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Men or mice with homozygous serine/threonine kinase 33 (STK33) mutations are sterile owing to defective sperm morphology and motility. To chemically evaluate STK33 for male contraception with STK33-specific inhibitors, we screened our multibillion-compound collection of DNA-encoded chemical libraries, uncovered potent STK33-specific inhibitors, determined the STK33 kinase domain structure bound with a truncated hit CDD-2211, and generated an optimized hit CDD-2807 that demonstrates nanomolar cellular potency (half-maximal inhibitory concentration = 9.2 nanomolar) and favorable metabolic stability. In mice, CDD-2807 exhibited no toxicity, efficiently crossed the blood-testis barrier, did not accumulate in brain, and induced a reversible contraceptive effect that phenocopied genetic STK33 perturbations without altering testis size. Thus, STK33 is a chemically validated, nonhormonal contraceptive target, and CDD-2807 is an effective tool compound.
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Anticoncepção , Anticoncepcionais Masculinos , Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases , Bibliotecas de Moléculas Pequenas , Animais , Humanos , Masculino , Camundongos , Barreira Hematotesticular/metabolismo , Anticoncepcionais Masculinos/química , Anticoncepcionais Masculinos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Testículo/efeitos dos fármacos , Anticoncepção/métodos , Relação Estrutura-AtividadeRESUMO
As a key regulator of the innate immune system, the NLRP3 inflammasome responds to a variety of environmental insults through activation of caspase-1 and release of the proinflammatory cytokines IL-1ß and IL-18. Aberrant NLRP3 inflammasome function is implicated in numerous inflammatory diseases, spurring drug discovery efforts at NLRP3 as a therapeutic target. A diverse array of small molecules is undergoing preclinical/clinical evaluation with a reported mode of action involving direct modulation of the NLRP3 pathway. However, for a subset of these ligands the functional link between live-cell target engagement and pathway inhibition has yet to be fully established. Herein we present a cohort of mechanistic assays to both query direct NLRP3 engagement in cells, and functionally interrogate different nodes of NLRP3 pathway activity. This system enabled the stratification of potency for five confirmed NLRP3 inhibitors, and identification of two reported NLRP3 inhibitors that failed to demonstrate direct pathway antagonism.
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Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Citocinas/metabolismo , Interleucina-1beta/metabolismoRESUMO
Kinases represent one of the most therapeutically tractable targets for drug discovery in the twenty-first century. However, confirming engagement and achieving intracellular kinase selectivity for small-molecule kinase inhibitors can represent noteworthy challenges. The NanoBRETTM platform enables broad-spectrum live-cell kinase selectivity profiling in most laboratory settings, without advanced instrumentation or expertise. However, the prototype workflow for this selectivity profiling is currently limited to manual liquid handling and 96-well plates. Herein, we describe a scalable workflow with automation and acoustic dispensing, thus dramatically improving the throughput. Such adaptations enable profiling of larger compound sets against 192 full-length protein kinases in live cells, with statistical robustness supporting quantitative analysis.
Assuntos
Ensaios de Triagem em Larga Escala , Proteínas Quinases , Proteínas Quinases/metabolismo , Descoberta de DrogasRESUMO
RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers. The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex. Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF complex in cells. RAF target engagement can be measured in the presence or absence of any mutant KRAS allele, enabling the high-affinity state of RAF dimer inhibitors to be quantified in the cellular milieu. The intracellular protomer selectivity of clinical-stage type II RAF inhibitors revealed that ARAF protomer engagement, but not engagement of BRAF or CRAF, is commensurate with inhibition of MAPK signaling in various mutant RAS cell lines. Our results support a fundamental role for ARAF in mutant RAS signaling and reveal poor ARAF protomer vulnerability for a cohort of RAF inhibitors undergoing clinical evaluation.
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Proteínas Proto-Oncogênicas B-raf , Transdução de Sinais , Humanos , Subunidades Proteicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Linhagem Celular Tumoral , Inibidores de Proteínas Quinases/farmacologia , Mutação , Sistema de Sinalização das MAP QuinasesRESUMO
DNA-encoded libraries (DELs) provide unmatched chemical diversity and starting points for novel drug modalities. Here, we describe a workflow that exploits the bifunctional attributes of DEL ligands as a platform to generate BRET probes for live cell target engagement studies. To establish proof of concept, we performed a DEL screen using aurora kinase A and successfully converted aurora DEL ligands as cell-active BRET probes. Aurora BRET probes enabled the validation and stratification of the chemical series identified from primary selection data. Furthermore, we have evaluated the effective repurposing of pre-existing DEL screen data to find suitable leads for BRET probe development. Our findings support the use of DEL workflows as an engine to create cell-active BRET probes independent of structure or compound SAR. The combination of DEL and BRET technology accelerates hit-to-lead studies in a live cell setting.
