Design and characterisation of a minibeam collimator utilising Monte Carlo simulation and a clinical linear accelerator.

Spatially fractionated radiotherapy is showing promise as a treatment modality. Research has primarily focused on applications in proton therapy, however, there is some evidence that minibeam effects may also be present in photon therapy. This study presents a 3D printed tungsten minibeam collimator intended to produce peak-to-valley dose ratios (PVDR) of between seven and ten with a 1 MeV, bremsstrahlung generated, photon beam. The design of the collimator is motivated by a Monte Carlo study estimating the peak to valley dose ratio for different collimator designs at different energies. This collimator was characterised on a clinical linear accelerator (Elekta VersaHD). It was found that 1 MeV photon sources may generate PVDRs in the region of seven. The performance of the fabricated collimator was measured on a 250 keV superficial treatment unit and an Elekta VersaHD running in unflattened mode with a 6 MV beam. On the superficial unit the measured PVDR was greater than 10. On the Elekta VersaHD units the PVDR was measured to be between approximately 1.5 and 2.0 at 3 cm deep.

Discovery of Nedd4 auto-ubiquitination inhibitors.

E3 ubiquitin ligases are critical to the protein degradation pathway by catalyzing the final step in protein ubiquitination by mediating ubiquitin transfer from E2 enzymes to target proteins. Nedd4 is a HECT domain-containing E3 ubiquitin ligase with a wide range of protein targets, the dysregulation of which has been implicated in myriad pathologies, including cancer and Parkinson's disease. Towards the discovery of compounds disrupting the auto-ubiquitination activity of Nedd4, we developed and optimized a TR-FRET assay for high-throughput screening. Through selective screening of a library of potentially covalent compounds, compounds 25 and 81 demonstrated apparent IC50 values of 52 µM and 31 µM, respectively. Tandem mass spectrometry (MS/MS) analysis confirmed that 25 and 81 were covalently bound to Nedd4 cysteine residues (Cys182 and Cys867). In addition, 81 also adducted to Cys627. Auto-ubiquitination assays of Nedd4 mutants featuring alanine substitutions for each of these cysteines suggested that the mode of inhibition of these compounds occurs through blocking the catalytic Cys867. The discovery of these inhibitors could enable the development of therapeutics for various diseases caused by Nedd4 E3 ligase dysregulation.

Discovery of a Novel DCAF1 Ligand Using a Drug-Target Interaction Prediction Model: Generalizing Machine Learning to New Drug Targets.

DCAF1 functions as a substrate recruitment subunit for the RING-type CRL4DCAF1 and the HECT family EDVPDCAF1 E3 ubiquitin ligases. The WDR domain of DCAF1 serves as a binding platform for substrate proteins and is also targeted by HIV and SIV lentiviral adaptors to induce the ubiquitination and proteasomal degradation of antiviral host factors. It is therefore attractive both as a potential therapeutic target for the development of chemical inhibitors and as an E3 ligase that could be recruited by novel PROTACs for targeted protein degradation. In this study, we used a proteome-scale drug-target interaction prediction model, MatchMaker, combined with cheminformatics filtering and docking to identify ligands for the DCAF1 WDR domain. Biophysical screening and X-ray crystallographic studies of the predicted binders confirmed a selective ligand occupying the central cavity of the WDR domain. This study shows that artificial intelligence-enabled virtual screening methods can successfully be applied in the absence of previously known ligands.

Improvements in Objective and Subjective Measures of Chronic Cough with Gefapixant: A Pooled Phase 3 Efficacy Analysis of Predefined Subgroups.

INTRODUCTION: In phase 3 trials (COUGH-1/COUGH-2), gefapixant 45 mg twice daily significantly reduced 24-h cough frequency vs placebo in refractory or unexplained chronic cough (RCC or UCC). METHODS: Here, the efficacy of gefapixant 45 mg vs placebo was evaluated across COUGH-1/COUGH-2 in predefined subgroups based on sex, region, age, cough duration, cough severity, cough frequency, and diagnosis (RCC, UCC). Awake cough frequency reductions at Week 12 and LCQ response rates (i.e., ≥ 1.3-point improvement) at Week 24 were assessed. RESULTS: Among 1360 participants analyzed, gefapixant 45 mg resulted in consistent awake cough frequency reductions overall and across predefined subgroups at Week 12. Gefapixant also resulted in improved LCQ scores across subgroups at Week 24; ≥ 70% of participants in each subgroup treated with gefapixant 45 mg had an LCQ response. CONCLUSION: These data suggest gefapixant 45 mg provides consistent objective and subjective efficacy across subgroups of individuals with RCC or UCC.

