Ascl1 phospho-status regulates neuronal differentiation in a Xenopus developmental model of neuroblastoma.

Neuroblastoma (NB), although rare, accounts for 15% of all paediatric cancer mortality. Unusual among cancers, NBs lack a consistent set of gene mutations and, excluding large-scale chromosomal rearrangements, the genome seems to be largely intact. Indeed, many interesting features of NB suggest that it has little in common with adult solid tumours but instead has characteristics of a developmental disorder. NB arises overwhelmingly in infants under 2 years of age during a specific window of development and, histologically, NB bears striking similarity to undifferentiated neuroblasts of the sympathetic nervous system, its likely cells of origin. Hence, NB could be considered a disease of development arising when neuroblasts of the sympathetic nervous system fail to undergo proper differentiation, but instead are maintained precociously as progenitors with the potential for acquiring further mutations eventually resulting in tumour formation. To explore this possibility, we require a robust and flexible developmental model to investigate the differentiation of NB's presumptive cell of origin. Here, we use Xenopus frog embryos to characterise the differentiation of anteroventral noradrenergic (AVNA) cells, cells derived from the neural crest. We find that these cells share many characteristics with their mammalian developmental counterparts, and also with NB cells. We find that the transcriptional regulator Ascl1 is expressed transiently in normal AVNA cell differentiation but its expression is aberrantly maintained in NB cells, where it is largely phosphorylated on multiple sites. We show that Ascl1's ability to induce differentiation of AVNA cells is inhibited by its multi-site phosphorylation at serine-proline motifs, whereas overexpression of cyclin-dependent kinases (CDKs) and MYCN inhibit wild-type Ascl1-driven AVNA differentiation, but not differentiation driven by a phospho-mutant form of Ascl1. This suggests that the maintenance of ASCL1 in its multiply phosphorylated state might prevent terminal differentiation in NB, which could offer new approaches for differentiation therapy in NB.This work was supported by a grant from the UK Neuroblastoma Society (A.P., L.A.W. and T.D.P.). C.J.T. and L.A.W. are supported by the intramural research program of the National Cancer Institute, National Institutes of Health. L.A.W. is an NIH-OxCam Scholar. L.J.A.H. is supported by a UK Medical Research Council Doctoral Training Award.This is the final version of the article. It first appeared from The Company of Biologists via http://dx.doi.org/10.1242/dmm.01863

Dephosphorylation of the Proneural Transcription Factor ASCL1 Re-Engages a Latent Post-Mitotic Differentiation Program in Neuroblastoma.

Pediatric cancers often resemble trapped developmental intermediate states that fail to engage the normal differentiation program, typified by high-risk neuroblastoma arising from the developing sympathetic nervous system. Neuroblastoma cells resemble arrested neuroblasts trapped by a stable but aberrant epigenetic program controlled by sustained expression of a core transcriptional circuit of developmental regulators in conjunction with elevated MYCN or MYC (MYC). The transcription factor ASCL1 is a key master regulator in neuroblastoma and has oncogenic and tumor-suppressive activities in several other tumor types. Using functional mutational approaches, we find that preventing CDK-dependent phosphorylation of ASCL1 in neuroblastoma cells drives coordinated suppression of the MYC-driven core circuit supporting neuroblast identity and proliferation, while simultaneously activating an enduring gene program driving mitotic exit and neuronal differentiation. IMPLICATIONS: These findings indicate that targeting phosphorylation of ASCL1 may offer a new approach to development of differentiation therapies in neuroblastoma. VISUAL OVERVIEW: http://mcr.aacrjournals.org/content/molcanres/18/12/1759/F1.large.jpg.Work was supported by Cancer Research UK Programme Grant RG91505 (AP), Wellcome Trust Investigator Award 212253/Z/18/Z (AP), MRC Research Grant MR/L021129/1 (F.A, A.P); Neuroblastoma UK (D.M, T.P, A.P), CRUK Cambridge Centre Paediatric Programme (L.P), The Terry Fox Foundation (FA), MBRU College of Medicine Internal grant award MBRU-CM-RG2019-14 (FA), MBRU-ALMAHMEED Collaborative Research Award ALM1909 (FA) and core support from the Wellcome Trust and the MRC Cambridge Stem Cell Institute (F.A, D.M, J.D., A.P.) and Cancer Research UK Cambridge Insititute (I.C, J.C)

Somatic mutations and clonal dynamics in healthy and cirrhotic human liver.

