A Direct Approach for Living Biomembrane Printing on a Nanoparticle.

Biomembrane coating technologies have been developed to equip synthetic nanomaterials with natural biointerfaces. We report a one-step method for nondestructively coating the biomembranes of "living" cells onto nanoparticle surfaces. By using simple centrifugation, nanoparticles pass through a concentrated layer of living cells. This process mimics exosome release via endocytosis and exocytosis, preserving the membrane integrity of the source cells. The resulting silica nanoparticles were efficiently coated with membrane components from Raw264.7 cells. Nanoflow-liquid chromatography-tandem mass spectrometry confirmed that the proteins composing the membrane originated from the source cells. Additionally, the biomembrane coating suppressed the phagocytosis of silica nanoparticles by Raw264.7 cells while enhancing their uptake by HeLa cells. Our simple and efficient method for living biomembrane coating holds promise for the development of nanoparticles for medical and pharmaceutical applications.

Stabilized Cell Membrane-Derived Vesicles by Lipid Anchoring for Enhanced Drug Delivery.

A cell membrane-derived vesicle (MV) that has cell-mimicking features with characteristic functionalities holds vast appeal for biomimetic nanomedicine and drug delivery but suffers from a major limitation of innate fragility and poor stability. Herein, we report a lipid-anchoring strategy for stabilizing MV for enhanced drug delivery. An array of amphiphilic mono-acyl phosphatidylcholines (MPCs) with specific hydrophobic moieties are synthesized and readily engineered on MV based on their commendable aqueous solubility and efficient membrane insertability. Incorporation of MPCs containing rigid ring structures in the hydrophobic segment demonstrates the potency of stabilizing MV by the combined ordering and condensing effects. The optimized MPC-stabilized MV exhibits prolonged circulation in the bloodstream, elevated accumulation within a tumor, and enhanced therapeutic effects of chemotherapeutic and photothermal drugs. Moreover, doxorubicin-loaded MV, engineered with mono-all-trans retinoyl phosphatidylcholine as an MV stabilizer and a therapeutic prodrug, potently suppresses growth and metastasis of high-stemness tumors.

Engineering Hyperechogenic Colloids with Clot-Targeting Capabilities from Platelet-Derived Membranes.

Thrombosis-related cardiovascular diseases remain the leading global cause of mortality and morbidity. In this study, we present a pioneering approach in the field of nanobiotechnology, with a focus on clinical translation, aimed at advancing early diagnosis and enhancing treatment options for thrombotic disorders. We introduce the fabrication of Platelet Membrane-Derived Bubbles (PMBs), which exhibit distinctive characteristics compared to conventional nanoparticles. These PMBs possess an average diameter of 700 nm and a negative ζ-potential, mirroring the attributes of parent platelet membranes. Utilizing diagnostic ultrasound imaging, we demonstrated the ability to visualize PMBs as hyperechogenic entities in agarose phantoms in vitro and in live mice in vivo. Furthermore, through confocal laser microscopy, we verified the retention of crucial transmembrane proteins, such as CD41 (GPIIb) and CD42 (GPIb), pivotal in conferring platelet-specific targeting functions. Importantly, our platelet aggregation studies confirmed that PMBs do not induce platelet aggregation but instead adhere to preformed platelet-rich in vitro thrombi. Overall, our work showcases the safe and precise utilization of PMBs to directly target acute thrombosis induced by laser injury in murine mesenteric veins in vivo, as visualized through intravital microscopy. In conclusion, we have successfully developed a rapid method for generating PMBs with unique ultrasound-directed and thrombus-targeting properties. These exceptional attributes of PMBs hold significant promise for advancing the field of ultrasound diagnostic thrombus imaging and clot-targeted therapy in the clinical context.

Cancer cell membrane-camouflaged curcumin nanoparticles trigger ferroptosis for accurate gastric cancer therapy.

