Purinergic receptor mediated calcium signalling in urothelial cells

Non-neuronal ATP released from the urothelium in response to bladder stretch is a key modulator of bladder mechanosensation. Whilst non-neuronal ATP acts on the underlying bladder afferent nerves to facilitate sensation, there is also the potential for ATP to act in an autocrine manner, modulating urothelial cell function. The aim of this study was to systematically characterise the functional response of primary mouse urothelial cells (PMUCs) to ATP. PMUCs isolated from male mice (14-16 weeks) were used for live-cell fluorescent calcium imaging and qRT-PCR to determine the expression profile of P2X and P2Y receptors. The majority of PMUCs (74-92%) responded to ATP (1 μM-1 mM), as indicted by an increase in intracellular calcium (iCa2+). PMUCs exhibited dose-dependent responses to ATP (10 nM-1 mM) in both calcium containing (2 mM, EC50 = 3.49 ± 0.77 μM) or calcium free (0 mM, EC50 = 9.5 ± 1.5 μM) buffers. However, maximum iCa2+ responses to ATP were significantly attenuated upon repetitive applications in calcium containing but not in calcium free buffer. qRT-PCR revealed expression of P2X1-6, and P2Y1-2, P2Y4, P2Y6, P2Y11-14, but not P2X7 in PMUCs. These findings suggest the major component of ATP induced increases in iCa2+ are mediated via the liberation of calcium from intracellular stores, implicating functional P2Y receptors that are ubiquitously expressed on PMUCs.Russell Chess-Williams, Donna J. Sellers, Stuart M. Brierley, David Grundy, Luke Grund

α_1L-adrenoceptors mediate contraction of human erectile tissue

α1-adrenoceptor antagonists can impact upon sexual function and have potential in the treatment of erectile dysfunction. Human erectile tissue contains predominantly α1A-adrenoceptors, and here we examined whether contractions of this tissue are mediated by the functional phenotype, the α1L-adrenoceptor. Functional experiments using subtype selective agonists and antagonists, along with radioligand ([3H]tamsulosin) binding assays, were used to determine the α1-adrenoceptor population. A61603, a α1A-adrenoceptor agonist, was a full agonist with a potency 21-fold greater than that of noradrenaline. The α1A- and α1D-adrenoceptor antagonist tamsulosin antagonized noradrenaline responses with high affinity (pKD = 9.7 ± 0.3), whilst BMY7378 (100 nM) (α1D-adrenoceptor antagonist) failed to antagonize responses. In contrast, relatively low affinity estimates were obtained for both prazosin (pKD = 8.2 ± 0.1) and RS17053 (pKD = 6.9 ± 0.2), antagonists which discriminate between the α1A- and α1L-adrenoceptors. [3H]Tamsulosin bound with high affinity to the receptors of human erectile tissue (pKD = 10.3 ± 0.1) with a receptor density of 28.1 ± 1.4 fmol mg−1 protein. Prazosin displacement of [3H]tamsulosin binding revealed a single homogenous population of binding sites with a relatively low affinity for prazosin (pKi = 8.9). Taken together these data confirm that the receptor mediating contraction in human erectile tissue has the pharmacological properties of the α1L-adrenoceptor. Keywords: Erectile tissue, α1-adrenoceptor subtypes, α1L-adrenoceptor, Tamsulosin, Prazosi

A Pilot retrospective analysis of alpha-blockers on recurrence in men with localised prostate cancer treated with radiotherapy

While alpha-blockers are commonly used to reduce lower urinary tract symptoms in prostate cancer patients receiving radiotherapy, their impact on response to radiotherapy remains unknown. Therefore, this pilot study aimed to retrospectively determine if alpha-blockers use, influenced response to radiotherapy for localised prostate cancer. In total, 303 prostate cancer patients were included, consisting of 84 control (alpha-blocker naïve), 72 tamsulosin and 147 prazosin patients. The main outcomes measured were relapse rates (%), time to biochemical relapse (months) and PSA velocity (ng/mL/year). Recurrence free survival was calculated using Kaplan-Meier analysis. Prazosin significantly reduced biochemical relapse at both two and five-years (2.72%, 8.84%) compared to control (22.61%, 34.52%). Recurrence free survival was also significantly higher in the prazosin group. This remained after multivariable analysis (HR: 0.09, 95% CI: 0.04–0.26, p < 0.001). Patients receiving prazosin had a 3.9 times lower relative risk of biochemical relapse compared to control. Although not statistically significant, tamsulosin and prazosin extended recurrence free survival by 13.15 and 9.21 months respectively. We show for the first time that prazosin may reduce risk of prostate cancer recurrence and delay time to biochemical relapse and provides justification for prospective studies to examine its potential as an adjunct treatment option for localised prostate cancer.Full Tex

