Susceptibility of pediatric acute lymphoblastic leukemia to STAT3 inhibition depends on p53 induction.

Advances in the clinical management of pediatric B-cell acute lymphoblastic leukemia (B-ALL) have dramatically improved outcomes for this disease. However, relapsed and high-risk disease still contribute to significant numbers of treatment failures. Development of new, broad range therapies is urgently needed for these cases. We previously reported the susceptibility of ETV6-RUNX1+ pediatric B-ALL to inhibition of signal transducer and activator of transcription 3 (STAT3) activity. In the present study, we demonstrate that pharmacological or genetic inhibition of STAT3 results in p53 induction and that CRISPR-mediated TP53 knockout substantially reverses susceptibility to STAT3 inhibition. Furthermore, we demonstrate that sensitivity to STAT3 inhibition in patient-derived xenograft (PDX) B-ALL samples is not restricted to any particular disease subtype, but rather depends on TP53 status, the only resistant samples being TP53 mutant. Induction of p53 following STAT3 inhibition is not directly dependent on MDM2 but correlates with degradation of MDM4. As such, STAT3 inhibition exhibits synergistic in vitro and in vivo anti-leukemia activity when combined with MDM2 inhibition. Taken together with the relatively low frequency of TP53 mutations in this disease, these data support the future development of combined STAT3/ MDM2 inhibition in the therapy of refractory and relapsed pediatric B-ALL.

High-Throughput Screening Platform To Identify Inhibitors of Protein Synthesis with Potential for the Treatment of Malaria.

Artemisinin-based combination therapies have been crucial in driving down the global burden of malaria, the world's largest parasitic killer. However, their efficacy is now threatened by the emergence of resistance in Southeast Asia and sub-Saharan Africa. Thus, there is a pressing need to develop new antimalarials with diverse mechanisms of action. One area of Plasmodium metabolism that has recently proven rich in exploitable antimalarial targets is protein synthesis, with a compound targeting elongation factor 2 now in clinical development and inhibitors of several aminoacyl-tRNA synthetases in lead optimization. Given the promise of these components of translation as viable drug targets, we rationalized that an assay containing all functional components of translation would be a valuable tool for antimalarial screening and drug discovery. Here, we report the development and validation of an assay platform that enables specific inhibitors of Plasmodium falciparum translation (PfIVT) to be identified. The primary assay in this platform monitors the translation of a luciferase reporter in a P. falciparum lysate-based expression system. Hits identified in this primary assay are assessed in a counterscreen assay that enables false positives that directly interfere with the luciferase to be triaged. The remaining hit compounds are then assessed in an equivalent human IVT assay. This platform of assays was used to screen MMV's Pandemic and Pathogen Box libraries, identifying several selective inhibitors of protein synthesis. We believe this new high-throughput screening platform has the potential to greatly expedite the discovery of antimalarials that act via this highly desirable mechanism of action.

Veterinary trypanocidal benzoxaboroles are peptidase-activated prodrugs.

Livestock diseases caused by Trypanosoma congolense, T. vivax and T. brucei, collectively known as nagana, are responsible for billions of dollars in lost food production annually. There is an urgent need for novel therapeutics. Encouragingly, promising antitrypanosomal benzoxaboroles are under veterinary development. Here, we show that the most efficacious subclass of these compounds are prodrugs activated by trypanosome serine carboxypeptidases (CBPs). Drug-resistance to a development candidate, AN11736, emerged readily in T. brucei, due to partial deletion within the locus containing three tandem copies of the CBP genes. T. congolense parasites, which possess a larger array of related CBPs, also developed resistance to AN11736 through deletion within the locus. A genome-scale screen in T. brucei confirmed CBP loss-of-function as the primary mechanism of resistance and CRISPR-Cas9 editing proved that partial deletion within the locus was sufficient to confer resistance. CBP re-expression in either T. brucei or T. congolense AN11736-resistant lines restored drug-susceptibility. CBPs act by cleaving the benzoxaborole AN11736 to a carboxylic acid derivative, revealing a prodrug activation mechanism. Loss of CBP activity results in massive reduction in net uptake of AN11736, indicating that entry is facilitated by the concentration gradient created by prodrug metabolism.

