Finback: a web-based data collection system at SSRF biological macromolecular crystallography beamlines.

An integrated computer software system for macromolecular crystallography (MX) data collection at the BL02U1 and BL10U2 beamlines of the Shanghai Synchrotron Radiation Facility is described. The system, Finback, implements a set of features designed for the automated MX beamlines, and is marked with a user-friendly web-based graphical user interface (GUI) for interactive data collection. The Finback client GUI can run on modern browsers and has been developed using several modern web technologies including WebSocket, WebGL, WebWorker and WebAssembly. Finback supports multiple concurrent sessions, so on-site and remote users can access the beamline simultaneously. Finback also cooperates with the deployed experimental data and information management system, the relevant experimental parameters and results are automatically deposited to a database.

Culture medium-associated physicochemical insights on the cytotoxicity of carbon nanomaterials.

Carbon nanomaterials are the most studied materials in nanotechnology. There have been numerous studies on cytotoxicity assessments of carbon nanomaterials, which, however, often lead to controversy. It is generally considered that chemical and physical properties of carbon nanomaterials should have specific biological outcomes. More recent studies have identified the significance of environmental factors surrounding nanomaterial-treated cells. In this perspective, we mainly review culture medium-associated physicochemical insights on the cytotoxicity of carbon nanomaterials, which are largely based on studies in our laboratory. These studies established the close relationship and interplay among the physicochemical properties of the nanomaterials, culture medium, and their toxicological responses.

Visualization of the hepatic and renal cell uptake and trafficking of tetrahedral DNA origami in tumour.

DNA nanostructures, known for their programmability, ease of modification, and favourable biocompatibility, have gained widespread application in the biomedical field. Among them, Tetrahedral DNA Origami (TDOs), as a novel DNA nanostructure, possesses well-defined structures, multiple modification sites, and large cavities, making it a promising drug carrier. However, current understanding of TDOs' interactions with biological systems, particularly with target cells and organs, remains unexplored, limiting its further applications in biomedicine. In this work, we prepared TDOs with an average particle size of 40 nm and labelled them with Cy5 fluorescent molecules. Following intravenous injection in mice, the uptake of TDOs by different types of liver and kidney cells was observed. Results indicated that TDOs accumulate in renal tubules and are metabolized by Kupffer cells, epithelial cells, and hepatocytes in the liver. Additionally, in a tumour-bearing mouse model, TDOs passively targeted tumour tissues and exhibited excellent tumour penetration and retention after rapid metabolism in hepatocytes. Our findings provide crucial insights for the development of TDO-based drug delivery systems.

Clickable X-ray Nanoprobes for Nanoscopic Bioimaging of Cellular Structures.

Synchrotron-based X-ray microscopy (XRM) has garnered widespread attention from researchers due to its high spatial resolution and excellent energy (element) resolution. Existing molecular probes suitable for XRM include immune probes and genetic labeling probes, enabling the precise imaging of various biological targets within cells. However, immune labeling techniques are prone to cross-interference between antigens and antibodies. Genetic labeling technologies have limited systems that allow express markers independently, and moreover, genetically encoded labels based on catalytic polymerization lack a fixed morphology. When applied to cell imaging, this can result in reduced localization accuracy due to the diffusion of labels within the cells. Therefore, both techniques face challenges in simultaneously labeling multiple biotargets within cells and achieving high-precision imaging. In this work, we applied the click reaction and developed a third category of imaging probes suitable for XRM, termed clickable X-ray nanoprobes (Click-XRN). Click-XRN consists of two components: an X-ray-sensitive multicolor imaging module and a particle-size-controllable morphology module. Efficient identification of intra- and extracellular biotargets is achieved through click reactions between the probe and biomolecules. Click-XRN possesses a controllable particle size, and its loading of various metal ions provides distinctive signals for imaging under XRM. Based on this, we optimized the imaging energy of Click-XRN with different particle sizes, enabling single-color and two-color imaging of the cell membrane, cell nucleus, and mitochondria with nanoscale spatial nanometers. Our work provides a potent molecular tool for investigating cellular activities through XRM.

Size-dependent cytotoxicity of nanocarbon blacks.

In this study, we investigated the toxic effects of nanocarbon blacks (NCBs) with different sizes to mouse macrophage RAW264.7 cells. MTT and fluorescence-based LIVE assays demonstrated that NCBs uptake caused a size and dose-dependent growth inhibition to the cells. Optical microscopy observations and (99m)Tc radionuclide labeling techniques were used to investigate the cellular uptake of NCBs with different sizes qualitatively and quantitatively, respectively. Results showed that the cellular uptake amounts of NCBs increased with their increasing size. Large quantities of internal NCBs induced oxidative stress and nuclear damage in cells; these effects may be the critical factors involved in the cytotoxicity of NCBs. The implications associated with these findings are discussed.

