Age-related changes in afferent pathways and urothelial function in the male mouse bladder

Key points •The prevalence of bladder conditions such as overactive bladder syndrome and urinary incontinence significantly increases with age, but how bladder function is altered by ageing is unclear. •Sensory nerves together with the epithelial lining of the bladder known as the urothelium play a key role in mediating bladder function. •In aged male mice we find a significant increase in natural bladder voiding, augmented afferent nerve firing during bladder filling and a significant increase in urothelial responses to purinergic receptor stimulation. •This suggests that with ageing there is increased purinergic transmission in the mouse bladder which may lead to increased sensation and result in bladder hypersensitivity. •These findings help us better understand how the function of the bladder may be affected by advancing age. Abstract The prevalence of lower urinary tract storage disorders such as overactive bladder syndrome and urinary incontinence significantly increase with age. Previous studies have demonstrated age-related changes in detrusor function and urothelial transmitter release but few studies have investigated how the urothelium and sensory pathways are affected. The aim of this study was to investigate the effect of ageing on urothelial-afferent signalling in the mouse bladder. Three-month-old control and 24-month-old aged male mice were used. In vivo natural voiding behaviour, sensory nerve activity, urothelial cell function, muscle contractility, transmitter release and gene and protein expression were measured to identify how all three components of the bladder (neural, contractile and urothelial) are affected by ageing. In aged mice, increased voiding frequency and enhanced low threshold afferent nerve activity was observed, suggesting that ageing induces overactivity and hypersensitivity of the bladder. These changes were concurrent with altered ATP and acetylcholine bioavailability, measured as transmitter overflow into the lumen, increased purinergic receptor sensitivity and raised P2X3 receptor expression in the urothelium. Taken together, these data suggest that ageing results in aberrant urothelial function, increased afferent mechanosensitivity, increased smooth muscle contractility, and changes in gene and protein expression (including of P2X3). These data are consistent with the hypothesis that ageing evokes changes in purinergic signalling from the bladder, and further studies are now required to fully validate this idea

Serotonin exerts a direct modulatory role on bladder afferent firing in mice

Key points Functional disorders (i.e., interstitial cystitis/painful bladder syndrome and irritable bowel syndrome) are associated with hyperexcitability of afferent nerves innervating the urinary tract and the bowel respectively. Various non‐5‐HT3 receptor mRNA transcripts are expressed in mouse urothelium and exert functional responses to 5‐HT. Whilst 5‐HT3 receptors were not detected in mouse urothelium, 5‐HT3 receptors expressed on bladder sensory neurons plays a role in bladder afferent excitability under both normal conditions and in a mouse model of chronic visceral hypersensitivity (CVH). These data suggest that the role 5‐HT3 receptors play in bladder afferent signaling warrants further study as a potential therapeutic target for functional bladder disorders. Abstract Serotonin (5‐HT) is an excitatory mediator, which in the gastrointestinal (GI) tract, plays a physiological role in gut‐brain signaling and which is dysregulated in functional GI disorders such as irritable bowel syndrome (IBS). Patients suffering from IBS frequently suffer from urological symptoms characteristic of interstitial cystitis/painful bladder syndrome, which manifests due to cross‐sensitization of shared innervation pathways between the bladder and colon. However, a direct modulatory role of 5‐HT in bladder afferent signaling and its role in colon‐bladder neuronal crosstalk remain elusive. The aim of this study was to investigate the action of 5‐HT on bladder afferent signaling in normal mice and mice with chronic visceral hypersensitivity (CVH) following trinitrobenzenesulfonic acid (TNBS) induced colitis. Bladder afferent activity was recorded directly using ex vivo afferent nerve recordings. Expression of 14 5‐HT receptor subtypes, the serotonin transporter (SERT) and 5‐HT producing enzymes were determined in the urothelium using RT‐PCR. Retrograde labelling of bladder projecting dorsal root ganglion (DRG) neurons was used to investigate expression of 5‐HT3 receptors using single cell RT‐PCR, while sensory neuronal and urothelial responses to 5‐HT were determined by live cell calcium imaging. 5‐HT elicited bladder afferent firing predominantly via 5‐HT3 receptors expressed on afferent terminals. CVH animals showed a downregulation of SERT mRNA expression in urothelium, suggesting increased 5‐HT bioavailability. Granisetron, a 5‐HT3 antagonist, reversed bladder afferent hypersensitivity in CVH mice. These data suggest 5‐HT exerts a direct effect on bladder afferents to enhance signaling.5‐HT3 antagonists could therefore be a potential therapeutic target to treat functional bladder and bowel disorders

