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Simple Summary: Applications of nanoparticles as well as their use have increased over the past decades without sufficient research on their environmental impact. Focusing on Daphnia magna, a widely adopted aquatic organism employed to evaluate the adverse effects of pollutants, we investigated several aspects of the experimental design used in D. magna toxicity testing which could impact the observed effect of exposure to nanoparticles. Furthermore, we evaluated the feasibility of a miniaturized version of D. magna toxicity test as a potential candidate for nanoparticle toxicity assays. Results showed that the exposure vessel and its characteristics can affect the impact of nanoparticles and, therefore, skew the observed effects. Additionally, the miniaturized exposure showed that for physiology markers such as toxicity and feeding rate it can be a good alternative to the traditional setups, as it vastly reduces the amount of media and number of nanoparticles required as well as generated waste. The rapid progress of the modern world has resulted in new materials and products created at an accelerating pace. As such, nanoparticles have widespread applications and often find their way into the aquatic ecosystem. In the case of freshwater ecosystems, one of the commonly used bioindicators species used for pollution assessment is Daphnid magna. The Organization for Economic Co-operation and Development (OECD), and other organizations such as the European Chemicals Agency (ECHA) and Environmental Protection Agency (EPA), have set guidelines for acute toxicity testing in daphnids that are severely lacking in terms of information on the characteristics of the exposure vessel when studying the adverse effects of nanoparticles (NPs). Understanding the toxicity mechanisms of nanomaterials is imperative given the scarcity of information on their adverse effects. Furthermore, miniaturization of nanotoxicity assays can reduce the number of daphnids used, as well as the cost and nanomaterial waste, and provide results even at the individual animal level with enhanced reproducibility of testing. In this study, the impact of the exposure vessel on the observed physiological changes of daphnids was investigated for a silver nano ink. Exposures in eleven commercially available vessels; nine made of plastic and two made of glass were compared for 24 h. The effect of surface to volume ratio of the exposure vessel and the animal number or "crowding" during exposure was investigated in the context of miniaturizing biomarker assays as alternatives to traditional experimental setups in Daphnid magna. Toxicity curves showed differences depending on the vessel used, while a novel feeding rate assay and the activity of key enzymes were assessed as physiology endpoints. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria are traditionally applied in a variety of fermented food products, and they have the ability to produce a wide range of bioactive ingredients during fermentation, including vitamins, bacteriocins, bioactive peptides, and bioactive compounds. The bioactivity and health benefits associated with these ingredients have garnered interest in applications in the functional dairy market and have relevance both as components produced in situ and as functional additives. This review provides a brief description of the regulations regarding the functional food market in the European Union, as well as an overview of some of the functional dairy products currently available in the Irish and European markets. A better understanding of the production of these ingredients excreted by lactic acid bacteria can further drive the development and innovation of the continuously growing functional food market. [ABSTRACT FROM AUTHOR]

The biodegradable biopolymer polylactic acid (PLA) has been used in the recent past in single-use packaging as a suitable replacement for non-biodegradable fossil fuel-based plastics, such as polyethylene terephthalate (PET). Under FDA and EU regulations, lactic acid (LA), the building block of PLA, is considered safe to use as a food contact material. The mechanical, thermal, and barrier properties of PLA are, however, major challenges for this material. PLA is a brittle material with a Young's modulus of 2996–3750 MPa and an elongation at break of 1.3–7%. PLA has a glass transition temperature (Tg) of 60 °C, exhibiting structural distortion at this temperature. The water permeability of PLA can lead to hydrolytic degradation of the material. These properties can be improved with biopolymer blending and composites. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), for instance, increases the thermal stability of PLA while decreasing the water permeability by up to 59%. Polypropylene (PP) is one of the most common plastics in reusable food containers. This study will compare PLA-based blends and composites to the currently used PP as a sustainable alternative to fossil fuel-based plastics. The end-of-life options for PLA-based food containers are considered, as is the commercial cost of replacing PP with PLA. [ABSTRACT FROM AUTHOR]

Several methods used for the quantification of DNA are based on UV absorbance or the fluorescence of complexes with intercalator dyes. Most of these intercalators are used in gels to visualize DNA and its structural integrity. Due to many extraterrestrial samples, such as meteorites or comets, which are likely to contain very small amounts of biological material, and because the ability to detect this material is crucial for understanding the origin and evolution of life in the universe, the development of assays that can detect DNA at low limits and withstand the rigors of space exploration is a pressing need in the field of astrobiology. In this study, we present a comparison of optimized protocols used for the fast and accurate quantification of DNA using common intercalator dyes. The sensitivity of assays exceeded that generated by any commercial kit and allowed for the accurate quantification of minimum concentrations of DNA. The methods were successful when applied to the detection and measurement of DNA spiked on soil samples. Furthermore, the impact of UV radiation as a harsh condition on the surface of Mars was assessed by DNA degradation and this was also confirmed by gel electrophoresis. Overall, the methods described provide economical, simple-step, and efficient approaches for the detection of DNA and can be used in future planetary exploration missions as tests used for the extraction of nucleic acid biosignatures. [ABSTRACT FROM AUTHOR]