The most common causes of chronic liver disease are excess alcohol intake, viral hepatitis and non-alcoholic fatty liver disease, with the clinical spectrum ranging in severity from hepatic inflammation to cirrhosis, liver failure or hepatocellular carcinoma (HCC). The genome of HCC exhibits diverse mutational signatures, resulting in recurrent mutations across more than 30 cancer genes1-7. Stem cells from normal livers have a low mutational burden and limited diversity of signatures8, which suggests that the complexity of HCC arises during the progression to chronic liver disease and subsequent malignant transformation. Here, by sequencing whole genomes of 482 microdissections of 100-500 hepatocytes from 5 normal and 9 cirrhotic livers, we show that cirrhotic liver has a higher mutational burden than normal liver. Although rare in normal hepatocytes, structural variants, including chromothripsis, were prominent in cirrhosis. Driver mutations, such as point mutations and structural variants, affected 1-5% of clones. Clonal expansions of millimetres in diameter occurred in cirrhosis, with clones sequestered by the bands of fibrosis that surround regenerative nodules. Some mutational signatures were universal and equally active in both non-malignant hepatocytes and HCCs; some were substantially more active in HCCs than chronic liver disease; and others-arising from exogenous exposures-were present in a subset of patients. The activity of exogenous signatures between adjacent cirrhotic nodules varied by up to tenfold within each patient, as a result of clone-specific and microenvironmental forces. Synchronous HCCs exhibited the same mutational signatures as background cirrhotic liver, but with higher burden. Somatic mutations chronicle the exposures, toxicity, regeneration and clonal structure of liver tissue as it progresses from health to disease.This work was supported by a Wellcome Trust and Cancer Research UK (CRUK) Grand Challenge Award (C98/A24032). P.J.C. is a Wellcome Trust Senior Clinical Fellow (WT088340MA); S.F.B. was supported by the Swiss National Science Foundation (P2SKP3-171753 and P400PB-180790); M.A.S. is supported by a Rubicon fellowship from NWO (019.153LW.038); the Cambridge Human Research Tissue Bank is supported by the NIHR Cambridge Biomedical Research Centre; and M.H. is supported by a CRUK Clinician Scientist Fellowship (C52489/A19924)

Comparative effectiveness and safety of non-vitamin K antagonists for atrial fibrillation in clinical practice: GLORIA-AF Registry

Background and purpose: Prospectively collected data comparing the safety and effectiveness of individual non-vitamin K antagonists (NOACs) are lacking. Our objective was to directly compare the effectiveness and safety of NOACs in patients with newly diagnosed atrial fibrillation (AF). Methods: In GLORIA-AF, a large, prospective, global registry program, consecutive patients with newly diagnosed AF were followed for 3 years. The comparative analyses for (1) dabigatran vs rivaroxaban or apixaban and (2) rivaroxaban vs apixaban were performed on propensity score (PS)-matched patient sets. Proportional hazards regression was used to estimate hazard ratios (HRs) for outcomes of interest. Results: The GLORIA-AF Phase III registry enrolled 21,300 patients between January 2014 and December 2016. Of these, 3839 were prescribed dabigatran, 4015 rivaroxaban and 4505 apixaban, with median ages of 71.0, 71.0, and 73.0 years, respectively. In the PS-matched set, the adjusted HRs and 95% confidence intervals (CIs) for dabigatran vs rivaroxaban were, for stroke: 1.27 (0.79–2.03), major bleeding 0.59 (0.40–0.88), myocardial infarction 0.68 (0.40–1.16), and all-cause death 0.86 (0.67–1.10). For the comparison of dabigatran vs apixaban, in the PS-matched set, the adjusted HRs were, for stroke 1.16 (0.76–1.78), myocardial infarction 0.84 (0.48–1.46), major bleeding 0.98 (0.63–1.52) and all-cause death 1.01 (0.79–1.29). For the comparison of rivaroxaban vs apixaban, in the PS-matched set, the adjusted HRs were, for stroke 0.78 (0.52–1.19), myocardial infarction 0.96 (0.63–1.45), major bleeding 1.54 (1.14–2.08), and all-cause death 0.97 (0.80–1.19). Conclusions: Patients treated with dabigatran had a 41% lower risk of major bleeding compared with rivaroxaban, but similar risks of stroke, MI, and death. Relative to apixaban, patients treated with dabigatran had similar risks of stroke, major bleeding, MI, and death. Rivaroxaban relative to apixaban had increased risk for major bleeding, but similar risks for stroke, MI, and death. Registration: URL: https://www.clinicaltrials.gov. Unique identifiers: NCT01468701, NCT01671007. Date of registration: September 2013

