Abstract The glucagon-like peptide-1 receptor (GLP-1R) plays a central role in metabolic regulation and is a major therapeutic target for obesity and diabetes. Peptide agonists, like semaglutide, targeting the GLP-1R remain among the most effective regulators of glucose metabolism and appetite. Nonetheless, recent reports about weight regain have limited the effectiveness of GLP1R peptide agonists, sustaining the interest in expanding the chemical diversity of GLP-1R ligands through drug discovery strategies. However, the structural complexity and conformational plasticity of class B1 GPCRs make conventional single-method virtual screening approaches prone to bias and limited chemotype recovery. Using an integrated ligand– and structure-based virtual screening pipeline, explicitly combining complementary ligand-based descriptors, multi-fingerprint similarity, electrostatic similarity, pharmacophore modeling, and multi-conformation docking under a consensus-driven selection strategy, we were able to identify three chemically distinct classes of GLP-1R agonist candidates: GQB47810, a non-peptidic molecule; neuromedin C, a peptide, and 2,5-Pen-enkephalin (DPDPE), a small peptide. From all of them, DPDPE showed the greatest effectiveness, reaching values similar to those of GLP-1, although with lower potency. Further in vitro characterization confirmed that pen-enkephalin behaved as a full agonist and exhibited dual GLP-1R/GIPR agonistic activity. These findings establish a consensus-driven and transferable computational framework for chemotype-diverse agonist discovery at conformationally flexible GPCR targets, and revealed a pentapeptide with GLP-1-like efficacy as a promising lead for next-generation small peptide therapeutics.

To the Editor: The glucagon-like peptide-1 receptor(GLP-1R)plays a central role in glucose homeostasis and energy balance through its acti-vation by the incretin hormone GLP-11.It is highly expressed in pancreatic islets and the gastrointestinal tract,and is also present in the brain and heart2.At the signaling level,GLP-1R primarily couples to Gs,activating adenylate cyclase,elevating intracellular cyclic AMP(cAMP),and stimulating PKA/EPAC pathways3.

Hyperthermia, a physical therapeutic modality with unique advantages in treating inflammatory diseases, has evolved rapidly from empirical practice to precision medicine. Technologically, it has expanded from whole-body hyperthermia and traditional moxibustion to localized approaches, including photothermal, magnetothermal, ultrasound, and implantable platforms. Traditional moxibustion acts through heat and near-infrared radiation, while nanomaterials and smart devices now enable precise control over temperature, spatial targeting, and treatment duration. these modalities converge on conserved mechanisms: TRPV channel activation, HSF1-HSP70 induction, and immune modulation, which collectively alleviate inflammation and promote tissue repair in inflammatory bowel disease, osteoarthritis, and Alzheimer's disease. This review summarizes technological evolution, mechanistic insights and therapeutic applications, thereby providing a framework for developing precision hyperthermia strategies and multifunctional platforms for inflammatory diseases.

Histamine H3 receptor (H3R) and H4 receptor (H4R) are key members of the histamine receptor family, with H3R as a potential target for narcolepsy treatments and H4R as a candidate for next-generation antihistamines for inflammatory and allergic diseases. Although progress has been made in understanding the structure of histamine receptors, the detailed mechanisms of ligand recognition and receptor antagonism for H3R and H4R remain unclear. In this study, using cryo-electron microscopy, we present an inactive structure of H4R bound to a selective antagonist, adriforant, and two Gi-coupled structures of H3R and H4R in complex with histamine. Our structural and mutagenesis analyses provide insights into the selective binding of adriforant to H4R and the recognition of histamine across histamine receptors. Our findings also uncovered distinct antagonistic mechanisms for H3R and H4R and identified the role of aromatic amino acids on extracellular loop 2 in modulating the constitutive activity of H3R and H4R. These findings advance our knowledge of the functional modulation of histamine receptors, providing a foundation for the development of targeted therapeutics for neurological and immune-related disorders.

One primary challenge in whole-brain imaging technology is to achieve high-resolution visualization of neuronal connectivity at large scales. Although the Golgi method allows for labeling random neurons in their entirety, visualizing individual dendritic trees, and tracing long-distance axonal projections, the lengthy processing time poses a limitation, as staining a mouse whole-brain sample of ∼500 mm3 takes over two weeks. Here, we developed a high-pressure-assisted Golgi-Cox (HP Golgi-Cox) method that reduced the staining time for mouse whole-brain neurons from 16 to 4 days. We demonstrated its applicability in zebrafish, mice, and rats, and further achieved rapid staining of hippocampal neurons in an intact pig brain, which is challenging with the classical Golgi-Cox method. By combining the HP Golgi-Cox method with synchrotron-based X-ray microscopy, we achieved high-resolution imaging of whole-brain neurons in mice. This HP Golgi-Cox method enables rapid and high-resolution neuronal imaging in large model organisms, showcasing its broad applicability for diverse applications.

