Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration

Satellite cells represent the stem cell population of adult skeletal muscle. The molecular mechanisms that control the proliferation of satellite cells are not well understood. In this study, we show that in response to injury, myofibres activate Wnt ligand transcription and activate a reporter cell line that is sensitive to the canonical Wnt-signalling pathway. Activated satellite cells on isolated cultured myofibres show robust expression of activated-β-catenin (Act-β-Cat), a key downstream transcriptional coactivator of canonical Wnt signalling. We provide evidence that the Wnt family of secreted glycoproteins act on satellite cells in a ligand-specific manner. Overexpression of Wnt1, Wnt3a or Wnt5a protein causes a dramatic increase in satellite-cell proliferation. By contrast, exposure of satellite cells to Wnt4 or Wnt6 diminishes this process. Moreover, we show that the prolonged satellite-cell quiescence induced by inhibitory Wnt is reversible and exposing inhibited satellite cells to stimulatory Wnt signalling restores their proliferation rate. Stimulatory Wnt proteins induce premature satellite cell BrdU incorporation as well as nuclear translocation of Act-β-Cat. Finally, we provide evidence that the Act-β-Cat translocation observed in single fibres during in vitro culture also occurs in cases of acute and chronic skeletal muscle regeneration in rodents and humans. We propose that Wnt proteins may be key factors that regulate the rate of satellite-cell proliferation on adult muscle fibres during the wound-healing response.

Modulation of endothelial cell KCa3.1 Channels during endothelium-derived hyperpolarizing factor signaling in mesenteric resistance arteries

Arterial hyperpolarization to acetylcholine (ACh) reflects coactivation of KCa3.1 (IKCa) channels and KCa2.3 (SKCa) channels in the endothelium that transfers through myoendothelial gap junctions and diffusible factor(s) to affect smooth muscle relaxation (endothelium-derived hyperpolarizing factor [EDHF] response). However, ACh can differentially activate KCa3.1 and KCa2.3 channels, and we investigated the mechanisms responsible in rat mesenteric arteries. KCa3.1 channel input to EDHF hyperpolarization was enhanced by reducing external [Ca2+]o but blocked either with forskolin to activate protein kinase A or by limiting smooth muscle [Ca2+]i increases stimulated by phenylephrine depolarization. Imaging [Ca2+]i within the endothelial cell projections forming myoendothelial gap junctions revealed increases in cytoplasmic [Ca2+]i during endothelial stimulation with ACh that were unaffected by simultaneous increases in muscle [Ca2+]i evoked by phenylephrine. If gap junctions were uncoupled, KCa3.1 channels became the predominant input to EDHF hyperpolarization, and relaxation was inhibited with ouabain, implicating a crucial link through Na+/K+-ATPase. There was no evidence for an equivalent link through KCa2.3 channels nor between these channels and the putative EDHF pathway involving natriuretic peptide receptor-C. Reconstruction of confocal z-stack images from pressurized arteries revealed KCa2.3 immunostain at endothelial cell borders, including endothelial cell projections, whereas KCa3.1 channels and Na+/K+-ATPase {alpha}2/{alpha}3 subunits were highly concentrated in endothelial cell projections and adjacent to myoendothelial gap junctions. Thus, extracellular [Ca2+]o appears to modify KCa3.1 channel activity through a protein kinase A-dependent mechanism independent of changes in endothelial [Ca2+]i. The resulting hyperpolarization links to arterial relaxation largely through Na+/K+-ATPase, possibly reflecting K+ acting as an EDHF. In contrast, KCa2.3 hyperpolarization appears mainly to affect relaxation through myoendothelial gap junctions. Overall, these data suggest that K+ and myoendothelial coupling evoke EDHF-mediated relaxation through distinct, definable pathways.

Regulation of platelet biology by platelet endothelial cell adhesion molecule-1.

