Screening and construction of nanobodies against human CD93 using phage libraries and study of their antiangiogenic effects

BackgroundCluster of Differentiation 93 (CD93) plays an important role in angiogenesis and is considered an important target for inhibiting tumor angiogenesis, but there are currently no therapeutic antibodies against CD93 in the clinic. Thus, we describe the screening of novel nanobodies (Nbs) targeting human CD93 from a phage library of shark-derived Nbs.MethodsScreening and enrichment of phage libraries by enzyme-linked immunosorbent assay (ELISA). Anti-CD93 Nbs were purified by expression in E. coli. The binding affinity of anti-CD93 Nbs NC81/NC89 for CD93 was examined by flow cytometry (FC) and ELISA. The thermal stability of NC81/NC89 was examined by ELISA and CD spectroscopy. Afterward, the anti-angiogenic ability of NC81/NC89 was examined by MTT, wound healing assay, and tube formation assay. The expression level of VE-cadherin (VE-Ca) and CD93 was detected by Western Blot (WB). The binding sites and binding forms of NC81/NC89 to CD93 were analyzed by molecular docking.ResultsThe anti-CD93 Nbs were screened in a phage library, expressed in E. coli, and purified to >95% purity. The results of FC and ELISA showed that NC81/NC89 have binding ability to human umbilical vein endothelial cells (HUVECs). The results of ELISA and CD spectroscopy showed that NC81/NC89 retained the ability to bind CD93 at 80°C and that the secondary structure remained stable. In vitro, the results showed that NC81 and NC89 significantly inhibited the proliferation and migration of human umbilical vein endothelial cells (HUVECs) as well as tube formation on Matrigel. Western Blot showed that NC81 and NC89 also inhibited the expression of VE-Ca thereby increasing vascular permeability. It was found during molecular docking that the CDR regions of NC81 and NC89 could be attached to CD93 by strong hydrogen bonds and salt bridges, and the binding sites were different.ConclusionWe have successfully isolated NC81 and NC89, which bind CD93, and both Nbs significantly inhibit angiogenesis and increase vascular permeability. These results suggest that NC81 and NC89 have potential clinical applications in angiogenesis-related therapies

JujubeNet: A high-precision lightweight jujube surface defect classification network with an attention mechanism

Surface Defect Detection (SDD) is a significant research content in Industry 4.0 field. In the real complex industrial environment, SDD is often faced with many challenges, such as small difference between defect imaging and background, low contrast, large variation of defect scale and diverse types, and large amount of noise in defect images. Jujubes are naturally growing plants, and the appearance of the same type of surface defect can vary greatly, so it is more difficult than industrial products produced according to the prescribed process. In this paper, a ConvNeXt-based high-precision lightweight classification network JujubeNet is presented to address the practical needs of Jujube Surface Defect (JSD) classification. In the proposed method, a Multi-branching module using Depthwise separable Convolution (MDC) is designed to extract more feature information through multi-branching and substantially reduces the number of parameters in the model by using depthwise separable convolutions. What’s more, in our proposed method, the Convolutional Block Attention Module (CBAM) is introduced to make the model concentrate on different classes of JSD features. The proposed JujubeNet is compared with other mainstream networks in the actual production environment. The experimental results show that the proposed JujubeNet can achieve 99.1% classification accuracy, which is significantly better than the current mainstream classification models. The FLOPS and parameters are only 30.7% and 30.6% of ConvNeXt-Tiny respectively, indicating that the model can quickly and effectively classify JSD and is of great practical value

A nanobody-based molecular toolkit for ubiquitin–proteasome system explores the main role of survivin subcellular localization

Targeted protein degradation is a powerful tool for determining the function of specific proteins nowadays. Survivin is the smallest member of the inhibitor of the apoptosis protein (IAP) family. It exists in the cytoplasm and nucleus of cells, but the exact function of survivin in different subcellular locations retained unclear updates due to the lack of effective and simple technical means. In this study, we created a novel nanoantibody-based molecular toolkit, namely, the ubiquitin–proteasome system (Nb4A-Fc-T2A-TRIM21), that can target to degrade survivin localized in cytoplasmic and cell nuclear by ubiquitinating, and by which to verify the potential roles of survivin subcellular localization. Also, the results showed that the cytoplasmic survivin mainly plays an anti-apoptotic function by directly or indirectly inhibiting the caspase pathway, and the nuclear survivin mainly promotes cell proliferation and participates in the regulation of the cell cycle. In addition, the Nb4A-Fc-T2A-TRIM21 system can degrade the endogenous survivin protein in a large amount by the ubiquitin–proteasome pathway, and the system can provide theoretical support for ubiquitination degradation targeting other endogenous proteins

Extreme suppression of antiferromagnetic order and critical scaling in a two-dimensional random quantum magnet