Behavioral activation for depression delivered by drug and alcohol treatment workers: A pilot randomized controlled trial.

BACKGROUND: One in two patients seeking help for substance use disorders (SUDs) has clinically significant depression symptoms. This co-occurrence impairs treatment outcomes, but limited evaluation of the implementation of evidence-based interventions has taken place. METHODS: This pilot randomized controlled trial (RCT) assessed the feasibility and potential efficacy of a brief, protocol-driven behavioral activation (BA) intervention delivered by drug and alcohol workers in a community drug and alcohol treatment (CDAT) service. Eligible participants (n = 34) were randomly allocated to either BA (n = 17) or treatment as usual (n = 17) and assessed at baseline and 6-, 12-, and 24-week follow-up. Feasibility outcomes were participant engagement and worker protocol adherence. The primary pilot outcome was severity of depression symptoms (PHQ-9) at 12-week follow-up. Secondary outcomes included percent days abstinent (PDA) in the past month, severity of dependence (SDS), anxiety symptoms (GAD-7), and valued living (VQ) at all follow-up points. RESULTS: Fifty-nine percent of BA participants attended at least one session and there was 95% adherence to the treatment protocol. BA was associated with significantly reduced depression at 12-week follow-up (PHQ-9 mean difference - 5.69, 95% CI -10.07 to -1.31). BA participants had significantly greater improvements in PDA (mean difference 17.9, 95% CI 0.99 to 34.82) and VQ-Progress (mean difference 5.34, 95% CI 1.47 to 9.22) at 6-week follow-up and PDA (mean difference 27.69, 95% CI 4.44 o 50.95) at 12-week follow-up. The study found no significant between-group differences at 24-week follow-up. CONCLUSION: BA implemented by drug and alcohol treatment workers in CDAT appears feasible and may add clinical benefit to usual care for SUD patients with elevated depressive symptoms accessing CDAT. Fully powered RCTs are warranted to better investigate the replicability of these preliminary findings. Methodological limitations are discussed and suggestions for future research are provided.

Efficacy and safety of gefapixant, a P2X3 receptor antagonist, in refractory chronic cough and unexplained chronic cough (COUGH-1 and COUGH-2): results from two double-blind, randomised, parallel-group, placebo-controlled, phase 3 trials.

BACKGROUND: Gefapixant is an oral P2X3 receptor antagonist that has previously shown efficacy and safety in refractory chronic cough and unexplained chronic cough. We therefore aim to confirm the efficacy and safety of gefapixant in participants with refractory chronic cough and unexplained chronic cough. METHODS: COUGH-1 and COUGH-2 were both double-blind, randomised, parallel-group, placebo-controlled, phase 3 trials. COUGH-1 was done in 156 sites in 17 countries and COUGH-2 in 175 sites in 20 countries. We enrolled participants who were 18 years or older with a diagnosis of refractory chronic cough or unexplained chronic cough of 1 year duration or more. Participants were also required to have a cough severity visual analogue scale score of 40 mm or more at screening and baseline. Eligible participants were randomly allocated (1:1:1), using a computer-generated allocation schedule, to one of three treatment groups: placebo, gefapixant 15 mg twice per day, or gefapixant 45 mg twice per day. All study treatments were given orally. Participants were treated over a 12-week main study period in COUGH-1 and a 24-week main study period in COUGH-2; followed by extension periods for a total of up to 52 weeks of treatment in both trials. The primary outcome was placebo-adjusted mean change in 24-h cough frequency at 12 weeks in COUGH-1 and 24 weeks in COUGH-2. Both studies were registered with ClinicalTrials.gov, NCT03449134 (COUGH-1) and NCT03449147 (COUGH-2). FINDINGS: From March 14, 2018, (first participant screened) to July 26, 2019, (last participant screened) 732 patients were recruited in COUGH-1 and 1317 in COUGH-2. COUGH-1 randomly assigned and treated 730 participants (243 [33×3%] with placebo, 244 [33×4%] with gefapixant 15 mg twice per day, and 243 [33×3%] with gefapixant 45 mg twice per day); COUGH-2 randomly assigned and treated 1314 participants (435 [33×1%] with placebo, 440 [33×5%] with gefapixant 15 mg twice per day, and 439 [33×4%] with gefapixant 45 mg twice per day). Participants were mostly female (542 [74×2%] of 730 in COUGH-1 and 984 [74×9%] of 1314 in COUGH-2). The mean age was 59×0 years (SD 12×6) in COUGH-1 and 58×1 years (12×1) in COUGH-2, and the mean cough duration was 11·6 years (SD 9·5) in COUGH-1 and 11·2 years (9·8) in COUGH-2. Gefapixant 45 mg twice per day showed significant reductions in 24-h cough frequency compared with placebo at week 12 in COUGH-1 (18·5% [95% CI 32·9-0·9]; p=0·041) and at week 24 in COUGH-2 (14·6% [26·1-1·4]; p=0·031). Gefapixant 15 mg twice per day did not show a significant reduction in cough frequency versus placebo in both studies. The most common adverse events were related to taste disturbance: ageusia (36 [4·9%] of 730 in COUGH-1 and 86 [6·5%] of 1314 in COUGH-2), dysgeusia (118 [16·2%] in COUGH-1 and 277 [21·1%] in COUGH-2), hypergeusia (3 [0·4%] in COUGH-1 and 6 [0×5%] in COUGH-2), hypogeusia (19 [2·6%] in COUGH-1 and 80 [6·1%] in COUGH-2), and taste disorder (28 [3·8%] in COUGH-1 and 46 [3·5%] in COUGH-2). INTERPRETATION: Gefapixant 45 mg twice per day is the first treatment to show efficacy with an acceptable safety profile in phase 3 clinical trials for refractory chronic cough or unexplained chronic cough. FUNDING: Merck Sharp & Dohme.

