DNA Molecular Glue Assisted Bacterial Conjugative Transfer.

Bacterial conjugation, a commonly used method to horizontally transfer functional genes from donor to recipient strains, plays an important role in the genetic manipulation of bacteria for basic research and industrial production. Successful conjugation depends on the donor-recipient cell recognition and a tight mating junction formation. However, the efficiency of conjugative transfer is usually very low. In this work, we developed a new technique that employed DNA molecule "glue" to increase the match frequency and the interaction stability between the donor and recipient cells. We used two E. coli strains, ETZ and BL21, as a model system, and modified them with the complementary ssDNA oligonucleotides by click chemistry. The conjugation efficiency of the modified bacteria was improved more than 4 times from 10% to 46%. This technique is simple and generalizable as it only relies on the active amino groups on the bacterial surface. It is expected to have broad applications in constructing engineered bacteria.

Effects of Silybum marianum, Pueraria lobate, combined with Salvia miltiorrhiza tablets on non-alcoholic fatty liver disease in adults: A triple-blind, randomized, placebo-controlled clinical trial.

BACKGROUND & AIMS: Several medicinal plant extracts have demonstrated hepatoprotective effects. However, data are scarce regarding their combined effects on non-alcoholic fatty liver disease (NAFLD). This study aimed to investigate the effects of tablets containing Silybum marianum, Pueraria lobata, and Salvia miltiorrhiza (SPS) on NAFLD progression in Chinese adults. METHODS: In this randomized, triple-blind, placebo-controlled clinical trial, 121 NAFLD patients (60 female and 61 male), diagnosed via magnetic resonance imaging (MRI) and aged 18-65 years, were enrolled. Participants were randomly allocated to receive SPS tablets (n = 60; three tablets per dose, twice daily) or placebo (n = 61) for 24 weeks. Each SPS tablet contained approximately 23.0 mg of silybin, 11.4 mg of puerarin, and 10.9 mg of salvianolic acid. There were no differences in appearance, taste and odour between the SPS tablets and placebo manufactured by BYHEALTH Co., LTD (Guangzhou, China). The primary endpoints were changes in the liver fat content (LFC) and steatosis grade from baseline to 24 weeks. Secondary outcomes included changes in biomarkers/scores of liver fibrosis and steatosis, oxidative stress, inflammatory cytokines, alcohol metabolism, and glucose metabolism. RESULTS: A total of 112 participants completed the research. The intention-to-treat results showed a trend toward reduction in both absolute LFC (-0.52%) and percentage of LFC (-4.57%) in the SPS group compared to the placebo group after 24 weeks, but these changes didn't reach statistical significance (p > 0.05). The SPS intervention (vs. placebo) significantly decreased hypersensitive C-reactive protein level (-6.76%) and increased aldehyde dehydrogenase activity (+18.1%) at 24 weeks post-intervention (all p < 0.05). Per-protocol analysis further supported these effects. This trial is registered at Clinical Trials.gov (NCT05076058). CONCLUSION: SPS supplementation may have potential benefits in improving NAFLD, but further larger-scale trials are necessary to confirm these findings.

Comparative Effects of Umbilical Cord Mesenchymal Stem Cell Treatment via Different Routes on Lipopolysaccharide-Induced Acute Lung Injury.

BACKGROUND: Although umbilical cord mesenchymal stem cell (UCMSC) infusion has been proposed as a promising strategy for the treatment of acute lung injury (ALI), the parameters of UCMSC transplantation, such as infusion routes and doses, need to be further optimized. METHODS: In this study, we compared the therapeutic effects of UCMSCs transplanted via intravenous injection and intratracheal instillation on lipopolysaccharide-induced ALI using a rat model. Following transplantation, levels of inflammatory factors in serum; neutrophils, total white blood cells, and lymphocytes in bronchoalveolar lavage fluid (BALF); and lung damage levels were analyzed. RESULTS: The results indicated that UCMSCs administered via both intravenous and intratracheal routes were effective in alleviating ALI, as determined by analyses of arterial blood gas, lung histopathology, BALF contents, and levels of inflammatory factors. Comparatively, the intratracheal instillation of UCMSCs was found to result in lower levels of lymphocytes and total proteins in BALF, whereas greater reductions in the serum levels of tumor necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß) were detected in rats receiving intravenously injected stem cells. CONCLUSIONS: Our findings in this study provide convincing evidence to indicate the efficacy of UCMSC therapy in the treatment of ALI mediated via different delivery routes, thereby providing a reliable theoretical basis for further clinical studies. Moreover, these findings imply that the effects obtained using the two assessed delivery routes for UCMSC transplantation are mediated via different mechanisms, which could be attributable to different cellular or molecular targets.

