Activation of the cGMP/PKG pathway inhibits electrical activity in rabbit urethral interstitial cells of Cajal by reducing the spatial spread of Ca2+ waves.

In the present study we used a combination of patch clamping and fast confocal Ca2+ imaging to examine the effects of activators of the nitric oxide (NO)/cGMP pathway on pacemaker activity in freshly dispersed ICC from the rabbit urethra, using the amphotericin B perforated patch configuration of the patch-clamp technique. The nitric oxide donor, DEA-NO, the soluble guanylyl cyclase activator YC-1 and the membrane-permeant analogue of cGMP, 8-Br-cGMP inhibited spontaneous transient depolarizations (STDs) and spontaneous transient inward currents (STICs) recorded under current-clamp and voltage-clamp conditions, respectively. Caffeine-evoked Cl- currents were unaltered in the presence of SP-8-Br-PET-cGMPs, suggesting that activation of the cGMP/PKG pathway does not block Cl- channels directly or interfere with Ca2+ release via ryanodine receptors (RyR). However, noradrenaline-evoked Cl- currents were attenuated by SP-8-Br-PET-cGMPs, suggesting that activation of cGMP-dependent protein kinase (PKG) may modulate release of Ca2+ via IP3 receptors (IP3R). When urethral interstitial cells (ICC) were loaded with Fluo4-AM (2 microm), and viewed with a confocal microscope, they fired regular propagating Ca2+ waves, which originated in one or more regions of the cell. Application of DEA-NO or other activators of the cGMP/PKG pathway did not significantly affect the oscillation frequency of these cells, but did significantly reduce their spatial spread. These effects were mimicked by the IP3R blocker, 2-APB (100 microm). These data suggest that NO donors and activators of the cGMP pathway inhibit electrical activity of urethral ICC by reducing the spatial spread of Ca2+ waves, rather than decreasing wave frequency.

Functional expression of KCNQ (Kv7) channels in guinea-pig bladder smooth muscle and their contribution to spontaneous activity

Background and Purpose: The aim of the study was to determine whether KCNQ channels are functionally expressed in bladder smooth muscle cells (SMC) and to investigate their physiological significance in bladder contractility. 

Experimental Approach: KCNQ channels were examined at the genetic, protein, cellular and tissue level in guinea pig bladder smooth muscle using RT-PCR, immunofluorescence, patch-clamp electrophysiology, calcium imaging, detrusor strip myography, and a panel of KCNQ activators and inhibitors. 

Key Results: KCNQ subtypes 1-5 are expressed in bladder detrusor smooth muscle. Detrusor strips typically displayed TTX-insensitive myogenic spontaneous contractions that were increased in amplitude by the KCNQ channel inhibitors XE991, linopirdine or chromanol 293B. Contractility was inhibited by the KCNQ channel activators flupirtine or meclofenamic acid (MFA). The frequency of Ca2+-oscillations in SMC contained within bladder tissue sheets was increased by XE991. Outward currents in dispersed bladder SMC, recorded under conditions where BK and KATP currents were minimal, were significantly reduced by XE991, linopirdine, or chromanol, and enhanced by flupirtine or MFA. XE991 depolarized the cell membrane and could evoke transient depolarizations in quiescent cells. Flupirtine (20M) hyperpolarized the cell membrane with a simultaneous cessation of any spontaneous electrical activity. 

Conclusions and Implications: These novel findings reveal the role of KCNQ currents in the regulation of the resting membrane potential of detrusor SMC and their important physiological function in the control of spontaneous contractility in the guinea pig bladder.

Hydration and properties of sodium sulfate activated slag

Interest in alkali-activated slag as a construction material is increasing, primarily due to its environmentally friendly nature. Although strong alkaline activators, such as sodium hydroxide and sodium silicate solution, are preferred for high strength, none of them exists naturally and their manufacturing process is quite energy intensive. Whilst sodium sulfate (NaSO ) can be obtained from natural resources, the early strength of NaSO activated slag is usually low. In this paper, the effects of slag fineness and NaSO dosage on strength, pH, hydration and microstructure were investigated and compared with those of a pure Portland cement (PC). Test results indicated that increasing the slag fineness is a more effective approach than increasing NaSO dosage for increasing both the early and long-term strength of NaSO activated slags. In addition, increasing the slag fineness can also increase the strength without increasing the pH of the hardened matrix, which is beneficial for immobilizing certain types of nuclear waste containing reactive metals and resins.© 2012 Elsevier Ltd. All rights reserved.