Vastus medialis motor unit properties in knee osteoarthritis

<p>Abstract</p> <p>Background</p> <p>Maximal isometric quadriceps strength deficits have been widely reported in studies of knee osteoarthritis (OA), however little is known about the effect of osteoarthritis knee pain on submaximal quadriceps neuromuscular function. The purpose of this study was to measure vastus medialis motor unit (MU) properties in participants with knee OA, during submaximal isometric contractions.</p> <p>Methods</p> <p>Vastus medialis motor unit potential (MUP) parameters were assessed in 8 patients with knee OA and 8 healthy, sex and age-matched controls during submaximal isometric contractions (20% of maximum isometric torque). Unpaired t-tests were used to compare groups for demographic and muscle parameters.</p> <p>Results</p> <p>Maximum knee extension torque was ~22% lower in the OA group, a difference that was not statistically significantly (p = 0.11). During submaximal contractions, size related parameters of the needle MUPs (e.g. negative peak duration and amplitude-to-area ratio) were greater in the OA group (p < 0.05), with a rightward shift in the frequency distribution of surface MUP negative peak amplitude. MUP firing rates were significantly lower in the OA group (p < 0.05).</p> <p>Conclusions</p> <p>Changes in MU recruitment and rate coding strategies in OA may reflect a chronic reinnervation process or a compensatory strategy in the presence of chronic knee pain associated with OA.</p

Ready ... Go: Amplitude of the fMRI Signal Encodes Expectation of Cue Arrival Time

What happens when the brain awaits a signal of uncertain arrival time, as when a sprinter waits for the starting pistol? And what happens just after the starting pistol fires? Using functional magnetic resonance imaging (fMRI), we have discovered a novel correlate of temporal expectations in several brain regions, most prominently in the supplementary motor area (SMA). Contrary to expectations, we found little fMRI activity during the waiting period; however, a large signal appears after the “go” signal, the amplitude of which reflects learned expectations about the distribution of possible waiting times. Specifically, the amplitude of the fMRI signal appears to encode a cumulative conditional probability, also known as the cumulative hazard function. The fMRI signal loses its dependence on waiting time in a “countdown” condition in which the arrival time of the go cue is known in advance, suggesting that the signal encodes temporal probabilities rather than simply elapsed time. The dependence of the signal on temporal expectation is present in “no-go” conditions, demonstrating that the effect is not a consequence of motor output. Finally, the encoding is not dependent on modality, operating in the same manner with auditory or visual signals. This finding extends our understanding of the relationship between temporal expectancy and measurable neural signals

Profiling allele-specific gene expression in brains from individuals with autism spectrum disorder reveals preferential minor allele usage.

One fundamental but understudied mechanism of gene regulation in disease is allele-specific expression (ASE), the preferential expression of one allele. We leveraged RNA-sequencing data from human brain to assess ASE in autism spectrum disorder (ASD). When ASE is observed in ASD, the allele with lower population frequency (minor allele) is preferentially more highly expressed than the major allele, opposite to the canonical pattern. Importantly, genes showing ASE in ASD are enriched in those downregulated in ASD postmortem brains and in genes harboring de novo mutations in ASD. Two regions, 14q32 and 15q11, containing all known orphan C/D box small nucleolar RNAs (snoRNAs), are particularly enriched in shifts to higher minor allele expression. We demonstrate that this allele shifting enhances snoRNA-targeted splicing changes in ASD-related target genes in idiopathic ASD and 15q11-q13 duplication syndrome. Together, these results implicate allelic imbalance and dysregulation of orphan C/D box snoRNAs in ASD pathogenesis

Human neutrophil clearance of bacterial pathogens triggers anti-microbial gamma delta T cell responses in early infection

Human blood Vc9/Vd2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vc9/Vd2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vc9/Vd2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-c and tumor necrosis factor (TNF)-a. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vc9/Vd2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-a dependent proliferation of Vc9/Vd2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting cd T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vc9/Vd2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The cd T celldriven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of cd T cells and TNF-a and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive cd T cells in early infection and suggest novel diagnostic and therapeutic approaches.Martin S. Davey, Chan-Yu Lin, Gareth W. Roberts, Sinéad Heuston, Amanda C. Brown, James A. Chess, Mark A. Toleman, Cormac G.M. Gahan, Colin Hill, Tanya Parish, John D. Williams, Simon J. Davies, David W. Johnson, Nicholas Topley, Bernhard Moser and Matthias Eber

