Bioactive chitosan/polydopamine nanospheres coating on carbon fiber towards strengthening epoxy composites.

This paper initially examines the feasibility and effectiveness on interfacial adhesion of composites when grafting nanoparticle-structured polydopamine (PDA) and chitosan around carbon fiber periphery. The resulting interfacial shear strength was maximized as 92.3 MPa, delivering 50.1 % and 15.7-16.2 % gains over those of control fiber and only polydopamine nanospheres (PDANPs) or only chitosan modified fiber composites. Measuring surface morphology and thermal stability of fibers found that abundant PDANPs well adhered with the help of chitosan, highlighting nanoscale size effects and intrinsic adhesiveness of PDA. Under good wettability, rich and dense interfacial interactions (covalent and hydrogen bond, electrostatic interaction, and π conjugation) caused by PDANPs/chitosan coating provides impetus for effective stress transfer. Additionally, the stable "soft-rigid" combination of chitosan and PDANPs adds the efficiency of crack passivation. As such, it is hoped that this work could fully explore the possibility of PDA geometry in interphase engineering of fiber composites.

HRS mediates tumor immune evasion by regulating proteostasis-associated interferon pathway activation.

By sorting receptor tyrosine kinases into endolysosomes, the endosomal sorting complexes required for transport (ESCRTs) are thought to attenuate oncogenic signaling in tumor cells. Paradoxically, ESCRT members are upregulated in tumors. Here, we show that disruption of hepatocyte growth factor-regulated tyrosine kinase substrate (HRS), a pivotal ESCRT component, inhibited tumor growth by promoting CD8+ T cell infiltration in melanoma and colon cancer mouse models. HRS ablation led to misfolded protein accumulation and triggered endoplasmic reticulum (ER) stress, resulting in the activation of the type I interferon pathway in an inositol-requiring enzyme-1α (IRE1α)/X-box binding protein 1 (XBP1)-dependent manner. HRS was upregulated in tumor cells with high tumor mutational burden (TMB). HRS expression associates with the response to PD-L1/PD-1 blockade therapy in melanoma patients with high TMB tumors. HRS ablation sensitized anti-PD-1 treatment in mouse melanoma models. Our study shows a mechanism by which tumor cells with high TMB evade immune surveillance and suggests HRS as a promising target to improve immunotherapy.

Simultaneous Detection of Two Extracellular Vesicle Subpopulations in Saliva Assisting Tumor T Staging of Oral Squamous Cell Carcinoma.

Extracellular vesicles (EVs), acting as important mediators of intercellular communication, play an essential role in physiological processes, which have unique potential in the medical field. However, the heterogeneity of EVs limits their development for disease diagnosis and therapy, making the EV subpopulation analysis extremely valuable. In this article, a simple microfluidic approach was presented for the on-chip specific isolation and detection of two phenotypes of EVs (Annexin V+ EGFR+ EVs and Annexin V- EGFR+ EVs) based on different biomolecule-modified magnetic nanospheres and a fluorescence labeling technique. Combined with the control of the magnetic field in the microzone and fluid flow, it was easy to form two separate functional regions in the chip to capture different EV subpopulations. This method was successfully applied to the tests of clinical saliva samples in 75 oral squamous cell carcinoma (OSCC) patients and 10 healthy people. The results showed that the total level of EGFR+ EVs was much higher in OSCC patients that in healthy people. Meantime, the ratio of Annexin V+ EGFR+ EVs to Annexin V- EGFR+ EVs was found to be negatively correlated with tumor T stage of OSCC patients with a statistical difference, which suggested the ratio as a clinical index for monitoring the progression of OSCC in real time based on a noninvasive method. The approach provided a novel idea for evaluating the tumor T stage of OSCC and a powerful tool for clinical application.

ALIX promotes cell migration and invasion of head and neck squamous cell carcinoma by regulating the expression of MMP9, MMP14, VEGF-C.

