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Seo, H.

Publications and source records attributed to Seo, H..

At least 19 recordsLinked to original sources

Predicting conformational changes induced by biomolecular interactions

Biomolecular interactions trigger structural changes of protein complexes essential for cellular signaling, regulation, and drug action. Modern co-folding models can now generate detailed structures of multi-component complexes directly from sequence. However, recent evaluations reveal that they frequently fail to capture specific conformational states induced by a ligand or partner. Here, we show that explicitly modeling the structural transition from an unbound to a bound state, rather than generating the bound complex alone, better resolves these interaction-induced states. Our generative learning framework, K-Fold, transforms independently predicted unbound component priors into an assembled complex through an ultrafast diffusion bridge, operating entirely without multiple sequence alignments. By eliminating co-evolutionary dependencies, K-Fold accurately recovers non-coevolved interfaces in antibody-antigen and targeted-degrader complexes, as well as ligand-directed regulatory states in GPCRs and kinases. Furthermore, by coupling this diffusion bridge with diverse monomeric equilibrium ensembles, K-Fold provides a computationally tractable foundation for sampling complex multi-state landscapes without retraining. These results demonstrate that formulating molecular assembly as an explicit structural transition moves structure prediction beyond static geometries toward capturing biomolecular dynamics.

molecular biology↗

Single-Cell and Spatial Analysis Reveals Retention of FCRL4⁺ Memory B Cells in Pediatric Tonsillar Hypertrophy

Pediatric tonsillar hypertrophy is a leading cause of sleep disordered breathing in children, yet the immune mechanisms sustaining tonsillar enlargement remain unclear. Using integrated single cell and spatial profiling of pediatric tonsils in a discovery cohort (n = 45) and independent validation cohort (n = 32), we identify a selective expansion and follicular retention of FCRL4+ memory B cells within the mantle zone of hypertrophic tonsils. These cells exhibit a quiescent, tissue-adapted phenotype characterized by NFATC1 repression and reciprocal RUNX1/RUNX2 regulation. Their accumulation is orchestrated by follicular type 1 regulatory T (Tr1) cells, which engage CTLA-4/CD86 checkpoint interactions to suppress plasma cell differentiation and reinforce a local immune tolerance program. Together, these interactions establish a persistent follicular niche enriched in regulatory FCRL4+ memory B cells. Our findings delineate a mechanistic Tr1/FCRL4+ B cell axis underlying follicular hypertrophy and highlight potential immunomodulatory targets for nonsurgical management of pediatric sleep-disordered breathing.

immunology↗

Dual-targeted nanoparticles enable the ex vivo generation of cytotoxic and phagocytic CAR effector cells

Chimeric antigen receptor (CAR)-T cells and CAR-macrophages (or CAR-monocytes) offer complementary antitumor functions. CAR-T cells exert potent cytotoxicity against cancer cells, whereas CAR-macrophages can infiltrate solid tumors, phagocytose cancer cells and promote antigen spreading. Antibody-conjugated lipid nanoparticles (LNPs) have emerged as non-viral mRNA delivery platforms for generating CAR-immune cells. However, these systems have generally been optimized to target a single immune-cell type. Here, to enable the ex vivo generation of CAR-T cells and CAR-monocytes, we developed CLCM08, an anti-CD222 antibody, and co-displayed it with an anti-CD5 antibody on LNPs. CD222 is predominantly expressed by monocytes, macrophages and T cells, and facilitates the internalization of CD222-binding molecules. CD5 is primarily expressed by T cells and has previously been used to enable LNP-mediated mRNA delivery to these cells. Delivery of CAR mRNA by dual CD222/CD5 antibody-conjugated LNPs to primary monocytes and T cells substantially enhanced the ex vivo generation of CAR-monocytes and CAR-T cells compared with non-conjugated LNPs. Moreover, CD19 CAR-T cells and CAR-monocytes generated using dual CD222/CD5 antibody-conjugated LNPs showed enhanced cytotoxic and phagocytic activity, respectively, relative to cells generated using non-conjugated LNPs, with anti-CD222 LNPs producing the largest phagocytic gain in monocytes. These findings establish an adaptable antibody-conjugated LNP-mRNA platform for the ex vivo generation of CAR-T cells and CAR-monocytes.

