealed that the snails were infected with Schistosoma haematobium. ConclusionWe report the first data on the identification of snails by MALDI-TOF MS in Gabon and show that Bu. truncatus and Bu. forskalii are potential intermediate hosts of S. haematobium. We also show a heterogeneous spatiotemporal distribution of snails in south-eastern Gabon. The role of G. costulatus in the transmission of schistosome remains unclear. Author SummarySchistosomiasis is a disease caused by trematodes of the genus Schistosoma. Freshwater snails of the genera Biomphalaria, Bulinus and Oncomelania release infectious cercariae, which are transmitted to the final host (human or animal). It is the second most widespread parasitic disease after malaria, and among the most important neglected tropical diseases in the world. Snail identification is extremely important for monitoring snail populations and schistosomiasis. Identification has long relied on morphological criteria and molecular biology, both of which have several drawbacks. However, numerous studies have reported the performance of MALDI-TOF MS, a technology that allows species to be identified based on their proteins, as a reliable, rapid and easy-to-use tool in many fields. Recently, it has been described as an effective tool for snail identification and even for tracing the origin of the intermediate hosts of schistosomiasis. The objective of this study was to accurately identify freshwater snails, including the intermediate hosts of schistosomiasis in Gabon.

ecology↗

bioRxiv2025

Investigating Cannabis sativa L. gene expression through housekeeping genes and gene coexpression networks

Cannabis sativa L. is a versatile crop with applications ranging from medicinal products to industrial materials. Despite growing interest in cannabis transcriptomics, comprehensive studies of gene expression in this species remain limited. Here, we explore cannabis transcriptomics through two complementary approaches: the identification and prioritization of housekeeping (HK) genes for qRT-PCR normalization and the construction of a genome-wide gene coexpression network (GCN) using publicly available RNA-Seq data. Based on expression stability criteria (i.e. log mean TPM [≥] 6, log variance < 0.3), we proposed the optimal HK genes and designed qRT-PCR primers for them. Primer specificity was verified in silico using the Jamaican Lion mother + Y genome. In parallel, to explore the non HK gene expression, we constructed a GCN comprising 32 coexpression modules, which was then analyzed for tissue-specific expression patterns, transcription factor (TF), and functional enrichment. Several modules were significantly correlated with cannabis tissues such as flowers and bast fibres and were linked to key processes, including photoperiod sensitivity, stress responses, and vegetative-to-reproductive phase transitions. Together, our results help understand the functions and maximize the use of HK genes in molecular biology and reveal coexpression modules that constitute critical genetic circuits underlying cannabis physiology.

plant biology↗

bioRxiv2025

A scalable computational framework for predicting gene expression from candidate cis-regulatory elements

Deciphering the relationships between cis-regulatory elements (CREs) and target gene expression has been a long-standing unsolved problem in molecular biology, and the dynamics of CREs in different cell types make this problem more challenging. To address this challenge, we propose a scalable computational framework for predicting gene expression (ScPGE) from discrete candidate CREs (cCREs). ScPGE assembles DNA sequences, transcription factor (TF) binding scores, and epigenomic tracks from discrete cCREs into 3-dimensional tensors, and then models the relationships between cCREs and genes by combining convolutional neural network with transformer. Compared with current state-of-the-art models, ScPGE exhibits superior performance in predicting gene expression and yields higher accuracy in identifying active enhancer-gene interactions through attention mechanisms. By comprehensively analyzing ScPGEs predictions, we find a pattern in true positives (TPs) that the regulatory effect of cCREs on genes decreases with distance. Inspired by the pattern, we design two methods to enhance the ability to capture distal cCRE-gene interactions by incorporating chromatin loops into the ScPGE model. Furthermore, ScPGE accurately discovers some crucial TF motifs within prioritized cCREs and reveals the different regulatory types of these cCREs.

bioinformatics↗

bioRxiv2025

Sex-specific hypothalamic axis disruption in the rNLS8 mouse model of amyotrophic lateral sclerosis (ALS)

