Histone deacetylases maintain expression of the pluripotent gene network via recruitment of RNA polymerase II to coding and noncoding loci.

Histone acetylation is a dynamic modification regulated by the opposing actions of histone acetyltransferases (HATs) and histone deacetylases (HDACs). Deacetylation of histone tails results in chromatin tightening, and therefore, HDACs are generally regarded as transcriptional repressors. Counterintuitively, simultaneous deletion of Hdac1 and Hdac2 in embryonic stem cells (ESCs) reduces expression of the pluripotency-associated transcription factors Pou5f1, Sox2, and Nanog (PSN). By shaping global histone acetylation patterns, HDACs indirectly regulate the activity of acetyl-lysine readers, such as the transcriptional activator BRD4. Here, we use inhibitors of HDACs and BRD4 (LBH589 and JQ1, respectively) in combination with precision nuclear run-on and sequencing (PRO-seq) to examine their roles in defining the ESC transcriptome. Both LBH589 and JQ1 cause a marked reduction in the pluripotent gene network. However, although JQ1 treatment induces widespread transcriptional pausing, HDAC inhibition causes a reduction in both paused and elongating polymerase, suggesting an overall reduction in polymerase recruitment. Using enhancer RNA (eRNA) expression to measure enhancer activity, we find that LBH589-sensitive eRNAs are preferentially associated with superenhancers and PSN binding sites. These findings suggest that HDAC activity is required to maintain pluripotency by regulating the PSN enhancer network via the recruitment of RNA polymerase II.

Prostate cancer-Exercise and Metformin Trial (Pre-EMpT): study protocol for a feasibility factorial randomized controlled trial in men with localised or locally advanced prostate cancer.

BACKGROUND: Evidence from observational studies have shown that moderate intensity physical activity can reduce risk of progression and cancer-specific mortality in participants with prostate cancer. Epidemiological studies have also shown participants taking metformin to have a reduced risk of prostate cancer. However, data from randomised controlled trials supporting the use of these interventions are limited. The Prostate cancer-Exercise and Metformin Trial examines that feasibility of randomising participants diagnosed with localised or locally advanced prostate cancer to interventions that modify physical activity and blood glucose levels. The primary outcomes are randomisation rates and adherence to the interventions over 6 months. The secondary outcomes include intervention tolerability and retention rates, measures of insulin-like growth factor I, prostate-specific antigen, physical activity, symptom-reporting, and quality of life. METHODS: Participants are randomised in a 2 × 2 factorial design to both a physical activity (brisk walking or control) and a pharmacological (metformin or control) intervention. Participants perform the interventions for 6 months with final measures collected at 12 months follow-up. DISCUSSION: Our trial will determine whether participants diagnosed with localised or locally advanced prostate cancer, who are scheduled for radical treatments or being monitored for signs of cancer progression, can be randomised to a 6 months physical activity and metformin intervention. The findings from our trial will inform a larger trial powered to examine the clinical benefits of these interventions. TRIAL REGISTRATION: Prostate Cancer Exercise and Metformin Trial (Pre-EMpT) is registered on the ISRCTN registry, reference number ISRCTN13543667 . Date of registration 2nd August 2018-retrospectively registered. First participant was recruited on 11th September 2018.

NeuroSAFE PROOF: study protocol for a single-blinded, IDEAL stage 3, multi-centre, randomised controlled trial of NeuroSAFE robotic-assisted radical prostatectomy versus standard robotic-assisted radical prostatectomy in men with localized prostate cancer.

