Biology subjects

Gao, A.

Publications and source records attributed to Gao, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Coevolutionary mining of prokaryotic non-coding elements with a genome language model

Microbial genomes encode compact molecular machines and diverse non-coding RNAs (ncRNAs) essential to gene regulation, pathogenesis, and many foundational biotechnologies. However, annotation remains largely protein-centric and homology-driven. Here, we introduce Minerva, a framework for coevolutionary mining that uses genome language models to predict local interactions directly from sequence as two-dimensional maps. Introducing two complementary techniques, categorical Jacobian fingerprinting and interaction heads, we demonstrate fast, accurate, alignment-free prediction of ncRNA base-pairing, monomeric protein contacts, and repetitive sequence motifs. Applied to 150 bacterial genomes, Minerva recovers known systems and predicts that 84.3% of predicted intergenic base-pairing falls outside of known annotations. In Pseudomonas, we find that the widespread TwoAYGGAY ncRNA family carries large secondary-structure extensions and is often flanked by short upstream repetitive motifs and larger downstream genomic repeats. Interpreting the coevolution maps, we find that Minerva emergently detects open-reading-frame (ORF) signatures at the DNA level despite never being trained to do so. In prophages within these genomes, we discover that Unknown Group 27 (UG27) reverse transcriptase systems encode arrays of structurally conserved yet sequence-diverse ncRNAs that template complementary DNA (cDNA) hairpin products. Together, these results establish coevolutionary mining as a scalable route to genome annotation and biological discovery across the rapidly expanding microbial universe.

bioinformatics↗

bioRxiv2026

TFE3 fusions drive expression of CD44 and SPP1 in Translocation Renal Cell Carcinoma

Xp11.2 translocation RCC [Xp11.2 tRCC]) is an underdiagnosed and aggressive subtype of RCC with few specific or targeted therapies. The transmembrane glycoprotein CD44 is an emerging target in many advanced malignancies, and with its ligand OPN (SPP1), is a crucial driver of cancer progression, stemness, metastasis, and immune suppression. Here we show that common TFE3-fusions [ including SFPQ-TFE3, PRCC-TFE3, ASPSCR1-TFE3, and NONO-TFE3] are associated with upregulated expression of CD44 and SPP1, as observed in multiple human and murine bulk transcriptomic studies and a murine tRCC snRNA-Seq dataset. CD44 and/or SPP1 protein expression were also upregulated in murine models of transgenic tRCC kidneys and urine specimens, patient-derived cell lines and human tRCC cases, by immunoblotting and/or IHC. Transient deletion of CD44 was associated with profound and specific suppression of tRCC cell line growth, with decreased mTOR signaling. These data suggest that CD44 and/or SPP1 may potentially drive tumorigenesis in tRCC.

cancer biology↗

bioRxiv2026

Single-cell profiling reveals disruption of cell-cycle homeostasis following JDP2 depletion in colorectal epithelial cells

Intestinal epithelial cells (IECs) undergo rapid and continuous renewal to maintain gut epithelial homeostasis and barrier integrity at the interface with luminal microbiota, dietary antigens, and enteric pathogens. IEC proliferation is tightly regulated by a complex machinery of cell cycle regulators and effectors, and genetic or transcriptional dysregulation of cell-cycle regulatory pathways is a hallmark of colorectal cancer development and progression. Previously, JDP2 (Jun dimerization protein 2) has been shown to be involved in cell cycle control and is associated with an array of cancers. JDP2 functions as a transcription factor or epigenetic regulator depending on cellular context, disease, and cancer types. However, the role of JDP2 in CRC, cell cycle homeostasis of colon cancer cells, and their fate remains poorly defined. In this study, we found that patients with higher JDP2 expression have significantly worse survival than patients with lower JDP2 expression. Our functional assays showed that JDP2 depletion increased EdU incorporation during S phase and PHH3 abundance, which is consistent with increased proliferative and mitotic activity. Our single-cell RNA sequencing and transcriptomic profiling further revealed reprogramming of cell-cycle-associated transcriptional states following JDP2 depletion. Differential-expression and pathway analysis identified changes in mitotic and cell-cycle regulatory genes, including programs involving cyclins, cyclin-dependent kinases, chromosome segregation, and mitotic progression. We also found that lower residual JDP2 expression was associated with escalated G2/M representation and a change toward later states along a G1 - S - G2/M-associated transcriptional trajectory. Furthermore, low-JDP2 cells showed significantly greater late-state occupancy than High-JDP2 cells (33.6% versus 24.0%; paired P=0.048). Among the JDP2-siRNA-treated cells, analysis of residual JDP2-expressing cells also revealed heterogeneous associations of JDP2 with cell-cycle, survival, apoptotic, and stress-response programs, consistent with JDP2's context-dependent transcriptional functions. Together, these data identify JDP2 as an important component of intestinal epithelial cell-cycle homeostasis and transcriptional landscape and suggest that JDP2 depletion perturbs the balance of proliferative cell states.