Platelet endothelial cell adhesion molecule-1 (PECAM-1), an immunoreceptor tyrosine-based inhibitory motif containing receptor, plays diverse and apparently contradictory roles in regulating the response of platelets to stimuli; inhibiting platelet response to immunoreceptor tyrosine-based activation motif and G protein-coupled receptor signalling following stimulation with collagen, adenosine diphosphate, and thrombin, as well as enhancing integrin outside-in signalling. These dual, and opposing, roles suggest an important and complex role for PECAM-1 in orchestrating platelet response to vascular damage. Indeed, during thrombus formation, the influence of PECAM-1 on the multiple signalling pathways combines leading to a relatively large inhibitory effect on thrombus formation.

Genetic predisposition to type 2 diabetes is associated with impaired insulin secretion but does not modify insulin resistance or secretion in response to an intervention to lower dietary saturated fat

Genome-wide association studies have identified SNPs reproducibly associated with type 2 diabetes (T2D). We examined the effect of genetic predisposition to T2D on insulin sensitivity and secretion using detailed phenotyping in overweight individuals with no diagnosis of T2D. Furthermore, we investigated whether this genetic predisposition modifies the responses in beta-cell function and insulin sensitivity to a 24-week dietary intervention. We genotyped 25 T2D-associated SNPs in 377 white participants from the RISCK study. Participants underwent an IVGTT prior to and following a dietary intervention that aimed to lower saturated fat intake by replacement with monounsaturated fat or carbohydrate. We composed a genetic predisposition score (T2D-GPS) by summing the T2D risk-increasing alleles of the 25 SNPs and tested for association with insulin secretion and sensitivity at baseline, and with the change in response to the dietary intervention. At baseline, a higher T2D-GPS was associated with lower acute insulin secretion (AIRg 4% lower/risk allele, P = 0.006) and lower insulin secretion for a given level of insulin sensitivity, assessed by the disposition index (DI 5% lower/risk allele, P = 0.002), but not with insulin sensitivity (Si). T2D-GPS did not modify changes in insulin secretion, insulin sensitivity or the disposition index in response to the dietary interventions to lower saturated fat. Participants genetically predisposed to T2D have an impaired ability to compensate for peripheral insulin resistance with insulin secretion at baseline, but this does not modify the response to a reduction in dietary saturated fat through iso-energetic replacement with carbohydrate or monounsaturated fat.

Development of an ex vivo human skin model for intradermal vaccination : tissue viability and Langerhans cell behaviour

The presence of resident Langerhans cells (LCs) in the epidermis makes the skin an attractive target for DNA vaccination. However, reliable animal models for cutaneous vaccination studies are limited. We demonstrate an ex vivo human skin model for cutaneous DNA vaccination which can potentially bridge the gap between pre-clinical in vivo animal models and clinical studies. Cutaneous transgene expression was utilised to demonstrate epidermal tissue viability in culture. LC response to the culture environment was monitored by immunohistochemistry. Full-thickness and split-thickness skin remained genetically viable in culture for at least 72 h in both phosphate-buffered saline (PBS) and full organ culture medium (OCM). The epidermis of explants cultured in OCM remained morphologically intact throughout the culture duration. LCs in full-thickness skin exhibited a delayed response (reduction in cell number and increase in cell size) to the culture conditions compared with split-thickness skin, whose response was immediate. In conclusion, excised human skin can be cultured for a minimum of 72 h for analysis of gene expression and immune cell activation. However, the use of split-thickness skin for vaccine formulation studies may not be appropriate because of the nature of the activation. Full-thickness skin explants are a more suitable model to assess cutaneous vaccination ex vivo.

Intracellular accumulation of high levels of gamma-aminobutyrate by Listeria monocytogenes 10403S in response to low pH: uncoupling of gamma-aminobutyrate synthesis from efflux in a chemically defined medium

