TBC1D1 Ser237 phosphorylation, but not insulin sensitivity, is higher following a bout of high-intensity interval exercise in healthy males as compared with females.

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.

Development of Cell Permeable NanoBRET Probes for the Measurement of PLK1 Target Engagement in Live Cells.

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.

Effect of Different Corticosteroid Dosing Regimens on Clinical Outcomes in Boys With Duchenne Muscular Dystrophy: A Randomized Clinical Trial.

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.

Intensive versus standard physical rehabilitation therapy in the critically ill (EPICC): a multicentre, parallel-group, randomised controlled trial.

BACKGROUND: Early physical rehabilitation in the intensive care unit (ICU) has been shown to improve short-term clinical outcomes but long-term benefit has not been proven and the optimum intensity of rehabilitation is not known. METHODS: We conducted a randomised, parallel-group, allocation-concealed, assessor-blinded, controlled trial in patients who had received at least 48 hours of invasive or non-invasive ventilation. Participants were randomised in a 1:1 ratio, stratified by admitting ICU, admission type and level of independence. The intervention group had a target of 90 min physical rehabilitation per day, the control group a target of 30 min per day (both Monday to Friday). The primary outcome was the Physical Component Summary (PCS) measure of SF-36 at 6 months. RESULTS: We recruited 308 participants over 34 months: 150 assigned to the intervention and 158 to the control group. The intervention group received a median (IQR) of 161 (67-273) min of physical rehabilitation on ICU compared with 86 (31-139) min in the control group. At 6 months, 62 participants in the intervention group and 54 participants in the control group contributed primary outcome data. In the intervention group, 43 had died, 11 had withdrawn and 34 were lost to follow-up, while in the control group, 56 had died, 5 had withdrawn and 43 were lost to follow-up. There was no difference in the primary outcome at 6 months, mean (SD) PCS 37 (12.2) in the intervention group and 37 (11.3) in the control group. CONCLUSIONS: In this study, ICU-based physical rehabilitation did not appear to improve physical outcomes at 6 months compared with standard physical rehabilitation. TRIAL REGISTRATION NUMBER: ISRCTN 20436833.

Feasibility of trial procedures for a randomised controlled trial of a community based group exercise intervention for falls prevention for visually impaired older people: the VIOLET study.

BACKGROUND: Visually impaired older people (VIOP) have a higher risk of falling than their sighted peers, and are likely to avoid physical activity. The aim was to adapt the existing Falls Management Exercise (FaME) programme for VIOP, delivered in the community, and to investigate the feasibility of conducting a definitive randomised controlled trial (RCT) of this adapted intervention. METHODS: Two-centre randomised mixed methods pilot trial and economic evaluation of the adapted group-based FaME programme for VIOP versus usual care. A one hour exercise programme ran weekly over 12 weeks at the study sites (Newcastle and Glasgow), delivered by third sector (voluntary and community) organisations. Participants were advised to exercise at home for an additional two hours over the week. Those randomised to the usual activities group received no intervention. Outcome measures were completed at baseline, 12 and 24 weeks. The potential primary outcome was the Short Form Falls Efficacy Scale - International (SFES-I). Participants' adherence was assessed by reviewing attendance records and self-reported compliance to the home exercises. Adherence with the course content (fidelity) by instructors was assessed by a researcher. Adverse events were collected in a weekly phone call. RESULTS: Eighteen participants, drawn from community-living VIOP were screened; 68 met the inclusion criteria; 64 participants were randomised with 33 allocated to the intervention and 31 to the usual activities arm. 94% of participants provided data at the 12 week visit and 92% at 24 weeks. Adherence was high. The intervention was found to be safe with 76% attending nine or more classes. Median time for home exercise was 50 min per week. There was little or no evidence that fear of falling, balance and falls risk, physical activity, emotional, attitudinal or quality of life outcomes differed between trial arms at follow-up. CONCLUSIONS: The intervention, FaME, was implemented successfully for VIOP and all progression criteria for a main trial were met. The lack of difference between groups on fear of falling was unsurprising given it was a pilot study but there may have been other contributory factors including suboptimal exercise dose and apparent low risk of falls in participants. These issues need addressing for a future trial. TRIAL REGISTRATION: Current Controlled Trials ISRCTN ID: 16949845 Registered: 21 May 2015.

Evaluation of treatment outcome in 175 patients with Hodgkin lymphoma aged 60 years or over: the SHIELD study.

The SHIELD program for Hodgkin lymphoma in patients 60 years of age or older, prospectively evaluated clinical features and outcome in a large patient cohort (n = 175). The central element was a phase 2 study of VEPEMB chemotherapy (n = 103, median age 73 years) incorporating comorbidity assessment. A total of 72 other patients were treated off-study but registered prospectively and treated concurrently with: ABVD (n = 35); CLVPP (n = 19), or other (n = 18). Of VEPEMB patients, 31 had early-stage disease (stage 1A/2A) and received VEPEMB 3 times plus radiotherapy. Median follow-up was 36 months. Complete remission (CR) rate (intention-to-treat) was 74% and 3-year overall survival (OS) and progression-free survival (PFS) were 81% and 74%, respectively. A total of 72 patients had advanced-stage disease (stage 1B/2B/3 or 4) and received VEPEMB 6 times. CR rate was 61% with 3-year OS and PFS of 66% and 58%, respectively. Of patients achieving CR, 13% with early-stage and 5% with advanced-stage disease progressed. Overall treatment-related mortality was 7%. In patients treated with curative intent with VEPEMB, ABVD, and CLVPP (n = 157), CR linked to several factors in univariate analysis. In a Cox regression model only, obtaining CR remained significant for OS and CR plus comorbidity and age for PFS. RS-EBV status had no significant effect on outcome.

