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.

Syringaresinol attenuates Tau phosphorylation and ameliorates cognitive dysfunction induced by sevoflurane in aged rats.

Cognitive dysfunction following anesthesia with agents such as sevoflurane is a significant clinical problem, particularly in elderly patients. This study aimed to explore the protective effects of the phytochemical syringaresinol (SYR) against sevoflurane-induced cognitive deficits in aged Sprague-Dawley rats and to determine the underlying mechanisms involved. We assessed the impact of SYR on sevoflurane-induced cognitive impairment, glial activation, and neuronal apoptosis through behavioral tests (Morris water maze), immunofluorescence, Western blotting for key proteins involved in apoptosis and inflammation, and enzyme-linked immunosorbent assays for interleukin-1ß, tumor necrosis factor-α, and interleukin-6. SYR treatment mitigated sevoflurane-induced cognitive decline, reduced microglial and astrocyte activation (decreased Iba-1 and GFAP expression), and countered neuronal apoptosis (reduced Bax, cleaved-caspase3, and cleaved-PARP expression). SYR also enhanced Sirtuin-1 (SIRT1) expression and reduced p-Tau phosphorylation; these effects were reversed by the SIRT1 inhibitor EX527. SYR exerts neuroprotective effects on sevoflurane-induced cognitive dysfunction by modulating glial activity, apoptotic signaling, and Tau phosphorylation through the SIRT1 pathway. These findings could inform clinical strategies to safeguard cognitive function in patients undergoing anesthesia.

Exploring the potential of white birch sap: A natural alternative to traditional skin whitening agents with reduced side effects.

Common tyrosinase (TYR) inhibitors used in cosmetics, such as hydroquinone, kojic acid, and arbutin, can cause side effects including erythema, skin peeling, and dryness. Therefore, the development of natural whitening agents that offer excellent permeability, minimal irritation, and high safety has become a primary focus in the field of TYR inhibitors. In this study, we demonstrate that White birch sap (WBS), within a safe concentration range, effectively reduces TYR activity and melanin content in both B16F10 mouse melanoma cells and zebrafish larvae. Importantly, WBS exhibits minimal irritation to neutrophils in fluorescent zebrafish and does not affect the behavior of adult zebrafish. Furthermore, WBS downregulates the gene expression levels of microphthalmia-associated transcription factor, TYR, tyrosinase-related protein-1, and tyrosinase-related protein-2 in B16F10 cells. In conclusion, our research confirms that WBS, a naturally derived substance, offers high safety and mild effects, making it a promising candidate for a skin-whitening agent.

An Aptamer-Functionalized DNA Circuit to Establish an Artificial Interaction between T Cells and Cancer Cells.

Nongenetic strategies that enable control over the cell-cell interaction network would be highly desired, particularly in T cell-based cancer immunotherapy. In this work, we developed an aptamer-functionalized DNA circuit to modulate the interaction between T cells and cancer cells. This DNA circuit was composed of recognition-then-triggering and aggregation-then-activation modules. Upon recognizing target cancer cells, the triggering strand was released to induce aggregation of immune receptors on the T cell surface, leading to an enhancement of T cell activity for effective cancer eradication. Our results demonstrated the feasibility of this DNA circuit for promoting target cancer cell-directed stimulation of T cells, which, consequently, enhanced their killing effect on cancer cells. This DNA circuit, as a modular strategy to modulate intercellular interactions, could lead to a new paradigm for the development of nongenetic T cell-based immunotherapy.

Recent progress of aptamer‒drug conjugates in cancer therapy.

Aptamers are single-stranded DNA or RNA sequences that can specifically bind with the target protein or molecule via specific secondary structures. Compared to antibody-drug conjugates (ADC), aptamer‒drug conjugate (ApDC) is also an efficient, targeted drug for cancer therapy with a smaller size, higher chemical stability, lower immunogenicity, faster tissue penetration, and facile engineering. Despite all these advantages, several key factors have delayed the clinical translation of ApDC, such as in vivo off-target effects and potential safety issues. In this review, we highlight the most recent progress in the development of ApDC and discuss solutions to the problems noted above.