BACKGROUND: Robotic radical prostatectomy (RARP) is a first-line curative treatment option for localized prostate cancer. Postoperative erectile dysfunction and urinary incontinence are common associated adverse side effects that can negatively impact patients' quality of life. Preserving the lateral neurovascular bundles (NS) during RARP improves functional outcomes. However, selecting men for NS may be difficult when there is concern about incurring in positive surgical margin (PSM) which in turn risks adverse oncological outcomes. The NeuroSAFE technique (intra-operative frozen section examination of the neurovascular structure adjacent prostate margin) can provide real-time pathological consult to promote optimal NS whilst avoiding PSM. METHODS: NeuroSAFE PROOF is a single-blinded, multi-centre, randomised controlled trial (RCT) in which men are randomly allocated 1:1 to either NeuroSAFE RARP or standard RARP. Men electing for RARP as primary treatment, who are continent and have good baseline erectile function (EF), defined by International Index of Erectile Function (IIEF-5) score > 21, are eligible. NS in the intervention arm is guided by the NeuroSAFE technique. NS in the standard arm is based on standard of care, i.e. a pre-operative image-based planning meeting, patient-specific clinical information, and digital rectal examination. The primary outcome is assessment of EF at 12 months. The primary endpoint is the proportion of men who achieve IIEF-5 score ≥ 21. A sample size of 404 was calculated to give a power of 90% to detect a difference of 14% between groups based on a feasibility study. Oncological outcomes are continuously monitored by an independent Data Monitoring Committee. Key secondary outcomes include urinary continence at 3 months assessed by the international consultation on incontinence questionnaire, rate of biochemical recurrence, EF recovery at 24 months, and difference in quality of life. DISCUSSION: NeuroSAFE PROOF is the first RCT of intra-operative frozen section during radical prostatectomy in the world. It is properly powered to evaluate a difference in the recovery of EF for men undergoing RARP assessed by patient-reported outcome measures. It will provide evidence to guide the use of the NeuroSAFE technique around the world. TRIAL REGISTRATION: NCT03317990 (23 October 2017). Regional Ethics Committee; reference 17/LO/1978.

Thulium laser transurethral vaporesection versus transurethral resection of the prostate for benign prostatic obstruction: the UNBLOCS RCT.

BACKGROUND: Transurethral resection of the prostate (TURP) is the standard operation for benign prostatic obstruction (BPO). Thulium laser transurethral vaporesection of the prostate (ThuVARP) vaporises and resects the prostate using a technique similar to TURP. The small amount of existing literature suggests that there may be potential advantages of ThuVARP over TURP. OBJECTIVE: To determine whether or not the outcomes from ThuVARP are equivalent to the outcomes from TURP in men with BPO treated in the NHS. DESIGN: A multicentre, pragmatic, randomised controlled parallel-group trial, with an embedded qualitative study and economic evaluation. SETTING: Seven UK centres - four university teaching hospitals and three district general hospitals. PARTICIPANTS: Men aged ≥ 18 years who were suitable to undergo TURP, presenting with bothersome lower urinary tract symptoms (LUTS) or urinary retention secondary to BPO. INTERVENTIONS: Patients were randomised 1 : 1 to receive TURP or ThuVARP and remained blinded. MAIN OUTCOME MEASURES: Two co-primary outcomes - patient-reported International Prostate Symptom Score (IPSS) and clinical measure of maximum urine flow rate (Qmax) at 12 months post surgery. RESULTS: In total, 410 men were randomised, 205 to each arm. The two procedures were equivalent in terms of IPSS [adjusted mean difference 0.28 points higher for ThuVARP (favouring TURP), 95% confidence interval (CI) -0.92 to 1.49 points]. The two procedures were not equivalent in terms of Qmax (adjusted mean difference 3.12 ml/second in favour of TURP, 95% CI 0.45 to 5.79 ml/second), with TURP deemed superior. Surgical outcomes, such as complications and blood transfusion rates, and hospital stay were similar for both procedures. Patient-reported urinary and sexual symptoms were also similar between the arms. Qualitative interviews indicated similar patient experiences with both procedures. However, 25% of participants in the ThuVARP arm did not undergo their randomised allocation, compared with 2% of participants in the TURP arm. Prostate cancer was also detected less frequently from routine histology after ThuVARP (65% lower odds of detection) in an exploratory analysis. The adjusted mean differences between the arms were similar for secondary care NHS costs (£9 higher for ThuVARP, 95% CI -£359 to £376) and quality-adjusted life-years (0.01 favouring TURP, 95% CI -0.04 to 0.01). LIMITATIONS: Complications were recorded in prespecified categories; those not prespecified were excluded owing to variable reporting. Preoperative Qmax and IPSS data could not be collected for participants with indwelling catheters, making adjustment for baseline status difficult. CONCLUSIONS: TURP was superior to ThuVARP in terms of Qmax, although both operations resulted in a Qmax considered clinically successful. ThuVARP also potentially resulted in lower detection rates of prostate cancer as a result of the smaller volume of tissue available for histology. Length of hospital stay after ThuVARP, anticipated to be a key benefit, was equal to that after TURP in this trial. Overall, both ThuVARP and TURP were effective procedures for BPO, with minor benefits in favour of TURP. Therefore, the results suggest that it may be appropriate that new treatment alternatives continue to be compared with TURP. FUTURE WORK: Longer-term follow-up to assess reoperation rates over time, and research into the comparative effectiveness of ThuVARP and TURP in large prostates. TRIAL REGISTRATION: Current Controlled Trials ISRCTN00788389. FUNDING: This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 41. See the NIHR Journals Library website for further project information.

