Glioblastomas are invasive brain tumors with high therapeutic resistance. Neuron-to-glioma synapses have been shown to promote glioblastoma progression. However, a characterization of tumor-connected neurons has been hampered by a lack of technologies. Here, we adapted retrograde tracing using rabies viruses to investigate and manipulate neuron-tumor networks. Glioblastoma rapidly integrated into neural circuits across the brain, engaging in widespread functional communication, with cholinergic neurons driving glioblastoma invasion. We uncovered patient-specific and tumor-cell-state-dependent differences in synaptogenic gene expression associated with neuron-tumor connectivity and subsequent invasiveness. Importantly, radiotherapy enhanced neuron-tumor connectivity by increased neuronal activity. In turn, simultaneous neuronal activity inhibition and radiotherapy showed increased therapeutic effects, indicative of a role for neuron-to-glioma synapses in contributing to therapeutic resistance. Lastly, rabies-mediated genetic ablation of tumor-connected neurons halted glioblastoma progression, offering a viral strategy to tackle glioblastoma. Together, this study provides a framework to comprehensively characterize neuron-tumor networks and target glioblastoma.

Indigenous maize varieties from eastern North America have played an outsized role in breeding programs, yet their early origins are not fully understood. We generated paleogenomic data to reconstruct how maize first reached this region and how it was selected during the process. Genomic ancestry analyses reveal recurrent movements northward from different parts of Mexico, likely culminating in at least two dispersals from the US Southwest across the Great Plains to the Ozarks and beyond. We find that 1,000-year-old Ozark specimens carry a highly differentiated wx1 gene, which is involved in the synthesis of amylose, highlighting repeated selective pressures on the starch metabolic pathway throughout maize's domestication. This population shows a close affinity with the lineage that ultimately became the Northern Flints, a major contributor to modern commercial maize.

Designer receptors exclusively activated by designer drugs (DREADDs) are chemogenetic tools for remotely controlling cellular signaling, neural activity, behavior, and physiology. Using a structure-guided approach, we provide a peripherally restricted Gi-DREADD, hydroxycarboxylic acid receptor DREADD (HCAD), whose native receptor is minimally expressed in the brain, and a chemical actuator that does not cross the blood-brain barrier (BBB). This was accomplished by combined mutagenesis, analoging via an ultra-large make-on-demand library, structural determination of the designed DREADD receptor via cryoelectron microscopy (cryo-EM), and validation of HCAD function. Expression and activation of HCAD in dorsal root ganglion (DRG) neurons inhibit action potential (AP) firing and reduce both acute and tissue-injury-induced inflammatory pain. The HCAD chemogenetic system expands the possibilities for studying numerous peripheral systems with little adverse effects on the central nervous system (CNS). The structure-guided approach used to generate HCAD also has the potential to accelerate the development of emerging chemogenetic tools for basic and translational sciences.

The mammalian pancreas consists of three epithelial compartments: the acini and ducts of the exocrine pancreas and the endocrine islets of Langerhans. Murine studies indicate that these three compartments derive from a transient, common pancreatic progenitor. Here, we report derivation of 18 human fetal pancreas organoid (hfPO) lines from gestational weeks 8-17 (8-17 GWs) fetal pancreas samples. Four of these lines, derived from 15 to 16 GWs samples, generate acinar-, ductal-, and endocrine-lineage cells while expanding exponentially for >2 years under optimized culture conditions. Single-cell RNA sequencing identifies rare LGR5+ cells in fetal pancreas and in hfPOs as the root of the developmental hierarchy. These LGR5+ cells share multiple markers with adult gastrointestinal tract stem cells. Organoids derived from single LGR5+ organoid-derived cells recapitulate this tripotency in vitro. We describe a human fetal tripotent stem/progenitor cell capable of long-term expansion in vitro and of generating all three pancreatic cell lineages.

The pathogenic mechanisms of many diseases are well understood at the molecular level, but there are prevalent syndromes associated with pathogenic signaling, such as diabetes and chronic inflammation, where our understanding is more limited. Here, we report that pathogenic signaling suppresses the mobility of a spectrum of proteins that play essential roles in cellular functions known to be dysregulated in these chronic diseases. The reduced protein mobility, which we call proteolethargy, was linked to cysteine residues in the affected proteins and signaling-related increases in excess reactive oxygen species. Diverse pathogenic stimuli, including hyperglycemia, dyslipidemia, and inflammation, produce similar reduced protein mobility phenotypes. We propose that proteolethargy is an overlooked cellular mechanism that may account for various pathogenic features of diverse chronic diseases.