A pH-Responsive Covalent Nanoscale Device Enhancing Temporal and Force Stability for Specific Tumor Imaging.

Approaches to DNA probe-mediated precision medicine have been extensively explored for the diagnosis and treatment of diverse types of cancer. Despite this, simple nanoscale devices with the required recognition specificity and sensitivity for clinical application have remained elusive until now. Here, we report a pH-driven covalent nanoscale device that integrates pH-responsive, switchable structure and proximity-driven covalent cross-linking. A tumor acidic, pH-driven mechanism eliminates "on-target, off-tumor" nonspecific recognition. By manipulating covalent binding to target molecule on the cell surface, this nanodevice avoids binding-then-shedding to improve the sensitivity of tumor recognition. We envision that this pH-driven covalent nanoscale device will inspire more clinical applications toward specific, long-term tumor imaging in the cancer microenvironment.

Concentration Prediction of Polymer Insulation Aging Indicator-Alcohols in Oil Based on Genetic Algorithm-Optimized Support Vector Machines.

The predictive model of aging indicator based on intelligent algorithms has become an auxiliary method for the aging condition of transformer polymer insulation. However, most of the current research on the concentration prediction of aging products focuses on dissolved gases in oil, and the concentration prediction of alcohols in oil is ignored. As new types of aging indicators, alcohols (methanol, ethanol) are becoming prevalent in the aging evaluation of transformer polymer insulation. To address this, this study proposes a prediction model for the concentration of alcohols based on a genetic-algorithm-optimized support vector machine (GA-SVM). Firstly, accelerated thermal aging experiments on oil-paper insulation are conducted, and the concentration of alcohols is measured. Then, the data of the past 4 days of aging are used as the input feature of SVM, and the GA algorithm is utilized to optimize the kernel function parameter and penalty factor of SVM. Moreover, the concentrations of methanol and ethanol are predicted, after which the prediction accuracy of other algorithms and GA-SVM are compared. Finally, an industrial software program for predicting the concentration of methanol and ethanol is established. The results show that the mean square errors (MSE) of methanol and ethanol concentration predictions of the model proposed in this paper are 0.008 and 0.003, respectively. The prediction model proposed in this paper can track changes in methanol and ethanol concentrations well, providing a theoretical basis for the field of alcohol concentration prediction in transformer oil.

Tumor microenvironment (TME)-activatable circular aptamer-PEG as an effective hierarchical-targeting molecular medicine for photodynamic therapy.

Photodynamic therapy (PDT) is an effective and noninvasive therapeutic strategy employing light-triggered singlet oxygen (SO) and reactive oxygen species (ROS) to kill lesional cells. However, for effective in vivo delivery of PDT agent into the cancer cells, various biological obstacles including blood circulation and condense extracellular matrix (ECM) in the tumor microenvironment (TME) need to be overcome. Furthermore, the enormous challenge in design of smart drug delivery systems is meeting the difference, even contradictory required functions, in different steps of the complicated delivery process. To this end, we present that TME-activatable circular pyrochlorophyll A (PA)-aptamer-PEG (PA-Apt-CHO-PEG) nanostructures, which combine the advantages of PEG and aptamer, would be able to realize efficient in vivo imaging and PDT. Upon intravenous (i.v.) injection, PA-Apt-CHO-PEG shows "stealth-like" long circulation in blood compartments without specific recognition capacity, but once inside solid tumor, PA-Apt-CHO-PEG nanostructures are cleaved and then form PA-Apt Aptamer-drug conjugations (ApDCs) in situ, allowing deep penetration into the solid tumor and specific recognition of cancer cells, both merits, considering anticipated future clinical translation of ApDCs.

Molecular domino reactor built by automated modular synthesis for cancer treatment.

