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Background & AimsThe extrahepatic bile duct is the primary tissue initially affected by the cholangiopathy biliary atresia. Biliary atresia affects neonates exclusively and current animal models suggest that the developing bile duct is uniquely susceptible to damage. In this study, we aimed to define the anatomical and functional differences between the neonatal and adult mouse extrahepatic bile ducts.MethodsWe studied mouse passaged cholangiocytes, mouse BALB/c neonatal and adult primary cholangiocytes and isolated extrahepatic bile ducts, and a collagen reporter mouse. Methods included transmission electron microscopy, lectin staining, immunostaining, rhodamine uptake assays, bile acid toxicity assays, and in vitro modeling of the matrix.ResultsThe cholangiocyte monolayer of the neonatal extrahepatic bile duct was immature, lacking the uniform apical glycocalyx and mature cell-cell junctions typical of adult cholangiocytes. Functional studies showed that the glycocalyx protected against bile acid injury and that neonatal cholangiocyte monolayers were more permeable than adult monolayers. In adult ducts, the submucosal space was filled with collagen I, elastin, hyaluronic acid, and proteoglycans. In contrast, the neonatal submucosa had little collagen I and elastin, although both increased rapidly after birth. In vitro modeling suggested that the composition of the neonatal submucosa relative to the adult submucosa led to increased diffusion of bile. A Col-GFP reporter mouse showed that cells in the neonatal but not adult submucosa were actively producing collagen.ConclusionWe identified four key differences between the neonatal and adult extrahepatic bile duct. We showed that these features may have functional implications, suggesting the neonatal extrahepatic bile ducts are particularly susceptible to injury and fibrosis.Lay SummaryBiliary atresia is a disease that affects newborns and is characterized by extrahepatic bile duct injury and obstruction with resulting liver injury. We identify four key differences between the epithelial and submucosal layers of the neonatal and adult extrahepatic bile duct and show that these may render the neonatal duct particularly susceptible to injury.HighlightsThe apical glycocalyx is thin and patchy in neonatal compared to adult cholangiocytesNeonatal cholangiocytes have immature cell-cell junctions and increased permeabilityThe neonatal submucosal space has minimal collagen I or elastinThe neonatal submucosal space contains many actively collagen-secreting cellsGraphical abstract

Industry, and in particular the energy-intensive extraction and production of basic materials such as chemicals, cement, steel, non-ferrous metals, ceramics, and so on, is responsible for approximately 50% of global greenhouse gas emissions. If EU industry makes a successful low-carbon transition, it can be a model for other regions proving that there are numerous pathways which can help reconcile a climate-neutral society with the large consumption of materials

Background and aims Chronic cholestatic liver diseases, such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC), are frequently associated with damage to the barrier function of the biliary epithelium. Here, we report on a bile duct-on-a-chip that phenocopies not only the tubular architecture of the bile duct in three dimensions, but also its barrier functions. Approach and results We showed that mouse cholangiocytes in the channel of the device became polarized and formed mature tight junctions, that the permeability of the cholangiocyte monolayer was comparable to ex vivo measurements, and that cholangiocytes in the device were mechanosensitive (as demonstrated by changes in calcium flux under applied luminal flow). Permeability decreased significantly when cells formed a compact monolayer with cell densities comparable to those observed in vivo. This device enabled independent access to the apical and basolateral surfaces of the cholangiocyte channel, allowing proof-of-concept toxicity studies with the biliary toxin, biliatresone, and the bile acid, glycochenodeoxycholic acid. The cholangiocyte basolateral side was more vulnerable than the apical side to treatment with either agent, suggesting a protective adaptation of the apical surface that is normally exposed to bile. Further studies revealed a protective role of the cholangiocyte apical glycocalyx, wherein disruption of the glycocalyx with neuraminidase increased the permeability of the cholangiocyte monolayer after treatment with glycochenodeoxycholic acid. Conclusions This bile duct-on-a-chip captured essential features of a simplified bile duct in structure and organ-level functions and represents an in vitro platform to study the pathophysiology of the bile duct using cholangiocytes from a variety of sources.

This article investigates the contribution of global governance to advancing the decarbonization of energy-intensive industries (EIIs – steel, chemicals, cement, aluminium, etc.). It explores to what extent relevant intergovernmental and transnational institutions have exploited the potentials of global governance to address related barriers and challenges, in particular competitiveness concerns, the need to incentivise investments in breakthrough technologies and enhance circularity across global value chains. We find that global governance's high potential to contribute to the decarbonization of EIIs has remained very much underexploited. Few international institutions contribute and there is no clear centre. Existing institutions have especially not delivered a sector-specific signal/vision and consequent international rules. In response, the formation of a central institution and/or subsector-specific initiatives might be considered. We argue that advancing global governance to tap into its considerable but so far underexploited potential ought to be an integral part of any strategy for the decarbonization of EIIs.

