Biology subjects

Park, J.

Publications and source records attributed to Park, J..

At least 145 records · Page 8Linked to original sources

bioRxiv2025

Unveiling the Role of PHF20 in TBC1D4-Mediated Glucose Uptake During Toxoplasma gondii Infection

BackgroundToxoplasma gondii relies on host cell nutrients, especially glucose, to support its intracellular growth. However, the mechanisms by which it enhances host glucose metabolism remain incompletely understood. MethodsWe used fluorescence-based glucose uptake assays, flow cytometry, metabolic flux analysis, and gene silencing in ARPE-19 cells, along with transgenic mouse models, to explore how T. gondii manipulates host glucose utilization and whether this promotes parasite proliferation. ResultsT. gondii infection significantly enhanced host glucose uptake in a dose-dependent manner and promoted GLUT4 translocation to the plasma membrane. Notably, extracellular acidification rate (ECAR) assays demonstrated a marked increase in glycolytic activity following infection. Mechanistically, we identified that the PI3K/AKT signaling pathway mediates the phosphorylation and transcriptional upregulation of TBC1D4, a Rab-GAP protein essential for GLUT4 trafficking. Genetic silencing of TBC1D4 impaired both glucose uptake and parasite replication. Furthermore, we uncovered a regulatory mechanism involving AKT-dependent nuclear signaling that modulates TBC1D4 transcription. In vivo experiments using Phf20 transgenic mice confirmed increased susceptibility to T. gondii and elevated glucose metabolic responses. ConclusionsOur study reveals that T. gondii hijacks a host PI3K/AKT-TBC1D4 axis to enhance glucose uptake and glycolysis, thereby ensuring metabolic support for its proliferation. These findings highlight host glucose metabolism as a potential therapeutic target for controlling toxoplasmosis.

cell biology↗

bioRxiv2025

Targeting the COP9 signalosome overcomes platinum resistance in ovarian cancer through two distinct genome stability mechanisms

Tubo-ovarian high-grade serous carcinoma (HGSC) is a leading cause of gynecologic cancer mortality, largely due to the emergence of platinum resistance, which serves as the mainstay of chemotherapy. Here, we identify COPS5 as a therapeutic target and use an available small molecule inhibitor to overcome platinum resistance. A genetic screen for platinum-induced DNA damage in a platinum resistant ovarian cancer model identified COPS5 and COPS6, two components of the COP9 signalosome. Consistently, high COPS5 expression correlated with poor clinical outcomes in patients with HGSC. In both in vitro and in vivo experiments, COPS5 depletion sensitized ovarian cancer cells to carboplatin. A small molecule COPS5 inhibitor, CSN5i-3, synergized with carboplatin in homologous recombination-deficient and -proficient cells. This combination was also effective in xenografts and in a syngeneic mouse model of carboplatin-resistant HGSC. Importantly, we demonstrate that CSN5i-3 is selective for cancer cells, with patient-derived HGSC cells exhibiting up to 50-fold greater sensitivity to CSN5i-3 than benign cells. Finally, we show that genetic or small molecule inhibition of COPS5 impaired both nucleotide excision repair (NER) and interstrand crosslink (ICL) repair, leading to increased DNA platinum adducts. Mechanistically, this was due to increased ubiquitination and degradation of DNA-specific DNA binding protein 1 (DDB1) and other key NER and ICL repair proteins, consistent with the role of COPS5 in the regulation of these factors. Our findings highlight the importance of NER and ICL regulation in chemotherapy response and indicate that targeting COPS5 can enhance the efficacy of platinum-based chemotherapy in HGSC. One Sentence SummaryCOPS5 depletion or inhibition using a small molecule COPS5 inhibitor CSN5i-3 sensitizes high-grade serous carcinoma to platinum chemotherapy through downregulation of nucleotide excision repair and interstrand crosslink repair.

cancer biology↗

bioRxiv2025

Optimizing twin prime editing components for scalable genome editing and therapy in spinocerebellar ataxia type 3