Rationale: A cascade, or domino, reaction consists of two, or more, consecutive reactions such that subsequent reactions occur only if some chemical functionality has first been established in the prior step. However, while construction of predesigned and desired molecular domino reactors in a tailored manner is a valuable endeavor, it is still challenging. Methods: To address this challenge, we herein report an aptamer-based photodynamic domino reactor built through automated modular synthesis. The engineering of this reactor takes advantage of the well-established solid-phase synthesis platform to incorporate a photosensitizer into G-quadruplex/ hemin DNAzyme at the molecular level. Results: As a proof of concept, our photodynamic domino reactor, termed AS1411/hemin- pyrochlorophyll A, achieves in vivo photodynamic domino reaction for efficient cancer treatment by using a high concentration of hydrogen peroxide (H2O2) in the tumor microenvironment (TME) to produce O2, followed by consecutive generation of singlet oxygen (1O2) using the pre-produced O2. More specifically, phosphoramidite PA (pyrochlorophyll A) is coupled to aptamer AS1411 to form AS1411-PA ApDC able to simultaneously perform in vivo targeted imaging and photodynamic therapy (PDT). The insertion of hemin into the AS1411 G-quadruplex was demonstrated to alleviate tumor hypoxia by decomposition of H2O2 to produce O2. This was followed by the generation of 1O2 by PA to trigger cascading amplified PDT. Conclusion: Therefore, this study provides a general strategy for building an aptamer-based molecular domino reactor through automated modular synthesis. By proof of concept, we further demonstrate a novel method of achieving enhanced PDT, as well as alleviating TME hypoxia at the molecular level.

Conjugating Aptamer and Mitomycin C with Reductant-Responsive Linker Leading to Synergistically Enhanced Anticancer Effect.

Mitomycin C (MMC) has been using for the treatment of a variety of digestive tract cancers. However, its nonspecific DNA-alkylating ability usually causes severe side effects, thus largely limiting its clinical applications. The utilization of an efficient active targeted drug delivery technique would address this issue. Accordingly, we report the design and development of aptamer-mitomycin C conjugates that use different cross-linking chemistry. The targeted delivery ability and cytotoxicity of these conjugates were carefully studied. It is worth noting that a linker-dependent cytotoxicity effect was observed for these conjugates. The use of a reductant-sensitive disulfide bond cross-linking strategy resulted in significantly enhanced cytotoxicity of MMC against the target cancer cell lines. Importantly, this cytotoxicity enhancement was suited to different types of aptamers, demonstrating the success of our design. Mechanistic studies of the enhanced cytotoxicity effect indicated that the target recognition, specific binding, and receptor-mediated internalization of aptamer were also critical for the observed effect.

Molecularly Engineering Triptolide with Aptamers for High Specificity and Cytotoxicity for Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) lacks three important receptors, ER, PR, and HER2. It is more aggressive and more likely to relapse after treatment, thus has been identified as one of the most malignant breast cancer types. The development of efficient targeted TNBC therapy is an important research topic in TNBC treatment. We report the development of a new aptamer-drug conjugate (ApDC), AS1411-triptolide conjugate (ATC), as targeted therapy for the treatment of TNBC with high efficacy. The conjugate possesses excellent specificity and high cytotoxicity against the MDA-MB-231 cell line. The advantages of our newly invented ATC are further highlighted by its excellent in vivo anti-TNBC efficacy and negligible side effects toward healthy organs.

Polymeric Engineering of Aptamer-Drug Conjugates for Targeted Cancer Therapy.

Nucleic acid aptamers, also known as "chemical antibodies", have been widely employed in targeted cancer therapy and diagnosis. For example, aptamer-drug conjugates (ApDCs), through covalent conjugation of cytotoxic warheads to aptamers, have demonstrated anticancer efficacy both in vitro and in vivo. However, a general strategy to endow ApDCs with enhanced biostability, prolonged circulation half-life, and high drug loading content remained elusive. Herein, we present a polymeric approach to engineer ApDCs via conjugation of cell-targeting aptamers with water-soluble polyprodrugs containing a reductive environmentally sensitive prodrug and biocompatible brush-like backbone. The resultant high-drug loading Aptamer-PolyproDrug Conjugates (ApPDCs) exhibited high nuclease resistance, extended in vivo circulation time, specific recognition, and cellular uptake to target cells, reduction-triggered and fluorescent-reporting drug release, and effective cytotoxicity. We could also further expand this design principle toward combination therapy by using two kinds of therapeutic drugs with distinct pharmacological mechanisms.