Sex-related differences have gained increasing attention in recent years due to evidence of varying prevalence, pathophysiology, survival rates, and disease progression between males and females in amyotrophic lateral sclerosis (ALS). Differences in brain metabolism between sexes in ALS patients have also been reported; however, the specific molecular mechanisms remain poorly understood. As growing evidence supports a strong metabolic component in ALS, this study investigates alterations in leptin, one of the key regulators of metabolism, that is known to be altered during ALS progression in rNLS8 mice, a transgenic mouse model of ALS which closely recapitulates the TAR DNA-binding protein (TDP-43) pathology observed in most patients with ALS. We also examined changes in hypothalamic neuronal genes involved in metabolic regulation through food intake in rNLS8 mice. Additionally, pathological alterations in the spinal cord, a primary site of ALS pathology, were also assessed. Using molecular biology techniques we analysed the expression levels of leptin, its long receptor (Ob-Rb), and its downstream signaling pathways (Akt and STAT3) in rNLS8 mice compared to age- and sex-matched wild-type littermates. Our results revealed significant sex- and disease stage-dependent differences in leptin and Ob-Rb expression in white adipose tissue and the hypothalamus, along with increased activation of the Akt signaling pathway in the spinal cord of rNLS8 mice. These findings suggest that ALS progression differs by sex in rNLS8 mice, which may impact overall disease progression and the effectiveness of potential therapeutic interventions targeting metabolism, such as nutritional strategies that influence leptin levels.

neuroscience↗

bioRxiv2025

KiF1a-regulated neuronal infrastructures in sensory and prefrontal cortices essential for fear memory and anxiety

Stress in social activities induces fears. Cellular infrastructures and molecular profiles underlying fear-relevant psychosis remain elusive. By giving psychological stress to mice that watched fear scenes in the resident/intruder paradigm, we have studied the features of neuronal infrastructures in visual, auditory and medial prefrontal cortices correlated to fear-induced anxiety by approaches of behavior task, neural tracing, molecular biology and electrophysiology in vivo. This psychological stress causes observational mice fears specific to resident mouse and anxiety. This social phobia is associated with the interconnections newly emerged among visual, auditory and medial prefrontal cortices as well as among intramodal neurons. These cortical neurons receive the new synapse innervations from their interconnected neurons alongside the innate synapse innervations, and become to encode the stress signals including battle image and battle sound. The KiF1a knockdown in the medial prefrontal cortex precludes the formations of neuronal infrastructures and social phobia. KiF1a-mediated intracellular transportation in the medial prefrontal cortex is essential for the recruitment of associative memory neurons that encode fear scenes and anxiety. HighlightO_LIPsychological stress induces fears and interconnections among sensory and prefrontal cortices. C_LIO_LIAssociative memory neurons in these cortices are recruited to encode fear scenes and anxiety. C_LIO_LIKiF1a-mediated transportation essential for these cellular infrastructures relevant to learned fear. C_LI

