Angewandte Chemie International Edition

Small interfering RNA (siRNA) enables precise post-transcriptional gene silencing for refractory diseases, yet its clinical translation remains limited by the lack of safe and efficient delivery vectors. Inspired by the dissymmetric alkyl chain architecture of natural membrane phospholipids, we designed and synthesized 34 novel ionizable lipids with dissymmetric hydrophobic tails and formulated them into lipid nanoparticles (LNPs). Through systematic physicochemical and biological assessments, we established clear structure-activity relationships and identified two lead LNPs (O14-LNP, H18a-LNP) with superior endosomal escape capacity, enhanced in vivo gene silencing potency, and favorable biosafety relative to the clinical benchmark MC3-LNP. In both streptozotocin-induced and spontaneous db/db type 2 diabetes (T2D) mouse models, lead LNPs delivering ferroptosis-related siRNAs effectively ameliorated glucose and lipid metabolic disorders, restored islet function, and alleviated hepatic steatosis. This study not only lays a theoretical foundation for the rational design of novel ionizable lipids, but also validates the therapeutic potential of siRNA therapy targeting ferroptosis, providing a versatile delivery platform and targeted therapeutic strategy for the treatment of T2D.

Protein synthesis supports most biological processes. In the brain in particular, protein synthesis plays a critical role in physiological and pathological states. Here, we describe Tellurophene-Alkyne Cycloaddition-mediated Amino acid Tagging (TeACAT), a versatile strategy for fast, facile, and flexible tagging of newly synthesized proteins in mice. TeACAT is based on metabolic incorporation of the non-canonical amino acid TePhe into proteins by the endogenous protein synthesis machinery. Due to their high similarity, TePhe can efficiently replace canonical Phe without dietary or genetic manipulation. The subsequent bio-orthogonal reaction of TePhe with either fluorescent dyes or affinity handles enables both visualization and affinity enrichment of proteins synthesized during TePhe exposure. TeACAT is compatible with immunofluorescence for cell-type specific visualization of protein synthesis with subcellular resolution and can be used in conjunction with routine proteomics to identify and quantify newly synthesized proteins. Robust incorporation into the mouse proteome was observed on the scale of hours to days, allowing the interrogation of various biological processes. In summary, TeACAT enables the visualization and quantification of protein synthesis with minimal perturbation for biological discoveries.

Cereblon (CRBN) is widely used in targeted protein degradation, but its ligand space has remained dominated by a narrow set of cyclic imide chemotypes. Here, we show that the accessible CRBN ligand space extends substantially beyond this canonical space. A high-throughput screen of > 40,000 compounds, followed by orthogonal biophysical validation, X-ray crystallography and SAR analyses, identified several chemically distinct ligand classes. These include linear acetyl-based motifs, a phthalide-derived scaffold, steroidal compounds, and a range of bicyclic ligands. They engage CRBN through distinct recognition modes, several of which deviate from the canonical hydrogen-bonding pattern. Steroidal scaffolds were particularly notable: cortisone binds the human CRBN thalidomide-binding domain with an affinity comparable to thalidomide, with its A-ring occupying the tri-tryptophan pocket in a glutarimide-like orientation despite lacking the canonical imide NH donor. SAR within this series showed substantial tolerance for chemical modification and scaffold simplification, raising the possibility that endogenous steroidal metabolites may contribute to the physiological ligand landscape of CRBN. Bicyclic lactams additionally provided synthetically accessible scaffolds with tunable affinity and promising sites for linker attachment. Across the identified ligand classes, none of the tested representatives induced detectable degradation of canonical CRBN neosubstrates, and several showed largely clean proteomic profiles. Together, these findings broaden the chemical, mechanistic and potential physiological landscape of CRBN recognition and provide diverse starting points for alternative, potentially neosubstrate-sparing CRBN recruiters.