Sr_2CuTeO_6 is a square-lattice Néel antiferromagnet with superexchange between first-neighbor S=1/2 Cu spins mediated by plaquette centered Te ions. Substituting Te by W, the affected impurity plaquettes have predominantly second-neighbor interactions, thus causing local magnetic frustration. Here we report a study of Sr_2CuTe_1-xW_xO_6 using neutron diffraction and μSR techniques, showing that the Néel order vanishes already at x=0.025±0.005. We explain this extreme order suppression using a two-dimensional Heisenberg spin model, demonstrating that a W-type impurity induces a deformation of the order parameter that decays with distance as 1/r^2 at temperature T=0. The associated logarithmic singularity leads to loss of order for any x>0. Order for small x>0 and T>0 is induced by weak interplane couplings. In the nonmagnetic phase of Sr_2CuTe_1-x W_x O_6, the μSR relaxation rate exhibits quantum critical scaling with a large dynamic exponent, z≈3, consistent with a random-singlet state.Accepted manuscrip

Modeling Autonomous Vehicles’ Altruistic Behavior to Human-Driven Vehicles in the Car following Events and Impact Analysis

To explore the impact of autonomous vehicles (AVs) on human-driven vehicles (HDVs), a solution for AV to coexist harmoniously with HDV during the car following period when AVs are in low market penetration rate (MPR) was provided. An extension car following framework with two possible soft optimization targets was proposed in this article to improve the experience of HDV followers with different following strategies by deep deterministic policy gradient (DDPG) algorithm. The pretreated Next Generation Simulation (NGSIM) dataset was used for the experiments. 1027 car following events with being redefined were extracted from it, in which 600 of the events were used for training and 427 of the events were used for testing. The different driving strategies obtained from the classical car following models were embedded into virtual environment built by OpenAI gym. The reward function combined safety, efficiency, jerk, and stability was used to encourage the agent with DDPG algorithm to maximize it. The final result reveals that disturbance of HDV followers decreases by 2.362% (strategy a), 8.184% (strategy b), and 13.904% (strategy c), respectively. The disturbance of HDV follower decreases by 14.961% (strategy a), 12.020% (strategy b), and 13.425% (strategy c), respectively. HDV followers with different strategies get less jerk in both soft optimizations. AV passengers get a loss on jerk and efficiency, but safety is enhanced. Also, AV car following performs better than HDV car following in both soft and brutal optimizations. Moreover, two possible solutions for harmonious coexistence of HDVs and AVs when AVs are in low MPR are proposed

doi: 10.1093/jpe/rtn005 Advanced Access published

available online at www.jpe.oxfordjournals.org GeoSVM: an efficient and effective tool to predict species ’ potential distribution

Split‐Cas9‐based targeted gene editing and nanobody‐mediated proteolysis‐targeting chimeras optogenetically coordinated regulation of Survivin to control the fate of cancer cells

Abstract Background Precise regulation of partial critical proteins in cancer cells, such as anti‐apoptotic proteins, is one of the crucial strategies for treating cancer and discovering related molecular mechanisms. Still, it is also challenging in actual research and practice. The widely used CRISPR/Cas9‐based gene editing technology and proteolysis‐targeting chimeras (PROTACs) have played an essential role in regulating gene expression and protein function in cells. However, the accuracy and controllability of their targeting remain necessary. Methods Construction of UMUC‐3‐EGFP stable transgenic cell lines using the Sleeping Beauty system, Flow cytometry, quantitative real‐time PCR, western blot, fluorescence microplate reader and fluorescence inverted microscope analysis of EGFP intensity. Characterization of Survivin inhibition was done by using Annexin V‐FITC/PI apoptosis, calcein/PI/DAPI cell viability/cytotoxicity assay, cloning formation assay and scratch assay. The cell‐derived xenograft (CDX) model was constructed to assess the in vivo effects of reducing Survivin expression. Results Herein, we established a synergistic control platform that coordinated photoactivatable split‐Cas9 targeted gene editing and light‐induced protein degradation, on which the Survivin gene in the nucleus was controllably edited by blue light irradiation (named paCas9‐Survivin) and simultaneously the Survivin protein in the cytoplasm was degraded precisely by a nanobody‐mediated target (named paProtacL‐Survivin). Meanwhile, in vitro experiments demonstrated that reducing Survivin expression could effectively promote apoptosis and decrease the proliferation and migration of bladder cancerous cells. Furthermore, the CDX model was constructed using UMUC‐3 cell lines, results from animal studies indicated that both the paCas9‐Survivin system and paProtacL‐Survivin significantly inhibited tumour growth, with higher inhibition rates when combined. Conclusions In short, the coordinated regulatory strategies and combinable technology platforms offer clear advantages in controllability and targeting, as well as an excellent reference value and universal applicability in controlling the fate of cancer cells through multi‐level regulation of key intracellular factors