molecular biology↗

bioRxiv2026

Campylobacter jejuni Infection Is Associated With Cell-Cycle, Redox, and Metabolic Remodeling of Human Intestinal Epithelial Cells at Single-Cell Resolution

Campylobacter jejuni is the leading cause of bacterial enteric infections worldwide, including in the US. It is also a zoonotic pathogen that is transmitted by food and water, causing diarrhea and intestinal inflammation, and is responsible for high morbidity and mortality in young children, the elderly, and immunocompromised patients. During infection, C. jejuni profoundly perturbs intestinal epithelial physiology, leading to widespread mucosal damage and inflammatory response. However, the heterogeneity and coordination of host transcriptional responses remain incompletely defined. We performed single-cell RNA sequencing of Caco-2 intestinal epithelial cells from uninfected Control and C. jejuni-infected biological samples using 10x Genomics GEM-X Flex chemistry. Our dataset comprised 159,088 cells and 18,142 measured features. A Seurat workflow resolved 19 transcriptional states and revealed a highly reproducible condition-associated redistribution of clusters across biological replicates. Our study demonstrated that C. jejuni infection was associated with increased representation of G2/M-classified cells and coordinated induction of checkpoint and mitotic-spindle genes, including CDKN1A, WEE1, MAD2L1, BUB1B, PLK1, CDC20, CDK1, and UBE2C. Furthermore, ranked enrichment independently identified a C. jejuni-enriched Reactome mitotic spindle checkpoint program (NES ~1.67, FDR ~0.013), while Hallmark analysis identified strong TNF/NF-kB, hypoxia, G2M checkpoint, TGF-beta, glycolysis, apoptosis, p53, and mTORC1-associated programs. Infection was also associated with significantly decreased JDP2 and higher NOX1 and DUOX2 gene expression. Furthermore, scMetabolism/AUCell identified 20 KEGG metabolic pathways at FDR <0.05 in a balanced 10,000-cell analysis, with 12 lower and 8 higher in infection. Together, these data support a model in which C. jejuni dramatically restructures epithelial transcriptional states and is accompanied by mitotic-checkpoint, inflammatory/stress, redox, and metabolic remodeling.

microbiology↗

bioRxiv2026

The role of mu opioid receptors on excitatory and inhibitory neurons in the rostral ventromedial medulla in neuropathic pain

Descending projections from the brain to the spinal cord can regulate painful stimulus processing and are modulated by endogenous and exogenous opioids. We investigated the role of mu opioid receptors (MORs) in GABAergic vs. glutamatergic neurons of the rostral ventral medulla (RVM) in a mouse model of chronic neuropathic pain. We found that activating glutamatergic and GABAergic neurons in the RVM both result in antinociception [BC1.1]at baseline, but glutamatergic neurons enhance pain responses after nerve injury. [BC2.1]We then interrogated the role of RVM MOR signaling on neuropathic pain by using CRISPR/Cas9 to delete MOR in glutamatergic or GABAergic RVM neurons. We found that MOR knockout in glutamatergic and GABAergic RVM neurons precipitates early neuropathic pain onset with no effect on chronic pain intensity. These results suggest that RVM MOR signaling modulates hypersensitivity in the early phase of injury, but chronic neuropathic pain is largely independent of mu opioid receptor signaling.