It is well established that the glutamate decarboxylase (GAD) system is central to the survival of Listeria monocytogenes at low pH, both in acidic foods and within the mammalian stomach. The accepted model proposes that under acidic conditions extracellular glutamate is transported into the cell in exchange for an intracellular gamma-aminobutyrate (GABA(i)). The glutamate is then decarboxylated to GABA(i), a reaction that consumes a proton, thereby helping to prevent acidification of the cytoplasm. In this study, we show that glutamate supplementation had no influence on either growth rate at pH 5.0 or survival at pH 2.5 when L. monocytogenes 10403S was grown in a chemically defined medium (DM). In response to acidification, cells grown in DM failed to efflux GABA, even when glutamate was added to the medium. In contrast, in brain heart infusion (BHI), the same strain produced significant extracellular GABA (GABA(e)) in response to acidification. In addition, high levels of GABA(i) (>80 mM) were found in the cytoplasm in response to low pH in both growth media. Medium-swap and medium-mixing experiments revealed that the GABA efflux apparatus was nonfunctional in DM, even when glutamate was present. It was also found that the GadT2D2 antiporter/decarboxylase system was transcribed poorly in DM-grown cultures while overexpression of gadD1T1 and gadD3 occurred in response to pH 3.5. Interestingly, BHI-grown cells did not respond with upregulation of any of the GAD system genes when challenged at pH 3.5. The accumulation of GABA(i) in cells grown in DM in the absence of extracellular glutamate indicates that intracellular glutamate is the source of the GABA(i). These results demonstrate that GABA production can be uncoupled from GABA efflux, a finding that alters the way we should view the operation of bacterial GAD systems.

The Aqua-Planet Experiment (APE): Response to changed meridional SST profile

This paper explores the sensitivity of Atmospheric General Circulation Model (AGCM) simulations to changes in the meridional distribution of sea surface temperature (SST). The simulations are for an aqua-planet, a water covered Earth with no land, orography or sea-ice and with specified zonally symmetric SST. Simulations from 14 AGCMs developed for Numerical Weather Prediction and climate applications are compared. Four experiments are performed to study the sensitivity to the meridional SST profile. These profiles range from one in which the SST gradient continues to the equator to one which is flat approaching the equator, all with the same maximum SST at the equator. The zonal mean circulation of all models shows strong sensitivity to latitudinal distribution of SST. The Hadley circulation weakens and shifts poleward as the SST profile flattens in the tropics. One question of interest is the formation of a double versus a single ITCZ. There is a large variation between models of the strength of the ITCZ and where in the SST experiment sequence they transition from a single to double ITCZ. The SST profiles are defined such that as the equatorial SST gradient flattens, the maximum gradient increases and moves poleward. This leads to a weakening of the mid-latitude jet accompanied by a poleward shift of the jet core. Also considered are tropical wave activity and tropical precipitation frequency distributions. The details of each vary greatly between models, both with a given SST and in the response to the change in SST. One additional experiment is included to examine the sensitivity to an off-equatorial SST maximum. The upward branch of the Hadley circulation follows the SST maximum off the equator. The models that form a single precipitation maximum when the maximum SST is on the equator shift the precipitation maximum off equator and keep it centered over the SST maximum. Those that form a double with minimum on the equatorial maximum SST shift the double structure off the equator, keeping the minimum over the maximum SST. In both situations only modest changes appear in the shifted profile of zonal average precipitation. When the upward branch of the Hadley circulation moves into the hemisphere with SST maximum, the zonal average zonal, meridional and vertical winds all indicate that the Hadley cell in the other hemisphere dominates.

Platelet endothelial cell adhesion molecule-1 signaling inhibits the activation of human platelets

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a 130-kd transmembrane glycoprotein and a member of the growing family of receptors with immunoreceptor tyrosine-based inhibitory motifs (ITIMs). PECAM-1 is expressed on platelets, certain T cells, monocytes, neutrophils, and vascular endothelial cells and is involved in a range of cellular processes, though the role of PECAM-1 in platelets is unclear. Cross-linking of PECAM-1 results in phosphorylation of the ITIM allowing the recruitment of signaling proteins that bind by way of Src-homology domain 2 interactions. Proteins that have been implicated in the negative regulation of cellular activation by ITIM-bearing receptors include the tyrosine phosphatases SHP-1 and SHP-2. Tyrosine phosphorylation of immunoreceptor tyrosine-based activatory motif (ITAM)-bearing receptors such as the collagen receptor GPVI-Fc receptor gamma-chain complex on platelets leads to activation. Increasing evidence suggests that ITIM- and ITAM-containing receptors may act antagonistically when expressed on the same cell. In this study it is demonstrated that cross-linking PECAM-1 inhibits the aggregation and secretion of platelets in response to collagen and the GPVI-selective agonist convulxin. In these experiments thrombin-mediated platelet aggregation and secretion were also reduced, albeit to a lesser degree than for collagen, suggesting that PECAM-1 function may not be restricted to the inhibition of ITAM-containing receptor pathways. PECAM-1 activation also inhibited platelet protein tyrosine phosphorylation stimulated by convulxin and thrombin; this was accompanied by inhibition of the mobilization of calcium from intracellular stores. These data suggest that PECAM-1 may play a role in the regulation of platelet function in vivo.