Shortening the preparation time of the single prolonged breath-hold for radiotherapy sessions.

OBJECTIVE: Single prolonged breath-holds of >5 min can be obtained in cancer patients. Currently, however, the preparation time in each radiotherapy session is a practical limitation for clinical adoption of this new technique. Here, we show by how much our original preparation time can be shortened without unduly compromising breath-hold duration. METHODS: 44 healthy subjects performed single prolonged breath-holds from 60% O2 and mechanically induced hypocapnia. We tested the effect on breath-hold duration of shortening preparation time (the durations of acclimatization, hyperventilation and hypocapnia) by changing these durations and or ventilator settings. RESULTS: Mean original breath-hold duration was 6.5 ± 0.2 (standard error) min. The total original preparation time (from connecting the facemask to the start of the breath-hold) was 26 ± 1 min. After shortening the hypocapnia duration from 16 to 5 min, mean breath-hold duration was still 6.1 ± 0.2 min (ns vs the original). After abolishing the acclimatization and shortening the hypocapnia to 1 min (a total preparation time now of 9 ± 1 min), a mean breath-hold duration of >5 min was still possible (now significantly shortened to 5.2 ± 0.6 min, p < 0.001). After shorter and more vigorous hyperventilation (lasting 2.7 ± 0.3 min) and shorter hypocapnia (lasting 43 ± 4 s), a mean breath-hold duration of >5 min (5.3 ± 0.2 min, p < 0.05) was still possible. Here, the final total preparation time was 3.5 ± 0.3 min. CONCLUSIONS: These improvements may facilitate adoption of the single prolonged breath-hold for a range of thoracic and abdominal radiotherapies especially involving hypofractionation. ADVANCES IN KNOWLEDGE: Multiple short breath-holds improve radiotherapy for thoracic and abdominal cancers. Further improvement may occur by adopting the single prolonged breath-hold of >5 min. One limitation to clinical adoption is its long preparation time. We show here how to reduce the mean preparation time from 26 to 3.5 min without compromising breath-hold duration.

Phosphorylation status of pyruvate dehydrogenase in the mousebird Colius striatus undergoing torpor.

Torpor is a heterothermic response that occurs in some animals to reduce metabolic expenditure. The speckled mousebird (Colius striatus) belongs to one of the few avian taxa possessing the capacity for pronounced torpor, entering a hypometabolic state with concomitant decreases in body temperature in response to reduced food access or elevated thermoregulatory energy requirements. The pyruvate dehydrogenase complex (PDC) is a crucial site regulating metabolism by bridging glycolysis and the Krebs cycle. Three highly conserved phosphorylation sites are found within the E1 enzyme of the complex that inhibit PDC activity and reduce the flow of carbohydrate substrates into the mitochondria. The current study demonstrates a marked increase in S232 phosphorylation during torpor in liver, heart, and skeletal muscle of C. striatus. The increase in S232 phosphorylation during torpor was particularly notable in skeletal muscle where levels were ~49-fold higher in torpid birds compared to controls. This was in contrast to the other two phosphorylation sites (S293 and S300) which remained consistently phosphorylated regardless of tissue. The relevant PDH kinase (PDHK1) known to phosphorylate S232 was found to be substantially upregulated (~5-fold change) in the muscle during torpor as well as increasing moderately in the liver (~2.2-fold increase). Additionally, in the heart, a slight (~23%) decrease in total PDH levels was noted. Taken together the phosphorylation changes in PDH suggest that inhibition of the complex is a common feature across several tissues in the mousebird during torpor and that this regulation is mediated at a specific residue.

