H3 histamine receptor-mediated activation of protein kinase calpha inhibits the growth of cholangiocarcinoma in vitro and in vivo

Histamine regulates functions via four receptors (HRH1, HRH2, HRH3, and HRH4). The D-myo-inositol 1,4,5-trisphosphate (IP(3))/Ca(2+)/protein kinase C (PKC)/mitogen-activated protein kinase pathway regulates cholangiocarcinoma growth. We evaluated the role of HRH3 in the regulation of cholangiocarcinoma growth. Expression of HRH3 in intrahepatic and extrahepatic cell lines, normal cholangiocytes, and human tissue arrays was measured. In Mz-ChA-1 cells stimulated with (R)-(alpha)-(-)-methylhistamine dihydrobromide (RAMH), we measured (a) cell growth, (b) IP(3) and cyclic AMP levels, and (c) phosphorylation of PKC and mitogen-activated protein kinase isoforms. Localization of PKC alpha was visualized by immunofluorescence in cell smears and immunoblotting for PKC alpha in cytosol and membrane fractions. Following knockdown of PKC alpha, Mz-ChA-1 cells were stimulated with RAMH before evaluating cell growth and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation. In vivo experiments were done in BALB/c nude mice. Mice were treated with saline or RAMH for 44 days and tumor volume was measured. Tumors were excised and evaluated for proliferation, apoptosis, and expression of PKC alpha, vascular endothelial growth factor (VEGF)-A, VEGF-C, VEGF receptor 2, and VEGF receptor 3. HRH3 expression was found in all cells. RAMH inhibited the growth of cholangiocarcinoma cells. RAMH increased IP(3) levels and PKC alpha phosphorylation and decreased ERK1/2 phosphorylation. RAMH induced a shift in the localization of PKC alpha expression from the cytosolic domain into the membrane region of Mz-ChA-1 cells. Silencing of PKC alpha prevented RAMH inhibition of Mz-ChA-1 cell growth and ablated RAMH effects on ERK1/2 phosphorylation. In vivo, RAMH decreased tumor growth and expression of VEGF and its receptors; PKC alpha expression was increased. RAMH inhibits cholangiocarcinoma growth by PKC alpha-dependent ERK1/2 dephosphorylation. Modulation of PKC alpha by histamine receptors may be important in regulating cholangiocarcinoma growth. (Mol Cancer Res 2009;7(10):1704-13

Meristematic connectome: A cellular coordinator of plant responses to environmental signals?

Mechanical stress in tree roots induces the production of reaction wood (RW) and the formation of new branch roots, both functioning to avoid anchorage failure and limb damage. The vascular cambium (VC) is the factor responsible for the onset of these responses as shown by their occurrence when all primary tissues and the root tips are removed. The data presented confirm that the VC is able to evaluate both the direction and magnitude of the mechanical forces experienced before coordinating the most fitting responses along the root axis whenever and wherever these are necessary. The coordination of these responses requires intense crosstalk between meristematic cells of the VC which may be very distant from the place where the mechanical stress is first detected. Signaling could be facilitated through plasmodesmata between meristematic cells. The mechanism of RW production also seems to be well conserved in the stem and this fact suggests that the VC could behave as a single structure spread along the plant body axis as a means to control the relationship between the plant and its environment. The observation that there are numerous morphological and functional similarities between different meristems and that some important regulatory mechanisms of meristem activity, such as homeostasis, are common to several meristems, supports the hypothesis that not only the VC but all apical, primary and secondary meristems present in the plant body behave as a single interconnected structure. We propose to name this structure \u201cmeristematic connectome\u201d given the possibility that the sequence of meristems from root apex to shoot apex could represent a pluricellular network that facilitates long-distance signaling in the plant body. The possibility that the \u201cmeristematic connectome\u201d could act as a single structure active in adjusting the plant body to its surrounding environment throughout the life of a plant is now proposed

VEGF signaling mediates bladder neuroplasticity and inflammation in response to BCG