High-throughput sequencing of peripheral blood for minimal residual disease monitoring in childhood precursor B-cell acute lymphoblastic leukemia: A prospective feasibility study.

BACKGROUND: Minimal residual disease (MRD) measured on end-of-induction bone marrow (BM) is the most important biomarker for guiding therapy in pediatric acute lymphoblastic leukemia (ALL). Due to limited sensitivity of current approaches, peripheral blood (PB) is not a reliable source for identifying patients needing treatment changes. We sought to determine if high-throughput sequencing (HTS) (next-generation sequencing) of rearranged immunoglobulin and T-cell receptor genes can overcome this and be used to measure MRD in PB. PROCEDURE: We employed a quantitative HTS approach to accurately measure MRD from one million cell equivalents of DNA from 17 PB samples collected at day 29 after induction therapy in patients with precursor B-cell ALL. We compared these results to the gold-standard real-time PCR result obtained from their paired BM samples, median follow-up 49 months. RESULTS: With the increased sensitivity, detecting up to one abnormal cell in a million normal cells, we were able to detect MRD in the PB by HTS in all those patients requiring treatment intensification (MRD ≥ 0.005% in BM). CONCLUSION: This is proof of principle that using the increased sensitivity of HTS, PB can be used to measure MRD and stratify children with ALL. The method is cost effective, rapid, accurate, and reproducible, with inherent advantages in children. Importantly, increasing the frequency testing by PB as opposed to intermittent BM sampling may allow extension of the dynamic range of MRD, giving a more complete picture of the kinetics of disease remission while improving relapse prediction and speed of detection.

Unitization of route knowledge.

There are many theories that explain how route knowledge is acquired. We examined here if the sequence of elements that are part of a route can become integrated into a single unit, to the extent that the processing of individual transitions may only be relevant in the context of this entire unit. In Experiments 1 and 2, participants learned a route for ten blocks. Subsequently, at test they were intermittently exposed to the same training route along with a novel route which contained partial overlap with the original training route. Results show that the very same stimulus, appearing in the very same location, requiring the very same response (e.g., left turn), was responded to significantly faster in the context of the original training route than in the novel route. In Experiment 3, we employed a modified paradigm containing landmarks and two matched routes which were both substantially longer and contained a greater degree of overlap than the routes in Experiments 1 and 2. Results were replicated, namely, the same overlapping route segment, common to both routes, was performed significantly slower when appearing in the context of a novel than the original route. Furthermore, the difference between the overlapping segments was similar to the difference observed for the non-overlapping segments, i.e., an old route segment in the context of a novel route was processed as if it were an entirely novel segment. We discuss the results in relation to binding, chunking, and transfer effects, as well as potential practical implications.

Unsupervised categorization with individuals diagnosed as having moderate traumatic brain injury: Over-selective responding.

PRIMARY OBJECTIVE: This study explored over-selectivity (executive dysfunction) using a standard unsupervised categorization task. Over-selectivity has been demonstrated using supervised categorization procedures (where training is given); however, little has been done in the way of unsupervised categorization (without training). METHODS AND PROCEDURE: A standard unsupervised categorization task was used to assess levels of over-selectivity in a traumatic brain injury (TBI) population. Individuals with TBI were selected from the Tertiary Traumatic Brain Injury Clinic at Swansea University and were asked to categorize two-dimensional items (pictures on cards), into groups that they felt were most intuitive, and without any learning (feedback from experimenter). This was compared against categories made by a control group for the same task. OUTCOMES AND RESULTS: The findings of this study demonstrate that individuals with TBI had deficits for both easy and difficult categorization sets, as indicated by a larger amount of one-dimensional sorting compared to control participants. Deficits were significantly greater for the easy condition. CONCLUSIONS: The implications of these findings are discussed in the context of over-selectivity, and the processes that underlie this deficit. Also, the implications for using this procedure as a screening measure for over-selectivity in TBI are discussed.