The inflation of viscoelastic balloons and hollow viscera

For the first time, the problem of the inflation of a nonlinear viscoelastic thick-walled spherical shell is considered. Specifically, the wall has quasilinear viscoelastic constitutive behaviour, which is of fundamental importance in a wide range of applications, particularly in the context of biological systems such as hollow viscera, including the lungs and bladder. Experiments are performed to demonstrate the efficacy of the model in fitting relaxation tests associated with the volumetric inflation of murine bladders . While the associated nonlinear elastic problem of inflation of a balloon has been studied extensively, there is a paucity of studies considering the equivalent nonlinear viscoelastic case. We show that, in contrast to the elastic scenario, the peak pressure associated with the inflation of a neo-Hookean balloon is not independent of the shear modulus of the medium. Moreover, a novel numerical technique is described in order to solve the nonlinear Volterra integral equation in space and time originating from the fundamental problem of inflation and deflation of a thick-walled nonlinear viscoelastic shell under imposed pressure. EPSRC Grant EP/R014604/

Development of a surveillance scheme for equine influenza in the UK and characterisation of viruses isolated in Europe, Dubai and the USA from 2010-2012

Equine influenza viruses are a major cause of respiratory disease in horses worldwide and undergo antigenic drift. Several outbreaks of equine influenza occurred worldwide during 2010-2012, including in vaccinated animals, highlighting the importance of surveillance and virus characterisation. Virus isolates were characterised from more than 20 outbreaks over a 3-year period, including strains from the UK, Dubai, Germany and the USA. The haemagglutinin-1 (HA1) sequence of all isolates was determined and compared with OIE-recommended vaccine strains. Viruses from Florida clades 1 and 2 showed continued divergence from each other compared with 2009 isolates. The antigenic inter-relationships among viruses were determined using a haemagglutination-inhibition (HI) assay with ferret antisera and visualised using antigenic cartography. All European isolates belonged to Florida clade 2, all those from the USA belonged to Florida clade 1. Two subpopulations of clade 2 viruses were isolated, with either substitution A144V or I179V. Isolates from Dubai, obtained from horses shipped from Uruguay, belonged to Florida clade 1 and were similar to viruses isolated in the USA the previous year. The neuraminidase (NA) sequence of representative strains from 2007 and 2009 to 2012 was also determined and compared with that of earlier isolates dating back to 1963. Multiple changes were observed at the amino acid level and clear distinctions could be made between viruses belonging to Florida clade 1 and clade 2

The 22q11.2 region regulates presynaptic gene-products linked to schizophrenia

How the 22q11.2 deletion predisposes to psychiatric disease is unclear. Here, the authors examine living human neuronal cells and show that 22q11.2 regulates the expression of genes linked to autism during early development, and genes linked to schizophrenia and synaptic biology in neurons. It is unclear how the 22q11.2 deletion predisposes to psychiatric disease. To study this, we generated induced pluripotent stem cells from deletion carriers and controls and utilized CRISPR/Cas9 to introduce the heterozygous deletion into a control cell line. Here, we show that upon differentiation into neural progenitor cells, the deletion acted in trans to alter the abundance of transcripts associated with risk for neurodevelopmental disorders including autism. In excitatory neurons, altered transcripts encoded presynaptic factors and were associated with genetic risk for schizophrenia, including common and rare variants. To understand how the deletion contributed to these changes, we defined the minimal protein-protein interaction network that best explains gene expression alterations. We found that many genes in 22q11.2 interact in presynaptic, proteasome, and JUN/FOS transcriptional pathways. Our findings suggest that the 22q11.2 deletion impacts genes that may converge with psychiatric risk loci to influence disease manifestation in each deletion carrier.Peer reviewe

Pharmacogenetic allele nomenclature: International workgroup recommendations for test result reporting

This manuscript provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward

Analysis of Rare, Exonic Variation amongst Subjects with Autism Spectrum Disorders and Population Controls