Recent advances in prime editing technologies using CRISPR modules fused with reverse transcriptase (RT) have enabled efficient and precise reprogramming of target genomic sequences. Twin prime editing using two coordinated prime editor complexes is a promising strategy for inducing extensive genomic modifications via reverse transcribed complementary templates. However, current twin prime editing systems still require improvements in editing efficiency, accuracy, and intended edit predictability. Here, efficiency and precision of twin prime editing were enhanced via engineering and optimizing conventional SpCas9(H840A)-RT-based prime editor components. A La domain-fused prime editor (La-SpCas9(H840A)-RT) and optimized pegRNAs were developed, achieving a 1.75 {+/-} 0.21-fold increase in gene editing efficiency at multiple genomic loci in human-derived cell lines without increasing off-target activity. La-SpCas9(H840A)-RT facilitated efficient [~]2.8 kb GFP transgene knock-in at target loci and eliminated the expanded polyQ tract in ATXN3 in patient-derived mutant cell lines modeling spinocerebellar ataxia type 3. The advanced twin prime editing platform expands genome engineering capabilities beyond existing CRISPR-based systems and holds great promise for diverse biotechnological and therapeutic applications.

bioengineering↗

bioRxiv2025

Prediction of bacterial protein-compound interactions with only positive samples

Prediction of Compound-Protein Interactions (CPI) in bacteria is crucial to advance various pharmaceutical and chemical engineering fields, including bio-catalysis, drug discovery, and industrial processing. However, current CPI models cannot be applied for bacterial CPI prediction due to the lack of curated negative interaction samples. This paper introduces a novel Positive-Unlabeled (PU) learning framework, named BIN-PU, to address this limitation. BIN-PU generates pseudo positive and negative labels from known positive interaction data, enabling effective training of deep learning models for CPI prediction. We also propose a weighted positive loss function that weights to truly positive samples. We have validated BIN-PU with multiple CPI backbone models, comparing the performance with the existing PU model using bacterial cytochrome P450 (CYP) data. Extensive experiments demonstrate the superiority of BIN-PU over the benchmark model in predicting CPIs with only truly positive samples. Furthermore, we have validated BIN-PU on additional bacterial proteins obtained from literature review, human CYP datasets, and uncurated data for its reproducibility. We have also validated the CPI prediction for the uncurated CYP data with biological and biophysical experiments. BIN-PU represents a significant advancement in CPI prediction for bacterial proteins, opening new possibilities for improving predictive models in related biological interaction tasks.

bioinformatics↗

bioRxiv2025

Copper-Containing ROS-Scavenging Nanozyme Paradoxically Drives Alpha-Synucleinopathy by Amplifying Nitrosative Stress

Reactive oxygen and nitrogen species (RONS) are implicated in neurodegeneration, but their specific pathogenic roles remain unclear. Here, we developed a pair of iridium-based nanozymes with opposing functionalities to dissect these pathways. We show that a copper-tuned iridium nanozyme (Ir{square}Cu), despite being a superior ROS scavenger, paradoxically and dramatically exacerbated -synuclein (Syn) pathology in vivo. This pathology was causally linked to its ability to amplify RNS, as pharmacological inhibition of nitric oxide synthase (NOS) with L-NAME completely abrogated the pathology and reversed a human Parkinsons disease (PD)-like transcriptomic signature. In contrast, a copper-free, broad-spectrum RONS-scavenging iridium (Ir) nanozyme demonstrated substantial therapeutic efficacy across diverse brain-first, body-first, and Alzheimers disease with Lewy body co-pathology models. Our findings uncover the importance of the RNS pathway in driving -synucleinopathies and establish a critical design principle for nanomedicine, mandating caution in the use of redox-active copper for neuroprotective applications.

pathology↗

bioRxiv2025

Machine learning enables de novo multi-epitope design of plasmodium falciparum circumsporozoite protein to target trimeric L9 antibody

