TRAF6 enhances PD-L1 expression through YAP1-TFCP2 signaling in melanoma.

Immunotherapy represented by programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1) monoclonal antibodies has led tumor treatment into a new era. However, the low overall response rate and high incidence of drug resistance largely damage the clinical benefits of existing immune checkpoint therapies. Recent studies correlate the response to PD-1/PD-L1 blockade with PD-L1 expression levels in tumor cells. Hence, identifying molecular targets and pathways controlling PD-L1 protein expression and stability in tumor cells is a major priority. In this study, we performed a Stress and Proteostasis CRISPR interference screening to identify PD-L1 positive modulators. Here, we identified TRAF6 as a critical regulator of PD-L1 in melanoma cells. As a non-conventional E3 ubiquitin ligase, TRAF6 is inclined to catalyze the synthesis and linkage of lysine-63 (K63) ubiquitin which is related to the stabilization of substrate proteins. Our results showed that suppression of TRAF6 expression down-regulates PD-L1 expression on the membrane surface of melanoma cells. We then used in vitro and in vivo assays to investigate the biological function and mechanism of TRAF6 and its downstream YAP1/TFCP2 signaling in melanoma. TRAF6 stabilizes YAP1 by K63 poly-ubiquitination modification, subsequently promoting the formation of YAP1/TFCP2 transcriptional complex and PD-L1 transcription. Inhibition of TRAF6 by Bortezomib enhanced cytolytic activity of CD8+ T cells by reduction of endogenous PD-L1. Notably, Bortezomib enhances anti-tumor immunity to an extent comparable to anti-PD-1 therapies with no obvious toxicity. Our findings reveal the potential of inhibiting TRAF6 to stimulate internal anti-tumor immunological effect for TRAF6-PD-L1 overexpressing cancers.

Ligation-Based High-Performance Mimetic Enzyme Sensing Platform for Nucleic Acid Detection.

G-quadruplex (G4)/hemin DNAzyme is a promising candidate to substitute horseradish peroxidase in biosensing systems, especially for the detection of nucleic acids. However, the relatively suboptimal catalytic capacity limits its potential applications. This makes it imperative to develop an ideal signal for the construction of highly sensitive biosensing platforms. Herein, we integrated a novel chimeric peptide-DNAzyme (CPDzyme) with the ligase chain reaction (LCR) for the cost-efficient and highly sensitive detection of nucleic acids. By employing microRNA (miRNA) and single-nucleotide polymorphism detection as the model, we designed a G4-forming sequence on the LCR probe with a terminally labeled amino group. Subsequently, asymmetric hemin with carboxylic arms allowed assembly with the LCR products and peptide to form CPDzyme, followed by the magnetic separation of the extraneous components and chemiluminescence detection. Compared with the conventional G4/hemin signaling-based method, the LCR-CPDzyme system demonstrated 3 orders of magnitude improved sensitivity, with accurate quantification of as low as 25 aM miRNA and differentiation of 0.1% of mutant DNA from the pool containing a large amount of wild-type DNA. The proposed LCR-CPDzyme strategy is a potentially powerful method for in vitro diagnostics and serves as a reference for the development of other ligation- or hybridization-based nucleic acid amplification assays.

Photocross-Linkable and Shape-Memory Biomaterial Hydrogel Based on Methacrylated Cellulose Nanofibres.

In the context of three-dimensional (3D) cell culture and tissue engineering, 3D printing is a powerful tool for customizing in vitro 3D cell culture models that are critical for understanding the cell-matrix and cell-cell interactions. Cellulose nanofibril (CNF) hydrogels are emerging in constructing scaffolds able to imitate tissue in a microenvironment. A direct modification of the methacryloyl (MA) group onto CNF is an appealing approach to synthesize photocross-linkable building blocks in formulating CNF-based bioinks for light-assisted 3D printing; however, it faces the challenge of the low efficiency of heterogenous surface modification. Here, a multistep approach yields CNF methacrylate (CNF-MA) with a decent degree of substitution while maintaining a highly dispersible CNF hydrogel, and CNF-MA is further formulated and copolymerized with monomeric acrylamide (AA) to form a super transparent hydrogel with tuneable mechanical strength (compression modulus, approximately 5-15 kPa). The resulting photocurable hydrogel shows good printability in direct ink writing and good cytocompatibility with HeLa and human dermal fibroblast cell lines. Moreover, the hydrogel reswells in water and expands to all directions to restore its original dimension after being air-dried, with further enhanced mechanical properties, for example, Young's modulus of a 1.1% CNF-MA/1% PAA hydrogel after reswelling in water increases to 10.3 kPa from 5.5 kPa.

Exosomal vimentin from adipocyte progenitors accelerates wound healing.

Adipose stem cell-derived exosomes have great potential in accelerating cutaneous wound healing by optimizing fibroblast activities. Recent studies have demonstrated that exosomes play an active role in the transport of functional cytoskeletal proteins such as vimentin. Previously we showed that vimentin serves as a coordinator of the healing process. Therefore, we hypothesized that vimentin incorporated into the exosomes may contribute to mediate fibroblast activities in wound healing. Our results revealed that exosomal vimentin from adipocyte progenitor cells acts as a promoter of fibroblast proliferation, migration, and ECM secretion. Furthermore, our in vitro and in vivo experiments provide evidence that exosomal vimentin shortens the healing time and reduces scar formation. These findings suggest the reciprocal roles of exosomes and vimentin in accelerating wound healing. Exosomes can serve as an efficient transportation system to deliver and internalize vimentin into target cells, while vimentin could have an impact on exosome transportation, internalization, and cell communication.

Surface Engineered Biomimetic Inks Based on UV Cross-Linkable Wood Biopolymers for 3D Printing.

Owing to their superior mechanical strength and structure similarity to the extracellular matrix, nanocelluloses as a class of emerging biomaterials have attracted great attention in three-dimensional (3D) bioprinting to fabricate various tissue mimics. Yet, when printing complex geometries, the desired ink performance in terms of shape fidelity and object resolution demands a wide catalogue of tunability on the material property. This paper describes surface engineered biomimetic inks based on cellulose nanofibrils (CNFs) and cross-linkable hemicellulose derivatives for UV-aided extrusion printing, being inspired by the biomimetic aspect of intrinsic affinity of heteropolysaccharides to cellulose in providing the ultrastrong but flexible plant cell wall structure. A facile aqueous-based approach was established for the synthesis of a series of UV cross-linkable galactoglucomannan methacrylates (GGMMAs) with tunable substitution degrees. The rapid gelation window of the formulated inks facilitates the utilization of these wood-based biopolymers as the feeding ink for extrusion-based 3D printing. Most importantly, a wide and tunable spectrum ranging from 2.5 to 22.5 kPa of different hydrogels with different mechanical properties could be achieved by varying the substitution degree in GGMMA and the compositional ratio between GGMMA and CNFs. Used as the seeding matrices in the cultures of human dermal fibroblasts and pancreatic tumor cells, the scaffolds printed with the CNF/GGMMA inks showed great cytocompatibility as well as supported the matrix adhesion and proliferative behaviors of the studied cell lines. As a new family of 3D printing feedstock materials, the CNF/GGMMA ink will broaden the map of bioinks, which potentially meets the requirements for a variety of in vitro cell-matrix and cell-cell interaction studies in the context of tissue engineering, cancer cell research, and high-throughput drug screening.