High-throughput screening identifies ibuprofen as an sEV PD-L1 inhibitor for synergistic cancer immunotherapy.

Programmed death-ligand 1 (PD-L1) on tumor-derived small extracellular vesicles (sEVs) limits therapeutic effectiveness by interacting with the PD-1 receptor on host immune cells. Targeting the secretion of sEV PD-L1 has emerged as a promising strategy to enhance immunotherapy. However, the lack of small-molecule inhibitors poses a challenge for clinical translation. In this study, we developed a target and phenotype dual-driven high-throughput screening strategy that combined virtual screening with nanoflow-based experimental verification. We identified ibuprofen (IBP) as a novel inhibitor that effectively targeted sEV PD-L1 secretion. IBP disrupted the biogenesis and secretion of PD-L1+ sEVs in tumor cells by physically interacting with a critical regulator of sEV biogenesis, hepatocyte growth factor-regulated tyrosine kinase substrate. Notably, the mechanism of action of IBP is distinct from its commonly known targets, cyclooxygenases. Administration of IBP stimulated antitumor immunity and enhanced the efficacy of anti-PD-1 therapy in melanoma and oral squamous cell carcinoma mouse models. To address potential adverse effects, we further developed an IBP gel for topical application, which demonstrated remarkable therapeutic efficacy when combined with anti-PD-1 treatment. The discovery of this specific small inhibitor provides a promising avenue for establishing durable, systemic antitumor immunity.

Temporal Micro-Action Localization for Videofluoroscopic Swallowing Study.

Videofluoroscopic swallowing study (VFSS) visualizes the swallowing movement by using X-ray fluoroscopy, which is the most widely used method for dysphagia examination. To better facilitate swallowing assessment, the temporal parameter is one of the most important indicators. However, most information of that acquire is hand-crafted and elaborated, which is time-consuming and difficult to ensure objectivity and accuracy. In this article, we propose to formulate this task as a temporal action localization task and solve it using deep neural networks. However, the action of VFSS has the following characteristics such as small motion targets, small action amplitudes, large sample variances, short duration, and variations in duration. Furthermore, all existing methods often rely on daily behaviors, which makes locating and recognizing micro-actions more challenging. To address the above issues, we first collect and annotate the VFSS micro-action dataset, which includes 847 VFSS data from 71 subjects, due to the lack of benchmarks. We then introduce a coarse-to-fine mechanism to handle the short and repeated nature of micro-actions, which can significantly enhancing micro-action localization accuracy. Moreover, we propose a Variable-Size Window Generator method, which improves the model's characterization performance and addresses the issue of different action timings, leading to further improvements in localization accuracy. The results of our experiments demonstrate the superiority of our method, with significantly improved performance (46.10% vs. 37.70%).

RNA aptamers with specific binding affinity to CD40 (CD40Apt) represents a promising antagonist of the CD40-CD40L signaling for thyroid-associated ophthalmopathy (TAO) treatment in mouse.

Thyroid-associated ophthalmopathy (TAO) is the most common autoimmune inflammatory diseases of the orbit. The CD40-CD40L pathway has been regarded as a potential molecular mechanism contributing to the development and progression of TAO, and RNA aptamers with specific binding affinity to CD40 (CD40Apt) represents a promising inhibitor of the CD40-CD40L signaling in TAO treatment. In this study, CD40Apt was confirmed to specifically recognize mouse CD40-positive ortibtal fibroblast. Mouse orbital fibroblasts were isolated from TAO mice model orbital tissues and validated. In TGF-ß-induced orbital fibroblast activation model in vitro, CD40Apt administration inhibited TGF-ß-induced cell viability, decreased TGF-ß-induced α-SMA, Collagen I, Timp-1, and vimentin levels, and suppressed TGF-ß-induced phosphorylation of Erk, p38, JNK, and NF-κB. In TAO mice model in vivo, CD40Apt caused no significant differences to the body weight of mice; furthermore, CD40Apt improved the eyelid broadening, ameliorated inflammatory infiltration and the hyperplasia in orbital muscle and adipose tissues in model mice. Concerning orbital fibroblast activation, CD40Apt reduced the levels of CD40, collagen I, TGF-ß, and α-SMA in orbital muscle and adipose tissues of model mice. Finally, CD40Apt administration significantly suppressed Erk, p38, JNK, and NF-κB phosphorylation. In conclusion, CD40Apt, specifically binds to CD40 proteins in their natural state on the cell surface with high affinity, could suppress mouse orbital fibroblast activation, therefore improving TAO in mice model through the CD40 and downstream signaling pathways. CD40Apt represents a promising antagonist of the CD40-CD40L signaling for TAO treatment.

