Employing antagonistic C-X-C motif chemokine receptor 4 antagonistic peptide functionalized NaGdF4 nanodots for magnetic resonance imaging-guided biotherapy of breast cancer.

C-X-C motif chemokine receptor 4 (CXCR4) is a promising therapeutic target of breast cancer because it is overexpressed on cell surface of all molecular subtypes of breast cancer including triplenegative breast cancer (TNBC). Herein, CXCR4 antagonistic peptide-NaGdF4 nanodot conjugates (termed as anti-CXCR4-NaGdF4 NDs) have been constructed for magnetic resonance imaging (MRI)-guided biotherapy of TNBC through conjugation of the C-X-C Motif Chemokine 12 (CXCL12)-derived cyclic peptide with tryptone coated NaGdF4 nanodots (5 ± 0.5 nm in diameter, termed as Try-NaGdF4 NDs). The as-prepared anti-CXCR4-NaGdF4 NDs exhibits high longitudinal relaxivity (r1) value (21.87 mM-1S-1), reasonable biocompatibility and good tumor accumulation ability. The features of anti-CXCR4-NaGdF4 NDs improve the tumor-MRI sensitivity and facilitate tumor biotherapy after injection in mouse-bearing MDA-MB-231 tumor model in vivo. MRI-guided biotherapy using anti-CXCR4-NaGdF4 NDs enables to suppress 46% tumor growth. In addition, about 47% injection dose of anti-CXCR4-NaGdF4 NDs is found in the mouse urine at 24 h post-injection. These findings demonstrate that anti-CXCR4-NaGdF4 NDs enable to be used as renal clearable nanomedicine for biotherapy and MRI of breast cancer.

Lectin microarray based glycan profiling of exosomes for dynamic monitoring of colorectal cancer progression.

BACKGROUND: Exosomes, as emerging biomarkers in liquid biopsies in recent years, offer profound insights into cancer diagnostics due to their unique molecular signatures. The glycosylation profiles of exosomes have emerged as potential biomarkers, offering a novel and less invasive method for cancer diagnosis and monitoring. Colorectal cancer (CRC) represents a substantial global health challenge and burden. Thus there is a great need for the aberrant glycosylation patterns on the surface of CRC cell-derived exosomes, proposing them as potential biomarkers for tumor characterization. RESULTS: The interactions of 27 lectins with exosomes from three CRC cell lines (SW480, SW620, HCT116) and one normal colon epithelial cell line (NCM460) have been analyzed by the lectin microarray. The result indicates that Ulex Europaeus Agglutinin I (UEA-I) exhibits high affinity and specificity towards exosomes derived from SW480 cells. The expression of glycosylation related genes within cells has been analyzed by high-throughput quantitative polymerase chain reaction (HT-qPCR). The experimental result of HT-qPCR is consistent with that of lectin microarray. Moreover, the limit of detection (LOD) of UEA-I microarray is calculated to be as low as 2.7 × 105 extracellular vehicles (EVs) mL-1 (three times standard deviation (3σ) of blank sample). The UEA-I microarray has been successfully utilized to dynamically monitor the progression of tumors in mice-bearing SW480 CRC subtype, applicable in tumor sizes ranging from 2 mm to 20 mm in diameter. SIGNIFICANCE: The results reveal that glycan expression pattern of exosome is linked to specific CRC subtypes, and regulated by glycosyltransferase and glycosidase genes of mother cells. Our findings illuminate the potential of glycosylation molecules on the surface of exosomes as reliable biomarkers for diagnosis of tumor at early stage and monitoring of cancer progression.

Sea Anemone-like Nanomachine Based on DNA Strand Displacement Composed of Three Boolean Logic Gates: Diversified Input for Intracellular Multitarget Detection.

Efficient and accurate acquisition of cellular biomolecular information is crucial for exploring cell fate, achieving early diagnosis, and the effective treatment of various diseases. However, current DNA biosensors are mostly limited to single-target detection, with few complex logic circuits for comprehensive analysis of three or more targets. Herein, we designed a sea anemone-like DNA nanomachine based on DNA strand displacement composed of three logic gates (YES-AND-YES) and delivered into the cells using gold nano bipyramid carriers. The AND gate activation depends on the trigger chain released by upstream DNA strand displacement reactions, while the output signal relies on the downstream DNAzyme structure. Under the influence of diverse inputs (including enzymes, miRNA, and metal ions), the interconnected logic gates simultaneously perform logical analysis on multiple targets, generating a unique output signal in the YES/NO format. This sensor can successfully distinguish healthy cells from tumor cells and can be further used for the diagnosis of different tumor cells, providing a promising platform for accurate cell-type identification.

