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Tall towers with large diameters on floating liquefied natural gas devices are highly sensitive to sway. If tower equipment is relatively high, swaying can easily cause uneven gas–liquid contact in the tower, inhibiting its absorption capacity. In this paper, gas–liquid counterflow triethylene glycol dehydration absorption towers are taken as the research object. A porous medium model was used to simplify the packing environment, and the Euler–Euler method was used to simulate the flow field in the tower. The flow field encompasses the effects of the gas–liquid phase dispersion force, gas–liquid phase diffusion coefficient, and interphase mass transfer. By introducing a dynamic grid model to establish sway boundary conditions, we quantitatively examine the influence of sway duration and angle on gas–liquid flow and mass transfer performance in absorption towers. The results show that, when the sloshing angle of the absorption tower is 9° and the sloshing period is 20 s, the influence of the disturbance of the absorption tower's internal flow field is increased by 85% and 78% respectively compared with normal working conditions. When the sloshing angle of the absorption tower is 9° and the sloshing period exceeds 21 s, the gas–liquid mass transfer inside the absorption tower diminishes. When the sloshing period of the regeneration tower is 6 s and the sloshing angle reaches 20°, the mass fraction of poor ethylene glycol in the regeneration tower fluctuates significantly in the first sloshing cycle, and unqualified products appear.

In order to study the effect of CFRP layers on the mechanical properties of coal cylinders. A discrete-continuous coupled meso-mechanical numerical model of CFRP passively confined coal cylinders was developed using a three-dimensional FLAC-PFC coupling method. The validity of the model was verified with experimental data of 0-2 layers of CFRP passively confined coal cylinders. On this basis, studies of the influence of 3-7 layers on mechanical properties and energy evolution of CFRP confined coal cylinders was carried out. The results show that the yield limit and the peak of the coal cylinders gradually coincide with the increase of CFRP layers, showing a state of yielding or failure without warning. With the increase of CFRP layers, the increment in the peak strength of coal cylinders first increases and then decreases. A function is obtained to characterize the evolution of the peak strength increment of CFRP confined coal cylinders under different layers. The increment in the peak strain shows a behavior of first increasing and then decreasing, and then increasing and decreasing again. When the elastic strain energy is constant, the axial strain of the coal cylinders has no correlation with CFRP layers, but its peak strain and ultimate strain both increase with the increase of CFRP layers. The dissipative energy conversion ratio at peak point increases first and then decreases with the increase of CFRP layers. A function is established to characterize the evolution of the dissipative energy conversion ratio of CFRP confined coal cylinders with different layers. The ductility characteristics of stress-strain curves, peak strength increment, peak strain increment, dissipative energy conversion ratio and total energy increment of coal cylinders are considered comprehensively, and taking into account the principles of economic cost and winding level, the three layers are determined as the optimal winding layers for CFRP confined coal circular-columns.

DNA methylation is a form of epigenetic regulation, having pivotal parts in controlling cellular expansion and expression levels within genes. Although blood DNA methylation has been studied in humans and other species, its prominence in cattle is largely unknown. This study aimed to methodically probe the genomic methylation map of Xinjiang brown (XJB) cattle suffering from bovine respiratory disease (BRD), consequently widening cattle blood methylome ranges. Genome-wide DNA methylation profiling of the XJB blood was investigated through whole-genome bisulfite sequencing (WGBS). Many differentially methylated regions (DMRs) obtained by comparing the cases and controls groups were found within the CG, CHG, and CHH (where H is A, T, or C) sequences (16,765, 7502, and 2656, respectively), encompassing 4334 differentially methylated genes (DMGs). Furthermore, GO/KEGG analyses showed that some DMGs were involved within immune response pathways. Combining WGBS-Seq data and existing RNA-Seq data, we identified 71 significantly differentially methylated (DMGs) and expressed (DEGs) genes (p < 0.05). Next, complementary analyses identified nine DMGs (LTA, STAT3, IKBKG, IRAK1, NOD2, TLR2, TNFRSF1A, and IKBKB) that might be involved in the immune response of XJB cattle infected with respiratory diseases. Although further investigations are needed to confirm their exact implication in the involved immune processes, these genes could potentially be used for a marker-assisted selection of animals resistant to BRD. This study also provides new knowledge regarding epigenetic control for the bovine respiratory immune process.

Atmospheric motion vectors, which can be used to infer wind speed and direction based on the trajectory of cloud movement, are instrumental in enhancing atmospheric wind-field insights, contributing notably to wind-field optimization and forecasting. However, a widespread problem with vector data is their inaccuracy, which, when coupled with the mediocre effectiveness of existing correction methods, limits their practical utility in forecasting, often falling short of expectations. Deep-learning techniques are used to refine atmospheric motion vector data from the FY-4A satellite, notably enhancing data quality. Post-training data undergoes a thorough analysis using a quality evaluation function, followed by its integration into a numerical weather prediction system in order to conduct forecasting experiments. Results indicate a marked improvement in data quality post-error correction by the model, characterized by a significant reduction in root mean square error and a notable increase in correlation coefficients. Furthermore, refined data demonstrate a considerable enhancement in the accuracy of meteorological element forecasts, particularly for Asian and Western Pacific regions.