Effect of Carbon Fiber and Potassium Titanate Whisker on the Mechanical and Impact Tribological Properties of Fe-Based Impregnated Diamond Bit Matrix.

Various contents of carbon fibers (CFs) and potassium titanate whiskers (PTWs) were added to an Fe-based impregnated diamond bit (IDB) matrix to enhance its adaptability to percussive-rotary drilling. A series of mechanical tests were conducted successively to find the effects of the reinforcing materials on the properties of the Fe-based IDB samples. Then, the fracture surfaces of the samples were analyzed via scanning electron microscopy (SEM) and energy-dispersive spectroscopy, and the worn surfaces and abrasive debris of the samples were analyzed using a laser scanning confocal microscope and SEM. The results show that both the CF and PTW can effectively improve the hardness and bending strength of an Fe-based IDB matrix, and those parameters reached their maximum values at the additive amount of 1 wt%. However, the CF had a better enhancement effect than the PTW. Furthermore, the CF improved the impact wear resistance of the IDB matrix, with a minimum wear rate of 2.38 g/min at the additive amount of 2 wt%. However, the PTW continuously weakened the impact wear resistance of the IDB matrix with increases in its content. Moreover, the morphologies of the worn surfaces indicated that the minimum roughness of the CF-reinforced IDB matrix decreased significantly to as low as 4.91 µm, which was 46.16% lower than that without CF, whereas the minimum roughness of the PTW-reinforced samples decreased by 11.31%. Meanwhile, the abrasive debris of the CF-reinforced samples was more uniform and continuous compared to that of the PTW-reinforced samples. Overall, the appropriate addition of CF or PTWs can enhance the mechanical properties of Fe-based IDB matrices, which can be used on different formations based on their impact wear resistance.

Perceptions, Attitudes, Beliefs, and Practices Toward Traditional Chinese Medicine Therapies for Herpes Zoster: A Cross-Sectional Study of Healthcare Professionals.

Introduction: Traditional Chinese medicine (TCM) holds great potential in promoting healing and relieving pain for herpes zoster (HZ) treatments. Evidence showed that both healthcare professionals' (HCPs) belief and knowledge influence their attitudes, which result in their expression and direct behavior. However, little is known in this area regarding TCM treatments for HZ. This study aimed to understand the HCPs' perceptions, attitudes, beliefs, and practices toward TCM and its services for HZ. Methods: During July 2021 and October 2022, a cross-sectional study of HCPs querying demographics, perceptions, attitudes, beliefs, and practices toward TCM and TCM services for HZ was conducted. The frequency and percentage or mean and standard deviation were used to present categorical data and continuous data, respectively. A Chi-square analysis compared nurses' and doctors' views on TCM treatments for HZ. Results: Out of 306 eligible respondents, 66.0% used TCM content in clinical practice less than 40% of the time. Respondents reported that there were three main advantages of TCM for HZ, including better crusting and healing, fewer side effects, and mitigating complications. A total of 41.3% (81/196) of the respondents who had cared for/treated HZ patients applied TCM treatments. The three factors most associated with referrals/providing TCM to patients were postherpetic neuralgia, early erythema or papules, and acute pain. Compared to nurses, doctors showed more endorsement of the efficacy and cost-effectiveness of TCM treatments for HZ patients. Conclusion: The study found that most healthcare professionals in HZ had a favorable view of TCM, but lacked practical experience administering it to patients. Programs should be developed to provide evidence-based TCM treatments and encourage combining TCM with Western medicine for better patient care.

