Peripheral KV7 channels regulate visceral sensory function in mouse and human colon

$\textbf{Background}$ Chronic visceral pain is a defining symptom of many gastrointestinal disorders. The K$_V$7 family (K$_V$7.1-K$_V$7.5) of voltage-gated potassium channels mediates the M current that regulates excitability in peripheral sensory nociceptors and central pain pathways. Here, we use a combination of immunohistochemistry, gut-nerve electrophysiological recordings in both mouse and human tissues, and single-cell qualitative real-time polymerase chain reaction of gut-projecting sensory neurons, to investigate the contribution of peripheral K$_V$7 channels to visceral nociception. $\textbf{Results}$ Immunohistochemical staining of mouse colon revealed labelling of K$_V$7 subtypes (K$_V$7.3 and K$_V$7.5) with CGRP around intrinsic enteric neurons of the myenteric plexuses and within extrinsic sensory fibres along mesenteric blood vessels. Treatment with the K$_V$7 opener retigabine almost completely abolished visceral afferent firing evoked by the algogen bradykinin, in agreement with significant co-expression of mRNA transcripts by single-cell qualitative real-time polymerase chain reaction for KCNQ subtypes and the B$_2$ bradykinin receptor in retrogradely labelled extrinsic sensory neurons from the colon. Retigabine also attenuated responses to mechanical stimulation of the bowel following noxious distension (0-80 mmHg) in a concentration-dependent manner, whereas the K$_V$7 blocker XE991 potentiated such responses. In human bowel tissues, K$_V$7.3 and K$_V$7.5 were expressed in neuronal varicosities co-labelled with synaptophysin and CGRP, and retigabine inhibited bradykinin-induced afferent activation in afferent recordings from human colon. $\textbf{Conclusions}$ We show that K$_V$7 channels contribute to the sensitivity of visceral sensory neurons to noxious chemical and mechanical stimuli in both mouse and human gut tissues. As such, peripherally restricted K$_V$7 openers may represent a viable therapeutic modality for the treatment of gastrointestinal pathologies.This work was supported by an EFIC-Grunenthal grant awarded to Madusha Peiris, CAG/CIHR/CCFR Fellowship to David E Reed, Rosetrees Postdoctoral Grant (A1296) to James RF Hockley/Ewan St. John Smith, Medical Research Council Grant (G0900907) to David C Bulmer and a Wellcome Trust University Award to L Ashley Blackshaw

Evidence for long-term sensitization of the bowel in patients with post-infectious-IBS.

Post-infectious irritable bowel syndrome (PI-IBS) is a common gastrointestinal disorder characterized by persistent abdominal pain despite recovery from acute gastroenteritis. The underlying mechanisms are unclear, although long-term changes in neuronal function, and low grade inflammation of the bowel have been hypothesized. We investigated the presence and mechanism of neuronal sensitization in a unique cohort of individuals who developed PI-IBS following exposure to contaminated drinking water 7 years ago. We provide direct evidence of ongoing sensitization of neuronal signaling in the bowel of patients with PI-IBS. These changes occur in the absence of any detectable tissue inflammation, and instead appear to be driven by pro-nociceptive changes in the gut micro-environment. This is evidenced by the activation of murine colonic afferents, and sensitization responses to capsaicin in dorsal root ganglia (DRGs) following application of supernatants generated from tissue biopsy of patients with PI-IBS. We demonstrate that neuronal signaling within the bowel of PI-IBS patients is sensitized 2 years after the initial infection has resolved. This sensitization appears to be mediated by a persistent pro-nociceptive change in the gut micro-environment, that has the capacity to stimulate visceral afferents and facilitate neuronal TRPV1 signaling

P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors

Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Na(v)1.9 in mediating murine responses. The application of UTP (P2Y(2) and P2Y(4) agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y(1), P2Y(12), and P2Y(13) agonist) also increased action potential firing, an effect blocked by the selective P2Y(1) receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y(1) and P2Y(2) transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Na(v)1.9 transcripts colocalized in 86% of P2Y(1)-positive and 100% of P2Y(2)-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Na(v)1.9(−/−) mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease. SIGNIFICANCE STATEMENT Chronic visceral pain is a debilitating symptom of many gastrointestinal disorders. The activation of pain-sensing nerves located in the bowel wall and their sensitization to physiological stimuli, including bowel movements, underpins the development of such pain, and is associated with mediators released during disease. This work addresses the unstudied role of purine and pyrimidine nucleotides in modulating colonic nociceptors via P2Y receptors using a combination of electrophysiological recordings from human ex vivo samples and a detailed functional study in the mouse. This is the first report to identify colonic purinergic signaling as a function of P2Y receptor activation, in addition to established P2X receptor activity, and the results contribute to our understanding of the development of visceral pain during gastrointestinal disease