neuroscience↗

bioRxiv2025

Metagenomics and qPCR analysis of aerobic metabolic TCE degrading bacteria

Chloroethenes are common groundwater contaminants. While they were initially considered non-biodegradable, different pathways for both, anaerobic and aerobic degradation have been discovered over the last 40 years. Anaerobic reductive dechlorination of tetrachloroethene and trichloroethene (TCE) leads to the formation and possible accumulation of the toxic intermediate products, dichloroethene (DCE) and vinyl chloride (VC). In contrast, aerobic metabolic processes result in mineralization of the contaminants without the formation of stable intermediates. Therefore, productive aerobic degradation processes can have a considerable advantage, depending on site conditions and chloroethene molecule. The potential for aerobic degradation processes can be assessed by molecular biological analysis. In our study, aerobic metabolic TCE enrichment cultures, bioaugmented groundwater, and a microcosm with intrinsic aerobic TCE degradation potential were analyzed. Amplicon sequencing and shotgun metagenomics were used to identify the Rhodocyclaceae bacterial family in all samples, as well as functional gene sequences, which were relatives of monooxygenases and dehalogenases. Based on these sequencing data, qPCR detection methods for TCE assimilating Rhodocyclaceae (Rho) as well as for different functional genes that were associated with monooxygenases (moABC) and haloacid dehalogenases like hydrolases (hdlh) have been established. The specificity of the qPCR methods was demonstrated with a variety of environmental samples and different chloroethene degrading bacterial cultures. The new metagenomic insights and qPCR methods facilitate the assessment of biodegradation potential and monitoring of aerobic metabolic TCE degradation at contaminated sites. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/667672v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@10b51feorg.highwire.dtl.DTLVardef@3414e0org.highwire.dtl.DTLVardef@14694ccorg.highwire.dtl.DTLVardef@d2e4e9_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIRhodocyclaceae bacteria were identified in all aerobic TCE degrading samples C_LIO_LIFunctional monooxygenase and dehalogenase like genes were detected C_LIO_LIqPCR methods for monitoring of aerobic metabolic TCE degradation were developed C_LIO_LISpecificity of qPCR methods was shown with different aerobic chloroethene degrading cultures and environmental samples C_LI

microbiology↗

bioRxiv2025

Separate-scan atomic force microscope for fast infrared scattering-type scanning near-field optical microscope

Pseudo-heterodyne scattering-type scanning near-field optical microscopy (sSNOM) is applied in the mid-infrared region to detect the chemical composition of biomolecules on the nanoscale. However, the application of sSNOM in molecular biology has been limited to static images in air. Recently, bottom illumination sSNOM (BI-sSNOM) was developed for operation in water. Yet, the scan rate of sSNOM remains a bottleneck to record protein structural changes in aqueous solution on the seconds time scale. We designed an optical and mechanical system consisting of a separate scan high-speed atomic force microscope (HS-AFM) coupled to the BI-sSNOM optics. The designed AFM scanner has a mechanical bandwidth of ca 70 kHz along the Z-axis, and ca 6 kHz along the XY-axis, equivalent to the sample scanning HS-AFM. The AFM performance is demonstrated by imaging actin filaments. The optical design is validated by sSNOM experiments on purple membranes and microtubules.

biophysics↗

bioRxiv2025

Accelerating protein design by scaling experimental characterization

Recent advances in de novo protein design have greatly outpaced standard protein biochemistry workflows, and experimental testing has been a bottleneck in the validation of new designs and methodologies. Here, we describe experimental and computational workflows to address the issues of scale, speed and reproducibility of common in vitro protein testing methods, enabling at least an order of magnitude increase in throughput while reducing wetlab time. Semi-Automated Protein Production (SAPP) is a rapid, modular, scalable and cost-effective protocol, enabling up to milligram-scale protein production, and standardized characterization including yield, dispersity, and oligomeric state of hundreds of designs per day, at the cost-equivalent of a few DNA oligos per construct. End-to-end protocol execution takes 48 hours, with about 6 hours spent benchside using mostly standard laboratory equipment. This protocol has become the standard at our institute, providing critical experimental validation for dozens of projects spanning tens of thousands of designs. We showcase the power of the platform by using it to rapidly characterize de novo designed inhibitors of respiratory syncytial virus. Since at least 80% of SAPPs total cost comes from synthetic DNA, we also developed a scalable demultiplexing protocol (DMX) to leverage oligo pools as input DNA, providing a further 5-fold reduction in costs, enabling >1000 designs to be purified and characterized in arrayed, clonal format at a cost of $5 per construct. By reframing standard molecular biology practices and orchestrating wetlab workflows with partial automation instead of complex end-to-end robotics, these protocols should be widely adoptable, accelerating protein design.

biochemistry↗

bioRxiv2025

CD27+ γδ T Cells Drive Plaque Instability in Advanced Atherosclerosis: Targeting CXCR3 for Therapeutic Intervention

