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Individual component testing is an integral part of vehicle development and can be performed in multiple stages of the product development process. One such component assembly that often requires extensive testing is the brake system. The brake system is crucial for vehicle safety and, thus, needs to be validated from a functional perspective.A test rig setup to accommodate a brake caliper fixture, as well as a funnel housing an anemometer, was purposefully designed and manufactured. This arrangement allows for measuring brake disc performance, surface temperature and quantifying mass airflow through the disc. The results show that the mass flow of air through the disc vanes highly depends on the disc temperature, but can also vary by more than 10 % for discs with the same specifications. Furthermore, the results highlight non-negligible differences in disc cool-down behaviour based on the braking scenario used to heat the disc up.

Recent research shows that integrin-mediated adhesion contributes to the regulation of cell division at two key steps: the formation of the mitotic spindle at the mitotic entry and the final cytokinetic abscission at the mitotic exit. Failure in either of these processes will have a direct impact on the other in each round of the cell cycle and on the genomic integrity. This review aims to present how integrin signals are involved at these cell cycle stages under normal conditions and some safety mechanisms that may counteract the generation of aneuploid cells in cases of defective integrin signals.

Our previous studies demonstrated that enzymatic removal of highly sulfated heparan sulfates with heparinase 1 impaired axonal excitability and reduced expression of ankyrin G at the axon initial segments in the CA1 region of the hippocampus ex vivo, impaired context discrimination in vivo, and increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity in vitro. Here, we show that in vivo delivery of heparinase 1 in the CA1 region of the hippocampus elevated autophosphorylation of CaMKII 24 h after injection in mice. Patch clamp recording in CA1 neurons revealed no significant heparinase effects on the amplitude or frequency of miniature excitatory and inhibitory postsynaptic currents, while the threshold for action potential generation was increased and fewer spikes were generated in response to current injection. Delivery of heparinase on the next day after contextual fear conditioning induced context overgeneralization 24 h after injection. Co-administration of heparinase with the CaMKII inhibitor (autocamtide-2-related inhibitory peptide) rescued neuronal excitability and expression of ankyrin G at the axon initial segment. It also restored context discrimination, suggesting the key role of CaMKII in neuronal signaling downstream of heparan sulfate proteoglycans and highlighting a link between impaired CA1 pyramidal cell excitability and context generalization during recall of contextual memories.

Abstract Background Integrin-mediated adhesion is normally required for cytokinetic abscission, and failure in the process can generate potentially oncogenic tetraploid cells. Here, detachment-induced formation of oncogenic tetraploid cells was analyzed in non-transformed human BJ fibroblasts and BJ expressing SV40LT (BJ-LT) ± overactive HRas. Results In contrast to BJ and BJ-LT cells, non-adherent BJ-LT-Ras cells recruited ALIX and CHMP4B to the midbody and divided. In detached BJ and BJ-LT cells regression of the cytokinetic furrow was suppressed by intercellular bridge-associated septin; after re-adhesion these cells divided by cytofission, however, some cells became bi-nucleated because of septin reorganization and furrow regression. Adherent bi-nucleated BJ cells became senescent in G1 with p21 accumulation in the nucleus, apparently due to p53 activation since adherent bi-nucleated BJ-LT cells passed through next cell cycle and divided into mono-nucleated tetraploids; the two centrosomes present in bi-nucleated BJ cells fused after furrow regression, pointing to the PIDDosome pathway as a possible mechanism for the p53 activation. Conclusions Several mechanisms prevent detached normal cells from generating tumor-causing tetraploid cells unless they have a suppressed p53 response by viruses, mutation or inflammation. Importantly, activating Ras mutations promote colony growth of detached transformed cells by inducing anchorage-independent cytokinetic abscission in single cells.

Intimal calcification and vascular stiffening are predominant features of end-stage atherosclerosis. However, their role in atherosclerotic plaque instability and how the extent and spatial distribution of calcification influence plaque biology remain unclear. We recently showed that extensive macro calcification can be a stabilizing feature of late-stage human lesions, associated with a reacquisition of more differentiated properties of plaque smooth muscle cells (SMCs) and extracellular matrix (ECM) remodeling. Here, we hypothesized that biomechanical forces related to macro-calcification within plaques influence SMC phenotype and contribute to plaque stabilization. We generated a finite element modeling (FEM) pipeline to assess plaque tissue stretch based on image analysis of preoperative computed tomography angiography (CTA) of carotid atherosclerotic plaques to visualize calcification and soft tissues (lipids and extracellular matrix) within the lesions. Biomechanical stretch was significantly reduced in tissues in close proximity to macro calcification, while increased levels were observed within distant soft tissues. Applying this data to an in vitro stretch model on primary vascular SMCs revealed upregulation of typical markers for differentiated SMCs and contractility under low stretch conditions but also impeded SMC alignment. In contrast, high stretch conditions in combination with calcifying conditions induced SMC apoptosis. Our findings suggest that the load bearing capacities of macro calcifications influence SMC differentiation and survival and contribute to atherosclerotic plaque stabilization.

