Agarose microgel culture delineates lumenogenesis in naive and primed human pluripotent stem cells.

Human periimplantation development requires the transformation of the naive pluripotent epiblast into a polarized epithelium. Lumenogenesis plays a critical role in this process, as the epiblast undergoes rosette formation and lumen expansion to form the amniotic cavity. Here, we present a high-throughput in vitro model for epiblast morphogenesis. We established a microfluidic workflow to encapsulate human pluripotent stem cells (hPSCs) into monodisperse agarose microgels. Strikingly, hPSCs self-organized into polarized epiblast spheroids that could be maintained in self-renewing and differentiating conditions. Encapsulated primed hPSCs required Rho-associated kinase inhibition, in contrast to naive hPSCs. We applied microgel suspension culture to examine the lumen-forming capacity of hPSCs and reveal an increase in lumenogenesis during the naive-to-primed transition. Finally, we demonstrate the feasibility of co-encapsulating cell types across different lineages and species. Our work provides a foundation for stem cell-based embryo models to interrogate the critical components of human epiblast self-organization and morphogenesis

Pharmacological inhibition of protein tyrosine kinases axl and fyn reduces TNF-α-induced endothelial inflammatory activation in vitro

Major surgery induces systemic inflammation leading to pro-inflammatory activation of endothelial cells. Endothelial inflammation is one of the drivers of postoperative organ damage, including acute kidney injury Tumour Necrosis Factor alpha (TNF-α) is an important component of surgery-induced pro-inflammatory activation of endothelial cells. Kinases, the backbone of signalling cascades, can be targeted by pharmacological inhibition. This is a promising treatment option to interfere with excessive endothelial inflammation. In this study, we identified activated kinases as potential therapeutic targets. These targets were pharmacologically inhibited to reduce TNF-α-induced pro-inflammatory signalling in endothelial cells. Kinome profiling using PamChip arrays identified 64 protein tyrosine kinases and 88 serine-threonine kinases, the activity of which was determined at various timepoints (5–240 min) following stimulation with 10 ng/ml TNF-α in Human umbilical vein endothelial cells in vitro. The PTKs Axl and Fyn were selected based on high kinase activity profiles. Co-localisation experiments with the endothelial-specific protein CD31 showed Axl expression in endothelial cells of glomeruli and Fyn in arterioles and glomeruli of both control and TNF-α-exposed mice. Pharmacological inhibition with Axl inhibitor BMS-777607 and Fyn inhibitor PP2 significantly reduced TNF-α-induced pro-inflammatory activation of E-selectin, VCAM-1, ICAM-1, IL-6 and IL-8 at mRNA and VCAM-1, ICAM-1, and IL-6 at protein level in HUVEC in vitro. Upon pharmacological inhibition with each inhibitor, leukocyte adhesion to HUVEC was also significantly reduced, however to a minor extent. In conclusion, pre-treatment of endothelial cells with kinase inhibitors BMS-777607 and PP2 reduces TNF-α-induced endothelial inflammation in vitro

Spatial and temporal variations in ammonia emissions – a freely accessible model code for Europe

Deriving a parameterisation of ammonia emissions for use in chemistry-transport models (CTMs) is a complex problem as the emission varies locally as a result of local climate and local agricultural management. In current CTMs such factors are generally not taken into account. This paper demonstrates how local climate and local management can be accounted for in CTMs by applying a modular approach for deriving data as input to a dynamic ammonia emission model for Europe. Default data are obtained from information in the RAINS system, and it is demonstrated how this dynamic emission model based on these input data improves the NH<sub>3</sub> calculations in a CTM model when the results are compared with calculations obtained by traditional methods in emission handling. It is also shown how input data can be modified over a specific target region resulting in even further improvement in performance over this domain. The model code and the obtained default values for the modelling experiments are available as supplementary information to this article for use by the modelling community on similar terms as the EMEP CTM model: the GPL licencse v3

Improved modelling of atmospheric ammonia over Denmark using the coupled modelling system DAMOS

A local-scale Gaussian dispersion-deposition model (OML-DEP) has been coupled to a regional chemistry-transport model (DEHM with a resolution of approximately 6 km × 6 km over Denmark) in the Danish Ammonia Modelling System, DAMOS. Thereby, it has been possible to model the distribution of ammonia concentrations and depositions on a spatial resolution down to 400 m × 400 m for selected areas in Denmark. DAMOS has been validated against measured concentrations from the dense measuring network covering Denmark. Here measured data from 21 sites are included and the validation period covers 2–5 years within the period 2005–2009. A standard time series analysis (using statistic parameters like correlation and bias) shows that the coupled model system captures the measured time series better than the regional- scale model alone. However, our study also shows that about 50% of the modelled concentration level at a given location originates from non-local emission sources. The local-scale model covers a domain of 16 km × 16 km, and of the locally released ammonia (NH<sub>3</sub>) within this domain, our simulations at five sites show that 14–27% of the locally (within 16 km × 16 km) emitted NH<sub>3</sub> also deposits locally. These results underline the importance of including both high-resolution local-scale modelling of NH<sub>3</sub> as well as the regional-scale component described by the regional model. The DAMOS system can be used as a tool in environmental management in relation to assessments of total nitrogen load of sensitive nature areas in intense agricultural regions. However, high spatio-temporal resolution in input parameters like NH<sub>3</sub> emissions and land-use data is required

Konsekvenser for emissioner og luftkvalitet af tiltag på transportområdet i Regeringens klima- og luftudspil

