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.

Selective Single-Molecule Nanopore Detection of mpox A29 Protein Directly in Biofluids.

Single-molecule antigen detection using nanopores offers a promising alternative for accurate virus testing to contain their transmission. However, the selective and efficient identification of small viral proteins directly in human biofluids remains a challenge. Here, we report a nanopore sensing strategy based on a customized DNA molecular probe that combines an aptamer and an antibody to enhance the single-molecule detection of mpox virus (MPXV) A29 protein, a small protein with an M.W. of ca. 14 kDa. The formation of the aptamer-target-antibody sandwich structures enables efficient identification of targets when translocating through the nanopore. This technique can accurately detect A29 protein with a limit of detection of ∼11 fM and can distinguish the MPXV A29 from vaccinia virus A27 protein (a difference of only four amino acids) and Varicella Zoster Virus (VZV) protein directly in biofluids. The simplicity, high selectivity, and sensitivity of this approach have the potential to contribute to the diagnosis of viruses in point-of-care settings.

Programmable Circular Multivalent Nanobody-Targeting Chimeras (mNbTACs) for Multireceptor-Mediated Protein Degradation and Targeted Drug Delivery.

Multispecific therapeutics hold significant promise in drug delivery, protein degradation, and cell recruitment to address clinical issues of tumor heterogeneity, resistance, and immune evasion. However, their modular engineering remains challenging. We developed a targeted degradation platform, termed multivalent nanobody-targeting chimeras (mNbTACs), by encoding diverse nanobody codons on a circular template using DNA printing technology. The homo- or hetero- mNbTACs specifically recognized membrane targets in a multivalent manner and simultaneously recruited scavenger receptors to favor clathrin-/caveolae-dependent endocytosis and lysosomal degradation of multiple proteins with high efficiency and selectivity. We demonstrated that a bispecific doxorubicin-loaded mNbTAC, named Doxo-mvNbsPPH, passively accumulated at tumor sites, specifically interacted with PD-L1 and HER2 targets, and was rapidly transported into lysosome, inducing potent immunogenic cell death and alleviating immune checkpoint evasion. The synergistic boosting of innate and adaptive immunity promoted the infiltration and proliferation of CD8+ T cells in tumor microenvironment (an 11-fold increase) with high toxicity and low exhaustion, eventually enhancing antitumor efficacy. Our mNbTAC platform provides multispecific therapeutics with variable valences and programmed species, whereas it induces targeted protein degradation through multireceptor-mediated endocytosis and lysosomal degradation without the need for lysosome-targeting receptors, representing a general and modular tool to harness extracellular proteome for disease treatment.

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.

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.

Synthesis and Anti-Oomycete Preliminary Mechanism of Sulfonate Derivatives of Ethyl Maltol.

To discover novel molecules with unique mechanism against plant pathogenic oomycetes, sixteen new sulfonate derivatives of ethyl maltol (3a-p) were synthesized by structural modification of 2-ethyl-3-hydroxy-4H-pyran-4-one, and their anti-oomycete activity against a serious agricultural disease, Phytophthora capsici Leonian was determined in this study. Among all tested compounds, derivatives 3e, 3m and 3p exhibited the most potent anti-oomycete activity against P. capsici with EC50 values of 19.40, 21.04 and 31.10 mg/L, respectively; especially 3e and 3m showed the best promising and pronounced anti-oomycete activity than zoxamide (EC50 =26.87 mg/L). The results further proved that 4-tert-butylphenylsulfonyl group, 3-nitro-4-chlorophenylsulfonyl group and 8-quinolinesulfonyl group introduced at the hydroxy position of ethyl maltol or maltol were necessary for obtaining the most potent compounds. Further mechanism studies of P. capsici treated with 3e demonstrated that this compound can affect the growth of mycelia by disrupting the integrity of the membrane, and the higher the concentration of the compound is, the greater the degree of membrane integrity damage. These important results will pave the way for further modification of ethyl maltol to develop potential new fungicides.

Synthesis and Anti-Oomycete Activity of Sulfonate Derivatives of Fenjuntong.

In order to discover highly active fungicides, sixteen novel sulfonate derivatives of Fenjuntong were synthesized by structural modification of 2'-hydroxybutyrophenone, and their anti-oomycete activity against Phytophthora capsici Leonian was determined in this study. Among all tested compounds, compound 3b displayed more significant anti-oomycete activity than the precursor Fenjuntong against P. capsici, and the EC50 values of 3b and Fenjuntong were 84.50 and 517.25 mg/L, respectively. By comparing the anti-oomycete activity of compounds 3a-p, I-a-p, and II-a-p, the following conclusions were drawn: (1) Hydroxy group is well tolerated, and sulfonylation of hydroxy group enhances its anti-oomycete activity. (2) The proper length of the ketone carbonyl chain is very important for their anti-oomycete activity. (3) The presence of a site methoxy group in the structural skeleton is closely related to the anti-oomycete activity. These important results will pave the way for further modification of Fenjuntong to develop potential new fungicides.