Cell cycle and cell adhesion are two interdependent cellular processes regulating each other, reciprocally, in every cell cycle phase. The cell adhesion to the extracellular matrix (ECM) via integrin receptors triggers signaling pathways required for the cell cycle progression; the passage from the G1 to S phase and the completion of cytokinesis are the best-understood events. Growing evidence, however, suggests more adhesion-dependent regulatory aspects of the cell cycle, particularly during G2 to M transition and early mitosis. Conversely, the cell cycle machinery regulates cell adhesion in manners recently shown driven mainly by cyclin-dependent kinase 1 (CDK1). This review summarizes the recent findings regarding the role of integrin-mediated cell adhesion and its downstream signaling components in regulating the cell cycle, emphasizing the cell cycle progression through the G2 and early M phases. Further investigations are required to raise our knowledge about the molecular mechanisms of crosstalk between cell adhesion and the cell cycle in detail.

Integrin-mediated adhesion to the extracellular matrix is a key regulator of the cell cycle, as demonstrated for the passage of the G1/S checkpoint and the completion of cytokinetic abscission. Here, integrin-dependent regulation of the cell cycle in G2 and early M phases was investigated. The progression through the G2 and M phases was monitored by live-cell imaging and immunofluorescence staining in adherent and non-adherent fibroblast cells. Non-adherent cells, as well as adherent cells lacking FAK activity due to suppressed expression or pharmacological inhibition, exhibited a prolonged G2 phase and severely defect centrosome separation, resulting in delayed progress through the early mitotic stages. The activation of the critical mitotic regulator PLK1 and its indirect target Eg5, a kinesin-family motor protein driving the centrosome separation, were reduced in the cells lacking FAK activity. Furthermore, the absence of integrin adhesion or FAK activity destabilized the structural integrity of centrosomes and often caused detachment of pericentriolar material from the centrioles. These data identify a novel adhesion-dependent mechanism by which integrins via FAK and PLK1 contribute to the regulation of the cell cycle in the G2 and early M phases, and to the maintenance of genome integrity.

The establishment of strategic coordination bodies with members from different agencies and that are governed by various laws and regulations can be understood as an answer to the demand for improved coordinated care for citizens with complex needs, such as frail older people. However, this demand raises fundamental questions of democratic control and accountability in the modern welfare state. Although these issues are addressed in current literature on network governance, they have not been investigated empirically very much. The aim of this paper is to investigate coordination bodies as important actors in integrated care, and especially to investigate how the members of these governance networks perceive their own influence and how they are held accountable by their principals. This study is conceptually built on theories of network governance and accountability. The empirical investigation is based on a survey with 545 respondents from 73 different coordination bodies in Sweden. The analysis shows that there seems to be an imbalance between perceived influence and perceived demands from different stakeholders to account for the services. This imbalance provides an opening for a discussion of how to improve the current situation for vulnerable groups and about new perspectives on accountability and power in the modern welfare state.

Anchorage-independent growth is a characteristic feature of cancer cells. However, it is unclear whether it represents a cause or a consequence of tumorigenesis. For normal cells, integrin-mediated adhesion is required for completion of the G1 and cytokinesis stages of the cell cycle. This study identified a mechanism that can drive anchorage-independent growth if the G1 checkpoint is suppressed. Cells with defective G1 checkpoint progressed through several rounds of the cell cycle in suspension in spite of uncompleted cytokinesis, thereby forming bi- and multilobular cells. Aurora B and CEP55 were localized to midbodies between the lobes, suggesting that the cytokinesis process reached close to abscission. Integrin-mediated re-attachment of such cells induced cytokinesis completion uncoupled from karyokinesis in most cells. However, a portion of the cells instead lost the constriction and became binucleated. Also, long-term suspension culture in soft agar produced colonies where the cytokinesis block was overcome. This process was fibronectin-dependent since fibronectin-deficient cells did not form colonies unless fibronectin was expressed or exogenously added. While fibronectin normally is not deposited on non-adherent single cells, bi/multilobular cells accumulated fibronectin in the intussusceptions. Based on our data we conclude: 1) Suppression of the G1 checkpoint allows multiple rounds of the cell cycle in detached cells and thereby enables matrix formation on their surface. 2) Uncompleted cytokinesis due to cell detachment resumes if integrin interactions are re-formed, allowing colony formation in soft agar 3) Such delayed cell division can generate binucleated cells, a feature known to cause chromosomal instability.