High expression of B7-H3 on monocyte/macrophages in tumor microenvironment promotes lung cancer progression by inhibiting apoptosis.

Monocyte/macrophages constitute a significant population of tumor-infiltrating immune cells and play a crucial role in tumor growth, invasion, and metastasis. B7-H3, has immune regulatory functions, however, it is unclear whether B7-H3 expressed on monocyte/macrophages plays a significance role in tumor progression. We found B7-H3 was high-expressed on monocyte/macrophages in tumor microenvironment compared with adjacent tissues in lung cancer, and its expression level was positively correlated with the number of monocyte/macrophages. Furthermore, the expression of B7-H3 was related to clinical stage and lymph node metastasis. Moreover, miR-29a-3p negatively regulated B7-H3, and the expression of B7-H3 on THP-1-derived macrophages was regulated by secreting exosomes containing miR-29a-3p. In addition, knockdown of B7-H3 promoted macrophage apoptosis under hypoxia. Mechanistically, B7-H3 enhanced the antiapoptotic ability of macrophage by up-regulating HIF-1ɑ via activating NF-κB. Taken together, these results imply that B7-H3 as a therapeutic target could hold promise for enhancing anti-tumor immune responses in individuals diagnosed with lung cancer.

Development of a peptide microarray-based metal-enhanced fluorescence assay for ultrasensitive detection of multiple matrix metalloproteinase activities by using a gold nanorod-polymer substrate.

Matrix metalloproteinases (MMPs) are attractive biomarkers for cancer diagnosis and treatment, while it is still a challenge to precise analysis of MMP activities owing to their very low abundance in the biological samples, especially at the early stages of tumors. Herein, a peptide microarray-based metal-enhanced fluorescence assay (PMMEFA) is proposed to simultaneously detect MMP-1, -2, -3, -7, -9, and -13 activities. The assay involves immobilization of Förster resonance energy transfer dye pair decorated peptides (FRET-peptides) on a poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) coated gold nanorod modified glass slide (GNR@P(GMA-HEMA)). To fabricate the GNR@P(GMA-HEMA) slide, GNRs are self-assembled onto an aminated glass slide, and a polymer brush (P(GMA-HEMA)) is grown through a surface-initiated atom transfer radical polymerization reaction (SI-ATRP). Upon the addition of MMPs, the FRET pairs are broken due to the specific cleavage of FRET-peptides by enzymes, resulting in the recovery of fluorescence signals and further enhancement by the MEF of GNRs. The fluorescence recovery degree provides a direct indicator for MMP activity. The PMMEFA exhibits excellent sensitivity, which enables to detect MMP-1, -2, -3, -7, -9, and -13 activities, with low limits of detection (LODs) of 1.7 fg mL-1, 0.3 fg mL-1, 2.0 fg mL-1, 1.8 fg mL-1, 2.2 fg mL-1 and 14.0 fg mL-1, respectively. To substantiate the practicability of PMMEFA, MMP activities were measured in a range of matrices, encompassing cell culture medium, serum, and tumor tissue homogenate, and MMP activities can be detected only in 0.15 µL serum and 0.025 mg tumor tissue.

Von Willebrand factor promotes radiation-induced intestinal injury (RIII) development and its cleavage enzyme rhADAMTS13 protects against RIII by reducing inflammation and oxidative stress.

Patients with abdominopelvic cancer undergoing radiotherapy commonly develop radiation-induced intestinal injury (RIII); however, its underlying pathogenesis remains elusive. The von Willebrand factor (vWF)/a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) axis has been implicated in thrombosis, inflammation, and oxidative stress. However, its role in RIII remains unclear. In this study, the effect of radiation on vWF and ADAMTS13 expression was firstly evaluated in patients with cervical cancer undergoing radiotherapy and C57BL/6J mice exposed to different doses of total abdominal irradiation. Then, mice with the specific deletion of vWF in the platelets and endothelium were established to demonstrate the contribution of vWF to RIII. Additionally, the radioprotective effect of recombinant human (rh) ADAMTS13 against RIII was assessed. Results showed that both the patients with cervical cancer undergoing radiotherapy and RIII mouse model exhibited increased vWF levels and decreased ADAMTS13 levels. The knockout of platelet- and endothelium-derived vWF rectified the vWF/ADAMTS13 axis imbalance; improved intestinal structural damage; increased crypt epithelial cell proliferation; and reduced radiation-induced apoptosis, inflammation, and oxidative stress, thereby alleviating RIII. Administration of rhADAMTS13 could equally alleviate RIII. Our results demonstrated that abdominal irradiation affected the balance of the vWF/ADAMTS13 axis. vWF exerted a deleterious role and ADAMTS13 exhibited a protective role in RIII progression. rhADAMTS13 has the potential to be developed into a radioprotective agent.