Phosphorylated lipid-conjugated oligonucleotide selectively anchors on cell membranes with high alkaline phosphatase expression.

Attachment of lipid tails to oligonucleotides has emerged as a powerful technology in constructing cell membrane-anchorable nucleic acid-based probes. In practice, however, conventional lipid-conjugated oligonucleotides fail to distinguish among different cell membranes. Herein, a phosphorylated lipid-conjugated oligonucleotide (DNA-lipid-P) is reported for alkaline phosphatase (ALP)-dependent cell membrane adhesion. In the absence of ALP, DNA-lipid-P with its poor hydrophobicity shows only weak interaction with cell membrane. However, in the presence of the highly expressed plasma membrane-associated ALP, DNA-lipid-P is converted to lipid-conjugated oligonucleotide (DNA-lipid) by enzymatic dephosphorylation. As a result of such conversion, the generated DNA-lipid has greater hydrophobicity than DNA-lipid-P and is thus able to insert into cell membranes in situ. Accordingly, DNA-lipid-P enables selective anchoring on cell membranes with elevated ALP level. Since elevated ALP level is a critical index of some diseases and even cancers, DNA-lipid-P holds promise for cell membrane engineering and disease diagnostics at the molecular level.

Long noncoding RNA PAGBC contributes to nitric oxide (NO) production by sponging miR-511 in airway hyperresponsiveness upon intubation.

BACKGROUND AND OBJECTIVES: In this study, we aimed to study the molecular mechanisms underlying the symptoms of hyperresponsiveness during intubation. METHOD: The value of circulating long noncoding RNA (lncRNA)-prognosis-associated gallbladder cancer (PAGBC) in the prediction of hyperresponsiveness upon intubation during general anesthesia was evaluated via the receiver operating characteristic analyses of serum miR-511, serum PAGBC, and serum nitric oxide (NO). In addition, the possible association between lncRNA-PAGBC/NOS1 messenger RNA (mRNA) and miR-511 was further validated via real-time quantitative polymerase chain reaction, immunohistochemistry assay, computational analysis, and luciferase assay. Enzyme-linked immunosorbent assay and Western blot analysis were also conducted to establish the regulatory relationship among PAGBC, miR-511, and NO synthase 1 (NOS1). RESULTS: Compared with circulating miR-511 and serum NO, circulating PAGBC was associated with a higher predictive value. In addition, a negative correlation was found between serum miR-511 and serum PAGBC (multicorrelation coefficient: -0.5) as well as between serum miR-511 and serum NO (multicorrelation coefficient: -0.6). In addition, both lncRNA-PAGBC and NO were decreased in patients with hyperresponsiveness, whereas the levels of miR-511 and NOS1 in these patients were similar to those in normal patients. Furthermore, our computational analyses and luciferase assays validated the direct binding between miR-511 and lncRNA-PAGBC, whereas NOS1 mRNA was identified as a virtual target gene of miR-511. Moreover, in the presence of lncRNA-PAGBC, we also observed an evident increase in the levels of NOS1 and NO accompanied by an obvious decrease of miR-511 expression. CONCLUSION: LncRNA-PAGBC downregulated the expression of miR-511, which in turn upregulated the expression of NOS1 mRNA and led to the increase in NOS1 expression, thus leading to the inhibited responsiveness (normal-responsiveness rather than hyperresponsiveness) to intubation in patients.

Pilose antler polypeptide protects against sevoflurane­mediated neurocyte injury.

