Real-time detection and resection of sentinel lymph node metastasis in breast cancer through a rare earth nanoprobe based NIR-IIb fluorescence imaging.

Sentinel lymph node (SLN) biopsy is a commonly employed procedure for the routine assessment of axillary involvement in patients with breast cancer. Nevertheless, conventional SLN mapping cannot reliably distinguish the presence and absence of metastatic disease. Additionally, the complex anatomical structures and lymphatic drainage patterns surrounding tumor sites pose challenges to the sensitivity of the near-infrared fluorescence imaging with subcutaneously injected probes. To identifying the SLN metastases, we developed a novel nanoprobe for in vivo fluorescence imaging within the second near-infrared (NIR-II) range. This nanoprobe utilizes rare-earth nanoparticles (RENPs) to emit bright fluorescence at 1525 nm and is conjugated with tumor-targeted hyaluronic acid (HA) to facilitate the detection of metastatic SLN. Upon intravenous administration, RENPs@HA effectively migrated to SLNs and selectively entered metastatic breast tumor cells through CD44-mediated endocytosis. The RENPs@HA nanoprobes exhibited rapid accumulation in metastatic inguinal lymph nodes in mouse model, displaying a 5.8-fold-stronger fluorescence intensity to that observed in normal SLNs. Consequently, these nanoprobes effectively differentiate metastatic SLNs from normal SLNs. Importantly, the probes accurately detected micrometastases. These findings underscore the potential of RENPs@HA for real-time visualization and screening of SLNs metastasis.

A Dual-Cascade Activatable Near-Infrared Fluorescent Probe for Precise Intraoperative Imaging of Tumor.

Accurate intraoperative tumor delineation is critical to achieving successful surgical outcomes. However, conventional techniques typically suffer from poor specificity and low sensitivity and are time-consuming, which greatly affects intraoperative decision-making. Here, we report a cascade activatable near-infrared fluorescent (NIRF) probe IR780SS@CaP that can sequentially respond to tumor acidity and elevated glutathione levels for accurate intraoperative tumor localization. Compared with nonactivatable and single-factor activatable probes, IR780SS@CaP with a cascade strategy can minimize nonspecific activation and false positive signals in a complicated biological environment, affording a superior tumor-to-normal tissue ratio to facilitate the delineation of abdominal metastases. Small metastatic lesions that were less than 1 mm in diameter can be precisely identified by IR780SS@CaP and completely excised under NIRF imaging guidance. This study could benefit tumor diagnosis and image-guided tumor surgery by providing real-time information and reliable decision support, thus reducing the risk of both recurrence and complications to improve patient outcomes.

[Preparation and characterization of a fluorogenic ddRFP-M biosensor as a specific SARS-CoV-2 main protease substrate].

The conventional peptide substrates of SARS-CoV-2 main protease (Mpro) are frequently associated with high cost, unstable kinetics, and multistep synthesis. Hence, there is an urgent need to design affordable and stable Mpro substrates for pharmacological research. Herein, we designed a functional Mpro substrate based on a dimerization-dependent red fluorescent protein (ddRFP) for the evaluation of Mpro inhibitors in vitro. The codon-optimized DNA fragment encoding RFP-A1 domain, a polypeptide linker containing Mpro cleavage sequence (AVLQS), and the RFP-B1 domain was subcloned into the pET-28a vector. After transformation into Escherichia coli Rosetta(DE3) cells, the kanamycin resistant transformants were selected. Using a low temperature induction strategy, most of the target proteins (ddRFP-M) presented in the supernatant fractions were collected and purified by a HisTrapTM chelating column. Subsequently, the inhibition of Mpro by ensitrelvir and baicalein was assessed using ddRFP-M assay, and the biochemical properties of ddRFP-M substrate were analyzed. Our results showed that the fluorogenic substrate ddRFP-M was successfully prepared from E. coli cells, and this biosensor exhibited the expected specificity, sensitivity, and reliability. In conclusion, the production of the fluorogenic substrate ddRFP-M provides an expedient avenue for the assessment of Mpro inhibitors in vitro.

An Activatable Near-Infrared Fluorescent Probe for Precise Detection of the Pulmonary Metastatic Tumors: A Traditional Molecule Having a Stunning Turn.

An accurate detection of lung metastasis is of great significance for making better treatment choices and improving cancer prognosis, but remains a big challenge in clinical practice. In this study, we propose a reinventing strategy to develop a pH-activatable near-infrared (NIR) fluorescent nanoprobe, pulmonary metastasis tracer (denoted as PMT), based on assembly of NIR dye IR780 and calcium phosphate (CaP). By delicately tuning the intermolecular interactions during the assembly process and dye doping content, as well as the synthetic condition of probe, the fluorescence of PMT could be finely adjusted via the tumor acidity-triggered disassembly. Notably, the selected PMT9 could sharply convert subtle pH variations into a distinct fluorescence signal to generate high fluorescence ON/OFF contrast, dramatically reducing the background signals. Benefiting from such preferable features, PMT9 is able to precisely identify not only the tumor sites in orthotopic lung cancer models but also the pulmonary metastases in mice with remarkable signal-to-background ratio (SBR). This study provides a unique strategy to turn shortcomings of traditional dye IR780 during in vivo imaging into advantages and further expand the application of fluorescent probe to image lung associated tumor lesions.

A Novel Estrone Degradation Gene Cluster and Catabolic Mechanism in Microbacterium oxydans ML-6.