Curcumin (CUR) is a hydrophobic polyphenol with considerable antitumor efficiency, but its clinical application is limited because of its poor solubility and low stability in aqueous solution and lack of targeting in vivo. Herein, we fabricated a tumor-targeting drug delivery system by loading CUR and cloaking homologous cancer cell membrane (CM) onto mesoporous silica NPs (MSN-CUR@CM). Characterization analysis showed that MSN-CUR@CM with a size of approximately 70 nm showed high water solubility and biocompatibility. Besides, MSN-CUR@CM exhibited tumor-targeting and excellent anti-gastric cancer efficiency both in vitro and in vivo owing to the cellular self-recognition of CM. In the established xenograft tumor nude mouse model, it was still significantly drug accumulated at the tumor site 72 h post administration. In addition, the mean tumor volume and weight of the MSN-CUR@CM group were was 3.97 and 7.47 times smaller than those of the CUR group. Ferroptosis, a type of non-apoptotic regulated cell death accompanied by iron-dependent lipid peroxidation, was triggered by MSN-CUR@CM. Further analysis demonstrated that MSN-CUR@CUR upregulated heme oxygenase (HO)-1 levels whereas it downregulated the expression of glutathione peroxidase 4 (GPX4) in SGC7901 cells in vitro, indicating that the canonical and noncanonical ferroptosis pathways were regulated by MSN-CUR@CM. In conclusion, our study demonstrated that MSN-CUR@CM with high water solubility, biocompatibility, and tumor-targeting properties inhibited gastric cancer both in vitro and in vivo by triggering ferroptosis and provided an admirable cancer therapy efficacy.

The interaction of galectin-8 C-terminal domain with cell surface glycoconjugates modulates membrane elasticity to stimulate antigen uptake and presentation to CD4 T cells.

Galectins constitute a family of soluble lectins with unique capacity to induce macroscale rearrangements upon interacting with cell membrane glycoconjugates. Galectin-8 (Gal-8) is acknowledged for its role in facilitating antigen uptake and processing upon engaging with cell surface glycoconjugates on antigen-presenting cells (APCs). Gal-8 consists of two covalently fused N- and C-terminal carbohydrate recognition domains (N- and C-CRD), each exhibiting distinct glycan specificity. In this study, we utilized single N- and C-CRD recombinant proteins to dissect the nature of Gal-8-glycan interactions during antigen internalization enhancement. Single C-CRD was able to replicate the effect of full-length Gal-8 (FLGal-8) on antigen internalization in BMDCs. Antigen uptake enhancement was diminished in the presence of lactose or when N-glycosylation-deficient macrophages served as APCs, underscoring the significance of glycan recognition. Measurement of the elastic modulus using Atomic Force Microscopy unveiled that FLGal-8- and C-CRD-stimulated macrophages exhibited heightened membrane stiffness compared to untreated cells, providing a plausible mechanism for their involvement in endocytosis. C-CRD proved to be as efficient as FLGal-8 in promoting antigen degradation, suggesting its implication in antigen-processing induction. Lastly, C-CRD was able to replicate FLGal-8-induced antigen presentation in the MHC-II context both in vitro and in vivo. Our findings support the notion that Gal-8 binds through its C-CRD to cell surface N-glycans, thereby altering membrane mechanical forces conducive to soluble antigen endocytosis, processing, and presentation to cognate CD4 T-cells. These findings contribute to a deeper comprehension of Gal-8 and its mechanisms of action, paving the way for the development of more efficacious immunotherapies.

Brain targeted biomimetic siRNA nanoparticles for drug resistance glioblastoma treatment.

Glioblastoma multiforme (GBM), the most aggressive intracranial neoplasm, remains incurable at present, primarily due to drug resistance, which significantly contributes to elevated recurrence rates and dismal prognosis. Signal transducer and activator of transcription 3 (STAT3) is a critical gene closely associated with GBM drug resistance and the progression of GBM stem cells (GSCs), making it a promising therapeutic target. In this study, we developed cancer cell membrane-cloaked biomimetic nanoparticles to deliver STAT3 siRNA to reverse drug resistance in homologous GBM. These biomimetic nanoparticles leverage homotypic targeting, rapid endosome escape, and fast siRNA release, leading to efficient in vitro STAT3 knockdown in both temozolomide-resistant U251-TR cells and X01 GSCs. Moreover, benefited from the membrane functionalization, significant prolonged blood circulation, improved blood brain barrier (BBB) penetration and GBM tumor accumulation are achieved by these siRNA biomimetic nanoparticles. Importantly, these nanoparticles effectively inhibit tumor proliferation, significantly extending median survival time in orthotopic U251-TR (43.5 d versus 20 d for PBS control) and X01 GSC-bearing mouse xenografts (52 d versus 19.5 d for PBS control). Altogether, this biomimetic siRNA platform offers a promising strategy for gene therapy targeting drug-resistant GBM.