Exploring the Potential of Three-Dimensional DNA Crystals in Nanotechnology: Design, Optimization, and Applications.

DNA has been used as a robust material for the building of a variety of nanoscale structures and devices owing to its unique properties. Structural DNA nanotechnology has reported a wide range of applications including computing, photonics, synthetic biology, biosensing, bioimaging, and therapeutic delivery, among others. Nevertheless, the foundational goal of structural DNA nanotechnology is exploiting DNA molecules to build three-dimensional crystals as periodic molecular scaffolds to precisely align, obtain, or collect desired guest molecules. Over the past 30 years, a series of 3D DNA crystals have been rationally designed and developed. This review aims to showcase various 3D DNA crystals, their design, optimization, applications, and the crystallization conditions utilized. Additionally, the history of nucleic acid crystallography and potential future directions for 3D DNA crystals in the era of nanotechnology are discussed.

Nature-Inspired Thylakoid-Based Photosynthetic Nanoarchitectures for Biomedical Applications.

"Drawing inspiration from nature" offers a wealth of creative possibilities for designing cutting-edge materials with improved properties and performance. Nature-inspired thylakoid-based nanoarchitectures, seamlessly integrate the inherent structures and functions of natural components with the diverse and controllable characteristics of nanotechnology. These innovative biomaterials have garnered significant attention for their potential in various biomedical applications. Thylakoids possess fundamental traits such as light harvesting, oxygen evolution, and photosynthesis. Through the integration of artificially fabricated nanostructures with distinct physical and chemical properties, novel photosynthetic nanoarchitectures can be catalytically generated, offering versatile functionalities for diverse biomedical applications. In this article, an overview of the properties and extraction methods of thylakoids are provided. Additionally, the recent advancements in the design, preparation, functions, and biomedical applications of a range of thylakoid-based photosynthetic nanoarchitectures are reviewed. Finally, the foreseeable challenges and future prospects in this field is discussed.

Implementation of level-2 biosafety for a macromolecular crystallography beamline at SSRF.

Macromolecular crystallography is commonly used to determine the structure of biological macromolecules. Currently the beamlines at synchrotron radiation facilities play an important role in macromolecular crystallography, and have produced an enormous number of molecular structures to help solve scientific questions and support applications. Structure information makes significant contributions to the virus-related research as well. However, it is mandatory to be protected the operators under a compatible biosafety infrastructure when a pathological agent is set up in a beamline. Here a level-2 biosafety protection for a macromolecular crystallography beamline at Shanghai Synchrotron Radiation Facility (SSRF) is introduced. To fulfill the biosafety in a radioactive environment, a dedicated design is implemented. Since the beamline will be opened to the external users from nationwide research units, the management process and experimental method are also drawn up.

Autophagy changes in lung tissues of mice at 30 days after carbon black-metal ion co-exposure.

OBJECTIVES: Accumulating studies have investigated the PM2.5-induced pulmonary toxicity, while gaps still remain in understanding its toxic mechanism. Due to its high specific surface area and adsorption capacity similar to nanoparticles, PM2.5 acts as a significant carrier of metals in air and then leads to altered toxic effects. In this study, we aimed to use CBs and Ni as model materials to investigate the autophagy changes and pulmonary toxic effects at 30 days following intratracheal instillation of CBs-Ni mixture. MATERIALS AND METHODS: Groups of mice were instilled with 100 µL normal saline (NS), 20 µg CBs, and 4 µg Ni or CBs-Ni mixture, respectively. At 7 and 30 days post-instillation, all the mice were weighed and then sacrificed. The evaluation system was composed of the following: (a) autophagy and lysosomal function assessment, (b) trace element biodistribution observation in lungs, (c) pulmonary lavage biomedical analysis, (d) lung histopathological evaluation, (e) coefficient analysis of major organs and (f) CBs-Ni interaction and cell proliferation assessment. RESULTS: We found that after CBs-Ni co-exposure, no obvious autophagy and lysosomal dysfunction or pulmonary toxicity was detected, along with complete clearance of Ni from lung tissues as well as recovery of biochemical indexes to normal range. CONCLUSIONS: We conclude that the damaged autophagy and lysosomal function, as well as physiological function, was repaired at 30 days after exposure of CBs-Ni. Our findings provide a new idea for scientific assessment of the impact of fine particles on environment and human health, and useful information for the comprehensive treatment of air pollution.