This paper explores climate-friendly projects that could be part of the COVID-19 recovery while jump-starting the transition of the European basic materials industry. Findings from a literature review on technology options in advanced development stages for climate-friendly production, enhanced sorting, and recycling of steel, cement, aluminium, and plastics, are combined with insights from interviews with 31 European stakeholders in these sectors about the practical and economic feasibility of these technology options. Results indicate that with an estimated investment of 28.9 billion Euros, up to 20% of EU’s basic materials could be produced through low-emission processes or additional recycling by 2025 with technologies that are commercially available or at pilot scale today. However, our stakeholder consultation also shows that in order to make these short-term investments viable, six main barriers need to be addressed, namely: (i) the lack of effective and predictable carbon pricing, (ii) the limited availability of affordable green electricity, (iii) the lack of a regulatory framework for circularity, (iv) low technology market readiness and funding, (v) the lack of infrastructure for hydrogen, CO2 and power, and (vi) the lack of demand for climate-friendly and recycled materials. Based on these insights, the paper proposes elements of a policy package that can create a framework favourable for investments in these technologies; these policies should ideally accompany the recovery package to give credibility to investors that the business case will last beyond the recovery period. Key policy insights: Technologies for climate-friendly materials production, sorting and recycling can be supported as part of the recovery package but require an enabling policy framework. Combining continued free allocation with a Climate Contribution within the EU ETS can enhance economic viability of climate-friendly options. Project-based Carbon Contracts for Difference can eliminate carbon price uncertainty for climate-friendly processes. Auctions for publicly backed Contracts for Difference and Power Purchasing Agreements can guarantee price-stability of low-emission electricity. Green public procurement and public-private partnerships can provide infrastructure for hydrogen, CO2 and electricity while creating demand for climate-friendly materials. Revising regulations on product design and end-of-life emissions can improve sorting and recycling incentives.

We appreciate the interest of Xiao et al. in our work. We believe, however, that these authors have misinterpreted our experimental design and findings. They question our conclusion that the basal surface of cholangiocyte monolayers is more sensitive to the biliary toxin biliatresone than the apical surface and raise the possibility that apical and basal biliatresone concentration and flow in our system are different.

The extracellular matrix (ECM) is a complex mixture composed of fibrillar collagens as well as additional protein and carbohydrate components. Proteoglycans (PGs) contribute to the heterogeneity of the ECM and play an important role in its structure and function. While the small leucine rich proteoglycans (SLRPs), including decorin and lumican, have been studied extensively as mediators of collagen fibrillogenesis and organization, the function of large matrix PGs in collagen matrices is less well known. In this study, we showed that different matrix PGs have distinct roles in regulating collagen behaviors. We found that versican, a large chondroitin sulfate PG, promotes collagen fibrillogenesis in a turbidity assay and upregulates cell-mediated collagen compaction and reorganization, whereas aggrecan, a structurally-similar large PG, has different and often opposing effects on collagen. Compared to versican, decorin and lumican also have distinct functions in regulating collagen behaviors. The different ways in which matrix PGs interact with collagen have important implications for understanding the role of the ECM in diseases such as fibrosis and cancer, and suggest that matrix PGs are potential therapeutic targets.

Chronic cholestatic liver diseases such as primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) are frequently associated with damage to the barrier function of the biliary epithelium, but barrier function is difficult to study in vivo and has not been recapitulated in vitro. Here we report the development of a bile duct-on-a-chip that phenocopies not only the tubular architecture of the bile duct in three dimensions, but also its barrier functions. We demonstrated that mouse cholangiocytes in the channel of the device became polarized and formed mature tight junctions, and that the permeability of the cholangiocyte monolayer was comparable to that measured ex vivo for the rat bile duct. Permeability decreased significantly when cells formed a compacted monolayer with cell densities comparable to that seen in vivo. This device enabled independent access to the apical and basolateral surfaces of the cholangiocyte channel, allowing proof-of-concept toxicity studies with the biliary toxin biliatresone and the bile acid glycochenodeoxycholic acid. The cholangiocyte basolateral side was more vulnerable than the apical side to treatment with either agent, suggesting a protective adaptation of the apical surface that is normally exposed to bile. Further studies revealed a protective role of the cholangiocyte apical glycocalyx, wherein disruption of the glycocalyx with neuraminidase increased the permeability of the cholangiocyte monolayer after treatment with glycochenodeoxycholic acid. Conclusion: This bile duct-on-a-chip captured essential features of a simplified bile duct in structure and organ-level functions and represents a novel in vitro platform to study the pathophysiology of the bile duct using cholangiocytes from a variety of sources.