Achieving detailed neuronal structural information in large-volume brain tissue has been a longstanding challenge in human brain imaging. A key obstacle arises from the trade-off between staining efficiency and tissue autolysis. Traditional Golgi staining, typically conducted at room temperature or 37 °C to optimize staining efficiency, leads to rapid autolysis of brain tissue, resulting in the loss of fine structural details. Here, a near-freezing temperature (NFT) staining strategy in post-mortem frozen (PMF) human brain samples are presented, using a mercury chloride-based method under ice-water bath conditions. In contrast to the 37 °C Golgi staining, this NFT-based method significantly reduces tissue autolysis, preserving fine neuronal structures. Notably, neuronal counts in the same field of view increased by 5.5-fold, and dendritic spine density increases by 22-fold. Using this approach, uniform staining of millimeter-thick is achieved, centimeter-scale human brain slices and integrated it with synchrotron-based X-ray microscopy to perform micrometer resolution 3D reconstructions of the cerebellum and frontal lobe. This novel technique offers a powerful tool for the fine-structural imaging of large-volume brain tissue, providing new insights into the intricate organization of neural networks.

Understanding the structural and functional organisation of brain networks is a fundamental objective in neuroscience, with three-dimensional (3D) reconstruction of single-neuron morphology serving as a critical foundation. The Golgi staining method, which enables random neuronal labeling and provides high-contrast signals in both optical and X-ray microscopy, remains a valuable tool for morphological analysis. However, its widespread application in large-scale neuronal reconstructions is hindered by signal discontinuities in neuronal branches, high-density labeling, and complex background interference. While automated reconstruction methods perform well in sparsely labelled and morphologically simple neuronal populations, their effectiveness is limited in Golgi-stained samples. Here we develop a semi-automated single-neuron reconstruction method for Golgi-stained mouse brain neurons (SNR-Golgi). By integrating three key technical modules-background denoising, single-neuron extraction, and branch repair-SNR-Golgi significantly enhances the accuracy and completeness of neuronal reconstruction. In fluorescence micro-optical sectioning tomography (fMOST) datasets, SNR-Golgi demonstrated superior performance in neuronal reconstruction within the mouse somatosensory cortex, achieving a 30% increase in reconstructed branch count, a 76% improvement in total branch length, and a 3.7-fold increase in axonal length. Additionally, in synchrotron-based X-ray imaging datasets, SNR-Golgi enabled submicron-resolution 3D reconstruction of single neurons. These results demonstrate that SNR-Golgi effectively addresses the complexity of Golgi-stained samples and provides robust technical support for the structural analysis of brain neurons across various imaging modalities.

The X-ray Imaging and Biomedical Application Beamline (BL13HB) at the Shanghai Synchrotron Radiation Facility (SSRF) is a general imaging beamline for micrometer-millisecond synchrotron X-ray imaging platform upgraded and relocated during SSRF Phase-II. This beamline employs an super-high magnetic field bending magnet light source combined with W/Si and Ru/C multilayer monochromators, offering an energy range of 8-40 keV and a newly implemented white-beam mode. With a field of view up to 48 mmx5.2 mm, the beamline meets routine imaging requirements for both soft biological tissues and metals/bones. It achieves remarkable performance metrics, including a maximum spatial resolution of 0.8 mu m, a maximum temporal resolution of 0.5 ms for single-frame imaging, and a 200 ms resolution for dynamic computerized tomography (CT) applications. The experimental hutch is divided into three dedicated zones-fast imaging, in situ/total scattering, and high-resolution imaging-equipped with flexible sample positioning systems and specialized detectors. BL13HB supports diverse techniques including two-dimensional absorption and phase-contrast imaging, three-dimensional static/dynamic micro-CT, atomic pair distribution function (PDF) analysis, and move-contrast X-ray imaging (MCXI). Since the operation in June 2021 after relocation and upgradation, BL13HB has provided about 15360 h user beamtime and served nearly 600 user proposals coming from universities, institutes, hospitals, and high-tech companies covering biomedicine, pharmacy, materials science, energy, archaeology, soil science, and other fields. And over 400 papers have been published based on the BL13HB. BL13HB of SSRF has become an important public experimental platform for cutting-edge research in multiple disciplines and technological breakthroughs in industry.

