Upregulation of Robo4 expression by SMAD signaling suppresses vascular permeability and mortality in endotoxemia and COVID-19 models.

There is an urgent need to develop novel drugs to reduce the mortality from severe infectious diseases with the emergence of new pathogens, including Coronavirus disease 2019 (COVID-19). Although current drugs effectively suppress the proliferation of pathogens, immune cell activation, and inflammatory cytokine functions, they cannot completely reduce mortality from severe infections and sepsis. In this study, we focused on the endothelial cell-specific protein, Roundabout 4 (Robo4), which suppresses vascular permeability by stabilizing endothelial cells, and investigated whether enhanced Robo4 expression could be a novel therapeutic strategy against severe infectious diseases. Endothelial-specific overexpression of Robo4 suppresses vascular permeability and reduces mortality in lipopolysaccharide (LPS)-treated mice. Screening of small molecules that regulate Robo4 expression and subsequent analysis revealed that two competitive small mothers against decapentaplegic (SMAD) signaling pathways, activin receptor-like kinase 5 (ALK5)-SMAD2/3 and ALK1-SMAD1/5, positively and negatively regulate Robo4 expression, respectively. An ALK1 inhibitor was found to increase Robo4 expression in mouse lungs, suppress vascular permeability, prevent extravasation of melanoma cells, and decrease mortality in LPS-treated mice. The inhibitor suppressed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced endothelial barrier disruption and decreased mortality in mice infected with SARS-CoV-2. These results indicate that enhancing Robo4 expression is an efficient strategy to suppress vascular permeability and mortality in severe infectious diseases, including COVID-19, and that small molecules that upregulate Robo4 can be potential therapeutic agents against these diseases.

Potential Therapeutic Strategies and Drugs That Target Vascular Permeability in Severe Infectious Diseases.

Severe infection pathogenicity is induced by processes such as pathogen exposure, immune cell activation, inflammatory cytokine production, and vascular hyperpermeability. Highly effective drugs, such as antipathogenic agents, steroids, and antibodies that suppress cytokine function, have been developed to treat the first three processes. However, these drugs cannot completely suppress severe infectious diseases, such as coronavirus disease 2019 (COVID-19). Therefore, developing novel drugs that inhibit vascular hyperpermeability is crucial. This review summarizes the mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced vascular hyperpermeability and identifies inhibitors that increase endothelial cell (EC) junction-related proteins and determines their efficacy in COVID-19 and endotoxemia models. Analyzing the effects of SARS-CoV-2 on vascular permeability revealed that SARS-CoV-2 suppresses Claudin-5 (CLDN5) expression, which is responsible for adhesion between ECs, thereby increasing vascular permeability. Inhibiting CLDN5 function in mice induced vascular hyperpermeability and pulmonary edema. In contrast, Enhancing CLDN5 expression suppressed SARS-CoV-2-induced endothelial hyperpermeability, suggesting that SARS-CoV-2-induced vascular hyperpermeability contributes to pathological progression, which can be suppressed by upregulating EC junction proteins. Based on these results, we focused on Roundabout4 (Robo4), another EC-specific protein that stabilizes EC junctions. EC-specific Robo4 overexpression suppressed vascular hyperpermeability and mortality in lipopolysaccharide-treated mice. An ALK1 inhibitor (a molecule that increases Robo4 expression), suppressed vascular hyperpermeability and mortality in lipopolysaccharide- and SARS-CoV-2-treated mice. These results indicate that Robo4 expression-increasing drugs suppress vascular permeability and pathological phenotype in COVID-19 and endotoxemia models.

Risk Assessment of Roundabout Scenarios in Virtual Testing Based on an Improved Driving Safety Field.

With the advancement of autonomous driving technology, scenario-based testing has become the mainstream testing method for intelligent vehicles. However, traditional risk indicators often fail in roundabout scenarios and cannot accurately define dangerous situations. To accurately quantify driving risks in roundabout scenarios, an improved driving safety field model is proposed in this paper. First, considering the unique traffic flow characteristics of roundabouts, the dynamic characteristics of vehicles during diverging or merging were taken into account, and the driving safety field model was improved to accurately quantify the driving risks in roundabout scenarios. Second, based on data from the rounD dataset, the model parameters were calibrated using the social force model. Finally, a DENCLUE-like method was used to extract collision systems, calculate vehicle risk degree, and analyze these risks for both the temporal and the spatial dimensions, providing guidance for virtual testing. The proposed method significantly improves detection efficiency, increasing the number of identified dangerous scenarios by 175% compared to the Time to Collision (TTC) method. Moreover, this method can more accurately quantify driving risks in roundabout scenarios and enhance the efficiency of generating dangerous scenarios, contributing to promoting the safety of autonomous vehicles.

