Aptamer-Based Enforced Phosphatase-Recruiting Chimeras Inhibit Receptor Tyrosine Kinase Signal Transduction.

Aberrant phosphorylation of receptor tyrosine kinases (RTKs) is usually involved in tumor initiation, progression, and metastasis. However, developing specific and efficient molecular tools to regulate RTK phosphorylation remains a considerable challenge. In this study, we reported novel aptamer-based chimeras to inhibit the phosphorylation of RTKs, such as c-Met and EGFR, by enforced recruitment of a protein tyrosine phosphatase receptor type F (PTPRF). Our studies revealed that aptamer-based chimeras displayed a generic and potent inhibitory effect on RTK phosphorylation induced by growth factor or auto-dimerization in different cell lines and modulated cell biological behaviors by recruiting PTPRF. Furthermore, based on angstrom accuracy of the DNA duplex, the maximum catalytic radius of PTPRF was determined as ∼25.84 nm, providing a basis for the development of phosphatase-recruiting strategies. Taken together, our study provides a generic methodology not only for selectively mediating RTK phosphorylation and cellular biological processes but also for developing novel therapeutic drugs.

Efficient Strategy to Discover DNA Aptamers Against Low Abundance Cell Surface Proteins in Scarce Samples.

Molecular recognition probes targeting cell surface proteins such as aptamers play crucial roles in precise diagnostics and therapy. However, the selection of aptamers against low-abundance proteins in situ on the cell surface, especially in scarce samples, remains an unmet challenge. In this study, we present a single-round, single-cell aptamer selection method by employing a digital DNA sequencing strategy, termed DiDS selection, to address this dilemma. This approach incorporates a molecular identification card for each DNA template, thereby mitigating biases introduced by multiple PCR amplifications and ensuring the accurate identification of aptamer candidates. Through DiDS selection, we successfully obtained a series of high-quality aptamers against cell lines, clinical specimens, and neurons. Subsequent analyses for target identification revealed that aptamers derived from DiDS selection exhibit recognition capabilities for proteins with varying abundance levels. In contrast, multiple rounds of selection resulted in the enrichment of only one aptamer targeting a high-abundance target. Moreover, the comprehensive profiling of cell surfaces at the single-cell level, utilizing an enriched aptamer pool, revealed unique molecular patterns for each cell line. This streamlined approach holds promise for the rapid generation of specific recognition molecules targeting cell surface proteins across a broad range of expression levels and expands its applications in cell profiling, specific probe identification, biomarker discovery, etc.

Streamlining RNA Aptamer Selection via Unique Molecular Identifiers and High-Throughput Sequencing.

Aptamers are valuable tools for applications such as cell imaging, drug delivery, and therapeutics. RNA aptamers, in particular, exhibit complex structural diversity and flexibility, affording higher affinity and specificity, broader target recognition, and easier chemical modification compared with DNA aptamers. However, traditional selection methods for RNA aptamers are time-consuming and involve numerous rounds of screening, thus limiting their widespread application. To overcome this challenge, we propose an efficient truncated selection approach termed ID-SELEX. This method incorporates a molecular identification marker whereby each template is labeled with a unique molecular identifier, or UMI. Such incorporation helps mitigate biases introduced by multiple polymerase chain reaction (PCR) amplification during high-throughput sequencing, ensuring accurate identification of aptamer candidates. Utilizing ID-SELEX, we successfully identified a panel of high-quality aptamers targeting the human colon cancer cell line HCT-8 in just 2 rounds of selection. Furthermore, we demonstrated the versatility of this strategy by selecting 6 RNA aptamers targeting mouse myoblast cell line C2C12 with only one round of selection. In summary, RNA aptamer selection based on ID-SELEX utilizes high-throughput sequencing and UMI labeling to enable the rapid screening of RNA aptamers across human and murine cell lines. As such, ID-SELEX has the potential to facilitate RNA aptamer discovery, providing a novel molecular tool for biomedical research, clinical applications, and precision medicine.

