New Wine in Old Bottle: Crown Ether-Functionalized Digital Polymer toward Efficient MALDI-TOF MS/MS Decoding via a Classical Supramolecular Interaction.

Digital polymers (DPs), which serve as promising molecular-level storage media, have increasingly garnered interest. Their application significantly depends on the efficiency of the information writing (synthesis) and reading processes (sequencing). For reading, rational incorporation of weak bonds in the main chain was applied in most cases in order to improve readability of the tandem mass spectra (MS/MS), which would limit the chain length of DPs, thus reducing the information storage capacity. In this study, the introduction of commercially available crown ether (CE) at the terminus of digital oligo(γ-butyrolactone)s (DOBLs) significantly enhances the predictability and fidelity of matrix-assisted laser desorption/ionization time-of-flight tandem mass spectra (MALDI-TOF MS/MS), thus improving the decoding process. The use of crown ether, leveraging a well-established supramolecular interaction with alkali cations known since 1967, offers a strong affinity between ionization agents and CE motifs, to form a selective effect of the desired fragments during the tandem MS. This method is particularly effective for long-chain DPs, extending up to 32-mer, and allows for customizable fragmentation patterns. The incorporation of CE at the DP chain end presents a novel and efficient strategy for enhancing MS/MS readability and amplifying the information storage capacity of polymers.

Exploration of the immune response of grass carp (Ctenopharyngodon idellus) erythrocytes during bacterial infection.

In teleost blood, red blood cells (RBCs) are the most common type of cell, and they differ from mammalian RBCs in having a nucleus and other organelles. As nucleated cells, teleost RBCs contribute to the immune response against pathogens, but their antibacterial mechanism remains unclear. Here, we utilized RNA-Seq to analyze gene expression patterns of grass carp (Ctenopharyngodon idellus) RBCs (GcRBCs) stimulated by Aeromonas hydrophila, Escherichia coli, and Staphylococcus aureus. Our transcriptomic data showed that bacterial stimulation generated many differentially expressed genes (DEGs). Furthermore, several inflammatory pathways responded to bacterial activation, and the TLR, IL-17, and tumor necrosis factor (TNF) signaling pathways were significantly activated based on Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Furthermore, the findings of qRT-PCR showed markedly elevated expression of various cytokines, including IL-1ß, IL4, IL6, IL8, IL12, and TNFα, in GcRBCs after incubation with bacteria. Reactive oxygen species (ROS) production in GcRBCs was markedly increased after the cells were stimulated with the three bacteria, and the expression of superoxide dismutase, glutathione peroxidase, and antioxidant enzymes, including catalase, was altered. Flow cytometry analysis showed that the apoptosis rate of GcRBCs was enhanced after stimulation with the three bacteria for different times. In summary, our findings reveal that bacterial stimulation activates the immune response of GcRBCs by regulating ROS release, cytokine expression, and the antioxidant system, leading to apoptosis of GcRBCs.

The infection of Aeromonas hydrophila activated Multiple programmed cell death pathways in red blood cells of Clarias fuscus.

In contrast to mammalian red blood cells (RBCs), Osteichthyes RBCs contain a nucleus and organelles, suggesting the involvement of more intricate mechanisms, particularly in the context of ferroptosis. In this study, we utilized RBCs from Clarias fuscus (referred to as Cf-RBCs) as a model system. We conducted RNA-seq analysis to quantify gene expression levels in Cf-RBCs after exposure to both Aeromonas hydrophila and lipopolysaccharides. Our analysis unveiled 1326 differentially expressed genes (DEGs) in Cf-RBCs following 4 h of incubation with A. hydrophila, comprising 715 and 611 genes with upregulated and downregulated expression, respectively. These DEGs were further categorized into functional clusters: 292 related to cellular processes, 241 involved in environmental information processing, 272 associated with genetic information processing, and 399 linked to organismal systems. Additionally, notable changes were observed in genes associated with the autophagy pathway at 4 h, and alterations in the ferroptosis pathway were observed at 8 h following A. hydrophila incubation. To validate these findings, we assessed the expression of cytokines (DMT1, TFR1, LC3, and GSS). All selected genes were significantly upregulated after exposure to A. hydrophila. Using flow cytometry, we evaluated the extent of ferroptosis, and the group incubated with A. hydrophila for 8 h exhibited higher levels of lipid peroxidation compared with the 4-h incubation group, even under baseline conditions. An evaluation of the glutathione redox system through GSSG/GSH ratios indicated an increased ratio in Cf-RBCs after exposure to A. hydrophila. In summary, our data suggest that A. hydrophila may induce ferroptosis in Cf-RBCs, potentially by triggering the cystine/glutamate antiporter system (system XC-), while Cf-RBCs counteract ferroptosis through the regulation of the glutathione redox system. These findings contribute to our understanding of the iron overload mechanism in Osteichthyes RBCs, provide insights into the management of bacterial diseases in Clarias fuscus, and offer potential strategies to mitigate economic losses in aquaculture.