AbstractCurrently approved vaccines for the prevention of malaria provide only partial protection against disease, due to high variability in the quality of induced antibodies. These vaccines present the unstructured central repeat region, as well as the C-terminal domain, of the circumsporozoite protein (PfCSP) of the malaria parasite, Plasmodium falciparum (1). A recently discovered protective monoclonal antibody, L9, recognizes three structured copies of the PfCSP minor repeat. Similarly to other highly potent anti-malarial antibodies, L9 relies on critical homotypic interactions between antibodies for its high protective efficacy (2, 3). Here, we report the design of immunogens scaffolding one copy of PfCSPs minor repeat capable of binding L9. To design immunogens capable of presenting multiple, structure-based epitopes in one scaffold, we developed a machine-learning-driven structural immunogen design pipeline, MESODID, tailored to focus on multi-epitope vaccine targets. We use this pipeline to design multiple scaffolds that present three copies of the PfCSP minor repeat. A 3.6 [A] cryo-EM structure of our top design, minor repeat targeting immunogen (M-TIM), demonstrates that M-TIM successfully orients three copies of L9, effectively recapitulating its critical homotypic interactions. The wide prevalence of repeated epitopes in key vaccine targets, such as HIV-1 Envelope, SARS-CoV-2 spike, and Influenza Hemagglutinin, suggests that MESODID will have broad utility in creating immunogens that incorporate such epitopes, offering a new powerful approach to developing vaccines against a range of challenging infections, including malaria. Significance StatementIn this study, we present a machine learning driven, structure-guided protein design pipeline built specifically for the design of multi-epitope vaccine immunogens. We employ the pipeline here to design and solve the cryo-EM structure of a de novo immunogen that binds and properly orients three copies of the anti-malarial monoclonal antibody L9, producing a promising next-generation malaria vaccine immunogen. This design pipeline could be employed to design any number of structurally constrained multi-epitope immunogens, as well as other proteins designed to bind multiple targets.

biophysics↗

bioRxiv2025

Modular striatal dynamics and reciprocal inhibition orchestrate skilled action sequencing

The basal ganglia, particularly the striatum, are critical for orchestrating skilled behavioral sequences, yet the precise mechanisms underlying this process remain unclear. Using high-resolution kinematic tracking and neural recordings in mice performing a water-reaching task, we found spatially distributed modules of striatal projection neurons whose activity corresponded to the generation of each element in the sequence--aiming, reaching, and drinking. They are activated sequentially and exhibit reciprocal inhibition, ensuring a strict serial order. Optogenetic activation of the direct pathway of the orofacial module promoted licking while suppressing reaching. Reaching could in turn suppress stimulation-evoked licking, revealing bidirectional inhibitory interactions. Our findings demonstrate that the striatums modular organization, coupled with lateral inhibition, sculpts the temporal progression of actions, providing a mechanistic framework for understanding how the basal ganglia coordinate complex behaviors.

neuroscience↗

bioRxiv2025

Synergistic effects of APOE ϵ4 and Alzheimer's pathology on the neural correlates of episodic remembering in cognitively unimpaired older adults

Amyloid-{beta} (A{beta}) and tau pathology begin accumulating decades before clinical symptoms and are influenced by APOE {varepsilon}4, a key genetic risk factor for Alzheimers disease (AD). Although the presence of A{beta}, tau, and APOE {varepsilon}4 are thought to impact brain function, their effects on the neural correlates of episodic memory retrieval in preclinical AD remains unknown. We investigated this question in 159 cognitively unimpaired older adults (mean age, 68.9{+/-}5.8 years; 57% female) in the Stanford Aging and Memory Study. Participants completed an associative memory task concurrent with functional MRI. A{beta} was measured using CSF A{beta}42/A{beta}40 or Florbetaben-PET imaging and tau was measured using CSF pTau181. Hippocampal univariate activity and cortical reinstatement - that is, reinstatement of patterns of neocortical activity that were present during memory encoding - were measured during successful memory retrieval. Analyses revealed that APOE {varepsilon}4 was independently associated with greater A{beta} and tau burden, and that associations of AD biomarkers with brain function and memory were moderated by APOE {varepsilon}4. Among APOE {varepsilon}4 non-carriers, A{beta} burden was linked to a pattern of hippocampal hyperactivity. Among APOE {varepsilon}4 carriers, CSF pTau181 was linked to weaker cortical reinstatement during memory retrieval and lower memory performance. Thus, abnormal AD biomarkers and genetic risk synergistically impact neural and behavioral expressions of memory in preclinical AD. These findings highlight the critical role of APOE {varepsilon}4 in moderating effects of AD pathology on brain function and identify candidate mechanisms that may contribute to increased risk of memory impairment in preclinical AD. Significance StatementHippocampus-dependent cortical reinstatement is a critical mechanism supporting episodic remembering that contributes to individual differences in memory performance in older adults. However, the contribution of early Alzheimers disease (AD) pathology to variability in this mechanism is unknown. We demonstrate that associations of AD biomarkers with hippocampal activity and cortical reinstatement are moderated by APOE {varepsilon}4 in cognitively unimpaired older adults. Amyloid-{beta}-related hyperactivity was observed in the hippocampus among APOE {varepsilon}4 non-carriers, while CSF pTau181 was linked to weaker cortical reinstatement during memory retrieval and lower memory performance among APOE {varepsilon}4 carriers. Our findings highlight synergistic effects of APOE and AD pathology on brain function and identify candidate mechanisms that may underlie increased risk of memory impairment in preclinical AD.