A Nedd4 E3 Ubiquitin Ligase Pathway Inhibits Robo1 Repulsion and Promotes Commissural Axon Guidance across the Midline.

Commissural axons initially respond to attractive signals at the midline, but once they cross, they become sensitive to repulsive cues. In insects and mammals, negative regulation of the surface expression of Roundabout (Robo) receptors prevents premature response to Slit. We previously identified two mammalian Nedd4 interacting proteins, Ndfip1 and Ndfip2, that act analogously to Drosophila Commissureless (Comm) to recruit mammalian Robo1 to late endosomes. However, whether Nedd4 E3 ubiquitin ligases are required for Ndfip-mediated Robo1 regulation and midline axon crossing in vivo is not known. Here, we show using in vitro biochemical techniques and genetic analysis using embryonic mice of either sex that Nedd4-1 and Nedd4-2 are specifically required for Robo1 regulation and spinal commissural axon guidance. Biochemical data indicate that Robo1, Ndfip and Nedd4 form a ternary protein complex that depends on the presence of Ndfip, and these interactions are required for Robo1 endosomal sorting, ubiquitylation and degradation. Nedd4-1 and Nedd4-2 are expressed in commissural neurons in the developing spinal cord, and conditional deletion of Nedd4-1 or Nedd4-2 results in dose-dependent defects in midline crossing. We propose that Nedd4 E3 Ubiquitin ligases and their adaptor proteins Ndfip1 and Ndfip2 constitute a vital intracellular trafficking pathway required to downregulate Robo1 and promote midline crossing of commissural axons.SIGNIFICANCE STATEMENT During the development of the nervous system, many neurons extend their axons across the midline to establish circuits that are important for sensory, motor and cognitive functions. In order to cross the midline, axon responses to midline-derived cues must be precisely regulated. Here, we characterize an important intracellular trafficking pathway that regulates the membrane expression of the conserved Roundabout (Robo) axon guidance receptor- the receptor for the midline repellant Slit. We show that Nedd4 E3 Ubiquitin ligases and their Ndfip adapter proteins inhibit premature responses to Slit by promoting Robo degradation in precrossing commissural neurons in the developing spinal cord.

Inhibition of MAPK/ERK pathway activation rescues congenital anomalies of the kidney and urinary tract (CAKUT) in Robo2PB/+ Gen1PB/+ mice.

Congenital anomalies of the kidney and urinary tract (CAKUT) have been attributed to genetic and environmental factors. However, monogenic and copy number variations cannot sufficiently explain the cause of the majority of CAKUT cases. Multiple genes through various modes of inheritance may lead to CAKUT pathogenesis. We previously showed that Robo2 and Gen1 coregulated the germination of ureteral buds (UB), significantly increasing CAKUT incidence. Furthermore, MAPK/ERK pathway activation is the central mechanism of these two genes. Thus, we explored the effect of the MAPK/ERK inhibitor U0126 in the CAKUT phenotype in Robo2PB/+Gen1PB/+ mice. Intraperitoneal injection of U0126 during pregnancy prevented the development of the CAKUT phenotype in Robo2PB/+Gen1PB/+ mice. Additionally, a single dose of 30 mg/kg U0126 on day 10.5 embryos (E10.5) was most effective for reducing CAKUT incidence and ectopic UB outgrowth in Robo2PB/+Gen1PB/+ mice. Furthermore, embryonic kidney mesenchymal levels of p-ERK were significantly decreased on day E11.5 after U0126 treatment, along with decreased cell proliferation index PHH3 and ETV5 expression. Collectively, Gen1 and Robo2 exacerbated the CAKUT phenotype in Robo2PB/+Gen1PB/+ mice through the MAPK/ERK pathway, increasing proliferation and ectopic UB outgrowth.

miR-152-3p impedes the malignant phenotypes of hepatocellular carcinoma by repressing roundabout guidance receptor 1.