Summary: Background: Drug-resistant epilepsy (DRE) is associated with distributed laminar disruptions due to cytoarchitectonic pathologies, which may be characterized by multimodal MRI approaches such as morphometric similarity networks (MSNs). However, the genetic and histological underpinning of MSN alterations in DRE remains poorly understood, hampering its clinical application. Methods: We enrolled 60 patients with DRE and 23 controls, acquiring T1 and diffusion spectrum imaging data with a 3.0T GE SIGNA Premier scanner. Morphometric similarity networks (MSNs) were constructed and analyzed to identify microstructure similarity differences between patients and controls. Subsequently, patient-specific MSN alteration patterns were associated with gene expression using the GAMBA tool, and layer-specific neuronal signature mapping were also applied. During these analyses, sex and age were adjusted as covariates and multiple comparisons corrections were applied when appropriate. Findings: We observed widespread MSN changes in patients with DRE and identified five distinct MSN alteration patterns. Major patterns presented pattern-specific associations with expressions of epilepsy-related genes, particularly involving the mTOR pathway. Histological analysis confirmed the presence of cortical microstructure changes in areas with MSN alterations and revealed cellular abnormalities matching the aforementioned genetic risks. Interpretation: Our findings highlight the potential of quantifying laminar-related microstructure integrity using MSN to uncover the cytoarchitectonic changes in the pathophysiology of DRE. This approach may facilitate the identification of genetic and histological underpinnings of MSN alterations in DRE, ultimately aiding in the development of targeted therapeutic strategies. Fundings: The National Natural Science Foundation of China, the Ministry of Science and Technology of the People's Republic of China, and the Beijing Municipal Health Commission.

In a past study, the team used specific-locus amplified fragment sequencing (SLAF sequencing) to detect single-nucleotide polymorphisms (SNPs) contributing to the differences in lambing numbers in Xinjiang sheep. This study verified the correlation between the COIL gene and lambing number characters in sheep and explored its possible mechanism of action. In this study, three SNPs in the COIL gene, namely COILSNP1 (rs7321466), COILSNP2 (rs7314134), and COILSNP3 (rs7321563), were explored in terms of their possible mechanism of action. A tissue expression profiling analysis revealed that the COIL gene was significantly more expressed in the uterus and ovaries than in other tissues (p < 0.05), whereas an association analysis revealed that the number of lambs born was significantly different among individuals with different genotypes of this COILSNP1 (p < 0.05). The Cell Counting Kit-8(CCK-8) revealed that the overexpression of the COIL gene significantly increased the proliferation of mouse ovarian fibroblasts and sheep fibroblasts (p < 0.05). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) revealed that the overexpression of the COIL gene significantly increased the activity of sheep fibroblasts (p < 0.01) and mouse ovarian fibroblasts (p < 0.05). The overexpression of the COIL gene affected the biogenesis pathway of spliceosomal U snRNPs by validating protein network connections. This activity affects ovulation, embryonic development, and changes in lambing size in sheep.

The heat transfer characteristics of porous rock layers (PRLs) have significant seasonal differences. This feature has been used to protect the permafrost subgrade under highways and railways from degeneration. However, in cold sandy environments, the transformation law of heat transfer characteristics of PRLs on account of climate warming and aeolian sand filling needs to be solved. This work developed a coupled heat transfer model for the soil–PRL system aimed at analyzing the convective heat transfer process and mechanism of a closed PRL. Furthermore, the impact of climate warming and sand filling on the cooling performance of the PRL under different mean annual air temperatures (MAATs) of −3.5, −4.5, and −5.5 °C was quantified. The numerical results indicated that the natural convection of the closed PRL occurred only in winter, and the effective convective height of the rock layer decreased with the sand-filling thickness. As the thickness of sand filling increased, the critical temperature difference for the occurrence of natural convection increased, accompanied by decreases in the Rayleigh number, the duration, and intensity of natural convection. When the sand-filling thickness exceeded 80 cm, natural convection would not occur in the PRL. Under a warming scenario of 0.052 °C·a−1, the cooling performance of the PRL could offset the adverse impact of climate warming and raise the permafrost table in the first 20 years. Moreover, the closed PRL can be more effective in permafrost regions with colder MAATs. For cold sandy permafrost zones, sand-control measures should be taken to maintain the long-term cooling performance of the PRL. This study is of great significance in guiding porous rock embankment design and road maintenance along the Qinghai–Tibetan Railway.

Bovine respiratory disease (BRD) is one of the major health issues in the cattle industry, resulting in significant financial crises globally. There is currently no good treatment, and cattle are made resistant to pneumonia through disease-resistant breeding. The serial blood samples from six Xinjiang brown (XJB) calves were collected for the RNA sequencing (RNA-seq). The obtained six samples were grouped into two groups, in each group as infected with BRD and healthy calves, respectively. In our study, the differential expression mRNAs were detected by using RNA-seq and constructed a protein–protein interaction (PPI) network related to the immunity in cattle. The key genes were identified by protein interaction network analysis, and the results from RNA-seq were verified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). A total of 488 differentially expressed (DE) mRNAs were identified. Importantly, the enrichment analysis of these identified DEGs classified them as mainly enriched in the regulation and immune response processes. The 16 hub genes were found to be related to immune pathways categorized by PPIs analysis. Results revealed that many hub genes were related to the immune response to respiratory disease. These results will provide the basis for a better understanding of the molecular mechanism of bovine resistance to BRD.