OBJECTIVE: The poor survival rate of head and neck squamous cell carcinoma (HNSCC), one of the most prevalent human cancer, is attributed to frequent locoregional recurrence and lymph node metastases. Though it is reported that the expression of ALG-2 interacting protein X (ALIX) closely correlates with the progression of various tumors, its role in HNSCC remains unclear. The present study aims to investigate the role of ALIX in the development of HNSCC. DESIGN: With immunohistochemical staining, the expression levels of ALIX and series of related functional proteins were compared in normal mucosal (n = 18), HNSCC tissues (n = 54), and metastatic lymph nodes (n = 11). Further, the correlation analysis was performed among the proteins detected. By knocking down ALIX in HNSCC cell lines, the correlation of ALIX with the proteins was verified in vitro. The role of ALIX in proliferation, migration, and invasion of HNSCC cells was further studied by flow cytometry, wounding healing, and transwell assays, respectively. RESULTS: Higher expression level of ALIX was revealed in HNSCC samples, especially in metastatic lymph nodes, than in normal mucosal tissues. Accordingly, increasing levels of MMP9, MMP14, and VEGF-C were also discovered in metastatic lymph nodes and significantly correlated with the expression of ALIX. In vitro assays demonstrated that the knockdown of ALIX reduced both the transcriptional and protein levels of MMP9, MMP14, and VEGF-C, together with suppressed migration and weakened invasion of HNSCC cell lines. CONCLUSIONS: ALIX up-regulated the expression of MMP9, MMP14 and VEGF-C, and promoted migration and invasion of HNSCC cells.

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.

Proteomic and phosphoproteomic landscape of salivary extracellular vesicles to assess OSCC therapeutical outcomes.

Circulating extracellular vesicles (EVs) have emerged as an appealing source for surrogates to evaluate the disease status. Herein, we present a novel proteomic strategy to identify proteins and phosphoproteins from salivary EVs to distinguish oral squamous cell carcinoma (OSCC) patients from healthy individuals and explore the feasibility to evaluate therapeutical outcomes. Bi-functionalized magnetic beads (BiMBs) with Ti (IV) ions and a lipid analog, 1,2-Distearoyl-3-sn-glycerophosphoethanolamine (DSPE) are developed to efficiently isolate EVs from small volume of saliva. In the discovery stage, label-free proteomics and phosphoproteomics quantification showed 315 upregulated proteins and 132 upregulated phosphoproteins in OSCC patients among more than 2500 EV proteins and 1000 EV phosphoproteins, respectively. We further applied targeted proteomics by coupling parallel reaction monitoring with parallel accumulation-serial fragmentation (prm-PASEF) to measure panels of proteins and phosphoproteins from salivary EVs collected before and after surgical resection. A panel of three total proteins and three phosphoproteins, most of which have previously been associated with OSCC and other cancer types, show sensitive response to the therapy in individual patients. Our study presents a novel strategy to the discovery of effective biomarkers for non-invasive assessment of OSCC surgical outcomes with small amount of saliva.

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.

USP8 inhibition reshapes an inflamed tumor microenvironment that potentiates the immunotherapy.

Anti-PD-1/PD-L1 immunotherapy has achieved impressive therapeutic outcomes in patients with multiple cancer types. However, the underlined molecular mechanism(s) for moderate response rate (15-25%) or resistance to PD-1/PD-L1 blockade remains not completely understood. Here, we report that inhibiting the deubiquitinase, USP8, significantly enhances the efficacy of anti-PD-1/PD-L1 immunotherapy through reshaping an inflamed tumor microenvironment (TME). Mechanistically, USP8 inhibition increases PD-L1 protein abundance through elevating the TRAF6-mediated K63-linked ubiquitination of PD-L1 to antagonize K48-linked ubiquitination and degradation of PD-L1. In addition, USP8 inhibition also triggers innate immune response and MHC-I expression largely through activating the NF-κB signaling. Based on these mechanisms, USP8 inhibitor combination with PD-1/PD-L1 blockade significantly activates the infiltrated CD8+ T cells to suppress tumor growth and improves the survival benefit in several murine tumor models. Thus, our study reveals a potential combined therapeutic strategy to utilize a USP8 inhibitor and PD-1/PD-L1 blockade for enhancing anti-tumor efficacy.