BACKGROUND: miR-152-3p functions as a tumour suppressor in the progression of hepatic tumorigenesis. Herein, we further discussed the prognostic significance and immune infiltration of miR-152-3p and its potential gene target in hepatocellular carcinoma (HCC). METHODS: The Cancer Genome Atlas (TCGA), Integrative Molecular Database of Hepatocellular Carcinoma (HCCDB), Human Protein Atlas (HPA) and Kaplan-Meier Plotter databases were used to evaluate miR-152-3p and roundabout guidance receptor 1 (ROBO1) expression, prognosis and immune infiltration. In vitro cell experiments, including cell proliferation and apoptosis, were evaluated using Cell Counting Kit 8 (CCK8) and terminal-deoxynucleotidyl transferase-mediated nick end labelling (TUNEL) assays. RESULTS: Up-regulation of ROBO1 functioned as an oncogene associated with poor prognosis, immune cell enrichment and cell proliferation in HCC. ROBO1 was significantly positively correlated with the enrichment of multiple immune cells and their biomarkers. Enrichment of type-2 T-helper (Th2) cells is an unfavourable biomarker of HCC prognosis. GSEA revealed that ROBO1 correlated with apoptosis, mitosis and carcinogenic signalling pathways. Suppression of cell proliferation and the enhancement of cell apoptosis by miR-152-3p mimics were counteracted by overexpression of ROBO1 in HCC cells. CONCLUSION: ROBO1 expression is positively correlated with multiple immune checkpoint molecules, suggesting that ROBO1 may be a potential drug target to enhance the potency of immunotherapy. The miR-152-3p/ROBO1 signalling axis contributes to malignant progression and provides a prospective immunotherapeutic target for HCC.

Simulation-Based Analysis of "What-If" Scenarios with Connected and Automated Vehicles Navigating Roundabouts.

Despite the potential of connected and automated vehicles (CAVs), there are still many open questions on how road capacity can be influenced and what methods can be used to assess its expected benefits in the progressive transition towards fully cooperative driving. This paper contributes to a better understanding of the benefits of CAV technologies by investigating mobility-related issues of automated vehicles operating with a cooperative adaptive cruise control system on roundabout efficiency using microscopic traffic simulation. The availability of the adjustment factors for CAVs provided by the 2022 Highway Capacity Manual allowed to adjust the entry capacity equations to reflect the presence of CAVs on roundabouts. Two mechanisms of entry maneuver based on the entry lane type were examined to compare the capacity target values with the simulated capacities. The microscopic traffic simulator Aimsun Next has been of great help in building the "what-if" traffic scenarios that we analysed to endorse hypothesis on the model parameters which affect the CAVs' capabilities to increase roundabouts' throughput. The results highlighted that the increasing penetration rates of CAVs have greater impacts on the operational performances of roundabouts, and provided a synthetic insight to assess the potential benefits of CAVs from an efficiency perspective.

Calculation of an Average Vehicle's Sideways Acceleration on Small Roundabouts.

The calculation of the average sideways acceleration, based on speed and angular velocity on small roundabouts for a vehicle of up to 3.5 t gross vehicle mass, is described in this paper. Calculations of the turning radius are derived from angular velocity and an automatic selection of events, based on the lateral acceleration of the coefficient of variation within a defined time window. The calculation of the turning radius based on speed and angular velocity yields almost identical results to the calculation of the turning radius by the three-point method using GPS coordinates, as described in previous research. This means that the calculation of the turning radius, derived from the speed of GNSS/INS dual-antenna sensor and gyroscope data, yields similar results to those from the computation of the turning radius derived from the coordinates of a GNSS/INS dual-antenna sensor. The research results can be used in the development of sensors to improve road safety.

Soluble TREM-1, as a new ligand for the membrane receptor Robo2, promotes hepatic stellate cells activation and liver fibrosis.