A cause-effect relationship between Graves' disease and the gut microbiome contributes to the thyroid-gut axis: A bidirectional two-sample Mendelian randomization study.

Background: An association between Graves' disease (GD) and the gut microbiome has been identified, but the causal effect between them remains unclear. Methods: Bidirectional two-sample Mendelian randomization (MR) analysis was used to detect the causal effect between GD and the gut microbiome. Gut microbiome data were derived from samples from a range of different ethnicities (18,340 samples) and data on GD were obtained from samples of Asian ethnicity (212,453 samples). Single nucleotide polymorphisms (SNPs) were selected as instrumental variables according to different criteria. They were used to evaluate the causal effect between exposures and outcomes through inverse-variance weighting (IVW), weighted median, weighted mode, MR-Egger, and simple mode methods. F-statistics and sensitivity analyses were performed to evaluate bias and reliability. Results: In total, 1,560 instrumental variables were extracted from the gut microbiome data (p< 1 × 105). The classes Deltaproteobacteria [odds ratio (OR) = 3.603] and Mollicutes, as well as the genera Ruminococcus torques group, Oxalobacter, and Ruminococcaceae UCG 011 were identified as risk factors for GD. The family Peptococcaceae and the genus Anaerostipes (OR = 0.489) were protective factors for GD. In addition, 13 instrumental variables were extracted from GD (p< 1 × 10-8), causing one family and eight genera to be regulated. The genus Clostridium innocuum group (p = 0.024, OR = 0.918) and Anaerofilum (p = 0.049, OR = 1.584) had the greatest probability of being regulated. Significant bias, heterogeneity, and horizontal pleiotropy were not detected. Conclusion: A causal effect relationship exists between GD and the gut microbiome, demonstrating regulatory activity and interactions, and thus providing evidence supporting the involvement of a thyroid-gut axis.

Effects of biochar on anaerobic treatment systems: Some perspectives.

Many anaerobic activities involve carbon, nitrogen, iron, and sulfur cycles. As a well-developed porous material with abundant functional groups, pyrolytic biochar has been widely researched in efforts to promote microbial activities. However, the lack of consensus on the biochar mechanism has limited its practical application. This review summarizes the effects of different pyrolysis temperatures, particle sizes, and dosages of biochar on microbial activities and community in Fe(III) reduction, anaerobic digestion, nitrogen removal, and sulfate reduction systems. It was found that biochar could promote anaerobic activities by stimulating electron transfer, alleviating toxicity, and providing suitable habitats for microbes. However, it inhibits microbial activities by releasing heavy metal ions or persistent free radicals and adsorbing signaling molecules. Finding a balance between the promotion and inhibition of biochar is therefore essential. This review provides valuable perspectives on how to achieve efficient and stable use of biochar in anaerobic systems.

The miR-103a-3p/TGFBR3 axis regulates TGF-ß-induced orbital fibroblast activation and fibrosis in thyroid-eye disease.