An enlarged prostate can make it difficult, or even impossible, for a man to pass urine by blocking the urine flow from the bladder. This can cause significant problems, and 25,000 men in the UK each year are treated with an operation to relieve their symptoms. The standard operation [transurethral resection of the prostate (TURP)], which uses electricity to shave off the enlarged prostate, is successful, but it can have some complications. There is some evidence to suggest that laser surgery can lead to less blood loss and a shorter stay in hospital, but laser operations can be difficult for surgeons to carry out. This trial has looked at a procedure using a new type of laser called thulium, which uses a very similar surgical technique to TURP and has shown promising results so far. A total of 410 men needing a prostate operation received either TURP or a laser operation. Participants were unaware of which operation they received until the end of the study to ensure a fair comparison. Seven hospitals across the UK were involved over 4 years. The trial mainly assessed the benefits of the operations using a urinary symptom questionnaire completed by participants, and by measuring the speed of passing urine after surgery. Overall, both procedures achieved positive results, and participants expressed high levels of satisfaction with the outcomes. Participants who had either operation reported a similar improvement in urinary symptoms in their questionnaires. However, although both operations did a good job of improving the speed of passing urine, TURP was better. Participants experienced few complications, and the complications that did occur were similar after both operations, including levels of bleeding and time spent in hospital. The cost of the two operations to the NHS was also similar. Overall, we concluded that both operations are suitable for patients with prostate enlargement, with TURP showing some minor additional benefits.

The cost-effectiveness of transurethral resection of the prostate vs thulium laser transurethral vaporesection of the prostate in the UNBLOCS randomised controlled trial for benign prostatic obstruction.

OBJECTIVE: To determine the cost-effectiveness of the current 'gold standard' operation of transurethral resection of the prostate (TURP) compared to the new laser technique of thulium laser transurethral vaporesection of the prostate (ThuVARP) in men with benign prostatic obstruction (BPO) within the UK National Health Service (NHS). PATIENTS AND METHODS: The trial was conducted across seven UK centres (four university teaching hospitals and three district general hospitals). A total of 410 men aged ≥18 years presenting with either bothersome lower urinary tract symptoms (LUTS) or urinary retention secondary to BPO, and suitable for surgery, were randomised (whilst under anaesthetic) 1:1 to receive the TURP or ThuVARP procedure. Resource use in relation to the operation, initial inpatient stay, and subsequent use of NHS services was collected for 12 months from randomisation (equivalent to primary effectiveness outcome) using hospital records and patient questionnaires. Resources were valued using UK reference costs. Quality adjusted life years (QALYs) were calculated from the EuroQoL five Dimensions five Levels (EQ-5D-5L) questionnaire completed at baseline, 3- and 12-months. Total adjusted mean costs, QALYs and incremental Net Monetary Benefit statistics were calculated: cost-effectiveness acceptability curves and sensitivity analyses addressed uncertainty. RESULTS: The total adjusted mean secondary care cost over the 12 months in the TURP arm (£4244) was £9 (95% CI -£376, £359) lower than the ThuVARP arm (£4253). The ThuVARP operation took on average 21 min longer than TURP. The adjusted mean difference of QALYs (0.01 favouring TURP, 95% CI -0.01, 0.04) was similar between the arms. There is a 76% probability that TURP is the cost-effective option compared with ThuVARP at the £20 000 per QALY willingness to pay threshold used by National Institute for Health and Care Excellence (NICE). CONCLUSION: One of the anticipated benefits of the laser surgery, reduced length of hospital stay with an associated reduction in cost, did not materialise within the study. The longer duration of the ThuVARP procedure is important to consider, both from a patient perspective in terms of increased time under anaesthetic, and from a service delivery perspective. TURP remains a highly cost-effective treatment for men with BPO.

A Monte Carlo framework for managing biological variability in manufacture of autologous cell therapy from mesenchymal stromal cells therapies.