Condensed droplets of protein regulate many cellular functions, yet the physiological conditions regulating their formation remain largely unexplored. Increasing our understanding of these mechanisms is paramount, as failure to control condensate formation and dynamics can lead to many diseases. Here, we provide evidence that matrix stiffening promotes biomolecular condensation in vivo. We demonstrate that the extracellular matrix links mechanical cues with the control of glucose metabolism to sorbitol. In turn, sorbitol acts as a natural crowding agent to promote biomolecular condensation. Using in silico simulations and in vitro assays, we establish that variations in the physiological range of sorbitol concentrations, but not glucose concentrations, are sufficient to regulate biomolecular condensates. Accordingly, pharmacological and genetic manipulation of intracellular sorbitol concentration modulates biomolecular condensates in breast cancer-a mechano-dependent disease. We propose that sorbitol is a mechanosensitive metabolite enabling protein condensation to control mechano-regulated cellular functions.

Metastatic dissemination to distant organs demands that cancer cells possess high morphological and metabolic adaptability. However, contributions of the cellular lipidome to metastasis remain elusive. Here, we uncover a correlation between metastasis potential and ferroptosis susceptibility in multiple cancers. Metastases-derived cancer cells exhibited higher ferroptosis sensitivity and polyunsaturated fatty acyl (PUFA)-lipid contents than primary-tumor-derived cells from ovarian cancer patients. Metabolism-focused CRISPR screens in a mouse model for ovarian cancer distant metastasis established via two rounds of in vivo selection revealed the PUFA-lipid biosynthesis enzyme acyl-coenzyme A (CoA) synthetase long-chain family member 4 (ACSL4) as a pro-hematogenous metastasis factor. ACSL4 promotes metastatic extravasation by enhancing membrane fluidity and cellular invasiveness. While promoting metastasis, the high PUFA-lipid state creates dependencies on abhydrolase-domain-containing 6, acylglycerol lipase (ABHD6), enoyl-CoA delta isomerase 1 (ECI1), and enoyl-CoA hydratase 1 (ECH1)-rate-limiting enzymes preparing unsaturated fatty acids (UFAs) for β-oxidation. ACSL4/ECH1 co-inhibition achieved potent suppression of metastasis. Our work establishes the dual functions of PUFA-lipids in tumor progression and metastasis that may be exploitable for therapeutic development.

Large-scale proteomics studies can refine our understanding of health and disease and enable precision medicine. Here, we provide a detailed atlas of 2,920 plasma proteins linking to diseases (406 prevalent and 660 incident) and 986 health-related traits in 53,026 individuals (median follow-up: 14.8 years) from the UK Biobank, representing the most comprehensive proteome profiles to date. This atlas revealed 168,100 protein-disease associations and 554,488 protein-trait associations. Over 650 proteins were shared among at least 50 diseases, and over 1,000 showed sex and age heterogeneity. Furthermore, proteins demonstrated promising potential in disease discrimination (area under the curve [AUC] > 0.80 in 183 diseases). Finally, integrating protein quantitative trait locus data determined 474 causal proteins, providing 37 drug-repurposing opportunities and 26 promising targets with favorable safety profiles. These results provide an open-access comprehensive proteome-phenome resource (https://proteome-phenome-atlas.com/) to help elucidate the biological mechanisms of diseases and accelerate the development of disease biomarkers, prediction models, and therapeutic targets.

Small nucleolar RNAs (snoRNAs) are non-coding RNAs known for guiding RNA modifications, including 2'-O-methylation (Nm) and pseudouridine (Ψ). While snoRNAs may also interact with other RNAs, such as mRNA, the full repertoire of RNAs targeted by snoRNA remains elusive due to the lack of effective technologies that identify snoRNA targets transcriptome wide. Here, we develop a chemical crosslinking-based approach that comprehensively detects cellular RNA targets of snoRNAs, yielding thousands of previously unrecognized snoRNA-mRNA interactions in human cells and mouse brain tissues. Many interactions occur outside of snoRNA-guided RNA modification sites, hinting at non-canonical functions beyond RNA modification. We find that one of these snoRNAs, SNORA73, targets mRNAs that encode secretory proteins and membrane proteins. SNORA73 also interacts with 7SL RNA, part of the signal recognition particle (SRP) required for protein secretion. The mRNA-SNORA73-7SL RNA interactions enhance the association of the SNORA73-target mRNAs with SRP, thereby facilitating the secretion of encoded proteins.