Identification of the Binding Site between Aptamer sgc8c and PTK7.

Aptamers are single-stranded RNA or DNA molecules that can specifically bind to targets and have found broad applications in cancer early-stage detection, accurate drug delivery, and precise treatment. Although various aptamer screening methods have been developed over the past several decades, the accurate binding site between the target and the aptamer cannot be characterized during a typical aptamer screening process. In this research, we chose a widely used aptamer screened by our group, sgc8c, and its target protein tyrosine kinase 7 (PTK7) as the model aptamer and target and tried to determine the binding site between aptamer sgc8c and PTK7. Through sequential protein truncation, we confirmed that the exact binding site of sgc8c was within the region of Ig 3 to Ig 4 in the extracellular domain of PTK7. Using in vitro expressed Ig (3-4), we successfully acquired the crystal of an sgc8c-Ig (3-4) binding complex. The possible sgc8c-binding amino acid residues on PTK7 and PTK7-binding nucleotide residues on sgc8c were further identified and simulated by mass spectrometry and molecular dynamics simulation and finally verified by aptamer/protein truncation and mutation.

A Bispecific Chimeric Aptamer Design Platform Based on c-MET Aptamer with a Replaceable Redundant Region.

Molecular engineering enables the creation of aptamers with novel functions, but the prerequisite is a deep understanding of their structure and recognition mechanism. The cellular-mesenchymal epithelial transition factor (c-MET) is garnering significant attention due to the critical role of the c-MET/HGF signaling pathway in tumor development and invasion. This study reports a strategy for constructing novel chimeric aptamers that bind to both c-MET and other specific proteins. c-MET was identified to be the molecular target of a DNA aptamer, HF3-58, selected through cell-SELEX. The binding structure and mechanism of HF3-58 with c-MET were systematically studied, revealing the scaffold, recognition, and redundancy regions. Through molecular engineering design, the redundancy region was replaced with other aptamers possessing stem-loop structures, yielding novel chimeric aptamers with bispecificity for binding to c-MET and specific proteins. A chimeric bispecific aptamer HF-3b showed the ability to mediate the adhesion of T-cells to tumor cells, suggesting the prospective utility in tumor immunotherapy. These findings suggest that aptamer HF3-58 can serve as a molecular engineering platform for the development of diverse multifunctional ligands targeting c-MET. Moreover, comprehensive understanding of the binding mechanisms of aptamers will provide guidance for the design of functional aptamers, significantly expanding their potential applications.

[Effects and mechanisms of Gegen Qinlian Decoction in inhibiting M1 polarization of macrophages under inflammatory hypoxia microenvironment].

To explore the effect and mechanism of Gegen Qinlian Decoction(GQD) in inhibiting M1 polarization of macrophages under inflammatory hypoxia by simulating intestinal hypoxia microenvironment in vitro. A tri-gas incubator was used to simulate normal physiological hypoxia of the colon and inflammatory hypoxia microenvironments of ulcerative colitis(UC). RAW264.7 macrophages were divided into 18.5% O_(2 )(normoxia group), 4% O_2(physiological hypoxia group), and 1% O_2(inflammatory hypoxia group), and they were induced by lipopolysaccharide(LPS) for 24 h. M1 polarization was detected by flow cytometry. Under the condition of 1% inflammatory hypoxia, they were divided into blank group, model group, and GQD-containing serum low, medium, and high dose groups. Flow cytometry was used to detect M1 polarization marker CD86, and ELISA was used to detect the expression of tumor necrosis factor-α(TNF-α) and interleukin-1ß(IL-1ß) in cell supernatant. The mRNA expression of hypoxia-inducible factor-1α(HIF-1α), TNF-α, and IL-1ß was detected by qRT-PCR. Western blot was used to detect the expression of HIF-1α/nuclear transcription factor-κB(NF-κB) signaling pathway-related proteins. The nuclear translocation of NF-κB p65 was detected by immunofluorescence. The results showed that the positive rate of CD86 in the 1% O_2 group was the highest. Under the condition of 1% inflammatory hypoxia, compared with the blank group, the expression of CD86, TNF-α, IL-1ß, and HIF-1α in the model group increased. Compared with the model group, each group of GQD could reduce the expression of CD86, TNF-α, IL-1ß, and HIF-1α. Compared with the blank group, the protein expression of HIF-1α, NF-κB p65, p-IKKα/ß, and p-IκBα in the model group increased. Compared with the model group, the protein expression of HIF-1α, NF-κB p65, p-IKKα/ß, and p-IκBα in GQD groups was significantly decreased. Compared with the blank group, NF-κB p65 in the model group entered the nucleus significantly. Compared with the model group, the nuclear expression of NF-κB p65 was decreased in each GQD group. Studies have shown that GQD may protect the intestine by down-regulating the HIF-1α/NF-κB signaling pathway to inhibit M1 polarization of macrophages and secretion of related inflammatory factors under 1% inflammatory hypoxia.