A Bimetallic Polymerization Network for Effective Increase in Labile Iron Pool and Robust Activation of cGAS/STING Induces Ferroptosis-Based Tumor Immunotherapy.

Due to the inherent low immunogenicity and immunosuppressive tumor microenvironment (TME) of malignant cancers, the clinical efficacy and application of tumor immunotherapy have been limited. Herein, a bimetallic drug-gene co-loading network (Cu/ZIF-8@U-104@siNFS1-HA) is developed that increased the intracellular labile iron pool (LIP) and enhanced the weakly acidic TME by co-suppressing the dual enzymatic activities of carbonic anhydrase IX (CA IX) and cysteine desulfurylase (NFS1), inducing a safe and efficient initial tumor immunogenic ferroptosis. During this process, Cu2+ is responsively released to deplete glutathione (GSH) and reduce the enzyme activity of glutathione peroxidase 4 (GPX4), achieving the co-inhibition of the three enzymes and further inducing lipid peroxidation (LPO). Additionally, the reactive oxygen species (ROS) storm in target cells promoted the generation of large numbers of double-stranded DNA breaks. The presence of Zn2+ substantially increased the expression of cGAS/STING, which cooperated with ferroptosis to strengthen the immunogenic cell death (ICD) response and remodel the immunosuppressive TME. In brief, Cu/ZIF-8@U-104@siNFS1-HA linked ferroptosis with immunotherapy through multiple pathways, including the increase in LIP, regulation of pH, depletion of GSH/GPX4, and activation of STING, effectively inhibiting cancer growth and metastasis.

Perillaldehyde Mitigates Ionizing Radiation-Induced Intestinal Injury by Inhibiting Ferroptosis via the Nrf2 Signaling Pathway.

SCOPE: Gastrointestinal toxicity is one of the major side effects of abdominopelvic tumor radiotherapy. Studies have shown that perillaldehyde (PAH) has antioxidant, antiinflammatory, antimicrobial activity, and antitumor effects. This study aims to determine whether PAH has radioprotective effects on radiation-induced intestinal injury and explore the underlying mechanisms. METHODS AND RESULTS: C57BL/6J mice are gavaged with PAH for 7 days, then exposed to a single dose of 13 Gy X-ray total abdominal irradiation (TAI). PAH treatment prolongs the survival time, promotes the survival of crypt cells, attenuates radiation-induced DNA damage, and mitigates intestinal barrier damage in the irradiated mice. PAH also shows radioprotective effects in intestinal crypt organoids and human intestinal epithelial cells (HIEC-6). PAH-mediated radioprotection is associated with the upregulation of nuclear factor erythroid-2 related factor 2 (Nrf2), activation of the antioxidant pathway, and inhibition of ferroptosis. Notably, treatment with the Nrf2 inhibitor ML385 abolishes the protective effects of PAH, indicating that Nrf2 activation is essential for PAH activity. CONCLUSION: PAH inhibits ionizing radiation (IR)-induced ferroptosis and attenuates intestinal injury after irradiation by activating Nrf2 signaling. Therefore, PAH is a promising therapeutic strategy for IR-induced intestinal injury.

Bacterial Magnetosome-Hitchhiked Quick-Frozen Neutrophils for Targeted Destruction of Pre-Metastatic Niche and Prevention of Tumor Metastasis.