Interrogating direct NLRP3 engagement and functional inflammasome inhibition using cellular assays.

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.

Reversible male contraception by targeted inhibition of serine/threonine kinase 33.

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.

Independent validation of genes and polymorphisms reported to be associated with radiation toxicity: a prospective analysis study.

BACKGROUND: Several studies have reported associations between radiation toxicity and single nucleotide polymorphisms (SNPs) in candidate genes. Few associations have been tested in independent validation studies. This prospective study aimed to validate reported associations between genotype and radiation toxicity in a large independent dataset. METHODS: 92 (of 98 attempted) SNPs in 46 genes were successfully genotyped in 1613 patients: 976 received adjuvant breast radiotherapy in the Cambridge breast IMRT trial (ISRCTN21474421, n=942) or in a prospective study of breast toxicity at the Christie Hospital, Manchester, UK (n=34). A further 637 received radical prostate radiotherapy in the MRC RT01 multicentre trial (ISRCTN47772397, n=224) or in the Conventional or Hypofractionated High Dose Intensity Modulated Radiotherapy for Prostate Cancer (CHHiP) trial (ISRCTN97182923, n=413). Late toxicity was assessed 2 years after radiotherapy with a validated photographic technique (patients with breast cancer only), clinical assessment, and patient questionnaires. Association tests of genotype with overall radiation toxicity score and individual endpoints were undertaken in univariate and multivariable analyses. At a type I error rate adjusted for multiple testing, this study had 99% power to detect a SNP, with minor allele frequency of 0·35, associated with a per allele odds ratio of 2·2. FINDINGS: None of the previously reported associations were confirmed by this study, after adjustment for multiple comparisons. The p value distribution of the SNPs tested against overall toxicity score was not different from that expected by chance. INTERPRETATION: We did not replicate previously reported late toxicity associations, suggesting that we can essentially exclude the hypothesis that published SNPs individually exert a clinically relevant effect. Continued recruitment of patients into studies within the Radiogenomics Consortium is essential so that sufficiently powered studies can be done and methodological challenges addressed. FUNDING: Cancer Research UK, The Royal College of Radiologists, Addenbrooke's Charitable Trust, Breast Cancer Campaign, Cambridge National Institute of Health Research (NIHR) Biomedical Research Centre, Experimental Cancer Medicine Centre, East Midlands Innovation, the National Cancer Institute, Joseph Mitchell Trust, Royal Marsden NHS Foundation Trust, Institute of Cancer Research NIHR Biomedical Research Centre for Cancer.

Development of Cell Permeable NanoBRET Probes for the Measurement of PLK1 Target Engagement in Live Cells.

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.

NanoBRET™ Live-Cell Kinase Selectivity Profiling Adapted for High-Throughput Screening.

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.

Protomer selectivity of type II RAF inhibitors within the RAS/RAF complex.

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.

DELs enable the development of BRET probes for target engagement studies in cells.

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.

A population-based cytogenetic study of adults with acute lymphoblastic leukemia.

Chromosomal abnormalities are increasingly used to risk stratify adults with acute lymphoblastic leukemia. Published data describing the age-specific incidence of chromosomal abnormalities and their prognostic relevance are largely derived from clinical trials. Trials frequently have age restrictions and low recruitment rates. Thus we investigated these factors in a population-based cohort of 349 patients diagnosed during the course of 19 years in the northern part of England. The incidence of most chromosomal abnormalities varied significantly with age. The incidence of t(9;22)(q34;q11) increased in each successive decade, up to 24% among 40- to 49-year-old subjects. Thereafter the incidence reached a plateau. t(4;11)(q21;q23) and t(1;19)(q23;p13) were a rare occurrence among patients older than 60 years of age. In contrast, the frequency of t(8;14)(q24;q32) and t(14;18)(q32;q21) increased with age. High hyperdiploidy occurred in 13% of patients younger than 20 years of age but in only 5% of older patients. The incidence of low hypodiploidy/near-triploidy and complex karyotype increased with age from 4% (15-29 years) to 16% (>or= 60 years). Overall survival varied significantly by age and cytogenetics. Older patients and those with t(9;22), t(4;11), low hypodiploidy/near-triploidy, or complex karyotype had a significantly inferior outcome. These population-based results demonstrate the cytogenetic heterogeneity of adult acute lymphoblastic leukemia. These data will inform the delivery of routine clinical services and the design of new age-focused clinical trials.

Characterization of neutral sphingomyelinase activity and isoform expression in rodent skeletal muscle mitochondria.

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.

The effect of cardiolipin side chain composition on cytochrome c protein conformation and peroxidase activity.

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.

Single tracer-based protocol for broad-spectrum kinase profiling in live cells with NanoBRET.

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).