Cells are exposed to several types of integrin stimuli, which generate responses generally referred to as "integrin signals", but the specific responses to different integrin stimuli are poorly defined. In this study, signals induced by integrin ligation during cell attachment, mechanical force from intracellular contraction, or cell stretching by external force were compared. The elevated phosphorylation levels of several proteins during the early phase of cell attachment and spreading of fibroblast cell lines were not affected by inhibition of ROCK and myosin II activity, i.e. the reactions occurred independently of intracellular contractile force acting on the adhesion sites. The contraction-independent phosphorylation sites included ERK1/2 T202/Y204, AKT S473, p130CAS Y410, and cofilin S3. In contrast to cell attachment, cyclic stretching of the adherent cells induced a robust phosphorylation only of ERK1/2 and the phosphorylation levels of the other investigated proteins were not or only moderately affected by stretching. No major differences between signaling via α5β1 or αvβ3 integrins were detected. The importance of mitochondrial ROS for the integrin-induced signaling pathways was investigated using rotenone, a specific inhibitor of complex I in the respiratory chain. While rotenone only moderately reduced ATP levels and hardly affected the signals induced by cyclic cell stretching, it abolished the activation of AKT and reduced the actin polymerization rate in response to attachment in both cell lines. In contrast, scavenging of extracellular ROS with catalase or the vitamin C analog Asc-2P did not significantly influence the attachment-derived signaling, but caused a selective and pronounced enhancement of ERK1/2 phosphorylation in response to stretching. In conclusion, the results showed that "integrin signals" are composed of separate sets of reactions triggered by different types of integrin stimulation. Mitochondrial ROS and extracellular ROS had specific and distinct effects on the integrin signals induced by cell attachment and mechanical stretching.

A tight control over AKT/PKB activation is essential for cells, and they realise this in part by regulating the phosphorylation of Ser473 in the "hydrophobic motif" of the AKT carboxy-terminal region. The RICTOR-mTOR complex (TORC2) is a major kinase for AKT Ser473 phosphorylation after stimulation by several growth factors, in a reaction proposed to require p21-activated kinase (PAK) as a scaffold. However, other kinases may catalyse this reaction in stimuli-specific manners. Here we characterised the requirement of RICTOR, ILK, and PAK for AKT Ser473 phosphorylation downstream of selected family members of integrins, G protein-coupled receptors, and tyrosine-kinase receptors and analysed the importance of this phosphorylation site for adhesion-mediated survival. siRNA-mediated knockdown in HeLa and MCF7 cells showed that RICTOR-mTOR was required for phosphorylation of AKT Ser473, and for efficient phosphorylation of the downstream AKT targets FOXO1 Thr24 and BAD Ser136, in response to β1 integrin-stimulation. ILK and PAK1/2 were dispensable for these reactions. RICTOR knockdown increased the number of apoptotic MCF7 cells on β1 integrin ligands up to 2-fold after 24 h in serum-free conditions. β1 integrin-stimulation induced phosphorylation of both AKT1 and AKT2 but markedly preferred AKT2. RICTOR-mTOR was required also for LPA-induced AKT Ser473 phosphorylation in MCF7 cells, but, interestingly, not in HeLa cells. PAK was needed for the AKT Ser473 phosphorylation in response to LPA and PDGF, but not to EGF. These results demonstrate that different receptors utilise different enzyme complexes to phosphorylate AKT at Ser473, and that AKT Ser473 phosphorylation significantly contributes to β1 integrin-mediated anchorage-dependent survival of cells.

The present study was conducted to characterize possible rapid effects of 17-β-estradiol on voltage-gated K(+) channels in preoptic neurons and, in particular, to identify the mechanisms by which 17-β-estradiol affects the K(+) channels. Whole-cell currents from dissociated rat preoptic neurons were studied by perforated-patch recording. 17-β-Estradiol rapidly (within seconds) and reversibly reduced the K(+) currents, showing an EC(50) value of 9.7 µM. The effect was slightly voltage dependent, but independent of external Ca(2+), and not sensitive to an estrogen-receptor blocker. Although 17-α-estradiol also significantly reduced the K(+) currents, membrane-impermeant forms of estradiol did not reduce the K(+) currents and other estrogens, testosterone and cholesterol were considerably less effective. The reduction induced by estradiol was overlapping with that of the K(V)-2-channel blocker r-stromatoxin-1. The time course of K(+) current in 17-β-estradiol, with a time-dependent inhibition and a slight dependence on external K(+), suggested an open-channel block mechanism. The properties of block were predicted from a computational model where 17-β-estradiol binds to open K(+) channels. It was concluded that 17-β-estradiol rapidly reduces voltage-gated K(+) currents in a way consistent with an open-channel block mechanism. This suggests a new mechanism for steroid action on ion channels.

