Shedding light on cellular dynamics: the progress in developing photoactivated fluorophores.

Photoactivated fluorophores (PAFs) are highly effective imaging tools that exhibit a removal of caging groups upon light excitation, resulting in the restoration of their bright fluorescence. This unique property allows for precise control over the spatiotemporal aspects of small molecule substances, making them indispensable for studying protein labeling and small molecule signaling within live cells. In this comprehensive review, we explore the historical background of this field and emphasize recent advancements based on various reaction mechanisms. Additionally, we discuss the structures and applications of the PAFs. We firmly believe that the development of more novel PAFs will provide powerful tools to dynamically investigate cells and expand the applications of these techniques into new domains.

NUAK1 promotes tumor metastasis through upregulating slug transcription in esophageal squamous cell carcinoma.

BACKGROUND: Metastasis is still a major cause of poor pathological outcome and prognosis in esophageal squamous cell carcinoma (ESCC) patients. NUAK1 has been reported highly expressed in many human cancers and is associated with the poor prognosis of cancer patients. However, the role of NUAK1 and its underlying signaling mechanism in ESCC metastasis remain unclear. METHODS: Expression of NUAK1 in ESCC was detected by real-time quantitative RT-PCR (qRT-PCR), Western blotting and immunohistochemical staining. MTT, colony formation, wound-healing and transwell assays were used to determine the role NUAK1 in vitro. Metastasis was evaluated by use of an experimental pulmonary metastasis model in BALB/c-nu/nu mice. The mechanisms were assessed by using coimmunoprecipitation, immunofluorescence and dual-luciferase reporter gene experiments. RESULTS: NUAK1 was highly expressed in ESCC tissues compared with the adjacent normal esophageal epithelial tissues. Moreover, the elevated expression of NUAK1 positively correlated with tumor invasion depth, lymph node metastasis, pathological TNM stage, and poor survival in ESCC patients. Further experiments showed that NUAK1 overexpression did not change the cell viability and colony formation of ESCC cells, while remarkably promoted the migration and invasion in vitro and experimental pulmonary metastasis in vivo. Mechanistically, NUAK1 enhanced the transcription level of Slug, which enhanced the migratory and invasive capability of ESCC cells. Consistently, silencing Slug almost completely diminished the migration and invasion of NUAK1-overexpressing ESCC cells. Further studies demonstrated that NUAK1 upregulated the transcription activity of Slug through activating the JNK/c-Jun pathway. CONCLUSION: These results demonstrated that NUAK1 promoted the metastasis of ESCC cells through activating JNK/c-Jun/Slug signaling, indicating NUAK1 is a promising therapeutic target for metastatic ESCC.

A Water-Soluble, Two-Photon Probe for Imaging Endogenous Hypochlorous Acid in Live Tissue.

Detection of hypochlorous acid (HClO) in the living system may help to uncover its essential biological functions. However, current imaging agents suffer from poor water solubility that limit their live-tissue applications. Here, a water-soluble probe (NNH) is devised through innovative hydrazone modification of 1,8-naphthalimide at 3' position. NNH was successfully applied to tracking endogenous HClO in both cultured macrophages and a liver injury model in mice. NNH demonstrated remarkably increased water solubility and multiple desirable features including two-photon absorbance, anti-bleaching capability, rapid cellular uptake, and low cytotoxicity. NNH is the first hydrazone-based probe for real-time imaging of HClO in live tissue.

Air pollution particles hijack peroxidasin to disrupt immunosurveillance and promote lung cancer.

Although fine particulate matter (FPM) in air pollutants and tobacco smoke is recognized as a strong carcinogen and global threat to public health, its biological mechanism for inducing lung cancer remains unclear. Here, by investigating FPM's bioactivities in lung carcinoma mice models, we discover that these particles promote lung tumor progression by inducing aberrant thickening of tissue matrix and hampering migration of antitumor immunocytes. Upon inhalation into lung tissue, these FPM particles abundantly adsorb peroxidasin (PXDN) - an enzyme mediating type IV collagen (Col IV) crosslinking - onto their surface. The adsorbed PXDN exerts abnormally high activity to crosslink Col IV via increasing the formation of sulfilimine bonds at the NC1 domain, leading to an overly dense matrix in the lung tissue. This disordered structure decreases the mobility of cytotoxic CD8+ T lymphocytes into the lung and consequently impairs the local immune surveillance, enabling the flourishing of nascent tumor cells. Meanwhile, inhibiting the activity of PXDN abolishes the tumor-promoting effect of FPM, indicating the key impact of aberrant PXDN activity on the tumorigenic process. In summary, our finding elucidates a new mechanism for FPM-induced lung tumorigenesis and identifies PXDN as a potential target for treatment or prevention of the FPM-relevant biological risks.