The blood-brain barrier (BBB) constitutes a major bottleneck for the systemic delivery of most therapeutic agents to the central nervous system. Here, we report near-infrared reversible optoacoustic modulation of the BBB permeability (NIR-ROAMBBB), leveraging endothelial tight junction targeting hyperbranched gold nanoconstructs (HBGNCs) to amplify localized optoacoustic transduction under femtosecond laser excitation. We first synthesized HBGNCs with tunable particle sizes (62-150 nm) and consistent branch morphologies via a seed-mediated growth approach, and uncovered a non-monotonic relationship between particle dimension and optoacoustic output, where the 62 nm HBGNCs generated nearly twofold stronger optoacoustic signal than gold nanorods and gold nanostars under matched excitations. Conjugation with BV11 antibodies against junctional adhesion molecule A increased HBGNC endothelial association and cerebral accumulation, enabling focal and fluence-dependent transient BBB opening (3-6 h) under 800 nm femtosecond pulsed laser excitation, as validated by in vitro trans-endothelial electrical resistance measurements, ex vivo Evans blue extravasation staining, and in vivo NIR imaging. Featuring deep tissue penetration of NIR light, robust optoacoustic conversion of HBGNCs, and negligible femtosecond laser-induced photothermal damage, this non-invasive strategy enables precise focal modulation of BBB permeability and potential drug delivery.

Methods for installing synthetic functions on living cell surfaces provide powerful approaches for imaging, sensing, and manipulating cell behavior, but many require genetic modification of the target cell or chemical modification of the plasma membrane. Here, we repurpose the glycosylphosphatidylinositol-anchored protein (GPI-AP)-binding toxin aerolysin as a modular chassis for non-genetic cell-surface functionalization. We show that a non-cytotoxic, monomeric aerolysin mutant retains high-affinity and GPI-AP-dependent cell binding when genetically fused to diverse protein cargos. Fluorescent protein-aerolysin fusions robustly label multiple cell types and remain predominantly associated with the cell surface for at least 24 h, in contrast to wheat germ agglutinin, which is extensively internalized. Aerolysin can also be equipped with SpyTag/SpyCatcher to enable modular assembly with independently expressed protein cargos. Importantly, aerolysin supports functional rather than solely optical modification of the cell surface: fusion to the proximity-labeling enzyme APEX2 enables extracellular protein biotinylation, while fusion to HUH endonuclease tags enables covalent attachment of synthetic DNA to living cells. Using this latter architecture, we developed a DNA hairpin sensor that converts cell-surface nuclease activity into a fluorescent signal and distinguishes cells with different levels of extracellular nuclease activity. Together, these results establish non-cytotoxic aerolysin as a genetically encoded, soluble adapter for installing proteins, enzymes, and programmable nucleic acids onto living cells without modification of the target-cell genome.

The navigational prowess of migratory birds is thought to arise from light-dependent radical-pair chemistry in cryptochrome 4 (CRY4), yet the slow structural transitions that couple photochemistry to signaling remain elusive. Here, we combine temperature-controlled steady-state UV-visible spectroscopy and hydrogen-deuterium exchange mass spectrometry (HDX-MS) to elucidate the photochemical and conformational dynamics of pigeon CRY4 (ClCRY4). Steady-state measurements at 5-25 {degrees}C reveal that lower temperatures slow FAD photoreduction and prolong the FAD neutral semiquinone signaling state. This occurs without a solvent kinetic isotope effect, implicating a conformational change rather than proton transfer as the rate determining step in FAD neutral semiquinone formation. Simultaneous HDX-MS under blue-light exposure identifies protection near the FAD-binding site and C-terminal region. To enhance sensitivity, we developed a pump-probe HDX-MS approach at 10 {degrees}C. This reveals eight peptides (within the phosphate-binding loop, protrusion motif, electron-transfer-chain loops and C-terminal tail) that exhibit rapid ([≤]10 s) and sustained light-induced protection, delineating early conformational rearrangements as a prerequisite for FAD neutral semiquinone accumulation. The findings of slower onset HDX protection as well as a bimodal pattern of deuterium uptake in the phosphate-binding loop further identify a local redistribution of conformational substates on the time scale of the accumulation of the signaling species. Site specific mutagenesis within the CTT supports the findings, which lead to a model in which blue light triggers rapid clamping down of protein near the two regions of spin pair separation, followed by a rate limiting closure of a surface loop. The resolution of time-dependent structural transitions that follow photoactivation of CRY4 resolves the interface between quantum radical-pair formation and classical conformational changes, while providing an enhanced structural framework for the molecular events that underlie avian magnetoreception.