Anticoagulant selection in relation to the SAMe-TT2R2 score in patients with atrial fibrillation. the GLORIA-AF registry

Aim: The SAMe-TT2R2 score helps identify patients with atrial fibrillation (AF) likely to have poor anticoagulation control during anticoagulation with vitamin K antagonists (VKA) and those with scores >2 might be better managed with a target-specific oral anticoagulant (NOAC). We hypothesized that in clinical practice, VKAs may be prescribed less frequently to patients with AF and SAMe-TT2R2 scores >2 than to patients with lower scores. Methods and results: We analyzed the Phase III dataset of the Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation (GLORIA-AF), a large, global, prospective global registry of patients with newly diagnosed AF and ≥1 stroke risk factor. We compared baseline clinical characteristics and antithrombotic prescriptions to determine the probability of the VKA prescription among anticoagulated patients with the baseline SAMe-TT2R2 score >2 and ≤ 2. Among 17,465 anticoagulated patients with AF, 4,828 (27.6%) patients were prescribed VKA and 12,637 (72.4%) patients an NOAC: 11,884 (68.0%) patients had SAMe-TT2R2 scores 0-2 and 5,581 (32.0%) patients had scores >2. The proportion of patients prescribed VKA was 28.0% among patients with SAMe-TT2R2 scores >2 and 27.5% in those with scores ≤2. Conclusions: The lack of a clear association between the SAMe-TT2R2 score and anticoagulant selection may be attributed to the relative efficacy and safety profiles between NOACs and VKAs as well as to the absence of trial evidence that an SAMe-TT2R2-guided strategy for the selection of the type of anticoagulation in NVAF patients has an impact on clinical outcomes of efficacy and safety. The latter hypothesis is currently being tested in a randomized controlled trial. Clinical trial registration: URL: https://www.clinicaltrials.gov//Unique identifier: NCT01937377, NCT01468701, and NCT01671007

Beetroot supplementation improves the physiological responses to incline walking

The final publication is available at Springer via http://dx.doi.org/10.1007/s00421-018-3843-xPurpose: We investigated the effects of an acute 24-h nitrate-rich beetroot juice supplement (BR) on the energy cost, exercise efficiency and blood pressure responses to intermittent walking at different gradients. Methods: In a double-blind, cross-over design, eight participants were provided with a total of 350 ml of nitrate-rich (~20.5 mmol nitrate) BR or placebo (PLA) across 24-h before completing intermittent walking at 3 km/h on treadmill at gradients of 1%, 5%, 10%, 15% and 20%. Results: Resting mean arterial pressure (MAP) was ~4.1% lower after BR (93 vs. 89 mmHg; P = 0.001), as well as during exercise (102 vs. 99 mmHg; P = 0.011) and recovery (97 vs. 94 mmHg; P = 0.001). Exercising (1227 vs. 1129 ml/min P < 0.001) and end-stage (1404 vs. 1249 ml/min; P = 0.002) oxygen uptake (O2) was lower in BR compared to PLA, which was accompanied by an average reduction in phase II ̇O2 amplitude (1067 vs. 940 ml/min; P = 0.025). Similarly, recovery O2 (509 vs. 458 ml/min; P = 0.001) was lower in BR. Whole-blood potassium concentration increased from pre-post exercise in PLA (4.1 ± 0.3 vs. 4.5 ± 0.3 mmol/L; P = 0.013) but not BR (4.1 ± 0.31 vs. 4.3 ± 0.2 mmol/L; P = 0.188). Conclusions: Energy cost of exercise, recovery of O2, MAP and blood markers were ameliorated after BR. Previously reported mechanisms explain these findings, which are more noticeable during less efficient walking at steep gradients (15-20%). These findings have practical implications for hill-walkers

Police-initiated diversion for youth to prevent future delinquent behavior: a systematic review