Sport-related concussion (SRC) management may be poor in community sports such as Ladies Gaelic Football (LGF). This study examined factors associated with SRC management behavior in adult LGF players. Participants (n = 657) answered an online survey on demographic factors, concussion knowledge, attitudes, and education, and SRC management behavior. Data from participants who reported sustaining an LGF-related SRC during the previous year (n = 115) were further analyzed. Being diagnosed with SRC was the main factor influencing subacute management behavior. Players with diagnosed SRCs had increased odds of following a graded return-to-play (RTP) programme (OR = 4.89), following a medically supervised graded RTP programme (OR = 10.16), and being medically cleared before full RTP (OR = 13.45) compared with those with suspected SRCs. Concussion history was associated with increased odds of informing a coach of a possible SRC (OR = 2.86). Demographic factors, previous use of Ladies Gaelic Football Association concussion education resources, and concussion knowledge and attitudes had minimal or no influence on management behaviors. Greater access to medical personnel at LGF training and matches is recommended. Due to limited medical resources in community sport, a clear referral pathway for players with SRC and comprehensive SRC education should be introduced to ensure players receive adequate medical care. [ABSTRACT FROM AUTHOR]

The Organization for Economic Co-operation and Development (OECD) has set widely used guidelines that are used as a standardized approach for assessing toxicity in a number of species. Given that various studies use different experimental setups, it is difficult to compare findings across them as a result of the lack of a universally used setup in nano-ecotoxicology. For freshwater species, Daphnia magna, a commonly used filter feeding crustacean, can generate significant molecular information in response to pollutant exposure. One factor that has an effect in toxicity induced from nanomaterials in daphnids is the surface-to-volume ratio of the exposure vessels; however, there is limited information available about its impact on the observed effect of exposure. In this study, daphnids were exposed to silver nanoparticle ink in falcon tubes and Petri dishes for 24 h. Toxicity curves revealed differences in the observed mortality of daphnids, with animals exposed in Petri dishes displaying significantly higher mortality. Differences in the activities of a number of key enzymes involved in the catabolism of macromolecules and phosphate were also observed across the exposure setups, indicating possible differences in the toxicity mechanism of silver nano-ink. Understanding the impact of factors relevant to experimental setups in ecotoxicology can increase the reproducibility of testing, and also reduce experimental costs, time, generated waste, and daphnids used in research. [ABSTRACT FROM AUTHOR]

Blood flow produces shear stress that homeostatically regulates the phenotype of pulmonary endothelial cells, exerting antiinflammatory and antithrombotic actions and maintaining normal barrier function. Hypoxia due to diseases, such as chronic obstructive pulmonary disease (COPD), causes vasoconstriction, increased vascular resistance, and pulmonary hypertension. Hypoxia-induced changes in endothelial function play a central role in the development of pulmonary hypertension. However, the interactive effects of hypoxia and shear stress on the pulmonary endothelial phenotype have not been studied. Human pulmonary microvascular endothelial cells were cultured in normoxia or hypoxia while subjected to physiological shear stress or in static conditions. Unbiased proteomics was used to identify hypoxia-induced changes in protein expression. Using publicly available single-cell RNA sequencing datasets, differences in gene expression between the alveolar endothelial cells from COPD and healthy lungs were identified. Sixty proteins were identified whose expression changed in response to hypoxia in conditions of physiological shear stress but not in static conditions. These included proteins that are crucial for endothelial homeostasis (e.g., JAM-A [junctional adhesion molecule A], ERG [ETS transcription factor ERG]) or implicated in pulmonary hypertension (e.g., thrombospondin-1). Fifty-five of these 60 have not been previously implicated in the development of hypoxic lung diseases. mRNA for 5 of the 60 (ERG, MCRIP1 [MAPK regulated corepressor interacting protein 1], EIF4A2 [eukaryotic translation initiation factor 4A2], HSP90AA1 [heat shock protein 90 alpha family class A member 1], and DNAJA1 [DnaJ Hsp40 (heat shock protein family) member A1]) showed similar changes in the alveolar endothelial cells of COPD compared with healthy lungs in females but not in males. These data show that the proteomic responses of the pulmonary microvascular endothelium to hypoxia are significantly altered by shear stress and suggest that these shear-hypoxia interactions are important in the development of hypoxic pulmonary vascular disease. [ABSTRACT FROM AUTHOR]

Food spoilage is an ongoing global issue that contributes to rising carbon dioxide emissions and increased demand for food processing. This work developed anti-bacterial coatings utilising inkjet printing of silver nano-inks onto food-grade polymer packaging, with the potential to enhance food safety and reduce food spoilage. Silver nano-inks were synthesised via laser ablation synthesis in solution (LaSiS) and ultrasound pyrolysis (USP). The silver nanoparticles (AgNPs) produced using LaSiS and USP were characterised using transmission electron microscopy (TEM), Fourier transform infrared (FTIR) spectroscopy, UV-Vis spectrophotometry and dynamic light scattering (DLS) analysis. The laser ablation technique, operated under recirculation mode, produced nanoparticles with a small size distribution with an average diameter ranging from 7–30 nm. Silver nano-ink was synthesised by blending isopropanol with nanoparticles dispersed in deionised water. The silver nano-inks were printed on plasma-cleaned cyclo-olefin polymer. Irrespective of the production methods, all silver nanoparticles exhibited strong antibacterial activity against E. coli with a zone of inhibition exceeding 6 mm. Furthermore, silver nano-inks printed cyclo-olefin polymer reduced the bacterial cell population from 1235 (±45) × 106 cell/mL to 960 (±110) × 106 cell/mL. The bactericidal performance of silver-coated polymer was comparable to that of the penicillin-coated polymer, wherein a reduction in bacterial population from 1235 (±45) × 106 cell/mL to 830 (±70) × 106 cell/mL was observed. Finally, the ecotoxicity of the silver nano-ink printed cyclo-olefin polymer was tested with daphniids, a species of water flea, to simulate the release of coated packaging into a freshwater environment. [ABSTRACT FROM AUTHOR]