Pilose antler polypeptide (PAP) is an active substance isolated from the traditional Chinese medicine pilose antler, which possesses multiple biological activities. In the present study, the role and mechanism of PAP in sevoflurane (SEV)­induced neurocyte injury was explored. Cell viability was determined by Cell Counting kit­8 assay. Cell proliferation and apoptosis were analyzed by flow cytometry. Western blotting and reverse transcription­quantitative polymerase chain reaction analysis were used to evaluate the protein and mRNA expression levels, respectively. The results revealed that PAP enhanced the cell viability of SEV­treated nerve cells. In addition, through modulation of apoptosis­associated protein expression, PAP suppressed SEV­induced nerve cell apoptosis. Furthermore, PAP activated the p38 mitogen­activated protein kinase (p38)/c­Jun N­terminal kinase (JNK) pathway in the neurocyte injury model, whereas inhibition of the p38/JNK pathway reversed the beneficial effects produced by PAP. In conclusion, PAP protected against SEV­mediated neurocyte injury via upregulation of the p38/JNK pathway. The present findings suggested that PAP may be an effective agent for neurocyte injury.

A basic insight into aptamer-drug conjugates (ApDCs).

Aptamers are often compared with antibodies since both types of molecules function as targeting ligands for specific cancer cell recognition. However, aptamers offer several advantages, including small size, facile chemical modification, high chemical stability, low immunogenicity, rapid tissue penetration, and engineering simplicity. Despite these advantages, several crucial factors have delayed their clinical translation, such as concerns over inherent physicochemical stability and safety. Meanwhile, steps have been taken to make aptamer-drug conjugates, or ApDCs, a clinically practical tool. In this review, we highlight the development of ApDCs and discuss how researchers are solving some problems associated with their clinical application for targeted therapy.

Molecular mechanism of estrogen-mediated neuroprotection in the relief of brain ischemic injury.

BACKGROUND: This study aimed to explore the molecular mechanism of estrogen-mediated neuroprotection in the relief of cerebral ischemic injury. The gene expression profiles were downloaded from Gene Expression Omnibus database, and differentially expressed genes (DEGs) were identified using limma package in R software. Further, DEGs were subjected to Gene Ontology (GO) cluster analysis using online Gene Ontology Enrichment Analysis Software Toolkit and to GO functional enrichment analysis using DAVID software. Using the Gene Set Analysis Toolkit V2, enrichment analysis of Kyoto Encyclopedia of Genes and Genomes pathways was performed. In addition, protein-protein interaction (PPI) network was constructed using STRING database, and submodule analysis of PPI network. Lastly, the significant potential target sites of microRNAs (miRNAs) were predicted using Molecular Signatures Database, and the function analysis of targets of predicted miRNA was also performed using DAVID software. RESULTS: In total, 321 DEGs were screened in the estrogen-treated sample. The DEGs were mainly associated with intracellular signaling and metabolic pathways, such as calcium channel, calcineurin complex, insulin secretion, low-density lipoprotein reconstruction, and starch or sugar metabolism. In addition, GO enrichment analysis indicated an altered expression of the genes related to starch and sucrose metabolism, retinol metabolism, anti-apoptosis (eg., BDNF and ADAM17) and response to endogenous stimulus. The constructed PPI network comprised of 243 nodes and 590 interaction pairs, and four submodules were obtained from PPI network. Among the module d, four glutamate receptors as Gria4, Gria3, Grin3a and Grik4 were highlighted. Further, 5 novel potential regulatory miRNAs were also predicted. MIR-338 and MIR520D were closely associated with cell cycle, while the targets of MIR-376A and MIR-376B were only involved in cell soma. CONCLUSIONS: The DEGs in estrogen-treated samples are closely associated with calcium channel, glutamate induced excitotoxicity and anti-apoptotic activity. In addition, some functionally significant DEGs such as BDNF, ADAM17, Gria4, Gria3, Grin3a, Grik4, Gys2 and Ugtla2, and new miRNAs like MIR-338 and MIR-376A were identified, which may serve as potential therapeutic targets for the effective treatment of cerebral ischemic injury.

TXNIP knockdown alleviates hepatocyte ischemia reperfusion injury through preventing p38/JNK pathway activation.