This chapter examines European perceptions of and priorities for ASEM as a platform for interaction between Europe and Asia at both the inter-regional level and among the group of countries involved. The chapter critically examines Europe’s objectives for ASEM, including how it seeks to utilize it as a tool to engage with an economically strong region, as well as an instrument to address solve regional and global challenges. Although ASEM was recognized as a strategic requisite at its inception, this chapter assesses whether Europe still sees ASEM through the same lens.

The ASEM6 summit (Helsinki 2006) proposed the creation of a number of issue/interest-based groups of ASEM member countries to lead projects that could eventually involve others. Since then, 20 thematic areas have been outlined under which such “coalitions of the willing” could work. This chapter, however, argues that such an orientation threatens to further loosen an already disbanded association. The chapter argues that creating an ASEM center on urbanization and a center on human security and climate action could add much-needed relevance to a forum struggling to compete in a surfeit of regional fora.

This chapter explores the limited progress made in ASEM economic cooperation since the forum’s inception in 1996. It argues that ASEM’s economic pillar has drastically lost pace as compared to its initial years, even if Europe and Asia are each other’s largest external trading partners. The chapter explains the complexities of the current state of economic affairs between Europe and Asia, the shift in geoeconomic patterns in Asia, and demonstrates why the current level of bilateral trade between European and Asian countries is far from optimum. The final section looks at possible, new directions and shifts in focus that could help ASEM deliver better in its third decade.

Factor VII, the initiator of the extrinsic coagulation cascade, circulates in human plasma mainly in its zymogen form, factor VII and in small amounts in its activated form, factor VIIa. However, the mechanism of initial generation of factor VIIa is not known despite intensive research using currently available model systems. Earlier findings suggested serine proteases factor VII activating protease and hepsin play a role in activating factor VII, however, it has remained controversial. In this paper we estimated the levels of factor VIIa and factor VII for the first time in zebrafish adult population and also reevaluated the role of the above two serine proteases in activating factor VII in vivo using zebrafish as a model system. Knockdown of factor VII activating protease and hepsin was performed followed by assaying for their effect on factor VIIa concentration and extrinsic coagulation as measured by the kinetic prothrombin time. Factor VII activating protease knockdown showed no change in kinetic prothrombin time and no effect on factor VIIa levels while hepsin knockdown increased the kinetic prothrombin time and significantly reduced the factor VIIa plasma levels. Our results thus indicate that hepsin plays a physiologically important role in factor VII activation and hemostasis in zebrafish.

Nearly a decade later, the EU-India relationship in its current form is yet to authenticate a 'strategic partnership' that was announced in 2004.Vast amounts of potential remains untapped while little synergies have been visible at the regional or multilateral level. Much relates to the fact that while the EU and India both acknowledge each other as strategic partners, they don't really recognize each other as such. Trade is critically important but there is more than trade to delivering a strategic partnership. A new, forward looking partnership based on urbanization, development and sustainable growth must be sought.

European Union-Asia relations have been progressively developing over the years. The European Union (EU) today is the leading trade partner of many Asian nations and the spectrum of issues falling under the EU’s bilateral relations with countries in Asia spans far and wide. The EU has been a development partner in Asia for decades and is one of Asia’s largest sources of foreign direct investment (FDI), aid and humanitarian assistance. The EU and Asia interact closely on regional and multilateral issues and within multilateral forums. The EU has even constructed developed deeper relationships with some key Asian countries through strategic partnerships: with China, India, Japan, South Korea and Russia. But potential of overall EU-Asia relations remains far from optimum. The EU has been rather late in recognising the rise of Asia and in gearing itself towards this important development. It has ambitions to play a greater political role in complement to its status as a global trading giant, but efforts fall short. In particular, the EU lacks visibility in Asia commensurate to its actual weight. The ongoing financial and economic crisis has further weakened the EU as an actor in Asia. The changing landscape of EU-Asia relations now depends on two important considerations – a dynamically ascendant and economically integrated Asia, and the US’ renewed focus to Asia. This paper examines the EU’s relations with Asia focussing on the Asian partners of the Asia-Europe Meeting (ASEM).