biochemistry↗

PTEN Subcellular Localization Dictates Function

Mutations in phosphatase and tensin homolog (PTEN) drive unregulated activation of the phosphatidylinositol-3-kinase (PI3K) pathway, resulting in neuronal hypertrophy, and are strongly associated with autism spectrum disorder (ASD). Several PTEN mutations alter subcellular localization, yet how localization governs PTEN function in developing neurons remains unclear. Although PTEN has been reported broadly distributed throughout neurons, here, live imaging of HaloTagged PTEN reveals dynamically regulated localization, suggesting spatial control of its signaling. We then used retroviral-mediated genetic manipulation to delete endogenous Pten in developing hippocampal neurons while simultaneously expressing PTEN fused to defined localization motifs, allowing us to directly test how subcellular targeting regulates neuronal morphology. Loss of Pten produces neurons characterized by enlarged somata, more elaborate dendritic arbors, and increased spine density, length, and head area. Nuclear-excluded PTEN fully rescued these phenotypes, whereas targeting PTEN to filopodia via fusion to the FBAR domain of srGAP3 or to the postsynaptic density via Homer1C corrected or corrected all morphological abnormalities in PTEN-deficient neurons and simplified dendritic arborization compared to wild-type. In contrast, nuclear-localized PTEN produced only partial rescue, normalizing soma size and spine head area but not dendritic complexity or spine density. These findings indicate that PTEN acts locally to restrain growth and structural connectivity, whereas regulation of spine head size can be mediated by PTEN both inside and outside the nucleus, potentially through transcriptional or splicing-dependent mechanisms. Together, our results identify subcellular localization as a critical determinant of PTEN function and reveal spatially distinct mechanisms through which PTEN sculpts neuronal development.

neuroscience↗

Reproducible transcriptional modules define glioblastoma ecosystems across independent cohorts.

Glioblastoma (GBM) comprises a complex ecosystem of malignant, immune, vascular and neural transcriptional states. However, it remains difficult to determine which gene expression programmes are reproducibly recovered across independent cohorts and profiling platforms, because programme-level analyses are sensitive to cohort composition, technical context and factorization rank. Here, we analyzed three public GBM datasets--GLASS and IVYGAP bulk RNA-seq cohorts and the HEILAND Visium spatial transcriptomics cohort--to examine whether deconvolution-derived programmes could be organized into shared cross-cohort modules. Integrating 279 programmes inferred by consensus non-negative matrix factorization identified eight transcriptional communities, including myeloid immune microenvironment, neuronal and synaptic, oligodendrocyte and myelin, developmental, tumour-associated mesenchymal or hypoxic, proliferative, and ciliated or ependymal-like modules, as well as one cohort-restricted community. Community activity showed coherent associations with independent annotations: the myeloid community correlated with ESTIMATE immune score and inversely with tumour purity; the oligodendrocyte and myelin community was reduced in recurrent tumours; and ciliated or ependymal-like and neuronal communities showed modest exploratory associations with overall survival. Spatial projection onto Visium data provided qualitative support for the histological coherence of several modules, while also highlighting the limits of spot-level interpretation. Together, these results provide a proof-of-concept that cross-cohort integration can recover recurrent transcriptional structure across heterogeneous GBM datasets and offer an interpretable framework for comparing gene expression programmes while preserving cohort-specific signal and uncertainty in biological assignment.

bioinformatics↗

B7-H3 Modulates Cell Adhesion and Immune Evasion to Promote Tumor Progression and Natural Killer Cell Resistance in Hepatocellular Carcinoma