This paper presents a parametric B-spline-based path planning approach for roundabout exit traverse planners to handle successively generated trajectories. A roundabout is a complex driving scene because of its large curvature and the varying intersection leg angles. A few studies focus on the relationship between roundabout intersection leg angle and path performance. This paper discusses B-spline advantages to curve road conditions and the impact of roundabout geometry elements on path planning performance. Knot vectors are defined, and control points are calculated to meet the requirements for generating paths that fit in different roundabout direction maneuvers. Four countries’ roundabout design standards are summarized with their unique geometry elements. Additionally, the Stanley tracking controller was used to calculate the steering behavior. The impacts of maneuver directions and intersection angles are discussed. It is explained stepwise how the maneuver system framework was built and what notable impacts the geometric structure of the roundabout has on the paths.

A successful pregnancy outcome is dependent on a delicate balance between inflammatory and anti-inflammatory processes throughout the different trimesters. Interruption in this balance can lead to an adverse outcome resulting in pregnancy loss. Since late 2019, the emergence of the new SARS-CoV-2 virus has affected lives worldwide, including pregnant women; therefore, there is an urgent need to address different approaches in relation to prevention, diagnostics, and therapeutics. Early pregnancy is affected by SARS-CoV-2 infection leading to fetal demise. Available evidence also suggests that 90% of pregnant women infected with the SARS-CoV-2 virus seem to be asymptomatic. Nonetheless, it is still unclear how COVID-19 affects exosome production in pregnant women recovered from COVID-19 and how these exosomes regulate the adaptive immune response. In this study, we found several exosomes including CD9, CD31, CD40, CD45, CD41b, CD42a, CD62P, CD69, CD81, CD105, and HLA-DRDPDQ in the plasma of COVID-19-recovered pregnant women were significantly less abundant than the control group. Furthermore, to understand how these exosomes affect the adaptive immune response, we co-cultured the peripheral blood mononuclear cells (PBMCs) from healthy control (HC) pregnant women with exosomes of either Preg-HC or Preg-recovered COVID-19 women. We identified that Preg-recovered COVID-19 women have reduced capacity for the inflammatory cytokine TNF-α from cytotoxic CD8+ T cells. In summary, our study highlights that pregnant recovered COVID-19 women have reduced production of several exosomes and possess fewer immunogenic properties. Our study implicates that exosomes can control inflammation and antigen presentation capacity of immune cells, thus limiting the infection in pregnant women.

Abstract Background Glioma, the most frequent primary tumor of the central nervous system, has poor prognosis. The epidermal growth factor receptor (EGFR) pathway and angiogenesis play important roles in glioma growth, invasion, and recurrence. The present study aimed to use proteomic methods to probe into the role of the EGF‐EGFR‐angiogenesis axis in the tumorigenesis of glioma and access the therapeutic efficacy of selumetinib on glioma. Methods Proteomic profiling was used to characterize 200 paired EGFR‐positive and EGFR‐negative glioma tissues of all pathological types. The quantitative mass spectrometry data were used for systematic analysis of the proteomic profiles of 10 EGFR‐positive and 10 EGFR‐negative glioma cases. Consensus‐clustering analysis was used to screen target proteins. Immunofluorescence analysis, cell growth assay, and intracranial xenograft experiments were used to verify and test the therapeutic effect of selumetinib on glioma. Results Advanced proteomic screening demonstrated that the expression of EGF‐like domain multiple 7 (EGFL7) was higher in EGFR‐positive tumor tissues than in EGFR‐negative tumor tissues. In addition, EGFL7 could act as an activator in vitro and in vivo to promote glioma cell proliferation. EGFL7 was associated strongly with EGFR and prognosis. EGFL7 knockdown effectively suppressed glioma cell proliferation. Selumetinib treatment showed tumor reduction effect in EGFR‐positive glioblastoma xenograft mouse model. Conclusions EGFL7 is a potential diagnostic biomarker and therapeutic target of glioma. Selumetinib could target the EGFR pathway and possibly improve the prognosis of EGFR‐positive glioma.

Abstract Background The presence of tumor-associated stroma and tumor-infiltrated immune cells have been largely reported across glioblastomas. Tumor purity, defined as the proportion of tumor cells in the tumor, was associated with the genomic and clinicopathologic features of the tumor and may alter the interpretation of glioblastoma biology. Methods We use an integrative approach to infer tumor purity based on multi-omic data and comprehensively evaluate the impact of tumor purity on glioblastoma (GBM) prognosis, genomic profiling, and the immune microenvironment in the Cancer Genome Atlas Consortium (TCGA) cohort. Results We found that low tumor purity was significantly associated with reduced survival time. Additionally, we established a purity-relevant 5-gene signature that was an independent prognostic biomarker and validated it in the TCGA, CGGA and GSE4412 cohort. Moreover, we correlated tumor purity with genomic characteristics and tumor microenvironment. We identified that gamma delta T cells in glioblastoma microenvironment were positively correlated with purity and served as a marker for favorable prognosis, which was validated in both TCGA and CGGA dataset. Conclusions We observe the potential confounding effects of tumor purity on GBM clinical and molecular information interpretation. GBM microenvironment could be purity-dependent, which provides new insights into the clinical implications of glioblastoma.

