Female Urgency, Trial of Urodynamics as Routine Evaluation (FUTURE study): a superiority randomised clinical trial to evaluate the effectiveness and cost-effectiveness of invasive urodynamic investigations in management of women with refractory overactive bladder symptoms.

BACKGROUND: Overactive bladder (OAB) syndrome is a symptom complex affecting 12-14% of the UK adult female population. Symptoms include urinary urgency, with or without urgency incontinence, increased daytime urinary frequency and nocturia. OAB has a negative impact on women's social, physical, and psychological wellbeing. Initial treatment includes lifestyle modifications, bladder retraining, pelvic floor exercises and pharmacological therapy. However, these measures are unsuccessful in 25-40% of women (refractory OAB). Before considering invasive treatments, such as Botulinum toxin injection or sacral neuromodulation, most guidelines recommend urodynamics to confirm diagnosis of detrusor overactivity (DO). However, urodynamics may fail to show evidence of DO in up to 45% of cases, hence the need to evaluate its effectiveness and cost-effectiveness. FUTURE (Female Urgency, Trial of Urodynamics as Routine Evaluation) aims to test the hypothesis that, in women with refractory OAB, urodynamics and comprehensive clinical assessment is associated with superior patient-reported outcomes following treatment and is more cost-effective, compared to comprehensive clinical assessment only. METHODS: FUTURE is a pragmatic, multi-centre, superiority randomised controlled trial. Women aged ≥ 18 years with refractory OAB or urgency predominant mixed urinary incontinence, and who have failed/not tolerated conservative and medical treatment, are considered for trial entry. We aim to recruit 1096 women from approximately 60 secondary/tertiary care hospitals across the UK. All consenting women will complete questionnaires at baseline, 3 months, 6 months and 15 months post-randomisation. The primary outcome is participant-reported success at 15 months post-randomisation measured using the Patient Global Impression of Improvement. The primary economic outcome is incremental cost per quality-adjusted life year gained at 15 months. The secondary outcomes include adverse events, impact on other urinary symptoms and health-related quality of life. Qualitative interviews with participants and clinicians and a health economic evaluation will also be conducted. The statistical analysis of the primary outcome will be by intention-to-treat. Results will be presented as estimates and 95% CIs. DISCUSSION: The FUTURE study will inform patients, clinicians and policy makers whether routine urodynamics improves treatment outcomes in women with refractory OAB and whether it is cost-effective. TRIAL REGISTRATION: ISRCTN63268739 . Registered on 14 September 2017.

Simultaneous in vivo comparison of water-filled and air-filled pressure measurement catheters: Implications for good urodynamic practice.

AIMS: This study aimed to evaluate whether the pressure readings obtained from air-filled catheters (AFCs) are the same as the readings from simultaneously inserted water-filled catheters (WFCs). It also aimed to make any possible recommendations for the use of AFCs to conform to International Continence Society (ICS) Good Urodynamic Practices (GUP). METHODS: Female patients undergoing urodynamic studies in a single center had water-filled and air-filled catheters simultaneously measuring abdominal and intravesical pressure during filling with saline and during voiding. The pressures recorded by each system at each event during the test were compared using paired t-test and Bland-Altman analyses. RESULTS: 62 patients were recruited, of whom 51 had pressures that could be compared during filling, and 23 during voiding. On average, the pressures measured by the two systems were not significantly different during filling and at maximum flow, but the values for a given patient were found to differ by up to 10 cmH2 O. CONCLUSIONS: This study shows that AFCs and WFCs cannot be assumed to register equal values of pressure. It has further shown that even when the pdet readings are compared with their value at the start of a test, a divergence of values of up to 10 cmH2 O remains. If AFCs are used, care must be taken to compensate for any pdet variations that occur during patient movement. Before AFCs are adopted, new normal values for resting pressures need to be developed to allow good quality AFC pressure readings to be made. Neurourol. Urodynam. 35:926-933, 2016. © 2015 Wiley Periodicals, Inc.

Quality control in urodynamics and the role of software support in the QC procedure.