Targeted blood-brain barrier penetration and precise imaging of infiltrative glioblastoma margins using hybrid cell membrane-coated ICG liposomes.

Surgical resection remains the primary treatment modality for glioblastoma (GBM); however, the infiltrative nature of GBM margins complicates achieving complete tumor removal. Additionally, the blood-brain barrier (BBB) poses a formidable challenge to effective probe delivery, thereby hindering precise imaging-guided surgery. Here, we introduce hybrid cell membrane-coated indocyanine green (ICG) liposomes (HM-Lipo-ICG) as biomimetic near-infrared (NIR) fluorescent probes for targeted BBB penetration and accurate delineation of infiltrative GBM margins. HM-Lipo-ICG encapsulates clinically approved ICG within its core and utilizes a hybrid cell membrane exterior, enabling specific targeting and enhanced BBB permeation. Quantitative assessments demonstrate that HM-Lipo-ICG achieves BBB penetration efficiency 2.8 times higher than conventional ICG liposomes. Mechanistically, CD44 receptor-mediated endocytosis facilitates BBB translocation of HM-Lipo-ICG. Furthermore, HM-Lipo-ICG enables high-contrast NIR imaging, achieving a signal-to-background ratio of 6.5 in GBM regions of an orthotopic glioma mouse model, thereby improving tumor margin detection accuracy fourfold (84.4% vs. 22.7%) compared to conventional ICG liposomes. Application of HM-Lipo-ICG facilitates fluorescence-guided precision surgery, resulting in complete resection of GBM cells. This study underscores the potential of hybrid cell membrane-coated liposomal probes in precisely visualizing and treating infiltrative GBM margins.

Extracellular vesicles-hitchhiking boosts the deep penetration of drugs to amplify anti-tumor efficacy.

Developing drug delivery systems capable of achieving deep tumor penetration is a challenging task, yet there is a significant demand for such systems in cancer treatment. Hitchhiking on tumor-derived extracellular vesicles (EVs) represents a promising strategy for enhancing drug penetration into tumors. However, the limited drug assembly on EVs restricts its further application. Here, we present a novel approach to efficiently attach antitumor drugs to EVs using an engineered cell membrane-based vector. This vector includes the AS1411 aptamer for tumor-specific targeting, the vesicular stomatitis virus glycoprotein (VSV-G) for tumor cell membrane fusion, and a photosensitizer as the therapeutic agent while ensuring optimal drug encapsulation and stability. Upon injection, photosensitizers are firstly transferred to the tumor cell membrane and subsequently piggybacked onto EVs with the inherent secretion process. By hitchhiking with EVs, photosensitizers can be transferred layer by layer deep into the solid tumors. The results suggest that this EVs-hitchhiking strategy enables photosensitizers to penetrate deeply into tumor tissue, thereby enhancing the efficacy of phototherapy. This study offers broad application prospects for delivering drugs deeply into tumor tissues.

Tebuconazole exacerbates co-occurrence and horizontal transfer of antibiotic resistance genes.

As one of the most widely used pesticides in the global fungicide market, tebuconazole has become heavily embedded in soil along with antibiotic resistance genes (ARGs). However, it remains unclear whether the selective pressure produced by tebuconazole affects ARGs and their horizontal transfer. In this experiment, we simulated a tebuconazole-contaminated soil ecosystem and observed changes in the abundance of ARGs and mobile genetic element (MGEs) due to tebuconazole exposure. We also established a plasmid RP4-mediated conjugative transfer system to investigate in depth the impact of tebuconazole on the horizontal transfer of ARGs and its mechanism of action. The results showed that under tebuconazole treatment at concentrations ranging from 0 to 10 mg/L, there was a gradual increase in the frequency of plasmid conjugative transfer, peaking at 10 mg/L which was 7.93 times higher than that of the control group, significantly promoting horizontal transfer of ARGs. Further analysis revealed that the conjugative transfer system under tebuconazole stress exhibited strong ability to form biofilm, and the conjugative transfer frequency ratio of biofilm to planktonic bacteria varied with the growth cycle of biofilm. Additionally, scanning electron microscopy and flow cytometry demonstrated increased cell membrane permeability in both donor and recipient bacteria under tebuconazole stress, accompanied by upregulation of ompA gene expression controlling cell membrane permeability. Furthermore, enzyme activity assays indicated significant increases in CAT, SOD activity, and GSH content in recipient bacteria under tebuconazole stress. Moreover, expression levels of transmembrane transporter gene trfAp as well as genes involved in oxidative stress and SOS response were found to be correlated with the frequency of plasmid conjugative transfer.

