Interferons (IFNs) are signaling proteins that play fundamental roles during health and disease. Although types I, II, and III IFNs are structurally and functionally different, all IFNs signal via an intricate network of Janus kinases, named after the Roman god of time and duality. IFNs are characterized by activities that vary over time and can lead to opposing outcomes. IFNs have protective roles during bacterial, viral, and fungal infections but can also drive numerous inflammatory and autoimmune diseases. In this review, we provide an overview of the cellular and molecular mechanisms governing IFN induction and responses, emphasizing their roles in infections, tumorigenesis, and inflammatory, autoimmune, and genetic diseases, with particular attention to mucosal tissues. Overall, we spotlight how the balanced production of distinct members of the IFN families over time is necessary to exert their protective functions and the detrimental consequences for the host when this balance is lost.

The temporospatial distribution of proteins within cilia is regulated by intraflagellar transport (IFT), wherein molecular trains shuttle between the cell body and cilium. Defects in this process impair various signal-transduction pathways and cause ciliopathies. Although K63-linked ubiquitination appears to trigger protein export from cilia, the mechanisms coupling polyubiquitinated proteins to IFT remain unclear. Using a multidisciplinary approach, we demonstrate that a complex of CFAP36, a conserved ciliary protein of previously unknown function, and ARL3, a GTPase involved in ciliary import, binds polyubiquitinated proteins and links them to retrograde IFT trains. CFAP36 uses a coincidence detection mechanism to simultaneously bind two IFT subunits accessible only in retrograde trains. Depleting CFAP36 accumulates K63-linked ubiquitin in cilia and disrupts hedgehog signaling, a pathway reliant on the retrieval of ubiquitinated receptors. These findings advance our understanding of ubiquitin-mediated protein transport and ciliary homeostasis and demonstrate how structural changes in IFT trains achieve cargo selectivity.

RNA contains diverse post-transcriptional modifications, and its catabolic breakdown yields numerous modified nucleosides requiring correct processing, but the mechanisms remain unknown. Here, we demonstrate that three RNA-derived modified adenosines, N6-methyladenosine (m6A), N6,N6-dimethyladenosine (m6,6A), and N6-isopentenyladenosine (i6A), are sequentially metabolized into inosine monophosphate (IMP) to mitigate their intrinsic cytotoxicity. After phosphorylation by adenosine kinase (ADK), they undergo deamination by adenosine deaminase-like (ADAL). In Adal knockout mice, N6-modified adenosine monophosphates (AMPs) accumulate and allosterically inhibit AMP-activated protein kinase (AMPK), dysregulating glucose metabolism. Furthermore, ADK deficiency, linked to human inherited disorders of purine metabolism, elevates levels of the three modified adenosines, resulting in early lethality in mice. Mechanistically, excessive m6A, m6,6A, and i6A impair lysosomal function by interfering with lysosomal membrane proteins, thereby disrupting lipid metabolism and causing cellular toxicity. Through this nucleotide metabolism pathway and mechanism, cells detoxify modified adenosines, linking modified RNA metabolism to human disease.

The pace of gene discovery in plants has slowed as forward genetic screens reach saturation. To address this, we built a unified single-cell atlas of shoot apices from six vascular plant species spanning major evolutionary groups. This cross-species resource allowed us to identify a core set of cell-type foundational genes linked to key tissues such as the epidermis, xylem, and phloem, streamlining gene discovery with greater accuracy. Among these, we uncovered a previously unrecognized clade of X8 domain proteins and JULGI-LIKE as regulators of phloem development. We also identified companion-cell-like cells in ferns and gymnosperms that cannot be distinguished by conventional histological methods and developed an automated cell-type annotation tool for vascular plant cell types, expanding the utility of our approach. Our findings establish a high-resolution framework for identifying key regulators of plant cell types and set an innovative paradigm for studying plant cell-type evolution.

Nuclear pore complexes (NPCs) bridge across the nuclear envelope and mediate nucleocytoplasmic exchange. They consist of hundreds of nucleoporin building blocks and exemplify the structural complexity of macromolecular assemblies. To ensure transport directionality, different nucleoporin complexes are attached to the cytoplasmic and nuclear face of the NPC. How those asymmetric structures are faithfully assembled onto the symmetric scaffold architecture that exposes the same interaction surfaces to either side remained enigmatic. Here, we combine cryo-electron tomography, subtomogram averaging, and template matching with live imaging to address this question in budding yeast and Drosophila. We genetically induce ectopic nuclear pores and show that pores outside the nuclear envelope are symmetric. We furthermore demonstrate that the peripheral NPC configuration is affected by the nucleotide state of the small GTPase Ran. Our findings indicate that the nuclear transport system is self-regulatory, namely that the same molecular mechanism controls both transport and transport channel composition.