AIMS: This article aims to identify quality control (QC) best practice, to review published QC audits in order to identify how closely good practice is followed, and to carry out a market survey of the software features that support QC offered by urodynamics machines available in the UK. METHODS AND RESULTS: All UK distributors of urodynamic systems were contacted and asked to provide information on the software features relating to data quality of the products they supply. The results of the market survey show that the features offered by manufacturers differ greatly. Automated features, which can be turned off in most cases, include: cough recognition, detrusor contraction detection, and high pressure alerts. There are currently no systems that assess data quality based on published guidelines. A literature review of current QC guidelines for urodynamics was carried out; QC audits were included in the literature review to see how closely guidelines were being followed. This review highlights the fact that basic QC is not being carried out effectively by urodynamicists. CONCLUSION: Based on the software features currently available and the results of the literature review there is both the need and capacity for a greater degree of automation in relation to urodynamic data quality and accuracy assessment. Some progress has been made in this area and certain manufacturers have already developed automated cough detection.

Bladder compliance what does it represent: can we measure it, and is it clinically relevant?

AIMS: To report the conclusion of the Think Thank 8 on Compliance Discussions during the second ICI-RS meeting in 2010. METHODS: During a 3-day meeting a group of specialists discussed bladder compliance, what it represents, how it can be measured and if it is clinically relevant. RESULTS: Bladder compliance is the result of a mathematical calculation of the volume required for a unit rise of pressure measured during a cystometric filling. It gives an indication on how the different mechanisms in the bladder wall react on stretching. There is a need of standardization of measurement and suggestions for this are given in the text. Pitfalls are described and how to avoid them. There is a wide range of compliance values in healthy volunteers and groups of patients. Poor compliance needs to be defined better as it can have significant clinical consequences. Prevention and treatment are discussed. CONCLUSION: If compliance is correctly measured and interpreted, it has importance in urodynamic testing and gives information relevant for clinical management.

Antibodies in crevicular fluid: an epidemiological tool for investigation of waterborne disease.

A significant challenge in the epidemiological investigation of recreational waterborne disease is the establishment of a definite association between exposure to a contaminated water and infection. An increase in specific antibodies as a result of infection is a potent measure of disease exposure and its determination would enhance epidemiological studies of waterborne diseases. We report on the automated detection of HAV antibodies in crevicular fluid and its use in a field study. The method is easy to use, non-invasive, could be applied to volunteers of all ages and is comparable in sensitivity to serological procedures. Application to an epidemiological study of water recreationalists demonstrated that surfers were three times more likely to be immune to hepatitis A virus than either wind-surfers or a control group without recreational water contact.

The two forms of karyogamy transcription factor Kar4p are regulated by differential initiation of transcription, translation, and protein turnover.

Kar4p is a transcription factor in Saccharomyces cerevisiae that is required for the expression of karyogamy-specific genes during mating, for the efficient transit from G1 during mitosis, and for essential functions during meiosis. Kar4p exists in two forms: a constitutive slower-migrating form, which predominates during vegetative growth, and a faster-migrating form, which is highly induced by mating pheromone. Transcript mapping of KAR4 revealed that the constitutive mRNA was initiated upstream of two in-frame ATG initiation codons, while the major inducible mRNA originated between them. Thus, the two forms of Kar4p are derived from the translation of alternative transcripts, which possess different AUG initiation codons. Site-directed mutations were constructed to inactivate one or the other of the initiation codons, allowing the expression of the two Kar4p forms separately. At normal levels of expression, the constitutive form of Kar4p did not support wild-type levels of mating. However, the two forms of Kar4p could also be expressed separately from the regulatable GAL1 promoter, and no functional difference was detected when they were expressed at equivalent levels. Pulse-chase experiments showed that the induced form of Kar4p was highly expressed and stable during mating but rapidly turned over in vegetative cells. In contrast, the constitutively expressed longer form showed the same rate of turnover regardless of the growth condition. Furthermore, overexpression of either form of Kar4p in vegetative cells was toxic. Thus, the elaborate regulation of the two forms of Kar4p at the levels of transcription, translation, and protein turnover reflects the requirement for high levels of the protein during mating and for low levels during the subsequent phases of the cell cycle.

The kinesin-related proteins, Kip2p and Kip3p, function differently in nuclear migration in yeast.

The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated. Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants. By indirect immunofluorescence, the cytoplasmic microtubules in kip2Delta were consistently short or absent throughout the cell cycle. In contrast, in kip3Delta strains, the cytoplasmic microtubules were significantly longer than wild type at telophase. Furthermore, in the kip3Delta cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud. Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules. Genetic analysis demonstrated that the kip2Delta kar9Delta double mutants were synthetically lethal, whereas kip3Delta kar9Delta double mutants were viable. Conversely, kip3Delta dhc1Delta double mutants were synthetically lethal, whereas kip2Delta dhc1Delta double mutants were viable. We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms. We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration. Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.