Mixed-methods feasibility outcomes for a novel ACT-based video game 'ACTing Minds' to support mental health.

OBJECTIVES: To determine the feasibility and acceptability of 'ACTing Minds', a novel single-player adventure video game based on acceptance and commitment therapy (ACT). DESIGN: A single-arm, mixed-methods repeated measures feasibility study. SETTING: Intervention and questionnaires were completed at home by participants. Semistructured interviews were also conducted at home via the Zoom platform. PARTICIPANTS: Thirty-six participants were recruited into the study, 29 completed all phases of the feasibility design. Eligibility criteria required participants to be over the age of 18 and self-reporting experiencing ongoing depression, anxiety or stress. INTERVENTION: Participants completed a single session of the 'ACTing Minds' video game, lasting approximately 1 hour, designed to educate users on key principles from ACT. PRIMARY OUTCOME MEASURES: Participant recruitment and retention, questionnaire completion, long-term intervention adherence and acceptability of the intervention. Reflexive thematic analysis was conducted on semistructured interviews run immediately postintervention and 3 weeks later. SECONDARY OUTCOME MEASURES: Measures of depression, anxiety, stress, psychological flexibility, social connectedness and well-being were assessed at baseline, immediately following intervention completion, and after a 3-week follow-up period. We used a standardised battery of questionnaires. PRIMARY RESULTS: Twenty-nine participants completed the study. A reflexive thematic analysis indicated that participants responded positively to the intervention and the study at all stages. Themes reflect participants' desire for an engaging therapeutic experience, use of game for exploring emotions, as well as their perspectives on how they had applied their learning to the real world. SECONDARY RESULTS: Quantitative results indicated small to large effect sizes associated with decreases in depression (ηp2 = 0.011), anxiety (ηp2 = 0.096) and stress (ηp2 = 0.108), and increases in psychological flexibility (ηp2 = 0.060), social connectedness (ηp2 = 0.021), well-being (ηp2 = 0.011) and participation in usual activities (ηp2 = .307). CONCLUSIONS: Implementation of the 'ACTing Minds' intervention is warranted, based on both qualitative and quantitative outcomes. TRIAL REGISTRATION NUMBER: NCT04566042 ClinicalTrials.gov.

Functional contextual implementation of an evolutionary, entropy-based, and embodied free energy framework: Utilizing Lagrangian mechanics and evolutionary game theory's truth vs. fitness test of the veridicality of phenomenological experience.

The Bayesian approach of cognitive science largely takes the position that evolution drives perception to produce precepts that are veridical. However, some efforts utilizing evolutionary game theory simulations have shown that perception is more likely based on a fitness function, which promotes survival rather than promoting perceptual truth about the environment. Although these findings do not correspond well with the standard Bayesian approach to cognition, they may correspond with a behavioral functional contextual approach that is ontologically neutral (a-ontological). This approach, formalized through a post-Skinnerian account of behaviorism called relational frame theory (RFT), can, in fact, be shown to correspond well with an evolutionary fitness function, whereby contextual functions form that corresponds to a fitness function interface of the world. This fitness interface approach therefore may help provide a mathematical description for a functional contextual interface of phenomenological experience. Furthermore, this more broadly fits with a neurological active inference approach based on the free-energy principle (FEP) and more broadly with Lagrangian mechanics. These assumptions of how fitness beats truth (FBT) and FEP correspond to RFT are then discussed within a broader multidimensional and evolutionary framework called the extended evolutionary meta-model (EEMM) that has emerged out of the functional contextual behavioral science literature to incorporate principles of cognition, neurobiology, behaviorism, and evolution and are discussed in the context of a novel RFT framework called "Neurobiological and Natural Selection Relational Frame Theory" (N-frame). This framework mathematically connects RFT to FBT, FEP, and EEMM within a single framework that expands into dynamic graph networking. This is then discussed for its implications of empirical work at the non-ergodic process-based idiographic level as applied to individual and societal level dynamic modeling and clinical work. This discussion is framed within the context of individuals that are described as evolutionary adaptive and conscious (observer-self) agents that minimize entropy and can promote a prosocial society through group-level values and psychological flexibility.