neuroscience↗

bioRxiv2025

Uncovering the hidden diversity and functional roles of root endophytic Streptomyces under drought stress

The genus Streptomyces has consistently been found to be enriched in drought-stressed plant root microbiomes, yet the ecological basis and functional variation underlying this enrichment at the strain and isolate level remain unclear. Using two 16S rRNA sequencing methods with different levels of taxonomic resolution, we confirmed drought-associated enrichment of Streptomyces in field-grown sorghum roots and identified five closely related but distinct ASVs belonging to the genus with variable drought enrichment patterns. From a culture collection of sorghum root endophytes, we selected 12 Streptomyces isolates representing these ASVs for phenotypic and genomic characterization. Whole-genome sequencing revealed substantial variation in gene content, even among closely related isolates, and exometabolomic profiling showed distinct metabolic responses to media supplemented with drought-versus well-watered root tissue. Traits linked to drought survival, including osmotic stress tolerance, siderophore production, and carbon utilization, varied widely among isolates and were not phylogenetically conserved. Using a broader panel of 48 Streptomyces, we demonstrate that drought enrichment (DE) scores, determined through mono-association experiments in gnotobiotic sorghum systems, showed high variability and lacked correlation with plant growth promotion. Pangenome-wide association identified gene clusters involved in osmolyte transport (e.g., proP) and membrane biosynthesis (e.g., fabG) as positively associated with DE, though most associations lacked phylogenetic signal. Collectively, these results demonstrate that Streptomyces enrichment under drought is not a conserved genus-level trait but is instead strain-specific and functionally heterogeneous. Furthermore, enrichment in the root microbiome does not predict beneficial effects on plant growth. This work underscores the need to resolve functional traits at the strain level and highlights the complexity of microbe-host-environment interactions under abiotic stress. ImportanceUnderstanding how beneficial microbes respond to drought is critical for developing microbiome-based strategies to enhance crop resilience. The Streptomyces genus is consistently enriched in drought-stressed root microbiomes, yet our study reveals that this enrichment is not a uniform isolate-level response. Instead, we show that drought enrichment is highly strain-specific and functionally diverse, with key associated traits like osmotic stress tolerance and siderophore production varying independently of phylogeny. Importantly, enrichment does not predict a microbes ability to promote plant growth under stress. By integrating high-resolution taxonomic, genomic, and metabolomic approaches, we uncover the ecological and functional complexity underlying Streptomyces responses to drought. These findings emphasize the importance of resolving microbial function at the strain level and caution against assuming functional traits based on taxonomic identity itself.

microbiology↗

bioRxiv2025

The Spatial Atlas of Human Anatomy (SAHA): A Multimodal Subcellular-Resolution Reference Across Human Organs

The Spatial Atlas of Human Anatomy (SAHA) represents the first multimodal, subcellular- resolution reference of healthy adult human tissues across multiple organ systems. Integrating spatial transcriptomics, proteomics, and histological features across over 15 million cells from more than 100 donors, SAHA maps conserved and organ-specific cellular niches in gastrointestinal and immune tissues. High-resolution profiling using CosMx SMI, 10x Xenium, RNAscope, GeoMx DSP, and single-nucleus RNA-seq reveals spatially organized cell states, rare adaptive immune populations, and tissue-specific cell-cell interactions and ligand-receptor pairs. Comparative analyses with colorectal cancer and inflammatory bowel disease demonstrate the power of SAHA to detect disease-associated spatial disruptions, including crypt dedifferentiation, perineural invasion, and therapy-resistant immune remodeling. All data are openly accessible through a FAIR-compliant interactive portal to support exploration, benchmarking, and machine learning model training. Through SAHA, we provide a foundational framework for spatial diagnostics and next-generation precision medicine grounded in a comprehensive human tissue atlas, enabling the development of context-aware models that simulate tissue behavior, decode complex pathologies, and accelerate therapeutic innovation at unprecedented scale.