Brown seaweeds are keystone species of coastal ecosystems, often forming extensive underwater forests, and are under considerable threat from climate change. In this study, analysis of multiple genomes has provided insights across the entire evolutionary history of this lineage, from initial emergence, through later diversification of the brown algal orders, down to microevolutionary events at the genus level. Emergence of the brown algal lineage was associated with a marked gain of new orthologous gene families, enhanced protein domain rearrangement, increased horizontal gene transfer events, and the acquisition of novel signaling molecules and key metabolic pathways, the latter notably related to biosynthesis of the alginate-based extracellular matrix, and halogen and phlorotannin biosynthesis. We show that brown algal genome diversification is tightly linked to phenotypic divergence, including changes in life cycle strategy and zoid flagellar structure. The study also showed that integration of large viral genomes has had a significant impact on brown algal genome content throughout the emergence of the lineage.

Stringent control of T cell activity in the tumor microenvironment is essential for the generation of protective antitumor immunity. However, the identity, differentiation, and functions of the cells that create critical fibroblastic niches promoting tumor-infiltrating T cells remain elusive. Here, we show that CCL19-expressing fibroblastic reticular cells (FRCs) generate interconnected T cell environments (TEs) in human non-small cell lung cancer, including tertiary lymphoid structures and T cell tracks. Analysis of the FRC-T cell interactome in TEs indicated molecular networks regulating niche-specific differentiation of CCL19-expressing fibroblasts and T cell activation pathways. Single-cell transcriptomics and cell fate-mapping analyses in mice confirmed that FRCs in TEs originate from mural and adventitial progenitors. Ablation of intratumoral FRC precursors decreased antitumor T cell activity, resulting in reduced tumor control during coronavirus vector-based immunotherapy. In summary, specialized FRC niches in the tumor microenvironment govern the quality and extent of antitumor T cell immunity.

The sense of taste generally shows diminishing sensitivity to prolonged sweet stimuli, referred to as sweet adaptation. Yet, its mechanistic landscape remains incomplete. Here, we report that glia-like type I cells provide a distinct mode of sweet adaptation via intercellular crosstalk with chemosensory type II cells. Using the microfluidic-based intravital tongue imaging system, we found that sweet adaptation is facilitated along the synaptic transduction from type II cells to gustatory afferent nerves, while type I cells display temporally delayed and prolonged activities. We identified that type I cells receive purinergic input from adjacent type II cells via P2RY2 and provide inhibitory feedback to the synaptic transduction of sweet taste. Aligning with our cellular-level findings, purinergic activation of type I cells attenuated sweet licking behavior, and P2RY2 knockout mice showed decelerated adaptation behavior. Our study highlights a veiled intercellular mode of sweet adaptation, potentially contributing to the efficient encoding of prolonged sweetness.

Cryptochromes (CRYs) are blue-light receptors that regulate diverse aspects of plant growth. However, whether and how non-photoexcited CRYs function in darkness or non-blue-light conditions is unknown. Here, we show that CRY2 affects the Arabidopsis transcriptome even in darkness, revealing a non-canonical function. CRY2 suppresses cell division in the root apical meristem to downregulate root elongation in darkness. Blue-light oligomerizes CRY2 to de-repress root elongation. CRY2 physically interacts with FORKED-LIKE 1 (FL1) and FL3, and these interactions are inhibited by blue light, with only monomeric but not dimeric CRY2 able to interact. FL1 and FL3 associate with the chromatin of cell division genes to facilitate their transcription. This pro-growth activity is inhibited by CRY2's physical interaction with FLs in darkness. Plants have evolved to perceive both blue-light and dark cues to coordinate activation and repression of competing developmental processes in above- and below-ground organs through economical and dichotomous use of ancient light receptors.

We present replication-aware single-molecule accessibility mapping (RASAM), a method to nondestructively measure replication status and protein-DNA interactions on chromatin genome-wide. Using RASAM, we uncover a genome-wide state of single-molecule "hyperaccessibility" post-replication that resolves over several hours. Combining RASAM with cellular models for rapid protein degradation, we demonstrate that histone chaperone CAF-1 reduces nascent chromatin accessibility by filling single-molecular "gaps" and generating closely spaced dinucleosomes on replicated DNA. At cis-regulatory elements, we observe unique modes by which nascent chromatin hyperaccessibility resolves: at CCCTC-binding factor (CTCF)-binding sites, CTCF and nucleosomes compete, reducing CTCF occupancy and motif accessibility post-replication; at active transcription start sites, high chromatin accessibility is maintained, implying rapid re-establishment of nucleosome-free regions. Our study introduces a new paradigm for studying replicated chromatin fiber organization. More broadly, we uncover a unique organization of newly replicated chromatin that must be reset by active processes, providing a substrate for epigenetic reprogramming.