Applying an osteopathic intervention to improve mild to moderate mental health symptoms: a mixed-methods feasibility study protocol.

INTRODUCTION: Mental health services are stretched in the UK and are in need of support. One approach that could improve mental health symptoms is osteopathy. Research suggests that osteopathy influences psychophysiological factors, which could lead to improvements in mental health. The first objective of this protocol is to investigate the feasibility and acceptability of four osteopathic interventions. A secondary aim is to evaluate the interventions' effectiveness for improving psychophysiological and mental health outcomes. METHODS AND ANALYSIS: This study will be an explanatory mixed-methods design. Participants will be 30 adults who have mild to moderate mental health symptoms and not experiencing any issues with pain. The feasibility and acceptability of the interventions will be the primary outcomes. Secondary outcomes will be physiological measures including heart rate variability, interoceptive accuracy and blood pressure. Psychological outcomes, collected preintervention and postintervention, will also be measured by five standardised questionnaires, which include: (1) the Depression, Anxiety and Stress Scale (DASS); (2) the International Positive and Negative Affect Schedule-Short-Form; (3) Acceptance and Action Questionnaire-II; (4) the Self as Context Scale and (5) and the Multidimensional Assessment of Interoceptive Awareness Version 2. Participants will be randomised to one of four intervention groups and receive a single intervention treatment session. These intervention groups are: (1) high-velocity and articulation techniques, (2) soft-tissue massage, (3) craniosacral techniques, and (4) a combination of these three approaches. Mixed design two (preintervention and postintervention) by the four interventions analysis of covariance models will be used to analyse the quantitative data for each quantitative measure. Participants will also be interviewed about their experiences of the study and interventions and a thematic analysis will be used to analyse this qualitative data. This will aid the assessment of the feasibility and acceptability of the study design. ETHICS AND DISSEMINATION: The protocol for this feasibility study has received ethical approval from the Department of Psychology Ethics Committee at Swansea University, ethical review reference number: 2022-5603-4810. Feasibility results from this protocol will be published in a peer review journal and presented at both national and international conferences. DISCUSSION: This study will assess the feasibility and acceptability of conducting osteopathic interventions for improving mental health outcomes. The results from this will help to inform the development of a future randomised controlled trial. The study will also produce original data which could provide preliminary evidence of whether osteopathic approaches are of benefit to individual's mental health in the form of effect sizes, even if they are pain-free. TRIAL REGISTRATION NUMBER: NCT05674071.

Structure-Guided Design and Synthesis of a Pyridazinone Series of Trypanosoma cruzi Proteasome Inhibitors.

There is an urgent need for new treatments for Chagas disease, a parasitic infection which mostly impacts South and Central America. We previously reported on the discovery of GSK3494245/DDD01305143, a preclinical candidate for visceral leishmaniasis which acted through inhibition of the Leishmania proteasome. A related analogue, active against Trypanosoma cruzi, showed suboptimal efficacy in an animal model of Chagas disease, so alternative proteasome inhibitors were investigated. Screening a library of phenotypically active analogues against the T. cruzi proteasome identified an active, selective pyridazinone, the development of which is described herein. We obtained a cryo-EM co-structure of proteasome and a key inhibitor and used this to drive optimization of the compounds. Alongside this, optimization of the absorption, distribution, metabolism, and excretion (ADME) properties afforded a suitable compound for mouse efficacy studies. The outcome of these studies is discussed, alongside future plans to further understand the series and its potential to deliver a new treatment for Chagas disease.