neuroscience↗

bioRxiv2026

Single-cell multi-omics analysis reveals heterogeneity and plasticity of neutrophil states in response to immunotherapies

BackgroundTumor-associated neutrophils (TANs) are emerging as functionally heterogeneous and plastic cells in the tumor microenvironment. In immunologically cold tumors, elevated neutrophil abundance correlates with poor prognosis and resistance to immune checkpoint inhibition (ICI). Whether distinct anti-tumoral neutrophil states can be induced by different immunotherapies and how they relate to treatment efficacy remains unclear. MethodsUsing the syngeneic MOC2-huEGFR (M2h) mouse model of head and neck squamous cell cancer (HNSCC), we treated tumor-bearing mice with agonistic anti-CD40 monoclonal antibody (mAb) (aCD40), TNF, Cetuximab, or a combination of all three, designated Neutrophil Activating Therapy (NAT). In addition to evaluating anti-tumor efficacy, we performed single-cell multiomics RNA and protein sequencing, followed by bioinformatics analyses and flow cytometry validation. NAT-induced anti-tumor efficacy and related neutrophil states were also assessed in another cold tumor model, 9464D-GD2 neuroblastoma. Murine treatment-induced neutrophil gene signatures were then evaluated using clinical, proteomic, and transcriptomic data from HNSCC patients. ResultsFive transcriptionally distinct neutrophil states (N0-N4), including precursor state CD49d+ N4, were identified using the M2h model. N0 neutrophils (immunosuppressive/quiescent) dominated untreated tumors, but not in successful treatments. ISG+ N1 neutrophils and CCR3+ N3 neutrophils expanded by aCD40, TNF, and NAT treatment with anti-tumoral gene signatures and found more interacting with CD8+ T cells from bioinformatics analysis. N2 neutrophils reflected a recently established hypoxia-adapted state found in all treatments. ICAM1 (CD54) emerged as a marker of treatment-induced neutrophil activation, discriminating N1, N2, and N3 neutrophils from N0 neutrophils, validated by flow cytometry. In the 9464D-GD2 neuroblastoma model, NAT treatment also reduced the N0 dominance seen in untreated tumors in the HNSCC model but failed to induce anti-tumoral neutrophil states. In 23 HNSCC patients who received ICI therapy, ICAM1 protein expression in neutrophils trended toward association with responder status (TMA-level p=0.029), and ICAM1 neutrophil gene expression also trended toward association with improved overall survival in TCGA data (HR=0.75, p=0.059). ConclusionsDistinct immunotherapy-induced neutrophil states are defined by transcriptional profiles enriched in different functional pathways, associated with both anti-tumor and pro-tumor signatures. ICAM1 identifies activated neutrophils and potentially serves as a biomarker of ICI response in HNSCC, warranting further clinical validation. WHAT IS ALREADY KNOWN ON THIS TOPICNeutrophil heterogeneity has received increasing attention, with studies identifying antitumoral neutrophil populations, either at baseline or induced by treatment. Several effective treatment regimens involve an anti-CD40 agonist (aCD40) antibody, among them Neutrophil Activating Therapy (NAT), which combines aCD40, TNF, and a tumor antigen binding antibody designed to reprogram neutrophils. NAT could thus be particularly effective in cold, myeloid-rich tumors that are largely unresponsive to conventional immunotherapies such as checkpoint blockade, enacting these anti-tumoral effects through similar and different mechanisms; however, this has not been tested. WHAT THIS STUDY ADDSThis study adds a single-cell multi-omics framework for defining treatment-induced neutrophil heterogeneity in MOC2-huEGFR and 9464D-GD2 tumors, two immunologically cold models. It highlights ICAM1/CD54 and interferon-stimulated genes as markers of a dominant antitumor neutrophil state, while showing that neutrophil state composition variy across tumor models. HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICYThese results support the efficacy of a myeloid-modulating therapy built around aCD40 and TNF in a cold murine head and neck cancer model, and to a lesser extent in a cold murine neuroblastoma model. ICAM1/CD54 expression in neutrophils was also identified as a promising marker of antitumor activity and treatment response. More broadly, this work suggests that incorporating aCD40 and/or TNF into existing treatment regimens could improve outcomes, while ICAM1/CD54-high neutrophils may serve as a useful therapeutic readout.