Assuntos
Pesquisa , LigantesRESUMO
PLK1 is a protein kinase that regulates mitosis and is both an important oncology drug target and a potential antitarget of drugs for the DNA damage response pathway or anti-infective host kinases. To expand the range of live cell NanoBRET target engagement assays to include PLK1, we developed an energy transfer probe based on the anilino-tetrahydropteridine chemotype found in several selective PLK inhibitors. Probe 11 was used to configure NanoBRET target engagement assays for PLK1, PLK2, and PLK3 and measure the potency of several known PLK inhibitors. In-cell target engagement for PLK1 was in good agreement with the reported cellular potency for the inhibition of cell proliferation. Probe 11 enabled the investigation of the promiscuity of adavosertib, which had been described as a dual PLK1/WEE1 inhibitor in biochemical assays. Live cell target engagement analysis of adavosertib via NanoBRET demonstrated PLK activity at micromolar concentrations but only selective engagement of WEE1 at clinically relevant doses.
Assuntos
Proteínas de Ciclo Celular , Proteínas Serina-Treonina Quinases , Proteínas de Ciclo Celular/metabolismo , Proteínas Quinases , Proliferação de Células , Mitose , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Interval training has been found to lower glucose concentrations and increase insulin sensitivity in males but not in females, which may be due to inherent sex-based differences in metabolism. Twenty-four (12/sex) participants completed a bout of high-intensity interval exercise (HIIE, 10 × 1 min at 90% HRmax) to evaluate whether sex influenced the physiological effects of HIIE on postexercise glycemic control during an oral glucose tolerance test (OGTT). Given that body anthropometrics influence postprandial glucose, data were also expressed as a function of the normalized glucose dose. In addition, we examined whether sex differences in postexercise glycemic control were related to sex differences in muscle metabolism and/or insulin signaling proteins. HIIE increased insulin sensitivity in both sexes as characterized by the Matsuda (P = 0.03, ηp2= 0.20) and HOMA-IR (P = 0.047, ηp2 = 0.17) indices. HIIE also lowered insulin concentration during the OGTT (P = 0.04, ηp2 = 0.18) as compared with control. When normalized for glucose dose relative to lean mass, glucose area under the curve (AUC) was lower in females than in males (P ≤ 0.001, ηp2 = 0.47). TBC1D1 Ser237 phosphorylation increased in males, but not in females, postexercise (P = 0.03, ηp2 = 0.19). There was no difference in total insulin signaling protein content, muscle glycogen utilization, or AMPK activation during exercise between the sexes. These findings indicate that when the glucose dose is normalized for differences in body composition glycemic handling is better in females and that an acute bout of HIIE improves insulin sensitivity equally in healthy males and females.
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Treinamento Intervalado de Alta Intensidade , Resistência à Insulina , Humanos , Feminino , Masculino , Fosforilação , Exercício Físico/fisiologia , Resistência à Insulina/fisiologia , Insulina/metabolismo , Glucose/metabolismo , Glicemia/metabolismo , Proteínas Ativadoras de GTPase/metabolismoRESUMO
PLK1 is a protein kinase that regulates mitosis and is both an important oncology drug target and a potential anti target of drugs for the DNA damage response pathway or anti-infective host kinases. To expand the range of live cell NanoBRET target engagement assays to include PLK1 we developed an energy transfer probe based on the anilino-tetrahydropteridine chemotype found in several selective PLK inhibitors. Probe 11 was used to configure NanoBRET target engagement assays for PLK1, PLK2, and PLK3 and measure the potency of several known PLK inhibitors. In cell target engagement for PLK1 was in good agreement with the reported cellular potency for inhibition of cell proliferation. Probe 11 enabled investigation of the promiscuity of adavosertib, which had been described as a dual PLK1/WEE1 inhibitor in biochemical assays. Live cell target engagement analysis of adavosertib by NanoBRET demonstrated PLK activity at micromolar concentrations but only selective engagement of WEE1 at clinically relevant doses.