Ion channels orchestrate electrical signaling in excitable cells. In nature, ion channel function is customized by modulatory proteins that have evolved to fulfill distinct physiological needs. Yet, engineering synthetic modulators that precisely tune ion channel function is challenging. One example involves the voltage-gated sodium (NaV) channel that initiates the action potential and whose dysfunction amplifies the late/persistent sodium current (INaL), a commonality that underlies various human diseases, including cardiac arrhythmias and epilepsy. Here, using a computational protein design platform, we engineered a de novo peptide modulator, engineered late-current inhibitor X by inactivation-gate release (ELIXIR), that binds NaV channels with submicromolar affinity. Functional analysis revealed unexpected selectivity in inhibiting "pathogenic" INaL and confirmed its effectiveness in reversing NaV dysfunction linked to both cardiac arrhythmias and epilepsy in cellular and murine models. These findings exemplify the efficacy of de novo protein design for engineering synthetic ion channel modulators and set the stage for the rational design of future therapeutic approaches.

RNA-guided RNA editing represents an attractive alternative to DNA editing. However, the prevailing tool, CRISPR-Cas13, has collateral RNA cleavage activity that causes undesirable cytotoxicity in human cells. Here, we report an ultracompact RNA-editing platform engineered from IscB, which has comparable or higher activity than Cas13 but without cytotoxicity concerns. We show that IscB, the evolutionary ancestor of Cas9, has an intrinsic affinity for complementary single-stranded (ss)DNA and RNA. This activity becomes dominant when its double-stranded DNA binding activity is switched off through the deletion of its target-adjacent motif domain. The resulting R-IscB is comparable to or better than Cas13, can efficiently alter splicing outcomes in human cells, and can further mediate trans-splicing to correct any mutation at the mRNA level. R-IscB also drives efficient A-to-I editing on mRNA when fused to adenosine deaminase acting on RNA 2 (ADAR2) and mediates cleavage-based mRNA knockdown upon HNH engineering. Finally, we show that the same approach converts some Cas9s to RNA-targeting tools.

RNA Pol II-mediated transcription is essential for eukaryotic life. Although loss of transcription is thought to be universally lethal, the associated mechanisms promoting cell death are not yet known. Here, we show that death following the loss of RNA Pol II activity does not result from dysregulated gene expression. Instead, it occurs in response to loss of the hypophosphorylated form of Rbp1 (also called RNA Pol IIA). Loss of RNA Pol IIA exclusively activates apoptosis, and expression of a transcriptionally inactive version of Rpb1 rescues cell viability. Using functional genomics, we identify the mechanisms driving lethality following the loss of RNA Pol IIA, which we call the Pol II degradation-dependent apoptotic response (PDAR). Using the genetic dependencies of PDAR, we identify clinically used drugs that owe their lethality to a PDAR-dependent mechanism. Our findings unveil an apoptotic signaling response that contributes to the efficacy of a wide array of anti-cancer therapies.

Hair loss affects millions globally, significantly impacting quality of life and psychological well-being. Despite its prevalence, effective strategies for promoting human hair growth remain elusive. By investigating congenital generalized hypertrichosis terminalis (CGHT), a rare genetic disorder characterized by excessive hair growth, we discover that chromatin deletions or an inverted duplication disrupt the topologically associating domain (TAD), leading to the upregulation of the potassium channel KCNJ2 in dermal fibroblasts. Mouse genetics demonstrate that KCNJ2-mediated membrane hyperpolarization in dermal fibroblasts promotes hair growth by enhancing fibroblasts Wnt signaling responses, involving a reduction in intracellular calcium levels. Notably, fibroblast membrane potential oscillates during the normal hair cycle, with hyperpolarization specifically associated with the growth phase. Inducing fibroblast membrane depolarization delays the growth phase, while KCNJ2-mediated hyperpolarization rescues hair loss in aging and androgenetic alopecia models. These results uncover a previously unrecognized role of fibroblast bioelectricity in tissue regeneration, offering novel therapeutic avenues for hair loss treatment.