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.

[Pollution Characteristics and Assessment of Heavy Metals in Atmospheric Deposition in Core Urban Areas,Chongqing].

Pollution characteristics and risk of heavy metals in atmospheric deposition in core urban areas of Chongqing were investigated for one year from December 2017 to November 2018.Six functional zones:suburb, education area, residential area, commercial area, transportation hub, and industrial-residential area in Chongqing were selected for monthly atmospheric deposition collection. Concentrations of Cd, Cr, Ni, and Pb were analyzed using AAS. The potential ecological risk index and geoaccumulation index were used to evaluate the heavy metals pollution. Results show that the concentrations of Cd, Cr, Ni, and Pb in the atmospheric deposition were 1.59, 72.68, 20.99, and 101.17 mg·kg-1, respectively, and their annual deposition fluxes were 0.39, 8.04, 2.41, and 10.41 mg·(m2·a)-1, respectively. Concentrations of heavy metals in autumn were significantly higher than those in the other three seasons, especially for Cd, and their deposition fluxes in winter were lowest. The potential ecological risk index of Cd was biggest, achieving a very high ecological hazard level, while the ecological risk of Cd and Pb in industrial-residential area was highest, and that of Cr and Ni, respectively, were highest in transportation hub and residential area. The geoaccumulation index indicated that the pollution of Cd was the highest, and that of Cr, Ni, and Pb was very low. The pollution in industrial-residential area and transportation hub was high, while that in the suburb was relatively low.

Anti-Inflammatory Activity of Adenosine 5'-Trisphosphate in Lipopolysaccharide-Stimulated Human Umbilical Vein Endothelial Cells Through Negative Regulation of Toll-Like Receptor MyD88 Signaling.

Activation of TLR4-MyD88-NF-κB signaling by lipopolysaccharide (LPS) evokes a proinflammatory immune response, and plays a pivotal role in initiation and progression of atherosclerosis (AS). ATP (adenosine 5'-trisphosphate), a powerful extracellular signal transduction molecule, functions to regulate immune inflammatory responses depending on the type of P2 receptors and cell lines. In this study, we first performed RT-PCR to detect the mRNA expression of monocyte chemoattractant protein-1 (MCP-1), IL-8, and IL-1ß induced by different concentrations of LPS in human umbilical vein endothelial cells (HUVECs). Protein level of TLR4 signaling including TLR4, myeloid differentiation factor (MyD88), and CD14 induced by LPS (1 µg/mL) at different times (0, 10, 30, 60, 120 min) was analyzed by Western blot. Then, RT-PCR was performed to detect the effect of different concentrations of ATP on mRNA expression of IL-1ß and MCP-1 induced by LPS (1 µg/mL) and the TLR4 signaling pathway. Western blot was performed to detect the effect of low concentrations of ATP on phosphorylation of p65 induced by 1 µg/mL LPS. Finally, we used P2Y receptor blocker Suramin to verify whether the role of ATP on LPS-induced inflammatory cytokine expression was through P2Y receptors. The results showed that LPS upregulated the expression of MCP-1, IL-8, and IL-1ß in a dose-dependent manner accompanied by the activation of TLR4-MyD88 signaling in HUVECs. Only low concentration ATP (1, 10 µM) inhibited LPS-induced mRNA expression of IL-1ß and MCP-1. ATP at low concentrations also downregulated the mRNA expression of TLR4, CD14, and MyD88 and inhibited LPS-induced phosphorylation of p65. Furthermore, Suramin, a nonspecific P2Y receptor antagonist, did not attenuate the inhibition of ATP on LPS-induced IL-1ß and MCP-1 expression. Taking this together, low concentration ATP inhibited LPS-induced inflammation in HUVECs by negatively regulating TLR4-MyD88 signaling, and P2Y receptors were not involved in this process, which might provide new ideas for prevention and treatment of inflammatory diseases such as AS.