Premetastatic niche (PMN) is a prerequisite for tumor metastasis. Destruction of PMN can significantly suppress the tumor metastasis. Bone marrow-derived cells are usually recruited into the premetastatic organs to support PMN formation, which can be orchestrated by tumor-derived secreted factors. Neutrophils can chemotactically migrate towards the inflammatory sites and consume tumor-derived secreted factors, capable of acting as therapeutic agents for a broad-spectrum suppression of PMN formation and metastasis. However, neutrophils in response to inflammatory signals can release neutrophil extracellular traps (NETs), promoting the tumor metastasis. Herein, live neutrophils are converted into dead neutrophils (C NE) through a quick-frozen process to maintain PMN-targeting and tumor-derived secreted factor-consuming abilities but eliminate NET-releasing shortcomings. Considering macrophages-regulated remodeling of the extracellular matrix in PMN, bacterial magnetosomes (Mag) are further hitchhiked on the surface of C NE to form C NEMag , which can repolarize macrophages from M2 to M1 phenotype for further disruption of PMN formation. A series of in vitro and in vivo assessments have been applied to confirm the effectiveness of C NEMag in suppression of PMN formation and metastasis. This study presents a promising strategy for targeted anti-metastatic therapy in clinics.

Mitochondrial Omi/HtrA2 signaling pathway is involved in neuronal apoptosis in patients with cerebral hemorrhage.

OBJECTIVE: This study aimed to analyze the role of mitochondrial Omi/HtrA2 signaling pathway in neuronal apoptosis in patients with cerebral hemorrhage (CH). METHODS: In this retrospective analysis, the clinical data of 60 patients with CH who received craniotomy or minimally invasive intracranial hematoma (MIIH) were included in the case group, which was sub-divided into a craniotomy group (n=22) and a minimally invasive group (n=38) depending on the type of surgery. The brain tissue specimens of the above patients were retained in the surgical specimen repository of Yuhuan Second People's Hospital. Another 15 normal brain tissue samples retained in the surgical specimen repository were included in the normal group. The expression levels of Omi/HtrA2, X-linked inhibitor of apoptosis protein (XIAP), poly-adenosine diphosphate-ribose polymerase (PARP), pro-caspase 3, and pro-caspase 9 were determined using Western blotting. RESULTS: The case group exhibited a higher proportion of neuronal apoptosis, higher expression levels of Omi/HtrA2, PARP, and pro-caspase 3 and 9, higher activities of caspase 3 and caspase 9 (P < 0.05), and lower XIAP expression (P < 0.05) in brain tissue than the normal group. The proportion of neuronal cell apoptosis in brain tissues was positively correlated with the expression of Omi/HtrA2, PARP, and pro-caspase 3 and pro-caspase 9 (r > 0, P < 0.05), and the activity of caspase 3 and caspase 9 was negatively correlated with XIAP expression (r < 0, P < 0.05). Compared with the craniotomy group, the minimally invasive group demonstrated higher efficacy and hematoma removal rate, shorter hematoma removal time, hematoma drainage time, operation time, and hospital stay, less intraoperative bleeding, and lower postoperative complication rates (P < 0.05). The minimally invasive group showed higher expression level of serum XIAP and lower levels of serum caspase 3 and caspase 9 than the craniotomy group (P < 0.05). CONCLUSIONS: Mitochondrial Omi/HtrA2 signaling pathway may be involved in neuronal apoptosis. MIIH has the advantages of high efficacy, high hematoma clearance rate, and few complications for the treatment of CH.

Development and clinical application of a liquid chromatography-tandem mass spectrometry-based assay to quantify eight tyrosine kinase inhibitors in human plasma.

Introduction: Tyrosine kinase inhibitors (TKIs) are widely used in tumor treatment. The detection of these medicines by liquid chromatography-tandem mass spectrometry (LC-MS/MS) can avoid the interference of structurally similar compounds. Objectives: This study aimed to develop and validate a new LC-MS/MS assay for the quantification of eight tyrosine kinase inhibitors in human plasma and to preliminarily evaluate the clinical utility of the therapeutic drug monitoring method. Methods: Plasma samples were prepared by simple protein precipitation and separated using an ultra-high-performance reversed phase column. Detection was achieved using a triple quadrupole mass spectrometer in the positive ionization mode. The assay was validated against standard guidelines. We reviewed and analyzed the results of 268 plasma samples obtained from patients administered imatinib and other TKIs collected from January 2020 to November 2021 at Zhongshan Hospital. The analytes were separated and quantified within 3.5 min. Results: The newly developed method demonstrated linearity for the detected drug concentration in the range of 20 to 2000 ng/ml for gefitinib (r2 = 0.991) and crizotinib (r2 = 0.992), 50 to 5000 ng/ml for nilotinib (r2 = 0.991) and imatinib (r2 = 0.995), 1500-150,000 ng/ml for vemurafenib (r2 = 0.998), 1000-100,000 ng/ml for pazopanib (r2 = 0.993), 0.5-100 ng/ml for axitinib (r2 = 0.992) and 5-500 ng/ml for sunitinib (r2 = 0.991) and N-desethyl sunitinib (r2 = 0.998). The lower limit of quantification (LLOQ) was 20 ng/ml for gefitinib and crizotinib, 50 ng/ml for nilotinib and imatinib, 1500 ng/ml for vemurafenib, 1000 ng/ml for pazopanib, 0.5, and 5 ng/ml for sunitinib and N-desethyl sunitinib, respectively. Specificity, precision, accuracy, and stability were tested, and met the requirements of the guidelines. At the same dose, there was no significant difference in plasma drug concentration between the original imatinib medicine and the generic medicine after patent expiration. Conclusion: We developed a sensitive and reliable method for the quantification of eight TKIs.