B7-H3 (CD276) is an immune checkpoint molecule frequently overexpressed in hepatocellular carcinoma (HCC) and represents a promising therapeutic target. However, its roles in tumor cell adhesion, metastatic behavior and immune evasion--particularly in interactions with natural killer (NK) cells--remain incompletely understood. In the present study, B7-H3 was depleted using small interfering RNA and CRISPR/Cas9 in epithelial (Huh7 and HepG2) and mesenchymal (SNU449) HCC cell lines. Tumor cell survival, apoptosis, adhesion, migration and invasion were evaluated using functional assays. Expression of adhesion molecules and immune checkpoint proteins was assessed by flow cytometry and western blotting. Oncogenic signaling pathways were analyzed by examining phosphorylation of Akt, ERK, FAK and STAT3. NK cell-mediated cytotoxicity was assessed using primary human NK cells. B7-H3 depletion reduced clonogenic survival and increased apoptosis in mesenchymal HCC cells under anchorage-independent conditions. Loss of B7-H3 impaired cell adhesion, migration and invasion, accompanied by downregulation of PTGFRN, E-cadherin, integrin 3 and integrin V, and reduced cell-to-cell aggregation under anchorage-independent conditions. B7-H3 depletion also decreased surface expression of PD-L1, PD-L2 and CD47. Notably, B7-H3-deficient cells exhibited enhanced susceptibility to primary NK cell-mediated cytotoxicity. Mechanistically, B7-H3 promoted tumorigenic signaling through Akt/S6, MVP/ERK and FAK/Src pathways in epithelial cells, and through FAK/Src and JAK2/STAT3 pathways in mesenchymal cells. Together, these findings reveal previously unrecognized roles for B7-H3 in coordinating adhesion and NK immune evasion in HCC, and support its therapeutic targeting for next-generation immunotherapies.

cancer biology↗

High-resolution single-cell RNA sequencing using canFam4 reveals novel immune subsets and checkpoint programs in healthy dogs

Single-cell RNA sequencing (scRNA-seq) enables high-resolution profiling of immune heterogeneity. Although previous studies have mapped the single-cell transcriptomic atlases of peripheral leukocytes in healthy dogs, the identification and functional characterization of distinct immune subsets remain incomplete. We constructed a single-cell atlas of peripheral leukocytes from six healthy small-breed dogs using the 10x Genomics platform and the updated canFam4 genome. Analysis of 30,040 high-quality transcriptomes revealed 51 distinct immune subsets, including CD14{square}CD33{square} monocytes, XCR1{square}CD1D{square} dendritic cells, CEACAM1{square}CD24{square} neutrophils, and IL32{square}BATF{square} regulatory T cells, which were underrepresented in canFam3.1-based studies. Interferon-enriched CD14{square} monocytes and CD4{square} T subsets associated with myxomatous mitral valve disease were also identified. Functional analysis revealed that PDCD1 attenuates TCR signaling, LAG3 modulates malate metabolism in CD4{square} T cells, and suppresses TBX21 in CD8{square} T cells associated with viral response. CD274 encoding PD-L1 was linked to IL-10 production in neutrophils, and CTLA4 represented an initial activation of double-negative T subsets. T cell exhaustion scores and proliferative fractions varied across cohorts, reflecting differences in environmental antigenic exposures. Our study represents the first comprehensive, gene-resolved single-cell analysis that reveals immunoregulatory checkpoint mechanisms underlying immune homeostasis in healthy dogs. Our dataset will serve as a valuable resource for future comparative and translational immunology research in dogs.

immunology↗

Enabling supratheoretical isopropanol yields from carbon-negative glucose fermentations with Clostridium acetobutylicum-Clostridium ljungdahlii cocultures