ObjectivesTo measure the metrics of glioma pre-operative MRI reports and build IDH prediction models.MethodsPre-operative MRI reports of 144 glioma patients in a single institution were collected retrospectively. Words were transformed to lowercase letters. White spaces, punctuations, and stop words were removed. Stemming was performed. A word cloud method applied to processed text matrix visualized language behavior. Spearman’s rank correlation assessed the correlation between the subjective descriptions of the enhancement pattern. The T1-contrast images associated with enhancement descriptions were selected. The keywords associated with IDH status were evaluated by χ2 value ranking. Random forest, k-nearest neighbors and Support Vector Machine algorithms were used to train models based on report features and age. All statistical analysis used two-tailed test with significance at p

The increasing demand for power, fuel efficiency, and safety of aeroengines has called for weight reduction and structural integrity examination of the critical components. This paper is aimed at performing a systematic investigation on the design of a high-speed Ti2AlNb blisk, including disc geometry optimization and burst speed prediction. Incorporating the design of the experimental approach and the commercial software has guaranteed that the optimization could be accomplished. Six key parameters were defined as variables with regard to the geometric dimensions whereas the safety factors were set as constraints to make the disc feasible. Sensitivity analysis has been conducted to study the effects of the variables on the safety factors and disc weight. Bore width, web width, and bore angle are identified to be the dominant factors regarding optimization. Results reveal that the bore width and web width are positively related to the safety factors at the cost of increasing the disc weight. On the contrary, the effects of the bore angle show the opposite trend. Finally, the achieved minimum disc weight is 15.2 kg with all the safety factors meeting the requirements. Upon completing the disc shape optimization, the burst speed was estimated using three elaborated methods. The comparisons between the numerical results and the experimental results indicate that the mean stress method is accurate when the correction coefficient is chosen properly. The local stress and strain method and the global plastic instability method also offer a precise prediction on the burst speed with errors of less than 5%. It could also be concluded that the predicted web failure in the radial direction of the disc is in good agreement with the experimental results.

Abstract The second version of Chinese Academy of Sciences Earth System Model (CAS‐ESM 2) is described with emphasis on the development process, strength and weakness, and climate sensitivities in simulations of the Coupled Model Intercomparison Project (CMIP6) DECK experiments. CAS‐ESM 2 was built as a numerical model to simulate both the physical climate system as well as atmospheric chemistry and carbon cycle. It is a newcomer in the international modeling community to provide sufficiently independent solutions of climate simulations from those of other models. Performances of the model in simulating the basic states of the radiation budget of the atmosphere and ocean, precipitation, circulations, variabilities, and the twentieth century warming are presented. Model biases and their possible causes are discussed. Strength includes horizontal heat transport in the atmosphere and oceans, vertical profile of the Atlantic Meridional Overturning Circulation; weakness includes the double intertropical convergence zone (ITCZ) and stronger amplitude of the El Niño–Southern Oscillation (ENSO) that are also common in many other models. The simulated the twentieth century warming shares a similar discrepancy with observations as in several other models—less warming in the 1920s and stronger cooling in the 1960s than in observation—at the time when there was a steep increase of anthropogenic aerosols. As a result, the twentieth century warming is about 60% of the observed warming despite that the model simulated a similar slope of warming trend after 1980 to observation. The model has an equilibrium climate sensitivity of 3.4 K with a positive cloud feedback from the shortwave radiation.

Clinical studies have demonstrated that exposure to the inhalational general anesthetic nitrous oxide (N2O) produces antidepressant effects in depressed patients. However, the mechanisms underlying the antidepressant effects of N2O remain largely unknown. Neuronal nitric oxide synthase (nNOS)–mediated nitric oxide (NO) synthesis is essential for brain function and underlies the molecular mechanisms of many neuromodulators. We hypothesized that activation of the nNOS/NO pathway in the medial prefrontal cortex (mPFC) might mediate the antidepressant effects of N2O. In this study, we revealed that repeated N2O exposure produced antidepressant-like responses in mice. Our mechanistic exploration showed that repeated N2O exposure increased burst firing activity and that the expression levels of BDNF with nNOS activation were dependent in the mPFC. In particular, the antidepressant-like effects of N2O were also antagonized by local nNOS inhibition in the mPFC. In summary, our results indicated that N2O exposure enhances BDNF expression levels and burst firing rates in an nNOS activation dependent manner, which might underlie the pharmacological mechanism of the antidepressant-like effects of N2O exposure. The present study appears to provide further mechanistic evidence supporting the antidepressant effects of N2O.

Background/Aims: The Hedgehog pathway disrupting drug sonidegib is used in the treatment of basal cell carcinoma. Side effects of sonidegib include anemia, which could result either from impaired erythropoiesis or from loss of erythrocytes e.g. due to suicidal erythrocyte death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is stimulated by cell stress, including energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether sonidegib exerts an effect on eryptosis. Methods: Human erythrocytes have been treated with energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of sonidegib (2-6 µg/ ml). After treatment flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter. Hemolysis was estimated from the hemoglobin concentration in the supernatant. Results: In the absence of cell stress exposure to sonidegib did not significantly modify annexin-V-binding or forward scatter, but triggered hemolysis. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Sonidegib significantly blunted the effect of energy depletion, hyperosmotic shock, and oxidative stress, but not of ionomycin on annexin-V-binding. Sonidegib further significantly blunted the effect of energy depletion, but not of hyperosmotic shock, oxidative stress, and ionomycin on forward scatter. Conclusions: Sonidegib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion, hyperosmotic shock and oxidative stress.