Inhibitory activity and antioomycete mechanism of citral against Phytophthora capsici.

The natural terpenoid citral has antifungal activity against multiple fungi, but its bioactivity against oomycetes is unclear. Therefore, this study investigated the antioomycete activity and mechanism of citral against Phytophthora capsici, a highly destructive invasive oomycete. Results showed that citral not only had a great inhibition on the mycelial growth of P. capsici (EC50 = 94.15 mg/L), but also had a significant inhibition on multiple spores, such as sporangia formation, zoospore discharge and zoospore germination. Citral at 4000 mg/L exhibited favorable protective (73.33%) and curative efficacy (55.11%) against pepper Phytophthora blight. Citral significantly damaged the hyphal morphology, disrupted the cell membrane integrity, increased the permeability of cell membrane, and increased the glycerol content in P. capsici. A total of 250 upregulated and 288 downregulated proteins were identified in iTRAQ-based quantitative proteomic analysis. Downregulated proteins were mostly enriched in pathways of ABC transporters, cyanoamino acid metabolism and starch and sucrose metabolism, suggesting an inhibition of citral on transmembrane transporter (e.g., ABC transporters) and pathogenicity (e.g., ß-glucosidases) proteins. Upregulated proteins were enriched in biosynthesis of unsaturated fatty acids, pyruvate metabolism and glycolysis/gluconeogenesis, suggesting an activation of citral on energy generation proteins, including acyl-CoA oxidase, D-lactate dehydrogenase, pyruvate kinase, acetyl-CoA synthetase and phosphoenolpyruvate carboxykinase. Biochemical and iTRAQ analysis suggested that cell membrane may be the target of citral in P. capsici.

Evaluation of the antifungal activity of novel bis-pyrazole carboxamide derivatives and preliminary investigation of the mechanism.

To facilitate the development of novel agricultural succinate dehydrogenase inhibitor (SDHI) fungicides, we synthesized three series of derivatives by introducing phenyl pyrazole fragments into the structure of pyrazol-4-yl amides. The results of the bioactivity assay showed that most of the target compounds possessed varying degrees of inhibitory activity against the tested fungi. At a concentration of 100 mg/L, the compound B8 exhibited effective protective activity against S. sclerotiorum in vivo. Molecular docking analysis and succinate dehydrogenase (SDH) inhibition assay indicated that B8 was not a potential SDHI. The preliminary antifungal mechanism of studies showed that B8 induced a large amount of reactive oxygen species (ROS) and severe lipid peroxidation damage in S. sclerotiorum mycelium, resulting in mycelial rupture and disruption of the integrity of the cell membrane and leakage of soluble proteins, soluble sugars and nucleic acids. Further transcriptome analysis showed that compound B8 blocked various metabolic pathways by downregulating the differentially expressed genes (DEGs) catalase, disrupting hydrogen peroxide hydrolysis, accelerating membrane oxidative damage, and upregulating neutral ceramidase, accelerating sphingolipid metabolism to disrupt cell membrane structure and cell proliferation and differentiation, potentially accelerating cell death. The above results indicated that the potential target of these dis-pyrazole carboxamide derivatives may be the cell membrane of pathogenic fungi.

A biomimic anti-neuroinflammatory nanoplatform for active neutrophil extracellular traps targeting and spinal cord injury therapy.