Manufacturing processes for autologous cell therapy need to reproducibly generate in specification (quality and quantity) clinical product. However, patient variability prevents the level of control of cell input material that could be achieved in a cell line or allogeneic-based process. We have applied literature data on bone marrow-derived mesenchymal stromal cells variability to estimate probability distributions for stem cell yields given underlying truncated normal distributions in total nucleated cell concentration, stem cell percentage and plausible aspirate volumes. Monte Carlo simulation identified potential variability in harvested stem cell number in excess of an order of magnitude. The source material variability was used to identify the proportion of donor manufacturing runs that would achieve a target yield specification of 2E7 cells in a fixed time window with given proliferative rates and different aspirate volumes. A rapid, screening, development approach was undertaken to assess culture materials and process parameters (T-flask surface, medium, feed schedule) to specify a protocol with identified proliferative rate and a consequent model-based target aspirate volume. Finally, four engineering runs of the candidate process were conducted and a range of relevant quality parameters measured including expression of markers CD105, CD73, CD44, CD45, CD34, CD11b, CD19, HLA-DR, CD146 (melanoma cell adhesion molecule), CD106 (vascular cell adhesion molecule) and SSEA-4, specific metabolic activity and vascular endothelial growth factor secretion, and osteogenic differentiation potential. Our approach of using estimated distributions from publicly available information provides a route for data-poor earl- stage developers to plan manufacture with defined risk based on rational assumptions; furthermore, the models produced by such assumptions can be used to evaluate candidate processes, and can be incrementally improved with accumulating distribution understanding or subdivision by new process variables.

Phase II randomised control feasibility trial of a nutrition and physical activity intervention after radical prostatectomy for prostate cancer.

OBJECTIVE: Dietary factors and physical activity may alter prostate cancer progression. We explored the feasibility of lifestyle interventions following radical prostatectomy for localised prostate cancer. DESIGN: Patients were recruited into a presurgical observational cohort; following radical prostatectomy, they were offered randomisation into a 2×3 factorial randomised controlled trial (RCT). SETTING: A single National Health Service trust in the South West of England, UK. PARTICIPANTS: Those with localised prostate cancer and listed for radical prostatectomy were invited to participate. RANDOMISATION: Random allocation was performed by the Bristol Randomised Trial Collaboration via an online system. INTERVENTIONS: Men were randomised into both a modified nutrition group (either increased vegetable and fruit, and reduced dairy milk; or lycopene supplementation; or control) and a physical activity group (brisk walking or control) for 6 months. BLINDING: Only the trial statistician was blind to allocations. PRIMARY OUTCOME MEASURES: Primary outcomes were measures of feasibility: randomisation rates and intervention adherence at 6 months. Collected at trial baseline, three and six months, with daily adherence reported throughout. Our intended adherence rate was 75% or above, the threshold for acceptable adherence was 90%. RESULTS: 108 men entered the presurgical cohort, and 81 were randomised into the postsurgical RCT (randomisation rate: 93.1%) and 75 completed the trial. Of 25 men in the nutrition intervention, 10 (40.0%; 95% CI 23.4% to 59.3%) adhered to the fruit and vegetable recommendations and 18 (72.0%; 95% CI 52.4% to 85.7%) to reduced dairy intake. Adherence to lycopene (n=28), was 78.6% (95% CI 60.5% to 89.8%), while 21/39 adhered to the walking intervention (53.8%; 95% CI 38.6% to 68.4%). Most men were followed up at 6 months (75/81; 92.6%). Three 'possibly related' adverse events were indigestion, abdominal bloating and knee pain. CONCLUSIONS: Interventions were deemed feasible, with high randomisation rates and generally good adherence. A definitive RCT is proposed. TRIAL REGISTRATION NUMBER: ISRCTN 99048944.

Results of a randomized, double blind, placebo controlled, crossover trial of melatonin for treatment of Nocturia in adults with multiple sclerosis (MeNiMS).

BACKGROUND: Nocturia is a common urinary symptom of multiple sclerosis (MS) which can affect quality of life (QoL) adversely. Melatonin is a hormone known to regulate circadian rhythm and reduce smooth muscle activity such as in the bladder. There is limited evidence supporting use of melatonin to alleviate urinary frequency at night in the treatment of nocturia. The aim of this study was to evaluate the effect of melatonin on the mean number of nocturia episodes per night in patients with MS. METHODS: A randomized, double blind, placebo controlled crossover trial was conducted. 34 patients with nocturia secondary to multiple sclerosis underwent a 4-day pre-treatment monitoring phase. The patients were randomized to receive either 2 mg per night (taken at bedtime) of capsulated sustained-release melatonin (Circadin®) or 1 placebo capsule for 6 weeks followed by a crossover to the other regimen for an additional 6 weeks after a 1-month washout period. RESULTS: From the 26 patients who completed the study, there was no significant difference observed in the signs or symptoms of nocturia when taking 2 mg melatonin compared to placebo. The primary outcome measure, mean number of nocturia episodes on bladder diaries, was 1.8/night at baseline, and 1.4/night on melatonin, compared with 1.6 for placebo (Medians 1.70, 1.50, and 1.30 respectively, p = 0.85). There was also no significant difference seen in LUTS, QoL and sleep quality when taking melatonin. No significant safety concerns arose. CONCLUSIONS: This small study suggests that a low dose of melatonin taken at bedtime may be ineffective therapy for nocturia in MS. TRIAL REGISTRATION: (EudraCT reference) 2012-00418321 registered: 25/01/13. ISRCTN Registry: ISRCTN38687869 .