Upconversion nanoparticle-based fluorescence resonance energy transfer sensing platform for the detection of cathepsin B activity in vitro and in vivo.

A simple fluorescence resonance energy transfer (FRET) sensing platform (termed as USP), comprised of upconversion nanoparticles (UCNPs) as the energy donor and Cy5 as the energy acceptor, has been synthesized for cathepsin B (CTSB) activity detection in vitro and in vivo. When Cy5-modified peptide substrate (peptide-Cy5) of CTSB is covalently linked on the surface of UCNPs, the FRET between the UCNPs (excitation: 980 nm; emission: 541 nm/655 nm) and Cy5 (excitation: 645 nm) leads to a reduction in the red upconversion luminescence (UCL) signal intensity of UCNPs. Cy5 can be liberated from UCNPs in the presence of CTSB through the cleavage of peptide-Cy5 by CTSB, leading to the recovery of the red UCL signal of UCNPs. Because the green UCL signal of UCNPs remains constant during the CTSB digestion, it can be considered as an internal reference. The findings demonstrate the ability of USP to detect CTSB with the linear detection ranges of 1 to 100 ng·mL-1 in buffer and 2 × 103 to 1 × 105 cells in 0.2 mL cell lysates. The limits of detection (LODs) are 0.30 ng·mL-1 in buffer and 887 cells in 0.2 mL of cell lysates (S/N = 3). The viability of USP to detect CTSB activity in tumor-bearing mice is has further been investigated using in vivo fluorescent imaging.

Universal Detection and Imaging for Multiple Targets by Coupling Target-Primed Nicking-Enhanced Rolling Circle Amplification with Self-Powered DNAzyme Walker.

Although promising in monitoring low-abundance analytes, most of the DNAzyme walker is only responsive to a specific target. Herein, a universal, ready-to-use platform is developed by coupling nicking-enhanced rolling circle amplification and a self-powered DNAzyme walker (NERSD). It addressed the issues that DNAzyme strands need to be specifically designed for different biosensing system, allowing highly sensitive analysis of various targets with the same DNAzyme walker components. It is also specific owing to target-dependent ligation of the padlock probe and precise cleavage of a substrate by a DNAzyme strand. As typically demonstrated, the strategy has an equivalent capacity with the qRT-PCR kit in distinguishing plasma miR-21 levels of breast cancer patients from normal subjects and is able to differentiate intracellular miR-21 and ATP levels by confocal imaging. The approach characteristic of programmability, flexibility, and generality indicated the potential in all kinds of biosensing and imaging platform.

Oxygen vacancy-enhanced catalytic activity of hyaluronic acid covered-biomineralization nanozyme for reactive oxygen species-augmented antitumor therapy.

Insufficient hydrogen peroxide content in tumor cells, unsuitable pH and low efficiency of commonly used metal catalysts severely affect the efficiency of chemodynamic therapy, resulting in unsatisfactory efficacy of chemodynamic therapy alone. For this purpose, we designed a composite nanoplatform capable of targeting tumors and selectively degrading in the tumor microenvironment (TME) to address these issues. In this work, we synthesized Au@Co3O4 nanozyme inspired by crystal defect engineering. The addition of Au determines the formation of oxygen vacancies, accelerates electron transfer, and enhances redox activity, thus significantly enhancing the superoxide dismutase (SOD)-like and catalase (CAT)-like catalytic activities of the nanozyme. Subsequently, we camouflaged the nanozyme using a biomineralized CaCO3 shell to avoid damage to normal tissues by the nanozyme while effectively encapsulating the photosensitizer IR820, and finally the tumor targeting ability of the nanoplatform was enhanced by the modification of hyaluronic acid. Under near-infrared (NIR) light irradiation, the Au@Co3O4@CaCO3/IR820@HA nanoplatform not only visualizes the treatment with multimodal imaging, but also plays a photothermal sensitizing role through various strategies, while enhancing the enzyme catalytic activity, cobalt ion-mediated chemodynamic therapy (CDT) and IR820-mediated photodynamic therapy (PDT), and achieving the synergistic enhancement of reactive oxygen species (ROS) generation.