<p>Abstract</p> <p>Background</p> <p>This work tests the hypothesis that increased levels of vascular endothelial growth factor (VEGF) observed during bladder inflammation modulates nerve plasticity.</p> <p>Methods</p> <p>Chronic inflammation was induced by intravesical instillations of Bacillus Calmette-Guérin (BCG) into the urinary bladder and the density of nerves expressing the transient receptor potential vanilloid subfamily 1 (TRPV1) or pan-neuronal marker PGP9.5 was used to quantify alterations in peripheral nerve plasticity. Some mice were treated with B20, a VEGF neutralizing antibody to reduce the participation of VEGF. Additional mice were treated systemically with antibodies engineered to specifically block the binding of VEGF to NRP1 (anti-NRP1^B) and NRP2 (NRP2^B), or the binding of semaphorins to NRP1 (anti-NRP1 ^A) to diminish activity of axon guidance molecules such as neuropilins (NRPs) and semaphorins (SEMAs). To confirm that VEGF is capable of inducing inflammation and neuronal plasticity, another group of mice was instilled with recombinant VEGF₁₆₅or VEGF₁₂₁into the urinary bladder.</p> <p>Results</p> <p>The major finding of this work was that chronic BCG instillation resulted in inflammation and an overwhelming increase in both PGP9.5 and TRPV1 immunoreactivity, primarily in the sub-urothelium of the urinary bladder. Treatment of mice with anti-VEGF neutralizing antibody (B20) abolished the effect of BCG on inflammation and nerve density.</p> <p>NRP1^Aand NRP1^Bantibodies, known to reduce BCG-induced inflammation, failed to block BCG-induced increase in nerve fibers. However, the NRP2^Bantibody dramatically potentiated the effects of BCG in increasing PGP9.5-, TRPV1-, substance P (SP)-, and calcitonin gene-related peptide (CGRP)-immunoreactivity (IR). Finally, instillation of VEGF₁₂₁or VEGF₁₆₅into the mouse bladder recapitulated the effects of BCG and resulted in a significant inflammation and increase in nerve density.</p> <p>Conclusions</p> <p>For the first time, evidence is being presented supporting that chronic BCG instillation into the mouse bladder promotes a significant increase in peripheral nerve density that was mimicked by VEGF instillation. Effects of BCG were abolished by pre-treatment with neutralizing VEGF antibody. The present results implicate the VEGF pathway as a key modulator of inflammation and nerve plasticity, introduces a new animal model for investigation of VEGF-induced nerve plasticity, and suggests putative mechanisms underlying this phenomenon.</p

RoSE: Roadmaps Towards Sustainable Energy Futures and Climate Protection: A Synthesis of Results from the Rose Project