Regeringens klima- og luftudspil (”Sammen om en grønnere fremtid”) indeholder en beskrivelse af 38 initiativer rettet mod at reducere udledningen af drivhusgasser og/eller luftforurening. DCE - Nationalt Center for Miljø og Energi under Aarhus Universitet har gennemgået de 38 initiativer med henblik på en vurdering af hvilke af disse initiativer, der har indflydelse på luftforureningen i 2030, samt den forventede størrelse af den reducerende effekt. DCE har kvantificeret effekten af følgende initiativer og benævnt det klimascenariet: (1) Stop for salg af nye benzin- og dieselbiler i 2030 og for nye plug-in hybridbiler fra 2035, (2) Slut med udledning af CO2 og luftforurening fra busser i byerne fra 2030, (3) Benzin og diesel skal ud af taxidriften inden 2030, (4) Gamle brændeovne skrottes ved ejerskifte, og (5) Skrotpræmie til gamle brændeovne. Artiklen fokuserer på de tre virkemidler rettet mod vejtransportsektoren og sammenligner med den eksisterende basisemissionsfremskrivning for vejtrafik i Danmark i 2030, og effektvurderer scenarierne mht. til emission og luftkvalitet.  Den største emissionsbesparelse opnås i Scenarie 1 (1 mio. elbiler), og emissions-reduktionerne for hele vejtrafikken i 2030 bliver på hhv. 13 %, 17 % og 2,1 % for NOx, PM2,5–udstødning og PM2,5–samlet (udstødning og ikke-udstødning). Hvis alle tre scenarier indføres bliver emissionsreduktionerne for NOx og PM2,5 –udstødning og PM2,5 –samlet (udstødning og ikke-udstødning) hhv. 15 %, 19 % og 2,3 %.  Effekten af klimascenariet er vurderet for luftkvaliteten på 98 gader i København i 2030 ved beregninger af ændringer i den regionale baggrund, bybaggrund og gadekoncentrationer. Basisudviklingen fra 2016 til 2030 viser et stort fald i koncentrationerne, og klimascenariet bidrager til et yderligere mindre fald. Effekten af klimascenariet for reduktion af NO2 gadekoncentrationerne er især bestemt af reduktionen i trafikkens emissioner, mens de mindre reduktioner af PM2,5 og PM10 i højere grad er bestemt af reduktioner i emissioner fra brændeovne end fra trafik.&nbsp

Geometric and Electronic Structures of the NiI and Methyl−NiIII Intermediates of Methyl-Coenzyme M Reductase†

ABSTRACT: Methyl-coenzyme M reductase (MCR) catalyzes the terminal step in the formation of biological methane from methyl-coenzyme M (Me-SCoM) and coenzyme B (CoBSH). The active site in MCR contains a Ni-F430 cofactor, which can exist in different oxidation states. The catalytic mechanism of methane formation has remained elusive despite intense spectroscopic and theoretical investigations. On the basis of spectroscopic and crystallographic data, the first step of the mechanism is proposed to involve a nucleophilic attack of the NiI active state (MCRred1) on Me-SCoM to form a NiIII-methyl intermediate, while computational studies indicate that the first step involves the attack of NiI on the sulfur of Me-SCoM, forming a CH3 radical and a NiII-thiolate species. In this study, a combination of Ni K-edge X-ray absorption spectroscopic (XAS) studies and density functional theory (DFT) calculations have been performed on the NiI (MCRred1), NiII (MCRred1-silent), and NiIII-methyl (MCRMe) states of MCR to elucidate the geometric and electronic structures of the different redox states. Ni K-edge EXAFS data are used to reveal a five-coordinate active site with an open upper axial coordination site in MCRred1. Ni K-pre-edge and EXAFS data and time-dependent DFT calculations unambiguously demonstrate the presence of a long Ni-C bond (∼2.04 Å) in the NiIII-methyl state of MCR. The formation and stability of this species support mechanism I, and the Ni-C bond length suggests a homolytic cleavage of the NiIII-methyl bon

Role of nickel in high rate methanol degradation in anaerobic granular sludge bioreactors

The effect of nickel deprivation from the influent of a mesophilic (30°C) methanol fed upflow anaerobic sludge bed (UASB) reactor was investigated by coupling the reactor performance to the evolution of the Methanosarcina population of the bioreactor sludge. The reactor was operated at pH 7.0 and an organic loading rate (OLR) of 5–15 g COD l−1 day−1 for 191 days. A clear limitation of the specific methanogenic activity (SMA) on methanol due to the absence of nickel was observed after 129 days of bioreactor operation: the SMA of the sludge in medium with the complete trace metal solution except nickel amounted to 1.164 (±0.167) g CH4-COD g VSS−1 day−1 compared to 2.027 (±0.111) g CH4-COD g VSS−1 day−1 in a medium with the complete (including nickel) trace metal solution. The methanol removal efficiency during these 129 days was 99%, no volatile fatty acid (VFA) accumulation was observed and the size of the Methanosarcina population increased compared to the seed sludge. Continuation of the UASB reactor operation with the nickel limited sludge lead to incomplete methanol removal, and thus methanol accumulation in the reactor effluent from day 142 onwards. This methanol accumulation subsequently induced an increase of the acetogenic activity in the UASB reactor on day 160. On day 165, 77% of the methanol fed to the system was converted to acetate and the Methanosarcina population size had substantially decreased. Inclusion of 0.5 μM Ni (dosed as NiCl2) to the influent from day 165 onwards lead to the recovery of the methanol removal efficiency to 99% without VFA accumulation within 2 days of bioreactor operation