Lactic acid bacteria (LAB) are capable of synthesising metabolites known as exopolysaccharides (EPS) during fermentation. Traditionally, EPS plays an important role in fermented dairy products through their gelling and thickening properties, but they can also be beneficial to human health. This bioactivity has gained attention in applications for functional foods, which leads them to have prebiotic, immunomodulatory, antioxidant, anti-tumour, cholesterol-lowering and anti-obesity activity. Understanding the parameters and conditions is crucial to optimising the EPS yields from LAB for applications in the food industry. This review provides an overview of the functional food market together with the biosynthesis of EPS. Factors influencing the production of EPS as well as methods for isolation, characterisation and quantification are reviewed. Finally, the health benefits associated with EPS are discussed. [ABSTRACT FROM AUTHOR]

Every year, the EU emits 13.4 Mt of CO2 solely from plastic production, with 99% of all plastics being produced from fossil fuel sources, while those that are produced from renewable sources use food products as feedstocks. In 2019, 29 Mt of plastic waste was collected in Europe. It is estimated that 32% was recycled, 43% was incinerated and 25% was sent to landfill. It has been estimated that life-sciences (biology, medicine, etc.) alone create plastic waste of approximately 5.5 Mt/yr, the majority being disposed of by incineration. The vast majority of this plastic waste is made from fossil fuel sources, though there is a growing interest in the possible use of bioplastics as a viable alternative for single-use lab consumables, such as petri dishes, pipette tips, etc. However, to-date only limited bioplastic replacement examples exist. In this review, common polymers used for labware are discussed, along with examining the possibility of replacing these materials with bioplastics, specifically polylactic acid (PLA). The material properties of PLA are described, along with possible functional improvements dure to additives. Finally, the standards and benchmarks needed for assessing bioplastics produced for labware components are reviewed. [ABSTRACT FROM AUTHOR]

Impairment of the arteries is a product of sustained exposure to various deleterious factors and progresses with time; a phenomenon inherent to vascular aging. Oxidative stress, inflammation, the accumulation of harmful agents in high cardiovascular risk conditions, changes to the extracellular matrix, and/or alterations of the epigenetic modification of molecules, are all vital pathophysiological processes proven to contribute to vascular aging, and also lead to changes in levels of associated circulating molecules. Many of these molecules are consequently recognized as markers of vascular impairment and accelerated vascular aging in clinical and research settings, however, for these molecules to be classified as biomarkers of vascular aging, further criteria must be met. In this paper, we conducted a scoping literature review identifying thirty of the most important, and eight less important, biomarkers of vascular aging. Herein, we overview a selection of the most important molecules connected with the above-mentioned pathological conditions and study their usefulness as circulating biomarkers of vascular aging. [ABSTRACT FROM AUTHOR]

Studies suggest that tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) has vasoprotective potential, as low levels of TRAIL cause accelerated vascular calcification, whereas exogenous TRAIL administration exhibits anti‐atherosclerotic activity. The mechanism of TRAIL‐mediated vasoprotection remains unclear. We studied the effects of TRAIL (100 ng/ml) on human aortic endothelial cells (HAECs) exposed to pro‐atherogenic conditions; (a) oscillatory shear stress (±10 dynes/cm2) using the ibidi µ‐slide fluidic system; (b) pro‐inflammatory injury, that is, tumor necrosis factor alpha (TNF‐α, 100 ng/ml) and hyperglycemia (30 mM d‐glucose). End‐points examined included inflammatory gene expression and reactive oxygen species (ROS) formation. TRAIL shifted the net gene expression toward an antioxidant phenotype in HAECs exposed to oscillatory shear stress. TRAIL significantly reduced ROS formation in HAECs exposed to both TNF‐α and hyperglycemia. Therefore, TRAIL appears to confer atheroprotective effects on the endothelium, at least in part, by reducing oxidative stress. [ABSTRACT FROM AUTHOR]