Synthetic microbial cocultures, which combine the unique capabilities of multiple microbes into one process, have significant potential for sustainable production of fuels and chemicals. Most studies of defined cocultures have tested relatively low cell densities in lab-scale batch cultures, not the high cell density fed-batch or continuous processes with cell retention typically required to achieve industrially-relevant volumetric productivities. Here, we explore the impact of increased cell density on isopropanol production from the syntrophic coculture of genetically-modified Clostridium acetobutylicum [CACas9 {Delta}hbd (p95ace02_atoB), with deleted 4-C metabolism expressing an acetone-formation pathway on the plasmid] with WT Clostridium ljungdahlii using first a pseudo-perfusion approach followed by perfusion culture. CACas9 {Delta}hbd (p95ace02_atoB) produces acetone without any 4-C metabolites and C. ljungdahlii converts that acetone to isopropanol. To explore the mechanism by which these cultures enable supratheoretical isopropanol yields, we first identified NADH-driven hydrogen conversion in CACas9 {Delta}hbd (p95ace02_atoB) as the thermodynamically-limiting step for acetone and thus isopropanol production. We then demonstrated the ability of C. ljungdahlii to mitigate this issue by eliminating detectable hydrogen accumulation in the coculture. Pseudo-perfusion cocultures showed that high cell densities combined with a high population fraction of C. ljungdahlii enable dramatic increases in isopropanol yields beyond the thermodynamic limitation imposed in CACas9 {Delta}hbd (p95ace02_atoB) monocultures. Finally, we demonstrate carbon-negative fermentation of glucose to isopropanol as the sole alcohol product in a perfusion bioreactor.

bioengineering↗

Inhibitory Circuit Compensations in Female and Male Mice: Increased Synaptic Output Offsets Reduced Parvalbumin Interneuron Density

Parvalbumin inhibitory interneurons (PV-INs) are critical regulators of excitatory/inhibitory balance in the cortex, and their dysfunction has been observed in various neurological disorders. Despite increasing recognition of sex differences in brain function, little is known about how PV-INs differ between males and females under healthy conditions. Previous work has pointed to sex differences in PV-IN vulnerability in disease and injury models. Here, we investigated sex differences in PV-IN characteristics, connectivity, and function in the retrosplenial cortex (RSC) of healthy mice. We found that female mice have significantly fewer PV-INs in the RSC compared to males, yet exhibit comparable memory induced neuronal activation (fos expression). Despite their lower numbers, female PV-INs displayed greater synaptic connectivity, as evidenced by increased synaptotagmin-2 (Syt-2) puncta per PV-IN and higher axonal bouton density. Additionally, fewer female PV-INs were surrounded by perineuronal nets (PNNs), suggesting greater plasticity in female inhibitory networks. From ex vivo slice electrophysiology recordings we observed greater excitability in female PV-INs compared to male PV-INs and, a reduced incidence of IPSCs. These findings indicate that female mice may compensate for reduced PV-IN numbers through enhanced synaptic output, preserving inhibitory function in the RSC. Finally, using spatial transcriptomic profiling of PV-INs we observed a number of differentially expressed genes that are consistent with the observed structural and functional differences between female and male PV-INs. Understanding these sex-specific inhibitory mechanisms is crucial for developing more targeted interventions for conditions involving PV-IN impairment and for understanding sex specific vulnerabilities to certain conditions such as Alzheimers disease.

neuroscience↗

Shared and distinct cortical mechanisms for working memory and decision-making.

The dorsolateral prefrontal cortex (DLPFC) and lateral intraparietal cortex (LIP) in the primate brain are critically involved in working memory during tasks that require the retention of information over a delay. These same regions have also been implicated in reinforcement learning (RL), where information about an animals choice and its outcome is retained to update future reward expectations based on past experiences. We investigated whether spatial memory, required across different behavioral contexts, relies on a shared neural mechanism. To explore this, we analyzed neural activity recorded from rhesus monkeys engaged in three distinct tasks--the oculomotor delayed response task (ODR), a visual search task, and the matching pennies game--each requiring the retention and use of similar spatial information under different cognitive demands. The ODR task demands only prospective memory, as the selection of action is dictated by the location of visual cue, and the subject must retain this intended action for execution after a temporal delay. In contrast, the matching pennies task engages both retrospective and prospective memory: retrospective memory of previous choice and its outcome to inform decision-making, while prospective memory is needed to carry out that decision. Visual search task, by comparison, does not explicitly require either retrospective or prospective memory. Our analysis revealed that neural signals encoding retrospective memory of the animals choice in the visual search and matching pennies tasks were not correlated with the prospective working memory signals of visually cued locations in the ODR task, in either the DLPFC or LIP. Moreover, retrospective choice signals in the visual search and matching pennies tasks were not correlated with each other. In contrast, neural activity related to upcoming choices (prospective memory) in the LIP showed significant correlations across all three tasks. In the DLPFC, prospective choice signals were correlated between the visual search and ODR tasks, but not between those tasks and matching pennies. Additionally, in the DLPFC, neural signals representing previously rewarded choices were significantly correlated with working memory signals during the ODR task. These results suggest that the LIP supports a consistent, shared mechanism for prospective memory linking a committed action to its eventual execution. In contrast, the DLPFC might mediate the transformation of retrospective memory-integrating past choices and outcomes - into a decision and its associated prospective memory.