Distinct morphological phenotypes of cell fusion mutants.

Cell fusion in yeast is the process by which two haploid cells fuse to form a diploid zygote. To dissect the pathway of cell fusion, we phenotypically and genetically characterized four cell fusion mutants, fus6/spa2, fus7/rvs161, fus1, and fus2. First, we examined the complete array of single and double mutants. In all cases but one, double mutants exhibited stronger cell fusion defects than single mutants. The exception was rvs161Delta fus2Delta, suggesting that Rvs161p and Fus2p act in concert. Dosage suppression analysis showed that Fus1p and Fus2p act downstream or parallel to Rvs161p and Spa2p. Second, electron microscopic analysis was used to define the mutant defects in cell fusion. In wild-type prezygotes vesicles were aligned and clustered across the cell fusion zone. The vesicles were associated with regions of cell wall thinning. Analysis of Fus- zygotes indicated that Fus1p was required for the normal localization of the vesicles to the zone of cell fusion, and Spa2p facilitated their clustering. In contrast, Fus2p and Rvs161p appeared to act after vesicle positioning. These findings lead us to propose that cell fusion is mediated in part by the localized release of vesicles containing components essential for cell fusion.

Rvs161p interacts with Fus2p to promote cell fusion in Saccharomyces cerevisiae.

FUS7 was previously identified by a mutation that causes a defect in cell fusion in a screen for bilateral mating defects. Here we show that FUS7 is allelic to RVS161/END6, a gene implicated in a variety of processes including viability after starvation, endocytosis, and actin cytoskeletal organization. Two lines of evidence indicate that RVS161/END6's endocytic function is not required for cell fusion. First, several other endocytic mutants showed no cell fusion defects. Second, we isolated five function-specific alleles of RVS161/FUS7 that were defective for endocytosis, but not mating, and three alleles that were defective for cell fusion but not endocytosis. The organization of the actin cytoskeleton was normal in the cell fusion mutants, indicating that Rvs161p's function in cell fusion is independent of actin organization. The three to fourfold induction of RVS161 by mating pheromone and the localization of Rvs161p-GFP to the cell fusion zone suggested that Rvs161p plays a direct role in cell fusion. The phenotypes of double mutants, the coprecipitation of Rvs161p and Fus2p, and the fact that the stability of Fus2p was strongly dependent on Rvs161p's mating function lead to the conclusion that Rvs161p is required to interact with Fus2p for efficient cell fusion.

Cell fusion during yeast mating requires high levels of a-factor mating pheromone.

During conjugation, two yeast cells fuse to form a single zygote. Cell fusion requires extensive remodeling of the cell wall, both to form a seal between the two cells and to remove the intervening material. The two plasma membranes then fuse to produce a continuous cytoplasm. We report the characterization of two cell fusion defective (Fus-) mutants, fus5 and fus8, isolated previously in our laboratory. Fluorescence and electron microscopy demonstrated that the fus5 and fus8 mutant zygotes were defective for cell wall remodeling/removal but not plasma membrane fusion. Strikingly, fus5 and fus8 were a specific; both mutations caused the mutant phenotype when present in the MATa parent but not in the MAT alpha parent. Consistent with an a-specific defect, the fus5 and fus8 mutants produced less a-factor than the isogenic wild-type strain. FUS5 and FUS8 were determined to be allelic to AXL1 and RAM1, respectively, two genes known to be required for biogenesis of a-factor. Several experiments demonstrated that the partial defect in a-factor production resulted in the Fus- phenotype. First, overexpression of a-factor in the fus mutants suppressed the Fus- defect. Second, matings to an MAT alpha partner supersensitive to mating pheromone (sst2 delta) suppressed the Fus- defect in trans. Finally, the gene encoding a-factor, MFA1, was placed under the control of a repressible promoter; reduced levels of wild-type a-factor caused an identical cell fusion defect during mating. We conclude that high levels of pheromone are required as one component of the signal for prezygotes to initiate cell fusion.

Kar4p, a karyogamy-specific component of the yeast pheromone response pathway.