Numerous studies have highlighted the causal link between over-production of reactive oxygen species (ROS) and cardiovascular complications such as vascular calcification (VC). Receptor-activator of nuclear factor-κB ligand (RANKL) has previously been shown to act on endothelial cells, eliciting the production/release of paracrine pro-calcific signals that act, in-turn, upon underlying vascular smooth muscle cells (VSMCs) to induce osteoblastic differentiation and VC. A role for endothelial ROS signaling in this process has not been established however. In the current paper, we investigate the possibility that RANKL leads to ROS signaling within the endothelial layer as part of the RANKL-driven VC signaling cascade. Human aortic endothelial cells (HAECs) were treated with RANKL (25 ng/ml, 72 h) and monitored for ROS production, in parallel with various pro-calcific signaling indices. Antioxidant co-treatments included TRAIL (5 ng/ml), apocynin (10 mM) and N-acetylcysteine (5 mM). Treatment of HAECs with RANKL-induced robust ROS production. This surge could be partially attenuated by TRAIL and strongly attenuated by apocynin and N-acetylcysteine. RANKL also elicited a range of signaling events in HAECs that we have previously demonstrated are coupled to osteoblastic differentiation in underlying VSMCs. These include non-canonical NF-κB/p52 activation, elevated BMP-2 release and increased alkaline phosphatase (ALP) enzyme activity (cellular and extracellular). Importantly, these RANKL-induced signaling events could be completely prevented by co-treatment of HAECs with antioxidants. In summary, RANKL elicits ROS generation in HAECs with direct consequences for generation of paracrine pro-calcific signals known to effect calcification in underlying VSMCs. [ABSTRACT FROM AUTHOR]

Fluid filtration in the pulmonary microcirculation depends on the hydrostatic and oncotic pressure gradients across the endothelium and the selective permeability of the endothelial barrier. Maintaining normal fluid balance depends both on specific properties of the endothelium and of the perfusing blood. Although some of the essential properties of blood needed to prevent excessive fluid leak have been identified and characterized, our understanding of these remains incomplete. The role of perfusate viscosity in maintaining normal fluid exchange has not previously been examined. We prepared a high-viscosity perfusion solution (HVS) with a relative viscosity of 2.5, i.e., within the range displayed by blood flowing in vessels of different diameters in vivo (1.5-4.0). Perfusion of isolated murine lungs with HVS significantly reduced the rate of edema formation compared with perfusion with a standard solution (SS), which had a lower viscosity similar to plasma (relative viscosity 1.5). HVS did not alter capillary filtration pressure. Increased endothelial shear stress produced by increasing flow rates of SS, to mimic the increased shear stress produced by HVS, did not reduce edema formation. HVS significantly reduced extravasation of Evans bluelabeled albumin compared with SS, indicating that it attenuated endothelial leak. These findings demonstrate for the first time that the viscosity of the solution perfusing the pulmonary microcirculation is an important physiological property contributing to the maintenance of normal fluid exchange. This has significant implications for our understanding of fluid homeostasis in the healthy lung, edema formation in disease, and reconditioning of donor organs for transplantation. [ABSTRACT FROM AUTHOR]

Background The intimal endothelium is known to condition the underlying medial smooth muscle cell (SMC) layer of the vessel wall, and is highly responsive to receptor-activator of nuclear factor-κB ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), pro-calcific and anti-calcific agents, respectively. In this paper, we tested the hypothesis that RANKL-induced activation of endothelial NF-κB signalling is essential for pro-calcific activation of the underlying SMCs. Methods For these studies, human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0–25 ng/ml ± 5 ng/ml TRAIL) for 72 h. Non-contact transwell HAEC:HASMC co-cultures were also employed in which the luminal HAECs were treated with RANKL (± 5 ng/ml TRAIL), followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. Results Treatment of either HAECs or HASMCs with RANKL activated the non-canonical NF-κB/p52 and canonical NF-κB/p65 pathways in both cell types. In RANKL ± TRAIL-treated HAECs, recombinant TRAIL, previously demonstrated by our group to strongly attenuate the pro-calcific signalling effects of RANKL, was shown to specifically block the RANKL-mediated activation of non-canonical NF-κB/p52, clearly pointing to the mechanistic relevance of this specific pathway to RANKL function within endothelial cells. In a final series of HAEC:HASMC transwell co-culture experiments, RANKL treatment of HAECs that had been genetically silenced (via siRNA) for the NF-κB2 gene (the molecular forerunner to NF-κB/p52 generation) exhibited strongly attenuated pro-calcific activation of underlying HASMCs relative to scrambled siRNA controls. Summary These in vitro observations provide valuable mechanistic insights into how RANKL may potentially act upon endothelial cells through activation of the alternative NF-κB pathway to alter endothelial paracrine signalling and elicit pro-calcific responses within underlying vascular smooth muscle cells. [ABSTRACT FROM AUTHOR]

Receptor activator of nuclear factor-κB ligand (RANKL) promotes vascular calcification, while osteoprotegerin (OPG) opposes it by blocking RANKL activity. It remains unclear which vascular cell populations produce and secrete OPG/RANKL. This study characterizes the production of OPG/RANKL from human aortic endothelial and smooth muscle cells (HAECs and HASMCs) under various conditions. HAECs and HASMCs were exposed to inflammatory stimuli (tumor necrosis factor-α or hyperglycemia) or physiological levels of hemodynamic (cyclic) strain. After 72 h, both cells and supernatant media were harvested for assessment of OPG/RANKL production. Based on initial findings, the experiments involving HASMCs were then repeated in the presence of exogenous RANKL. OPG was produced and secreted by HASMCs and (to a considerably lesser degree) HAECs under basal conditions. Inflammatory stimuli upregulated OPG production in both cell populations. Cyclic strain significantly upregulated OPG production in HASMCs. Neither cell population produced RANKL. Exposing HASMCs to exogenous RANKL inhibited basal OPG production and completely abrogated the strain-mediated upregulation of OPG. These data suggest that HASMCs are a significant source of OPG within the vasculature but that RANKL, once present, downregulates this production and appears capable of preventing the “protective” upregulation of OPG seen with HASMCs exposed to physiological levels of cyclic strain. [ABSTRACT FROM AUTHOR]