neuroscience↗

Targeted Molecular MRI of Colorectal Cancer by Antibody Functionalized Hyperpolarized Silicon Particles

The development of non-invasive, non-ionizing sensitive molecular targeting approaches to detect colorectal cancer (CRC) lesions is warranted to improve high risk patient outcomes. Hyperpolarized silicon nanoparticles and microparticles are potentially well-suited to act as targeted molecular imaging agents because of their overall biocompatibility and long-lasting enhanced magnetic resonance imaging (MRI) signals. In this study, dynamic nuclear polarization was performed on silicon particles functionalized with an antibody to Mucin-1 (MUC1), a surface mucin glycoprotein aberrantly expressed in CRC. Antibody conjugation to the particle surface did not affect 29Si hyperpolarization characteristics. Similarly, conjugation and the dynamic nuclear polarization process did not adversely affect the affinity of the targeting antibody. In vivo MRI scans performed 10-15 minutes after luminal administration of targeted hyperpolarized particles into human MUC1-expressing orthotopic CRC mouse models showed that particles actively targeted tumor sites. These results were supported by chemical and biological controls and blocking experiments as well as correlative immunohistochemical analysis. These surface-functionalized silicon particles are under development as a platform technology that will allow non-invasive molecular targeting of CRC using hyperpolarized MRI. Single Sentence SummaryTargeted molecular MR imaging of colorectal cancer by hyperpolarized silicon particles functionalized with mucin 1 antibody.

cancer biology↗

Evaluating gene representation in spatial transcriptomics across pre-designed panels

Spatial transcriptomics (ST) has revolutionized our understanding of gene expression within tissues by preserving spatial context. Over the past few years, this technology has led to a number of paradigm-shifting discoveries that have enabled a more comprehensive understanding of cellular functions and interactions in normal and diseased states. However, ST technologies still face challenges related to resolution, sensitivity, and technical variability. In this study, we evaluate the read coverage of commercialized pre-designed panels using publicly available ST datasets generated from the 10X Genomics and Nanostring platforms. We introduce the Coverage Index (CI) as a quantitative metric to assess the representation of established gene signatures across multiple ST datasets. Our findings reveal that cancer-related gene lists exhibit the highest CI values, while genes encoding for ligands and receptors tend to have low coverage. Additionally, CI analysis can help highlight intrinsic biases in gene panel design, influencing the detection capacity and thereby downstream comprehension of certain biological pathways. The insights gained from this study provide a framework for assessing ST panel performance and optimizing gene panels for future spatial transcriptomic applications.

bioinformatics↗

Cytosine-to-uracil RNA editing is upregulated by pro-inflammatory stimulation of myeloid cells

Myeloid cells undergo large changes to their gene expression profile in response to inflammatory stimulation. This includes an increase in post-transcriptional modifications carried out by adenosine-to-inosine (A-to-I) and cytosine-to-uracil (C-to-U) RNA editing enzymes. However, the precise RNA editing targets altered by stimulation and the consequences of RNA editing on gene expression and the proteome have been understudied. We present a comprehensive RNA editing analysis of stimulated myeloid cells across three independent cohorts totalling 297 samples, including monocytes and IPS-derived microglia. We observed that C-to-U editing, while less abundant, has a higher effect size in response to stimulation than A-to-I, and has a greater potential to recode the proteome. We investigated the consequences of RNA editing on RNA stability and gene expression using in silico and in vitro reporter methods, and identified a recoding C-to-U site in ARSB that mimics a reported lysosomal storage disorder mutation.

genomics↗

LAG3 marks activated but hyporesponsive NK cells.