Hepatic ischemia and reperfusion (I/R) injury is a major cause of liver damage during liver transplantation, resection surgery, shock, and trauma. It has been reported that TXNIP expression was upregulated in a rat model of hepatic I/R injury. However, the role of TXNIP in the hepatic I/R injury is little known. In our study, we investigated the biological role of TXNIP and its potential molecular mechanism in the human hepatic cell line (HL7702 cells). Using oxygen-glucose deprivation and reoxygenation (OGD/R) to create a cell model of hepatic I/R injury, we found that the mRNA and protein expression levels of TXNIP were upregulated in HL7702 cells exposed to OGD/R. TXNIP overexpression remarkably promoted OGD/R-induced cell apoptosis and lactate dehydrogenase (LDH) release, both of which were significantly decreased by TXNIP knockdown. The production of malondialdehyde (MDA) was also increased by TXNIP overexpression, but was reduced by TXNIP knockdown. Moreover, TXNIP overexpression significantly upregulated the phosphorylation of p38 and JNK, which was remarkably inhibited by TXNIP knockdown. Additionally, p38-specific inhibitor SB203580 abrogated the effect of TXNIP overexpression on OGD/R-induced cell injury. Taken together, these results indicated that TXNIP knockdown alleviated hepatocyte I/R injury through preventing p38/JNK pathway activation. Thus, TXNIP might offer a novel potential therapeutic target for the treatment of hepatic I/R injury.

Tea polyphenols may attenuate the neurocognitive impairment caused by global cerebral ischemia/reperfusion injury via anti-apoptosis.

BACKGROUND/AIMS: Global cerebral ischemia/reperfusion (GCIR) may incur neurocognitive impairment. Tea polyphenols (TP) have strong anti-oxidant capacity. This study planned to investigate the protective effect of TP against the neurocognitive impairment caused by GCIR and its mechanism. METHODS: One-stage anterior approach for cerebral four-vessel occlusion (4VO) was used to construct the GCIR model. Sprague Dawley rats were randomly classified into Sham group, GCIR group, and TP group (n = 50 per group). Besides receiving the same 4VO, the rats in TP group were treated with TP (6.4%) injection from the tail vein 30 minutes before cerebral ischemia. Morris water-maze test was used to evaluate the changes in space recognition and memory and open field activity test to assess the activity and motor function of rats. The cell apoptotic study in hippocampal CA1 region at specified time points (12, 24, 48, and 72 hours after surgery) was carried out by the flow cytometry, histology (hematoxylin and eosin staining), and immunohistochemical (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining) examinations. One-way analysis of variance and least significant difference t-test were used and statistical significance considered at P < 0.05. RESULTS: Compared with the GCIR group, the TP group was significantly attenuated in the impairment of space recognition and memory caused by GCIR and so was the neuronal apoptosis in the hippocampal CA1 region (P < 0.05). CONCLUSION: TP may attenuate the impairment of space recognition and memory caused by GCIR via anti-apoptosis.

Identification of the potential molecular targets for human intervertebral disc degeneration based on bioinformatic methods.

The present study aimed to explore potential molecular targets and gain further insights into the mechanism of intervertebral disc degeneration (IDD) progression. Microarray datasets of GSE19943, GSE15227 and GSE34095 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) in 3 IDD specimens compared with 3 controls in GSE34095, DEGs in 7 grade III and 3 grade IV samples compared with 5 grade II samples in GSE19943, and differentially expressed miRNAs in 3 degenerated samples compared with 3 controls in GSE15227 were screened. Grade III­ and IV­specific networks were constructed and grade­specific genes were extracted. The network features were analyzed, followed by Gene Ontology (GO) enrichment analysis and pathway enrichment analysis of grade­specific genes and DEGs identified in GSE34095. Furthermore, miRNA­pathway interactions were analyzed using Fisher's exact test. Tumor protein p53 (TP53) was a hub gene in the grade III­specific network and ubiquitin C (UBC) was identified to be a hub gene in the grade IV­specific network. Six significant features were identified by grade­specific network topology analysis. Grade­specific genes and DEGs were involved in different GO terms and pathways. Differentially expressed miRNAs were identified to participate in 35 pathways, among which 6 pathways were significantly enriched by DEGs, including apoptosis. The present study identified that key genes (TP53 and UBC) and miR­129­5p may participate in the mechanism of IDD progression. Thus, they may be potential therapeutic targets for IDD.