systems biology↗

bioRxiv2025

STARR-CRAAVT: A platform to design cell type-specific regulatory elements for next-generation gene therapy

Precise control of transgene expression through novel enhancer-promoter combinations is a promising strategy for advancing gene therapy mediated by adeno-associated virus (AAV). We present STARR-CRAAVT, a novel STARR-Seq-derived platform to screen for enhancers in the AAV context. Using HepG2 and HaCaT cells as screening models, we applied STARR-CRAAVT for the identification of cell type-specific enhancers. We integrated epigenetic datasets into an in-silico library of putative HepG2 enhancers and captured corresponding fragments from genomic DNA. The fragments were processed into AAV libraries and applied to HepG2 and HaCaT cells. STARR-CRAAVT analysis revealed a selective activity of the libraries confirming the HepG2-directed in-silico design and the specificity of single enhancer-promoter combinations could be validated using luciferase reporter assays. In addition, we scrutinized the impact of key experimental parameters on enhancer identification and found that the used promoter type had significant influence on the ability of candidates to act as enhancers. Furthermore, switching the location of identified enhancers in reporter assays revealed that the level of enhancer activity is highly dependent on the position in the AAV genome. Taken together, our study yields novel insights into enhancer function and demonstrates that STARR-CRAAVT can be employed to identify cell type-specific enhancers, highlighting promoter preference and enhancer positioning as key considerations for enhancer screening campaigns.

genomics↗

bioRxiv2025

An artificial nervous system for communication between wearable and implantable therapeutics

Bioelectronics have transformed our capacity to monitor and treat diseases; however, a lack of micrometer-scale, energy efficient communication options limit these devices from forming integrated networks that enable full-body, sensor driven, physiological control. Inspired by our nervous systems ability to transmit information via ionic conduction, we engineered a Smart Wireless Artificial Nervous System (SWANS) that utilizes the bodys own tissue to transmit signals between wearables and implantables. When SWANS emits signals, it generates voltage gradients throughout the body that selectively turn on implanted transistor switches when exceeding their gate threshold voltages. SWANS implantable communication components maintain syringe-injectable footprints and >15x greater power efficiencies than Bluetooth and Near Field Communication. In vivo studies in rats demonstrate SWANS ability to wirelessly regulate dual hind leg motor control by connecting electronic-skin sensors to implantable neural interfaces via ionic signaling as well as coordinate bioelectronics throughout the epidermal, subcutaneous, intraperitoneal, and gastrointestinal spaces.

bioengineering↗

bioRxiv2025

Cancer-associated metabolic alterations facilitated by astrocyte glycogenolysis mediate neuropathic pain chronicity