Molecular pathways that contribute to orbital fibroblast activation during thyroid-eye disease (TED) may promote TED progression. Non-coding RNAs, especially miRNAs, play a critical role in the pathogenesis of TED. In the present study, miR-103a-3p was dramatically upregulated and TGFBR3 was downregulated within TED orbital tissue samples and TGF-ß-stimulated TED orbital fibroblasts. miR-103a-3p inhibition in TGF-ß-stimulated TED orbital fibroblasts partially abolished TGF-ß-induced fibrotic alterations, as manifested by the impaired fibroblast cell viability and decreased vimentin and fibronectin levels. miR-103a-3p directly targeted TGFBR3 in TED orbital samples and TGF-ß-stimulated TED orbital fibroblasts. In TGF-ß-stimulated TED orbital fibroblasts, TGFBR3 overexpression inhibited fibroblast cell viability and decreased vimentin and fibronectin levels. TGFBR3 overexpression partially attenuated the inhibitory effects of miR-103a-3p overexpression on TGFBR3 expression and the promotive effects of miR-103a-3p overexpression on TGF-ß-induced fibrotic alterations. Under TGF-ß stimulation, miR-103a-3p overexpression significantly promoted, whereas TGFBR3 overexpression inhibited the phosphorylation of Erk1/2, JNK, Smad2, and Smad3. TGFBR3 overexpression also partially abolished the effects of miR-103a-3p overexpression on Erk1/2, JNK, Smad2, and Smad3 phosphorylation. In conclusion, the miR-103a-3p/TGFBR3 axis regulated TGF-ß-induced TED orbital fibroblast activation and fibrosis in TED, with the possible involvement of the Erk/JNK and TGF-ß/Smad signaling pathways.

HRS Regulates Small Extracellular Vesicle PD-L1 Secretion and Is Associated with Anti-PD-1 Treatment Efficacy.

PD-L1 localized to immunosuppressive small extracellular vesicles (sEV PD-L1) contributes to tumor progression and is associated with resistance to immune-checkpoint blockade (ICB) therapy. Here, by establishing a screening strategy with a combination of tissue microarray (TMA), IHC staining, and measurement of circulating sEV PD-L1, we found that the endosomal sorting complex required for transport (ESCRT) member protein hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was the key regulator of circulating sEV PD-L1 in head and neck squamous cell carcinoma (HNSCC) patients. Increased HRS expression was found in tumor tissues and positively correlated with elevated circulating sEV PD-L1 in patients with HNSCC. The expression of HRS was also negatively correlated to the infiltration of CD8+ T cells. Knockdown of HRS markedly reduced PD-L1 expression in HNSCC cell-derived sEVs, and these sEVs from HRS knockdown cells showed decreased immunosuppressive effects on CD8+ T cells. Knockout of HRS inhibited tumor growth in immunocompetent mice together with PD-1 blockade. Moreover, a higher HRS expression was associated with a lower response rate to anti-PD-1 therapy in patients with HNSCC. In summary, our study reveals HRS, the core component of ESCRT-0, regulates sEV PD-L1 secretion, and is associated with the response to ICB therapy in patients with HNSCC, suggesting HRS is a promising target to improve cancer immunotherapy.

Targeted inhibition of tumor-derived exosomes as a novel therapeutic option for cancer.

Mounting evidence indicates that tumor-derived exosomes (TDEs) play critical roles in tumor development and progression by regulating components in the tumor microenvironment (TME) in an autocrine or paracrine manner. Moreover, due to their delivery of critical molecules that react to chemotherapy and immunotherapy, TDEs also contribute to tumor drug resistance and impede the effective response of antitumor immunotherapy, thereby leading to poor clinical outcomes. There is a pressing need for the inhibition or removal of TDEs to facilitate the treatment and prognosis of cancer patients. Here, in the present review, we systematically overviewed the current strategies for TDE inhibition and clearance, providing novel insights for future tumor interventions in translational medicine. Moreover, existing challenges and potential prospects for TDE-targeted cancer therapy are also discussed to bridge the gaps between progress and promising applications.

The risk factors for Graves' ophthalmopathy.