A phase-transfer catalyst-based nanoreactor for accelerated hydrogen sulfide bio-imaging.

Hydrogen sulfide (H2S) is an important signaling molecule in various biological processes; however, its real-time monitoring in living cells is hampered by long detection time for current fluorescent probes. To overcome this challenge, we designed a phase-transfer catalyst (PTC) approach to accelerate the reaction between the probe and the analyte by conjugating common fluorescent probes - mostly hydrophobic small molecules - with an amphiphilic PEG-PPG-PEG polymer, enabling the controllable assembly of H2S nanoprobes in an aqueous solution. The PEG block helps to establish a PTC microenvironment that endows the assembled nanoprobes with a significantly reduced detection time (3-10 min; versus 20-60 min for small-molecule probes). Based on this approach, we synthesised two nanoprobes of different wavelengths, DS-Blue-nano and DN-Green-nano, which can sensitively detect H2S in living macrophage cells with bright fluorescence starting at as early as 7 min and reaching stability at 15 min. These data suggest PTC-based nanoprobes as a new and generic approach for constructing sensitive fluorescent probes for the real-time imaging of H2S, and perhaps other molecules in future, under biological conditions.

Engineering a microcarrier based on a polysaccharide-growth factor complex for enhancing the proliferation of mesenchymal stem cells.

Mesenchymal stem cell (MSC) delivery has been broadly investigated as a cell-based therapy strategy towards various diseases and tissue injury. In these applications, the cell-delivery vehicle plays a crucial role in determining the therapeutic performance of MSCs and their fate post-implantation. We report here the development of a microcarrier system combining platelet-derived growth factor-BB (PDGF-BB) and a PDGF-BB-binding polysaccharide - Eucommia ulmoides (EUP3) - for MSC cultivation. First, we investigated the optimal conditions to prepare the EUP3-PDGF-BB complex, by comparing its i) diameter, ii) morphology, and iii) bioactivity to promote MSC proliferation and fibroblast migration in vitro, under different PDGF-BB/EUP3 ratios. Then, we fabricated microspheres using gelatin and EUP3 as the matrix while stabilizing PDGF-BB at the optimal ratio for MSC adhesion and growth. Live staining and SEM observation indicated that the prepared microspheric carrier supported MSC growth and maintained cell stemness. We suggest that the EUP3/PDGF-gelatin microcarriers can potentially serve as a cell-delivery vehicle for tissue engineering.

An "all-in-one" scaffold targeting macrophages to direct endogenous bone repair in situ.