Background/Aims: The anaplastic lymphoma (tyrosine) kinase (ALK) inhibitor ceritinib triggers apoptosis of tumor cells and eryptosis of erythrocytes. Blood platelets may similarly enter a state resembling apoptosis, which could be triggered by activation with collagen related peptide (CRP). CRP-induced platelet apoptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the platelet surface and cell shrinkage, preceded by externalization of Ca2+ channel Orai1, increase of cytosolic Ca2+-activity ([Ca2+]i), formation of reactive oxygen species (ROS), and caspase activation. The present study explored whether ceritinib triggers platelet apoptosis and/or modifies the CRP induced apoptosis. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to ceritinib (1.5 µg/ml) without or with 2.5 – 15 min pretreatment with CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, ROS abundance from 2’,7’-dichlorodihydrofluorescein diacetate fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of CRP, ceritinib slightly, but significantly decreased [Ca2+]i without significantly modifying the other measured parameters. CRP significantly increased [Ca2+]i, ROS abundance, P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, caspase activity as well as aggregation and decreased cell volume, all effects significantly blunted in the presence of ceritinib. Conclusions: The present observations uncover a novel, unexpected effect of ceritinib, i.e. inhibition of CRP-induced platelet activation and apoptosis.

Background/Aims: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is used for the treatment of several malignancies. Afatinib is at least partially effective by triggering apoptosis of tumor cells. Platelets may similarly undergo apoptosis, which is characterized by caspase 3 activation, cell shrinkage and phosphatidylserine translocation. However, an effect of afatinib on platelets has never been reported. The present study explored whether treatment of platelets with afatinib modifies platelet activation and apoptosis in the absence and presence of platelet activators thrombin or collagen related peptide (CRP). Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to afatinib (18 µg/ml) without or with subsequent treatment with thrombin (0.005 U/ml or 0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate Orai1 abundance at the platelet surface with specific antibodies, cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of thrombin and CRP, the administration of afatinib (18 µg/ml) slightly, but significantly, increased [Ca2+]i and annexin-V-binding, but did not significantly modify Orai1 abundance, P-selectin abundance, activated αIIbβ3 integrin, cell volume, caspase activity and aggregation. Exposure of platelets to 0.005 U/ml or 0.01 U/ml thrombin or 2 µg/ml or 5 µg/ ml CRP was followed by a significant increase of Orai1 abundance, increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as a significant decrease of forward scatter, all effects significantly blunted (thrombin) or virtually abolished (CRP) by afatinib. Conclusions: Afatinib is a powerful inhibitor of platelet activation, platelet apoptosis and platelet aggregation.

The work is proposed to design a controller of the form known as the roundabout scenario trajectory tracking problem. The road condition is a four-leg, single-lane roundabout; the reference path is given. Due to the decision choice of exits, the MPC tracking controller is used to test the effect of weight parameter and target speed on the performance of the tracking controller. Two sets of test cases are proposed to make the experimental comparison, see the relationship between the control parameters and road conditions (different curvature path), and also see how the weight parameters Q and R and sample time affect the tracking performance. Our work, MPC controller utilization in a roundabout, plays an essential role with the increasing autonomy of vehicles.

Background/Aims: The anaplastic lymphoma kinase (ALK) inhibitor ASP3026 is in clinical development for the treatment of ALK expressing non-small cell lung carcinoma (NSCLC). ASP3026 is in part effective by inducing apoptosis of tumor cells. Erythrocytes lack mitochondria and nuclei, key organelles in the execution of apoptosis, but are nevertheless able to enter suicidal death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Eryptosis is triggered by cell stress, such as energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether ASP3026 impacts on eryptosis. Methods: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ loading with Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of ASP3026 (1-4 µg/ml). Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, and cell volume from forward scatter. Results: Treatment with ASP3026 alone did not significantly modify annexin-V-binding or forward scatter. Energy depletion, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. ASP3026 significantly blunted the effect of energy depletion and oxidative stress, but not of ionomycin on annexin-V-binding. ASP3026 did not significantly influence the effect of any maneuver on forward scatter. Conclusions: ASP3026 is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and oxidative stress.

Background/Aims: The 3-hydroxy-3-methyl-glutaryl-Coenzyme A (HMG-CoA) reductase inhibitor simvastatin has been shown to trigger apoptosis of several cell types. The substance has thus been proposed as an additional treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the extracellular face of the erythrocyte cell membrane. Signaling contributing to stimulation of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, increase of ceramide abundance, and activation of SB203580-sensitive p38 kinase. The present study explored, whether simvastatin induces eryptosis and aimed to shed light on cellular mechanisms involved. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 48 h exposure of human erythrocytes to simvastatin (1 µg/ml) significantly decreased the forward scatter, significantly augmented the percentage of annexin-V-binding cells, significantly increased Fluo3-fluorescence, and significantly enhanced DCFDA fluorescence. Simvastatin tended to increase ceramide abundance, an effect, however, escaping statistical significance. The effect of simvastatin on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of SB203580 (2 µM). Conclusions: Simvastatin stimulates eryptosis, an effect at least in part due to Ca2+ entry, oxidative stress, and p38 kinase.

