Ca 2+ signalling in urethral interstitial cells of Cajal

Interstitial cells of Cajal (ICC) in the urethra have been proposed as specialized pacemakers that are involved in the generation of urethral tone and therefore the maintenance of urinary continence. Recent studies on freshly dispersed ICC from the urethra of rabbits have demonstrated that pacemaker activity in urethra ICC is characterized by spontaneous transient depolarizations (STDs) under current clamp and spontaneous transient inward currents (STICs) under voltage clamp. When these events were simultaneously recorded with changes in intracellular Ca 2+ (using a Nipkow spinning disk confocal microscope) they were found to be associated with global Ca 2+ oscillations. In this short review we will consider some of these recent findings regarding the contribution of intracellular Ca 2+ stores and Ca 2+ influx to the generation of pacemaker activity in urethral ICC with particular emphasis on the contribution of reverse Na + /Ca 2+ exchange (NCX)

Cystic fibrosis gene repair: correction of ΔF508 using ZFN and CRISPR/Cas9 guide RNA gene editing tools

Cystic Fibrosis (CF) is an autosomal recessive monogenic disorder caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene with the ΔF508 mutation accounting for approximately 70% of all CF cases worldwide. This thesis investigates whether existing zinc finger nucleases designed in this lab and CRISPR/gRNAs designed in this thesis can mediate efficient homology-directed repair (HDR) with appropriate donor repair plasmids to correct CF-causing mutations in a CF cell line. Firstly, the most common mutation, ΔF508, was corrected using a pair of existing ZFNs, which cleave in intron 9, and the donor repair plasmid pITR-donor-XC, which contains the correct CTT sequence and two unique restriction sites. HDR was initially determined to be 80% of CF-causing mutations. The ZFN cut site was used as the site for HDR of a mini-gene construct comprising exons 10-24 from CFTR cDNA (with appropriate splice acceptor and poly A sites) to allow production of full length corrected CFTR mRNA. Finally, the ability to cleave closer to the mutation and mediate repair of CFTR using the latest gene editing tool CRISPR/Cas9 was explored. Two CRISPR gRNAs were tested; CRISPR ex10 was shown to cleave at an efficiency of 15% and CRISPR in9 cleaved at 3%. Both CRISPR gRNAs mediated HDR with appropriate donor plasmids at a rate of ~1% as determined by NGS. This is the first evidence of CRISPR induced HDR in CF cell lines

T- and L-type Ca 2+ currents in freshly dispersed smooth muscle cells from the human proximal urethra

The purpose of the present study was to characterise Ca 2+ currents in smooth muscle cells solated from biopsy samples taken from the proximal urethra of patients undergoing surgery for bladder or prostate cancer. Cells were studied at 37°C using the amphotericin B perforated-patch onfiguration of the patch-clamp technique. Currents were recorded using Cs + -rich pipette solutions to block K + currents. Two components of current, with electrophysiological and pharmacological properties typical of T- and L-type Ca 2+ currents, were present in these cells. When steady-state inactivation curves for the L current were fitted with a Boltzmann equation, this yielded a VÎ of _45 ± 5 mV. In contrast, the T current inactivated with a VÎ of _80 ± 3 mV. The L currents were reduced in a concentration-dependent manner by nifedipine (ED50 = 159 ± 54 nM) and Ni 2+ (ED50 = 65 ± 16 mM) but were enhanced when external Ca 2+ was substituted with Ba 2+ . The T current was little affected by TTX, reduction in external Na + , application of nifedipine at concentrations below 300 nM or substitution of external Ca 2+ with Ba 2+ , but was reduced by Ni 2+ with an ED50 of 6 ± 1 mM. When cells were stepped from _100 to _30 mV in Ca 2+ -free conditions, small inward currents could be detected. These were enhanced 40-fold in divalent-cation-free solution and blocked in a concentration-dependent manner by Mg 2+ with an ED50 of 32 ± 16 mM. These data support the idea that human urethral myocytes possess currents with electrophysiological and pharmacological properties typical of T- and L-type Ca 2+ currents

Contribution of K v 2.1 channels to the delayed rectifier current in freshly dispersed smooth muscle cells from rabbit urethra

