Tissue factor pathway inhibitors disrupt structures of rhabdovirus/ranairidovirus and inhibit viral infection in Chinese perch, Siniperca chuatsi.

Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.

Tracking the effects of PLGA-based nanoparticles on protein expression in living cells through quantitative proteomics.

Mass spectrometry (MS)-based proteomics can identify and quantify the differential abundance of expressed proteins in parallel, and bottom-up proteomic approaches are even approaching comprehensive coverage of the complex eukaryotic proteome. Protein-nanoparticle (NP) interactions have been extensively studied owing to their importance in biological applications and nanotoxicology. However, the proteome-level effects of NPs on cells have received little attention, although changes in protein abundance can reflect the direct effects of nanocarriers on protein expression. Herein, we investigated the effect of PLGA-based NPs on protein expression in HepG2 cells using a label-free quantitative proteomics approach with data independent acquisition (DIA). The percentage of two-fold change in the protein expression of cells treated with PLGA-based NPs was less than 10.15% during a 6 hour observation period. Among the changed proteins, we found that dynamic proteins involved in cell division, localization, and transport are more likely to be more susceptible to PLGA-based NPs.

K+-Dependent Photocycle and Photocurrent Reveal the Uptake of K+ in Light-Driven Sodium Pump.

Engineering light-controlled K+ pumps from Na+-pumping rhodopsins (NaR) greatly expands the scope of optogenetic applications. However, the limited knowledge regarding the kinetic and selective mechanism of K+ uptake has significantly impeded the modification and design of light-controlled K+ pumps, as well as their practical applications in various fields, including neuroscience. In this study, we presented K+-dependent photocycle kinetics and photocurrent of a light-driven Na+ pump called Nonlabens dokdonensis rhodopsin 2 (NdR2). As the concentration of K+ increased, we observed the accelerated decay of M intermediate in the wild type (WT) through flash photolysis. In 100 mM KCl, the lifetime of the M decay was approximately 1.0 s, which shortened to around 0.6 s in 1 M KCl. Additionally, the K+-dependent M decay kinetics were also observed in the G263W/N61P mutant, which transports K+. In 100 mM KCl, the lifetime of the M decay was approximately 2.5 s, which shortened to around 0.2 s in 1 M KCl. According to the competitive model, in high KCl, K+ may be taken up from the cytoplasmic surface, competing with Na+ or H+ during M decay. This was further confirmed by the K+-dependent photocurrent of WT liposome. As the concentration of K+ increased to 500 mM, the amplitude of peak current significantly dropped to approximately ~60%. Titration experiments revealed that the ratio of the rate constant of H+ uptake (kH) to that of K+ uptake (kK) is >108. Compared to the WT, the G263W/N61P mutant exhibited a decrease of approximately 40-fold in kH/kK. Previous studies focused on transforming NaR into K+ pumps have primarily targeted the intracellular ion uptake region of Krokinobacter eikastus rhodopsin 2 (KR2) to enhance K+ uptake. However, our results demonstrate that the naturally occurring WT NdR2 is capable of intracellular K+ uptake without requiring structural modifications on the intracellular region. This discovery provides diverse options for future K+ pump designs. Furthermore, we propose a novel photocurrent-based approach to evaluate K+ uptake, which can serve as a reference for similar studies on other ion pumps. In conclusion, our research not only provides new insights into the mechanism of K+ uptake but also offers a valuable point of reference for the development of optogenetic tools and other applications in this field.

Efficient Enrichment Method for N-Phosphorylation Peptides in Mouse Brain Tissue.

In addition to O-phosphorylation, protein N-phosphorylation was proven to play important roles in multiple biological processes. Although affinity methods were developed for the enrichment of N-phosphorylation peptides in cells, it was still difficult to enrich N-phosphorylation peptides in tissue due to its complexity. In this study, we established a workflow for the identification of N-phosphorylation peptides in mouse brain tissue by direct enrichment in high concentration urea. In total, 989 N-phosphorylation sites were obtained using 0.5 M urea as enrichment buffer. Among all identified N-phosphorylation sites, the localization probability over 0.75 was as high as 80%, suggesting the reliability of the method. Furthermore, the sequence motif analysis and gene ontology analysis results showed a good match to previous studies. The method was successfully used for N-phosphorylation analysis of mouse hippocampus from Alzheimer's disease model, and 533 N-phosphorylation sites were identified in 5 × FAD mouse hippocampus tissue. Biological process analysis results showed that "brain development", "cellular response to reactive oxygen species", "microtubule cytoskeleton organization", and "peptidyl-serine phosphorylation" were especially enriched in 5 × FAD mouse. It is suggested that N-phosphorylation may be related to Alzheimer's disease in these aspects.