Ethnic differences in beta-cell function, dietary intake and expression of the metabolic syndrome among UK adults of south Asian, black African-Caribbean and white-European origin at high risk of metabolic syndrome

A cross-sectional analysis of ethnic differences in dietary intake, insulin sensitivity and beta-cell function, using the intravenous glucose tolerance test (IVGTT), was conducted on 497 healthy adult participants of the ‘Reading, Imperial, Surrey, Cambridge, and Kings’ (RISCK) study. Insulin sensitivity (Si) was significantly lower in African-Caribbean (AC) and South Asian (SA) participants [IVGTT-Si; AC: 2.13 vs SA: 2.25 vs white-European (WE): 2.84 (×10−4 mL µU min)2, p < 0.001]. AC participants had a higher prevalence of anti-hypertensive therapy (AC: 19.7% vs SA: 7.5%), the most cardioprotective lipid profile [total:high-density lipoprotein (HDL); AC: 3.52 vs SA: 4.08 vs WE: 3.83, p = 0.03] and more pronounced hyperinsulinaemia [IVGTT–acute insulin response (AIR)] [AC: 575 vs SA: 428 vs WE: 344 mL/µU/min)2, p = 0.002], specifically in female participants. Intake of saturated fat and carbohydrate was lower and higher in AC (10.9% and 50.4%) and SA (11.1% and 52.3%), respectively, compared to WE (13.6% and 43.8%, p < 0.001). Insulin resistance in ACs is characterised by ‘normal’ lipid profiles but high rates of hypertension and pronounced hyperinsulinaemia.

Response of the middle atmosphere to CO2 doubling: results from the Canadian Middle Atmosphere Model

The Canadian Middle Atmosphere Model (CMAM) has been used to examine the middle atmosphere response to CO2 doubling. The radiative-photochemical response induced by doubling CO2 alone and the response produced by changes in prescribed SSTs are found to be approximately additive, with the former effect dominating throughout the middle atmosphere. The paper discusses the overall response, with emphasis on the effects of SST changes, which allow a tropospheric response to the CO2 forcing. The overall response is a cooling of the middle atmosphere accompanied by significant increases in the ozone and water vapor abundances. The ozone radiative feedback occurs through both an increase in solar heating and a decrease in infrared cooling, with the latter accounting for up to 15% of the total effect. Changes in global mean water vapor cooling are negligible above ~30 hPa. Near the polar summer mesopause, the temperature response is weak and not statistically significant. The main effects of SST changes are a warmer troposphere, a warmer and higher tropopause, cell-like structures of heating and cooling at low and middlelatitudes in the middle atmosphere, warming in the summer mesosphere, water vapor increase throughout the domain, and O3 decrease in the lower tropical stratosphere. No noticeable change in upwardpropagating planetary wave activity in the extratropical winter–spring stratosphere and no significant temperature response in the polar winter–spring stratosphere have been detected. Increased upwelling in the tropical stratosphere has been found to be linked to changed wave driving at low latitudes.