PEGylated pH-responsive peptide-mRNA nano self-assemblies enhance the pulmonary delivery efficiency and safety of aerosolized mRNA.

Inhalable messenger RNA (mRNA) has demonstrated great potential in therapy and vaccine development to confront various lung diseases. However, few gene vectors could overcome the airway mucus and intracellular barriers for successful pulmonary mRNA delivery. Apart from the low pulmonary gene delivery efficiency, nonnegligible toxicity is another common problem that impedes the clinical application of many non-viral vectors. PEGylated cationic peptide-based mRNA delivery vector is a prospective approach to enhance the pulmonary delivery efficacy and safety of aerosolized mRNA by oral inhalation administration. In this study, different lengths of hydrophilic PEG chains were covalently linked to an amphiphilic, water-soluble pH-responsive peptide, and the peptide/mRNA nano self-assemblies were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in vitro mRNA binding and release, cellular uptake, transfection, and cytotoxicity were studied, and finally, a proper PEGylated peptide with enhanced pulmonary mRNA delivery efficiency and improved safety in mice was identified. These results showed that a proper N-terminus PEGylation strategy using 12-monomer linear monodisperse PEG could significantly improve the mRNA transfection efficiency and biocompatibility of the non-PEGylated cationic peptide carrier, while a longer PEG chain modification adversely decreased the cellular uptake and transfection on A549 and HepG2 cells, emphasizing the importance of a proper PEG chain length selection. Moreover, the optimized PEGylated peptide showed a significantly enhanced mRNA pulmonary delivery efficiency and ameliorated safety profiles over the non-PEGylated peptide and LipofectamineTM 2000 in mice. Our results reveal that the PEGylated peptide could be a promising mRNA delivery vector candidate for inhaled mRNA vaccines and therapeutic applications for the prevention and treatment of different respiratory diseases in the future.

Saikosaponin-D Mitigates Oxidation in SH-SY5Y Cells Stimulated by Glutamate Through Activation of Nrf2 Pathway: Involvement of PI3K.

Alzheimer's disease (AD) is a typical neurodegenerative disease. Well-established studies have shown an elevated level of ROS (reactive oxygen species) that induces oxidative stress in AD. Saikosaponin-D exhibited significant therapeutic effects on neurodegenerative diseases. However, its in-depth molecular mechanisms against neurotoxicity remain not fully uncovered. Herein, the possible protective effects of saikosaponin-D on glutamate-induced neurotoxicity in SH-SY5Y cells and the underlying mechanism were elucidated. Saikosaponin-D pretreatment could ameliorate glutamate-induced cytotoxicity according to MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and depress apoptosis according to Hoechst 33,342 staining and Annexin V-FITC/PI double staining in SH-SY5Y cells. Additionally, saikosaponin-D administration suppressed oxidative stress in response to glutamate indicated by diminished intracellular ROS formation and reduced MDA (malondialdehyde) content in SH-SY5Y cells. These phenomena, appeared to correlate with the recovered cellular antioxidant enzyme activities and inducted HO-1 (heme oxygenase-1) expression accompanying the nuclear translocation of Nrf2 conduct by saikosaponin-D preconditioning which had been altered by glutamate, were correlated with its neuroprotective. Furthermore, addition of LY294002, a selective inhibitor of PI3K (phosphatidylinositol 3 kinase), blocked saikosaponin-D-caused Nrf2 nuclear translocation and reversed the protection of saikosaponin-D against glutamate in SH-SY5Y cells. Moreover, saikosaponin-D exhibited antioxidant potential with high free radical-scavenging activity as confirmed by a DPPH (2,2-diphenyl-1-picrylhydrazyl) and TEAC (Trolox equivalent antioxidant capacity) in a cell-free system in vitro. Taken together, our results indicated that saikosaponin-D enhanced cellular antioxidant capacity through not only intrinsic free radical-scavenging activity but also induction of endogenous antioxidant enzyme activities and HO-1 expression mediated, at least in part, by activating PI3K and subsequently Nrf2 nuclear translocation, thereby protecting the SH-SY5Y cells from glutamate-induced oxidative cytotoxicity. In concert, these data raise the possibility that saikosaponin-D may be an attractive candidate for prevention and treatment of AD and other diseases related to oxidation in the future.