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Importance: Corticosteroids improve strength and function in boys with Duchenne muscular dystrophy. However, there is uncertainty regarding the optimum regimen and dosage. Objective: To compare efficacy and adverse effects of the 3 most frequently prescribed corticosteroid regimens in boys with Duchenne muscular dystrophy. Design, Setting, and Participants: Double-blind, parallel-group randomized clinical trial including 196 boys aged 4 to 7 years with Duchenne muscular dystrophy who had not previously been treated with corticosteroids; enrollment occurred between January 30, 2013, and September 17, 2016, at 32 clinic sites in 5 countries. The boys were assessed for 3 years (last participant visit on October 16, 2019). Interventions: Participants were randomized to daily prednisone (0.75 mg/kg) (n = 65), daily deflazacort (0.90 mg/kg) (n = 65), or intermittent prednisone (0.75 mg/kg for 10 days on and then 10 days off) (n = 66). Main Outcomes and Measures: The global primary outcome comprised 3 end points: rise from the floor velocity (in rise/seconds), forced vital capacity (in liters), and participant or parent global satisfaction with treatment measured by the Treatment Satisfaction Questionnaire for Medication (TSQM; score range, 0 to 100), each averaged across all study visits after baseline. Pairwise group comparisons used a Bonferroni-adjusted significance level of .017. Results: Among the 196 boys randomized (mean age, 5.8 years [SD, 1.0 years]), 164 (84%) completed the trial. Both daily prednisone and daily deflazacort were more effective than intermittent prednisone for the primary outcome (P < .001 for daily prednisone vs intermittent prednisone using a global test; P = .017 for daily deflazacort vs intermittent prednisone using a global test) and the daily regimens did not differ significantly (P = .38 for daily prednisone vs daily deflazacort using a global test). The between-group differences were principally attributable to rise from the floor velocity (0.06 rise/s [98.3% CI, 0.03 to 0.08 rise/s] for daily prednisone vs intermittent prednisone [P = .003]; 0.06 rise/s [98.3% CI, 0.03 to 0.09 rise/s] for daily deflazacort vs intermittent prednisone [P = .017]; and -0.004 rise/s [98.3% CI, -0.03 to 0.02 rise/s] for daily prednisone vs daily deflazacort [P = .75]). The pairwise comparisons for forced vital capacity and TSQM global satisfaction subscale score were not statistically significant. The most common adverse events were abnormal behavior (22 [34%] in the daily prednisone group, 25 [38%] in the daily deflazacort group, and 24 [36%] in the intermittent prednisone group), upper respiratory tract infection (24 [37%], 19 [29%], and 24 [36%], respectively), and vomiting (19 [29%], 17 [26%], and 15 [23%]). Conclusions and Relevance: Among patients with Duchenne muscular dystrophy, treatment with daily prednisone or daily deflazacort, compared with intermittent prednisone alternating 10 days on and 10 days off, resulted in significant improvement over 3 years in a composite outcome comprising measures of motor function, pulmonary function, and satisfaction with treatment; there was no significant difference between the 2 daily corticosteroid regimens. The findings support the use of a daily corticosteroid regimen over the intermittent prednisone regimen tested in this study as initial treatment for boys with Duchenne muscular dystrophy. Trial Registration: ClinicalTrials.gov Identifier: NCT01603407.
Assuntos
Glucocorticoides , Distrofia Muscular de Duchenne , Prednisona , Criança , Pré-Escolar , Feminino , Glucocorticoides/administração & dosagem , Glucocorticoides/efeitos adversos , Glucocorticoides/uso terapêutico , Humanos , Masculino , Distrofia Muscular de Duchenne/tratamento farmacológico , Prednisona/administração & dosagem , Prednisona/efeitos adversos , Prednisona/uso terapêutico , Pregnenodionas/efeitos adversosRESUMO
This protocol is used to profile the engagement of kinase inhibitors across nearly 200 kinases in a live-cell context. This protocol utilizes one single kinase tracer (NanoBRET(TM) Tracer K10) that operates quantitatively at four different concentrations. Minimizing the number of tracers offers a significant workflow improvement over the previous protocol that utilized a combination of 6 tracers. Each NanoBRET(TM) kinase assay is built using commercially available plasmids and has been optimized for NanoLuc tagging orientation, diluent DNA, and tracer concentration. For complete details on the use and execution of this protocol, please refer to Vasta et al. (2018).