Pushpin-like nanozyme for plasmon-enhanced tumor targeted therapy.

Relatively low catalytic activity and poor targeting limit the applications of nanoceria (CeO2) nanozymes in the treatment of tumors. Here, we designed a unique pushpin-like Au/CeO2 hybrid nanozyme with high catalytic activity by combining site-selective growth and steric restriction strategies. The enhanced enzyme activity was attributed to plasmon-induced hot electrons. Furthermore, the pushpin-like structure facilitated targeting molecule modification. The nanozyme exhibited superior antitumor effects both in vitro and in vivo due to its high catalytic activity and targeting effects. Importantly, its potential mechanism of anti-tumor therapy was studied by quantitative proteomics. The reactive oxygen species (ROS) generated by folic acid-PEG thiol-Au/CeO2 (FA-Au/CeO2) caused mitochondrial and proteasomal damage in tumor cells and further evoked a response to oxidative stress and innate immunity in vivo. This study provided a spatiotemporal approach to enhance the antitumor activity of nanozymes by structural design. The designed pushpin-like Au/CeO2 could be utilized as a multifunctional nanoplatform for in vitro and in vivo plasmon-enhanced cancer therapy with active targeting effects. Moreover, this study systematically explored the anti-tumor mechanism of the nanozyme in both cell and mouse models, promoting its translation to the clinic. STATEMENT OF SIGNIFICANCE: A strategy combining the principles of site-selective growth and steric restriction was developed to prepare a unique pushpin-like Au/CeO2 hybrid nanozyme with high catalytic activity and low steric hindrance. The hybrid nanozyme showed superior antitumor activity at both the cellular and tissue levels. Furthermore, the antitumor mechanism was investigated in terms of the differential proteins and their pathways using quantitative proteomics, thus promoting the translation of nanozymes to the clinic.

Targeted killing of tumor cells based on isoelectric point suitable nanoceria-rod with high oxygen vacancies.

Nanozymes have great potential applications in tumor treatment due to their good stability, high biocompatibility, easy preparation and versatility. However, it remains a challenge to design highly active nanozymes with tumor cell targeting. Herein, three nanoceria structures (nanoceria-rod; nanoceria polyhedra, abbreviated as nanoceria-poly.; and nanoceria-cube) with different surface oxygen vacancy concentrations are designed. Among them, nanoceria-rod shows the highest enzyme activity and tumor cell toxicity because of its highest concentration of oxygen vacancies on the surface. Further study shows that nanoceria-rod can selectively enter tumor cells because nanoceria-rod with a suitable isoelectric point (IEP) remains positively charged in the acidic microenvironment of the tumor but negatively charged in the physiological microenvironment of normal cells. Nanoceria-rod distributes in lysosomes and phagosomes to produce reactive oxygen species (ROS) in tumor cells. Finally, the mitochondrial membrane potential (MMP) was reduced, which caused cell apoptosis. This study provides an interesting new tumor-targeting therapy method, which could also be extended to other drug nanocarriers and diagnostic imaging nanomaterials for tumors.

Antibody-Free Hydrogel with the Synergistic Effect of Cell Imprinting and Boronate Affinity: Toward the Selective Capture and Release of Undamaged Circulating Tumor Cells.

The selective and highly efficient capture of circulating tumor cells (CTCs) from blood and their subsequent release without damage are very important for the early diagnosis of tumors and for understanding the mechanism of metastasis. Herein, a universal strategy is proposed for the fabrication of an antibody-free hydrogel that has a synergistic effect by featuring microinterfaces obtained by cell imprinting and molecular recognition conferred by boronate affinity. With this artificial antibody, highly efficient capture of human hepatocarcinoma SMMC-7721 cells is achieved: as many as 90.3 ± 1.4% (n = 3) cells are captured when 1 × 105 SMMC-7721 cells are incubated on a 4.5 cm2 hydrogel, and 99% of these captured cells are subsequently released without any loss of proliferation ability. In the presence of 1000 times as many nontarget cells, namely, leukaemia Jurkat cells, the SMMC-7721 cells can be captured with an enrichment factor as high as 13.5 ± 3.2 (n = 3), demonstrating the superior selectivity of the artificial antibody for the capture of the targeted CTCs. Most importantly, the SMMC-7721 cells can be successfully captured even when spiked into whole blood, indicating the great promise of this approach for the further molecular characterization of CTCs.