Adenosine diphosphate-sensitive P2Y11 receptor inhibits endothelial cell proliferation by induction of cell cycle arrest in the S phase and induces the expression of inflammatory mediators.

Extracellular adenosine diphosphate (ADP) mediates a wide range of physiological effects as an extracellular signaling molecule, including platelet aggregation, vascular tone, cell proliferation, and apoptosis by interacting with plasma membrane P2 receptors. However, the effect of ADP on cell proliferation was contradictory. In this study, we found that ADP significantly inhibited cell proliferation of human umbilical vein endothelial cells at high concentrations (50 to 100 µM). Treatment with ADP did not induce cell apoptosis but instead induced cell cycle arrest in the S phase, which may be partly due to the downregulation of cyclin B1. The inhibition of cell proliferation was blocked by suramin, a nonspecific antagonist of the P2 receptors, and high concentrations of ADP significantly upregulated the messenger RNA (mRNA) and protein expression of P2Y11 in endothelial cells. Moreover, the downregulation of P2Y11 by RNA interference reversed the inhibition of cell proliferation. In addition, ADP (100 µM) can induce the formation of cytosolic autophagy in endothelial cells and a rapid phosphorylation of extracellular signal regulated kinase (ERK) 1/2, which is a canonical signal molecule downstream of P2Y receptors, accompanied by a mRNA expression of proinflammatory cytokines such as intercellular adhesion molecule 1 and vascular cell adhesion molecule 1. Taken together, our study excludes a mechanism for extracellular ADP impairing endothelial cells proliferation via P2Y11 receptor by downregulating cyclin B1 and arresting cell cycle at the S phase, besides, ADP induces cell autophagy and mRNA expression of inflammatory cytokines, whether it is mediated by Erk signaling pathways needs further studies to confirm.

[Effects of ATP on expression of inflammatory factors in endothelial progenitor cells induced by LPS and the mechanisms].

OBJECTIVE: To investigate the effects of adenosine triphosphate (ATP) on expression of inflammatory factors induced by lipopolysaccharide (LPS) in endothelial progenitor cells (EPCs), and to elucidate the possible mechanisms.
 Methods: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation, RT-PCR was performed to detect the expression of inflammatory factors induced by LPS (1 mg/mL) in EPCs, the effect of low concentration (5 µmol/L) of ATP on expression of IL-1ß, MCP-1 and ICAM-1, and the effect of different concentrations (5, 50 µmol/L) of ATP on the expression of Toll-like receptor (TLR) 4, myeloid differentiation primary response protein 88 (MyD88) and CD14. Western blot was performed to detect expression of TLR4 regulated proteins MyD88 and CD14 or to detect the low concentration (1, 5 µmol/L) of ATP on the expression of TLR4, MyD88 and CD14 and the NF-κB signaling pathway.
 Results: EPCs highly expressed TLR4, and its ligand LPS (1 mg/mL) significantly upregulated mRNA expression of IL-1ß, MCP-1 and ICAM-1 and protein expression of MyD88 and CD14 in a time-dependent manner (P<0.01), accompanied by activation of ERK and NF-κB signal pathway. ATP at low concentration (5 µmol/L) significantly inhibited LPS-induced mRNA expression of IL-1ß, MCP-1 and ICAM-1(P<0.05), downregulated the LPS-induced protein expression of TLR4, MyD88 and CD14 in EPCs (P<0.05), and suppressed LPS-induced activation of NF-κB signaling pathway (P<0.05).
 Conclusion: ATP at low concentration may suppress LPS-induced expression of inflammatory factors in EPCs through negative regulation of the TLR4 signaling pathway.