Natural Killer (NK) cells are critical for immunosurveillance yet become dysfunctional in contexts such as chronic stimulation by viral infections or cancer. This phenomenon is similar to T cell exhaustion but less well characterized, which limits therapeutic interventions. As shown for T cells, NK cells often display an increased expression of immune checkpoint proteins (ICP) following chronic stimulation, and ICP blockade therapies are currently being explored for several cancer types, which have remarkable patient benefits. Nevertheless, the nature of ICP expression in NK cells is still poorly documented. In this study, we aimed to identify the conditions that lead to and the phenotype of immune checkpoint LAG3 (Lymphocyte-activation gene 3) expressing NK cells. Using various experimental models, we found that LAG3 is expressed by murine NK cells upon activation in different contexts, including in response to cancer and acute viral infections. LAG3 marks a subset of immature, proliferating and activated cells, which, despite activation, have a reduced capacity to respond to a broad range of stimuli. Further characterization also revealed that LAG3+ NK cells exhibit a transcriptional signature similar to that of exhausted CD8+ T cells. Taken together, our results support the use of LAG3 as a marker of dysfunctional NK cells across diverse chronic and acute inflammatory conditions.

immunology↗

Butyrate as a growth factor of Clostridium acetobutylicum

The butyrate biosynthetic pathway not only contributes to electron management and energy generation in butyrate forming bacteria, but also confers evolutionary advantages to the host by inhibiting the growth of surrounding butyrate-sensitive microbes. Proteomic data suggest that butyrate may lead to lysine butyrylation, a lesser-known post-translational modification, which might affect enzyme catalysis and thus cellular metabolism. Although high levels of butyrate induce toxic stress responses, it is not known if butyrate at non-toxic levels influences cellular processes such as growth, health, metabolism, and sporulation. Here, we show that butyrate stimulates cellular processes of Clostridium acetobutylicum, a model butyrate forming Firmicute. First, we deleted the 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from the C. acetobutylicum chromosome in order to eliminate the butyrate synthetic pathway and thus butyrate formation. For rapid genome engineering, a xylose inducible Cas9 cassette was chromosomally integrated and utilized for the one-step markerless gene deletions. The addition of non-toxic levels of butyrate revealed that butyrate has a profound effect on the growth, health, and sporulation of C. acetobutylicum. By further deleting spo0A, the gene of the master regulator of sporulation, and followed by butyrate addition experiments, we conclude that butyrate affects cellular metabolism through both Spo0A dependent and independent mechanisms. We also deleted the hbd gene from the chromosome of the asporogenous C. acetobutylicum M5 strain lacking the pSOL1 plasmid to examine the potential involvement of pSOL1 genes on the observed butyrate effects. Addition of the precursor of butyrate biosynthesis crotonate to the hbd deficient M5 strain was used to probe the role of butyrate biosynthesis pathway in electron and metabolic fluxes. Finally, we found that butyrate addition can enhance the growth of the non-butyrate forming Clostridium saccharolyticum. Our data suggest that butyrate functions as a stimulator of cellular processes, like a growth factor, in C. acetobutylicum and other Clostridium organisms, and may thus be as a modulator of microbial population dynamics. HighlightsO_LIDeployed chromosomally integrated spCas9 for markerless one-step Clostridium acetobutylicum genome engineering. C_LIO_LIDeleted 3-hydroxybutyryl-CoA dehydrogenase gene (hbd) from Clostridium acetobutylicum to elucidate the roles of butyrate in cellular processes. C_LIO_LIDemonstrated butyrate as a growth factor stimulating cellular processes in Clostridium acetobutylicum and potentially other Clostridium species. C_LIO_LISuggested butyrate as a potential modulator of microbial population based on different responses of microbes against butyrate. C_LI

bioengineering↗

Landscape profiling of PET depolymerases using a natural sequence cluster framework