Assuntos
Bioensaio , Fosfotransferases , LuciferasesRESUMO
Skeletal muscle is composed of fiber types that differ in mitochondrial content, antioxidant capacity, and susceptibility to apoptosis. Ceramides have been linked to oxidative stress-mediated apoptotic intracellular signalling and the enzyme neutral sphingomyelinase (nSMase) is, in part, responsible for generating these ceramides through the hydrolysis of sphingomyelin. Despite the role of ceramides in mediating apoptosis, there is a gap in the literature regarding nSMase in skeletal muscle mitochondria. This study aimed to characterize total nSMase activity and individual isoform expression in isolated subsarcolemmal (SS) mitochondria from soleus, diaphragm, plantaris, and extensor digitorum longus (EDL). Total nSMase activity did not differ between muscle types. nSMase2 content was detectable in all muscles and higher in EDL, soleus, and plantaris compared to diaphragm whereas nSMase3 was undetectable in all muscles. Finally, total nSMase activity positively correlated to nSMase2 protein content in soleus but not the other muscles. These findings suggest that nSMase associated with SS mitochondria may play a role in intracellular signalling processes involving ceramides in skeletal muscle and nSMase2 may be the key isoform, specifically in slow twitch muscle like soleus. Further studies are needed to fully elucidate the specific contribution of nSMase, along with the role of the various isoforms and mitochondrial subpopulation in generating mitochondrial ceramides in skeletal muscle, and its potential effects on mediating apoptosis.
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Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Diafragma/metabolismo , Regulação da Expressão Gênica , Masculino , Oxazinas/metabolismo , RatosRESUMO
Skeletal muscle, a highly active tissue, makes up 40% of the total body weight. This tissue relies on mitochondria for ATP production, calcium homeostasis, and programed cell death. Mitochondrial phospholipid composition, namely, cardiolipin (CL), influences the functional efficiency of mitochondrial proteins, specifically cytochrome c. The interaction of CL with cytochrome c in the presence of free radicals induces structural and functional changes promoting peroxidase activity and cytochrome c release, a key event in the initiation of apoptosis. The CL acyl chain degree of saturation has been implicated in the cytochrome c to cytochrome c peroxidase transition in liposomal models. However, mitochondrial membranes are composed of differing CL acyl chain composition. Currently, it is unclear how differing CL acyl chain composition utilizing liposomes will influence the cytochrome c form and function as a peroxidase. Thus, this study examined the role of CL acyl chain saturation within liposomes broadly reflecting the relative CL composition of mitochondrial membranes from healthy and dystrophic mouse muscle on cytochrome c conformation and function. Despite no differences in protein conformation or function between healthy and dystrophic liposomes, cytochrome c's affinity to CL increased with greater unsaturation. These findings suggest that increasing CL acyl chain saturation, as implicated in muscle wasting diseases, may not influence cytochrome c transformation and function as a peroxidase but may alter its interaction with CL, potentially impacting further downstream effects.
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Cardiolipinas/metabolismo , Citocromos c/metabolismo , Peroxidases/metabolismo , Conformação Proteica , Animais , Antioxidantes/farmacologia , Lipossomos/metabolismo , Camundongos , Mitocôndrias/metabolismo , Peroxidases/farmacologia , Fosfolipídeos/metabolismo , Conformação Proteica/efeitos dos fármacosRESUMO
Muscle disuse rapidly induces insulin resistance (IR). Despite a relationship between intramyocellular lipid (IMCL) content and IR, during muscle-disuse IR develops before IMCL accumulation, suggesting that IMCL are not related to disuse-induced IR. However, recent studies show that it is not total IMCL content, but IMCL size and location that are related to IR. Changes in these IMCL parameters may occur prior to increases in IMCL content, thus contributing to disuse-induced IR. Omega-3 fatty acids may mitigate the effects of disuse on IR by preventing a decline in insulin signaling proteins. Twenty women (age 22 ± 3 yr) received either 5 g·day-1 omega-3 fatty acid or isoenergetic sunflower oil for 4 wk prior to, throughout 2 wk of single-leg immobilization, and during 2 wk of recovery. Changes in IMCL characteristics and insulin signaling proteins were examined in vastus lateralis samples taken before supplementation and immobilization, and following immobilization and recovery. Omega-3 supplementation had no effect. IMCL area density decreased in the subsarcolemmal region during immobilization and recovery (-19% and -56%, respectively, P = 0.009). IMCL size increased in the central intermyofibrillar region during immobilization (43%, P = 0.007), returning to baseline during recovery. PLIN5 and AKT increased during immobilization (87%, P = 0.002; 30%, P = 0.007, respectively). PLIN 5 remained elevated and AKT increased further (15%) during recovery. IRS1, AS160, and GLUT4 decreased during immobilization (-35%, P = 0.001; -44%, P = 0.03; -56%, P = 0.02, respectively), returning to baseline during recovery. Immobilization alters IMCL storage characteristics while negatively affecting unstimulated insulin signaling protein content in young women.NEW & NOTEWORTHY We report that the subcellular storage location of IMCL is altered by limb immobilization, highlighting the need to evaluate IMCL storage location when assessing the effects of disuse on IMCL content. We also found that AKT content increased during immobilization in our female population, contrary to studies in males finding that AKT decreases during disuse, highlighting that men and women may respond differently to disuse and the necessity to include women in all research.