cancer biology↗

bioRxiv2026

Phage terminase recognition by the bacterial immune sensors Avs2 and Upx

Prokaryotes employ diverse defense strategies to detect and halt the progression of phage infection. Multiple defense systems sense phage proteins through direct binding, including antiviral STAND NTPases (Avs), which oligomerize upon target recognition to induce programmed cell death. The widespread Avs2 family was previously shown to detect the large terminase subunit of tailed phages, but the mechanism of terminase sensing was unknown. Here, we determine the structural basis of terminase recognition by Avs2 from Escherichia coli (EcAvs2). A cryo-EM structure at 2.3 [A] resolution reveals that EcAvs2 forms a flat, C4-symmetric tetramer in which each protomer is bound to a single terminase monomer. Terminase recognition is mediated by a large, shape complementary binding pocket in the EcAvs2 sensor domain, including specific contacts with an unexpected ATP molecule at the interface of EcAvs2 and terminase. Furthermore, we demonstrate that the defense protein Upx also recognizes diverse phage terminases, despite lacking sequence and structural homology to Avs. AlphaFold 3 models indicate that Upx binds an unfolded state of the core terminase ATPase domain, mediated by {beta}-augmentation. These findings highlight the distinct modes of terminase recognition across structurally diverse defense proteins.

molecular biology↗

bioRxiv2026

Shape Factor Analysis as a Quantitative Framework for Assessing Spheroid and Organoid Morphology and Invasiveness

Morphological changes of spheroids and organoids are widely used as in vitro indicators of healthy and diseased tissue functions; however, quantitative methods to classify spheroid and organoid morphology are limited. In clinical breast imaging, radiologists use tumor shape as a prognostic marker, with irregular margins associated with invasive disease and increased malignancy. Here, we adapted this approach for translational research and developed a custom MATLAB algorithm to quantify the variance in radial lengths of invasive protrusions in spheroids and organoids. First, we analyzed digital phantoms by both ImageJ/FIJI shape descriptors and our radial length analysis to evaluate the capabilities of each measurement method. Subsequently, we performed the same comparisons with images from experimental spheroid and organoid datasets. We demonstrate that multivariate shape factor analysis, including radial length analysis, enables more reliable and comprehensive quantification of spheroid and organoid morphologies than standard shape descriptors alone. By enabling numerical morphological readouts, shape factor analysis can enhance phenotypic profiling of spheroids and organoids and provide valuable metrics for in vitro studies including high-throughput and drug screening workflows.

bioengineering↗

bioRxiv2026

SR2P: an efficient stacking method to predict protein abundance from gene expression in spatial transcriptomics data

Spatial transcriptomics data are largely available with RNA expression alone, limiting the detection of cell states defined by surface protein abundance. The lack of multi-omics spatial data limits the ability to identify immune cells and their signaling in the tumor microenvironment, as most solid tumors are immunologically poor and exhibit protein-RNA abundance discordance in critical immune cell surface markers. Although emerging technologies enable spatial multi-omics profiling, technical and cost constraints remain a hurdle. We introduce SR2P, a stacking-based machine-learning framework for predicting spatial protein abundance from RNA expression. SR2P integrates 11 complementary predictive models and consistently outperforms existing methods across multiple spatial multi-omics benchmark. We showcased an application of SR2P recovered macrophage-enriched regions and identified potential immune markers associated with therapeutic response from head-and-neck squamous cell carcinoma patients. SR2P enables protein-abundance inference from RNA-only spatial data, extending the analytical capabilities of current spatial platforms for studies of tumor immunology.

bioinformatics↗

bioRxiv2026

Activating Ras-MAPK pathway variants drive hippocampal clonal competition in human epilepsy