Reading both physical and chemical properties of a microenvironment from a single fluorophore remains a challenge. Here we demonstrate that two coexisting molecular equilibria within one near-infrared cyanine, CyC4, encode two mechanistically distinct ratiometric reporting channels. A meso-amino group and a pendant carboxylate form a tunable intramolecular hydrogen bond that toggles the dye between closed (700 nm) and open (780 nm) emissive conformers. Time-dependent density functional theory (TD-DFT) calculations show that the hydrogen bond raises the LUMO and blue-shifts the emission, establishing the 700/780 emission ratio as a local reporter of hydrogen bonding and polarity. Independently, the chromophore self-associates under crowding- and cosolvent-rich conditions into an aggregate with a blue-shifted, H-type absorption signature near 530-540 nm and a distinct emission near 610 nm upon 540 nm excitation. The intensity of this aggregate band relative to the monomer emission (Ra) serves as a ratiometric reporter of crowding and self-association. Because the two channels arise from distinct molecular equilibria (intramolecular hydrogen bonding vs. intermolecular self-association) they are largely decoupled: a glycerol titration series confirms that the self-association channel (Ra) can be moved while the hydrogen-bonding channel stays essentially fixed. Applied to protein-PEG biomolecular condensates, the two ratios move oppositely with increasing salt, showing that the interior's chemical (polarity, hydrogen bonding) and physical (packing, self-association) environments co-vary across the salt series; a single CyC4 measurement thereby maps this coupled microenvironment, providing a general strategy for multiparametric, ratiometric sensing of crowded microenvironments.

Lysine acylations, including lactylation (Klac), are pivotal regulators of cellular physiology. However, their analysis is currently bottlenecked by antibody enrichment strategies that suffer from sequence bias and require milligram-scale protein inputs, severely precluding the profiling of scarce clinical biopsies and rare cell populations. Here we present ChemIntelligence, an acyl-NHS chemistry-empowered derivatization strategy that rapidly generates unprecedented acylation-specific spectral libraries, exemplified by over 2.5x10^9 human Klac peptides, enabling cross-species reference atlases. Integrated with Prosit-based rescoring, these libraries substantially increase Klac identifications across diverse proteomic datasets. Leveraging this spectral resource, we devised ChemIntelligence Scope, a reproducible, multiplexed parallel reaction monitoring (PRM) platform that quantifies hundreds of Klac peptides per injection from as little as ~200 ng of cell lysates, clinical biopsies, and even true single cells - revealing functional Klac signatures inaccessible to conventional methods. The ChemIntelligence pipeline also extends seamlessly to lysine nicotinylation, underscoring its broad adaptability for discovering and profiling new acylations. Together, these chemical and computational advances establish a scalable, antibody-free framework for acyl-proteome mapping that overcomes input constraints and enables deep functional insights from otherwise intractable biological samples.

Rational design of covalent protein-labeling reagents in complex biological environments requires a molecular-level understanding of how the protein microenvironment governs chemical reactivity; yet, such mechanistic details remain inaccessible to experimental methods alone. In living neurons, Ligand-Directed Acyl Imidazole (LDAI) chemistry has been used to label AMPA receptors as a traceless, affinity-based protein labeling method. Although LDAI labeling reagents have been optimized in the lab, the atomic details of their interactions with the protein and the underlying mechanism remain elusive. In this work, we combined Quantum Mechanical (QM) calculations and molecular dynamics (MD) simulations to propose a detailed reaction mechanism for AMPAR labeling by LDAI reagents and to clarify how the protein microenvironment governs reactivity. Although Lys residues are usually protonated at physiological pH and therefore less nucleophilic in water, our QM results show that Lys labeling is energetically more favorable than competing reactions with Ser or water. MD simulations reveal that PFQX ---the LDAI reagent precursor--- binds dynamically to the GluA2 AMPAR as an antagonist, inducing conformational changes that reshape the local environment of the acyl imidazole (AI) warhead, underscoring that ligand identity strongly affects labeling outcomes. We also identified intra and intermolecular hydrogen bond networks that may contribute to further immobilize and pre-organize the LDAI reagent. Moreover, the probe's chemical nature shapes its interactions with the Ligand Binding Domain (LBD), offering a plausible rationale for the previously experimentally observed ligand-dependent fluorescent response. Taken together, our results establish design principles for exploiting the reagent geometry and binding pocket hydrogen-bonding networks for the rational design of LDAI reagents.