Background and objectives: Vascular calcification (VC) is a major risk factor for elevated cardiovascular morbidity/mortality. Underlying this process is osteoblastic signalling within the vessel wall involving complex and interlinked roles for receptor-activator of nuclear factor-κB ligand (RANKL), osteoprotegerin (OPG), and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL). RANKL promotes vascular cell osteoblastic differentiation, whilst OPG acts as a neutralizing decoy receptor for RANKL (and TRAIL). With respect to TRAIL, much recent evidence points to a vasoprotective role for this ligand, albeit via unknown mechanisms. In order to shed more light on TRAILs vasoprotective role therefore, we employed in vitro cell models to test the hypothesis that TRAIL can counteract the RANKL-mediated signalling that occurs between the vascular cells that comprise the vessel wall. Methods and results: Human aortic endothelial and smooth muscle cell mono-cultures (HAECs, HASMCs) were treated with RANKL (0–25 ng/mL ± 5 ng/mL TRAIL) for 72 hr. Furthermore, to better recapitulate the paracrine signalling that exists between endothelial and smooth muscle cells within the vessel wall, non-contact transwell HAEC:HASMC co-cultures were also employed and involved RANKL treatment of HAECs (±TRAIL), subsequently followed by analysis of pro-calcific markers in the underlying subluminal HASMCs. RANKL elicited robust osteoblastic signalling across both mono- and co-culture models (e.g. increased BMP-2, alkaline phosphatase/ALP, Runx2, and Sox9, in conjunction with decreased OPG). Importantly, several RANKL actions (e.g. increased BMP-2 release from mono-cultured HAECs or increased ALP/Sox9 levels in co-cultured HASMCs) could be strongly blocked by co-incubation with TRAIL. In summary, this paper clearly demonstrates that RANKL can elicit pro-osteoblastic signalling in HAECs and HASMCs both directly and across paracrine signalling axes. Moreover, within these contexts we present clear evidence that TRAIL can block several key signalling actions of RANKL in vascular cells, providing further evidence of its vasoprotective potential. [ABSTRACT FROM AUTHOR]

Blood-brain barrier (BBB) disruption constitutes a hallmark event during pathogen-mediated neurological disorders such as bacterial meningitis. As a prevalent opportunistic pathogen, Staphylococcus aureus (SA) is of particular interest in this context, although our fundamental understanding of how SA disrupts the BBB is very limited. This paper employs in vitro infection models to address this. Human brain microvascular endothelial cells (HBMvECs) were infected with formaldehyde-fixed (multiplicity of infection [MOI] 0-250, 0-48 hr) and live (MOI 0-100, 0-3 hr) SA cultures. Both Fixed-SA and Live-SA could adhere to HBMvECs with equal efficacy and cause elevated paracellular permeability. In further studies employing Fixed-SA, infection of HBMvECs caused dose-dependent release of cytokines/chemokines (TNF-α, IL-6, MCP-1, IP-10, and thrombomodulin), reduced expression of interendothelial junction proteins (VE-Cadherin, claudin-5, and ZO-1), and activation of both canonical and non-canonical NF-κB pathways. Using N-acetylcysteine, we determined that these events were coupled to the SA-mediated induction of reactive oxygen species (ROS) within HBMvECs. Finally, treatment of HBMvECs with Fixed-ΔSpA (MOI 0-250, 48 hr), a gene deletion mutant of Staphylococcal protein A associated with bacterial infectivity, had relatively similar effects to Newman WT Fixed-SA. In conclusion, these findings provide insight into how SA infection may activate proinflammatory mechanisms within the brain microvascular endothelium to elicit BBB failure. [ABSTRACT FROM AUTHOR]

An intact functioning blood-brain barrier (BBB) is fundamental to proper homoeostatic maintenance and perfusion of the central nervous system (CNS). Inflammatory damage to the unique microvascular endothelial cell monolayer that constitutes the luminal BBB surface, leading to elevated capillary permeability, has been linked to various neurological disorders ranging from ischaemic stroke and traumatic brain injury, to neurodegenerative disease and CNS infections. Moreover, the neuroinflammatory cascade that typically accompanies BBB failure in these circumstances has been strongly linked to elevated levels of proinflammatory cytokines such as tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). This mini review will examine our current knowledge of how cytokines may dysregulate the interendothelial paracellular pathway leading to elevated BBB permeability. The mechanistic role of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase)-induced oxidative stress in these events will also be addressed. [ABSTRACT FROM AUTHOR]