Traumatic spinal cord injury (SCI) always leads to severe neurological deficits and permanent damage. Neuroinflammation is a vital process of SCI and have become a promising target for SCI treatment. However, the neuroinflammation-targeted therapy would hinder the functional recovery of spinal cord and lead to the treatment failure. Herein, a biomimic anti-neuroinflammatory nanoplatform (DHCNPs) was developed for active neutrophil extracellular traps (NETs) targeting and SCI treatment. The curcumin-loaded liposome with the anti-inflammatory property acted as the core of the DHCNPs. Platelet membrane and neutrophil membrane were fused to form the biomimic hybrid membrane of the DHCNPs for hijacking neutrophils and neutralizing the elevated neutrophil-related proinflammatory cytokines, respectively. DNAse I modification on the hybrid membrane could achieve NETs degradation, blood spinal cord barrier, and neuron repair. Further studies proved that the DHCNPs could reprogram the multifaceted neuroinflammation and reverse the SCI process via nuclear factor kappa-B (NF-κB) pathway. We believe that the current study provides a new perspective for neuroinflammation inhibition and may shed new light on the treatment of SCI.

Progress of the Impact of Terahertz Radiation on Ion Channel Kinetics in Neuronal Cells.

In neurons and myocytes, selective ion channels in the plasma membrane play a pivotal role in transducing chemical or sensory stimuli into electrical signals, underpinning neural and cardiac functionality. Recent advancements in biomedical research have increasingly spotlighted the interaction between ion channels and electromagnetic fields, especially terahertz (THz) radiation. This review synthesizes current findings on the impact of THz radiation, known for its deep penetration and non-ionizing properties, on ion channel kinetics and membrane fluid dynamics. It is organized into three parts: the biophysical effects of THz exposure on cells, the specific modulation of ion channels by THz radiation, and the potential pathophysiological consequences of THz exposure. Understanding the biophysical mechanisms underlying these effects could lead to new therapeutic strategies for diseases.

Bacterial Secondary Metabolites Embedded in Producer Cell Membranes and Antibiotics Targeting Their Biosynthesis.

The bacterial cell membrane primarily houses lipids, carbohydrates, and proteins forming a barrier and interface that maintains cellular integrity, supports homeostasis, and senses environmental changes. Compared to lipid components and excreted secondary metabolites, compounds embedded in the producer cell membrane are often overlooked due to their low abundance and niche-specific functions. The accumulation of findings has led to an increased appreciation of their crucial roles in bacterial cell biochemistry, physiology, and ecology, as well as their impact on mutualistic and pathogenic bacteria-eukaryote interactions. This review highlights the structures, biosynthesis, regulation, and ecological functions of membrane-embedded secondary metabolites. It also discusses antibiotics that target their biosynthetic pathways, aiming to inspire the development of antibiotics specific to pathogenic bacteria without harming human cells.

Biomimetic Nanosensitizer Potentiates Efficient Glioblastoma Gene-Radiotherapy through Synergistic Hypoxia Mitigation and PLK1 Silencing.

Postoperative radiotherapy currently stands as the cornerstone of glioblastoma (GBM) treatment. Nevertheless, low-dose radiotherapy has been proven ineffective for GBM, due to hypoxia in the GBM microenvironment, which renders the resistance to radiation-induced cell death. Moreover, the overexpression of the PLK1 gene in glioma cells enhances GBM proliferation, invasion, metastasis, and resistance to radiation. This study introduced a hybrid membrane-camouflaged biomimetic lipid nanosensitizer (CNL@miPA), which efficiently encapsulated gold nanoclusters (PA) and miR-593-5p by a chimeric membrane derived from lipids, cancer cells, and natural killer cells. CNL@miPA exhibited exceptional blood-brain barrier and tumor tissue penetration, effectively ameliorating hypoxia and synergizing with radiotherapy. By enabling prolonged miRNA circulation in the bloodstream and achieving high enrichment at the tumor site, CNL@miPA significantly suppressed tumor growth in combination treatment, thereby significantly extending the survival period of treated mice. Overall, the developed biomimetic nanosensitizer represented an efficient and multifunctional targeted delivery system, offering a novel strategy for gene-radiotherapy of GBM.

Isobavachalcone enhances sensitivity of colistin-resistant Klebsiella pneumoniae: in vitro and in vivo proof-of-concept studies.