Stress induces aversive memory overgeneralization, a hallmark of many psychiatric disorders. Memories are encoded by a sparse ensemble of neurons active during an event (an engram ensemble). We examined the molecular and circuit processes mediating stress-induced threat memory overgeneralization in mice. Stress, acting via corticosterone, increased the density of engram ensembles supporting a threat memory in lateral amygdala, and this engram ensemble was reactivated by both specific and non-specific retrieval cues (generalized threat memory). Furthermore, we identified a critical role for endocannabinoids, acting retrogradely on parvalbumin-positive (PV+) lateral amygdala interneurons in the formation of a less-sparse engram and memory generalization induced by stress. Glucocorticoid receptor antagonists, endocannabinoid synthesis inhibitors, increasing PV+ neuronal activity, and knocking down cannabinoid receptors in lateral amygdala PV+ neurons restored threat memory specificity and a sparse engram in stressed mice. These findings offer insights into stress-induced memory alterations, providing potential therapeutic avenues for stress-related disorders.

Underlying variation in height are regulatory changes to chondrocytes, cartilage cells comprising long-bone growth plates. Currently, we lack knowledge on epigenetic regulation and gene expression of chondrocytes sampled across the human skeleton, and therefore we cannot understand basic regulatory mechanisms controlling height biology. We first rectify this issue by generating extensive epigenetic and transcriptomic maps from chondrocytes sampled from different growth plates across developing human skeletons, discovering novel regulatory networks shaping human bone/joint development. Next, using these maps in tandem with height genome-wide association study (GWAS) signals, we disentangle the regulatory impacts that skeletal element-specific versus global-acting variants have on skeletal growth, revealing the prime importance of regulatory pleiotropy in controlling height variation. Finally, as height is highly heritable, and thus often the test case for complex-trait genetics, we leverage these datasets within a testable omnigenic model framework to discover novel chondrocyte developmental modules and peripheral-acting factors shaping height biology and skeletal growth.

Transcription-coupled DNA repair (TCR) removes bulky DNA lesions impeding RNA polymerase II (RNAPII) transcription. Recent studies have outlined the stepwise assembly of TCR factors CSB, CSA, UVSSA, and transcription factor IIH (TFIIH) around lesion-stalled RNAPII. However, the mechanism and factors required for the transition to downstream repair steps, including RNAPII removal to provide repair proteins access to the DNA lesion, remain unclear. Here, we identify STK19 as a TCR factor facilitating this transition. Loss of STK19 does not impact initial TCR complex assembly or RNAPII ubiquitylation but delays lesion-stalled RNAPII clearance, thereby interfering with the downstream repair reaction. Cryoelectron microscopy (cryo-EM) and mutational analysis reveal that STK19 associates with the TCR complex, positioning itself between RNAPII, UVSSA, and CSA. The structural insights and molecular modeling suggest that STK19 positions the ATPase subunits of TFIIH onto DNA in front of RNAPII. Together, these findings provide new insights into the factors and mechanisms required for TCR.

In transcription-coupled nucleotide excision repair (TC-NER), stalled RNA polymerase II (RNA Pol II) binds CSB and CRL4CSA, which cooperate with UVSSA and ELOF1 to recruit TFIIH. To explore the mechanism of TC-NER, we recapitulated this reaction in vitro. When a plasmid containing a site-specific lesion is transcribed in frog egg extract, error-free repair is observed that depends on CSB, CRL4CSA, UVSSA, and ELOF1. Repair also requires STK19, a factor previously implicated in transcription recovery after UV exposure. A 1.9-Å cryo-electron microscopy structure shows that STK19 binds the TC-NER complex through CSA and the RPB1 subunit of RNA Pol II. Furthermore, AlphaFold predicts that STK19 interacts with the XPD subunit of TFIIH, and disrupting this interface impairs cell-free repair. Molecular modeling suggests that STK19 positions TFIIH ahead of RNA Pol II for lesion verification. Our analysis of cell-free TC-NER suggests that STK19 couples RNA Pol II stalling to downstream repair events.

Targeted protein degradation strategies leverage endogenous cellular degradation machinery to selectively eliminate a protein of interest. Emerging technologies are opening avenues in drug discovery and functional characterization of intracellular, membrane, and extracellular proteins. To view this SnapShot, open or download the PDF.