Zonula occludens-1 (ZO-1) is essential to the proper assembly of interendothelial junction complexes that control blood–brain barrier (BBB) integrity. The goal of the current paper was to improve our understanding of how proinflammatory cytokines modulate ZO-1 properties within the human BBB microvascular endothelium. In this respect, we investigated the effects of TNF-α and IL-6 on ZO-1 using human brain microvascular endothelial cells (HBMvECs). Following treatment of HBMvECs with either cytokine (0–100 ng/ml, 18 h), we observed significantly decreased ZO-1 expression and ZO-1:occludin co-association, in parallel with increased ZO-1 phosphorylation (pTyr and pThr). All effects were dose-dependent. Either cytokine also caused extensive cell-cell border delocalization of ZO-1 in parallel with elevated HBMvEC permeability. Furthermore, pre-treatment of HBMvECs with antioxidants (superoxide dismutase, catalase, apocynin, N -acetylcysteine), or employing targeted inhibition of NADPH oxidase activation (NSC23766, gp91/p47 siRNA), were all found to comparably attenuate the cytokine-dependent decrease in ZO-1 protein expression. In summary, we present an in vitro model of how different proinflammatory cytokines can dysregulate ZO-1 properties in HBMvECs. A causal role for NADPH oxidase activation and oxidant signalling is also confirmed. Our findings add mechanistic depth to current in vivo models of BBB injury manifesting ZO-1 dysregulation. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) provide an essential link between innate and adaptive immunity. At the site of infection, antigens recognized by DCs via pattern-recognition receptors, such asToll-like receptors (TLRs), initiate a specific immune response. Depending on the nature of the antigen, DCs secrete distinct cytokines with which they orchestrate homeostasis and pathogen clearance. Dysregulation of this process can lead to unnecessary inflammation, which can result in a plethora of inflammatory diseases. Therefore, the secretion of cytokines from DCs is tightly regulated and this regulation is facilitated by highly conserved trafficking protein families.These proteins control the transport of vesicles from the Golgi complex to the cell surface and between organelles. In this review, we will discuss the role of soluble n-ethylmaleimide-sensitive factor attachment protein receptor proteins (SNAREs) in DCs, both as facilitators of secretion and as useful tools to determine the pathways of secretion through their definite locations within the cells and inherent specificity in opposing binding partners on vesicles and target membranes.The role of SNAREs in DC function may present an opportunity to explore these proteins as novel targets in inflammatory disease. [ABSTRACT FROM AUTHOR]

Thrombomodulin (TM), an important determinant of blood vessel homeostasis, is expressed on the luminal vascular endothelial cell surface and is released into serum in response to circulatory signals. This includes the cerebrovascular endothelium, where the anti-coagulant and anti-inflammatory properties of TM are thought to be critical to the brain microcirculation and blood–brain barrier (BBB) integrity. Much is still unknown however about how circulatory stimuli may regulate TM activity within the brain microvasculature. To address this, the current short paper investigated the effects of opposing regulatory signals, namely cytokines (TNF-α, IL-6) and laminar shear stress, on the cellular levels and release of TM in cultured human brain microvascular endothelial cells (HBMvECs). Treatment of confluent HBMvECs with either TNF-α or IL-6 (100 ng/ml, 18 h) reduced TM protein levels by up to 70%, whilst inducing TM release into media by up to 4.4 and 5.5 fold, respectively. The effects of either cytokine (0–100 ng/ml) on TM protein levels (6 or 18 h) and release (0–18 h) were also found to be concentration- and time-dependent. Either cytokine (100 ng/ml, 24–72 h) also reduced TM mRNA levels by > 50%. When exposed to laminar shear stress for 24 h at 8 dyn/cm 2 (SI unit equivalent = 0.8 Pa), TM protein levels were upregulated by 65% in parallel with a 2-fold increase in TM mRNA levels. Shear stress also proved to be a much more potent stimulus for TM release from HBMvECs, yielding media TM levels of 1000 pg/10 5 cells, when compared to 175 and 210 pg/10 5 cells for TNF-α and IL-6, respectively, after parallel 18 h treatments. Finally, shear-conditioned media was found to completely block thrombin-induced permeabilization of HBMvECs, confirming the functional efficacy of released TM. In summary, our data indicate that TM is differentially regulated within cultured HBMvECs by humoral and biomechanical signals. [ABSTRACT FROM AUTHOR]

The role of dendritic cells (DCs) in directing the immune response is due in part to their capacity to produce a range of cytokines. Importantly, DCs are a source of cytokines, which can promote T cell survival and T helper cell differentiation. While it has become evident that soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptors (SNAREs) are involved in membrane fusion and ultimately cytokine release, little is known about which members of this family facilitate the secretion of specific cytokines from DCs. We profiled mRNA of 18 SNARE proteins in DCs in response to activation with a panel of three Toll-like receptors (TLR) ligands and show differential expression of SNAREs in response to their stimulus and subsequent secretion patterns. Of interest, STX3 mRNA was up-regulated in response to TLR4 and TLR7 activation but not TLR2 activation. This correlated with secretion of IL-6 and MIP-1α. Abolishment of STX3 from DCs by RNAi resulted in the attenuation of IL-6 levels and to some extent MIP-1α levels. Analysis of subcellular location of STX3 by confocal microscopy showed translocation of STX3 to the cell membrane only in DCs secreting IL-6 or MIP-1α, indicating a role for STX3 in trafficking of these immune mediators. Given the role of IL-6 in Th17 differentiation, these findings suggest the potential of STX3 as therapeutic target in inflammatory disease. [ABSTRACT FROM AUTHOR]