Since the demonstration that rapid polyethylene terephthalate (PET) decomposition using enzymes is feasible, a number of efficient depolymerases have been reported with the aim of resolving the plastic pollution issues. However, sporadic studies on enzymes with PET hydrolysis activity hinder the understanding of the distribution of potential PETases hidden in natures repertoire, and subsequently, the identification of potent enzymes. Here, we present the clustering of 1,894 PETase candidates, which include the majority of known PETases, and describe their profiling. An archipelago landscape of 170 lineages shows distribution of 289 representative sequences with features associated with PET-degrading capabilities. A birds-eye view of the landscape identifies three highly promising yet unexplored PETase lineages and two potent PETases, Mipa-P and Kubu-P. The engineered Mipa-PM19 and Kubu-PM12 variants exhibit both high PET hydrolysis activity and thermal stability. In particular, Kubu-PM12 outperformed the engineered benchmarks in terms of PET depolymerization in harsh environments, such as with high substrate load and ethylene glycol as the solvent. Our landscape framework and the identified variants assist in the understanding of how biological processes respond to solid-state and non-natural PET plastics.

systems biology↗

Spatiotemporal modeling reveals high-resolution invasion states in glioblastoma

Diffuse invasion of glioblastoma cells through normal brain tissue is a key contributor to tumor aggressiveness, resistance to conventional therapies, and dismal prognosis in patients. A deeper understanding of how components of the tumor microenvironment (TME) contribute to overall tumor organization and to programs of invasion may reveal opportunities for improved therapeutic strategies. Towards this goal, we applied a novel computational workflow to a spatiotemporally profiled GBM xenograft cohort, leveraging the ability to distinguish human tumor from mouse TME to overcome previous limitations in analysis of diffuse invasion. Our analytic approach, based on unsupervised deconvolution, performs reference-free discovery of cell types and cell activities within the complete GBM ecosystem. We present a comprehensive catalogue of 15 tumor cell programs set within the spatiotemporal context of 90 mouse brain and TME cell types, cell activities, and anatomic structures. Distinct tumor programs related to invasion were aligned with routes of perivascular, white matter, and parenchymal invasion. Furthermore, sub-modules of genes serving as program network hubs were highly prognostic in GBM patients. The compendium of programs presented here provides a basis for rational targeting of tumor and/or TME components. We anticipate that our approach will facilitate an ecosystem-level understanding of immediate and long-term consequences of such perturbations, including identification of compensatory programs that will inform improved combinatorial therapies.

cancer biology↗

Chemical inhibition of stomatal differentiation by perturbation of the master-regulatory bHLH heterodimer via an ACT-Like domain

Selective perturbation of protein interactions with chemical compounds enables dissection and control of developmental processes. Differentiation of stomata, cellular valves vital for plant growth and survival, is specified by the basic-helix-loop-helix (bHLH) heterodimers. Harnessing a new amination reaction, we here report a synthesis, derivatization, target identification, and mode of action of an atypical doubly-sulfonylated imidazolone, Stomidazolone, which triggers stomatal stem cell arrest. Our forward chemical genetics followed by biophysical analyses elucidated that Stomidazolone directly binds to the C-terminal ACT-Like (ACTL) domain of MUTE, a master regulator of stomatal differentiation, and perturbs its heterodimerization with a partner bHLH, SCREAM in vitro and in plant cells. On the other hand, Stomidazolone analogs that are biologically inactive did not bind to MUTE or disrupt the SCREAM-MUTE heterodimers. Guided by structural docking modeling, we rationally designed MUTE with reduced Stomidazolone binding. These engineered MUTE proteins are fully functional and confer Stomidazolone resistance in vivo. Our study identifies doubly-sulfonylated imidazolone as a direct inhibitor of the stomatal master regulator, further expanding the chemical space for perturbing bHLH-ACTL proteins to manipulate plant development.

plant biology↗