Generation of an Aptamer Targeting Receptor-Type Tyrosine-Protein Phosphatase F.

Cell-SELEX is a powerful tool to generate aptamers that specifically bind the native molecules on living cells. Here, we report an aptamer ZAJ4a generated by cell-SELEX. The molecular target of ZAJ4a was pulled down by the enriched cell-SELEX pool and identified to be the receptor-type tyrosine-protein phosphatase F (PTPRF) through a stable isotope labeling using amino acids in cell culture (SILAC)-based quantitative proteomic method. ZAJ4a showed high binding affinity with nanomolar range to cancer cells expressing PTPRF. Meanwhile, PTPRF was proven to highly express on several cancer cell lines using ZAJ4a as a molecular probe and to highly express in many kinds of cancer samples using gene expression profiling interactive analysis (GEPIA2) from the TCGA and GTEx databases. These results indicate that the aptamer generated by cell-SELEX showed good specificity at the molecular level. This cell-SELEX and target identification strategies show great potential for identifying biomarkers on the cell surface.

Distance-Triggered Distinct Aryl Migrations on Azidodiboranes.

Derived from structurally similar precursors, two different azidodiboranes went through distinct aryl migration reactions triggered by different boron-boron separation distances. Biphenylene based diborane with a shorter boron-boron distance underwent heterolateral aryl migration to form a seven-membered azadiborepin, while xanthrene based diborane with a longer boron-boron distance afforded a stable bis-azidoborane scaffold. The pyrolysis of such a bis-azidoborane led to eight-membered oxazadiborocine through homolateral aryl migration and subsequent [3+2] cycloaddition. Density functional theory (DFT) calculations unveiled that the boron-boron separation distances were the intrinsic factors for the distinct migrations.

Neutral Boryl Radicals in Mixed-Valent B(III) Br-B(II) Adducts.

The general strategies to stabilize a boryl radical involve single electron delocalization by π-system and the steric hinderance from bulky groups. Herein, a new class of boryl radicals is reported, with intramolecular mixed-valent B(III) Br-B(II) adducts ligated by a cyclic (alkyl)(amino)carbene (CAAC). The radicals feature a large spin density on the boron center, which is ascertained by EPR spectroscopy and DFT calculations. Structural and computational analyses revealed that the stability of radical species was assisted by the CAAC ligand and a weak but significant B(III)Br-B(II) interaction, suggesting a cooperative avenue for stabilization of boryl radicals. Two-electron reduction of these new boryl radicals provides C-H insertion products via a borylene intermediate.

Nitrogen fixation and transformation with main group elements.

Nitrogen fixation is essential for the maintenance of life and development of society, however, the large bond dissociation energy and nonpolarity of the triple bond constitute a considerable challenge. The transition metals, by virtue of their combination of empty and occupied d orbitals, are prevalent in the nitrogen fixation studies and are continuing to receive a significant focus. The main group metals have always been considered incapable in dinitrogen activation owing to the absence of energetically and symmetrically accessible orbitals. The past decades have witnessed significant breakthroughs in the dinitrogen activation with the main group elements and compounds via either matrix isolation, theoretical calculations or synthetic chemistry. The successful reactions of the low-valent species of the main group elements with inert dinitrogen have been reported via the π back-donation from either the d orbitals (Ca, Sr, Ba) or p orbitals (Be, B, C…). Herein, the significant achievements have been briefly summarized, along with predicting the future developments.