Genetically encoded X-ray cellular imaging for nanoscale protein localization.

Spatial resolution defines the physical limit of microscopes for probing biomolecular localization and interactions in cells. Whereas synchrotron-based X-ray microscopy (XRM) represents a unique approach for imaging a whole cell with nanoscale resolution due to its intrinsic nanoscale resolution and great penetration ability, existing approaches to label biomolecules rely on the use of exogenous tags that are multi-step and error-prone. Here, we repurpose engineered peroxidases as genetically encoded X-ray-sensitive tags (GXET) for site-specific labeling of protein-of-interest in mammalian cells. We find that 3,3'-diaminobenzidine (DAB) polymers that are in-situ catalytically formed by fusion-expressed peroxidases are visible under XRM. Using this new tag, we imaged the protein location associated with the alteration of a DNA-methylation pathway with an ultra-high resolution of 30 nanometers. Importantly, the excellent energy resolution of XRM enables multicolor imaging using different peroxidase tags. The development of GXET enlightens the way to nanoscopic imaging for biological studies.

Nanodiamond autophagy inhibitor allosterically improves the arsenical-based therapy of solid tumors.

Arsenic trioxide (ATO) is a successful chemotherapeutic drug for blood cancers via selective induction of apoptosis; however its efficacy in solid tumors is limited. Here we repurpose nanodiamonds (NDs) as a safe and potent autophagic inhibitor to allosterically improve the therapeutic efficacy of ATO-based treatment in solid tumors. We find that NDs and ATO are physically separate and functionally target different cellular pathways (autophagy vs. apoptosis); whereas their metabolic coupling in human liver carcinoma cells remarkably enhances programmed cell death. Combination therapy in liver tumor mice model results in ~91% carcinoma decrease as compared with ~28% without NDs. Treated mice show 100% survival rate in 150 days with greatly reduced advanced liver carcinoma-associated symptoms, and ~80% of post-therapy mice survive for over 20 weeks. Our work presents a novel strategy to harness the power of nanoparticles to broaden the scope of ATO-based therapy and more generally to fight solid tumors.

Serum protein corona-responsive autophagy tuning in cells.

Autophagy represents an important cellular response to nanoparticles (NPs), whose modulation holds great promise for developing nanomedicine. Here, we systematically studied cell autophagy responses elicited by the NP-protein corona with diverse protein corona types surrounding NPs with different sizes, shapes, and compositions. We demonstrated that these physicochemical properties of NP-protein coronas exerted a remarkable influence on cell autophagy responses. Particularly, for surface protein type-associated modulation of cell autophagy, we correlated the autophagy level to adsorbed protein type on Fe3O4 NPs. Accordingly, we could modulate cell autophagy in response to various levels of protein adsorption. Our work provides new clues to modulate cell autophagy by rational designing NP-protein complexes, which could aid in further biological and therapeutic applications.

Systematic Study in Mammalian Cells Showing No Adverse Response to Tetrahedral DNA Nanostructure.

The advent of DNA technology has demonstrated great potential in a wide range of applications, especially in the field of biology and biomedicine. However, current understanding of the toxicological effects and cellular responses of DNA nanostructures remains to be improved. Here, we chose tetrahedral DNA nanostructures (TDNs), a type of nanocarriers for delivering molecular drugs, as a model for systematic live-cell analysis of the biocompatibility of TDNs to normal bronchial epithelial cells, carcinoma cells, and macrophage. We found that the interaction behaviors of TDNs in different cell lines were very different, whereas after internalization, most of the TDNs in diverse cell lines were positioned to lysosomes. By a systematic assessment of cell responses after TDN exposure to various cells, we demonstrate that internalized TDNs have good innate biocompatibility. Interestingly, we found that TDN-bearing cells would not affect the cell cycle progression and accompany cell division and that TDNs were separated equally into two daughter cells. This study improves our understanding of the interaction of DNA nanostructures with living systems and their biocompatibility, which will be helpful for further designing DNA nanostructures for biomedical applications.

One-Shot Immunomodulatory Nanodiamond Agents for Cancer Immunotherapy.

The use of functional nanodiamonds (fNDs) to deliver CpG oligonucleotides (ODNs) for sustained immunostimulation is reported. It is demonstrated that monotherapy using this immunostimulatory agent significantly suppresses the tumor growth in two murine tumor models. This fND-based nanoagent opens new opportunities for immunotherapy, as well as clinical applications of various types of therapeutic nucleic acids.