Triggering receptor expressed on myeloid cells-1 (TREM-1) exists in two forms: a transmembrane form and a soluble form (sTREM-1). The levels of sTREM-1 are elevated in supernatants of activated HSCs. However, the role of sTREM-1 in HSC activation and liver fibrosis remains undefined. Previous studies have primarily focused on the transmembrane form of TREM-1; we innovatively observed the function of sTREM-1 as a ligand in liver fibrosis and screened its receptor. Here, recombinant sTREM-1 was used as a stimulator which induced HSC activation and further aggravated liver fibrosis. Then, screening for sTREM-1 interacting membrane receptors was performed using pull-down assay followed by mass spectrometry, and the membrane receptor roundabout guidance receptor 2 (Robo2) was identified as a candidate receptor for sTREM-1. The interaction between sTREM-1 and Robo2 was verified by pull-down and immunofluorescence. The role of Robo2 on sTREM-1-induced HSC activation and its downstream signal pathways was assessed by knockdown of Robo2 in LX-2 cells. Furthermore, HSC-specific knockdown of Robo2 was achieved in a mouse model of liver fibrosis by using a recombinant adeno-associated virus (AAV) vector to confirm the role of the receptor, and we proved that Robo2 knockdown inhibited the activation of HSC and liver fibrosis, which also led to the inactivation of Smad2/3 and PI3K/Akt pathways in sTREM-1-induced HSC activation and liver fibrosis. In conclusion, sTREM-1 acts as a new ligand of Robo2; the binding of sTREM-1 to Robo2 initiates the activation of the downstream Smad2/3 and PI3K/Akt signalling pathways, thereby promoting HSC activation and liver fibrosis.

Vascular Leakage Prevention by Roundabout 4 under Pathological Conditions.

Vascular permeability is regulated mainly by the endothelial barrier and controls vascular homeostasis, proper vessel development, and immune cell trafficking. Several molecules are involved in regulating endothelial barrier function. Roundabout 4 (Robo4) is a single-pass transmembrane protein that is specifically expressed in vascular endothelial cells. Robo4 is an important regulator of vascular leakage and angiogenesis, especially under pathological conditions. The role of Robo4 in preventing vascular leakage has been studied in various disease models, including animal models of retinopathy, tumors, diabetes, and endotoxemia. The involvement of Robo4 in vascular endothelial growth factor and inflammation-mediated signaling pathways has been well studied, and recent evidence suggests that Robo4 modulates endothelial barrier function via distinct mechanisms. In this review, we discuss the role of Robo4 in endothelial barrier function and the underlying molecular mechanisms.

Multivariable Optimisation for Waiting-Time Minimisation at Roundabout Intersections in a Cyber-Physical Framework.

The evolution of communication networks offers new possibilities for development in the automotive industry. Smart vehicles will benefit from the possibility of connecting with the infrastructure and from an extensive exchange of data between them. Furthermore, new control strategies can be developed that benefit the advantages of these communication networks. In this endeavour, the main purposes considered by the automotive industry and researchers from academia are defined by: (i) ensuring people's safety; (ii) reducing the overall costs, and (iii) improving the traffic by maximising the fluidity. In this paper, a cyber-physical framework (CPF) to control the access of vehicles in roundabout intersections composed of two levels is proposed. Both levels correspond to the cyber part of the CPF, while the physical part is composed of the vehicles crossing the roundabout. The first level, i.e., the edge-computing layer, is based on an analytical solution that uses multivariable optimisation to minimise the waiting times of the vehicles entering a roundabout intersection and to ensure a safe crossing. The second level, i.e., the cloud-computing layer, stores information about the waiting times and trajectories of all the vehicles that cross the roundabout and uses them for long-term analysis and prediction. The simulated results show the efficacy of the proposed method, which can be easily implemented on an embedded device for real-time operation.

Bi-Level Coordinated Merging of Connected and Automated Vehicles at Roundabouts.

Traditional uncoordinated traffic flows in a roundabout can lead to severe traffic congestion, travel delay, and the increased fuel consumption of vehicles. An interesting way to mitigate this would be through cooperative control of connected and automated vehicles (CAVs). In this paper, we propose a novel solution, which is a roundabout control system (RCS), for CAVs to attain smooth and safe traffic flows. The RCS is essentially a bi-level framework, consisting of higher and lower levels of control, where in the higher level, vehicles in the entry lane approaching the roundabout will be made to form clusters based on traffic flow volume, and in the lower level, the vehicles' optimal sequences and roundabout merging times are calculated by solving a combinatorial optimization problem using a receding horizon control (RHC) approach. The proposed RCS aims to minimize the total time taken for all approaching vehicles to enter the roundabout, whilst minimally affecting the movement of circulating vehicles. Our developed strategy ensures fast optimization, and can be implemented in real-time. Using microscopic simulations, we demonstrate the effectiveness of the RCS, and compare it to the current traditional roundabout system (TRS) for various traffic flow scenarios. From the results, we can conclude that the proposed RCS produces significant improvement in traffic flow performance, in particular for the average velocity, average fuel consumption, and average travel time in the roundabout.