With the construction and operational launch of third-generation synchrotron radiation sources, X-ray molecular probe technology is actively explored and developed, playing an increasingly significant role in modern life sciences. This review provides an overview of synchrotron-based X-ray imaging techniques and emphasizes the applications of X-ray molecular probes across various fields associated with intelligent biomedicine. Building on this foundation, it further discusses future advancements in X-ray molecular probes and forecasts their application trends in intelligent biomedicine, particularly in the realm of personalized treatment.

In this study, we have developed a dual-mode probe based on click chemistry for spherical nucleic acids (SNAs), achieving extremely specific fluorescence imaging and synchrotron X-ray microscopy of neuronal cells.

To the Editor:G protein-coupled receptors （GPCRs） are the largest group of membrane proteins with over 800 members, characteristic of a seven transmembrane domain1. By playing crucial roles in regulation of various physiological processes, GPCRs have been implicated in many diseases including diabetes, obesity,

Glucagon-like peptide-1 receptor (GLP-1R) is a prime drug target for type 2 diabetes and obesity. The ligand initiated GLP-1R interaction with G protein has been well studied, but not with β-arrestin 1/2. Therefore, bioluminescence resonance energy transfer (BRET), mutagenesis and an operational model were used to evaluate the roles of 85 extracellular surface residues on GLP-1R in β-arrestin 1/2 recruitment triggered by three representative GLP-1R agonists (GLP-1, exendin-4 and oxyntomodulin). Residues selectively regulated β-arrestin 1/2 recruitment for diverse ligands, and β-arrestin isoforms were identified. Mutation of residues K130-S136, L142 and Y145 on the transmembrane helix 1 (TM1)-extracellular domain (ECD) linker decreased β-arrestin 1 recruitment but increased β-arrestin 2 recruitment. Other extracellular loop (ECL) mutations, including P137A, Q211A, D222A and M303A selectively affected β-arrestin 1 recruitment while D215A, L217A, Q221A, S223A, Y289A, S301A, F381A and I382A involved more in β-arrestin 2 recruitment for the ligands. Oxyntomodulin engaged more broadly with GLP-1R extracellular surface to drive β-arrestin 1/2 recruitment than GLP-1 and exendin-4; I147, W214 and L218 involved in β-arrestin 1 recruitment, while L141, D215, L218, D293 and F381 in β-arrestin 2 recruitment for oxyntomodulin particularly. Additionally, the non-conserved residues on β-arrestin 1/2 C-domains contributed to interaction with GLP-1R. Further proteomic profiling of GLP-1R stably expressed cell line upon ligand stimulation with or without β-arrestin 1/2 overexpression demonstrated both commonly and biasedly regulated proteins and pathways associated with cognate ligands and β-arrestins. Our study offers valuable information about ligand induced β-arrestin recruitment mediated by GLP-1R and consequent intracellular signaling events.

Synchrotron-based X-ray microscopy (XRM) has garnered widespread attention from researchers due to its high spatial resolution and excellent energy (element) resolution. Existing molecular probes suitable for XRM include immune probes and genetic labeling probes, enabling the precise imaging of various biological targets within cells. However, immune labeling techniques are prone to cross-interference between antigens and antibodies. Genetic labeling technologies have limited systems that allow express markers independently, and moreover, genetically encoded labels based on catalytic polymerization lack a fixed morphology. When applied to cell imaging, this can result in reduced localization accuracy due to the diffusion of labels within the cells. Therefore, both techniques face challenges in simultaneously labeling multiple biotargets within cells and achieving high-precision imaging. In this work, we applied the click reaction and developed a third category of imaging probes suitable for XRM, termed clickable X-ray nanoprobes (Click-XRN). Click-XRN consists of two components: an X-ray-sensitive multicolor imaging module and a particle-size-controllable morphology module. Efficient identification of intra- and extracellular biotargets is achieved through click reactions between the probe and biomolecules. Click-XRN possesses a controllable particle size, and its loading of various metal ions provides distinctive signals for imaging under XRM. Based on this, we optimized the imaging energy of Click-XRN with different particle sizes, enabling single-color and two-color imaging of the cell membrane, cell nucleus, and mitochondria with nanoscale spatial nanometers. Our work provides a potent molecular tool for investigating cellular activities through XRM.