EXECUTIVE SUMMARY Exploring energy demand and supply uncertainty: An exploration of uncertainty on drivers of energy demand and supply is indispensable for better understanding the prospects of long-tern climate stabilization. The RoSE study is the first of its kind to systematically explore the impact of economic growth, population and fossil fuel scarcity, in scenarios with and without climate policy, using a model ensemble. A feature of RoSE is the participation of five established integrated assessment modelling teams from three important regions in international climate policy negotiations: the EU, the USA and China. Economic growth: Neither slow nor rapid economic growth solves the climate problem by itself. In the absence of climate policy and if energy intensity improvements continue along historical trends, higher economic activity implies higher energy demand and greenhouse gas emissions. The increase in energy and carbon intensity improvements with higher economic growth is overcompensated by the larger growth in per capita income. Even under slow economic growth assumptions, GDP will rise significantly above today’s level, leading to an increase in greenhouse gas emissions. Fossil fuel availability: Fossil fuel scarcity is insufficient to slow global warming significantly. Low fossil fuel availability leads to levels of greenhouse gas emissions that are higher than those under climate change stabilization. Nevertheless, fossil fuel availability significantly influences the energy mix and the CO2 emissions in scenarios without climate policy. Energy use: There are robust patterns in projections of energy use in the absence of climate policy. Higher economic growth increases the scale of the energy system, which continues to be mostly supplied by fossil fuels. Structural differences in the energy supply mix occur for variations in fossil resource availability, particularly coal and oil supply. Models unanimously show an electrification of energy end use independently of economic growth and fossil resource assumptions. Emissions phase out: Climate stabilization requires a phase out of global greenhouse gas emissions in the long run. For a stringent stabilization target compatible with the 2°C goal (a level of 450 ppm CO2 equivalent in the atmosphere), net emissions have to be nearly phased out by 2100. For a less ambitious, but still stringent stabilization level of 550 ppm CO2e, emissions would need to be more than halved by the end of the century, and decline towards zero in the 22nd century. Energy systems transformation: Climate stabilization implies a fundamental transformation of global energy systems. Climate stabilization requires a transformation to a low carbon energy system in the 21st century with historically unprecedented decarbonization rates. Models tell different stories when and what to reduce, but some robust patterns emerge. On the supply side, coal is rapidly replaced with non-fossil energy sources. On the demand side, models foresee a larger share of electricity and gases coupled with a strong reduction of solids. The structure of the energy transformation is largely unaffected by variations in fossil fuel availability and economic growth. The effect of fossil fuel availability on fossil fuel use is negligible in climate stabilization scenarios. Thus, climate policy effectively limits uncertainty about future fossil fuel use. Carbon prices and mitigation costs: Variations in economic growth and fossil fuel availability can alter carbon prices and mitigation costs substantially. A supply push of fossil energy can be more easily neutralized with a carbon price signal than a demand pull due to higher levels of economic output. Thus, carbon prices vary more strongly with growth projections than with fossil fuel availability. Mitigation cost estimates are sensitive to economic growth and fossil fuel assumptions. Costs increase by approximately 30 to 100% from low to high economic growth, and from low to high fossil fuel availability Weak policies: Current climate policies are insufficient for 2°C stabilization With the currently planned climate policies and pledged emissions reductions the world is not on track towards the 2°C target. If current trends of weak and globally uncoordinated climate policies continue, global mean temperatures are likely to increase by more than 3°C by 2100. Delayed action: Delaying action greatly increases the challenge of keeping warming below 2°C. In case of a further delay in the implementation of comprehensive global emissions reductions the transformation effort needs to be compressed into a shorter period of time. These higher emission reduction rates required in such later-action scenarios imply, inter alia, i) faster decarbonization of the energy system, ii) faster reductions of energy demand, iii) more stranded investments due to pre-maturely retired fossil capacities, and iv) higher transitory economic losses during the phase-in of climate policies. The implications of delaying action until 2030 are considerably more severe than those of a delay until 2020. While the models are able to compute low-stabilization scenarios with a prolonged delay of action, the dramatic increase in mitigation challenges in case of policy delay until 2030 make it seem unlikely that such pathways can be implemented in the real world. China: Climate stabilization implies a fundamental energy transformation for China. Carbon emissions from fossil fuel combustion in China are expected to double from 2005 levels by 2020. Different assumptions on climate policy driven carbon intensity reductions lead to a large range of 6-12 Gt CO2 emissions by 2050, as calculated with an energy system model of the Chinese economy (China-TIMES model). Climate stabilization scenarios from global models show emissions in China below or at the low end of this range in 2050. The emission trajectories differ across models but all peak during 2020-2025 for the 450 ppm CO2e target and 2025-2030 for the 550 ppm CO2e target. This indicates that stringent climate targets would imply ambitious emission reductions in China. Africa: The rates of economic and population growth in Africa have profound implications for energy use and greenhouse gas emissions. Today Africa accounts for a modest 3% of global energy system CO2 emissions. The evolution of Africa’s emissions over the coming century depends critically on future population and income. Absent any climate policy, Africa could become a major emitter in the second half of the 21st century if economic growth in this part of the world is steady. In the shorter term, the extent of energy poverty and improvements in access to modern energy in Africa are also driven by assumptions regarding future population and economic growth. Slower economic growth and larger population growth result in a significantly slower transition to modern energy access and use on the continent. Climate policies have a strong impact on energy resource markets, resource rents and energy security. Climate policies interfere with fossil fuel markets and reallocate rent incomes from providing scarce goods. The global losses of fossil fuel rents are overcompensated by revenues from carbon pricing. The losses of rents from coal are much smaller than those for oil, though coal is the fossil fuel that needs to be reduced the most. Achieving the 2°C target still allows using conventional and unconventional oil reserves. Large part of the coal reserve needs to be left underground. Energy security is significantly improved by climate policy under all assumptions about resource availability and GDP growth. That is due to a reduction of risks associated with energy trade and an increase in the resilience of energy systems through higher diversity. Climate policy also makes total energy supply, the energy mix and energy trade more predictable and possibly easier to manage. Climate policies may also entail certain risks for energy security. In particular, deep penetration of solar energy in the electricity sector or biofuels in the liquid fuels sector may reduce the diversity of these energy systems by the end of the century. Land use: Population, economic growth, and fossil fuel scarcity all have implications for land use. Larger populations require more food, increasing the extent of cropland area. Wealthier populations tend to eat more meat, a landintensive commodity, increasing cropland and pasture cover. Growing, wealthier populations also demand more energy. Fossil fuel scarcity drives increased consumption of bioenergy and land devoted to its production. All three of these effects lead to reductions in forest cover and increases in land-use change CO2 emissions. Investments and innovation: Economic growth and fossil fuel scarcity can both stimulate clean energy innovation and non-fossil-fuel investments. When economies grow faster energy resources are used more efficiently, but fossil fuels would remain the prevalent source of energy. In contrast, the expectation of high energy prices could redirect ample financial resources to R&D programs aimed at developing new energy sources. Although economic growth and fossil fuel prices can create an economic opportunity for more investments in non-fossil energy technologies and clean energy R&D, still they would lag behind the levels observed in stabilization scenarios and would not induce emission reductions compatible with climate stabilization objectives. On average, baseline total R&D investments amount to about 67 billion 2005 US$/yr while they increase to almost twice as much (113 billion 2005 US$/yr) in the 450 ppm CO2e stabilization scenario. The availability of cheap gas resources would increase gas investments, mostly to substitute coal especially in coal-intensive countries. Yet, it would only marginally displace investments in clean energy innovation

