A designed angiopoietin-2 variant, pentameric COMP-Ang2, strongly activates Tie2 receptor and stimulates angiogenesis

AbstractDespite that angiopoietin-2 (Ang2) produces more versatile and dynamic functions than angiopoietin-1 (Ang1) in angiogenesis and inflammation, the molecular mechanism that underlies this difference is still unknown. To define the role of oligomerization of Ang2 in activation of its receptor, Tie2, we designed and generated different oligomeric forms of Ang2 by replacement of the amino-terminal domains of Ang2 with dimeric, tetrameric, and pentameric short coiled-coil domains derived from GCN4, matrillin-1, and COMP. COMP-Ang2 strongly binds and activates Tie2, whereas GCN4-Ang2 and MAT-Ang2 weakly to moderately bind and activate Tie2. Although native Ang2 strongly binds to Tie2, it does not activate Tie2. Accordingly, COMP-Ang2 strongly promotes endothelial cell survival, migration, and tube formation in a Tie2-dependent manner, and the potency of COMP-Ang2 is almost identical to that of COMP-Ang1. Furthermore, the potency of COMP-Ang2-induced enhanced angiogenesis in the wound healing region is almost identical to the potency of COMP-Ang1-induced enhanced angiogenesis. Overall, there is no obvious difference between COMP-Ang2 and COMP-Ang1 in in vitro and in vivo angiogenesis. Our results provide compelling evidence that proper oligomerization of Ang2 is a critical determinant of its binding and activation of Tie2

Toll-Like Receptor 4 Decoy, TOY, Attenuates Gram-Negative Bacterial Sepsis

Lipopolysaccharide (LPS), the Gram-negative bacterial outer membrane glycolipid, induces sepsis through its interaction with myeloid differentiation protein-2 (MD-2) and Toll-like receptor 4 (TLR4). To block interaction between LPS/MD-2 complex and TLR4, we designed and generated soluble fusion proteins capable of binding MD-2, dubbed TLR4 decoy receptor (TOY) using ‘the Hybrid leucine-rich repeats (LRR) technique’. TOY contains the MD-2 binding ectodomain of TLR4, the LRR motif of hagfish variable lymphocyte receptor (VLR), and the Fc domain of IgG1 to make it soluble, productive, and functional. TOY exhibited strong binding to MD-2, but not to the extracellular matrix (ECM), resulting in a favorable pharmacokinetic profile in vivo. TOY significantly extended the lifespan, when administered in either preventive or therapeutic manners, in both the LPS- and cecal ligation/puncture-induced sepsis models in mice. TOY markedly attenuated LPS-triggered NF-κB activation, secretion of proinflammatory cytokines, and thrombus formation in multiple organs. Taken together, the targeting strategy for sequestration of LPS/MD-2 complex using the decoy receptor TOY is effective in treating LPS- and bacteria-induced sepsis; furthermore, the strategy used in TOY development can be applied to the generation of other novel decoy receptor proteins

Microbial and molecular differences according to the location of head and neck cancers

Microbiome has been shown to substantially contribute to some cancers. However, the diagnostic implications of microbiome in head and neck squamous cell carcinoma (HNSCC) remain unknown. To identify the molecular difference in the microbiome of oral and non-oral HNSCC, primary data was downloaded from the Kraken-TCGA dataset. The molecular differences in the microbiome of oral and non-oral HNSCC were identified using the linear discriminant analysis effect size method. In the study, the common microbiomes in oral and non-oral cancers were Fusobacterium, Leptotrichia, Selenomonas and Treponema and Clostridium and Pseudoalteromonas, respectively. We found unique microbial signatures that positively correlated with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in oral cancer and positively and negatively correlated KEGG pathways in non-oral cancer. In oral cancer, positively correlated genes were mostly found in prion diseases, Alzheimer disease, Parkinson disease, Salmonella infection, and Pathogenic Escherichia coli infection. In non-oral cancer, positively correlated genes showed Herpes simplex virus 1 infection and Spliceosome and negatively correlated genes showed results from PI3K-Akt signaling pathway, Focal adhesion, Regulation of actin cytoskeleton, ECM-receptor interaction and Dilated cardiomyopathy. These results could help in understanding the underlying biological mechanisms of the microbiome of oral and non-oral HNSCC. Microbiome-based oncology diagnostic tool warrants further exploration.This work was supported by the National Research Foundation of Korea (NRF-2018R1A5A2023879, 2020R1A2C1005203, 2020R1C1C1003741, and 2021R1A2C4001466). This research was supported by a grant of the Medical data-driven hospital support project through the Korea Health Information Service (KHIS), funded by the Ministry of Health & Welfare, Republic of Korea. A portion of the data used for this study were obtained from the GenomeInfraNet (IDs: 1711020733, 1711032008, and 1711028992) of the Korea Bioinformation Center

Recent advances in intravital microscopy for investigation of dynamic cellular behavior in vivo

Currently, most biological research relies on conventional experimental techniques that allow only static analyses at certain time points in vitro or ex vivo. However, if one could visualize cellular dynamics in living organisms, that would provide a unique opportunity to study key biological phenomena in vivo. Intravital microscopy (IVM) encompasses diverse optical systems for direct viewing of objects, including biological structures and individual cells in live animals. With the current development of devices and techniques, IVM addresses important questions in various fields of biological and biomedical sciences. In this mini-review, we provide a general introduction to IVM and examples of recent applications in the field of immunology, oncology, and vascular biology. We also introduce an advanced type of IVM, dubbed real-time IVM, equipped with video-rate resonant scanning. Since the real-time IVM can render cellular dynamics with high temporal resolution in vivo, it allows visualization and analysis of rapid biological processes.Y