Timing of Surgery and Treatment Options for Congenital Ptosis in Children: A Narrative Review of the Literature.

Congenital ptosis has varying degrees of impact on the visual development or psychological health of a child depending on its severity. Some controversies and misconceptions remain regarding the management of congenital ptosis in children. Particularly, the accurate diagnosis of the severity of congenital ptosis in younger children, assessment of the visual developmental status of the child, optimal timing of surgery, and treatment choice are still issues in clinical practice that need to be explored. This report presents a comprehensive review of these aspects of the correction of congenital ptosis to provide a valuable reference for clinical practice. Our review shows that currently used surgical procedures for ptosis may result in over- or under-correction to varying degrees. The differences may be due to the physical condition and age of the child and the degree of cooperation during preoperative examination and assessment, resulting in inaccurate results. Alternatively, intraoperative swelling and bleeding may lead to errors in the values recorded by the surgeon. LEVEL OF EVIDENCE IV: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Structural Optimization and Interaction Study of a DNA Aptamer to L1 Cell Adhesion Molecule.

The L1 cell adhesion molecule (L1CAM) plays important roles in the development and plasticity of the nervous system as well as in tumor formation, progression, and metastasis. New ligands are necessary tools for biomedical research and the detection of L1CAM. Here, DNA aptamer yly12 against L1CAM was optimized to have much stronger binding affinity (10-24 fold) at room temperature and 37 °C via sequence mutation and extension. This interaction study revealed that the optimized aptamers (yly20 and yly21) adopted a hairpin structure containing two loops and two stems. The key nucleotides for aptamer binding mainly located in loop I and its adjacent area. Stem I mainly played the role of stabilizing the binding structure. The yly-series aptamers were demonstrated to bind the Ig6 domain of L1CAM. This study reveals a detailed molecular mechanism for the interaction between yly-series aptamers and L1CAM and provides guidance for drug development and detection probe design against L1CAM.

[Optimization of ethanol reflux extraction process of Ziziphi Spinosae Semen- Schisandrae Sphenantherae Fructus based on network pharmacology combined with response surface methodology].

The present study optimized the ethanol extraction process of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus drug pair by network pharmacology and Box-Behnken method. Network pharmacology and molecular docking were used to screen out and verify the potential active components of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus, and the process evaluation indexes were determined in light of the components of the content determination under Ziziphi Spinosae Semen and Schisandrae Sphenantherae Fructus in the Chinese Pharmacopoeia(2020 edition). The analytic hierarchy process(AHP) was used to determine the weight coefficient of each component, and the comprehensive score was calculated as the process evaluation index. The ethanol extraction process of Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus was optimized by the Box-Behnken method. The core components of the Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus drug pair were screened out as spinosin, jujuboside A, jujuboside B, schisandrin, schisandrol, schisandrin A, and schisandrin B. The optimal extraction conditions obtained by using the Box-Behnken method were listed below: extraction time of 90 min, ethanol volume fraction of 85%, and two times of extraction. Through network pharmacology and molecular docking, the process evaluation indexes were determined, and the optimized process was stable, which could provide an experimental basis for the production of preparations containing Ziziphi Spinosae Semen-Schisandrae Sphenantherae Fructus.

[Metabolomic study on urine of chronic inflammation rats treated with Buyang Huanwu Decoction based on UPLC-Q-TOF-MS].