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Resistência à Insulina , Perna (Membro) , Adulto , Feminino , Transportador de Glucose Tipo 4/metabolismo , Humanos , Imobilização/efeitos adversos , Metabolismo dos Lipídeos , Lipídeos , Masculino , Músculo Esquelético/metabolismo , Músculo Quadríceps/metabolismo , Adulto JovemRESUMO
Repurposing biological samples collected for required diagnostic purposes into suitable biobanking projects is a particularly useful method for enabling research in vulnerable populations. This approach is especially appropriate for the neonate in the neonatal intensive care unit (NICU), where blood volume reductions can quickly increase beyond minimal risk for adverse events, such as iatrogenic anemia, and proxy consent provided by parents or guardians is required. The method described in this study provides a framework to prospectively collect and store blood-derived clinical samples after all clinical and regulatory requirements are fulfilled. The consent approach incorporated a 30-day window to allow parents and guardians ample consideration time with follow-up involvement with NICU embedded study team members. The study enrolled 875 participants over a 3-year period. This established a critically needed biobank to support investigator-initiated research with explicit study aims requiring samples at defined day of life frequencies within the NICU and created a normative control reference bank for case comparisons for premature and full-term neonates with brain injury.
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Bancos de Espécimes Biológicos , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Unidades de Terapia Intensiva Neonatal , Pais , Estudos ProspectivosRESUMO
BACKGROUND: Men who suffer recurrence of bulbar urethral stricture have to decide between endoscopic urethrotomy and open urethroplasty to manage their urinary symptoms. Evidence of relative clinical effectiveness and cost-effectiveness is lacking. OBJECTIVES: To assess benefit, harms and cost-effectiveness of open urethroplasty compared with endoscopic urethrotomy as treatment for recurrent urethral stricture in men. DESIGN: Parallel-group, open-label, patient-randomised trial of allocated intervention with 6-monthly follow-ups over 24 months. Target sample size was 210 participants providing outcome data. Participants, clinicians and local research staff could not be blinded to allocation. Central trial staff were blinded when needed. SETTING: UK NHS with recruitment from 38 hospital sites. PARTICIPANTS: A total of 222 men requiring operative treatment for recurrence of bulbar urethral stricture who had received at least one previous intervention for stricture. INTERVENTIONS: A centralised randomisation system using random blocks allocated participants 1 : 1 to open urethroplasty (experimental group) or endoscopic urethrotomy (control group). MAIN OUTCOME MEASURES: The primary clinical outcome was control of urinary symptoms. Cost-effectiveness was assessed by cost per quality-adjusted life-year (QALY) gained over 24 months. The main secondary outcome was the need for reintervention for stricture recurrence. RESULTS: The mean difference in the area under the curve of repeated measurement of voiding symptoms scored from 0 (no symptoms) to 24 (severe symptoms) between the two groups was -0.36 [95% confidence interval (CI) -1.78 to 1.02; p = 0.6]. Mean voiding symptom scores improved between baseline and 24 months after randomisation from 13.4 [standard deviation (SD) 4.5] to 6 (SD 5.5) for urethroplasty group and from 13.2 (SD 4.7) to 6.4 (SD 5.3) for urethrotomy. Reintervention was less frequent and occurred earlier in the urethroplasty group (hazard ratio 0.52, 95% CI 0.31 to 0.89; p = 0.02). There were two postoperative complications requiring reinterventions in the group that received urethroplasty and five, including one death from pulmonary embolism, in the group that received urethrotomy. Over 24 months, urethroplasty cost on average more than urethrotomy (cost difference £2148, 95% CI £689 to £3606) and resulted in a similar number of QALYs (QALY difference -0.01, 95% CI -0.17 to 0.14). Therefore, based on current evidence, urethrotomy is considered to be cost-effective. LIMITATIONS: We were able to include only 69 (63%) of the 109 men allocated to urethroplasty and 90 (80%) of the 113 men allocated to urethrotomy in the primary complete-case intention-to-treat analysis. CONCLUSIONS: The similar magnitude of symptom improvement seen for the two procedures over 24 months of follow-up shows that both provide effective symptom control. The lower likelihood of further intervention favours urethroplasty, but this had a higher cost over the 24 months of follow-up and was unlikely to be considered cost-effective. FUTURE WORK: Formulate methods to incorporate short-term disutility data into cost-effectiveness analysis. Survey pathways of care for men with urethral stricture, including the use of enhanced recovery after urethroplasty. Establish a pragmatic follow-up schedule to allow national audit of outcomes following urethral surgery with linkage to NHS Hospital Episode Statistics. TRIAL REGISTRATION: Current Controlled Trials ISRCTN98009168. FUNDING: This project was funded by the NIHR Health Technology Assessment programme and will be published in full in Health Technology Assessment; Vol. 24, No. 61. See the NIHR Journals Library website for further project information.