Antibiotic resistance poses a considerable worldwide concern, particularly in clinical environments where drug-resistant Gram-negative bacteria like Klebsiella pneumoniae present a major challenge. The objective of this research was to investigate the mechanisms by which isobavachalcone (IBC) restores the sensitivity of K. pneumoniae to colistin in vitro and to validate the synergistic therapeutic effect in vivo. The results indicate that the combined administration of colistin and IBC exhibits a potent antibacterial effect both in vitro and in vivo. The in vitro concurrent administration of colistin and IBC resulted in increased membrane permeability, compromised cell integrity, diminished membrane fluidity, and disrupted membrane homeostasis. Additionally, this combination reduced biofilm production, inhibited the synthesis of the AI-2 factor, altered membrane potential, and affected levels of reactive oxygen species and adenosine triphosphate synthesis, ultimately leading to bacterial death. In vivo experiments on Galleria mellonella and mice demonstrated that the co-administration of colistin and IBC increased the survival rate and significantly reduced pathological damage compared to colistin alone. These results suggested that IBC effectively restores the sensitivity of colistin by inducing physical disruption of bacterial membranes and oxidative stress. The combination therapy of colistin and IBC presents a viable and safe strategy to combat drug-resistant K. pneumoniae-associated infections.

Elastase-targeting biomimic nanoplatform for neurovascular remodeling by inhibiting NETosis mediated AlM2 inflammasome activation in ischemic stroke.

Neutrophil elastase (NE) is a protease released by activated neutrophils in the brain parenchyma after cerebral ischemia, which plays a pivotal role in the regulation of neutrophil extracellular traps (NETs) formation. The excess NETs could lead to blood-brain barrier (BBB) breakdown, overwhelming neuroinflammation, and neuronal injury. While the potential of targeting neutrophils and inhibiting NE activity to mitigate ischemic stroke (IS) pathology has been recognized, effective strategies that inhibit NETs formation remain under-explored. Herein, a biomimic multifunctional nanoplatform (HM@ST/TeTeLipos) was developed for active NE targeting and IS treatment. The core of the HM@ST/TeTeLipos consisted of sivelestat-loaded ditelluride-containing liposomes with ROS-responsive and NE-inhibiting properties. The outer shell was composed of platelet-neutrophil hybrid membrane vesicles (HMVs), which acted to hijack neutrophils and neutralize proinflammatory cytokines. Our studies revealed that HM@ST/TeTeLipos could effectively inhibit NE activity, thereby suppressing the release of NETs, impeding the activation of the AIM2 inflammasome, and consequently redirecting the immune response away from a pro-inflammatory M1 microglia phenotype. This resulted in enhanced neurovascular remodeling, reduced BBB disruption, and diminished neuroinflammation, ultimately promoting neuron survival. We believe that this innovative approach holds significant potential for improving the treatment of IS and various NE-mediated inflammatory diseases.

Carcinomembrane-Camouflaged Perfluorochemical Dual-Layer Nanopolymersomes Bearing Indocyanine Green and Camptothecin Effectuate Targeting Photochemotherapy of Cancer.

Photochemotherapy has been recognized as a promising combinational modality for cancer treatment. However, difficulties such as off-target drug delivery, systemic toxicity, and the hypoxic nature of the tumor microenvironment remain hindrances to its application. To overcome these challenges, cancer cell membrane camouflaged perfluorooctyl bromide (PFOB) dual-layer nanopolymersomes bearing indocyanine green (ICG) and camptothecin (CPT), named MICFNS, were developed in this study, and melanoma was exploited as the model for MICFNS manufacture and therapeutic application. Our data showed that MICFNS were able to stabilize both ICG and CPT in the nanocarriers and can be quickly internalized by B16F10 cells due to melanoma membrane-mediated homology. Upon NIR irradiation, MICFNS can trigger hyperthermia and offer enhanced singlet oxygen production due to the incorporation of PFOB. With ≥10/2.5 µM ICG/CPT, MICFNS + NIR can provide comparable in vitro cancericidal effects to those caused by using an 8-fold higher dose of encapsulated CPT alone. Through the animal study, we further demonstrated that MICFNS can be quickly brought to tumors and have a longer retention time than those of free agents in vivo. Moreover, the MICFNS with 40/10 µM ICG/CPT in combination with 30 s NIR irradiation can successfully inhibit tumor growth without systemic toxicity in mice within the 14 day treatment. We speculate that such an antitumoral effect was achieved by phototherapy followed by chemotherapy, a two-stage tumoricidal process performed by MICFNS. Taken together, we anticipate that MICFNS, a photochemotherapeutic nanoplatform, has high potential for use in clinical anticancer treatment.