We report on results from whole-exome sequencing (WES) of 1,039 subjects diagnosed with autism spectrum disorders (ASD) and 870 controls selected from the NIMH repository to be of similar ancestry to cases. The WES data came from two centers using different methods to produce sequence and to call variants from it. Therefore, an initial goal was to ensure the distribution of rare variation was similar for data from different centers. This proved straightforward by filtering called variants by fraction of missing data, read depth, and balance of alternative to reference reads. Results were evaluated using seven samples sequenced at both centers and by results from the association study. Next we addressed how the data and/or results from the centers should be combined. Gene-based analyses of association was an obvious choice, but should statistics for association be combined across centers (meta-analysis) or should data be combined and then analyzed (mega-analysis)? Because of the nature of many gene-based tests, we showed by theory and simulations that mega-analysis has better power than meta-analysis. Finally, before analyzing the data for association, we explored the impact of population structure on rare variant analysis in these data. Like other recent studies, we found evidence that population structure can confound case-control studies by the clustering of rare variants in ancestry space; yet, unlike some recent studies, for these data we found that principal component-based analyses were sufficient to control for ancestry and produce test statistics with appropriate distributions. After using a variety of gene-based tests and both meta- and mega-analysis, we found no new risk genes for ASD in this sample. Our results suggest that standard gene-based tests will require much larger samples of cases and controls before being effective for gene discovery, even for a disorder like ASD. © 2013 Liu et al

Patterns and rates of exonic de novo mutations in autism spectrum disorders

Autism spectrum disorders (ASD) are believed to have genetic and environmental origins, yet in only a modest fraction of individuals can specific causes be identified1,2. To identify further genetic risk factors, we assess the role of de novo mutations in ASD by sequencing the exomes of ASD cases and their parents (n= 175 trios). Fewer than half of the cases (46.3%) carry a missense or nonsense de novo variant and the overall rate of mutation is only modestly higher than the expected rate. In contrast, there is significantly enriched connectivity among the proteins encoded by genes harboring de novo missense or nonsense mutations, and excess connectivity to prior ASD genes of major effect, suggesting a subset of observed events are relevant to ASD risk. The small increase in rate of de novo events, when taken together with the connections among the proteins themselves and to ASD, are consistent with an important but limited role for de novo point mutations, similar to that documented for de novo copy number variants. Genetic models incorporating these data suggest that the majority of observed de novo events are unconnected to ASD, those that do confer risk are distributed across many genes and are incompletely penetrant (i.e., not necessarily causal). Our results support polygenic models in which spontaneous coding mutations in any of a large number of genes increases risk by 5 to 20-fold. Despite the challenge posed by such models, results from de novo events and a large parallel case-control study provide strong evidence in favor of CHD8 and KATNAL2 as genuine autism risk factors

Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes.

Pancreatic cancer is a highly lethal malignancy with few effective therapies. We performed exome sequencing and copy number analysis to define genomic aberrations in a prospectively accrued clinical cohort (n = 142) of early (stage I and II) sporadic pancreatic ductal adenocarcinoma. Detailed analysis of 99 informative tumours identified substantial heterogeneity with 2,016 non-silent mutations and 1,628 copy-number variations. We define 16 significantly mutated genes, reaffirming known mutations (KRAS, TP53, CDKN2A, SMAD4, MLL3, TGFBR2, ARID1A and SF3B1), and uncover novel mutated genes including additional genes involved in chromatin modification (EPC1 and ARID2), DNA damage repair (ATM) and other mechanisms (ZIM2, MAP2K4, NALCN, SLC16A4 and MAGEA6). Integrative analysis with in vitro functional data and animal models provided supportive evidence for potential roles for these genetic aberrations in carcinogenesis. Pathway-based analysis of recurrently mutated genes recapitulated clustering in core signalling pathways in pancreatic ductal adenocarcinoma, and identified new mutated genes in each pathway. We also identified frequent and diverse somatic aberrations in genes described traditionally as embryonic regulators of axon guidance, particularly SLIT/ROBO signalling, which was also evident in murine Sleeping Beauty transposon-mediated somatic mutagenesis models of pancreatic cancer, providing further supportive evidence for the potential involvement of axon guidance genes in pancreatic carcinogenesis