Background and Objectives: Thrombomodulin (TM), an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain) intrinsic to endothelial-mediated vascular homeostasis. Methods: This study employed human aortic endothelial cells (HAECs) to investigate the effects of equibiaxial cyclic strain (7.5%, 60 cycles/min, 24 hrs), and to a lesser extent, laminar shear stress (10 dynes/cm2, 24 hrs), on TM expression and release. Time-, dose- and frequency-dependency studies were performed. Results: Our initial studies demonstrated that cyclic strain strongly downregulated TM expression in a p38- and receptor tyrosine kinase-dependent manner. This was in contrast to the upregulatory effect of shear stress. Moreover, both forces significantly upregulated TM release over a 48 hr period. With continuing focus on the cyclic strain-induced TM release, we noted both dose (0–7.5%) and frequency (0.5–2.0 Hz) dependency, with no attenuation of strain-induced TM release observed following inhibition of MAP kinases (p38, ERK-1/2), receptor tyrosine kinase, or eNOS. The concerted impact of cyclic strain and inflammatory mediators on TM release from HAECs was also investigated. In this respect, both TNFα (100 ng/ml) and ox-LDL (10–50 µg/ml) appeared to potentiate strain-induced TM release. Finally, inhibition of neither MMPs (GM6001) nor rhomboids (3,4-dichloroisocoumarin) had any effect on strain-induced TM release. However, significantly elevated levels (2.1 fold) of TM were observed in isolated microparticle fractions following 7.5% strain for 24 hrs. Conclusions: A preliminary in vitro investigation into the effects of cyclic strain on TM in HAECs is presented. Physiologic cyclic strain was observed to downregulate TM expression, whilst upregulating in a time-, dose- and frequency-dependent manner the release of TM. [ABSTRACT FROM AUTHOR]

Background and Objectives: Blood-brain barrier (BBB) dysfunction is an integral feature of neurological disorders and involves the action of multiple proinflammatory cytokines on the microvascular endothelial cells lining cerebral capillaries. There is still however, considerable ambiguity throughout the scientific literature regarding the mechanistic role(s) of cytokines in this context, thereby warranting a comprehensive in vitro investigation into how different cytokines may cause dysregulation of adherens and tight junctions leading to BBB permeabilization. Methods: The present study employs human brain microvascular endothelial cells (HBMvECs) to compare/contrast the effects of TNF-α and IL-6 on BBB characteristics ranging from the expression of interendothelial junction proteins (VE-cadherin, occludin and claudin-5) to endothelial monolayer permeability. The contribution of cytokine-induced NADPH oxidase activation to altered barrier phenotype was also investigated. Results: In response to treatment with either TNF-α or IL-6 (0–100 ng/ml, 0–24 hrs), our studies consistently demonstrated significant dose- and time-dependent decreases in the expression of all interendothelial junction proteins examined, in parallel with dose- and time-dependent increases in ROS generation and HBMvEC permeability. Increased expression and co-association of gp91 and p47, pivotal NADPH oxidase subunits, was also observed in response to either cytokine. Finally, cytokine-dependent effects on junctional protein expression, ROS generation and endothelial permeability could all be attenuated to a comparable extent using a range of antioxidant strategies, which included ROS depleting agents (superoxide dismutase, catalase, N-acetylcysteine, apocynin) and targeted NADPH oxidase blockade (gp91 and p47 siRNA, NSC23766). Conclusion: A timely and wide-ranging investigation comparing the permeabilizing actions of TNF-α and IL-6 in HBMvECs is presented, in which we demonstrate how either cytokine can similarly downregulate the expression of interendothelial adherens and tight junction proteins leading to elevation of paracellular permeability. The cytokine-dependent activation of NADPH oxidase leading to ROS generation was also confirmed to be responsible in-part for these events. [ABSTRACT FROM AUTHOR]