Synthesis of novel 3/5(3,5)-(di)nitropaeonol hydrazone derivatives as nematicidal agents.

Eighteen novel 3/5(3,5)-(di)nitropaeonol hydrazone derivatives were prepared, and their structures well characterized by 1H NMR, HRMS, and mp. Due to the steric hindrance, the substituents on the C = N double bond of all hydrazine compounds (except E/Z = 4/1 for IV-1g, IV-1l, IV-2b, and E/Z = 3/2 for IV-1n, IV-3a) adopted E configuration. Among all compounds, four compounds 2, 4, IV-1j, and IV-1n exhibited potent nematicidal activity than their precursor paeonol, especially 5-nitropaeonol (2) and 3,5-dinitropaeonol (4) displayed the most potent nematicidal activity Heterodera glycines in vivo with LC50 values of 32.3307 and 36.7074 mg/L, respectively.

Synthesis and Anti-Oomycete Activity of 1-Sulfonyloxy/Acyloxydihydroeugenol Derivatives.

Endeavor to discover biorational natural products-based fungicides, two series (26) of novel 1-sulfonyloxy/acyloxydihydroeugenol derivatives (3a-p and 5a-j) were prepared and assessed for their fungicidal activity against P. capsici Leonian, in vitro. Results of fungicidal activity revealed that, among all compounds, especially compounds 3a, 5c, and 5e displayed the most potent anti-oomycete activity against P. capsici with EC50 values of 69.33, 68.81, and 67.77 mg/L, respectively. Overall, the anti-oomycete activities of 1-acyloxydihydroeugenol derivatives (5a-j) were higher than that of 1-sulfonyloxydihydroeugenol derivatives (3a-p). It is proved that the introduction of the acyl group at hydroxy position of dihydroeugenol is more beneficial to improve its anti-oomycete activity than that of the sulfonyl group. These preliminary results will pave the way for further modification of dihydroeugenol in the development of potential new fungicides.

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.

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.

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.

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.

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.

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.

Collapsin Response Mediator Protein-2 Ameliorates Sevoflurane-Mediated Neurocyte Injury by Targeting PI3K-mTOR-S6K Pathway.

BACKGROUND Collapsin response mediator protein-2 (CRMP-2) is the first member of the CRMP family that has been identified in primary neuronal cells; it was originally found and identified in the regulation of microtubule dimerization into microtubules. MATERIAL AND METHODS In the present study, we aimed to investigate the roles and mechanisms of CRMP-2 in sevoflurane-induced neurocyte injury. Cell viability, proliferation, and apoptosis were measured by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Colorimetry was performed to measure the activity of caspase-3. Western blot and quantitative real-time reverse transcription assays were used to evaluate the related mRNAs and proteins expression. RESULTS We found that CRMP-2 reversed the inhibitory effect of sevoflurane on the viability of nerve cells. Moreover, CRMP-2 accelerated the proliferation and suppressed the apoptosis of sevoflurane-induced nerve cells. CRMP-2 modulated the expression levels of apoptosis-associated protein in sevoflurane-induced nerve cells. Furthermore, it was demonstrated that CRMP-2 impacted the PI3K-mTOR-S6K pathway. CONCLUSIONS CRMP2 ameliorated sevoflurane-mediated neurocyte injury by targeting the PI3K-mTOR-S6K pathway. Thus, CRMP2 might be an effective target for sevoflurane-induced neurocyte injury therapies.

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.

Effect of Desflurane versus Sevoflurane in Pediatric Anesthesia: A Meta-Analysis.

PURPOSE: To compare the effect of desflurane versus sevoflurane in pediatric anesthesia by conducting meta-analysis. METHODS: Studies were searched from PubMed, Medline, Springer, Elsevier Science Direct, Cochrane Library and Google Scholar up to July 2014. Weighted mean difference (WMD) or risk ratio (RR) and 95% confidence intervals (CIs) were considered as effect sizes. Heterogeneity across studies was assessed by Cochran Q test and I2 statistic. The random effects model was performed in the meta-analysis when heterogeneity was observed, or the fixed effect model was used. Review Manager 5.1 software was applied for the meta-analysis. RESULTS: A total of 11 studies (13 comparisons) involving 1,273 objects were included in this meta-analysis. No heterogeneity was observed between studies for any comparison but for postoperative extubation time. The results showed significant differences between desflurane and sevoflurane groups for postoperative extubation time (WMD = -3.87, 95%CI = -6.14 to -1.60, P < 0.01), eye opening time (WMD = -1.11, 95%CI = -1.49 to -0.72, P < 0.01), awakening time (WMD = -4.27, 95%CI = -5.28 to -3.26, P < 0.01) and agitation (RR = 1.44, 95%CI = 1.05 to 1.96, P = 0.02). No significant differences (P > 0.05) were detected for discharge from the recovery room, oculocardiac reflex, nausea and vomiting and severe pain. CONCLUSIONS: Desflurane may have less adverse effects than sevoflurane when used in pediatric anesthesia with significantly shorter postoperative extubation time, eye opening time and awakening time as well as slighter agitation.

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.