Karyogamy is the process whereby two haploid nuclei fuse to form a diploid nucleus during mating in Saccharomyces cerevisiae. Here, we describe the characterization of the KAR4 gene, previously identified in a screen for new nuclear fusion-defective mutants. During mating, kar4 mutants were defective for the microtubule-dependent movement of nuclei, a phenotype identical to that of mutations in KAR3 and CIK1. Consistent with its mutant phenotype, we found that the kar4 mutation resulted in failure to induce KAR3 and CIK1 mRNA during mating. Expression of KAR3 and CIK1 under independent regulatory control suppressed the kar4 defect, indicating that KAR4 is required primarily for the induction of KAR3 and CIK1. KAR4 was also required for meiosis, during which it may regulate KAR3; however, mitotic expression of KAR3 and CIK1 during S/G2 phase was independent of KAR4. A 30-bp region upstream of KAR3 conferred both KAR4- and STE12-dependent induction by mating pheromone. This region contained one moderate and two weak matches to the consensus pheromone response element to which the Ste12p transcriptional activator binds and five repeats of the sequence CAAA(A). Overproduction of Ste12p suppressed the kar4 defect in KAR3 induction and nuclear fusion. In contrast, Ste12p-independent expression of Kar4p did not alleviate the requirement for Ste12p during KAR3 induction. We propose that Kar4p assists Ste12p in the pheromone-dependent expression of KAR3 and CIK1. KAR4 defines a novel level of regulation for the pheromone response pathway, acting at a subset of Stel2p-inducible genes required for karyogamy.

Identification and characterization of CEN12 in the budding yeast Saccharomyces cerevisiae.

In this paper we report the cloning, sequencing and functional characterization of CEN12 and an associated autonomously replicating sequence (ARS) from the budding yeast Saccharomyces cerevisiae. In the course of studying a dynamin-related gene, DNM1, we previously physically mapped the gene to chromosome 12. Genetic mapping showed that the gene was tightly linked (0.35 cM) to the centromere. Subcloning experiments revealed that a centromere-like activity was included in a small segment of DNA immediately downstream from the DNM1 gene. Mitotic centromere activity was discerned by the ability of the region to de-stabilize a centromere-containing plasmid, and to stabilize an ARS-containing plasmid. Meiotic centromere activity was determined by the first-division segregation in crosses of ARS plasmids containing this region. The DNA sequence of this region revealed a sequence with strong homology to the consensus for yeast centromeres.

DNM1, a dynamin-related gene, participates in endosomal trafficking in yeast.

We identified DNM1, a novel dynamin-related gene in Saccharomyces cerevisiae. Molecular and genetic mapping showed that DNM1 is the most proximal gene to the right of centromere 12, and is predicted to encode a protein of 85 kD, designated Dnm1p. The protein exhibits 41% overall identity with full-length dynamin I and 55% identity with the most highly conserved 400-amino acid GTPase region. Our findings show that like mammalian dynamin, Dnm1p participates in endocytosis; however, it is unlikely to be a cognate homologue. Cells with a disruption in the DNM1 gene showed mating response defects consistent with a delay in receptor-mediated endocytosis. The half-life of the Ste3p pheromone receptor was increased two- to threefold in the dnm1 mutant, demonstrating that Dnm1p participates in the constitutive turnover of the receptor. To define the step in the endocytic pathway at which Dnm1p acts, we analyzed mutant strains at both early and late steps of the process. Initial internalization of epitope-tagged pheromone receptor or of labeled pheromone proceeded with wild-type kinetics. However, delivery of the internalized receptor to the vacuole was greatly impeded during ligand-induced endocytosis. These data suggest that during receptor-mediated endocytosis, Dnm1p acts after internalization, but before fusion with the vacuole. The dnm1 mutant was not defective for sorting of vacuolar proteins, indicating that Dnm1p is not required for transport from the late endosome to the vacuole. Therefore, we suggest that Dnm1p participates at a novel step before fusion with the late endosome.

Functional characterization of a replication initiator protein.

Functional domains in the RepI replication initiator protein have been identified by classical and site-directed mutagenesis techniques. Mutations conferring an increase in plasmid copy number contained alterations in a key position of a putative helix-turn-helix DNA binding motif. The mutations did not appear to affect autorepressing functions. Regions of RepI important for autorepression were localized as well. Two classes of mutations resulting in diminished autorepression functions were identified. One class was distinguished by an elevated copy number, while the other class remained at the wild-type copy number level. Analysis of the various mutations leading to changes in copy number or autorepressing functions suggest that in some cases the autorepression and initiating functions of the RepI protein are separable. Finally, analysis with deletion clones suggests that the trans-acting autorepressing functions of RepI might depend on intermolecular coupling control.

Characterization of the recA gene of Vibrio anguillarum.