Chemosensor with Ultra-High Fluorescence Enhancement for Assisting in Diagnosis and Resection of Ovarian Cancer.

Fluorescence imaging-guided diagnostics is one of the most promising approaches for facile detection of tumors in situ owing to its simple operation and non-invasiveness. As a crucial biomarker for primary ovarian cancers, ß-galactosidase (ß-gal) has been demonstrated to be the significant molecular target for visualization of ovarian tumors. Herein, a membrane-permeable fluorescent chemosensor (namely, LAN-ßgal) was synthesized for ß-gal-specific detection using the d-galactose residue as a specific recognition unit and LAN-OH (ΦF = 0.47) as a fluorophore. After ß-gal was digested, the fluorescence of the initially quenched LAN-ßgal (ΦF < 0.001) was enhanced by up to more than 2000-fold, which exceeded the fluorescence enhancement of other previously reported probes. We also demonstrated that the chemosensor LAN-ßgal could visualize endogenous ß-gal and distinguish ovarian cancer cells from normal ovarian cells. Further, the chemosensor LAN-ßgal was successfully applied to visualize the back tumor-bearing mouse model and peritoneal metastatic ovarian cancer model in vivo. More importantly, through in situ spraying, the proposed chemosensor was successfully employed to assist in the surgical resection of ovarian cancer tumors due to its high tumor-to-normal (T/N) tissue fluorescence ratio of 218. To the best of our knowledge, this is the highest T/N tissue fluorescence ratio ever reported. We believe that the LAN-ßgal chemosensor can be utilized as a new tool for the clinical diagnosis and treatment of ovarian cancer.

Profiling of Multiple Matrix Metalloproteinases Activities in the Progression of Osteosarcoma by Peptide Microarray-Based Fluorescence Assay on Polymer Brush-Coated Zinc Oxide Nanorod Substrate.

Peptide microarray provides the ability to miniaturize, parallelize, and automate high-throughput screening substrate specificities of enzymes, profiling of multiple enzyme activities, discovery of disease biomarkers, and development of drugs. Matrix metalloproteinases (MMPs) are demonstrated as important biomarkers of tumor invasion and metastasis. Herein, a peptide microarray-based fluorescence assay is proposed to profile multiple MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13) activities in the culture medium of four human osteosarcoma (OS) cells and in the progression of OS by using the mouse-bearing xenograft OSs including U-2OS and Saos-2 human. This method has excellent selectivity and sensitivity, which enables to detect the activities of cellular secreted MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and MMP-13 with limit of detection downs to 10 pM, 30 pM, 113 pM, 13 pM, 93 pM, and 12 pM, respectively. Furthermore, it is demonstrated that the activity pattern of MMPs is serum closely relevant to the disease progression and type of tumor.

Peptide Array-Based In Situ Fluorescence Assay for Profiling Multiple Matrix Metalloproteinase Activities.

Peptide array-based in situ fluorescence assay is a reliable and efficient technique for high-throughput profiling and localization of enzyme activity. Here, peptide array is fabricated by spotting five specific MMPs (MMP-2, MMP-3, MMP-7, MMP-9, and MMP-14) peptide substrates containing FAM/Dabcyl fluorescent resonance energy transfer (FRET) pair on the surface of cell monolayers or tissue sections. MMP activities are determined in situ by the fluorescence intensity of stained cells/tissues due to the cellular internalization of hydrolyzed peptide fragments with FAM moieties. Identification of MMP expression patterns of cells, highly sensitive determination of MMP activities in cell monolayer (as low as hundreds of cells per square centimeter), and evaluation of inhibition potencies of six compounds toward five MMPs are achieved by this method. Five MMP activities in the localized parts of 32 thyroid tissues are also well profiled without separation or extraction procedures.

Selective profiling of liver-related specific proteins based on sofosbuvir-modified magnetic separation material.