Activation of the ACE2/Angiotensin-(1-7)/Mas Receptor Axis Enhances the Reparative Function of Dysfunctional Diabetic Endothelial Progenitors

We tested the hypothesis that activation of the protective arm of the renin angiotensin system, the angiotensin-converting enzyme 2 (ACE2)/angiotensin-(1-7) [Ang-(1-7)]/Mas receptor axis, corrects the vasoreparative dysfunction typically seen in the CD34(+) cells isolated from diabetic individuals. Peripheral blood CD34(+) cells from patients with diabetes were compared with those of nondiabetic controls. Ang-(1-7) restored impaired migration and nitric oxide bioavailability/cGMP in response to stromal cell-derived factor and resulted in a decrease in NADPH oxidase activity. The survival and proliferation of CD34(+) cells from diabetic individuals were enhanced by Ang-(1-7) in a Mas/phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner. ACE2 expression was lower, and ACE2 activators xanthenone and diminazine aceturate were less effective in inducing the migration in cells from patients with diabetes compared with controls. Ang-(1-7) overexpression by lentiviral gene modification restored both the in vitro vasoreparative functions of diabetic cells and the in vivo homing efficiency to areas of ischemia. A cohort of patients who remained free of microvascular complications despite having a history of longstanding inadequate glycemic control had higher expression of ACE2/Mas mRNA than patients with diabetes with microvascular complications matched for age, sex, and glycemic control. Thus, ACE2/Ang-(1-7)\Mas pathway activation corrects existing diabetes-induced CD34(+) cell dysfunction and also confers protection from development of this dysfunction.

Alkali Activated Fuel Ash and Slag Mixes:Optimization Study from Mortars to Concrete Building Blocks

Alkali activated binders, based on ash and slag, also known as geopolymers, can play a key role in reducing the carbon footprint of the construction sector by replacing ordinary Portland cement in some concretes. Since 1970s, research effort has been ongoing in many research institutions. In this study, pulverized fuel ash (PFA) from a UK power plant, ground granulated blast furnace slag (GGBS) and combinations of the two have been investigated as geopolymer binders for concrete applications. Activators used were sodium hydroxide and sodium silicate solutions. Mortars with sand/binder ratio of 2.75 with several PFA and GGBS combinations have been mixed and tested. The optimization of alkali dosage (defined as the Na2O/binder mass ratio) and modulus (defined as the Na2O/SiO2 mass ratio) resulted in strengths in excess of 70 MPa for tested mortars. Setting time and workability have been considered for the identification of the best combination of PFA/GGBS and alkali activator dosage for different precast concrete products. Geopolymer concrete building blocks have been replicated in laboratory and a real scale factory trial has been successfully carried out. Ongoing microstructural characterization is aiming to identify reaction products arising from PFA/GGBS combinations.

Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation

Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.

Domestic fridge-freezer load aggregation to support ancillary services

Grid operators and electricity retailers in Ireland manage peak demand, power system balancing and grid congestion by offering relevant incentives to consumers to reduce or shift their load. The need for active consumers in the home using smart appliances has never been greater, due to increased variable renewable generation and grid constraints. In this paper an aggregated model of a single compressor fridge-freezer population is developed. A price control strategy is examined to quantify and value demand response savings during a representative winter and summer week for Ireland in 2020. The results show an average reduction in fridge-freezer operating cost of 8.2% during winter and significantly lower during summer in Ireland. A peak reduction of at least 68% of the average winter refrigeration load is achieved consistently during the week analysed using a staggering control mode. An analysis of the current ancillary service payments confirms that these are insufficient to ensure widespread uptake by the small consumer, and new mechanisms need to be developed to make becoming an active consumer attractive. Demand response is proposed as a new ancillary service called ramping capability, as the need for this service will increase with more renewable energy penetration on the power system.

Selection and characterisation of geological materials for use as geopolymer precursors

Geopolymer binders are generally formed by reacting powdered aluminosilicate precursors with alkali silicate activators. Most research to date has concentrated on using either pulverised fuel ash or high purity dehydroxylated kaolin (metakaolin) in association with ground granulated blast furnace slag as the main precursor material. However, recently, attention has turned to alternative calcined clays that are abundant throughout the globe and have lower kaolinite contents than commercially available metakaolins. Due to the lack of clear and simple screening protocols enabling assessment of such geological resources for use as precursors in geopolymer systems, the present paper presents results from experimental work that was carried out to develop a functional binder using materials containing kaolinite taken from the Interbasaltic Formation of Northern Ireland. The influence of mineralogy has been examined, and a screening process, using three Interbasaltic materials as examples, that will assist in the rapid selection of suitable geopolymeric precursors from such materials is outlined.