Relational versus absolute representation in categorization.

This study explores relational-like and absolute-like representations in categorization. Although there is much evidence that categorization processes can involve information about both the particular physical properties of studied instances and abstract (relational) properties, there has been little work on the factors that lead to one kind of representation as opposed to the other. We tested 370 participants in 6 experiments, in which participants had to classify new items into predefined artificial categories. In 4 experiments, we observed a predominantly relational-like mode of classification, and in 2 experiments we observed a shift toward an absolute-like mode of classification. These results suggest 3 factors that promote a relational-like mode of classification: fewer items per group, more training groups, and the presence of a time delay. Overall, we propose that less information about the distributional properties of a category or weaker memory traces for the category exemplars (induced, e.g., by having smaller categories or a time delay) can encourage relational-like categorization.

Going beyond the DSM in predicting, diagnosing, and treating autism spectrum disorder with covarying alexithymia and OCD: A structural equation model and process-based predictive coding account.

Background: There is much overlap among the symptomology of autistic spectrum disorders (ASDs), obsessive compulsive disorders (OCDs), and alexithymia, which all typically involve impaired social interactions, repetitive impulsive behaviors, problems with communication, and mental health. Aim: This study aimed to identify direct and indirect associations among alexithymia, OCD, cardiac interoception, psychological inflexibility, and self-as-context, with the DV ASD and depression, while controlling for vagal related aging. Methodology: The data involved electrocardiogram (ECG) heart rate variability (HRV) and questionnaire data. In total, 1,089 participant's data of ECG recordings of healthy resting state HRV were recorded and grouped into age categories. In addition to this, another 224 participants completed an online survey that included the following questionnaires: Yale-Brown Obsessive Compulsive Scale (Y-BOCS); Toronto Alexithymia Scale 20 (TAS-20); Acceptance and Action Questionnaire (AAQII); Depression, Anxiety, and Stress Scale 21 (DAS21); Multi-dimensional Assessment of Interoceptive Awareness Scale (MAIA); and the Self-as-Context Scale (SAC). Results: Heart rate variability was shown to decrease with age when controlling for BMI and gender. In the two SEMs produced, it was found that OCD and alexithymia were causally associated with autism and depression indirectly through psychological inflexibility, SAC, and ISen interoception. Conclusion: The results are discussed in relation to the limitations of the DSM with its categorical focus of protocols for syndromes and provide support for more flexible ideographic approaches in diagnosing and treating mental health and autism within the Extended Evolutionary Meta-Model (EEMM). Graph theory approaches are discussed in their capacity to depict the processes of change potentially even at the level of the relational frame.

A Functional Contextual Account of Background Knowledge in Categorization: Implications for Artificial General Intelligence and Cognitive Accounts of General Knowledge.

Psychology has benefited from an enormous wealth of knowledge about processes of cognition in relation to how the brain organizes information. Within the categorization literature, this behavior is often explained through theories of memory construction called exemplar theory and prototype theory which are typically based on similarity or rule functions as explanations of how categories emerge. Although these theories work well at modeling highly controlled stimuli in laboratory settings, they often perform less well outside of these settings, such as explaining the emergence of background knowledge processes. In order to explain background knowledge, we present a non-similarity-based post-Skinnerian theory of human language called Relational Frame Theory (RFT) which is rooted in a philosophical world view called functional contextualism (FC). This theory offers a very different interpretation of how categories emerge through the functions of behavior and through contextual cues, which may be of some benefit to existing categorization theories. Specifically, RFT may be able to offer a novel explanation of how background knowledge arises, and we provide some mathematical considerations in order to identify a formal model. Finally, we discuss much of this work within the broader context of general semantic knowledge and artificial intelligence research.

The metabolism of the synthetic cannabinoids ADB-BUTINACA and ADB-4en-PINACA and their detection in forensic toxicology casework and infused papers seized in prisons.