Advanced quantitative imaging and biomechanical analyses of periosteal fibers in accelerated bone growth.

PURPOSE: The accepted mechanism explaining the accelerated growth following periosteal resection is that the periosteum serves as a mechanical restraint to restrict physeal growth. To test the veracity of this mechanism we first utilized Second Harmonic Generation (SHG) imaging to measure differences of periosteal fiber alignment at various strains. Additionally, we measured changes in periosteal growth factor transcription. Next we utilized SHG imaging to assess the alignment of the periosteal fibers on the bone both before and after periosteal resection. Based on the currently accepted mechanism, we hypothesized that the periosteal fibers adjacent to the physis should be more aligned (under tension) during growth and become less aligned (more relaxed) following metaphyseal periosteal resection. In addition, we measured the changes in periosteal micro- and macro-scale mechanics. METHODS: 30 seven-week old New Zealand White rabbits were sacrificed. The periosteum was imaged on the bone at five regions using SHG imaging. One centimeter periosteal resections were then performed at the proximal tibial metaphyses. The resected periosteal strips were stretched to different strains in a materials testing system (MTS), fixed, and imaged using SHG microscopy. Collagen fiber alignment at each strain was then determined computationally using CurveAlign. In addition, periosteal strips underwent biomechanical testing in both circumferential and axial directions to determine modulus, failure stress, and failure strain. Relative mRNA expression of growth factors: TGFß-1, -2, -3, Ihh, PTHrP, Gli, and Patched were measured following loading of the periosteal strips at physiological strains in a bioreactor. The periosteum adjacent to the physis of six tibiae was imaged on the bone, before and after, metaphyseal periosteal resection, and fiber alignment was computed. One-way ANOVA statistics were performed on all data. RESULTS: Imaging of the periosteum at different regions of the bone demonstrated complex regional differences in fiber orientation. Increasing periosteal strain on the resected strips increased periosteal fiber alignment (p<0.0001). The only exception to this pattern was the 10% strain on the tibial periosteum, which may indicate fiber rupture at this non-physiologic strain. Periosteal fiber alignment adjacent to the resection became less aligned while those adjacent to the physes remained relatively unchanged before and after periosteal resection. Increasing periosteal strain on the resected strips increased periosteal fiber alignment (p<0.0001). The only exception to this pattern was the 10% strain on the tibial periosteum, which may indicate fiber rupture (and consequent retraction) at this non-physiologic strain. Increasing periosteal strain revealed a significant increase in relative mRNA expression for Ihh, PTHrP, Gli, and Patched, respectively. CONCLUSION: Periosteal fibers adjacent to the growth plate do not appear under tension in the growing limb, and the alignments of these fibers remain unchanged following periosteal resection. SIGNIFICANCE: The results of this study call into question the long-accepted role of the periosteum acting as a simple mechanical tether restricting growth at the physis.

Acquisition of operant behavior in rats with delayed reinforcement: A retractable-lever procedure.

Experimentally naïve rats acquired lever pressing with delayed reinforcement when the immediate programmed consequence for lever pressing was the simultaneous retraction of two identical levers. Presses on one lever also produced access to sweetened condensed milk after a delay of 10s following retraction. Presses on the second lever resulted in retraction only. Lever retraction prevented the possibility of adventitious reinforcement of contacting the operanda during the reinforcement delays. Several measures indicated that the delayed reinforcers strengthened behavior. The majority of responses for all rats were on the lever that initiated reinforcer delivery. Responding for seven out of eight rats decreased during a subsequent extinction phase in which retraction was the only consequence arranged for lever pressing. Responding recovered rapidly when food reinforcement was available again. Furthermore, when contingencies on the two levers were switched, rats allocated their behavior accordingly, showing control by the delayed reinforcers.