The urethra carries urine from the bladder to the tip of the penis. Men can develop a condition called urethral stricture when part of the urethra narrows due to scarring. This can lead to difficulties in passing urine and can recur. There are two operations for urethral stricture. The standard approach is endoscopic urethrotomy. The alternative is open urethroplasty. This study wanted to find out which operation was preferable in terms of symptom control, time before further surgery and which operation was best value for the NHS. All aspects of the study were informed by patients. Two hundred and twenty-two men who had received at least one previous operation for stricture took part. The choice of operation was decided by chance (randomisation). Of these men, 113 were randomised to urethrotomy and 109 were randomised to urethroplasty. Following their operation, the men filled in questionnaires every 36 months for 2 years about their symptoms and if any further surgery was needed. The two groups were then compared. Of the 222 men who took part, 159 provided enough information for inclusion in the comparison (90 were in the urethrotomy group and 69 were in the urethroplasty group). The improvement over time in urinary symptoms was similar for the two groups. Men in the urethrotomy group were twice as likely to need a further operation over the 2-year study period. Very few men experienced serious complications. This study showed that both operations led to symptom improvement for men with recurrent urethral stricture. Urethroplasty, however, appears unlikely to offer good value for money for the NHS. Men needing treatment for recurrent urethral stricture can use this information to weigh up the pros and cons of each operation to decide with their clinical team which one to undergo.
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Estreitamento Uretral/cirurgia , Procedimentos Cirúrgicos Urológicos Masculinos/economia , Procedimentos Cirúrgicos Urológicos Masculinos/métodos , Adulto , Idoso , Análise Custo-Benefício , Endoscopia/efeitos adversos , Endoscopia/economia , Endoscopia/métodos , Humanos , Entrevistas como Assunto , Masculino , Pessoa de Meia-Idade , Modelos Econômicos , Qualidade de Vida , Anos de Vida Ajustados por Qualidade de Vida , Medicina Estatal , Avaliação da Tecnologia Biomédica , Reino Unido , Procedimentos Cirúrgicos Urológicos Masculinos/efeitos adversosRESUMO
BACKGROUND: Urethral stricture affects 0.9% of men. Initial treatment is urethrotomy. Approximately, half of the strictures recur within 4â¯yr. Options for further treatment are repeat urethrotomy or open urethroplasty. OBJECTIVE: To compare the effectiveness and cost-effectiveness of urethrotomy with open urethroplasty in adult men with recurrent bulbar urethral stricture. DESIGN, SETTING, AND PARTICIPANTS: This was an open label, two-arm, patient-randomised controlled trial. UK National Health Service hospitals were recruited and 222 men were randomised to receive urethroplasty or urethrotomy. INTERVENTION: Urethrotomy is a minimally invasive technique whereby the narrowed area is progressively widened by cutting the scar tissue with a steel blade mounted on a urethroscope. Urethroplasty is a more invasive surgery to reconstruct the narrowed area. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was the profile over 24 mo of a patient-reported outcome measure, the voiding symptom score. The main clinical outcome was time until reintervention. RESULTS AND LIMITATIONS: The primary analysis included 69 (63%) and 90 (81%) of those allocated to urethroplasty and urethrotomy, respectively. The mean difference between the urethroplasty and urethrotomy groups was -0.36 (95% confidence interval [CI] -1.74 to 1.02). Fifteen men allocated to urethroplasty needed a reintervention compared with 29 allocated to urethrotomy (hazard ratio [95% CI] 0.52 [0.31-0.89]). CONCLUSIONS: In men with recurrent bulbar urethral stricture, both urethroplasty and urethrotomy improved voiding symptoms. The benefit lasted longer for urethroplasty. PATIENT SUMMARY: There was uncertainty about the best treatment for men with recurrent bulbar urethral stricture. We randomised men to receive one of the following two treatment options: urethrotomy and urethroplasty. At the end of the study, both treatments resulted in similar and better symptom scores. However, the urethroplasty group had fewer reinterventions.