NGFS Climate Scenario Database: Technical Documentation V2.2

This document provides technical information on the two datasets behind the NGFS scenarios. It is intended to answer technical questions for those who want to perform analyses on the datasets themselves. It is an update of the Technical Documentation published in June 2020 alongside the first set of NGFS Scenarios. It is therefore aligned with the second set of NGFS Scenarios, released in June 2021

Transfection of IL-10 expression vectors into endothelial cultures attenuates α4β7-dependent lymphocyte adhesion mediated by MAdCAM-1

BACKGROUND: Enhanced expression of MAdCAM-1 (mucosal addressin cell adhesion molecule-1) is associated with the onset and progression of inflammatory bowel disease. The clinical significance of elevated MAdCAM-1 expression is supported by studies showing that immunoneutralization of MAdCAM-1, or its ligands reduce inflammation and mucosal damage in models of colitis. Interleukin-10 (IL-10) is an endogenous anti-inflammatory and immunomodulatory cytokine that has been shown to prevent inflammation and injury in several animal studies, however clinical IL-10 treatment remains insufficient because of difficulties in the route of IL-10 administration and its biological half-life. Here, we examined the ability of introducing an IL-10 expression vector into endothelial cultures to reduce responses to a proinflammatory cytokine, TNF-α METHODS: A human IL-10 expression vector was transfected into high endothelial venular ('HEV') cells (SVEC4-10); we then examined TNF-α induced lymphocyte adhesion to lymphatic endothelial cells and TNF-α induced expression of MAdCAM-1 and compared these responses to control monolayers. RESULTS: Transfection of the IL-10 vector into endothelial cultures significantly reduced TNF-α induced, MAdCAM-1 dependent lymphocyte adhesion (compared to non-transfected cells). IL-10 transfected endothelial cells expressed less than half (46 ± 6.6%) of the MAdCAM-1 induced by TNF-α (set as 100%) in non-transfected (control) cells. CONCLUSION: Our results suggest that gene therapy of the gut microvasculature with IL-10 vectors may be useful in the clinical treatment of IBD