Intrauterine low-protein diet aggravates developmental abnormalities of the urinary system via the Akt/Creb3 pathway in Robo2 mutant mice.

The offspring of Robo2 mutant mice usually present with variable phenotypes of congenital anomalies of the kidney and urinary tract (CAKUT). An intrauterine low-protein diet can also cause CAKUT in offspring, dominated by the duplicated collecting system phenotype. A single genetic or environment factor can only partially explain the pathogenesis of CAKUT. The present study aimed to establish an intrauterine low-protein diet roundabout 2 (Robo2) mutant mouse model and found that the intrauterine low-protein diet led to significantly increased CAKUT phenotypes in Robo2PB/+ mice offspring, dominant by a duplicated collecting system. At the same time, more ectopic and lower located ureteric buds (UBs) were observed in the intrauterine low-protein diet-fed Robo2 mutant mouse model, and the number of UB branches was reduced in the serum-free culture. During UB protrusion, intrauterine low-protein diet reduced the expression of Slit2/Robo2 in Robo2 mutant mice and affected the expression of glial cell-derived neurotrophic factor/Ret, which is a key molecule for metanephric development, with increasing phospho-Akt and phospho-cAMP responsive element-binding protein 3 activity and a reduction of apoptotic cells in embryonic day 11.5 UB tissues. The mechanism by which an intrauterine low-protein diet aggravates CAKUT in Robo2 mutant mice may be related to the disruption of Akt/cAMP responsive element-binding protein 3 signaling and a reduction in apoptosis in UB tissue.

PRC2 Components Maintain DNA Hypermethylation of the Upstream Promoter and Regulate Robo4 Expression in Endothelial Cells.

Roundabout4 (Robo4) is an endothelial cell-specific protein that stabilizes the vasculature in pathological angiogenesis and inflammation. We previously determined a 3-kb Robo4 promoter and demonstrated the importance of the upstream region for nuclear factor-kappaB (NF-κB)-mediated promoter activation induced by tumor necrosis factor α (TNFα). This region contains unique genomic features, including promoter region-specific DNA hypermethylation and chromatin condensation; however, the function of the region remains poorly understood. In this study, we analyzed the DNA sequences of the region and identified a motif for polycomb repressive complex 2 (PRC2). Chromatin immunoprecipitation assay indicates the binding of the PRC2 component, SUZ12, to the motif. A mutation in the motif decreased DNA methylation in embryonic stem cells and increased Robo4 promoter activity in endothelial cells. An inhibitor for the PRC2 component, EZH2, induced the promoter activity and expression of Robo4 in endothelial cells treated with or without TNFα. Taken together, these results indicate that the PRC2 components maintain DNA hypermethylation and suppress Robo4 expression via the PRC2 binding motif in the upstream promoter.

Dataset Construction from Naturalistic Driving in Roundabouts.

A proper driver characterization in complex environments using computational techniques depends on the richness and variety of data obtained from naturalistic driving. The present article proposes the construction of a dataset from naturalistic driving specific to maneuvers in roundabouts and makes it open and available to the scientific community for performing their own studies. The dataset is a combination of data gathered from on-board instrumentation and data obtained from the post-processing of maps as well as recorded videos. The approach proposed in this paper consists of handling roundabouts as a stretch of road that includes 100 m before the entrance, the internal part, and 100 m after the exit. This stretch of road is then spatially sampled in small sections to which data are associated.

[Association of plasma roundabout 4 concentration with pulmonary ventilation function decline in COPD patients].