Disulfide-rich peptides possess exceptional potency and selectivity but are often limited by the instability and synthetic challenges associated with native disulfide bonds. Here, we report the design, synthesis, pharmacological evaluation, and structural characterisation of triazole-based peptidomimetics of the -GI conotoxin, a selective antagonist of the muscle-type nicotinic acetylcholine receptor (nAChR). A series of 1,4- and 1,5-disubstituted triazole analogues were prepared entirely on resin using CuAAC and RuAAC chemistry to replace the native Cys3/13 disulfide bridge. Functional evaluation against human muscle nAChRs revealed that 1,5-triazole analogues retained low-nanomolar potency, with the lead mimetic exhibiting activity comparable to native -GI. Cryo-electron microscopy of the lead compound bound to the muscle-type nAChR provided the first structure of a disulfide-isostere peptidomimetic in complex with a membrane receptor. The structure demonstrates that the 1,5-triazole reproduces the native peptide fold with high fidelity while contributing receptor-facing interactions not available to the native disulfide bridge. Molecular dynamics simulations further revealed conserved hydration networks and similar conformational sampling between the native peptide and lead mimetic. Together, these findings establish triazoles as effective disulfide surrogates and provide a structural framework for the rational design of stabilised conotoxin therapeutics.

Cyclic oligosaccharides possess industrial advantages, including molecular encapsulation capability and high physicochemical stability, owing to the absence of a reducing end. Recently, a novel cyclic tetrasaccharide, cycloisomaltotetraose (CI4), consisting of four -1,6-linked glucose units, and the enzymes responsible for its synthesis, cycloisomaltotetraose glucanotransferases (CI4Tases), were discovered. Unlike known cycloisomaltooligosaccharide glucanotransferases (CITases) that yield a wide distribution of cyclic products with a degree of polymerization (DP) of 7 or higher, CI4Tases strictly produce CI4. To elucidate the molecular mechanism underlying this strict DP4 specificity, we determined the crystal structures of CI4Tase from Agreia sp. D1110, in its ligand-free form, as well as in complex with the linear hydrolysis product isomaltotetraose (IG4) and with CI4. Structural comparisons revealed that a loop (M247 to R251) blocks the region corresponding to the -5 subsite of typical CITases, narrowing the substrate-binding pocket. This "molecular ruler" mechanism ensures that only a glycan chain of exactly four glucose units is accommodated for cyclization. Among mutants of the residue positioned at the center of bound CI4, the formation of by-products other than CI4 was significantly suppressed in F245L, F245A, and F245W. While the cyclization activity of all F245 mutants decreased, the CI4 hydrolysis activity of these three mutants was also significantly reduced, resulting in an increased specificity for cyclic sugar production. These findings elucidate the strict size-control mechanism of CI4Tase and provide a structural foundation for engineering cycloisomaltooligosaccharide-producing enzymes with optimized transglycosylation efficiency and specificity for industrial applications.

To combat refractory diseases, such as cancer, multitarget-directed ligands (MTDLs) have become an emerging area of research to exploit synthetic lethality (SL) relationships associated with drug resistance. Herein, we present the in silico design of MTDLs for the polypharmaceutical treatment of endometrial adenocarcinoma (EAC) and our discovery of a novel SL in EAC; PTEN loss of function (LOF) and the inhibition of CDK9. We used high-resolution x-ray crystallographic data to chemically engineer, LCI133, to inhibit CDK9, CDK4/6-and AURKA/B kinases. PTEN LOF in EAC results in augmented deregulated transcription and a massive increase in nascent RNA, a phenotype which encodes a high level of apoptotic sensitivity to LCI133 and CDK9 inhibitors. Treatment with LCI133 results in a rapid decline nose-dive in global nRNA, MYC nRNA levels and TS elongation (TE) in PTEN LOF EAC. PTEN LOF is necessary and sufficient to confer sensitivity of EAC cells to LCI133 and other CDK9 inhibitors.