Functional and post-translational characterization of pyruvate dehydrogenase demonstrates repression of activity in the liver but not skeletal muscle of the Richardson's ground squirrel (Urocitellus richardsonii) during hibernation.

Hibernation consists of a series of physiological and biochemical alterations in an animal that allows for reduced body temperatures down to near ambient levels and substantial fuel conservation allowing it to survive on stored fat supplies accumulated during the summer. The Richardson's ground squirrel is one such hibernator that undergoes such changes for as long as 9 months of the year. This study examines the role of regulation of the pyruvate dehydrogenase complex (PDC) during hibernation in the skeletal muscle and liver of the Richardson's ground squirrel. The current study demonstrates a great reduction in the activity of PDC in the hibernating liver, but not in the skeletal muscle. This was matched by a significant increase in the phosphorylation on a regulatory serine residue (S300) of the pyruvate dehydrogenase (PDH) E1α subunit. Examining the expression patterns of the relevant kinases for PDH and the associated phosphatase demonstrated some unexpected results. Specifically, an increase in PDKs 1 and 2 and a decrease in PDK4 was noted in the skeletal muscle tissue in response to hibernation and no alterations in the expression patterns of any of these enzymes were noted in the liver. This suggests that alternative modes of regulation of the kinases may be at play in hibernation to bring about the observed effects. Taken together this study demonstrates that PDH regulatory responses differ markedly between tissues and emphasize the importance of inhibition of the complex in the liver during hibernation.

Skeletal muscle of torpid Richardson's ground squirrels (Urocitellus richardsonii) exhibits a less active form of citrate synthase associated with lowered lysine succinylation.

Hibernation is a metabolic/physiological strategy employed by many mammals to cope with periods when energy usage is greater than its input. Animals undergoing hibernation need to greatly reduce their metabolic rate and reshape their catabolic processes to survive on stored triglycerides. Citrate synthase (CS) is one of only two irreversible steps in the citric acid cycle (CAC) and forms an important regulatory checkpoint that gates the entry of acetyl-CoA formed in glycolysis or fatty acid catabolism into this critical central metabolic hub. This study investigated the regulation of citrate synthase in the muscle tissue of a small mammalian hibernator through comparison of functional and structural properties. The results demonstrated a significant decrease in the Vmax of purified torpid CS compared to the control euthermic enzyme (1.2-1.7 fold greater in the control) that was evident over a wide range of temperatures (8, 22 and 37 °C) that are encountered by the enzyme in hibernation. This was also reflected in the specific activity of the enzyme in crude muscle protein extracts. Analyzing the purified CS through immunoblotting demonstrated that the enzyme contained noticeably less lysine succinylation in the torpid state (about 50% of euthermic levels) and this was correlated with an increase in total levels of SIRT5, the enzyme responsible for mediating desuccinylation in the mitochondria (2.2 fold increase). Taken together, the results of this study support the idea that CS is inhibited during hibernation in the ground squirrel skeletal muscle and that this alteration could be mediated by decreases in succinylation.

Safely achieving single prolonged breath-holds of > 5 minutes for radiotherapy in the prone, front crawl position.

OBJECTIVE: Breast cancer radiotherapy is increasingly delivered supine with multiple, short breath-holds. There may be heart and lung sparing advantages for locoregional breast cancer of both prone treatment and in a single breath-hold. We test here whether single prolonged breath-holds are possible in the prone, front crawl position. METHODS: 19 healthy volunteers were trained to deliver supine, single prolonged breath-holds with pre-oxygenation and hypocapnia. We tested whether all could achieve the same durations in the prone, front crawl position. RESULTS: 19 healthy volunteers achieved supine, single prolonged breath-holds for mean of 6.2 ± 0.3 min. All were able to hold safely for the same duration while prone (6.1 ± 0.2 min ns. by paired ANOVA). With prone, the increased weight on the chest did not impede chest inflation, nor the ability to hold air in the chest. Thus, the rate of chest deflation (mean anteroposterior deflation movement of three craniocaudally arranged surface markers on the spinal cord) was the same (1.2 ± 0.2, 2.0 ± 0.4 and 1.2 ± 0.4 mm/min) as found previously during supine prolonged breath-holds. No leakage of carbon dioxide or air was detectable into the facemask. CONCLUSION: Single prolonged (>5 min) breath-holds are equally possible in the prone, front crawl position. ADVANCES IN KNOWLEDGE: Prolonged breath-holds in the front crawl position are possible and have the same durations as in the supine position. Such training would therefore be feasible for some patients with breast cancer requiring loco-regional irradiation. It would have obvious advantages for hypofractionation.