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus stimulates apoptosis of tumor cells and is thus therapeutically used for the treatment of diverse malignancies. On the other hand, temsirolimus has been shown to protect against apoptosis of hippocampal neurons. Similar to nucleated cells, blood platelets may enter suicidal death characterized by cell shrinkage and cell membrane scrambling. Platelet apoptosis is frequently preceded by Ca2+ entry, degranulation, integrin activation and stimulation of caspases. Those events could be triggered by collagen related peptide (CRP). The present study explored whether treatment of platelets with temsirolimus modifies platelet activation, caspase activity, platelet shrinkage, and phosphatidylserine abundance. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to temsirolimus (40 µg/ml) without or with additional CRP (2 µg/ ml or 5 µg/ml) treatment. Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fuorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding and relative platelet volume from forward scatter. Results: In the absence of CRP, the administration of temsirolimus (40 µg/ml) significantly decreased [Ca2+]i, but did not significantly modify P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, caspase activity and aggregation. Exposure of platelets to CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, ROS, caspase activity and aggregation, effects significantly blunted in the presence of temsirolimus. CRP further decreased forward scatter, an effect again significantly blunted by temsirolimus. Conclusions: Temsirolimus is a powerful inhibitor of platelet activation and suicidal platelet death.

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus is utilized for the treatment of malignancy. Temsirolimus is at least in part effective by triggering suicidal tumor cell death. The most common side effect of temsirolimus treatment is anemia. At least in theory, the anemia following temsirolimus treatment could result from stimulation of eryptosis, the suicidal erythrocyte death. Hallmarks of eryptosis include cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the orchestration of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, as well as activation of staurosporine and chelerythrine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The purpose of the present study was to test whether temsirolimus influences eryptosis and, if so, to shed light on the signaling involved. Methods: Flow cytometry was employed to estimate cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was determined from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to temsirolimus (5 – 20 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Temsirolimus significantly increased Fluo3-fluorescence, DCFDA fluorescence and ceramide abundance at the erythrocyte surface. The effect of temsirolimus on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+ and by addition of staurosporine (1 µM) or chelerythrine (10 µM) but not significantly modified by addition of SB203580 (2 µM), D4476 (10 µM), or zVAD (10 µM). Chelerythrine (10 µM) further significantly blunted the effect of temsirolimus on DCFDA fluorescence but not ceramide formation. Removal of extracellular Ca2+ had no effect on temsirolimus induced ROS formation or ceramide abundance. Conclusions: Temsirolimus triggers eryptosis with cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, ceramide and activation of staurosporine/Chelerythrine sensitive kinase(s).

Background/Aims: The retinoid X receptor (RXRs) stimulator Bexarotene ((4-[1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)ethynyl] benzoic acid) is used for the treatment of several malignancies. Bexarotene is at least in part effective by stimulation of apoptosis of tumor cells. Moreover, Bexarotene triggers eryptosis, the suicidal death of erythrocytes. Similar to erythrocytes, blood platelets lack nuclei but are nevertheless able to enter an apoptosis-like phenotype, characterized by caspase activation, cell shrinkage and cell membrane scrambling with phospha-tidylserine translocation to the cell surface. Platelet apoptosis is triggered by increase of cytosolic Ca2+-activity ([Ca2+]i), which further leads to degranulation and integrin activation. Platelet activation and apoptosis could be elicited by thrombin or collagen related peptide (CRP). The present study explored whether treatment of platelets with bexarotene modifies platelet activation and apoptosis following exposure to thrombin or CRP. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to bexarotene (6 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, and relative platelet volume from forward scatter. Results: In the absence of thrombin or CRP, the administration of bexarotene slightly but significantly increased [Ca2+]i, but did not significantly modify P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, or caspase activity. Exposure of platelets to thrombin or CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, active αIIbβ3 integrin, annexin-V-binding, and caspase activity. The effects of thrombin on [Ca2+]i, annexin-V-binding, cell volume, and caspase activity as well as the effects of CRP on [Ca2+]i, P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, and caspase activity were significantly augmented in the presence of bexarotene. Conclusions: Bexarotene sensitizes blood platelets for thrombin and/or CRP induced activation and apoptosis.

Background/Aims: The steroidal aromatase inactivator exemestane blocks estrogen biosynthesis and is thus employed for the prevention and treatment of breast cancer. Exemestane is in part effective by stimulation of suicidal cell death or apoptosis. Side effects of exemestane treatment include anemia. At least in theory, exemestane induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the stimulation of eryptosis includes increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, several kinases and caspases. The present study explored, whether exemestane is able to trigger eryptosis and, if so, to shed some light on the signaling involved. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCF fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to exemestane (≥ 10 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying forward scatter. Exemestane significantly increased Fluo3-fluorescence (10 and 20, but not 40 µg/ml), DCF fluorescence (40 µg/ml), and ceramide abundance (40 µg/ml). The effect of exemestane (40 µg/ml) on annexin-V-binding was significantly blunted by antioxidant N-acetylcysteine (1mM), but was not significantly modified by removal or increase of extracellular Ca2+, by p38 kinase inhibitor SB203580 (2 µM), casein kinase inhibitor D4476 (10 µM) and caspase inhibitor zVAD (10 µM). Conclusions: Exemestane triggers phospholipid scrambling of the erythrocyte cell membrane, an effect paralleled by enhanced [Ca2+]i, oxidative stress, and increased ceramide abundance.