Baicalein: A potential GLP-1R agonist improves cognitive disorder of diabetes through mitophagy enhancement.

There is increasing evidence that the activation of glucagon-like peptide-1 receptor (GLP-1R) can be used as a therapeutic intervention for cognitive disorders. Here, we have screened GLP-1R targeted compounds from Scutellaria baicalensis, which revealed baicalein is a potential GLP-1R small-molecule agonist. Mitophagy, a selective autophagy pathway for mitochondrial quality control, plays a neuroprotective role in multiple cognitive impairment diseases. We noticed that Glp1r knock-out (KO) mice present cognitive impairment symptoms and appear worse in spatial learning memory and learning capacity in Morris water maze (MWM) test than their wide-type (WT) counterparts. Our mechanistic studies revealed that mitophagy is impaired in hippocampus tissue of diabetic mice and Glp1r KO mice. Finally, we verified that the cognitive improvement effects of baicalein on diabetic cognitive dysfunction occur through the enhancement of mitophagy in a GLP-1R-dependent manner. Our findings shed light on the importance of GLP-1R for cognitive function maintenance, and revealed the vital significance of GLP-1R for maintaining mitochondrial homeostasis. Furthermore, we identified the therapeutic potential of baicalein in the treatment of cognitive disorder associated with diabetes.

Membrane RRM2-positive cells represent a malignant population with cancer stem cell features in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (iCCA) is one of the most lethal malignancies and highly heterogeneous. We thus aimed to identify and characterize iCCA cell subpopulations with severe malignant features. METHODS: Transcriptomic datasets from three independent iCCA cohorts (iCCA cohorts 1-3, n = 382) and formalin-fixed and paraffin-embedded tissues from iCCA cohort 4 (n = 31) were used. An unbiased global screening strategy was established, including the transcriptome analysis with the activated malignancy/stemness (MS) signature in iCCA cohorts 1-3 and the mass spectrometry analysis of the sorted stemness reporter-positive iCCA cells. A group of cellular assays and subcutaneous tumor xenograft assay were performed to investigate functional roles of the candidate. Immunohistochemistry was performed in iCCA cohort 4 to examine the expression and localization of the candidate. Molecular and biochemical assays were used to evaluate the membrane localization and functional protein domains of the candidate. Cell sorting was performed and the corresponding cellular molecular assays were utilized to examine cancer stem cell features of the sorted cells. RESULTS: The unbiased global screening identified RRM2 as the top candidate, with a significantly higher level in iCCA patients with the MS signature activation and in iCCA cells positive for the stemness reporter. Consistently, silencing RRM2 significantly suppressed iCCA malignancy phenotypes both in vitro and in vivo. Moreover, immunohistochemistry in tumor tissues of iCCA patients revealed an unreported cell membrane localization of RRM2, in contrast to its usual cytoplasmic localization. RRM2 cell membrane localization was then confirmed in iCCA cells via immunofluorescence with or without cell membrane permeabilization, cell fractionation assay and cell surface biotinylation assay. Meanwhile, an unclassical signal peptide and a transmembrane domain of RRM2 were revealed experimentally. They were essential for RRM2 trafficking to cell membrane via the conventional endoplasmic reticulum (ER)-Golgi secretory pathway. Furthermore, the membrane RRM2-positive iCCA cells were successfully sorted. These cells possessed significant cancer stem cell malignant features including cell differentiation ability, self-renewal ability, tumor initiation ability, and stemness/malignancy gene signatures. Patients with membrane RRM2-positive iCCA cells had poor prognosis. CONCLUSIONS: RRM2 had an alternative cell membrane localization. The membrane RRM2-positive iCCA cells represented a malignant subpopulation with cancer stem cell features.