PURPOSE: This review aimed to provide an overview of current research into the risk factors for Graves' ophthalmopathy (GO). METHODS: To find information about the risk factors for GO, the research database PubMed was searched and relevant articles were obtained to extract information about risk factors. RESULTS: Smoking has been widely accepted as an important risk factor and cigarette smoking cessation has been shown to improve the outcome and decrease the onset of GO. Radioactive iodine on the thyroid may induce hyperthyroidism and increase the occurrence of GO. Selenium deficiency is a risk factor for GO and the supplementation of selenium has been an adjuvant therapy. Decreasing stressful life events (SLE) may help improve GO. Imbalance in intestinal flora is essential to GO, with Yersinia enterocolitica and Escherichia coli both increased in the digestive tract of the individual with GO. In addition, controlling serum cholesterol may help improve GO since adipogenesis is an important pathological change in its pathogenesis. Considering the correlation between Graves' disease and GO, maintaining normal thyroid function hormone level is the first-line therapeutic strategy to prevent progression of GO. An increase in antibodies such as TSHR and IGF-1R is the main predictor of GO. Besides, gender and gene polymorphism are also risk factors towards GO. CONCLUSIONS: Risk factors for GO arise from five sources: physical and chemical environment, social-psychological environment, biological environment, the human organism, and genetic codes. Risk factors within these categories may interact with each other and their mechanisms in promoting the development of GO are complex. Research into risk factors for GO may promote emerging fields related to GO such as control of autoantibodies and intestinal microbiota.

In Situ Membrane Biotinylation Enables the Direct Labeling and Accurate Kinetic Analysis of Small Extracellular Vesicles in Circulation.

Circulating small extracellular vesicles (sEVs) are naturally occurring nanosized membrane vesicles that convey bioactive molecules between cells. Conventionally, to evaluate their behaviors in vivo, circulating sEVs have to be isolated from the bloodstream, then labeled with imaging materials in vitro, and finally injected back into the circulation of animals for subsequent detection. The tedious isolation-labeling-reinfusion procedures might have an undesirable influence on the natural properties of circulating sEVs, thereby changing their behaviors and the detected kinetics in vivo. Herein, we proposed an in situ biotinylation strategy to directly label circulating sEVs with intravenously injected DSPE-PEG-Biotin, aiming to evaluate the in vivo kinetics of circulating sEVs more biofriendly and accurately. Such an analysis strategy is free of isolation-labeling-reinfusion procedures and has no unfavorable influence on the natural behaviors of sEVs. The results showed that the lifetime of generic circulating sEVs in mice was around 3 days. Furthermore, we, for the first time, revealed the distinct in vivo kinetics of circulating sEV subpopulations with different cell sources, among which erythrocyte-derived sEVs showed the longest lifespan. Moreover, compared with circulating sEVs in situ or used as autograft, circulating sEVs used as allograft had the shortest lifetime. In addition, the in situ biotinylation strategy also provides a way for the enrichment of biotinylated circulating sEVs. In summary, this study provides a novel strategy for in situ labeling of circulating sEVs, which would facilitate the accurate characterization of their kinetics in vivo, thereby accelerating their future application as biomarkers and theranositic vectors.

One-step quantification of salivary exosomes based on combined aptamer recognition and quantum dot signal amplification.

As promising fluid biomarkers for non-invasive diagnosis, naturally-occurring exosomes in saliva have attracted a wide interest for their potential application in oral diseases especially oral cancers. However, accurate quantification of salivary exosomes is still challenging due to the current difficulties in simultaneous identification and measurement of these nano-sized vesicles. In this study, we developed a novel fluorescent biosensor for one-step sensitive quantification of salivary exosomes based on magnetic and fluorescent bio-probes (MFBPs). Within the MFBPs, self-assembled DNA concatamers loaded with numerous quantum dots (QDs) were ingeniously tethered to aptamers, which were anchored on the surface of magnetic microspheres (MMs). Efficient recognition and capture of an exosome by the aptamer would simultaneously trigger the release of a DNA concatamer as the detection signal carrier, thereby generating a "one exosome-numerous QDs" amplification effect. As the result, this biosensor allowed one-step quantification with less assay time and achieved a high sensitivity with low limit of detection. Moreover, unique fluorescent properties of QDs and the superparamagnetism of MMs offered a strong anti-interference ability, enabling a robust quantification in complex matrices. Furthermore, this biosensor exhibited a good clinical feasibility with favorable accuracy comparable to nanoscale flow cytometry, and a superiority in label-free analysis and convenient operation. This study provides a novel and general strategy for one-step sensitive quantification of exosomes from body fluids, facilitating the development of exosome-based liquid biopsy for disease diagnosis.