Class B1 G protein-coupled receptors (GPCRs) are important regulators of many physiological functions such as glucose homeostasis, which is mainly mediated by three peptide hormones, i.e., glucagon-like peptide-1 (GLP-1), glucagon (GCG), and glucose-dependent insulinotropic polypeptide (GIP). They trigger a cascade of signaling events leading to the formation of an active agonist–receptor–G protein complex. However, intracellular signal transducers can also activate the receptor independent of extracellular stimuli, suggesting an intrinsic role of G proteins in this process. Here, we report cryo-electron microscopy structures of the human GLP-1 receptor (GLP-1R), GCG receptor (GCGR), and GIP receptor (GIPR) in complex with G s proteins without the presence of cognate ligands. These ligand-free complexes share a similar intracellular architecture to those bound by endogenous peptides, in which, the G s protein alone directly opens the intracellular binding cavity and rewires the extracellular orthosteric pocket to stabilize the receptor in a state unseen before. While the peptide-binding site is partially occupied by the inward folded transmembrane helix 6 (TM6)–extracellular loop 3 (ECL3) juncture of GIPR or a segment of GCGR ECL2, the extracellular portion of GLP-1R adopts a conformation close to the active state. Our findings offer valuable insights into the distinct activation mechanisms of these three important receptors. It is possible that in the absence of a ligand, the intracellular half of transmembrane domain is mobilized with the help of G s protein, which in turn rearranges the extracellular half to form a transitional conformation, facilitating the entry of the peptide N-terminus.

Phosphorylation of G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) desensitizes G-protein signalling and promotes arrestin signalling, which is also modulated by biased ligands1–6. The molecular assembly of GRKs on GPCRs and the basis of GRK-mediated biased signalling remain largely unknown owing to the weak GPCR–GRK interactions. Here we report the complex structure of neurotensin receptor 1 (NTSR1) bound to GRK2, Gαq and the arrestin-biased ligand SBI-5537. The density map reveals the arrangement of the intact GRK2 with the receptor, with the N-terminal helix of GRK2 docking into the open cytoplasmic pocket formed by the outward movement of the receptor transmembrane helix 6, analogous to the binding of the G protein to the receptor. SBI-553 binds at the interface between GRK2 and NTSR1 to enhance GRK2 binding. The binding mode of SBI-553 is compatible with arrestin binding but clashes with the binding of Gαq protein, thus providing a mechanism for its arrestin-biased signalling capability. In sum, our structure provides a rational model for understanding the details of GPCR–GRK interactions and GRK2-mediated biased signalling. Structural studies on the complex containing G-protein-coupled receptor kinase 2 (GRK2), neurotensin receptor 1 (NTSR1), Gαq and the arrestin-biased ligand SBI-553 provide insights into these interactions and a foundation for the design of arrestin-biased ligands for G-protein-coupled receptors.

One of the basic goals of cell biology is to identify multiple biological molecules within cells and understand the complex interactions between biological molecules in cellular life activities. Synchrotron-based X-ray microscopy has high spatial resolution and good energy (element) resolution, which has great application potential in the recognition and imaging of intracellular biomolecules. At present, the probes that have been developed for synchrotron-based X-ray microscopy are mainly immunostaining probes and genetic labeling probes. Immunostaining probes are prone to lead to cross- reactions due to their dependence on antigen-antibody reactions. The genetic labeling probes, based on gene coding tags, catalyze the generation of X-ray sensitive polymers. However, the polymers used to provide X-ray imaging signals have no fixed morphology. When it is applied to cell imaging, the positioning accuracy will be reduced due to the diffusion of tags in cells, which is an inherent defect of such imaging tags. In addition, there are few existing systems that can express each other independently and step by step for this type of probe. Therefore, both types of X-ray probes mentioned above are difficult to achieve simultaneous high-resolution imaging observation of multiple biological target molecules in cellular life activities. In this research paper, by using the characteristics of X-ray that has good energy resolution and does not interfere with each other between element spectra, we can synthesize polydopamine (PDA) nanoparticles in a controlled manner, modify azide groups on PDA nanoparticles and chelate metal ions, develop a click chemistry based synchronous X-ray imaging tag (PDA-N3-Metal), and conduct synchrotron radition X-ray imaging on the tag with an imaging resolution of 30 nm. The research results lay a good foundation for further preparation of X-ray probes based on click chemistry, and for realizing the specific recognition and imaging of multiple biological molecules in cells at the same time.