Chronic pain remains a major unmet medical challenge, yet the metabolic checkpoints that govern its persistence are poorly defined. Astrocytes are increasingly recognized as chemically programmable hubs that tune neuronal excitability through metabolic circuits. Building on reports that astrocyte-neuron lactate shuttling (ANLS) in the anterior cingulate cortex (ACC) supports chronic pain, we asked how astrocytic metabolic states evolve over the course of pain chronification. Using untargeted metabolomics of the ACC combined with GFAP-RiboTag-based astrocyte-specific transcriptomics, we provide a time-resolved map of astrocytic metabolism across the transition from acute nociception to chronic neuropathic pain. This analysis reveals a biphasic glycogen program--an acute glycogenolysis-triggered glycogen supercompensation--that culminates in the emergence of a Warburg-like metabolic signature with tissue acidification associated with sustained lactate shuttling and persistent circuit activation. Using glycogen phosphorylase inhibitors (GPI-1, GPI-2) as pharmacological probes, we show that early glycogenolysis blockade attenuates this Warburg-like shift, partially normalizes ACC metabolic signatures, and reduces long-lasting mechanical hypersensitivity, without impairing acute nociceptive sensitization. These findings identify astrocytic metabolic reprogramming as a pharmacologically tractable circuit-level process and nominate glycogenolysis as an upstream biochemical gate and potential therapeutic control point in neuropathic pain. HIGHLIGHTSMost experimental models of chronic pain emphasize progressive sensitization, yet few have identified a chemically tractable switch that converts transient nociception into a persistent cortical state. Here, we propose astrocytic glycogenolysis as such a switch--a gatekeeper process whose brief engagement can reconfigure neuroglial metabolism toward a Warburg-like signature that stabilizes hyperexcitable circuit dynamics. By leveraging small-molecule glycogen phosphorylase inhibitors as mechanistic precision probes, we move beyond correlation to delineate a pathway-level control architecture that can be transiently perturbed to dissociate acute nociceptive processing from pain chronification. This framework reframes astrocytes from metabolic support cells to state-setting regulators of cortical excitability and positions glycogenolysis as a therapeutically actionable leverage point. More broadly, our findings underscore a methodological opportunity: applying chemical biology to neuroglial metabolism can expose governing checkpoints that are often obscured by descriptive physiology, enabling mechanism-first, pathway-directed intervention strategies across neurological disease contexts.

neuroscience↗

bioRxiv2025

Tac1 Deficiency Reduces the Severity of Enteric Bacterial Infection

BackgroundInfection with enteric bacterial pathogens continues to cause significant morbidity and mortality throughout the world. These pathogens include enterohemorrhagic and enteropathogenic Escherichia coli, which transit the intestinal tract, efface microvilli, and attach firmly to intestinal epithelial cells predominantly in the colon. Investigation of these human-adapted pathogens has been greatly aided by mouse models of infection. The mouse-adapted attaching and effacing pathogen Citrobacter rodentium utilizes many similar mechanisms of pathogenesis, including the use of a type III secretion system, and virulence factors encoded in a locus of enterocyte effacement. Although this model has allowed for assessing the complexity of the host response, the complex interplay between the nervous and immune systems in response to infection remains incomplete. MethodsWe assessed the role of sensory neurotransmitters encoded by the Tac1 gene in the host response to C. rodentium. ResultsTac1-deficient mice had significantly reduced pathogen shedding and colonic bacterial burden, accompanied by decreased expression of inflammatory cytokines and chemokines. In accordance with reduced chemokine production, we observed reduced colonic recruitment of specific immune cell populations in Tac1-/- compared to WT mice. ConclusionsSensory neuropeptides regulate key aspects of enteric bacterial infection and may serve as unique targets in the treatment of enteric disease.

immunology↗

bioRxiv2025

PEDOT:PSS Microparticles for Extrudable and Bioencapsulating Conducting Granular Hydrogel Bioelectronics

Conducting hydrogels are promising materials for forming physiomimetic bioelectronic interfaces to monitor and stimulate biological activity. However, most developed materials are non-microporous and possess fixed shapes, both of which can limit the integration of cells and tissues with devices. In non-conducting biomaterials, materials fabrication strategies imparting microporosity and dynamic mechanical properties have been shown to support cell infiltration and support biointerfaces of various geometries. Specifically, granular hydrogels have enabled encapsulating, conformal, and injectable interfaces through these features. However, granular hydrogels remain largely unexplored as conducting biomaterials. We present methods for fabricating spherical, poly(3,4-ethylenedioxythiophene):poly(styrene sulfonate) (PEDOT:PSS) hydrogel microparticles. When densely packed, these microparticles form a conducting granular hydrogel with microporosity as well as shear-thinning and self-healing dynamic mechanical properties. The PEDOT:PSS granular hydrogel can be extruded and maintain structure post-3D printing. Modulating microparticle PSS content achieves high granular hydrogel conductivity (137 S/m), and microparticles exhibit excellent cytocompatibility (>98% viability). Finally, we demonstrate utility as bioencapsulating electrodes for electrophysiological monitoring. These results highlight the functionality of our PEDOT:PSS conducting granular hydrogel, suggesting its potential as 3D printed bioencapsulating electrodes, 3D tissue engineering scaffolds for monitoring encapsulated cells, and injectable therapies for enhanced cell recruitment and tissue regeneration combined with electronic stimulation.