Global-scale estrone (E1) contamination of soil and aquatic environments results from the widespread use of animal manure as fertilizer, threatening both human health and environmental security. A detailed understanding of the degradation of E1 by microorganisms and the associated catabolic mechanism remains a key challenge for the bioremediation of E1-contaminated soil. Here, Microbacterium oxydans ML-6, isolated from estrogen-contaminated soil, was shown to efficiently degrade E1. A complete catabolic pathway for E1 was proposed via liquid chromatography-tandem mass spectrometry (LC-MS/MS), genome sequencing, transcriptomic analysis, and quantitative reverse transcription-PCR (qRT-PCR). In particular, a novel gene cluster (moc) associated with E1 catabolism was predicted. The combination of heterologous expression, gene knockout, and complementation experiments demonstrated that the 3-hydroxybenzoate 4-monooxygenase (MocA; a single-component flavoprotein monooxygenase) encoded by the mocA gene was responsible for the initial hydroxylation of E1. Furthermore, to demonstrate the detoxification of E1 by strain ML-6, phytotoxicity tests were performed. Overall, our findings provide new insight into the molecular mechanism underlying the diversity of E1 catabolism in microorganisms and suggest that M. oxydans ML-6 and its enzymes have potential applications in E1 bioremediation to reduce or eliminate E1-related environmental pollution. IMPORTANCE Steroidal estrogens (SEs) are mainly produced by animals, while bacteria are major consumers of SEs in the biosphere. However, the understanding of the gene clusters that participate in E1 degradation is still limited, and the enzymes involved in the biodegradation of E1 have not been well characterized. The present study reports that M. oxydans ML-6 has effective SE degradation capacity, which facilitates the development of strain ML-6 as a broad-spectrum biocatalyst for the production of certain desired compounds. A novel gene cluster (moc) associated with E1 catabolism was predicted. The 3-hydroxybenzoate 4-monooxygenase (MocA; a single-component flavoprotein monooxygenase) identified in the moc cluster was found to be necessary and specific for the initial hydroxylation of E1 to generate 4-OHE1, providing new insight into the biological role of flavoprotein monooxygenase.

A metabolic acidity-activatable calcium phosphate probe with fluorescence signal amplification capabilities for non-invasive imaging of tumor malignancy.

Dysregulated energy metabolism has recently been recognized as an emerging hallmark of cancer. Tumor cells, which are characterized by abnormal glycolysis, exhibit a lower extracellular pH (6.5-7.0) than normal tissues (7.2-7.4), providing a promising target for tumor-specific imaging and therapy. However, most pH-sensitive materials are unable to distinguish such a subtle pH difference owing to their wide and continuous pH-responsive range. In this study, we developed an efficient strategy for the fabrication of a tumor metabolic acidity-activatable calcium phosphate (CaP) fluorescent probe (termed MACaP9). Unlike traditional CaP-based biomedical nanomaterials, which only work within more acidic organelles, such as endosomes and lysosomes (pH 4.0-6.0), MACaP9 could not only specifically respond to the tumor extra-cellular pH but also rapidly convert pH variations into a distinct fluorescence signal to visually distinguish tumor from normal tissues. The superior sensitivity and specificity of MACaP9 enabled high-contrast visualization of a broad range of tumors, as well as small tumor lesions.

Protocol for high-throughput screening of SARS-CoV-2 main protease inhibitors using a robust fluorescence polarization assay.

Discovery of efficacious antiviral agents targeting SARS-CoV-2 main protease (Mpro) is of the highest importance to fight against COVID-19. Here, we describe a simple protocol for high-throughput screening of Mpro inhibitors using a robust fluorescence polarization (FP) assay. Candidate Mpro inhibitors from large compound libraries could be rapidly identified by monitoring the change of millipolarization unit value. This affordable FP assay can be modified to screen antiviral agents targeting virus protease. For complete details on the use and execution of this protocol, please refer to Li et al. (2022), Yan et al. (2021), and Yan et al. (2022c).

[Identifying SARS-CoV-2 main protease inhibitors by a novel sandwich-like fluorescence polarization screening assay].

SARS-CoV-2 main protease (Mpro) is responsible for polyprotein cleavage to release non-structural proteins (nsps) for viral genomic RNA replication, and its homologues are absent in human cells. Therefore, Mpro has been regarded as one of the ideal drug targets for the treatment of coronavirus disease 2019 (COVID-19). In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel sandwich-like FP screening assay for quick discovery of SARS-CoV-2 Mpro inhibitors from a natural product library. With this screening assay, anacardic acid (AA) and 1, 2, 3, 4, 6-O-pentagalloylglucose (PGG) were found to be the competitive inhibitor and mixed-type inhibitor targeting Mpro, respectively. Importantly, our results showed that the majority of the reported Mpro inhibitors are promiscuous cysteine inhibitors that are not specific to Mpro. In summary, this novel sandwich-like FP screening assay is simple, sensitive, and robust, which is ideal for large-scale screening. Natural products AA and PGG will be the promising lead compounds for generating more potent antiviral agents targeting Mpro, and the stringent hit validation at the early stage of drug discovery is urgently needed.

A robust high-throughput fluorescence polarization assay for rapid screening of SARS-CoV-2 papain-like protease inhibitors.

The global scourge of COVID-19 is a serious threat to public health, but effective therapies remain very limited for this disease. Therefore, the discovery of novel antiviral agents is urgently needed to fight against COVID-19. In the lifecycle of SARS-CoV-2, the causing pathogen of COVID-19, papain-like protease (PLpro) is responsible for the cleavage of polyprotein into functional units as well as immune evasion of vaccines. Hence, PLpro has been regarded as an attractive target to develop antiviral agents. Herein, we first developed a robust and simple sandwich-like fluorescence polarization (FP) screening assay for the discovery of PLpro inhibitors, and identified anacardic acid as a novel competitive inhibitor against PLpro in vitro with an IC50 value of 24.26 ± 0.4 µM. This reliable FP screening assay could provide a prospective avenue for rapid discovery of antiviral agents targeting PLpro in a large-scale screening.