Heavy charged particle beam therapy and related new radiotherapy technologies: The clinical potential, physics and technical developments required to deliver benefit for patients with cancer.

In the UK, one in two people will develop cancer during their lifetimes and radiotherapy (RT) plays a key role in effective treatment. High energy proton beam therapy commenced in the UK National Health Service in 2018. Heavier charged particles have potential advantages over protons by delivering more dose in the Bragg peak, with a sharper penumbra, lower oxygen dependence and increased biological effectiveness. However, they also require more costly equipment including larger gantries to deliver the treatment. There are significant uncertainties in the modelling of relative biological effectiveness and the effects of the fragmentation tail which can deliver dose beyond the Bragg peak. These effects need to be carefully considered especially in relation to long-term outcomes.In 2019, a group of clinicians, clinical scientists, engineers, physical and life scientists from academia and industry, together with funding agency stakeholders, met to consider how the UK should address new technologies for RT, especially the use of heavier charged particles such as helium and carbon and new modes of delivery such as FLASH and spatially fractionated radiotherapy (SFRT).There was unanimous agreement that the UK should develop a facility for heavier charged particle therapy, perhaps constituting a new National Ion Research Centre to enable research using protons and heavier charged particles. Discussion followed on the scale and features, including which ions should be included, from protons through helium, boron, and lithium to carbon, and even oxygen. The consensus view was that any facility intended to treat patients must be located in a hospital setting while providing dedicated research space for physics, preclinical biology and clinical research with beam lines designed for both in vitro and in vivo research. The facility should to be able to investigate and deliver both ultra-high dose rate FLASH RT and SFRT (GRID, minibeams etc.). Discussion included a number of accelerator design options and whether gantries were required. Other potential collaborations might be exploited, including with space agencies, electronics and global communications industries and the nuclear industry.In preparation for clinical delivery, there may be opportunities to send patients overseas (for 12C or 4He ion therapy) using the model of the National Health Service (NHS) Proton Overseas Programme and to look at potential national clinical trials which include heavier ions, FLASH or SFRT. This could be accomplished under the auspices of NCRI CTRad (National Cancer Research Institute, Clinical and Translational Radiotherapy Research Working Group).The initiative should be a community approach, involving all interested parties with a vision that combines discovery science, a translational research capability and a clinical treatment facility. Barriers to the project and ways to overcome them were discussed. Finally, a set of different scenarios of features with different costs and timelines was constructed, with consideration given to the funding environment (prer-Covid-19) and need for cross-funder collaboration.

Purification and Regulation of Pyruvate Kinase from the Foot Muscle of the Anoxia and Freeze Tolerant Marine Snail, Littorina littorea.

The intertidal marine snail, Littorina littorea, has evolved to survive bouts of anoxia and extracellular freezing brought about by changing tides and subsequent exposure to harsh environmental conditions. Survival in these anoxic conditions depends on the animals entering a state of metabolic rate depression in order to maintain an appropriate energy production-consumption balance during periods of limited oxygen availability. This study investigated the kinetic, physical, and regulatory properties of pyruvate kinase (PK), which catalyzes the final reaction of aerobic glycolysis, from foot muscle of L. littorea to determine if the enzyme is differentially regulated in response to anoxia and freezing exposure. PK purified from foot muscle of anoxic animals exhibited a lower affinity for its substrate phosphoenolpyruvate than PK from control and frozen animals. PK from anoxic animals was also more sensitive to a number of allosteric regulators, including alanine and aspartate, which are key anaerobic metabolites in L. littorea. Furthermore, PK purified from anoxic and frozen animals exhibited greater stability compared to the non-stressed control animals, determined through high-temperature incubation studies. Phosphorylation of threonine and tyrosine residues was also assessed and demonstrated that levels of threonine phosphorylation of PK from anoxic animals were significantly higher than those of PK from control and frozen animals, suggesting a potential mechanism for regulating PK activity. Taken together, these results suggest that PK plays a role in suppressing metabolic rate in these animals during environmental anoxia exposure.