Objective: To investigate the association of plasma roundabout 4 concentration with pulmonary ventilation function decline in chronic obstructive pulmonary disease (COPD) patients. Methods: To get the effective data, the study was conducted in the outpatient department of West China Hospital from September 2017 to September 2018. The subjects meeting the inclusion and exclusion criteria were continuously included. Among them, the COPD group (75 cases) was from the respiratory outpatient department, and the healthy control group (57 cases) was from the health examination center at the same time. Data of basic demographic characteristics, clinical characteristics, pulmonary ventilation function parameters and blood samples were collected. The concentrations of roundabout 4, C reactive protein (CRP), interleukin (IL)-6, IL-8, IL-1b and tumor necrosis factor (TNF)-α in plasma were detected, and the differences among groups were compared, the correlation between plasma roundabout 4 and pulmonary ventilation function parameters and inflammatory factors was analyzed. The diagnostic efficiency of roundabout 4 to COPD was analyzed according to receiver operating characteristic (ROC) curve. Results: The plasma concentration of roundabout 4 in COPD group was significantly higher than that in healthy control group [(41.3±14.2) vs (27.7±13.3) ng/L; P<0.001], the sensitivity and specificity of roundabout 4 in the diagnosis of COPD were 0.827 and 0.702 respectively. Correlation analysis showed that the plasma concentration of roundabout 4 was negatively correlated with lung function parameters forced expiratory volume in one second/forced vital capacity (FEV(1)/FVC), the first second forced expiratory volume as a percentage of the estimated value (FEV(1)%pred), forced exhalation of 50% and 25% lung capacity (MEF50, MEF25) and maximal mid-expiratory flow (MMEF) (r=-0.399, -0.321, -0.439, -0.363, -0.458; all P<0.001), positively correlated with CRP (adjusted r=0.311, P<0.001). Conclusion: The increased concentration of roundabout 4 in plasma leads to the decline of pulmonary ventilation function in COPD patients.

Enhanced ROBO4 is mediated by up-regulation of HIF-1α/SP1 or reduction in miR-125b-5p/miR-146a-5p in diabetic retinopathy.

Retinal cell damage caused by diabetes leads to retinal microvascular injury. Roundabout 4 (ROBO4) is involved in angiogenesis, which varies with the development of diabetic retinopathy (DR). Here, we explored the transcriptional regulation and microRNA-mediated modulation of ROBO4 expression and related retinal cell function in DR. A streptozotocin-induced type I diabetic animal model was established to detect the expression of hypoxia inducible factor-1α (HIF-1α), specificity protein 1 (SP1) and ROBO4. Retinal pigment epithelium (RPE) cells were cultured under hyperglycaemia or hypoxia and used for mechanistic analysis. Furthermore, roles of miR-125b-5p and miR-146a-5p were evaluated, and their targets were identified using luciferase assays. The cell functions were evaluated by MTS assays, permeability analysis and migration assays. The development of DR increased the levels of HIF-1α, SP1 and ROBO4 both in the DR model and in hyperglycaemic/hypoxic RPE cells. They were co-expressed and up-regulated in diabetic retinas and in RPE cells under hyperglycaemia/hypoxia. Knockdown of HIF-1α significantly inhibited SP1 and ROBO4, whereas SP1 down-regulation abolished ROBO4 expression in RPE cells under hyperglycaemia/hypoxia. miR-125b-5p and miR-146a-5p were down-regulated by hyperglycaemia and/or hypoxia. Up-regulation of miRNAs reversed these changes and resulted in recovery of target gene expression. Moreover, luciferase assays confirmed miR-125b-5p targeted SP1 and ROBO4, and miR-146a-5p targeted HIF-1α and ROBO4 directly. The decreased cell viability, enhanced permeability, and increased cell migration under DR conditions were mitigated by knockdown of HIF-1α/SP1/ROBO4 or up-regulation of miR-125b-5p/miR-146a-5p. In general, our results identified a novel mechanism that miR-125b-5p/miR-146a-5p targeting HIF-1α/SP1-dependent ROBO4 expression could retard DR progression.

Deletion of Robo4 prevents high-fat diet-induced adipose artery and systemic metabolic dysfunction.