MarcoPolo: a method to discover differentially expressed genes in single-cell RNA-seq data without depending on prior clustering

The standard analysis pipeline for single-cell RNA-seq data consists of sequential steps initiated by clustering the cells. An innate limitation of this pipeline is that an imperfect clustering result can irreversibly affect the succeeding steps. For example, there can be cell types not well distinguished by clustering because they largely share the global structure, such as the anterior primitive streak and mid primitive streak cells. If one searches differentially expressed genes (DEGs) solely based on clustering, marker genes for distinguishing these types will be missed. Moreover, clustering depends on many parameters and can often be subjective to manual decisions. To overcome these limitations, we propose MarcoPolo, a method that identifies informative DEGs independently of prior clustering. MarcoPolo sorts out genes by evaluating if the distributions are bimodal, if similar expression patterns are observed in other genes, and if the expressing cells are proximal in a low-dimensional space. Using real datasets with FACS-purified cell labels, we demonstrate that MarcoPolo recovers marker genes better than competing methods. Notably, MarcoPolo finds key genes that can distinguish cell types that are not distinguishable by the standard clustering. MarcoPolo is built in a convenient software package that provides analysis results in an HTML file.Y

Mobility of Nucleostemin in Live Cells Is Specifically Related to Transcription Inhibition by Actinomycin D and GTP-Binding Motif

In vertebrates, nucleostemin (NS) is an important marker of proliferation in several types of stem and cancer cells, and it can also interact with the tumor-suppressing transcription factor p53. In the present study, the intra-nuclear diffusional dynamics of native NS tagged with GFP and two GFP-tagged NS mutants with deleted guanosine triphosphate (GTP)-binding domains were analyzed by fluorescence correlation spectroscopy. Free and slow binding diffusion coefficients were evaluated, either under normal culture conditions or under treatment with specific cellular proliferation inhibitors actinomycin D (ActD), 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), or trichostatin A (TSA). When treated with ActD, the fractional ratio of the slow diffusion was significantly decreased in the nucleoplasm. The decrease was proportional to ActD treatment duration. In contrast, DRB or TSA treatment did not affect NS diffusion. Interestingly, it was also found that the rate of diffusion of two NS mutants increased significantly even under normal conditions. These results suggest that the mobility of NS in the nucleoplasm is related to the initiation of DNA or RNA replication, and that the GTP-binding motif is also related to the large change of mobility

Direct Electron Transfer of Glucose Oxidase and Carbon Nanotubes Entrapped with Biocompatible Organic Materials

Efficient electron transfer between redox enzymes and electrodes is essential for enzyme-based biosensors, biofuel cells, and bioelectronic devices. Generally glucose oxidase (GOx) requires mediators for electrical communication with electrodes because the redox center of GOx is deeply buried in the insulating protein shell. In the present work, direct electron transfer (DET) between GOx and electrodes was attempted. GOx and carbon nanotubes (CNTs) were immobilized on a glassy carbon (GC) electrode by using biocompatible polymer, chitosan (CHI). Cyclic voltammograms revealed that the CHI/GOx/CNT-GC electrode showed a pair of well-defined redox peaks in 0.1M phosphate buffer solution (pH 7.0) saturated with argon. Under the same conditions, no redox peak was observed in the absence of CNTs. The formal redox potential was similar to 450mV (vs. Ag/AgCl), which agreed well with that of FAD/FADH(2), the redox center of GOx. This result clearly shows that the DET between the GOx and the electrode was achieved. The use of thin CNTs significantly improved the DET efficiency of the GOx. It was found that the GOx immobilized on the electrode retained catalytic activity for the oxidation of glucose.close5

Flexible Piezoelectric Energy Harvesting from Mouse Click Motions

In this paper, we study energy harvesting from the mouse click motions of a robot finger and a human index finger using a piezoelectric material. The feasibility of energy harvesting from mouse click motions is experimentally and theoretically assessed. The fingers wear a glove with a pocket for including the piezoelectric material. We model the energy harvesting system through the inverse kinematic framework of parallel joints in a finger and the electromechanical coupling equations of the piezoelectric material. The model is validated through energy harvesting experiments in the robot and human fingers with the systematically varying load resistance. We find that energy harvesting is maximized at the matched load resistance to the impedance of the piezoelectric material, and the harvested energy level is tens of nJ

KULEX-Hand: An Underactuated Wearable Hand for Grasping Power Assistance

In this paper, we present KULEX-hand, a novel underactuated hand exoskeleton for grasping power assistance for patients having a partially paralyzed hand or the elderly with weakened muscle strength. This mechanism consists of an underactuated finger for grasp motion generation, a spherical four-bar linkage for power transmission, and a passive thumb link with a flexure hinge structure. Based on the natural closing motion of the human index finger, the motion generation linkage was synthesized as a planar five-bar in which two input links were coupled with a four-bar linkage. Therefore, after contact occurs on the proximal link, the synthesized linkage can mimic the grasping motion of the human middle and distal phalanges. The kinetoelastic relation of the underactuated finger was derived using the theory of screws. Based on this relation, guidelines were proposed for selecting the springs for achieving a stable pinch grasp. A prototype was designed, and the naturalness of motion was evaluated from an experiment with five subjects.11Nsciescopu