Genome wide interactions of wild-type and activator bypass forms of σ54

Enhancer-dependent transcription involving the promoter specificity factor σ54 is widely distributed amongst bacteria and commonly associated with cell envelope function. For transcription initiation, σ54-RNA polymerase yields open promoter complexes through its remodelling by cognate AAA+ ATPase activators. Since activators can be bypassed in vitro, bypass transcription in vivo could be a source of emergent gene expression along evolutionary pathways yielding new control networks and transcription patterns. At a single test promoter in vivo bypass transcription was not observed. We now use genome-wide transcription profiling, genome-wide mutagenesis and gene over-expression strategies in Escherichia coli, to (i) scope the range of bypass transcription in vivo and (ii) identify genes which might alter bypass transcription in vivo. We find little evidence for pervasive bypass transcription in vivo with only a small subset of σ54 promoters functioning without activators. Results also suggest no one gene limits bypass transcription in vivo, arguing bypass transcription is strongly kept in check. Promoter sequences subject to repression by σ54 were evident, indicating loss of rpoN (encoding σ54) rather than creating rpoN bypass alleles would be one evolutionary route for new gene expression patterns. Finally, cold-shock promoters showed unusual σ54-dependence in vivo not readily correlated with conventional σ54 binding-sites.

In vitro and in vivo methodologies for studying the Sigma 54-dependent transcription.

Here we describe approaches and methods to assaying in vitro the major variant bacterial sigma factor, Sigma 54 (σ54), in a purified system. We include the complete transcription system, binding interactions between σ54 and its activators, as well as the self-assembly and the critical ATPase activity of the cognate activators which serve to remodel the closed promoter complexes. We also present in vivo methodologies that are used to study the impact of physiological processes, metabolic states, global signalling networks, and cellular architecture on the control of σ54-dependent gene expression.

Alkali activated fuel ash and slag mixes:optimization study from paste to concrete building blocks

Alkali activated binders, based on ash and slag, also known as geopolymers, can play a key role in reducing the carbon footprint of the construction sector by replacing ordinary Portland cement in some concretes. Since 1970s, research effort has been ongoing in many research institutions. In this study, pulverized fuel ash (pfa) from a UK power plant, ground granulated blast furnace slag (ggbs) and combinations of the two have been investigated as geopolymer binders for concrete applications. Activators used were sodium hydroxide and sodium silicate solutions. Mortars with sand/binder ratio of 2.75 with several pfa and ggbs combinations have been mixed and tested. The optimization of alkali dosage (defined as the Na2O/binder mass ratio) and modulus (defined as the Na2O/SiO2 mass ratio) resulted in strengths in excess of 70 MPa for tested mortars. Setting time and workability have been considered for the identification of the best combination of pfa/ggbs and alkali activator dosage for different precast concrete products. Geopolymer concrete building blocks have been replicated in laboratory and a real scale factory trial has been successfully carried out. Ongoing microstructural characterization is aiming to identify reaction products arising from pfa/ggbs combinations.

Molecular subtyping of primary prostate cancer reveals specific and shared target genes of different ETS rearrangements

This work aimed to evaluate whether ETS transcription factors frequently involved in rearrangements in prostate carcinomas (PCa), namely ERG and ETV1, regulate specific or shared target genes. We performed differential expression analysis on nine normal prostate tissues and 50 PCa enriched for different ETS rearrangements using exon-level expression microarrays, followed by in vitro validation using cell line models. We found specific deregulation of 57 genes in ERG-positive PCa and 15 genes in ETV1-positive PCa, whereas deregulation of 27 genes was shared in both tumor subtypes. We further showed that the expression of seven tumor-associated ERG target genes (PLA1A, CACNA1D, ATP8A2, HLA-DMB, PDE3B, TDRD1, and TMBIM1) and two tumor-associated ETV1 target genes (FKBP10 and GLYATL2) was significantly affected by specific ETS silencing in VCaP and LNCaP cell line models, respectively, whereas the expression of three candidate ERG and ETV1 shared targets (GRPR, KCNH8, and TMEM45B) was significantly affected by silencing of either ETS. Interestingly, we demonstrate that the expression of TDRD1, the topmost overexpressed gene of our list of ERG-specific candidate targets, is inversely correlated with the methylation levels of a CpG island found at -66 bp of the transcription start site in PCa and that TDRD1 expression is regulated by direct binding of ERG to the CpG island in VCaP cells. We conclude that ETS transcription factors regulate specific and shared target genes and that TDRD1, FKBP10, and GRPR are promising therapeutic targets and can serve as diagnostic markers for molecular subtypes of PCa harboring specific fusion gene rearrangements.