We have cloned and sequenced the recA gene from two strains, 775 and 531A, of the fish pathogen, Vibrio anguillarum. Although both strains showed different sensitivities to methyl methanesulfonate (MMS), the recA genes were identical. In vitro expression of the V. anguillarum recA gene produced a polypeptide of about 40 kDa, in agreement with the value obtained from the nucleotide sequence. We identified the transcription start point by primer extension. The promoter for the recA gene mapped to an SOS regulatory element. The presence of an SOS box suggests that a LexA-like mediated response system may exist in V. anguillarum. The deduced RecA amino acid sequence is highly homologous with Escherichia coli RecA and other RecA proteins. Domains important in RecA function are conserved. We provide a comparative analysis of the activities and features of RecA analogs from a variety of species. We observed that certain residues that could be important in protein conformation are conserved in RecA proteins across a diverse range of bacterial species.

Co-operative autoregulation of a replication protein gene.

In this work we present the localization and characterization of the repl promoter (Prepl) and show aspects of the regulation. Comparison of Prepl with other autoregulated replication protein gene promoters revealed similarities, but Prepl differs from some of these characterized promoters in not being regulated by the heat-shock RNA polymerase. Primer extension analysis showed that Prepl is contained within five helically aligned 18 base pair repeats, or 18-mers of the previously defined minimal origin. In addition, we find that Prepl is autoregulated by a trans-acting product encoded in the REPI region. Purified Repl protein binds to the 18-mer region of the origin, suggesting that the repl gene is autoregulated by the protein product. The autoregulation appears to be co-operative since decreasing the 18-mer binding site region results in a concomitant non-linear loss of autorepression. The deletion derivatives show a decreased ability to bind the Repl protein when compared with origin DNA containing all of the binding region. The diminished capacity of the various deletion derivatives to bind Repl in vitro correlates with the loss of autorepression seen in vivo.

Current capabilities of rehabilitation robots.

Predictions are often made of intelligent and independently mobile robots for the disabled, and researchers are continually improving laboratory systems. Reductions in the cost of the technology involved may lead to affordable devices by the end of the decade. Less ambitious goals must be adopted by those projects wishing to distribute robotic aids to the disabled in the next few years. A modest selling price dictates the use of existing components. Even with the advent of more advanced robots, cost considerations may still make simpler devices on attractive alternative. Excessive optimism of future capabilities should be avoided, lest unrealistic expectations of current robotic aids hamper their development. Progress at all levels of rehabilitation robotics is complementary.

Clinical experience in rehabilitation robotics.

A robotic workstation system for the disabled, based on a commercially available arm, was tested with six patients at the Spinal Injuries Unit, Odstock Hospital, Salisbury. A questionnaire was administered to those who used the system. Users evaluated the usefulness and performance of the system and commented on their reactions to the use of robots in rehabilitation. The users were generally favourable as regards the ease of use of the system using a two-switch input, operating a scanning menu. All users wanted the robot to be able to replay previously created routines, and the majority also wanted to be able to directly control the robot as well. The users were unsure about the potential usefulness of the system. Because a robot is by definition a flexible device, the context in which it is introduced will effect the way it is received by potential users. Tests in a hospital environment are useful because there is a high concentration of users in their own home situations will give a better idea of the usefulness of such devices. The system was not ideal from the point of visibility and layout, and was too large for use in a domestic environment. The layout was largely dictated by the geometry of the manipulator. Therefore a new workstation system has been constructed using a purpose built manipulator. This new system particularly aims to overcome the poor layout of the earlier workstation and benefits from feedback from users.

Roles of DNA adenine methylation in controlling replication of the REPI replicon of plasmid pColV-K30.

DNA adenine methylation controls DNA replication of plasmids containing the prototypic REPI replicon by affecting protein recognition and by altering the helical stability of the origin. Denaturing gradient gel electrophoresis shows that adenine methylated origin DNA is more easily melted than unmethylated. However, because an added DNA adenine methylation (dam) site at the origin, whether in or out of phase with other helically aligned dam sites, actually prevents replication, we conclude that destabilization of the helix is not the exclusive function of adenine methylation in REPI replication. We find that the conformation and degree of methylation at the origin, features which are important for protein recognition, are essential for replication. In fact, RepI, a protein required for replication initiation at REPI replicons, contains a region homologous with a domain in proteins which specifically recognize and bind 5'-GATC-3'. We propose that the dam sites in the origin play a dual role: one is destabilization of the helix, and the other is protein recognition.