The antimicrobial resistance crisis necessitates structurally distinct antibiotics. While deep learning approaches can identify antibacterial compounds from existing libraries, structural novelty remains limited. Here, we developed a generative artificial intelligence framework for designing de novo antibiotics through two approaches: a fragment-based method to comprehensively screen >107 chemical fragments in silico against Neisseria gonorrhoeae or Staphylococcus aureus, subsequently expanding promising fragments, and an unconstrained de novo compound generation, each using genetic algorithms and variational autoencoders. Of 24 synthesized compounds, seven demonstrated selective antibacterial activity. Two lead compounds exhibited bactericidal efficacy against multidrug-resistant isolates with distinct mechanisms of action and reduced bacterial burden in vivo in mouse models of N. gonorrhoeae vaginal infection and methicillin-resistant S. aureus skin infection. We further validated structural analogs for both compound classes as antibacterial. Our approach enables the generative deep-learning-guided design of de novo antibiotics, providing a platform for mapping uncharted regions of chemical space.

The loss of cellular and tissue identity is a hallmark of aging and numerous diseases, but the underlying mechanisms are not well understood. Our analysis of gene expression data from over 40 human tissues and 20 diseases reveals a pervasive upregulation of mesenchymal genes across multiple cell types, along with an altered composition of stromal cell populations, denoting a "mesenchymal drift" (MD). Increased MD correlates with disease progression, reduced patient survival, and an elevated mortality risk, whereas suppression of key MD transcription factors leads to epigenetic rejuvenation. Notably, Yamanaka factor-induced partial reprogramming can markedly reduce MD before dedifferentiation and gain of pluripotency, rejuvenating the aging transcriptome at the cellular and tissue levels. These findings provide mechanistic insight into the underlying beneficial effects of partial reprogramming and offer a framework for developing interventions to reverse age-related diseases using the partial reprogramming approach.

Speech brain-computer interfaces (BCIs) show promise in restoring communication to people with paralysis but have also prompted discussions regarding their potential to decode private inner speech. Separately, inner speech may be a way to bypass the current approach of requiring speech BCI users to physically attempt speech, which is fatiguing and can slow communication. Using multi-unit recordings from four participants, we found that inner speech is robustly represented in the motor cortex and that imagined sentences can be decoded in real time. The representation of inner speech was highly correlated with attempted speech, though we also identified a neural "motor-intent" dimension that differentiates the two. We investigated the possibility of decoding private inner speech and found that some aspects of free-form inner speech could be decoded during sequence recall and counting tasks. Finally, we demonstrate high-fidelity strategies that prevent speech BCIs from unintentionally decoding private inner speech.

Artificial intelligence (AI) and robots offer vast opportunities in shifting toward precision agriculture to enhance crop yields, reduce costs, and promote sustainable practices. However, many crop traits obstruct the application of AI-based robots. One bottleneck is flower morphology with recessed stigmas, which hinders emasculation and pollination during hybrid breeding. We developed a crop-robot co-design strategy in tomatoes by combining genome editing with artificial-intelligence-based robots (GEAIR). We generated male-sterile lines bearing flowers with exserted stigmas, and then trained a mobile robot to automatically recognize and cross-pollinate those stigmas. GEAIR enables automated F1 hybrid breeding with efficiency comparable to manual pollination and facilitates the rapid breeding of stress-resilient and flavorful tomatoes when combined with de novo domestication under speed-breeding conditions. Multiplex gene editing in soybean recapitulated the male-sterile, exserted-stigma phenotype, potentially unlocking robotized hybrid breeding. We demonstrate the potential of GEAIR in boosting efficiency and lowering costs through automated, faster breeding of climate-resilient crops.

Most human pathogens are of zoonotic origin. Many emerged during prehistory, coinciding with domestication providing more opportunities for spillover into human populations. However, we lack direct DNA evidence linking animal and human infections during prehistory. Here, we present a Yersinia pestis genome recovered from a 3rd-millennium BCE domesticated sheep from the Eurasian Steppe belonging to the Late Neolithic Bronze Age (LNBA) lineage, until now exclusively identified in ancient humans across Eurasia. We show that this ancient lineage underwent ancestral gene decay paralleling extant lineages, but evolved under distinct selective pressures, contributing to its lack of geographic differentiation. We collect evidence supporting a scenario where the LNBA lineage, unable to efficiently transmit via fleas, spread from an unidentified reservoir to sheep and likely other domesticates, elevating human infection risk. Collectively, our results connect prehistoric livestock with infectious disease in humans and showcase the power of moving paleomicrobiology into the zooarchaeological record.