G protein-coupled receptors (GPCRs) are regulated by various downstream proteins, of which the melanocortin receptor accessory protein 1 (MRAP1) is closely involved in the regulation of melanocortin receptor 2 (MC2R). Assisted by MRAP1, MC2R responds to adrenocorticotropic hormone (ACTH) and stimulates glucocorticoid biogenesis and cortisol secretion. MC2R activation plays an essential role in the hypothalamic-pituitary-adrenal (HPA) axis that regulates stress response, while its dysfunction causes glucocorticoid insufficiency- or cortisol excess-associated disorders. Here, we present a cryo-electron microscopy (cryo-EM) structure of the ACTH-bound MC2R–Gs–MRAP1 complex. Our structure, together with mutagenesis analysis, reveals a unique sharp kink at the extracellular region of MRAP1 and the ‘seat-belt’ effect of MRAP1 on stabilizing ACTH binding and MC2R activation. Mechanisms of ACTH recognition by MC2R and receptor activation are also demonstrated. These findings deepen our understanding of GPCR regulation by accessory proteins and provide valuable insights into the ab initio design of therapeutic agents targeting MC2R.

Members of the melanocortin receptor (MCR) family that recognize different melanocortin peptides mediate a broad spectrum of cellular processes including energy homeostasis, inflammation and skin pigmentation through five MCR subtypes (MC1R–MC5R). The structural basis of subtype selectivity of the endogenous agonist γ-MSH and non-selectivity of agonist α-MSH remains elusive, as the two agonists are highly similar with a conserved HFRW motif. Here, we report three cryo-electron microscopy structures of MC3R–G s in complex with γ-MSH and MC5R–G s in the presence of α-MSH or a potent synthetic agonist PG-901. The structures reveal that α-MSH and γ-MSH adopt a “U-shape” conformation, penetrate into the wide-open orthosteric pocket and form massive common contacts with MCRs via the HFRW motif. The C-terminus of γ-MSH occupies an MC3R-specific complementary binding groove likely conferring subtype selectivity, whereas that of α-MSH distances itself from the receptor with neglectable contacts. PG-901 achieves the same potency as α-MSH with a shorter length by rebalancing the recognition site and mimicking the intra-peptide salt bridge in α-MSH by cyclization. Solid density confirmed the calcium ion binding in MC3R and MC5R, and the distinct modulation effects of divalent ions were demonstrated. Our results provide insights into ligand recognition and subtype selectivity among MCRs, and expand the knowledge of signal transduction among MCR family members.

Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a potential drug target for metabolic disorders. It works with glucagon-like peptide-1 receptor and glucagon receptor in humans to maintain glucose homeostasis. Unlike the other two receptors, GIPR has at least 13 reported splice variants (SVs), more than half of which have sequence variations at either C or N terminus. To explore their roles in endogenous peptide-mediated GIPR signaling, we determined the cryoelectron microscopy (cryo-EM) structures of the two N terminus-altered SVs (referred as GIPR-202 and GIPR-209 in the Ensembl database, SV1 and SV2 here, respectively) and investigated the outcome of coexpressing each of them in question with GIPR in HEK293T cells with respect to ligand binding, receptor expression, cAMP (adenosine 3,5-cyclic monophosphate) accumulation, β-arrestin recruitment, and cell surface localization. It was found that while both N terminus-altered SVs of GIPR neither bound to the hormone nor elicited signal transduction per se, they suppressed ligand binding and cAMP accumulation of GIPR. Meanwhile, SV1 reduced GIPR-mediated β-arrestin 2 responses. The cryo-EM structures of SV1 and SV2 showed that they reorganized the extracellular halves of transmembrane helices 1, 6, and 7 and extracellular loops 2 and 3 to adopt a ligand-binding pocket-occupied conformation, thereby losing binding ability to the peptide. The results suggest a form of signal bias that is constitutive and ligand-independent, thus expanding our knowledge of biased signaling beyond pharmacological manipulation (i.e., ligand specific) as well as constitutive and ligand-independent (e.g., SV1 of the growth hormone-releasing hormone receptor).

Members of the insulin superfamily regulate pleiotropic biological processes through two types of target-specific but structurally conserved peptides, insulin/insulin-like growth factors and relaxin/insulin-like peptides. The latter bind to the human relaxin family peptide receptors (RXFPs). Here, we report three cryo-electron microscopy structures of RXFP4–G i protein complexes in the presence of the endogenous ligand insulin-like peptide 5 (INSL5) or one of the two small molecule agonists, compound 4 and DC591053. The B chain of INSL5 adopts a single α-helix that penetrates into the orthosteric pocket, while the A chain sits above the orthosteric pocket, revealing a peptide-binding mode previously unknown. Together with mutagenesis and functional analyses, the key determinants responsible for the peptidomimetic agonism and subtype selectivity were identified. Our findings not only provide insights into ligand recognition and subtype selectivity among class A G protein-coupled receptors, but also expand the knowledge of signaling mechanisms in the insulin superfamily.