Background/Aims: The epidermal growth factor receptor-tyrosine kinase inhibitor gefitinib is effective against several malignancies and is mainly utilized in the treatment of epidermal growth factor receptor mutation positive non-small cell lung cancer. The anti-cancer effect of the drug involves stimulation of apoptosis. Side effects of gefitinib include anemia. At least in theory, the development of anemia during gefitinib treatment could result from triggering of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i) and generation of oxidative stress. The present study explored, whether gefitinib stimulates eryptosis and, if so, whether its effect involves Ca2+ entry and/or oxidative stress. Methods: Flow cytometry was employed to quantify cell volume from forward scatter, phosphatidylserine exposure at the cell surface from annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, and reactive oxygen species (ROS) abundance from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. Results: A 48 hours exposure of human erythrocytes to gefitinib (≥ 2 µg/ml) significantly decreased forward scatter and significantly increased the percentage of annexin-V-binding cells. Gefitinib did not significantly increase Fluo3-fluorescence but the effect of gefitinib on annexin-V-binding was significantly blunted by removal of extracellular Ca2+. Gefitinib further significantly increased DCFDA fluorescence. Conclusions: Gefitinib triggers erythrocyte shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part dependent on extracellular Ca2+ and paralleled by oxidative stress.

Background/Aims: The 8-aminoquinoline tafenoquine has been shown to be effective against Plasmodia, Leishmania and Trypanosoma. The substance is at least in part effective by triggering apoptosis of the parasites. Moreover, tafenoquine has been shown to trigger eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. The effect of tafenoquine on eryptosis is in part due to stimulation of Ca2+ entry and oxidative stress. Ca2+ entry is a critical event in the activation of blood platelets by thrombin and collagen related peptide (CRP). The present study explored, whether tafenoquine influences Ca2+ entry, activation and apoptosis of blood platelets. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to tafenoquine (2.5 µg/ml) without or with an additional treatment with thrombin (0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, reactive oxygen species (ROS) from DCF fluorescence, caspase 3 activity with an active caspase-3 Staining kit, and aggregation utilizing staining with CD9-APC and CD9-PE. Results: Both, thrombin (0.01 U/ml) and CRP (2 µg/ml or 5 µg/ml), significantly increased [Ca2+]i, P-selectin abundance, active αIIbβ3 integrin, and annexin-V-binding, and both significantly decreased platelet volume, activated caspase 3 and stimulated aggregation. Administration of tafenoquine (2.5 µg/ml, 30 min) significantly decreased [Ca2+]i both, in the absence and presence of thrombin and CRP. Tafenoquine significantly blunted the effect of thrombin and CRP on [Ca2+]i, P-selectin abundance, and active αIIbβ3 integrin, but significantly increased ROS and annexin-V-binding, significantly augmented the effect of thrombin on caspase 3 activity and platelet volume and significantly enhanced platelet aggregation. Conclusions: Tafenoquine counteracts thrombin and CRP induced increase of cytosolic Ca2+ activity and platelet activation, but enhances platelet apoptosis and platelet aggregation.

Background/Aims: The retinoid X receptor agonist bexarotene is utilized for the treatment of cutaneous T-cell lymphoma and is effective in several further malignancies. The substance counteracts tumor growth in part by triggering suicidal death or apoptosis of tumor cells. Side effects of bexarotene treatment include anemia. Theoretically, bexarotene induced anemia could be secondary to stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling potentially stimulating eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), induction of oxidative stress, increase of ceramide abundance, as well as activation of staurosporine sensitive protein kinase C, SB203580 sensitive p38 kinase, D4476 sensitive casein kinase 1, and zVAD sensitive caspases. The present study explored, whether bexarotene induces eryptosis and, if so, whether its effect involves Ca2+ entry, oxidative stress, ceramide, kinases and/or caspases. Methods: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) abundance from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to bexarotene (≥ 0.4 µg/ml) significantly increased the percentage of annexin-V-binding cells without significantly modifying forward scatter. Bexarotene significantly increased Fluo3-fluorescence and DCFDA fluorescence. Bexarotene tended to increase ceramide abundance, an effect, however, not reaching statistical significance. The effect of bexarotene on annexin-V-binding was significantly blunted by removal of extracellular Ca2+ and by addition of D4476 (10 µM), but not by addition of staurosporine (1 µM), SB203580 (2 µM), or zVAD (10 µM). Conclusions: Bexarotene triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to Ca2+ entry, oxidative stress, and activation of D4476 sensitive casein kinase.

The gut microbiota influences several biological functions including immune responses. Inflammatory bowel disease is favorably influenced by consumption of several dietary natural plant products such as pomegranate, walnuts, and berries containing polyphenolic compounds such as ellagitannins and ellagic acid. The gut microbiota metabolizes ellagic acid resulting in the formation of bioactive urolithins A, B, C, and D. Urolithin A (UA) is the most active and effective gut metabolite and acts as a potent anti-inflammatory and anti-oxidant agent. However, whether gut metabolite UA affects the function of immune cells remains incompletely understood. T cell proliferation is stimulated by store operated Ca2+ entry (SOCE) resulting from stimulation of Orai1 by STIM1/STIM2. We show here that treatment of murine CD4+ T cells with UA (10 μM, 3 days) significantly blunted SOCE in CD4+ T cells, an effect paralleled by significant downregulation of Orai1 and STIM1/2 transcript levels and protein abundance. UA treatment further increased miR-10a-5p abundance in CD4+ T cells in a dose dependent fashion. Overexpression of miR-10a-5p significantly decreased STIM1/2 and Orai1 mRNA and protein levels as well as SOCE in CD4+ T cells. UA further decreased CD4+ T cell proliferation. Thus, the gut bacterial metabolite UA increases miR-10a-5p levels thereby downregulating Orai1/STIM1/STIM2 expression, store operated Ca2+ entry, and proliferation of murine CD4+ T cells.