We have characterized the native voltage-dependent K + (K v ) current in rabbit urethral smooth muscle cells (RUSMC) and compared its pharmacological and biophysical properties with K v 2.1 and K v 2.2 channels cloned from the rabbit urethra and stably expressed in HEK 293 cells (HEK Kv2.1 and HEK Kv2.2 ). RUSMC were perfused with Hanks' solution at 37°C and studied using the patch clamp technique with K + -rich pipette solutions. Cells were bathed in 100 nM penitrem A (Pen A) to block large conductance Ca 2+ -activated K + (BK) currents and depolarized to +40 mV for 500 ms to evoke K v currents. These were unaffected by margatoxin, κ-dendrotoxin or α-dendrotoxin (100 nM, n=3-5), but were blocked by stromatoxin-1 (ScTx, IC 50 ~130 nM), consistent with the idea that the currents were carried through K v 2 channels. RNA was detected for K v 2.1 K v 2.2 and the silent subunit K v 9.3 in urethral smooth muscle. Immunocytochemistry showed membrane staining for both K v 2 subtypes and K v 9.3 in isolated RUSMC. HEK Kv2.1 and HEK Kv2.2 currents were blocked in a concentration dependent manner by ScTx with estimated IC 50 values of ~150 nM (K v 2.1, n=5) and 70 nM (K v 2.2, n=6). The mean V 1/2 of inactivation of the USMC K v current was – 56±3 mV (n=9). This was similar to the HEK Kv2.1 current (–55 ± 3 mV, n=13) but significantly different from the HEK Kv2.2 currents (-30 ± 3 mV, n=11). Action potentials (AP) evoked from RUSMC studied under current clamp mode were unaffected by ScTx. However when ScTx was applied in the presence of Pen A, the AP duration was significantly prolonged. Similarly, ScTx increased the amplitude of spontaneous contractions threefold, but only after Pen A application. These data suggest that K v 2.1 channels contribute significantly to the K v current in RUSMC

Bereavement through substance use: findings from an interview study with adults in England and Scotland

Background Deaths associated with alcohol and/or drugs belong to a category of ‘special’ deaths due to three characteristics: traumatic circumstances of the death, stigma directed to both the bereaved and the deceased, and resulting disenfranchised grief experienced by the bereaved. These factors can impede those who are bereaved in this way from both grieving and accessing support. In response to a lack of research in this area this paper reports on an interview study that has aimed to better understand the experiences and needs of this neglected group of bereaved people. Method Interviews with 106 adults (parents, children, spouses, siblings, nieces and friends) bereaved through substance use in Scotland and England. Results Five themes describe interviewee experiences: possibility of death, official processes, stigma, grief and support. These findings suggest what is dominant or unique in this group of bereaved people; namely, that living with substance use (including anticipatory grief), experiencing the subsequent death (often traumatic and stigmatised) and the responses of professionals and others (more likely negative than positive) can disenfranchise grief and negatively impact bereavement and seeking support. Conclusions This article describes a large and unique sample, the largest in the world to be recruited from this population. Our study raises awareness of a hitherto largely ignored and marginalised group of bereaved people, highlighting what might be particular to their bereavement experience and how this may differ from other bereavements, thereby providing an evidence base for improving the availability, level and quality of support. © 2016 Taylor & Franci

Barriers and facilitators to cooking from 'scratch' using basic or raw ingredients: A qualitative interview study

BACKGROUND: Previous research has highlighted an ambiguity in understanding cooking related terminology and a number of barriers and facilitators to home meal preparation. However, meals prepared in the home still include convenience products (typically high in sugars, fats and sodium) which can have negative effects on health. Therefore, this study aimed to qualitatively explore: (1) how individuals define cooking from 'scratch', and (2) their barriers and facilitators to cooking with basic ingredients. METHODS: 27 semi-structured interviews were conducted with participants (aged 18-58 years) living on the island of Ireland, eliciting definitions of 'cooking from scratch' and exploring the reasons participants cook in a particular way. The interviews were professionally transcribed verbatim and Nvivo 10 was used for an inductive thematic analysis. RESULTS: Our results highlighted that although cooking from 'scratch' lacks a single definition, participants viewed it as optimal cooking. Barriers to cooking with raw ingredients included: 1) time pressures; (2) desire to save money; (3) desire for effortless meals; (4) family food preferences; and (5) effect of kitchen disasters. Facilitators included: 1) desire to eat for health and well-being; (2) creative inspiration; (3) ability to plan and prepare meals ahead of time; and (4) greater self-efficacy in one's cooking ability. CONCLUSIONS: Our findings contribute to understanding how individuals define cooking from 'scratch', and barriers and facilitators to cooking with raw ingredients. Interventions should focus on practical sessions to increase cooking self-efficacy; highlight the importance of planning ahead and teach methods such as batch cooking and freezing to facilitate cooking from scratch