Label-free Raman imaging of living cells is photon limited: at exposures compatible with cellular dynamics, single-pixel spectra carry about one count per channel on a dominant smooth background. We present an unmixing framework in which the decoder of a physics-constrained autoencoder is restricted to a data-driven spectroscopic dictionary: band centers,widths, and pseudo-Voigt shapes are measured from the dataset and fixed, and the network learns only nonnegative band amplitudes, a smooth B-spline background, and a per-pixel gain.First, on slit-scanning images of HeLa cells (532 nm) the dictionary yields spike-free component spectra that read as band tables, including a resonance-enhanced cytochrome-c-associated component matching literature spectra, and the most stable decomposition against the component number. Second, the dictionary and initialization calibrated at 1 s exposure perline transfer to 100 ms per line (12 s sweeps): cytochrome-c spectral identity survives a single sweep (correlation 0.92) while its map remains photon limited; the dictionary provides spectral physicality, and the transferred initialization prevents a structural collapse that global map correlations miss; in a measurement-derived phantom the dictionary estimator holds thecytochrome-c spectrum to 17-19{degrees} spectral angle at 100 ms, where classical factorizations and free decoders lose it (55-64{degrees}). Estimation on the count-equivalent detector output uses a calibrated shifted-Poisson quasi-likelihood. Third, evaluation must be time matched:correlation against a separately acquired reference saturates through slow specimen drift and acquisition mismatch rather than photon noise, and the self-consistency of learned denoisers is inflated by shared bias; time-matched self-consistency and independent cross-checks areproposed.

More than one thousand bifunctional terpene synthases combining prenyltransferase and terpene cyclase activities have been identified in bacteria and fungi, but only a handful of enzymes have been identified that combine terpene cyclase activity with a downstream processing activity. Drimenol synthase from the marine bacterium Aquimarina spongiae (AsDMS) consists of a class II terpene cyclase that converts farnesyl diphosphate into drimenyl diphosphate, and a haloacid dehalogenase-like phosphatase that hydrolyzes drimenyl diphosphate to generate the sesquiterpene alcohol drimenol. The first crystal structure of AsDMS to be reported revealed the architecture of domain assembly as well as dimeric quaternary structure, establishing a structural chemical foundation for cyclization and hydrolysis mechanisms [K. R. Osika, M. N. Gaynes, D. W. Christianson (2025) Proc. Natl. Acad. Sci. U.S.A. 122, e2506584122]. Here, we report crystal structures of the catalytically-inactive double mutant, D33A-D323A AsDMS, complexed with farnesyl diphosphate, geranyl diphosphate, and dimethylallyl diphosphate, which bind in the active sites of both the cyclase and phosphatase domains. Molecular recognition of the diphosphate group dominates binding interactions in both active sites. In the cyclase active site, only farnesyl diphosphate is sufficiently long for its terminal isoprenoid C=C bond to bind adjacent to the catalytic general acid that would initiate the cyclization cascade in the wild-type enzyme. In the phosphatase active site, all isoprenoid diphosphate groups bind similarly, but isoprenoid chain conformations vary. These structures provide a foundation for understanding substrate recognition and catalysis in both active sites. Finally, we present kinetic evidence suggesting that substrate channeling is operative in wild-type AsDMS.