Mobility and environmental impact of cadmium (Cd) during weathering of carbonaceous black shales in western Hunan, China.

Carbonaceous black shale generally contains high concentration of Cd, with weathering leading to Cd release to environment. In this study, the mobility of Cd during weathering was quantified using geochemical assessment on black shale from western Hunan, China. Results suggested that Cd was heterogeneously distributed in shale profiles with concentrations ranging from 0.16 to 109.9 (mg/kg). Cd distribution was heterogeneous resulting from the parent shale inheritance and the mobility of Cd during weathering. Black shales weathered to a moderate degree with Cd mobility characterized by both enrichment in and release from weathered shales. Cd enrichment in weathered shales resulted from the re-enrichment of Cd in secondary minerals formed during the initial stage of carbonate (and phosphorite) dissolution, and the secondary stage of sulfide oxidation. The release of Cd was caused by decomposition of the secondary Cd-bearing minerals. Cadmium was extensively released during pedogenesis, and Cd release mass flux was estimated to range from 1.26 to 9.50 (g/m2) with a mean of 6.60 g/m2. Thus, black shale weathering may lead to the releasing of large amount of Cd resulting in Cd contamination to local environments.

Monodispersed and Monofunctionalized DNA-Caged Au Nano-Clusters with Enhanced Optical Properties for STED Imaging.

The performance of Stimulated Emission Depletion (STED) microscopy depends critically on the fluorescent probe. Ultrasmall Au nanoclusters (Au NCs) exhibit large Stokes shift, and good stimulated emission response, which are potentially useful for STED imaging. However, Au NCs are polydispersed in size, sensitive to the surrounding environment, and difficult to control surface functional group stoichiometry, which results in reduced density and high heterogeneity in the labeling of biological structures. Here, this limitation is overcome by developing a method to encapsulate ultrasmall Au NCs with DNA cages, which yielded monodispersed, and monofunctionalized Au NCs that are long-term stable. Moreover, the DNA-caging also greatly improved the fluorescence quantum yield and photostability of Au NCs. In STED imaging, the DNA-caged Au NCs yielded ≈40 nm spatial resolution and are able to resolve microtubule line shapes with good labeling density and homogeneity. In contrast, without caging, the Au NCs-DNA conjugates only achieved ≈55 nm resolution and yielded spotted, poorly resolved microtubule structures, due to the presence of aggregates. Overall, a method is developed to achieve precise surface functionalization and greatly improve the monodispersity, stability, as well as optical properties of Au NCs, providing a promising class of fluorescent probes for STED imaging.

Ultrasensitive Point-of-Care Detection of Protein Markers Using an Aptamer-CRISPR/Cas12a-Regulated Liquid Crystal Sensor (ALICS).

Despite extensive efforts, point-of-care testing (POCT) of protein markers with high sensitivity and specificity and at a low cost remains challenging. In this work, we developed an aptamer-CRISPR/Cas12a-regulated liquid crystal sensor (ALICS), which achieved ultrasensitive protein detection using a smartphone-coupled portable device. Specifically, a DNA probe that contained an aptamer sequence for the protein target and an activation sequence for the Cas12a-crRNA complex was prefixed on a substrate and was released in the presence of target. The activation sequence of the DNA probe then bound to the Cas12a-crRNA complex to activate the collateral cleavage reaction, producing a bright-to-dark optical change in a DNA-functionalized liquid crystal interface. The optical image was captured by a smartphone for quantification of the target concentration. For the two model proteins, SARS-CoV-2 nucleocapsid protein (N protein) and carcino-embryonic antigen (CEA), ALICS achieved detection limits of 0.4 and 20 pg/mL, respectively, which are higher than the typical sensitivity of the SARS-CoV-2 test and the clinical CEA test. In the clinical sample tests, ALICS also exhibited superior performances compared to those of the commercial ELISA and lateral flow test kits. Overall, ALICS represents an ultrasensitive and cost-effective platform for POCT, showing a great potential for pathogen detection and disease monitoring under resource-limited conditions.