Scaffolds for tissue repair are designed in an increasingly complicated manner to meet multi-facet biological needs during the healing process. However, overly sophisticated design, especially the use of multiple components and delivery of exogenous cells, hampers the bench-to-bedside translation. Here, a multi-functional - yet mono-compositional - bioactive scaffold is devised to mediate the full-range, endogenous bone repair. Based on immunoactivity screening, a chemically-modified glucomannan polysaccharide is selected and processed into an anisotropic porous scaffold, which accurately stimulates macrophages to produce pro-regenerative cytokines. These cytokines effectively enhance the recruitment ("R") and induced osteogenesis ("IO") of the bone progenitor cells in situ. Meanwhile, the anisotropic porosity and carbohydrate signal of the scaffold facilitate differential adhesion ("A") and distribution ("D") of the macrophages and bone progenitor cells - enabling the former's accumulation at the surface while encouraging the latter's infiltration into the scaffold. Implanted in a rat calvarial defect model, this "RADIO" system effectively promotes healing over 12 weeks, with the obvious formation of hard callus through the scaffold. In summary, RADIO integrates multiple functions into one single scalable system ("all-in-one") to govern the dynamic bone-repair process, by harnessing the power of host macrophages. RADIO represents an open platform to solving the long-lasting complexity-versus-simplicity dilemma in biomaterials design. STATEMENT OF SIGNIFICANCE: Biomaterials as versatile tools for tissue repair are becoming increasingly complicated, yet overly sophisticated design - especially the use of multiple components, exogenous cells, and overdosed growth factors - hampers their clinical application. The pre-requisite for designing a successful integrative scaffold is to identify an inherent biological target responding to biomaterial signals, thereby efficiently and safely promoting tissue repair via the endogenous healing capability instead of extra multifarious biochemical components. For bone regeneration, the pivotal regulator is macrophages. Through activating host macrophages, our single-component scaffold system coordinates the entire bone regenerative cascade in situ and induces successful bone regeneration in a calvarial defect model. This scaffold represents a scalable and multi-functional approach to effectively simplify the sophisticated design in regenerative medicine.

A pocket-escaping design to prevent the common interference with near-infrared fluorescent probes in vivo.

Near-infrared (NIR) fluorescent probes are among the most attractive chemical tools for biomedical imaging. However, their in vivo applications are hindered by albumin binding, generating unspecific fluorescence that masks the specific signal from the analyte. Here, combining experimental and docking methods, we elucidate that the reason for this problem is an acceptor (A) group-mediated capture of the dyes into hydrophobic pockets of albumin. This pocket-capturing phenomenon commonly applies to dyes designed under the twisted intramolecular charge-transfer (TICT) principle and, therefore, represents a generic but previously unidentified backdoor problem. Accordingly, we create a new A group that avoids being trapped into the albumin pockets (pocket-escaping) and thereby construct a NIR probe, BNLBN, which effectively prevents this backdoor problem with increased imaging accuracy for liver fibrosis in vivo. Overall, our study explains and overcomes a fundamental problem for the in vivo application of a broad class of bioimaging tools.

A toll-like receptor agonist mimicking microbial signal to generate tumor-suppressive macrophages.

Switching macrophages from a pro-tumor type to an anti-tumor state is a promising strategy for cancer immunotherapy. Existing agents, many derived from bacterial components, have safety or specificity concerns. Here, we postulate that the structures of the bacterial signals can be mimicked by using non-toxic biomolecules of simple design. Based on bioactivity screening, we devise a glucomannan polysaccharide with acetyl modification at a degree of 1.8 (acGM-1.8), which specifically activates toll-like receptor 2 (TLR2) signaling and consequently induces macrophages into an anti-tumor phenotype. For acGM-1.8, the degree of acetyl modification, glucomannan pattern, and acetylation-induced assembly are three crucial factors for its bioactivity. In mice, intratumoral injection of acGM-1.8 suppresses the growth of two tumor models, and this polysaccharide demonstrates higher safety than four classical TLR agonists. In summary, we report the design of a new, safe, and specific TLR2 agonist that can generate macrophages with strong anti-tumor potential in mice.

Bioactive polysaccharides from natural resources including Chinese medicinal herbs on tissue repair.

BACKGROUND: Functional polysaccharides can be derived from plants (including herbs), animals and microorganisms. They have been widely used in a broad of biomedical applications, such as immunoregulatory agents or drug delivery vehicles. In the past few years, increasing studies have started to develop natural polysaccharides-based biomaterials for various applications in tissue engineering and regenerative medicine. MAIN BODY: We discuss in this article the emerging applications of natural polysaccharides-particularly those derived from Chinese medicine-for wound healing. First, we introduce natural polysaccharides of three natural sources and their biological activities. Then, we focus on certain natural polysaccharides with growth factor-binding affinities and their inspired polymeric tools, with an emphasis on how these polysaccharides could possibly benefit wound healing. Finally, we report the latest progress in the discovery of polysaccharides from Chinese medicinal herbs with identified activities favouring tissue repair. CONCLUSION: Natural polysaccharides with clearly elucidated compositions/structures, identified cellular activities, as well as desirable physical properties have shown the potential to serve as therapeutic tools for tissue regeneration.