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Uretra/cirurgia , Estreitamento Uretral/cirurgia , Adulto , Análise Custo-Benefício , Endoscopia/economia , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva , Resultado do Tratamento , Procedimentos Cirúrgicos Urológicos Masculinos/economia , Procedimentos Cirúrgicos Urológicos Masculinos/métodosRESUMO
Concerted multidisciplinary efforts have led to the development of Cyclin-Dependent Kinase inhibitors (CDKi's) as small molecule drugs and chemical probes of intracellular CDK function. However, conflicting data has been reported on the inhibitory potency of CDKi's and a systematic characterization of affinity and selectivity against intracellular CDKs is lacking. We have developed a panel of cell-permeable energy transfer probes to quantify target occupancy for all 21 human CDKs in live cells, and present a comprehensive evaluation of intracellular isozyme potency and selectivity for a collection of 46 clinically-advanced CDKi's and tool molecules. We observed unexpected intracellular activity profiles for a number of CDKi's, offering avenues for repurposing of highly potent molecules as probes for previously unreported targets. Overall, we provide a broadly applicable method for evaluating the selectivity of CDK inhibitors in living cells, and present a refined set of tool molecules to study CDK function.
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Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proteínas Inibidoras de Quinase Dependente de Ciclina/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Proteína Quinase CDC2 , Quinase 2 Dependente de Ciclina , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Quinase 9 Dependente de Ciclina , Inibidores Enzimáticos/farmacologia , Células HEK293 , Humanos , Fosforilação , Relação Estrutura-AtividadeRESUMO
INTRODUCTION: Although cardiologists were 'late-comers' to the multidisciplinary team-contributing to the complex care of patients with Duchenne muscular dystrophy (DMD), they now recognise the importance of systematic cardiac surveillance and timely therapy to prolonged survival in patients with DMD. Empirical deployment of cardioactive medications has already improved outcomes, but the evidence base for clinical decision making is weak. Fundamental questions remain as to whether prophylactic therapy is justified and convincingly superior to prompt deployment of the same therapies once left ventricular (LV) dysfunction is detected. Even if it were, at what age should therapy be introduced and with what specific drugs? METHODS AND ANALYSIS: We are conducting a multicentre, parallel group, randomised, placebo-controlled study of combination therapy with an ACE inhibitor (perindopril) and a beta-blocker (bisoprolol) in boys with DMD aged 5-13 years, with normal LV function by echocardiographic criteria at the time of recruitment. Boys are being followed-up for a minimum of 3 years and a maximum of 5 years and undergo repeat assessments of LV function, heart rate and ECG, forced expiratory volume in the 1 s and forced vital capacity, adverse event reporting and quality of life at 6 monthly intervals.The primary outcome is change in LV function between active and placebo-treated participants over the course of the study. ETHICS AND DISSEMINATION: The study was approved by 'NRES Committee East Midlands - Derby'. The results will be disseminated through manuscript publications, an international workshop and presentations to scientific meetings and parent forums. TRANSLATIONAL ASPECTS: The study seeks to establish the evidence for prophylactic heart therapies for children with DMD, define the optimum age for their introduction and identify any safety concerns. ARTICLE SUMMARY: The protocol describes the design of an ongoing multicentre, double-blind, randomised placebo-controlled study to establish the evidence for the use of prophylactic heart therapies in children with DMD, define the optimum age for their introduction and identify any safety concerns. TRIAL REGISTRATION NUMBERS: EudraCT2007-005932-10 and ISRCTN50395346; Pre-results.