bioengineering↗

bioRxiv2025

The interaction of XPG with TFIIH through p62 and XPD is required for the completion of nucleotide excision repair

Nucleotide excision repair (NER) is the key pathway for the removal of DNA damage induced by UV irradiation and chemotherapeutic reagents. Protein-protein interactions are crucial for the dynamic and coordinated assembly of the proteins involved at DNA lesion. Here we focus on the role of interactions between the multi-subunit helicase/translocase complex TFIIH and the 3 endonuclease XPG. We show that XPG interacts with the p62 and XPD subunits of TFIIH through its long spacer region located in the middle of its split active site. We show the interactions between three acidic regions of XPG with the pleckstrin homology (PH) domain of p62 are of moderate importance for NER, while interactions with XPD are critical for activity. Combining mutations in the p62 and XPD interaction domains leads to additive defects in NER activity. Unexpectedly, we show that these interactions are not required for the recruitment of XPG, but rather for the formation of a catalytically competent NER complex and for triggering the incision 5 to the lesion by ERCC1-XPF. Our studies provide fundamental insights into how interactions between TFIIH and XPG contribute to the NER pathway and more generally how modular protein-protein interactions control each step along the NER reaction coordinate.

biochemistry↗

bioRxiv2025

Immune Spatial Organization Predicts Metastasis Risk in Aggressive Localized Prostate Cancer

Risk stratification in localized prostate cancer (PCa) remains imprecise. Computational pathology has emerged as an attractive option for improving risk stratification, but current approaches either lack interpretability or focus solely on tumor morphology. Here, we identify, validate, and provide interpretability for an immune microenvironment-derived computational pathology biomarker for high-grade PCa. Using digitized hematoxylin and eosin-stained (H&E) slides from two independent cohorts of patients treated with radical prostatectomy (n=490), we found that spatial clustering of immune clusters, but not immune cell abundance, was independently associated with reduced risk of distant metastasis for high-grade disease but not low-grade. Joint analysis of H&E images and RNA sequencing (n=326) revealed that in high-grade disease, high-cluster samples were enriched in CD8+ T cells, activated memory CD4+ T cells, and Tregs, as well as clonal T cell populations, overall suggestive of an underlying T cell-mediated antitumor response. No such enrichment was found in low-grade tumors. These findings establish immune spatial architecture as a novel, interpretable computational pathology biomarker and provide insight into the immune landscape of high-grade PCa.

bioinformatics↗

bioRxiv2025

Tracking Conditioned Fear in Pair-Housed Mice Using Deep Learning and Real-Time Cue Delivery

Post-traumatic stress disorder (PTSD) is a complex and prevalent neuropsychiatric condition that arises in response to exposure to a traumatic event. A common diagnostic criterion for PTSD includes heightened physiological reactivity to trauma-related sensory cues, in safe or familiar environments. Understanding complex PTSD criteria requires new pre-clinical paradigms and technologies that integrate sensory physiology (e.g., auditory, visual, olfactory) with behavior. Here we present a novel Pavlovian-based paradigm using an open-source software plus deep learning-based pose estimation to investigate the effects of a recurrent conditioned stimulus (CS) on fear behaviors in pair-housed mice within the home cage. Simultaneous home cage video recording and analysis of CS-evoked freezing behaviors were performed using a deep learning model, with consideration for light-dark circadian cycles. Fear- conditioned dyad mice exhibited high CS-evoked freezing, with evidence of extinction learning (characterized by low freezing) during the mid-phase of the 2-week paradigm. Females exhibited reduced CS-evoked home cage freezing compared to males with circadian differences between the light (low freezing) and dark (high freezing) periods. Following the 2-week paradigm, fear-conditioned mice, compared to controls, exhibited heightened context-dependent freezing, while males but not females showed heightened startle reactivity. Taken together, these results demonstrate a novel software application for examining conditioned defensive and fear behaviors over time in mouse dyads within an ethologically relevant environment. Future applications could be used for more integrative analysis and understanding of neural circuits and heightened sensory threat reactivity, potentially improving the understanding and treatment of PTSD.

animal behavior and cognition↗