The study investigated the effect of Buyang Huanwu Decoction(BYHWD) on endogenous biomarkers in the urine of rats with chronic inflammation induced by lipopolysaccharide(LPS) using ultra-high performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry(UPLC-Q-TOF-MS), aiming to elucidate the molecular mechanism underlying the therapeutic effect of BYHWD on chronic inflammation from a metabolomics perspective. Male SD rats were randomly divided into a normal group, a model group, and low-, medium-, and high-dose BYHWD groups(7.5, 15, and 30 g·kg~(-1)). The model group and BYHWD groups received tail intravenous injection of LPS(200 µg·kg~(-1)) on the first day of each week, followed by oral administration of BYHWD once a day for four consecutive weeks. Urine samples were collected at the end of the administration period, and UPLC-Q-TOF-MS was used to analyze the metabolic profiles of the rat urine in each group. Multivariate statistical analysis methods such as principal component analysis(PCA), partial least squares-discriminant analysis(PLS-DA), and orthogonal partial least squares-discriminant analysis(OPLS-DA) were used to analyze the effect of BYHWD on endogenous metabolites. One-way ANOVA and variable importance for the projection(VIP) were used to screen for potential biomarkers related to chronic inflammation. The identified biomarkers were subjected to pathway and enrichment analysis using MetaboAnalyst 5.0. A total of 25 potential biomarkers were screened and identified in the rat urine in this experiment. Compared with the normal group, the model group showed significant increases in the levels of 14 substances(P<0.05) and significant decreases in the levels of 11 substances(P<0.05). BYHWD was able to effectively reverse the trend of most endogenous biomarkers. Compared with the model group, BYHWD significantly down-regulated 13 biomarkers(P<0.05) and up-regulated 10 biomarkers(P<0.05). The metabolic products were mainly related to the biosynthesis of pantothenic acid and coenzyme A, tryptophan metabolism, retinol metabolism, and propionate metabolism. BYHWD has therapeutic effect on chronic inflammation induced by LPS, which may be related to its ability to improve the levels of endogenous metabolites, enhance the body's anti-inflammatory and antioxidant capabilities, and restore normal metabolic activity.

Cesium Amide-Catalyzed Selective Deuteration of Benzylic C-H Bonds with D2 and Application for Tritiation of Pharmaceuticals.

Hydrogen isotope exchange (HIE) represents one of the most attractive labeling methods to synthesize deuterium- and tritium-labeled compounds. Catalytic HIE methods that enable site-selective C-H bond activation and exchange labeling with gaseous isotopes D2 and T2 are of vital importance, in particular for high-specific-activity tritiation of pharmaceuticals. As part of our interest in exploring s-block metals for catalytic transformations, we found CsN(SiMe3 )2 to be an efficient catalyst for selective HIE of benzylic C-H bonds with D2 gas. The reaction proceeds through a kinetic deprotonative equilibrium that establishes an exchange pathway between C-H bonds and D2 gas. By virtue of multiple C-H bonds activation and high activity (isotope enrichment up to 99 %), the simple cesium amide catalyst provided a very powerful and practically convenient labeling protocol for synthesis of highly deuterated compounds and high-specific-activity tritiation of pharmaceuticals.

Aptamer-Based Cell Nucleus Imaging via Expansion Microscopy.

Expansion microscopy (ExM) is a newly developed technology in recent years that enables nanoscale imaging under conventional microscopes. Herein, we report an aptamer-based ExM imaging strategy. A nucleus-targeting aptamer Ch4-1 was chemically labeled with a dye and an acrydite at each end to perform the functions of molecular recognition, fluorescence reporting, and gel anchoring. After binding cell nucleus, the dual labeled aptamer Ac-Ch4-1-FAM directly participated in gelation and anchored in polyacrylamide gel. After expanding the gel, high-resolution imaging was achieved by confocal microscopy. Multicolor ExM imaging was also realized by combining Ac-Ch4-1-FAM, antibodies and fluorescent dyes. This aptamer-based ExM could clearly image the chromatin morphology at different mitotic stages. The expansion process is simple and the aptamer labeling is easy. The aptamer-based ExM holds great promise in super-resolution imaging of cells and tissues.

Urokinase-type plasminogen activator receptor (uPAR) as a therapeutic target in cancer.