Determinants of translation efficiency and accuracy

A given protein sequence can be encoded by an astronomical number of alternative nucleotide sequences. Recent research has revealed that this flexibility provides evolution with multiple ways to tune the efficiency and fidelity of protein translation and folding

General Rules for Optimal Codon Choice

Different synonymous codons are favored by natural selection for translation efficiency and accuracy in different organisms. The rules governing the identities of favored codons in different organisms remain obscure. In fact, it is not known whether such rules exist or whether favored codons are chosen randomly in evolution in a process akin to a series of frozen accidents. Here, we study this question by identifying for the first time the favored codons in 675 bacteria, 52 archea, and 10 fungi. We use a number of tests to show that the identified codons are indeed likely to be favored and find that across all studied organisms the identity of favored codons tracks the GC content of the genomes. Once the effect of the genomic GC content on selectively favored codon choice is taken into account, additional universal amino acid specific rules governing the identity of favored codons become apparent. Our results provide for the first time a clear set of rules governing the evolution of selectively favored codon usage. Based on these results, we describe a putative scenario for how evolutionary shifts in the identity of selectively favored codons can occur without even temporary weakening of natural selection for codon bias

Minor shift in background substitutional patterns in the Drosophila saltans and willistoni lineages is insufficient to explain GC content of coding sequences

BACKGROUND: Several lines of evidence suggest that codon usage in the Drosophila saltans and D. willistoni lineages has shifted towards a less frequent use of GC-ending codons. Introns in these lineages show a parallel shift toward a lower GC content. These patterns have been alternatively ascribed to either a shift in mutational patterns or changes in the definition of preferred and unpreferred codons in these lineages. RESULTS AND DISCUSSION: To gain additional insight into this question, we quantified background substitutional patterns in the saltans/willistoni group using inactive copies of a novel, Q-like retrotransposable element. We demonstrate that the pattern of background substitutions in the saltans/willistoni lineage has shifted to a significant degree, primarily due to changes in mutational biases. These differences predict a lower equilibrium GC content in the genomes of the saltans/willistoni species compared with that in the D. melanogaster species group. The magnitude of the difference can readily account for changes in intronic GC content, but it appears insufficient to explain changes in codon usage within the saltans/willistoni lineage. CONCLUSION: We suggest that the observed changes in codon usage in the saltans/willistoni clade reflects either lineage-specific changes in the definitions of preferred and unpreferred codons, or a weaker selective pressure on codon bias in this lineage

Twin births, sex of children and maternal risk of ovarian cancer: a cohort study in Norway

In a follow-up of 1 208 001 women aged 20–74 years, no significant association was found between twin births (112 cases) and risk, though those with twin girls had a non-significantly higher risk than those with singleton births; among the latter, those with girls only had a higher risk of endometrioid tumours (incidence rate ratio 1.35; 95% confidence interval 1.03–1.76, based on 475 cases) than women with boys only

Molecular Evolution of Ultraspiracle Protein (USP/RXR) in Insects

Ultraspiracle protein/retinoid X receptor (USP/RXR) is a nuclear receptor and transcription factor which is an essential component of a heterodimeric receptor complex with the ecdysone receptor (EcR). In insects this complex binds ecdysteroids and plays an important role in the regulation of growth, development, metamorphosis and reproduction. In some holometabolous insects, including Lepidoptera and Diptera, USP/RXR is thought to have experienced several important shifts in function. These include the acquisition of novel ligand-binding properties and an expanded dimerization interface with EcR. In light of these recent hypotheses, we implemented codon-based likelihood methods to investigate if the proposed shifts in function are reflected in changes in site-specific evolutionary rates across functional and structural motifs in insect USP/RXR sequences, and if there is any evidence for positive selection at functionally important sites. Our results reveal evidence of positive selection acting on sites within the loop connecting helices H1 and H3, the ligand-binding pocket, and the dimer interface in the holometabolous lineage leading to the Lepidoptera/Diptera/Trichoptera. Similar analyses conducted using EcR sequences did not indicate positive selection. However, analyses allowing for variation across sites demonstrated elevated non-synonymous/synonymous rate ratios (dN/dS), suggesting relaxed constraint, within the dimerization interface of both USP/RXR and EcR as well as within the coactivator binding groove and helix H12 of USP/RXR. Since the above methods are based on the assumption that dS is constant among sites, we also used more recent models which relax this assumption and obtained results consistent with traditional random-sites models. Overall our findings support the evolution of novel function in USP/RXR of more derived holometabolous insects, and are consistent with shifts in structure and function which may have increased USP/RXR reliance on EcR for cofactor recruitment. Moreover, these findings raise important questions regarding hypotheses which suggest the independent activation of USP/RXR by its own ligand