BackgroundOverly punitive responses to youth misconduct may have the unintended consequence ofincreasing the likelihood of future delinquency; yet, overly lenient responses may fail to serveas a corrective for the misbehavior. Police diversion schemes are a collection of strategiespolice can apply as an alternative to court processing of youth. Police-initiated diversionschemes aim to reduce reoffending by steering youth away from deeper penetration into thecriminal justice system and by providing an alternative intervention that can help youthaddress psychosocial development or other needs that contribute to their problem behavior.ObjectivesThe objective of this review was to synthesize the evidence on the effectiveness of pre-courtinterventions involving police warning or counseling and release, and cautioning schemes inreducing delinquent behavior.Search methodsA combination of 26 databases and websites were searched. References of relevant reviewswere also scanned to identify studies. We also consulted with experts in the field. Searcheswere executed by two reviewers and conducted between August 2016 and January 2017.Selection criteriaOnly experimental and quasi-experimental designs were eligible for this review. All quasiexperimentaldesigns must have had a comparison group similar to the police diversionintervention group with respect to demographic characteristics and prior involvement indelinquent behavior (i.e., at similar risk for future delinquent behavior). Additionally, studiesmust have included youth participants between 12 and 17 years of age who either underwenttraditional system processing or were diverted from court processing through a police-leddiversion program. Studies were also eligible if delinquency-related outcomes, includingofficial and non-official (self-report or third-party reporting) measures of delinquency werereported.Data collection and analysisThis study used meta-analysis to synthesize results across studies. This method involvedsystematic coding of study features and conversion of study findings into effect sizesreflecting the direction and magnitude of any police-led diversion effect. There were 19independent evaluations across the 14 primary documents coded for this review. From this,we coded 67 effect sizes of delinquent behavior post diversion across 31 diversion-traditionalprocessing comparisons. We analyzed these comparisons using two approaches. The firstapproach selected a single effect size per comparison based on a decision rule and the secondused all 67 effect sizes, nesting these within comparison condition and evaluation design.ResultsThe general pattern of evidence is positive, suggesting that police-led diversion modestlyreduces future delinquent behavior of low-risk youth relative to traditional processing.Authors’ conclusionsThe findings from this systematic review support the use of police-led diversion for low-riskyouth with limited or no prior involvement with the juvenile justice system. Thus, policedepartments and policy-makers should consider diversionary programs as part of the mix ofsolutions for addressing youth crime

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Ascl1 phospho-status regulates neuronal differentiation in a Xenopus developmental model of neuroblastoma

Neuroblastoma (NB), although rare, accounts for 15% of all paediatric cancer mortality. Unusual among cancers, NBs lack a consistent set of gene mutations and, excluding large-scale chromosomal rearrangements, the genome seems to be largely intact. Indeed, many interesting features of NB suggest that it has little in common with adult solid tumours but instead has characteristics of a developmental disorder. NB arises overwhelmingly in infants under 2 years of age during a specific window of development and, histologically, NB bears striking similarity to undifferentiated neuroblasts of the sympathetic nervous system, its likely cells of origin. Hence, NB could be considered a disease of development arising when neuroblasts of the sympathetic nervous system fail to undergo proper differentiation, but instead are maintained precociously as progenitors with the potential for acquiring further mutations eventually resulting in tumour formation. To explore this possibility, we require a robust and flexible developmental model to investigate the differentiation of NB's presumptive cell of origin. Here, we use Xenopus frog embryos to characterise the differentiation of anteroventral noradrenergic (AVNA) cells, cells derived from the neural crest. We find that these cells share many characteristics with their mammalian developmental counterparts, and also with NB cells. We find that the transcriptional regulator Ascl1 is expressed transiently in normal AVNA cell differentiation but its expression is aberrantly maintained in NB cells, where it is largely phosphorylated on multiple sites. We show that Ascl1's ability to induce differentiation of AVNA cells is inhibited by its multi-site phosphorylation at serine-proline motifs, whereas overexpression of cyclin-dependent kinases (CDKs) and MYCN inhibit wild-type Ascl1-driven AVNA differentiation, but not differentiation driven by a phospho-mutant form of Ascl1. This suggests that the maintenance of ASCL1 in its multiply phosphorylated state might prevent terminal differentiation in NB, which could offer new approaches for differentiation therapy in NB