Background/Aims: The polyamine oxidase inhibitor MDL-72527 (N1,N4-bis(2,3-butadienyl)-1,4-butanediamine) were expected to increase the abundance of spermine, a powerful inhibitor of platelet activation. Nothing is known, however, on the sensitivity of platelet function and survival to MDL-72527 exposure. The present study thus explored whether MDL-72527 modifies function and survival of platelets without and with platelet activation by collagen related peptide (CRP). Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to MDL-72527 (100 µM) with or without subsequent activation with CRP (2-5 µg/ml). Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, generation of reactive oxygen species (ROS) from DCFDA fluorescence, phospholipid scrambling of the cell membrane from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of CRP, exposure of platelets to MDL-72527 did not significantly modify [Ca2+]i, P-selectin abundance, αIIbβ3 integrin abundance, ROS, annexin-V-binding, and forward scatter. The addition of 2-5 µg/ml CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activation, ROS abundance, annexin-V-binding, and aggregation as well as a significant decrease of forward scatter, all effects significantly blunted or virtually abolished in the presence of MDL-72527. Conclusions: MDL-72527 is a powerful inhibitor of platelet activation, apoptosis and aggregation.

Garcinol, an anti-inflammatory and anti-carcinogenic polyisoprenylated benzophenone isolated from Garcinia plants, stimulates tumor cell apoptosis and suicidal erythrocyte death, but supports the survival of hepatocytes and neurons. The present study explored whether the substance influences platelet function and/or apoptosis. To this end, we exposed murine blood platelets to garcinol (33 µM, 30 min) without and with activation by collagen-related peptide (CRP) (2−5 µg/mL) or thrombin (0.01 U/mL); flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, relative platelet volume from forward scatter, and aggregation utilizing staining with CD9-APC and CD9-PE. As a result, in the absence of CRP and thrombin, the exposure of the platelets to garcinol did not significantly modify [Ca2+]i, P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, caspase activity, and aggregation. Exposure of platelets to CRP or thrombin was followed by a significant increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as significant cell shrinkage. All effects of CRP were strong and significant; those of thrombin were only partially and slightly blunted in the presence of garcinol. In conclusion, garcinol blunts CRP-induced platelet activity, apoptosis and aggregation.

In order to systematically study the rutting resistance performance of High-Modulus Asphalt Concrete (HMAC) pavements, a finite element method model of HMAC pavement was established using ABAQUS software. Based on the viscoelasticity theory of asphalt, the stress and deformation distribution characteristics of HMAC pavement were studied and compared to conventional asphalt pavement under moving loads. Then, the pavement temperature field model was established to study the temperature variation and the thermal stress in HMAC pavement. Finally, under the condition of continuous temperature variation, the creep behavior and permanent deformation of HMAC pavement were investigated. The results showed that under the action of moving loads, the strain and displacement generated in HMAC pavement were lower than those in conventional asphalt pavement. The upper surface layer was most obviously affected by outside air temperature, resulting in maximum thermal stress. Lastly, under the condition of continuous temperature change, HMAC pavement could greatly reduce the deformation of asphalt material in each surface layer compared to conventional asphalt pavement.

Background/Aims: The Polo-like kinase 1 (Plk1) inhibitor volasertib is used in the treatment of malignancy. Volasertib is partially effective by triggering suicidal death or apoptosis of tumor cells. Similar to apoptosis of nucleated cells, erythrocytes may enter suicidal cell death or eryptosis, which is characterized by cell membrane scrambling with phosphatidylserine translocation to the cell surface and by cell shrinkage. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress and excessive increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether volasertib impacts on eryptosis. Methods: Human erythrocytes have been exposed to energy depletion (glucose withdrawal for 48 hours), hyperosmotic shock (addition of 550 mM sucrose for 6 hours), oxidative stress (addition of 0.3 mM tert-butylhydroperoxide [tBOOH] for 50 min) or Ca2+ ionophore ionomycin (1 µM for 60 min) in absence and presence of volasertib (0.5-1.5 µg/ml) and flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3 fluorescence, reactive oxygen species from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing antibodies. For comparison, annexin-V-binding and forward scatter were determined following a 48 hours exposure of human leukemic K562 cells in RPMI-1640 medium to volasertib. Results: Treatment with volasertib alone did not significantly modify annexin-V-binding or forward scatter in mature erythrocytes. Energy depletion, hyperosmotic shock, oxidative stress and ionomycin, all markedly and significantly increased the percentage of annexin-V-binding erythrocytes, and decreased the forward scatter. Volasertib significantly blunted the effect of energy depletion and hyperosmotic shock, but not of oxidative stress and ionomycin on annexin-V-binding. Volasertib did not significantly influence the effect of any maneuver on forward scatter. In K562 cells, volasertib enhanced annexin-V-binding and decreased the forward scatter. Conclusions: Volasertib is a novel inhibitor of erythrocyte cell membrane scrambling following energy depletion and hyperosmotic shock, effects contrasting the stimulation of K562 cell apoptosis.