Nearly all mitochondrial proteins are translated on cytosolic ribosomes. How these proteins are subsequently delivered to mitochondria remains poorly understood. Using selective ribosome profiling, we show that nearly 20% of mitochondrial proteins can be imported cotranslationally in human cells. Cotranslational import requires an N-terminal presequence on the nascent protein and contributes to localized translation at the mitochondrial surface. This pathway does not favor membrane proteins but instead prioritizes large, multi-domain, topologically complex proteins, whose import efficiency is enhanced when targeted cotranslationally. In contrast to the early onset of cotranslational protein targeting to the endoplasmic reticulum (ER), the presequence on mitochondrial proteins is inhibited from initiating targeting early during translation until a large globular domain emerges from the ribosome. Our findings reveal a multi-layered protein sorting strategy that controls the timing and specificity of mitochondrial protein targeting.

Abscisic acid (ABA) is the most crucial phytohormone for plants in adapting to environmental conditions. While the ABA signaling network in plants has been extensively explored, our understanding of the diverse ABA sensing systems remains limited. Here, we found that the transcriptional response to ABA is suppressed under high-nitrate conditions but substantially increases under low-nitrate conditions, suggesting a tight integration of ABA signaling with nutrient conditions. Interestingly, NRT1.1B, traditionally recognized as a nitrate transporter and receptor, exhibits a markedly higher affinity for ABA, leading to the formation of an ABA-facilitated NRT1.1B-SPX4 complex. This complex triggers the release of SPX4-sequestered transcription factor NLP4, thereby initiating the transcriptional response to ABA. These findings establish that NRT1.1B functions as an ABA receptor. Notably, the competitive binding of nitrate and ABA to NRT1.1B unveils a mechanism that enables a flexible ABA response to fluctuating nutrient conditions, illustrating a sophisticated strategy for integrating compound environmental cues.

Many animal groups are organized hierarchically, which generates behavioral states that facilitate social interactions. Although generally stable, social status can change, underscoring the plasticity of underlying neural circuits. We examined competition among unfamiliar male mice and uncovered how the molecular and biophysical characteristics of a forebrain-thalamocortical circuit affect hierarchy. We identify the mediodorsal thalamus (MDT) as a hub receiving inputs from the orbitofrontal cortex and basal forebrain and projecting to the caudal anterior cingulate cortex (cACC) to regulate competitive performance. This circuit becomes potentiated or depressed in high- and low-rank males, respectively, in part through altered expression of the voltage-gated ion channel Trpm3 and synaptic plasticity. In high-rank mice, MDT projections drive inhibition of cACC pyramidal cells, promoting winning, in a pattern strikingly opposite to the dorsomedial prefrontal cortex, where winners display increased pyramidal cell activity. Our data suggest a model in which hierarchy modulation relies on coordinated remodeling of multiple forebrain-thalamocortical circuits.

Biological structures across scales integrate seamlessly to perform essential functions. While various histological methods have been developed to reveal these intricate structures, preserving the integrity of large-volume architectures while revealing microstructures with high resolution remains a major challenge. Here, we introduce vitreous ionic-liquid-solvent-based volumetric inspection of trans-scale biostructure (VIVIT), a 3D histological method leveraging the chemical properties of ionic liquids. VIVIT transforms biological tissue into an ionic glassy state, which enables optical clearing with minimal distortion and high transparency, preserves tissue from low-temperature crystal damage, and amplifies fluorescent signals from both genetically encoded and immunostained labels, thus yielding precise and reliable mapping of fluorescent signals within intact 3D architectures. Using VIVIT, we demonstrate the link between the modality of synaptic inputs to multisensory thalamic neurons and the targets of their brain-wide outputs and identified aspects of inhibitory control in the human cortex. VIVIT thus offers opportunities to elucidate the organizational principles underlying trans-scale biostructures.

The modifications of bile acids (BAs) are fundamental to their role in host physiology and pathology. Identifying their synthetases is crucial for uncovering the diversity of BAs and developing targeted interventions, yet it remains a significant challenge. To address this hurdle, we developed an artificial intelligence (AI)-assisted workflow, bile acid enzyme announcer unit tool (BEAUT), which predicted over 600,000 candidate BA metabolic enzymes that we compiled into the human generalized microbial BA metabolic enzyme (HGBME) database (https://beaut.bjmu.edu.cn). We identified a series of uncharacterized BA enzymes, including monoacid acylated BA hydrolase (MABH) and 3-acetoDCA synthetase (ADS). Notably, ADS can produce an unreported skeleton BA, 3-acetoDCA, with a carbon-carbon bond extension. After determining its bacterial source and catalytic mechanism, we found that 3-acetoDCA is widely distributed among populations and regulates the microbial interactions in the gut. In conclusion, our work offers alternative insights into the relationship between microbial BAs and the host from an enzymatic perspective.