Abstract Background Researchers applying compositional data analysis to time-use data (e.g., time spent in physical behaviors) often face the problem of zeros, that is, recordings of zero time spent in any of the studied behaviors. Zeros hinder the application of compositional data analysis because the analysis is based on log-ratios. One way to overcome this challenge is to replace the zeros with sensible small values. The aim of this study was to compare the performance of three existing replacement methods used within physical behavior time-use epidemiology: simple replacement, multiplicative replacement, and log-ratio expectation-maximization (lrEM) algorithm. Moreover, we assessed the consequence of choosing replacement values higher than the lowest observed value for a given behavior. Method Using a complete dataset based on accelerometer data from 1310 Danish adults as reference, multiple datasets were simulated across six scenarios of zeros (5–30% zeros in 5% increments). Moreover, four examples were produced based on real data, in which, 10 and 20% zeros were imposed and replaced using a replacement value of 0.5 min, 65% of the observation threshold, or an estimated value below the observation threshold. For the simulation study and the examples, the zeros were replaced using the three replacement methods and the degree of distortion introduced was assessed by comparison with the complete dataset. Results The lrEM method outperformed the other replacement methods as it had the smallest influence on the structure of relative variation of the datasets. Both the simple and multiplicative replacements introduced higher distortion, particularly in scenarios with more than 10% zeros; although the latter, like the lrEM, does preserve the ratios between behaviors with no zeros. The examples revealed that replacing zeros with a value higher than the observation threshold severely affected the structure of relative variation. Conclusions Given our findings, we encourage the use of replacement methods that preserve the relative structure of physical behavior data, as achieved by the multiplicative and lrEM replacements, and to avoid simple replacement. Moreover, we do not recommend replacing zeros with values higher than the lowest observed value for a behavior.

Abstract Background It is unclear whether walking can decrease cardiovascular disease (CVD) risk or if high intensity physical activity (HIPA) is needed, and whether the association is modified by age. We investigated how sedentary behaviour, walking, and HIPA, were associated with systolic blood pressure (SBP), waist circumference (WC), and low-density lipoprotein cholesterol (LDL-C) among adults and older adults in a general population sample using compositional data analysis. Specifically, the measure of association was quantified by reallocating time between sedentary behaviour and 1) walking, and 2) HIPA. Methods Cross-sectional data from the fifth examination of the Copenhagen City Heart Study was used. Using the software Acti4, we estimated daily time spent in physical behaviours from accelerometer data worn 24 h/day for 7 days (i.e., right frontal thigh and iliac crest; median wear time: 6 days, 23.8 h/day). SBP, WC, and LDL-C were measured during a physical examination. Inclusion criteria were ≥ 5 days with ≥16 h of accelerometer recordings per day, and no use of antihypertensives, diuretics or cholesterol lowering medicine. The 24-h physical behaviour composition consisted of sedentary behaviour, standing, moving, walking, HIPA (i.e., sum of climbing stairs, running, cycling, and rowing), and time in bed. We used fitted values from linear regression models to predict the difference in outcome given the investigated time reallocations relative to the group-specific mean composition. Results Among 1053 eligible participants, we found an interaction between the physical behaviour composition and age. Age-stratified analyses (i.e.,

Abstract Background Information about how much time adults spend cycling, walking and running can be used for planning and evaluating initiatives for active, healthy societies. The objectives of this study were to describe how much time adult Copenhageners cycle, walk, run, stand and spend sedentary using accelerometers, and to describe differences between population groups. Methods In the fifth examination of the Copenhagen City Heart Study, 2335 individuals gave consent to wear accelerometers (skin-attached; right thigh and iliac crest; 24 h/day, 7 consecutive days) of which 1670 fulfilled our inclusion criteria (≥16 h/day for ≥5 days; median wear time: 23.8 h/day). Daily time spent cycling, walking, running, standing and sedentary was derived from accelerometer-based data using the Acti4 software, and differences between sex, age groups, level of education and BMI were investigated using Kruskal-Wallis rank sum tests. Results Among those cycling (61%), the median cycling time was 8.3 min/day. The median time walking, running, standing and sedentary was 82.6, 0.1, 182.5 and 579.1 min/day, respectively. About 88% walked fast (i.e., ≥100 steps/min) ≥30 min/day. The shortest duration and lowest prevalence of cycling, walking and running were found among older individuals, those with a low level of education, and individuals being overweight or obese. Conclusions We found a long duration and high prevalence of cycling and walking, but also that many adult Copenhageners spent much time sedentary. Population groups with low participation in physical activities such as cycling and walking should be targeted in future initiatives towards an active, healthy society.