It has great significance in profiling specific proteins throughout for better understanding of complex pathological processes and in-depth pharmacological studies. In this work, an efficient protein profiling strategy was developed based on the specific protein-drug interaction. Sofosbuvir (SOF), as a first-line drug for the treatment of hepatitis C, was modified onto the surface of nanoparticles through stable chemical bonds to fabricate a novel magnetic separation material denoted as Fe3O4@SiO2@PAA@SOF. With sequence coverage as the screening parameter, nine proteins were profiled from fetal bovine serum (FBS) of which eight were liver related. Similarly, the strategy was applied to hepatocellular carcinoma (HCC) patient serum. Eight proteins were profiled and all of them were liver related, demonstrating the superb specificity and selectivity of this strategy for profiling liver-related proteins by virtue of protein-SOF interaction. When serum proteins from HCC patients were compared to those from healthy people, one unique differential protein (D3DQX7) was profiled, which was liver related and was a potential target for ameliorating liver diseases. For further research, this material design concept and protein profiling strategy can be extended to employ other drugs for corresponding studies. Sofosbuvir, as a therapeutic drug for liver diseases, was modified onto the surface of magnetic nanoparticles to fabricate the specific selective separation material (Fe3O4@SiO2@PAA@SOF). Based on protein-SOF interaction, the material was applied to adsorb specific proteins from different serum samples. After MS analysis, specific proteins, most of which were liver related, were successfully profiled from FBS and HCC patient serum, fully demonstrating the superb specificity and selectivity of this protein profiling strategy.

Cancer Serum Atlas-Supported Precise Pan-Targeted Proteomics Enable Multicancer Detection.

The wide dynamic range of serum proteome restrained discovery of clinically interested proteins in large cohort studies. Herein, we presented a high-sensitivity, high-throughput, and precise pan-targeted serum proteomic strategy for highly efficient cancer serum proteomic research and biomarker discovery. We constructed a resource of over 2000 cancer-secreted proteins, and the standard MS assays and spectra of at least one synthetic unique peptide per protein were acquired and documented (Cancer Serum Atlas, www.cancerserumatlas.com). Then, the standard peptide-anchored parallel reaction monitoring (SPA-PRM) method was developed with support of the Cancer Serum Atlas, achieving precise quantification of cancer-secreted proteins with high throughput and sensitivity. We directly quantified 325 cancer-related serum proteins in 288 serums of four cancer types (liver, stomach, lung, breast) and controls with the pan-targeted strategy and discovered considerable potential biomarker benefits for early detection of cancer. Finally, a proteomic-based multicancer detection model was built, demonstrating high sensitivity (87.2%) and specificity (100%), with 73.8% localization accuracy for an independent test set. In conclusion, the Cancer Serum Atlas provides a wide range of potential biomarkers that serve as targets and standard assays for systematic and highly efficient serological studies of cancer. The Cancer Serum Atlas-supported pan-targeted proteomic strategy enables highly efficient biomarker discovery and multicancer detection and thus can be a powerful tool for liquid biopsy.

Genomic and Metabolomic Analyses of the Medicinal Fungus Inonotus hispidus for Its Metabolite's Biosynthesis and Medicinal Application.

Inonotus hispidus mushroom is a traditional medicinal fungus with anti-cancer, antioxidation, and immunomodulatory activities, and it is used in folk medicine as a treatment for indigestion, cancer, diabetes, and gastric illnesses. Although I. hispidus is recognized as a rare edible medicinal macrofungi, its genomic sequence and biosynthesis potential of secondary metabolites have not been investigated. In this study, using Illumina NovaSeq combined with the PacBio platform, we sequenced and de novo assembled the whole genome of NPCB_001, a wild I. hispidus isolate from the Aksu area of Xinjiang Province, China. Comparative genomic and phylogenomic analyses reveal interspecific differences and evolutionary traits in the genus Inonotus. Bioinformatics analysis identified candidate genes associated with mating type, polysaccharide synthesis, carbohydrate-active enzymes, and secondary metabolite biosynthesis. Additionally, molecular networks of metabolites exhibit differences in chemical composition and content between fruiting bodies and mycelium, as well as association clusters of related compounds. The deciphering of the genome of I. hispidus will deepen the understanding of the biosynthesis of bioactive components, open the path for future biosynthesis research, and promote the application of Inonotus in the fields of drug research and functional food manufacturing.