Hyperbranched Polymer Dots with Strong Absorption and High Fluorescence Quantum Yield for In Vivo NIR-II Imaging.

In order to improve the fluorescence quantum yield (QY) of NIR-II-emitting nanoparticles, D-A-D fluorophores are typically linked to intramolecular rotatable units to reduce aggregation-induced quenching. However, incorporating such units often leads to a twisted molecular backbone, which affects the coupling within the D-A-D unit and, as a result, lowers the absorption. Here, we overcome this limitation by cross-linking the NIR-II fluorophores to form a 2D polymer network, which simultaneously achieves a high QY by well-controlled fluorophore separation and strong absorption by restricting intramolecular distortion. Using the strategy, we developed polymer dots with the highest NIR-II single-particle brightness among reported D-A-D-based nanoparticles and applied them for imaging of hindlimb vasculatures and tumors as well as fluorescence-guided tumor resection. The high brightness of the polymer dots offered exceptional image quality and excellent surgical results, showing a promising performance for these applications.

Distribution, source and contamination of rare earth elements in sediments from lower reaches of the Xiangjiang River, China.

Rare earth elements (REEs) are emerging micropollutants in aquatic environments. In this study, concentrations of REEs and major elements, and mineralogical compositions of sediments from lower reaches of the Xiangjiang River (China) were analyzed using ICP-MS technique. The results suggested that sediments were characterized by terrigenous compositions TiO2, SiO2, Al2O3, K2O, Na2O and P2O, and contained high concentrations of REEs with mean total REE concentrations (∑REE) of 318.7 mg/kg. REEs were moderately enriched in upper river sediments, and slightly or less enriched in downriver sediments. The normalized REE distribution pattern for sediments was characterized by flat shalelike and Eu depleted V-shape REE patterns, which indicated REEs in sediments were lithologically contributed from sedimentary rocks and granites distributed in the watershed respectively. REEs in sediments were hosted mainly in Fe-Mn oxides, and sulfide and organic matters that were characterized by middle REEs (MREE) enrichments relative to light REEs (LREE) and heavy REEs (HREE), and the distribution and differentiation of REEs in sediments were controlled by clays, Fe-Mn oxides, organic matters and finer grains; and also by accessory minerals (e.g., zircon) from granite. The distribution features of REEs in sediments and BCR extraction results suggested that the sediment REE enrichment resulted from additional REE input from anthropogenic sources, including those in discharges from sulfide-ore smelting industries at Zhuzhou city and from phosphate fertilizer plants at Xiangtan city along the river. Thus, sediments were contaminated with REEs in moderate degree in upper river area, and REE contamination was then formed by superimposing anthropogenic REEs on lithological residues. Finally, concentrations of Ce > 100 mg/kg, Gd > 8.12 mg/kg, ∑REE >274.9 mg/kg, ∑LREE >252.3 mg/kg and ∑HREE >28.8 mg/kg here were recommended as the REE contamination levels that represented as REE indices for identifying and rating REE contamination in this mining impacted river.

The Effects of Covert Narcissism on Chinese College Students Cyberbullying: The Mediation of Hostile Attribution Bias and the Moderation of Self-Control.

Purpose: The prevalence of cyberbullying has increased along with the growth of social media, which has brought about many adverse effects on individual development. The current study aimed to explore the connection between covert narcissism and cyberbullying and to test the roles of hostile attribution bias and self-control in the relationship between covert narcissism and cyberbullying. Materials and Methods: A total of 672 Chinese college students filled up questionnaires measuring covert narcissism, cyberbullying, hostile attribution bias, and self-control. Results: The results indicated that covert narcissism positively and significantly predicted cyberbullying. Hostile attribution bias partially mediated the relationship between covert narcissism and cyberbullying. Additionally, self-control moderated the relationship between covert narcissism and cyberbullying. Specifically, the positive predictive effect of covert narcissism on cyberbullying gradually weakened as self-control improved. Conclusion: This study explored the underlying mechanism of cyberbullying and found that covert narcissism could affect cyberbullying through hostile attribution bias. Self-control moderated the relationship between covert narcissism and cyberbullying. The results have significant implications for the intervention and prevention of cyberbullying and additional evidence for the relationship between covert narcissism and cyberbullying.

