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Inefficient sample preparation methods hinder the performance of high-throughput single-molecule force spectroscopy (H-SMFS) for viscous damping among reactants and unstable linkage. Here, we demonstrated a sample preparation method for H-SMFS systems to achieve a higher ratio of effective target molecules per sample cell by gas-phase silanization and reactant hydrophobization. Digital holographic centrifugal force microscopy (DH-CFM) was used to verify its performance. The experimental result indicated that the DNA stretching success ratio was improved from 0.89% to 13.5%. This enhanced efficiency preparation method has potential application for force-based DNA stretching experiments and other modifying procedures.
Bacterial outer membrane vesicle (OMV) is a kind of spherical lipid bilayer nanostructure naturally secreted by bacteria, which has diverse functions such as intracellular and extracellular communication, horizontal gene transfer, transfer of contents to host cells, and eliciting an immune response in host cells. In this review, several methods including ultracentrifugation and precipitation for isolating OMVs were summarized. The latest progresses of OMVs in biomedical fields, especially in vaccine development, cancer treatment, infection control, and bioimaging and detection were also summarized in this review. We highlighted the importance of genetic engineering for the safe and effective application and in facilitating the rapid development of OMVs. Finally, we discussed the bottleneck problems about OMVs in preparation and application at present and put forward our own suggestions about them. Some perspectives of OMVs in biomedical field were also provided.
Dendritic cells (DCs) are professional antigen-presenting cells (APCs). The key functions of DCs include engulfing, processing and presenting antigens to T cells and regulating the activation of T cells. There are two major DC subtypes in human blood:plasmacytoid DCs (pDCs) and conventional DCs. To define the differences between the adult and infant immune systems, especially in terms of DC constitution, we enriched DCs from human cord blood and generated single-cell RNA sequencing data from about 7000 cells using the 10x Genomics Single Cell 3' Solution. After incorporating the differential expression analysis method in our clustering process, we identified all the known dendritic cell subsets. Interestingly, we also found a group of DCs with gene expression that was a mix of megakaryocytes and pDCs. Further, we verified the expression of selected genes at both the RNA level by PCR and the protein level by flow cytometry. This study further demonstrates the power of single-cell RNA sequencing in dendritic cell research.
Cerebral ischemia triggers a cascade of events that contribute to ischemic brain damages. Zinc release and accumulation has been shown to lead to brain cell death following cerebral ischemia. However, the mechanism underlying remains to be elucidated. Our recently published work showed that suppression of mitochondrial-derived reactive oxygen species (ROS) production significantly reduced ischemic stroke related brain damage within 6 h. Herein, we investigated the relationship between zinc accumulation and mitochondrial-derived ROS production in astrocytes after 3-h hypoxia. We found that inhibition of mitochondrial-derived ROS significantly decreased total amount of ROS generation and cell death in primary astrocytes during hypoxia when zinc was overload. In contrast, the inhibition of NADPH oxidase-derived ROS had less of an effect. Our results also showed that zinc and mitochondria were colocalized in hypoxic astrocytes. Moreover, extracellular zinc addition caused zinc accumulation in the mitochondria and decreased mitochondrial membrane potential, leading to mitochondria dysfunction. These findings provide a novel mechanism that zinc accumulation contributes to hypoxiainduced astrocytes death by disrupting mitochondria function, following cerebral ischemia.