Urokinase-type plasminogen activator receptor (uPAR) is an attractive target for the treatment of cancer, because it is expressed at low levels in healthy tissues but at high levels in malignant tumours. uPAR is closely related to the invasion and metastasis of malignant tumours, plays important roles in the degradation of extracellular matrix (ECM), tumour angiogenesis, cell proliferation and apoptosis, and is associated with the multidrug resistance (MDR) of tumour cells, which has important guiding significance for the judgement of tumor malignancy and prognosis. Several uPAR-targeted antitumour therapeutic agents have been developed to suppress tumour growth, metastatic processes and drug resistance. Here, we review the recent advances in the development of uPAR-targeted antitumor therapeutic strategies, including nanoplatforms carrying therapeutic agents, photodynamic therapy (PDT)/photothermal therapy (PTT) platforms, oncolytic virotherapy, gene therapy technologies, monoclonal antibody therapy and tumour immunotherapy, to promote the translation of these therapeutic agents to clinical applications.

Review targeted drug delivery systems for norcantharidin in cancer therapy.

Norcantharidin (NCTD) is a demethylated derivative of cantharidin (CTD), the main anticancer active ingredient isolated from traditional Chinese medicine Mylabris. NCTD has been approved by the State Food and Drug Administration for the treatment of various solid tumors, especially liver cancer. Although NCTD greatly reduces the toxicity of CTD, there is still a certain degree of urinary toxicity and organ toxicity, and the poor solubility, short half-life, fast metabolism, as well as high venous irritation and weak tumor targeting ability limit its widespread application in the clinic. To reduce its toxicity and improve its efficacy, design of targeted drug delivery systems based on biomaterials and nanomaterials is one of the most feasible strategies. Therefore, this review focused on the studies of targeted drug delivery systems combined with NCTD in recent years, including passive and active targeted drug delivery systems, and physicochemical targeted drug delivery systems for improving drug bioavailability and enhancing its efficacy, as well as increasing drug targeting ability and reducing its adverse effects.

Cell-SELEX-based selection of ssDNA aptamers for specifically targeting BRAF V600E-mutated melanoma.

Malignant melanoma is regarded as the most aggressive form of skin cancer, and is responsible for most death caused by skin cancer. BRAF mutations occur in approximately 40-50% of melanomas, with V600E being the most common mutation. Testing for BRAF mutations and targeted therapy have markedly improved long-term survival for patients with BRAF-mutated melanoma. Here, we report two aptamers, CH1 and CH5 generated by Cell-SELEX, against BRAF V600E-mutated human melanoma cells A375. The two aptamers exhibited high affinity to target cells with low dissociation constants (Kd) in the nanomolar range. Furthermore, the binding of two aptamers to target cells was independent of incubation temperature, and their molecular targets were demonstrated to be membrane proteins on the cell surface. We also demonstrated that aptamer CH1 was able to bind to metastatic colorectal cancer cells harboring BRAF V600E mutation, indicating a relationship between aptamer CH1 and BRAF V600E-related metastatic cancer. Owing to the structure stability and high selectivity to BRAF V600E-mutated targeting cells, aptamer CH1 holds great potential as a molecular probe for the detection of BRAF V600E-mutated metastatic melanoma.

A DNA Aptameric Ligand of Human Transferrin Receptor Generated by Cell-SELEX.

General cancer-targeted ligands that can deliver drugs to cells have been given considerable attention. In this paper, a high-affinity DNA aptamer (HG1) generally binding to human tumor cells was evolved by cell-SELEX, and was further optimized to have 35 deoxynucleotides (HG1-9). Aptamer HG1-9 could be taken up by live cells, and its target protein on a cell was identified to be human transferrin receptor (TfR). As a man-made ligand of TfR, aptamer HG1-9 was demonstrated to bind at the same site of human TfR as transferrin with comparable binding affinity, and was proved to cross the epithelium barrier through transferrin receptor-mediated transcytosis. These results suggest that aptamer HG1-9 holds potential as a promising ligand to develop general cancer-targeted diagnostics and therapeutics.