Mesial (a.k.a., medial) temporal lobe epilepsy (MTLE) is the most common focal epilepsy1,2 and, in drug-resistant cases, is treated by surgical removal of the anterior temporal lobe, which often shows neuronal loss and gliosis consistent with hippocampal sclerosis (HS)2. MTLE with HS has minimal contribution from germline genetic variation3, and is associated with prior precipitating insults such as prolonged childhood seizures and head trauma4-6. Somatic variants in Ras-MAPK pathway genes were recently reported in a few MTLE surgical specimens7,8, but their prevalence, clinical relevance, and underlying biological mechanisms remain unknown. Targeted duplex sequencing of hippocampal DNA from 462 surgical resections revealed significant enrichment of deleterious somatic variants in MTLE versus controls, with >40% of MTLE specimens harboring activating Ras-MAPK variants in PTPN11, NF1, BRAF, KRAS, and twelve genes not previously associated with focal epilepsy. Eight Ras-MAPK genes showed positive clonal selection in MTLE. Increased somatic variant burden predicted worse surgical outcome. Somatic Ras-MAPK variants at ultra-low (<0.5%) variant allele fractions were associated with older seizure onset and HS pathology, supporting a late prenatal or postnatal origin. Ras-MAPK variants in MTLE were enriched in cells derived from hippocampal progenitors--neurons, astrocytes, oligodendrocytes--in line with the known neuronal hyperexcitability and seizures induced by Ras-MAPK overactivation9,10; in contrast, Alzheimer disease hippocampi exhibited microglial enrichment of Ras-MAPK variants, consistent with prior reports11. Single-nucleus RNA sequencing showed increased expression of Ras-MAPK genes in neurons and upregulation of pathways mediating neurogenesis and neural development in MTLE. Functional validation of novel, recurrent PTPN11 variants confirmed gain-of-function, while cellular modeling in induced pluripotent stem cells demonstrated proliferative/survival advantages for mutant cells in mosaic culture. Overall, our data suggest that somatic Ras-MAPK variants and acquired risk factors may converge on clonal competition in the hippocampus to modulate epilepsy risk.

genetics↗

bioRxiv2026

The ER membrane protein complex acts as a chaperone to promote the biogenesis of multi-bundle membrane proteins

Nearly half of the [~]5,000 human membrane proteins need to assemble into stoichiometric complexes as part of their biogenesis at the endoplasmic reticulum (ER) membrane. How ER resident biogenesis factors coordinate membrane insertion, folding and assembly is not well understood. Here, we demonstrate that the ER membrane protein complex (EMC) insertase additionally acts as a chaperone to facilitate the assembly of heterotrimeric voltage-gated calcium channels (Cav). Using function-separating mutations and inhibitory nanobodies we show that nascent Cav channels are degraded prematurely when EMCs chaperone function is selectively perturbed. Blocking EMCs chaperone function strongly impaired Cav-dependent cardiomyocyte contraction. EMC engagement of the pore-forming Cav-subunit occurred co-translationally and required Cavs first transmembrane domain bundle to protrude from the nascent ribosome*Sec61*multipass translocon complex. Our findings establish a chaperone function for the EMC and reveal that biogenesis of multi-bundle membrane proteins requires a highly orchestrated, co-translational interplay between ER biogenesis factors.

cell biology↗

bioRxiv2026

Analyzing an organisms sensors using Maximum Entropy models with bias, variance, and confusion matrices