Effect of the ketogenic diet on gut microbiome composition and metabolomics in polycystic ovarian syndrome rats induced by letrozole and a high-fat diet.

OBJECTIVES: The ketogenic diet (KD) is recommended to improve polycystic ovary syndrome (PCOS); however, its mechanisms of action are unclear. We aimed to study the effects and mechanisms of action of the KD on the gut microbiome and metabolites in PCOS rats and determine whether the sex hormone regulatory effects are related to modulations of the gut microbiota and metabolites. METHODS: PCOS was induced with a high-fat diet and letrozole in the rats. A KD was fed to rats for 8 wk, serum samples were collected for biochemical analysis, and the rats' fecal samples were subjected to 16S ribosomal RNA sequencing and metabolomic analysis. RESULTS: Feeding with a KD for 8 wk suppressed body weight gain, decreased luteinizing hormone and androgen levels, and improved insulin levels. Furthermore, the KD reversed the dysregulation of the gut microbiota in PCOS rats by adjusting the ratio of firmicutes and bacteroidetes. Also, the KD was involved in hormonal metabolic pathways by reducing the levels of some metabolites (such as testosterone and 7α-hydroxytestosterone) that are closely related to gut microbes. CONCLUSIONS: The KD improved the clinical phenotype and insulin resistance in PCOS rats and altered the composition of the gut microbiome and metabolites, which were associated with androgen metabolism, representing a potential mechanism for mediating the effects of the KD on sex hormone metabolism in PCOS. However, our study found contradictory effects of KD on the gut microbiome in PCOS, which need further research.

The RNA-binding protein LRPPRC promotes resistance to CDK4/6 inhibition in lung cancer.

Kinase inhibitors against Cyclin Dependent Kinase 4 and 6 (CDK4/6i) are promising cancer therapeutic drugs. However, their effects are limited by primary or acquired resistance in virtually all tumor types. Here, we demonstrate that Leucine Rich Pentatricopeptide Repeat Containing (LRPPRC) controls CDK4/6i response in lung cancer by forming a feedback loop with CDK6. LRPPRC binds to CDK6-mRNA, increasing the stability and expression of CDK6. CDK6 and its downstream E2F Transcription Factor 1 (E2F1), bind to the LRPPRC promoter and elevate LRPPRC transcription. The activation of the LRPPRC-CDK6 loop facilitates cell cycle G1/S transition, oxidative phosphorylation, and cancer stem cell generation. Gossypol acetate (GAA), a gynecological medicine that has been repurposed as a degrader of LRPPRC, enhances the CDK4/6i sensitivity in vitro and in vivo. Our study reveals a mechanism responsible for CDK4/6i resistance and provides an enlightening approach to investigating the combinations of CDK4/6 and LRPPRC inhibitors in cancer therapy.

Investigation of the Mechanisms and Experimental Verification of Yulin Formula in the Treatment of Diminished Ovarian Reserve via Network Pharmacology.