Response kinetics in the complex chemotaxis signalling pathway of Rhodobacter sphaeroides

Chemotaxis is one of the best characterised signalling systems in biology. It is the mechanism by which bacteria move towards optimal environments and is implicated in biofilm formation, pathogenesis and symbiosis. The properties of the bacterial chemosensory response have been described in detail for the single chemosensory pathway of Escherichia coli. We have characterised the properties of the chemosensory response of Rhodobacter sphaeroides, an -proteobacterium with multiple chemotaxis pathways, under two growth conditions allowing the effects of protein expression levels and cell architecture to be investigated. Using tethered cell assays we measured the responses of the system to step changes in concentration of the attractant propionate and show that, independently of the growth conditions, R. sphaeroides is chemotactic over at least five orders of magnitude and has a sensing profile following Weber’s law. Mathematical modelling also shows that, like E. coli, R. sphaeroides is capable of showing Fold-Change Detection (FCD). Our results indicate that general features of bacterial chemotaxis such as the range and sensitivity of detection, adaptation times, adherence to Weber’s law and the presence of FCD may be integral features of chemotaxis systems in general, regardless of network complexity, protein expression levels and cellular architecture across different species.

Exposure to parabens at the concentration of maximal proliferative response increases migratory and invasive activity of human breast cancer cells in vitro

Alkyl esters of p–hydroxybenzoic acid (parabens) are widely used as preservatives in personal care products, foods and pharmaceuticals. Their oestrogenic activity, their measurement in human breast tissue and their ability to drive proliferation of oestrogen-responsive human breast cancer cells has opened a debate on their potential to influence breast cancer development. Since proliferation is not the only hallmark of cancer cells, we have investigated the effects of exposure to parabens at concentrations of maximal proliferative response on migratory and invasive properties using three oestrogen-responsive human breast cancer cell lines (MCF-7, T-47-D, ZR-75-1). Cells were maintained short-term (1 week) or long-term (20±2 weeks) in phenol-red-free medium containing 5% charcoal-stripped serum with no addition, 10-8M 17-oestradiol, 1-5x10-4M methylparaben, 10-5M n-propylparaben or 10-5M n-butylparaben. Long-term exposure (20±2 weeks) of MCF-7 cells to methylparaben, n-propylparaben or n-butylparaben increased migration as measured using a scratch assay, time-lapse microscopy and xCELLigence technology: invasive properties were found to increase in matrix degradation assays and migration through matrigel on xCELLigence. Western immunoblotting showed an associated downregulation of E-cadherin and -catenin in the long-term paraben-exposed cells which could be consistent with a mechanism involving epithelial to mesenchymal transition. Increased migratory activity was demonstrated also in long-term paraben-exposed T-47-D and ZR-75-1 cells using a scratch assay and time-lapse microscopy. This is the first report that in vitro, parabens can influence not only proliferation but also migratory and invasive properties of human breast cancer cells.

Plasma free fatty acids do not provide the link between obesity and insulin resistance or β-cell dysfunction: results of the Reading, Imperial, Surrey, Cambridge, Kings (RISCK) study

Aims To investigate the relationship between adiposity and plasma free fatty acid levels and the influence of total plasma free fatty acid level on insulin sensitivity and β-cell function. Methods An insulin sensitivity index, acute insulin response to glucose and a disposition index, derived from i.v. glucose tolerance minimal model analysis and total fasting plasma free fatty acid levels were available for 533 participants in the Reading, Imperial, Surrey, Cambridge, Kings study. Bivariate correlations were made between insulin sensitivity index, acute insulin response to glucose and disposition index and both adiposity measures (BMI, waist circumference and body fat mass) and total plasma free fatty acid levels. Multivariate linear regression analysis was performed, controlling for age, sex, ethnicity and adiposity. Results After adjustment, all adiposity measures were inversely associated with insulin sensitivity index (BMI: β = −0.357; waist circumference: β = −0.380; body fat mass: β = −0.375) and disposition index (BMI: β = −0.215; waist circumference: β = −0.248; body fat mass: β = −0.221) and positively associated with acute insulin response to glucose [BMI: β = 0.200; waist circumference: β = 0.195; body fat mass β = 0.209 (P values <0.001)]. Adiposity explained 13, 4 and 5% of the variation in insulin sensitivity index, acute insulin response to glucose and disposition index, respectively. After adjustment, no adiposity measure was associated with free fatty acid level, but total plasma free fatty acid level was inversely associated with insulin sensitivity index (β = −0.133), acute insulin response to glucose (β = −0.148) and disposition index [β = −0.218 (P values <0.01)]. Plasma free fatty acid concentration accounted for 1.5, 2 and 4% of the variation in insulin sensitivity index, acute insulin response to glucose and disposition index, respectively. Conclusions Plasma free fatty acid levels have a modest negative association with insulin sensitivity, β-cell secretion and disposition index but no association with adiposity measures. It is unlikely that plasma free fatty acids are the primary mediators of obesity-related insulin resistance or β-cell dysfunction.