Biological organisms have sensors that communicate information about the environment. Analyzing how well these biological sensors function has usually been done with mutual information between the sensor signal and the environment, but that can be computationally intractable and summarize something quite complex with just a single number. We suggest that alternatively, one may profitably analyze these biosensors using bias and variance or confusion matrices, depending on the kind of environment. Stimulus-dependent Maximum Entropy models are used to develop estimators of the environmental state given the sensor state, and these estimators in turn are then used to calculate either the bias and variance of the estimator or confusion matrices. We focus on several examples to understand the utility of non-information-based analyses: ligand-receptor binding models spanning genetic regulation to neuronal communication to bacterial chemotaxis, and spin-glass Ising models for neural activity in cultured neurons. These new computationally-efficient analyses add insight to existing analyses based on mutual information; in particular, mutual information estimates give one number to characterize responses to all environmental inputs, and this analysis method characterizes how sensors respond to each environmental input. Categorical analyses, meanwhile, indicate the presence of memory without much prediction in confusion matrix elements in cultured neural networks, adding to previous understanding from mutual information estimates. Author summaryAll living organisms use external stimuli to navigate their environment via their sensors. Because encoding information costs energy, organisms retain only a fraction of the information received from their sensors, ideally information that maximizes their ability to remember past environmental states or predict future ones, key functions that support survival. To better understand how well sensor systems absorb stimulus information, we used stimulus-dependent Maximum Entropy (MaxEnt) models with maximum likelihood estimation and typical statistical metrics, such as confusion matrices or bias and variance. This approach provides two primary benefits over previous approaches: it is more computationally efficient, and it provides a more information-rich picture on how sensors interact with stimulus.

biophysics↗

bioRxiv2026

Diverse bacterial pattern recognition receptors sense the conserved phage proteome

Recognition of foreign molecules inside cells is critical for immunity across all domains of life. Proteins of the STAND NTPase superfamily1,2, including eukaryotic NOD-like receptors (NLRs), play a central role in this process3,4. In bacteria and archaea, although several STAND families sense phage proteins5-9, their functional diversity remains largely unexplored. We conducted a systematic phylogenetic analysis of prokaryotic STAND NTPases and identified at least 90 structurally distinct families associated with antiviral defense. We first show that the uncharacterized Avs7 family recognizes the major capsid protein (MCP) of tailed phages. Three cryo-EM structures of Salmonella enterica Avs7 reveal an asymmetric, butterfly-shaped tetramer that assembles stepwise via large, MCP-induced conformational changes, incorporating bacterial elongation factor Tu (EF-Tu) as a structural component that enhances defense. Using genetic screens with a library of 687 phage genes, we further show that 13 additional STAND families sense 13 conserved phage proteins, encompassing most of the core structural and replicative components of tailed phages. These include two distinct MCP-sensing families--Avs8 (PD-{lambda}-4) and Avs10 (Erebus/Hypnos/bNACHT64)--and 11 others (Avs11-21) recognizing the portal, portal adaptor, tail nozzle, head-tail connector, tail terminator, tail tube protein, tail assembly chaperone, tape measure protein, DNA polymerase, helicase/RecA-type ATPase, and single-stranded DNA annealing protein, respectively. Together, our findings highlight structurebased pattern recognition and host factor repurposing as fundamental strategies of bacterial immunity.

molecular biology↗

bioRxiv2025

Immune-mediated Engagement of T Regulatory Cells with Tumor Cells Results in Trogocytosis and Tumor Cell Killing.

Despite advances in treatment, >50% of patients with advanced melanoma are unresponsive to current therapies. Using the B78-D14 melanoma model (GD2+/MHC-I-/MHC-II+), we can cure mice with a regimen that includes radiation therapy (RT) in combination with immunocytokine (IC; anti-GD2 monoclonal antibody linked to IL-2) while establishing immunological memory. We interrogated the role of T cells in the antitumor and memory responses following RT+IC. We show a requirement for CD4, but not CD8 T cells, to achieve both the initial and memory responses. Upon IC-induced cell-cell contact, subsets of CD4 T cells, including Foxp3 T regulatory cells, trogocytose GD2 from tumor cells, acquire cytotoxic granules, and kill tumor cells. These results were confirmed using human tumor cell lines. These findings reveal that CD4 T regulatory cells, upon immunologically-induced binding to tumor cells, can trogocytose tumor antigens and directly kill tumor cells, redefining their potential role in antitumor immunity.

cancer biology↗

bioRxiv2025

Isoflavones impair response to anti-PD1 therapy in murine breast cancer models, irrespective of dietary fiber and fecal short chain fatty acid levels