Purpose: The aim of this study is to examine, using network pharmacology analysis and experimental validation, the pharmacological processes by which Yulin Formula (YLF) reduces cyclophosphamide-induced diminished ovarian reserve (DOR). Methods: First, information about the active components, associated targets, and related genes of YLF and DOR was gathered from open-access databases. The primary targets and pathways of YLF to reduce DOR were predicted using studies of functional enrichment from the Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and Protein-Protein Interaction (PPI) networks. Second, we built a cyclophosphamide-induced diminished ovarian reserve (DOR) rat model to verify the primary target proteins implicated in the predicted signaling pathway to explore the mechanism of improve ovarian function of YLF. Results: 98 targets met the targets of the 82 active ingredients in YLF and DOR after searching the intersection of the active ingredient targets and DOR targets. Fourteen targets, including AKT and Caspase-3 among others, were hub targets, according to the PPI network study. The PI3K/AKT pathway was revealed to be enriched by numerous targets by the GO and KEGG enrichment studies, and it was used as a target for in vivo validation. Animal studies showed that YLF administration not only reduced the number of atretic follicles, the proportion of TUNEL-positive ovarian cells, the rate of apoptosis of granulosa cells (GCs) and the proportion of abnormal mitochondria in DOR rats, but also reversed the high expression of Caspase-3, Caspase-9, BAX, cytochrome C, PI3K and P-AKT, improving the ovarian reserve in cyclophosphamide (CTX)-induced DOR rats. Conclusion: Our research results predicted the active ingredients and potential targets of YLF-interfering DOR by an integrated network pharmacology approach, and experimentally validated some key target proteins participated in the predicted signaling pathway. A more comprehensive understanding of the pharmacological mechanism of YLF for DOR treatment was obtained.

Mitochondrial Protease Targeting Chimeras for Mitochondrial Matrix Protein Degradation.

Targeted protein degradation (TPD) is an emerging technique for protein regulation. Currently, all TPD developed in eukaryotic cells relies on either ubiquitin-proteasome or lysosomal systems, thus are powerless against target proteins in membrane organelles lacking proteasomes and lysosomes, such as mitochondria. Here, we developed a mitochondrial protease targeting chimera (MtPTAC) to address this issue. MtPTAC is a bifunctional small molecule that can bind to mitochondrial caseinolytic protease P (ClpP) at one end and target protein at the other. Mechanistically, MtPTAC activates the hydrolase activity of ClpP while simultaneously bringing target proteins into proximity with ClpP. Taking mitochondrial RNA polymerase (POLRMT) as a model protein, we have demonstrated the powerful proteolytic ability and antitumor application prospects of MtPTAC, both in vivo and in vitro. This is the first modularly designed TPD that can specifically hydrolyze target proteins inside mitochondria.

RuCu Nanosheets with Ultrahigh Nanozyme Activity for Chemodynamic Therapy.

Nanoenzymes have been widely explored for chemodynamic therapy (CDT) in cancer treatment. However, poor catalytic efficiency of nanoenzymes, especially in the tumor microenvironment with insufficient H2 O2 and mild acidity, limits the effect of CDT. Herein, a new ultrathin RuCu nanosheet (NS) based nanoenzyme which has a large specific surface area and abundant channels and defects is developed. The RuCu NSs show superb catalytic efficiency for the oxidation of peroxidase substrate H2 O2 at a wide range of pH and their catalytic efficiency (kcat /Km = 177.2 m-1  s-1 ) is about 14.9 times higher than that of the single-atom catalyst FeN3 P. Besides being an efficient nanozyme as peroxidase, the RuCu NSs possess other two enzyme activities, not only disproportionating superoxide anion to produce H2 O2 but also consuming glutathione to keep a high concentration of H2 O2 in the tumor microenvironment for Fenton reaction. With these advantages, the RuCu NSs exhibit good performance to kill cancer cells and inhibit tumor growth in mice, demonstrating a promising potential as new CDT reagent.

Engineering Circular Aptamer Assemblies with Tunable Selectivity to Cell Membrane Antigens In Vitro and In Vivo.

The strategy of enhancing molecular recognition by improving the binding affinity of drug molecules against targets has generated a lot of successful therapeutic applications. However, one critical consequence of such affinity improvement, generally called "on-target, off-tumor" toxicity, emerged as a major obstacle limiting their clinical usage. Herein, we provide a modular assembly strategy that affords affinity-tunable DNA nanostructures allowing for immobilizing multiple aptamers that bind to the example antigen of EpCAM with different affinities. We develop a theoretical model proving that the apparent affinity of aptamer assemblies to target cells varies with antigen density as well as aptamer valency. More importantly, we demonstrate experimentally that the theoretical model can be used to predict the least valency required for discrimination between EpCAMhigh and EpCAMlow cells in vitro and in vivo. We believe that our strategy will have broad applications in an engineering nucleic acid-based delivery platform for targeted and cell therapy.