An ortho-aldehyde modified probe to image thiols in living cells with enhanced selectivity.

Developing fluorescent probes to image thiols in the living system may provide powerful tools to study the functions of thiol-containing biological molecules. In this study, we report the design and evaluation of a novel turn-on fluorescent probe NQNO for selective detection of thiols in living cells. By introducing an ortho-aldehyde group to NNO, a conventional compound representing a class of thiol-imaging strategy, we obtained NQNO with enhanced selectivity for thiols over the major interferent hydrogen sulfide (H2S). NQNO could be applied in phosphate-buffered saline (PBS), where the efficacy of NNO was usually weakened. Notably, NQNO demonstrated solid performance in imaging endogenous thiols in living cells without exerting cytotoxicity. In summary, NQNO has the potential to serve as a safe, sensitive and effective fluorescent probe for thiol imaging in biological systems.

Water solubility is essential for fluorescent probes to image hypochlorous acid in live cells.

We synthesised four probes and compared their HClO-detecting ability in different solvents. The data showed that only hydrophilic probes could sensitively and accurately detect HClO in live cells. Meanwhile, the addition of organic solvents, as is commonly practised, weakens the oxidising capacity of HClO and thus generates inaccurate outcomes.

Modulating the phenotype of host macrophages to enhance osteogenesis in MSC-laden hydrogels: Design of a glucomannan coating material.

The biomaterials-host interaction is a dynamic process in which macrophages play a vital role of regulation. Depending on the biochemical signals they sense, these highly plastic cells can mediate the immune response against the implanted scaffolds and/or exert regenerative potency to varying extent. Designing appropriate 'exterior signals' for scaffolds may exploit the power of endogenous macrophages to aid the regeneration of engineered tissues. To realise this goal, this study devised an injectable, instantaneously-solidifying coating material (acBSP) based on a unique, macrophage-affinitive glucomannan polysaccharide. Coating of three-dimensional hydrogel constructs with acBSP was rapid, neat and complete, requiring neither chemical reactions nor harsh conditions. Comprehensive in vitro analyses indicated that acBSP efficiently facilitated the adhesion and activation of macrophages and notably induced the macrophages to express pro-osteogenic/-angiogenic genes. Further in vivo assessment of acBSP-coated, mesenchymal stem cells-laden hydrogels in a murine dorsal subcutaneous pocket model demonstrated efficient macrophage activation, desirable scaffold-tissue integration and improved osteogenic differentiation in the delivered cells. In summary, by activating macrophages into a pro-osteogenic phenotype, the acBSP coating has demonstrated its competency as an innovative, open and efficacious platform to harness the power of host immunity for enhancing the regenerative performance of engineered tissue constructs.

A dinuclear ruthenium(II) complex as turn-on luminescent probe for hypochlorous acid and its application for in vivo imaging.

A dinuclear ruthenium(II) complex Ruazo was designed and synthesized, in which oxidative cyclization of the azo and o-amino group was employed for the detection of hypochlorous acid (HClO) in aqueous solution. The non-emissive Ruazo formed highly luminescent triazole-ruthenium(II) complex in presence of HClO and successfully imaged HClO in living cell and living mouse.

HEPES is not suitable for fluorescence detection of HClO: a novel probe for HClO in absolute PBS.

HEPES is not suitable for fluorescence detection of HClO because it can be oxidized by HClO. A novel probe for HClO, which can selectively and sensitively detect HClO in absolute PBS, was developed on the basis of an oxidation reaction with an azo moiety. Furthermore, it works well in live mouse imaging.

Ratiometric and colorimetric near-infrared sensors for multi-channel detection of cyanide ion and their application to measure ß-glucosidase.

A near-infrared sensor for cyanide ion (CN(-)) was developed via internal charge transfer (ICT). This sensor can selectively detect CN(-) either through dual-ratiometric fluorescence (logarithm of I414/I564 and I803/I564) or under various absorption (356 and 440 nm) and emission (414, 564 and 803 nm) channels. Especially, the proposed method can be employed to measure ß-glucosidase by detecting CN(-) traces in commercial amygdalin samples.