BackgroundFermentable dietary fibers, also called microbiota-accessible carbohydrates (MAC), and the consequent increase in fecal short-chain fatty acids (SCFAs) are linked to improved responsiveness to immune checkpoint blockade (ICB) therapy in human and mouse studies. However, experimental diets high in MAC also often contain estrogenic isoflavones, which may counter fibers beneficial effects by causing immunosuppression. MethodsWe studied the effects of feeding female C57BL/6Tac mice low-MAC (AIN93G), low-MAC supplemented with isoflavone genistein, high-MAC (5V5M) or high-MAC isoflavone (high-MACi; 5058D) diet on their gut microbiome and response to anti-PD1 therapy against E0771 allografted triple negative breast cancer (TNBC) and 7,12-dimethylbenz[a]anthracene (DMBA)-initiated estrogen receptor positive (ER+) mammary tumors. We also determined whether blocking ER with tamoxifen (TAM) impacted responsiveness to anti-PD1 therapy in mice fed different diets. The effect of diet and treatments on immune cell signaling pathways was investigated using NanoString PanCancer Immune Profiling Panel. ResultsHigh-MAC and high-MACi diets increased fecal microbial alpha-diversity and the abundances of SCFA producing families Lachnospiraceae, and Oscillospiraceae as well as fecal SCFA levels, compared with low-MAC diet. E0771 tumors responded to anti-PD1 in mice fed high-MAC, while mice fed high-MACi did not respond. Low-MAC fed mice with single E0771 allograft also responded to anti-PD1, but genistein supplementation eliminated responsiveness. E0771 tumors in high-MAC fed mice contained elevated levels of exhausted CD8+ T cells, which were decreased after anti-PD1 therapy. Opposite effects were seen in mice fed high-MACi diet. Mice with DMBA-initiated ER+ mammary tumors did not respond to anti-PD1. TAM converted TNBC and ER+ tumors to become sensitive to anti-PD1 therapy in mice fed high-MACi or low-MAC diets, respectively. Genes in TH17 differentiation pathways were linked to TAM-induced improved anti-PD1 response both in TNBC and ER+ mammary tumors. ConclusionsOur results highlight the role of diet in impacting the effectiveness of ICB therapies. We found that increased SCFA levels alone are not predictive of response to anti-PD1, but if tumor expresses ER or if diet contains ER activating compounds, such as isoflavones, blocking ER+ might convert unresponsive tumors responsive to anti-PD1. Word count: 339 What is already known on this topicDietary fiber is proposed to improve response to checkpoint inhibitor therapy against melanoma, but this has been challenged by a recent preclinical study in which different mouse tumor models were used. None of the studies have been done in breast cancer or preclinical breast cancer models. What this study addsOur study showed, using triple negative breast cancer (TNBC) and estrogen receptor positive (ER+) breast cancer models, that indeed high levels of microbiota accessible carbohydrates (MACs) in diet did not alone determine responsiveness to anti-PD1 therapy, but diet high in plant isoflavones/ hormones impaired anti-PD1 effectiveness, regardless of whether diet contained high fiber levels or not. We also found that the adverse effects of isoflavones were counteracted by tamoxifen, partial estrogen receptor antagonist. How this study might affect research, practice or policyOur findings could indicate that breast cancer patients, both those with TNBC and ER+ disease, should not consume diets high in isoflavones when treated with anti-PD1.

cancer biology↗

bioRxiv2025

Proteolytic activation of diverse antiviral defense modules in prokaryotes

Linked protease-effector modules are widespread in prokaryotic antiviral defense, yet the mechanisms of most remain poorly understood. Here we show that four of the most prevalent modules--metallo-{beta}-lactamase (MBL)-fold hydrolase, /{beta}-hydrolase, Pepco, and EACC1--form latent death effectors that are unleashed by site-specific proteolysis. Genetic, biochemical, and structural analyses reveal novel modes of effector licensing. MBL and /{beta}-hydrolase are zymogens activated by cleavage at two distinct sites, and upon proteolysis, MBL becomes a Zn2+-dependent double-stranded DNA nuclease. In contrast, Pepco and EACC1 act as pore-forming toxins via distinct mechanisms: Pepco constitutively oligomerizes into a denaturation-resistant beta barrel that is activated by cleavage after a specific isoleucine in its C-terminal tail, whereas EACC1 monomers assemble into large membrane pores following removal of an autoinhibitory domain. All four modules are fused to diverse sensors to detect a wide range of phage signals, and EACC1-DnaK chaperone fusions suggest a convergence between defense and general stress responses. These findings establish proteolysisgated activation as a dominant, modular logic for anti-phage defense and reveal parallels with eukaryotic innate immunity.