Ionospheric convection response to slow, strong variations in a northward interplanetary magnetic field: A case study for January 14, 1988

We analyze ionospheric convection patterns over the polar regions during the passage of an interplanetary magnetic cloud on January 14, 1988, when the interplanetary magnetic field (IMF) rotated slowly in direction and had a large amplitude. Using the assimilative mapping of ionospheric electrodynamics (AMIE) procedure, we combine simultaneous observations of ionospheric drifts and magnetic perturbations from many different instruments into consistent patterns of high-latitude electrodynamics, focusing on the period of northward IMF. By combining satellite data with ground-based observations, we have generated one of the most comprehensive data sets yet assembled and used it to produce convection maps for both hemispheres. We present evidence that a lobe convection cell was embedded within normal merging convection during a period when the IMF By and Bz components were large and positive. As the IMF became predominantly northward, a strong reversed convection pattern (afternoon-to-morning potential drop of around 100 kV) appeared in the southern (summer) polar cap, while convection in the northern (winter) hemisphere became weak and disordered with a dawn-to-dusk potential drop of the order of 30 kV. These patterns persisted for about 3 hours, until the IMF rotated significantly toward the west. We interpret this behavior in terms of a recently proposed merging model for northward IMF under solstice conditions, for which lobe field lines from the hemisphere tilted toward the Sun (summer hemisphere) drape over the dayside magnetosphere, producing reverse convection in the summer hemisphere and impeding direct contact between the solar wind and field lines connected to the winter polar cap. The positive IMF Bx component present at this time could have contributed to the observed hemispheric asymmetry. Reverse convection in the summer hemisphere broke down rapidly after the ratio |By/Bz| exceeded unity, while convection in the winter hemisphere strengthened. A dominant dawn-to-dusk potential drop was established in both hemispheres when the magnitude of By exceeded that of Bz, with potential drops of the order of 100 kV, even while Bz remained northward. The later transition to southward Bz produced a gradual intensification of the convection, but a greater qualitative change occurred at the transition through |By/Bz| = 1 than at the transition through Bz = 0. The various convection patterns we derive under northward IMF conditions illustrate all possibilities previously discussed in the literature: nearly single-cell and multicell, distorted and symmetric, ordered and unordered, and sunward and antisunward.

The impact of date palm fruits and their component polyphenols, on gut microbial ecology, bacterial metabolites and colon cancer cell proliferation

The fruit of the date palm (Phoenix dactylifera L.) is a rich source of dietary fibre and polyphenols. We have investigated gut bacterial changes induced by the whole date fruit extract (digested date extract; DDE) and its polyphenol-rich extract (date polyphenol extract; DPE) using faecal, pH-controlled, mixed batch cultures mimicking the distal part of the human large intestine, and utilising an array of microbial group-specific 16S rRNA oligonucleotide probes. Fluorescence microscopic enumeration indicated that there was a significant increase in the growth of bifidobacteria in response to both treatments, whilst whole dates also increased bacteroides at 24 h and the total bacterial counts at later fermentation time points when compared with DPE alone. Bacterial metabolism of whole date fruit led to the production of SCFA, with acetate significantly increasing following bacterial incubation with DDE. In addition, the production of flavonoid aglycones (myricetin, luteolin, quercetin and apigenin) and the anthocyanidin petunidin in less than 1 h was also observed. Lastly, the potential of DDE, DPE and metabolites to inhibit Caco-2 cell growth was investigated, indicating that both were capable of potentially acting as antiproliferative agents in vitro, following a 48 h exposure. This potential to inhibit growth was reduced following fermentation. Together these data suggest that consumption of date fruits may enhance colon health by increasing beneficial bacterial growth and inhibiting the proliferation of colon cancer cells. This is an early suggestion that date intake by humans may aid in the maintenance of bowel health and even the reduction of colorectal cancer development.