Carbapenems are the most potent {beta}-lactams, key antibiotics for healthcare-associated infections by Gram-negative bacteria and evade hydrolysis by most {beta}-lactamases, but are increasingly threatened by emergence of enzymes exhibiting hydrolytic activity towards them. Of the four recognised {beta}-lactamase subclasses, class A (active-site serine enzymes that hydrolyse {beta}-lactams via a covalent acylenzyme intermediate) is the most widely disseminated and, while the majority of such enzymes react with carbapenems to form long-lasting acylenzyme complexes, several possess carbapenem-hydrolyzing activity (carbapenemases). Here, we investigate the basis for these differences in a panel of class A {beta}-lactamases using molecular dynamics (MD) simulations of the respective acylenzyme complexes and tetrahedral intermediates (TI). The simulations reveal multiple features associated with catalytic activity across the spectrum of enzymes tested, including more extensive interactions of the carbapenem acylenzyme carbonyl and generally increased lifetimes of active site water molecules positioned for deacylation. Analysis of the dynamic trajectories shows carbapenemases to have reduced root mean-squared fluctuation (RMSF) differences between the acylenzyme and TI, that are not limited to the active site, indicating that the acylenzyme complex is pre-organised for reaction in carbapenemases but not in carbapenem-inhibited enzymes. Similarly, Principal Component Analysis (PCA) of acylenzyme and TI dynamics shows greater overlap between the two states in carbapenemases, providing further evidence for acylenzyme pre-organisation. Such simulations may represent an effective computational assay able to identify enzymes with carbapenemase activity at relatively modest computational cost.

UCH-L1 is a monomeric deubiquitinating enzyme whose native structure embeds a shallow $5_2$ knot located near the N-terminus, placing the knotted topology in direct proximity to both the substrate-binding pocket and the catalytic site. While our previous work established that N-terminal integrity is critical for catalytic activity, the energetic cost of unknotting and its structural consequences remained unquantified. Here, we combine steered molecular dynamics with an umbrella sampling scheme to generate topologically modified variants of UCH-L1 and, for the first time, reconstruct the free-energy profile of UCH-L1 unknotting. The potential of mean force reveals a steep energetic barrier to knot disruption, consistent with knotting being a late, rate-limiting folding step that is effectively locked in once the native structure is established. Long unbiased MD simulations of fully unknotted variants in both apo and holo states show that knot removal increases local flexibility at the N-terminus without inducing significant global structural destabilization. Binding energy calculations indicate that the unknotted variant binds to ubiquitin less tightly than the wild-type ($\sim$-62~vs~$\sim$-76~kcal/mol), suggesting that topological integrity contributes to substrate affinity. Together, these results show that the $5_2$ knot in UCH-L1 is not a passive structural feature but a functional element that fine-tunes folding kinetics and contributes to substrate binding efficiency.

Online structural characterization during microfluidic lipid self-assembly is important for understanding and controlling the formation of nonlamellar liquid crystalline nanodispersions. Here, we report a 3D-printed, X-ray-compatible hydrodynamic flow-focusing microfluidic chip with variable channel dimensions, integrated with synchrotron small-angle X-ray scattering (SAXS), for position-resolved SAXS-on-chip monitoring of Ca2+-triggered hexosome formation. Hexosomes were produced under continuous flow by mixing ethanolic solutions of docosahexaenoic acid monoglyceride (MAG-DHA), the negatively charged phosphatidylglycerol DOPG, and -tocopherol with Ca2+-containing PIPES buffer. Online SAXS-on-chip measurements detected three Bragg reflections characteristic of the internal inverse hexagonal (H2) phase on a tens-of-milliseconds residence-time scale, revealing rapid structural evolution during microfluidic mixing. Complementary ex situ SAXS identified the DOPG/Ca2+ molar ratio as a key parameter modulating the direct vesicle-to-hexosome transformation and the compactness of the internal H2 nanostructures. Dynamic light scattering showed that the flow-rate ratio modulated nanoparticle size, yielding hexosomes with mean hydrodynamic diameters in the range of approximately 120-175 nm and polydispersity index values down to 0.14 at a total flow rate of 200 {micro}L min-1. Cryo-TEM revealed coexistence of hexosomes and vesicular nanostructures, highlighting morphological heterogeneity, while Coarse-Grained Molecular Dynamics simulations supported a central role of Ca2+-DOPG association in promoting a direct lamellar-H2 phase transition. Overall, this work shows that 3D-printed SAXS-compatible microfluidics can integrate continuous production with online structural characterization, providing a basis for future formulation and process optimization of drug-loaded cubosomes, hexosomes, and related nonlamellar liquid crystalline nanodispersions.