BackgroundAtherosclerosis is a chronic inflammatory disease of the arterial wall that underlies most myocardial Ischaemic events. While multiple immune subsets contribute to plaque progression and instability, the role of {gamma}{delta} T cells remains poorly understood. We examined the contribution of {gamma}{delta} T cells to lesion development, progression and instability, and explored the therapeutic potential of their pharmacological blockade. MethodsTo investigate the role of {gamma}{delta} T cells, chimeric atherosclerosis-prone mice lacking {gamma}{delta} T cells were utilized in both loss- and gain-of-function experiments. Mixed bone marrow chimeras were generated to assess the role of {gamma}{delta} T cell-derived interferon-{gamma} (IFN-{gamma}) and perforin (Pfp). The therapeutic efficacy of AMG487 on plaque stability was evaluated in a preclinical tandem-stenosis mouse model. Lesion size, plaque composition, and stability were assessed using histology, immunoassays, and molecular biology techniques. ResultsCD27+ {gamma}{delta} T cells accumulated in atherosclerotic lesions and promoted plaque progression and instability via IFN-{gamma}- and Pfp-dependent manners. As early infiltrators, they amplified necrosis and inflammation by enhancing immune cell recruitment, thereby exacerbating lesion vulnerability. CXCR3 antagonism with AMG487 inhibited {gamma}{delta} T cell recruitment to plaques, reduced lesion size, and promoted features of plaque stability, including increased smooth muscle cell content and thicker fibrous caps. ConclusionsCD27 {gamma}{delta} T cells, which promote inflammation and necrosis through both direct and indirect mechanisms, are key drivers of plaque progression and instability. Targeting their recruitment via CXCR3 blockade enhances plaque stability and may represent a promising therapeutic strategy to reduce the risk of myocardial infarction.

immunology↗

bioRxiv2025

LieOTDock: A Protein Docking Framework via Lie Group Optimization and Optimal Transport

Protein-protein interactions (PPIs) are fundamental to cellular function, and structurally characterizing them is a key goal in molecular biology. Computational docking, a crucial tool for predicting the structure of protein complexes, faces significant challenges in search efficiency and scoring accuracy. Here, we introduce LieOTDock, a novel framework that leverages the mathematics of Lie groups and Optimal Transport (OT) for highly efficient protein docking pose generation. The core of our method is a data-driven surface sampling technique that identifies salient geometric features for initial pairing. These initial poses are then refined via a gradient-based optimization directly on the Lie group SE(3), the natural space of rigid-body motions. This optimization is guided by a differentiable score based on Sinkhorn-regularized Optimal Transport, which measures the geometric complementarity of surface patches. We tested LieOTDock on the 1AVW benchmark, where it successfully generated a near-native pose of 3.43 [A] RMSD from a 5000-candidate run in under three minutes on a consumer-grade Apple M4 Mini. Our results demonstrate that LieOTDock, by its principled use of Lie theory and OT, provides a powerful and efficient framework for generating high-quality candidate structures. This work decouples the challenge of geometric sampling from final scoring, offering a robust and modular approach to the protein docking problem, though future work is needed to integrate more physically realistic scoring functions for improved candidate ranking.

bioinformatics↗

bioRxiv2025

Enabling wider access to human molecular neuroscience research in pain: A simple preservation method for human dorsal root ganglion neurons in Hibernate A media

The use of human dorsal root ganglion (DRG) from organ donors opens the door for research into the molecular biology and physiology of human nociceptors; however, there are barriers to working with this tissue including logistical difficulties and limited access. We present an approach using Hibernate media to temporarily store either whole DRGs or dissociated DRG neurons prior to culturing and functional testing. Dissociation of DRGs following temporary storage (4-16hrs) in Hibernate media resulted in similar neuronal and immune cell yield as acutely dissociated DRGs. Neurons derived from DRGs stored in Hibernate media prior to dissociation exhibited similar electrophysiological properties and capsaicin responses as acutely dissociated DRG neurons. Similarly, neurons from acutely dissociated DRGs stored in Hibernate media (>24hrs) and shipped to geographically distant laboratories produced neuronal cultures displaying comparable electrophysiological properties as acutely cultured neurons. This approach overcomes insurmountable logistical burdens and increases access to freshly recovered human DRGs.

neuroscience↗