AAV Exploits Subcellular Stress Associated with Inflammation, Endoplasmic Reticulum Expansion, and Misfolded Proteins in Models of Cystic Fibrosis

Barriers to infection act at multiple levels to prevent viruses, bacteria, and parasites from commandeering host cells for their own purposes. An intriguing hypothesis is that if a cell experiences stress, such as that elicited by inflammation, endoplasmic reticulum (ER) expansion, or misfolded proteins, then subcellular barriers will be less effective at preventing viral infection. Here we have used models of cystic fibrosis (CF) to test whether subcellular stress increases susceptibility to adeno-associated virus (AAV) infection. In human airway epithelium cultured at an air/liquid interface, physiological conditions of subcellular stress and ER expansion were mimicked using supernatant from mucopurulent material derived from CF lungs. Using this inflammatory stimulus to recapitulate stress found in diseased airways, we demonstrated that AAV infection was significantly enhanced. Since over 90% of CF cases are associated with a misfolded variant of Cystic Fibrosis Transmembrane Conductance Regulator (ΔF508-CFTR), we then explored whether the presence of misfolded proteins could independently increase susceptibility to AAV infection. In these models, AAV was an order of magnitude more efficient at transducing cells expressing ΔF508-CFTR than in cells expressing wild-type CFTR. Rescue of misfolded ΔF508-CFTR under low temperature conditions restored viral transduction efficiency to that demonstrated in controls, suggesting effects related to protein misfolding were responsible for increasing susceptibility to infection. By testing other CFTR mutants, G551D, D572N, and 1410X, we have shown this phenomenon is common to other misfolded proteins and not related to loss of CFTR activity. The presence of misfolded proteins did not affect cell surface attachment of virus or influence expression levels from promoter transgene cassettes in plasmid transfection studies, indicating exploitation occurs at the level of virion trafficking or processing. Thus, we surmised that factors enlisted to process misfolded proteins such as ΔF508-CFTR in the secretory pathway also act to restrict viral infection. In line with this hypothesis, we found that AAV trafficked to the microtubule organizing center and localized near Golgi/ER transport proteins. Moreover, AAV infection efficiency could be modulated with siRNA-mediated knockdown of proteins involved in processing ΔF508-CFTR or sorting retrograde cargo from the Golgi and ER (calnexin, KDEL-R, β-COP, and PSMB3). In summary, our data support a model where AAV exploits a compromised secretory system and, importantly, underscore the gravity with which a stressed subcellular environment, under internal or external insults, can impact infection efficiency

Regulation of pH During Amelogenesis

During amelogenesis, extracellular matrix proteins interact with growing hydroxyapatite crystals to create one of the most architecturally complex biological tissues. The process of enamel formation is a unique biomineralizing system characterized first by an increase in crystallite length during the secretory phase of amelogenesis, followed by a vast increase in crystallite width and thickness in the later maturation phase when organic complexes are enzymatically removed. Crystal growth is modulated by changes in the pH of the enamel microenvironment that is critical for proper enamel biomineralization. Whereas the genetic bases for most abnormal enamel phenotypes (amelogenesis imperfecta) are generally associated with mutations to enamel matrix specific genes, mutations to genes involved in pH regulation may result in severely affected enamel structure, highlighting the importance of pH regulation for normal enamel development. This review summarizes the intra- and extracellular mechanisms employed by the enamel-forming cells, ameloblasts, to maintain pH homeostasis and, also, discusses the enamel phenotypes associated with disruptions to genes involved in pH regulation