Screening Candidates for Refractive Surgery With Corneal Tomographic-Based Deep Learning.

Importance: Evaluating corneal morphologic characteristics with corneal tomographic scans before refractive surgery is necessary to exclude patients with at-risk corneas and keratoconus. In previous studies, researchers performed screening with machine learning methods based on specific corneal parameters. To date, a deep learning algorithm has not been used in combination with corneal tomographic scans. Objective: To examine the use of a deep learning model in the screening of candidates for refractive surgery. Design, Setting, and Participants: A diagnostic, cross-sectional study was conducted at the Zhongshan Ophthalmic Center, Guangzhou, China, with examination dates extending from July 18, 2016, to March 29, 2019. The investigation was performed from July 2, 2018, to June 28, 2019. Participants included 1385 patients; 6465 corneal tomographic images were used to generate the artificial intelligence (AI) model. The Pentacam HR system was used for data collection. Interventions: The deidentified images were analyzed by ophthalmologists and the AI model. Main Outcomes and Measures: The performance of the AI classification system. Results: A classification system centered on the AI model Pentacam InceptionResNetV2 Screening System (PIRSS) was developed for screening potential candidates for refractive surgery. The model achieved an overall detection accuracy of 94.7% (95% CI, 93.3%-95.8%) on the validation data set. Moreover, on the independent test data set, the PIRSS model achieved an overall detection accuracy of 95% (95% CI, 88.8%-97.8%), which was comparable with that of senior ophthalmologists who are refractive surgeons (92.8%; 95% CI, 91.2%-94.4%) (P = .72). In distinguishing corneas with contraindications for refractive surgery, the PIRSS model performed better than the classifiers (95% vs 81%; P < .001) in the Pentacam HR system on an Asian patient database. Conclusions and Relevance: PIRSS appears to be useful in classifying images to provide corneal information and preliminarily identify at-risk corneas. PIRSS may provide guidance to refractive surgeons in screening candidates for refractive surgery as well as for generalized clinical application for Asian patients, but its use needs to be confirmed in other populations.

The hierarchical micro-/nanotextured topographies promote the proliferation and angiogenesis-related genes expression in human umbilical vein endothelial cells by initiation of Hedgehog-Gli1 signaling.

The hierarchical microtextured/nanotextured topographies have been recognized to have better tissue integration properties, but the underlying mechanisms are only partially understood. Hedgehog signaling plays a pivotal role in developmental and homeostatic angiogenesis. We suppose that the Hedgehog-Gli1 signaling may play a significant role in the response of endothelial cells to microtextured/nanotextured topographies (MNTs). To confirm this hypothesis, we produced the MNTs decorated with TiO2 nanotubes of two different diameters (25 and 70 nm), and the proliferation, apoptosis, angiogenesis-related genes expression and Hedgehog signaling activity of human umbilical vein endothelial cells (HUVECs) grown onto these MNTs were measured. Our results showed that the MNTs induced significantly high expression of Sonic Hedgehog (SHH), Smoothened (SMO) and GLI1 in the HUVECs as well as high activation of Hedgehog-Gli1 signaling, compared to the smooth surface. The HUVECs grown on the MNTs showed significantly high levels of adhesion, proliferation and expression of angiogenesis-related genes, including angiopoietin-1 (ANG-1), vascular endothelial growth factor (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2) and endothelial nitric oxide synthase (ENOS); these enhancements were attenuated by siRNA-mediated depletion of SMO, which indicated a significant role of Hedgehog-Gli1 signaling in mediating the enhanced effect of the MNTs on the angiogenic potential of HUVECs. This study may contribute to the modification of biomaterial surfaces for better tissue integration and clinical performance.