PIAS1 is increased in human prostate cancer and enhances proliferation through inhibition of p21

Prostate cancer development and progression are associated with alterations in expression and function of elements of cytokine networks, some of which can activate multiple signaling pathways. Protein inhibitor of activated signal transducers and activators of transcription (PIAS)1, a regulator of cytokine signaling, may be implicated in the modulation of cellular events during carcinogenesis. This study was designed to investigate the functional significance of PIAS1 in models of human prostate cancer. We demonstrate for the first time that PIAS1 protein expression is significantly higher in malignant areas of clinical prostate cancer specimens than in normal tissues, thus suggesting a growth-promoting role for PIAS1. Expression of PIAS1 was observed in the majority of tested prostate cancer cell lines. In addition, we investigated the mechanism by which PIAS1 might promote prostate cancer and found that down-regulation of PIAS1 leads to decreased proliferation and colony formation ability of prostate cancer cell lines. This decrease correlates with cell cycle arrest in the G0/G1 phase, which is mediated by increased expression of p21(CIP1/WAF1). Furthermore, PIAS1 overexpression positively influences cell cycle progression and thereby stimulates proliferation, which can be mechanistically explained by a decrease in the levels of cellular p21. Taken together, our data reveal an important new role for PIAS1 in the regulation of cell proliferation in prostate cancer.

An aggregated fridge-freezer peak shaving and valley filling control strategy for enhanced grid operations

The need for fast response demand side participation (DSP) has never been greater due to increased wind power penetration. White domestic goods suppliers are currently developing a `smart' chip for a range of domestic appliances (e.g. refrigeration units, tumble dryers and storage heaters) to support the home as a DSP unit in future power systems. This paper presents an aggregated population-based model of a single compressor fridge-freezer. Two scenarios (i.e. energy efficiency class and size) for valley filling and peak shaving are examined to quantify and value DSP savings in 2020. The analysis shows potential peak reductions of 40 MW to 55 MW are achievable in the Single wholesale Electricity Market of Ireland (i.e. the test system), and valley demand increases of up to 30 MW. The study also shows the importance of the control strategy start time and the staggering of the devices to obtain the desired filling or shaving effect.

Protease activated receptor 2 expression and functionality in caries

Introduction: Protease activated receptors (PARs) are G-protein-coupled transmembrane receptors that are expressed on many cell types and implicated in various inflammatory processes in vivo. The induction of PAR2 as a result of the inflammatory response associated with dental caries remains to be determined. Objectives: The aim was to localise the expression of PAR2 in human dental pulp from carious teeth and to confirm receptor functionality using an in vitro assay. Methods: Dental pulp sections from decalcified carious teeth were examined by immunocytochemsitry. Membrane preparations from cultured pulp fibroblasts were subject to SDS-PAGE and immunoblotting to confirm fibroblast-associated immunoreactivity. The functionality of PAR2 on dental pulp fibroblasts was studied using calcium imaging in the presence of several potential activators including a PAR2 agonist (PAR2-AP), trypsin and pulpal enzymes from a carious tooth. Results: Immunocytochemistry revealed intense PAR2 immunoreactivity on pulpal fibroblasts subjacent to carious lesions but not in surrounding regions of the dental pulp. Pulp specimens from a dental injury model showed no expression of PAR2, suggesting its expression was related to cellular changes associated with ongoing caries. The localisation of PAR2 staining to pulpal fibroblasts in carious teeth was confirmed by Western blotting which revealed PAR2 immunoreactive bands in membrane fractions prepared from pulp fibroblasts. In functional studies, challenge of cultured pupal fibroblasts with PAR2-AP, trypsin and an extract of proteolytic enzymes from a carious dental pulp, showed specific activation of PAR2. Conclusions: This work demonstrates that PAR2 is functional and inducible in human dental pulp fibroblasts in response to caries and that endogenous pulpal enzymes can activate PAR2.