Early warning systems detect new psychoactive substances (NPS), while dedicated monitoring programs and routine drug and toxicology testing identify fluctuations in prevalence. We report the increasing prevalence of the synthetic cannabinoid receptor agonist (SCRA) ADB-BUTINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-butyl-1H-indazole-3-carbox-amide). ADB-BUTINACA was first detected in a seizure in Sweden in 2019, and we report its detection in 13 routine Swedish forensic toxicology cases soon after. In January 2021, ADB-BUTINACA was detected in SCRA-infused papers seized in Scottish prisons and has rapidly increased in prevalence, being detected in 60.4% of the SCRA-infused papers tested between January and July 2021. In this work, ADB-BUTINACA was incubated with human hepatocytes (HHeps), and 21 metabolites were identified in vitro, 14 being detected in authentic case samples. The parent drug and metabolites B9 (mono-hydroxylation on the n-butyl tail) and B16 (mono-hydroxylation on the indazole ring) are recommended biomarkers in blood, while metabolites B4 (dihydrodiol formation on the indazole core), B9, and B16 are suitable biomarkers in urine. ADB-4en-PINACA (N-[1-amino-3,3-dimethyl-1-oxobutan-2-yl]-1-[pent-4-en-1-yl]-1H-indazole-3-carboxamide) was detected in Scottish prisons in December 2020, but, unlike ADB-BUTINACA, prevalence has remained low. ADB-4en-PINACA was incubated with HHeps, and 11 metabolites were identified. Metabolites E3 (dihydrodiol formed in the tail moiety) and E7 (hydroxylation on the linked/head group) are the most abundant metabolites in vitro and are suggested as urinary biomarkers. The in vitro potencies of ADB-BUTINACA (EC50 , 11.5 nM and ADB-4en-PINACA (EC50 , 11.6 nM) are similar to that of MDMB-4en-PINACA (EC50 , 4.3 nM). A third tert-leucinamide SCRA, ADB-HEXINACA was also detected in prison samples and warrants further investigation.

Pharmacokinetic optimisation of novel indole-2-carboxamide cannabinoid CB1 antagonists.

The pharmacokinetic based optimisation of a novel series of indole-2-carboxamide antagonists of the cannabinoid CB(1) receptor is disclosed. Compound 24 was found to be a highly potent and selective cannabinoid CB(1) antagonist with high predicted human oral bioavailability.

The discovery of novel indole-2-carboxamides as cannabinoid CB(1) receptor antagonists.

The discovery and structure-activity relationship of a novel series of indole-2-carboxamide antagonists of the cannabinoid CB(1) receptor is disclosed. Compound 26i was found to be a high potency, selective cannabinoid CB(1) antagonist.

Identification of potent, soluble, and orally active TRPV1 antagonists.

Optimization of a water soluble, moderately potent lead series of isoxazole-3-carboxamides was conducted, affording a compound with the requisite balance of potency, solubility and physicochemical properties for in vivo use. Compound 8e was demonstrated to be efficacious in a rat model of inflammatory pain, following oral administration.

Discovery of potent and orally bioavailable heterocycle-based cannabinoid CB1 receptor agonists.

Novel 3-(1H-indol-3-yl)-1,2,4-oxadiazoles and -thiadiazoles were synthesized and found to be potent CB1 cannabinoid receptor agonists. The oral bioavailability of these compounds could be dramatically improved by optimization studies of the side chains attached to the indole and oxadiazole cores, leading to identification of a CB1 receptor agonist with good oral activity in a range of preclinical models of antinociception and antihyperalgesia.

Discovery of potent, soluble and orally active TRPV1 antagonists. Structure-activity relationships of a series of isoxazoles.

Systematic optimisation of a poorly soluble lead series of isoxazole-3-carboxamides was conducted. Substitution of the 4-position with specific polar functionality afforded the requisite balance of potency, solubility and physicochemical properties. Compound 21a was found to be efficacious in the rat Capsaicin Hargreaves assay following oral administration.