Blood-brain barrier (BBB) regulation involves the coordinated interaction of intercellular adherens and tight junctions in response to stimuli. One such stimulus, shear stress, has been shown to upregulate brain microvascular endothelial cell (BMvEC) barrier function, although our knowledge of the signaling mechanisms involved is limited. In this article, we examined the hypothesis that VE-cadherin can transmit shear signals to tight junction occludin with consequences for pTyr-occludin and barrier function. In initial studies, chronic shear enhanced membrane localization of ZO-1 and claudin-5, decreased pTyr-occludin (in part via a dephostatin-sensitive mechanism), and reduced BMvEC permeability, with flow reduction in pre-sheared BMvECs having converse effects. In further studies, VE-cadherin inhibition (VE-cad ΔEXD) blocked shear-induced Rac1 activation, pTyr-occludin reduction, and barrier upregulation, consistent with an upstream role for VE-cadherin in transmitting shear signals to tight junctions through Rac1. As VE-cadherin is known to mediate Rac1 activation via Tiam1 recruitment, we subsequently confirmed that Tiam1 inhibition (Tiam1-C580) could elicit effects similar to VE-cad ΔEXD. Finally, the observed attenuation of shear-induced changes in pTyr-occludin level and barrier phenotype following Rac1 inhibition (NSC23766, T17N) establishes a downstream role for Rac1 in this pathway. In summary, we describe for the first time in BMvECs a role for VE-cadherin in the transmission of physiological shear signals to tight junction occludin through engagement of Tiam1/Rac1 leading to barrier stabilization. A downstream role is also strongly indicated for a protein tyrosine phosphatase in pTyr-occludin modulation. Importantly, these findings suggest an important route of inter-junctional signaling cross-talk during BBB response to flow. J. Cell. Physiol. 226: 3053-3063, 2011. © 2011 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Key points It is unclear how skeletal muscle metabolism and mitochondrial function adapt to long duration bed rest and whether changes can be prevented by nutritional intervention. The present study aimed (1) to assess the effect of prolonged bed rest on skeletal muscle mitochondrial function and dynamics and (2) to determine whether micronutrient supplementation would mitigate the adverse metabolic effect of bed rest. Participants were maintained in energy balance throughout 60 days of bed rest with micronutrient supplementation (INT) (body mass index: 23.747 ± 1.877 kg m–2; 34.80 ± 7.451 years; n = 10) or without (control) (body mass index: 24.087 ± 2.088 kg m–2; 33.50 ± 8.541 years; n = 10). Indirect calorimetry and dual‐energy x‐ray absorptiometry were used for measures of energy expenditure, exercise capacity and body composition. Mitochondrial respiration was determined by high‐resolution respirometry in permeabilized muscle fibre bundles from vastus lateralis biopsies. Protein and mRNA analysis further examined the metabolic changes relating to regulators of mitochondrial dynamics induced by bed rest. INT was not sufficient in preserving whole body metabolic changes conducive of a decrease in body mass, fat‐free mass and exercise capacity within both groups. Mitochondrial respiration, OPA1 and Drp1 protein expression decreased with bed rest, with an increase pDrp1s616. This reduction in mitochondrial respiration was explained through an observed decrease in mitochondrial content (mtDNA:nDNA). Changes in regulators of mitochondrial dynamics indicate an increase in mitochondrial fission driven by a decrease in inner mitochondrial membrane fusion (OPA1) and increased pDrp1s616. Sixty days of −6° head down tilt bed rest leads to significant changes in body composition, exercise capacity and whole‐body substrate metabolism. Micronutrient supplementation throughout bed rest did not preserve whole body metabolic changes. Bed rest results in a decrease in skeletal muscle mitochondrial respiratory capacity, mainly as a result of an observed decrease in mitochondrial content. Prolonged bed rest ensues changes in key regulators of mitochondrial dynamics. OPA1 and Drp1 are significantly reduced, with an increase in pDrp1s616 following bed rest indicative of an increase in mitochondrial fission. Given the reduction in mitochondrial content following 60 days of bed rest, the maintenance of regulators of mitophagy in line with the increase in regulators of mitochondrial fission may act to maintain mitochondrial respiration to meet energy demands. [ABSTRACT FROM AUTHOR]

The glycosyl-phosphatidyl-inositol (GPI)-anchored urokinase receptor (uPAR) has no intracellular domain, but nevertheless initiates signalling through proximal interactions with other membrane receptors including integrins. The relationships between uPAR and ezrin/radixin/moesin (ERM) proteins, moesin and merlin have never been explored. Moesin and merlin are versatile membrane-actin links and regulators of receptors signalling, respectively. We show that uPAR controls moesin and merlin, which propagate uPAR-initiated signals and modulate integrin functions, thereby regulating uPAR activity. uPAR rapidly de-phosphorylates moesin and phosphorylates merlin inactivating both proteins, and enhancing cell migration and angiogenesis. Moesin behaves as a molecular switch turning either on or off uPAR signalling through cycles of de-activation/activation, or sustained activation, respectively. Furthermore, moesin is at the crossroads of uPAR-initiated outside-in and inside-out signalling promoting integrin-dependent cell adhesion suggesting that uPAR also activates integrins distally through moesin. Knocking down merlin expression enhanced cell migration and adhesion through different regulation of fibronectin- and vitronectin-binding integrins. [ABSTRACT FROM AUTHOR]

Introduction Receptor activator of nuclear factor kappa beta-ligand (RANKL) is thought to promote vascular calcification (VC) by inducing osteoblastic behaviour in vascular smooth muscle cells (VSMC) in an ill-defined process. The present study assessed whether RANKL affects pro-osteoblastic paracrine signalling between human aortic endothelial cells (HAEC) and human aortic smooth muscle cells (HASMC) using both conditioned media transfer and cell co-culture experimental approaches. Methods and results For initial experiments (6-well format), HAEC-conditioned media was harvested following 72 h exposure to RANKL, and transferred to reporter HASMCs with/without noggin, an inhibitor of pro-osteoblastic bone morphogenetic protein (BMP) paracrine signalling. In further experiments, HAECs and HASMCs were co-cultured within the CellMax ® Duo, a perfusing bioreactor unit that mimics the flow-mediated co-interaction of these cells within the arterial wall, and RANKL was added to the perfusing media for 72 h. At the conclusion of each experiment markers of osteoblastic activity were measured in HASMCs, including alkaline phosphatase (ALP) activity, mRNA levels of ALP and Runx2, as well as BMP-2 and BMP-4 concentrations. RANKL increased BMP-2 release from HAECs, while exposure of HASMCs to RANKL-treated HAEC-conditioned media induced osteoblastic behaviour in HASMCs, an effect prevented by noggin. Within the CellMax ® Duo bioreactor, the addition of RANKL to the intraluminal HAECs also produced an increase in BMP-2 and increased osteoblastic behaviour within the co-cultured HASMC population. Conclusions RANKL promotes VC by inducing BMP-2 release from HAECs, which in turn appears to act in a paracrine fashion on the adjacent HASMC population to increase osteoblastic activity. [ABSTRACT FROM AUTHOR]