molecular biology↗

bioRxiv2025

Divergent role of CD8 T cells with distinct metabolic phenotypes during curative radio-immunotherapy in hot versus cold tumors

Immunotherapy has potential for impactful cancer cures by empowering patients own immune cells. We developed a radio-immunotherapy regimen that can cure large immunologically hot and cold murine tumors. Here, we explored the divergent role of CD8 T cells during this radio-immunotherapy in contrasting hot colon carcinoma versus cold melanoma. We introduced an immunocompetent mouse model with mCherry-expressing CD8 T cells to provide cell tracking in vivo. We investigated single-cell function, metabolism, and gene expression temporal changes using flow cytometry, in vivo multiphoton imaging, single-cell RNA sequencing, and multiplexed immunofluorescence to determine the underlying mechanisms. We found that in contrast to the hot colon carcinoma model, CD8 T cells from the cold melanoma model do not drive tumor cures, despite getting activated, possibly due to a static oxidative metabolism and exhausted phenotype plus down regulation of tumor MHC-I expression. These findings have implications for improving immunotherapy response in immunologically cold cancers.

immunology↗

bioRxiv2025

Pro-restitutive Bacteroides thetaiotaomicron reprograms the transcriptome of intestinal epithelial cells by modulating the expression of genes essential for proliferation and migration.

The mammalian intestine harbors a highly complex, very diverse, and numerically vast community of symbiotic microorganisms, which profoundly influence the development and maintenance of the intestinal barrier function. Alterations in microbial composition, known as dysbiosis, are observed in Inflammatory Bowel Disease (IBD), colorectal cancer (CRC), and gastrointestinal infections; however, the exact causal relationship between these changes and the resolution of intestinal inflammation and the repair of damaged mucosa remains unclear. Notably, IBD is not only marked by dysbiosis but also by changes in microbial metabolic pathways and metabolite landscape in the intestinal lumen. The small molecules and microbial metabolites present in the intestinal lumen have emerged as potential regulators of gut pathology, cancer, and mucosal repair. Investigating how altered microbiota and microbial metabolic activities influence intestinal epithelial cells (IEC) can provide insights into their role in the regeneration of mucosal epithelia and restoration of gut barrier functions. This knowledge can be harnessed to promote intestinal homeostasis, prevent relapse, and prolong remission of IBD. To dissect the complex interplay between the gut microbiome and IEC, we focused on the overrepresented bacterium Bacteroides thetaiotaomicron. Here, we show that B. thetaiotaomicron and Akkermansia muciniphila, the dominant members of gut microbiota, expand during the repair & resolution phase of the chemically induced acute murine colitis. Furthermore, our bioinformatics analysis demonstrated that the elevated relative abundance of B. thetaiotamicron was also accompanied by rewiring of bacterial metabolic programs towards the essential amino acid metabolism, polyamine synthesis and utilization, stress response mechanisms, cell envelope biogenesis, and nutrient scavenging. Our RNA sequencing and transcriptomic analysis of primary human colonic epithelial cells cocultured with B. thetaiotaomicron showed that B. thetaiotaomicron stimulates the expression of genes and pathways involved in different cellular functions, including proliferation, differentiation, adhesion, lipid metabolism, migration, chemotaxis, and receptor expression. Our study emphasizes the crucial functions of the gut microbiome and metabolic activities in regulating the functions of intestinal epithelial cells during the repair of injured gut mucosa. Thus, these microorganisms and their metabolism hold promise as potential therapeutic agents.

microbiology↗