ω-3 Polyunsaturated fatty acids prevent pressure overload-induced ventricular dilation and decrease in mitochondrial enzymes despite no change in adiponectin

<p>Abstract</p> <p>Background</p> <p>Pathological left ventricular (LV) hypertrophy frequently progresses to dilated heart failure with suppressed mitochondrial oxidative capacity. Dietary marine ω-3 polyunsaturated fatty acids (ω-3 PUFA) up-regulate adiponectin and prevent LV dilation in rats subjected to pressure overload. This study 1) assessed the effects of ω-3 PUFA on LV dilation and down-regulation of mitochondrial enzymes in response to pressure overload; and 2) evaluated the role of adiponectin in mediating the effects of ω-3 PUFA in heart.</p> <p>Methods</p> <p>Wild type (WT) and adiponectin-/- mice underwent transverse aortic constriction (TAC) and were fed standard chow ± ω-3 PUFA for 6 weeks. At 6 weeks, echocardiography was performed to assess LV function, mice were terminated, and mitochondrial enzyme activities were evaluated.</p> <p>Results</p> <p>TAC induced similar pathological LV hypertrophy compared to sham mice in both strains on both diets. In WT mice TAC increased LV systolic and diastolic volumes and reduced mitochondrial enzyme activities, which were attenuated by ω-3 PUFA without increasing adiponectin. In contrast, adiponectin-/- mice displayed no increase in LV end diastolic and systolic volumes or decrease in mitochondrial enzymes with TAC, and did not respond to ω-3 PUFA.</p> <p>Conclusion</p> <p>These findings suggest ω-3 PUFA attenuates cardiac pathology in response to pressure overload independent of an elevation in adiponectin.</p

Safety and pharmacokinetics of multiple dose myo-inositol in preterm infants

BACKGROUND: Preterm infants with respiratory distress syndrome (RDS) given inositol had reduced bronchopulmonary dysplasia (BPD), death and severe retinopathy of prematurity (ROP). We assessed the safety and pharmacokinetics of daily inositol to select a dose providing serum levels previously associated with benefit, and to learn if accumulation occurred when administered throughout the normal period of retinal vascularization. METHODS: Infants ≤ 29 wk GA (n = 122, 14 centers) were randomized and treated with placebo or inositol at 10, 40, or 80 mg/kg/d. Intravenous administration converted to enteral when feedings were established, and continued to the first of 10 wk, 34 wk postmenstrual age (PMA) or discharge. Serum collection employed a sparse sampling population pharmacokinetics design. Inositol urine losses and feeding intakes were measured. Safety was prospectively monitored. RESULTS: At 80 mg/kg/d mean serum levels reached 140 mg/l, similar to Hallman's findings. Levels declined after 2 wk, converging in all groups by 6 wk. Analyses showed a mean volume of distribution 0.657 l/kg, clearance 0.058 l/kg/h, and half-life 7.90 h. Adverse events and comorbidities were fewer in the inositol groups, but not significantly so. CONCLUSION: Multiple dose inositol at 80 mg/kg/d was not associated with increased adverse events, achieves previously effective serum levels, and is appropriate for investigation in a phase III trial

Plasmodium falciparum Merozoite Associated Armadillo Protein (PfMAAP) Is Apically Localized in Free Merozoites and Antibodies Are Associated With Reduced Risk of Malaria.

Understanding the functional role of proteins expressed by Plasmodium falciparum is an important step toward unlocking potential targets for the development of therapeutic or diagnostic interventions. The armadillo (ARM) repeat protein superfamily is associated with varied functions across the eukaryotes. Therefore, it is important to understand the role of members of this protein family in Plasmodium biology. The Plasmodium falciparum armadillo repeats only (PfARO; Pf3D7_0414900) and P. falciparum merozoite organizing proteins (PfMOP; Pf3D7_0917000) are armadillo-repeat containing proteins previously characterized in P. falciparum. Here, we describe the characterization of another ARM repeat-containing protein in P. falciparum, which we have named the P. falciparum Merozoites-Associated Armadillo repeats protein (PfMAAP). Antibodies raised to three different synthetic peptides of PfMAAP show apical staining of free merozoites and those within the mature infected schizont. We also demonstrate that the antibodies raised to the PfMAAP peptides inhibited invasion of erythrocytes by merozoites from different parasite isolates. In addition, naturally acquired human antibodies to the N- and C- termini of PfMAAP are associated with a reduced risk of malaria in a prospective cohort analysis