Background. The beneficial effects of imagined movements on motor learning and performance suggest that motor imagery is functionally close to preparatory and executive motor processes. Objective. The purpose of this study was to examine the cortical processes associated with imagery of movement of the wrist in subjects with severe hemiparesis. Methods. During fMRI, subjects with stroke performed alternating blocks of imagining wrist-tracking movements with the hemiparetic hand, active wrist-tracking movements with the unaffected hand, and resting. Control subjects performed the same tasks using an assigned hand. Cortical activation in the primary motor (M1), primary sensory (S1), supplementary motor area (SMA), and pre-SMA regions was determined through a laterality index of active voxels and signal intensity. Ability to imagine was assessed with an Imagery Rating Scale. Results.All subjects displayed primarily contralateral control during the track condition. Healthy subjects demonstrated contralateral control in all areas during the imagine condition, whereas subjects with stroke displayed primarily contralateral activation in S1 but ipsilateral in M1 and SMA. The percentage change in signal intensity was greater in the ipsilateral hemisphere in subjects with stroke than in the ipsilateral hemisphere in healthy subjects during the imagine condition. Additionally, subjects with self-reported low ability to imagine displayed no difference in activation compared to those with high imagery ability. Conclusions. These findings are consistent with other works demonstrating primarily ipsilateral control of the hemiparetic hand in subjects with functional movement and lay the groundwork for further investigation into the ability of mental imagery to affect functionally relevant cortical control in subjects recovering from stroke.

Morpholino and vivo-morpholino gene knockdown methods have been used to study thrombocyte function in zebrafish. However, a large-scale knockdown of the entire zebrafish genome using these technologies to study thrombocyte function is prohibitively expensive. We have developed an inexpensive gene knockdown method, which uses a hybrid of a control vivo-morpholino and a standard antisense oligonucleotide specific for a gene. This hybrid molecule is able to deliver antisense deoxyoligonucleotides into zebrafish thrombocytes because it piggybacks on a control vivo-morpholino. To validate use of this hybrid molecule in gene knockdowns, we targeted the thrombocyte specific αIIb gene with a hybrid of a control vivo-morpholino and an oligonucleotide antisense to αIIb mRNA. The use of this piggyback technology resulted in degradation of αIIb mRNA and led to thrombocyte functional defect. This piggyback method to knockdown genes is inexpensive since one control vivo-morpholino can be used to target many different genes by making many independent gene-specific oligonucleotide hybrids. Thus, this novel piggyback technology can be utilized for cost-effective large-scale knockdowns of genes to study thrombocyte function in zebrafish.

Platelets play an important role in mammalian hemostasis. Thrombocytes of early vertebrates are functionally equivalent to mammalian platelets. A substantial amount of research has been done to study platelet function in humans as well as in animal models. However, to date only limited functional genomic studies of platelets have been performed but are low throughput and are not cost-effective. Keeping this in mind we introduced zebrafish, a vertebrate genetic model to study platelet function. We characterized zebrafish thrombocytes and established functional assays study not only their hemostatic function but to also their production. We identified a few genes which play a role in their function and production. Since we introduced the zebrafish model for the study of hemostasis and thrombosis, other groups have adapted this model to study genes that are associated with thrombocyte function and a few novel genes have also been identified. Furthermore, transgenic zebrafish with GFP-tagged thrombocytes have been developed which helped to study the production of thrombocytes and their precursors as well as their functional roles not only in hemostasis but also hematopoiesis. This paper integrates the information available on zebrafish thrombocyte function and its formation.

Functional MRI (fMRI) has become one of the most commonly used neuroimaging tools to assess the cortical effects associated with rehabilitation, learning, or disease recovery in subjects with stroke. Despite this, there has been no systematic study of the reliability of the fMR signal in this population. The purpose of this study was to examine the within- and between-session reliability of fMRI in cortical and cerebellar structures in subjects with stroke during a complex, continuous visual motor task performed with the less affected hand. Nine subjects with stroke underwent four testing trials during two sessions separated by three weeks. Subjects performed a drawing task using an MRI compatible joystick while in the MRI. Methods of analysis evaluated included: percent signal intensity change, active voxel count and a voxel by voxel stat value analysis within and between testing sessions. Reliability was determined with Interclass correlation coefficients (ICC) in the following regions of interest: primary motor (M1), primary sensory (S1), premotor cortex (PMC), medial cerebellum (MCB), and lateral cerebellum (LCB). Results indicate that intensity change has superior reliability to the other methods of analysis (Average ICC across brain regions and trials: intensity change: 0.73, voxel count: 0.58, voxel by voxel: 0.67) and that generally with any analysis method, within-session reliability was higher than between-session, as indicated by higher ICC values across brain regions. Overall, when comparing between-session results, moderate to good reliability was obtained with intensity change (ICC: M1: 0.52, S1: 0.80, SMA: 0.78, PMC: 0.94, MCB: 0.86, and LCB: 0.59). These results show good reliability in subjects with stroke when performing a continuous motor task. These findings give confidence for interpreting fMRI test/retest research in subjects with stroke.