OBJECTIVE: Accumulating evidence suggests the vascular endothelium plays a fundamental role in the pathophysiology of obesity by regulating the functional status of white adipose and systemic metabolism. Robo4 is expressed specifically in endothelial cells and increases vascular stability and inhibits angiogenesis. We sought to determine the role of Robo4 in modulating cardiometabolic function in response to high-fat feeding. METHODS: We examined exercise capacity, glucose tolerance, and white adipose tissue artery gene expression, endothelium-dependent dilation (EDD), and angiogenesis in wild type and Robo4 knockout (KO) mice fed normal chow (NC) or a high-fat diet (HFD). RESULTS: We found Robo4 deletion enhances exercise capacity in NC-fed mice and HFD markedly increased the expression of the Robo4 ligand, Slit2, in white adipose tissue. Deletion of Robo4 increased angiogenesis in white adipose tissue and protected against HFD-induced impairments in white adipose artery vasodilation and glucose intolerance. CONCLUSIONS: We demonstrate a novel functional role for Robo4 in endothelial cell function and metabolic homeostasis in white adipose tissue, with Robo4 deletion protecting against endothelial and metabolic dysfunction associated with a HFD. Our findings suggest that Robo4-dependent signaling pathways may be a novel target in anti-obesity therapy.

Predicting Agent Behaviour and State for Applications in a Roundabout-Scenario Autonomous Driving.

As human drivers, we instinctively employ our understanding of other road users' behaviour for enhanced efficiency of our drive and safety of the traffic. In recent years, different aspects of assisted and autonomous driving have gotten a lot of attention from the research and industrial community, including the aspects of behaviour modelling and prediction of future state. In this paper, we address the problem of modelling and predicting agent behaviour and state in a roundabout traffic scenario. We present three ways of modelling traffic in a roundabout based on: (i) the roundabout geometry; (ii) mean path taken by vehicles inside the roundabout; and (iii) a set of reference trajectories traversed by vehicles inside the roundabout. The roundabout models are compared in terms of exit-direction classification and state (i.e., position inside the roundabout) prediction of query vehicles inside the roundabout. The exit-direction classification and state prediction are based on a particle-filter classifier algorithm. The results show that the roundabout model based on set of reference trajectories is better suited for both the exit-direction and state prediction.

[Killing effect of Robo1 targeted Chimeric Antigen Receptor modified NK92 cells against glioma and neuroblastoma cells].

Objective: To study the cytotoxicity of Robo1-CAR-NK92 cells against U87-MG and SH-SY5Y cells, to explore the effects of IL-15, IL-21 and dexamethasone on the proliferation, survival and cytotoxicity of Robo1-CAR-NK92 cells and to optimize the culture protocol. Methods: Robo1-CAR-NK92 cells were constructed by lentivirus transfection.The Robo1 car positive cells were sorted, expanded and detected by flow cytometry.The levels of Robo1 expression in SH-SY5Y and U87-MG cells were examined by flow cytometry.The cytotoxicity of Robo1-CAR-NK92 or NK92 cells against target cells was tested by CCK-8 and live cell imaging. The levels of cytokines in the supernatant of cultured cells during the cytotoxicity assay were quantified by the multiplex bead-array assay.NK92 and Robo1-CAR-NK92 cells (4×10(4)/ml) were treated with 25 ng/ml of IL-15, 25 ng/ml of IL-21 and/or 50 nmol/L dexamethasone for 3 days and were stained with trypan blue to acquire the viable cell numbers and survival rates. Results: Robo1-CAR-NK92 cells were constructed and tested 98.89% positive after sorting and expansion. While 88.14% of U87-MG cells were Robo1 positive, there were 99.75% of Robo1 positive SH-SY5Y cells.The specific lysis of Robo1-CAR-NK92 cells against target cells was significantly higher than that of NK92 cells (P<0.05). Robo1-CAR-NK92 cells obviously secreted more cytokines including IL-6, IL-10, TNF-α and IFN-γ than parental NK92 cells during cytotoxic activity against U87-MG cells (P<0.05). IL-15 significantly increased the proliferation and survival of Robo1-CAR-NK92 cells, but IL-21 played the opposite role.Remarkably, IL-21 and IL-15+ IL-21 enhanced the cytotoxicity of NK92 and Robo1-CAR-NK92 cells.The combination of dexamethasone and interleukins dramatically promoted the proliferation and survival but obviously impaired the cytotoxicity of NK92 and Robo1-CAR-NK92 cells (except that IL+ 15 and dexamethasone have no effect on the cytotoxicity of Robo1-CAR-NK92 cells). Conclusions: Compared to parental NK92 cells, Robo1-CAR-NK92 cells exhibited more potent targeted killing against glioma and neuroblastoma cells.Collectively, treatment of IL-15 and dexamethasone was the optimized protocol for culture of Robo1 CAR NK cells during our experimental time.