Engineering of a Multi-Modular DNA Nanodevice for Spatioselective Imaging and Evaluation of NK Cell-Mediated Cancer Immunotherapy.

Granzyme A (GzmA) secreted by natural killer (NK) cells has garnered considerable interest as a biomarker to evaluate the efficacy of cancer immunotherapy. However, current methodologies to selectively monitor the spatial distribution of GzmA in cancer cells during NK cell-targeted therapy are extremely challenging, primarily due to the existence of diverse cell populations, the low levels of GzmA expression, and the limited availability of GzmA probes. Herein we develop a multi-modular, structurally-ordered DNA nanodevice for evaluating NK cell-mediated cancer immunotherapy (MODERN), that permits spatioselective imaging of GzmA in cancer cells through GzmA-induced apurinic/apyrimidinic endonuclease 1 (APE1) inactivation. The MODERN incorporates multiple functional modules, including an APE1-gated recognition module, a photo-activated amplification module, an aptamer-mediated tumor-target module, and a polycatenane DNA module, enabling improved sensitivity and specificity towards intracellular GzmA. The MODERN was activated (on) in cancer cells due to the overexpression of APE1, whereas it remained silent (off) in the NK-treated cancer cells owing to the GzmA-induced APE1 inactivation. Furthermore, we demonstrated that GzmA-induced APE1 inactivation blocks the cellular repair of target cells, resulting in efficient cell death. This MODERN that relies on the specific inactivation of APE1 by GzmA should be beneficial for evaluating the efficacy of cancer immunotherapy.

Multienzymatic Orthogonal Activation of DNA Codec Enables Tumor-Specific Imaging of Base Excision Repair Activity.

Accurate monitoring of base excision repair (BER) activity in cancer cells is critical for advancing the comprehension of DNA repair processes, gaining insights into cancer development, and guiding treatment strategies. However, current assay techniques for assessing BER activity in cancer cells face challenges due to the heterogeneous origins and diversity of BER enzymes. In this work, we present a highly reliable triple loop-interlocked DNA codec (GATED) that enables precise assessment of BER activity in cancer cells through signal amplification mediated by multienzyme orthogonal activation. The GATED device features a dumbbell-shaped DNA probe to encode two BER enzymes for BER-related signal conversion as well as two bound circular DNA to decode the apurinic/apyrimidinic sites for apurinic/apyrimidinic endonuclease 1 (APE1)-mediated signal amplification. Importantly, GATED is orthogonally activated by multiple target BER enzymes (i.e., uracil DNA glycosylase, thymine DNA glycosylase, and APE1), resulting in a unified fluorescent signal that significantly improves the detection specificity and sensitivity to BER enzymes. Additionally, we demonstrate that the GATED has exceptional biostability within complex biological systems, where it was successfully employed to monitor BER activity in cancer cells with high specificity and enabled cell-based high-throughput screening for BER inhibitors. The GATED provides a much-needed tool for the real-time monitoring of BER activity and the screening of BER inhibitors in cancer cells, potentially advancing both the investigation and clinical application of BER biology.

In Situ Controllable Generation of Copper Nanoclusters Confined in a Poly-l-Cysteine Porous Film with Enhanced Electrochemiluminescence for Alkaline Phosphatase Detection.

Copper nanoclusters (Cu NCs) as emerging luminescent metal NCs are gaining increasing attention owing to the comparatively low cost and high abundance of the Cu element in nature. However, it remains challenging to manipulate the optical properties of Cu NCs. Unlike most dispersed Cu NCs, whose luminescence efficiency was restricted by nonexcited relaxation, the Cu NCs confined in a porous poly-l-cysteine (poly-l-Cys) film were generated controllably with enhanced electrochemiluminescence (ECL) by in situ electrochemical reduction. Specifically, poly-l-Cys provided a porous structure to regulate the generation of Cu NCs within its holes, which not only increased the restriction on the intramolecular vibration and rotation of the ligands but also expedited the electron transfer near the electrode surface, reflecting in an enhancement of the ECL signal and efficiency. As an application of the confined Cu NCs, an ECL biosensor with high performance was constructed skillfully for highly sensitive detection of alkaline phosphatase (ALP), which adopted Cu NCs as the ECL luminophore and poly-l-Cys as a coreaction accelerator in a novel ECL ternary system (Cu NCs/S2O82-/poly-l-Cys). Furthermore, an ingenious target amplification based on the combination of a DNA walker and click chemistry was developed to convert ALP to DNA strands efficiently, achieving great improvement in the recognition efficiency. As a result, the biosensor had a low detection limit (9.5 × 10-7 U·L-1) and a wide linear range (10-8-10-2 U·L-1) for ALP detection, which showed great promise for the detection of non-nucleic acid targets and the diagnosis of diseases.

Mannose-binding lectin inhibits monocyte proliferation through transforming growth factor-ß1 and p38 signaling pathways.

Mannose-binding lectin (MBL), a plasma C-type lectin, plays an important role in innate immunity. However, the interaction, and the consequences of it, between MBL and the immune system remain ill defined. We have investigated the contributing mechanisms and effects of MBL on the proliferation of human monocytes. At lower concentrations (≤4 µg/ml) MBL was shown to partially enhance monocyte proliferation. By contrast, at higher concentrations (8-20 µg/ml) of MBL, cell proliferation was markedly attenuated. MBL-induced growth inhibition was associated with G0/G1 arrest, down-regulation of cyclin D1/D3, cyclin-dependent kinase (Cdk) 2/Cdk4 and up-regulation of the Cdk inhibitory protein Cip1/p21. Additionally, MBL induced apoptosis, and did so through caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage. Moreover, transforming growth factor (TGF)-ß1 levels increased in the supernatants of MBL-stimulated monocyte cultures. We also found that MBL-dependent inhibition of monocyte proliferation could be reversed by the TGF-ß receptor antagonist SB-431542, or by anti-TGF-ß1 antibody, or by the mitogen-activated protein kinase (MAPK) inhibitors specific for p38 (SB203580), but not ERK (U0126) or JNK (SP600125). Thus, at high concentrations, MBL can affect the immune system by inhibiting monocyte proliferation, which suggests that MBL may exhibit anti-inflammatory effects.