Hyperglycaemia disrupts mitochondrial homeostasis, leading to oxidative stress, ATP depletion, lysosomal dysfunction, impaired stress signalling, and proteostasis collapse. Although static magnetic fields (SMFs) have shown therapeutic potential in diabetic models, the optimal field strength for restoring subcellular organelle integrity post high glucose associated dysfunction remains unknown. Here, using Caenorhabditis elegans exposed to 40 mM glucose, we systematically evaluated SMFs ranging from 20 to 100 mT. Hyperglycaemia induced mitochondrial fragmentation, elevated reactive oxygen species, lysosomal abnormalities, reduced ATP levels, caused developmental delay, suppression of cytoprotective stress reporters, and increased polyglutamine aggregation. Among all field strengths tested, 70 mT produced the most robust recovery, restoring mitochondrial network architecture, reducing oxidative stress, normalizing lysosomal morphology, recovering ATP homeostasis, improving developmental progression, enhancing stress-responsive signalling, reducing proteotoxic aggregates, and increasing mitochondrial resilience to secondary hypoxic stress. These findings identify an optimal therapeutic SMF window and provide the first whole-organism demonstration that appropriately tuned static magnetic fields restore mitochondrial homeostasis and reverse multiple downstream consequences of hyperglycaemic stress.

Ammonia oxidation initiates nitrification and is closely linked to microbial N2O production. Ammonia monooxygenase (AMO) catalyzes the first and rate-limiting step of nitrification and is widespread across evolutionarily distinct ammonia-oxidizing archaea (AOA) and bacteria (AOB). The ocean is the largest biome for AOA and AOB, which have distinct ecological niches and markedly different sensitivities to nitrification inhibitors. However, the lack of archaeal AMO structures and inhibitor-bound AMO complexes has hindered mechanistic understanding of the architectural, catalytic, and inhibitory divergence between these two enzyme systems. Here, we report high-resolution cryo-electron microscopy (cryo-EM) structures of marine archaeal AMO captured in active and inactivated states within its native membrane environment, together with inhibitor-bound structures of estuarine bacterial AMO. Archaeal AMO forms an unexpected cup-shaped homotrimer composed of eight subunits per protomer and exhibits substantial architectural divergence from bacterial AMO. Integrated structural, biochemical, kinetic, and computational analyses reveal distinct periplasmic architectures, copper-center organization, and hydrophobic channels between archaeal and bacterial AMOs for ammonium acquisition, catalysis and inhibitor response. These findings provide a structural and mechanistic framework for understanding how archaeal and bacterial AMOs have diverged to distinct ammonia-oxidizing strategies and inhibitor susceptibilities across environmentally important ammonia oxidizers.

SynNotch-CAR circuits improve T cell specificity by coupling antigen recognition to inducible CAR expression. However, basal CAR expression without receptor activation, termed here as leakiness, can reduce the separation between killing of intended target cells and sparing of antigen-positive off-target cells, limiting target-cell discrimination. Here, we systematically quantified basal CAR expression for several synNotch-CAR designs and developed a coupled ordinary differential equation model to show that discrimination depends on basal output, CAR potency, and effector-to-target ratio. We introduced C-terminal tags such as fluorescent proteins, degron domains, endocytosis signals, and endoplasmic reticulum retention motifs as a strategy to reduce CAR leakiness. We found that fluorescent proteins and degron-containing tags reduced basal CAR surface expression while preserving antigen-induced CAR expression, improving discrimination of antigen-density sensing and combinatorial circuits in vitro. In xenograft models, fluorescent protein-tagged CARs improved discrimination by reducing activity against off-target cells while retaining activity against high-antigen tumors. Degron-containing constructs reduced basal CAR expression in vitro but showed suboptimal performance in vivo, revealing a trade-off between basal CAR suppression and induced CAR persistence. Together, these findings demonstrate that basal output expression is a key parameter for inducible genetic circuit designs and establish layered transcriptional and post-translational regulation as a strategy to improve the fidelity of inducible T cell circuits.