Reprogramming Hypoxic Tumor-Associated Macrophages by Nanoglycoclusters for Boosted Cancer Immunotherapy.

The tumor-associated macrophages (TAMs) in intratumoral hypoxic regions are key drivers of immune escape. Reprogramming the hypoxic TAMs to antitumor phenotype holds great therapeutic benefits but remains challenging for current drugs. Here, an in situ activated nanoglycocluster is reported to realize effective tumor penetration and potent repolarization of hypoxic TAMs. Triggered by the hypoxia-upregulated matrix metalloproteinase-2 (MMP-2), the nanoglycocluster is self-assembled from the administered mannose-containing precursor glycopeptides and presents densely-arrayed mannoses to multivalently engage with mannose receptors on M2-like TAMs for efficient phenotype switch. By virtue of the high diffusivity of precursor glycopeptides due to their low molecular mass and weak affinity with TAMs in perivascular regions, the nanoglycoclusters are capable of substantially accumulating in hypoxic areas to strongly interact with local TAMs. This enables the efficient repolarization of overall TAMs with a higher rate than the small-molecule drug R848 and CD40 antibody, and beneficial therapeutic effects in mouse tumor models especially when combining with PD-1 antibody. This on-demand activated immunoagent is endowed with tumor-penetrating properties and inspires the design of diverse intelligent nanomedicines for hypoxia-related cancer immunotherapy.

Single-Molecule Imaging Reveals Differential AT1R Stoichiometry Change in Biased Signaling.

G protein-coupled receptors (GPCRs) represent promising therapeutic targets due to their involvement in numerous physiological processes mediated by downstream G protein- and ß-arrestin-mediated signal transduction cascades. Although the precise control of GPCR signaling pathways is therapeutically valuable, the molecular details for governing biased GPCR signaling remain elusive. The Angiotensin II type 1 receptor (AT1R), a prototypical class A GPCR with profound implications for cardiovascular functions, has become a focal point for biased ligand-based clinical interventions. Herein, we used single-molecule live-cell imaging techniques to evaluate the changes in stoichiometry and dynamics of AT1R with distinct biased ligand stimulations in real time. It was revealed that AT1R existed predominantly in monomers and dimers and underwent oligomerization upon ligand stimulation. Notably, ß-arrestin-biased ligands induced the formation of higher-order aggregates, resulting in a slower diffusion profile for AT1R compared to G protein-biased ligands. Furthermore, we demonstrated that the augmented aggregation of AT1R, triggered by activation from each biased ligand, was completely abrogated in ß-arrestin knockout cells. These findings furnish novel insights into the intricate relationship between GPCR aggregation states and biased signaling, underscoring the pivotal role of molecular behaviors in guiding the development of selective therapeutic agents.

Bispecific Aptamer-Based Recognition-then-Conjugation Strategy for PD1/PDL1 Axis Blockade and Enhanced Immunotherapy.

Cytotoxic T cells initiate antitumor effects mainly through direct interactions with tumor cells. As a counter to this, tumor cells can put the brakes on such T-cell activity via specific linkage between programmed death ligand 1 (PDL1) and its receptor programmed cell death protein 1 (PD1). Bispecific inhibitors that enabled synchronous blockade of PD1 and PDL1, thereby releasing the brakes on T-cell antitumor activity, should significantly improve the efficacy of immune checkpoint blockade (ICB) therapy. In this work, we identified a DNA aptamer, Ap3, that could specifically recognize PDL1 on tumor cells and competed with the binding of PD1. By integrating Ap3 with an anti-PD1 aptamer, the bispecific aptamer Ap3-7c was constructed, and it showed promise for improving the T-cell immune response. We further designed a dibenzocyclooctyne (DBCO)-labeled bispecific aptamer, D-Ap3-7c, allowing covalent conjugation of aptamers onto PD1 and PDL1 after specific cell recognition. Our in vivo studies showed that this recognition-then-conjugation strategy could induce a potent immunological effect against tumors. This work is expected to provide clues for antitumor immunotherapy.