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Heather L Martin,1 Laura Smith,2 Darren C Tomlinson11BioScreening Technology Group, Leeds Institutes of Molecular Medicine, University of Leeds, Leeds, UK; 2Leeds Institute of Cancer and Pathology, University of Leeds, Leeds, UKAbstract: Breast cancer is the most common cancer in women worldwide, and resistance to the current therapeutics, often concurrently, is an increasing clinical challenge. By understanding the molecular mechanisms behind multidrug-resistant breast cancer, new treatments may be developed. Here we review the recent advances in this understanding, emphasizing the common mechanisms underlying resistance to both targeted therapies, notably tamoxifen and trastuzumab, and traditional chemotherapies. We focus primarily on three molecular mechanisms, the phosphatidylinositide 3-kinase/Akt pathway, the role of microRNAs in gene silencing, and epigenetic alterations affecting gene expression, and discuss how these mechanisms can interact in multidrug resistance. The development of therapeutics targeting these mechanisms is also addressed.Keywords: PI3K/Akt, epigenetics, miRNA, ER, HER2, triple negative

Abstract: Hypofibrinolysis is a documented abnormality in conditions with high risk of vascular occlusion. A key inhibitor of fibrinolysis is α2-antiplasmin (α2AP), and we hypothesize that the Affimer technology, comprising small conformational proteins with 2 9-amino-acid variable regions, can be used to modulate α2AP activity and facilitate fibrinolysis. Using a phage display system, a library of Affimers was screened against α2AP. A total of 28 α2AP-specific Affimers were isolated, of which 1, termed Affimer A11, inhibited protein function and enhanced fibrinolysis. Affimer A11 displayed a monomeric form and consistently reduced the lysis time of clots made from plasma samples of individuals with type 2 diabetes mellitus (n = 15; from 150.8 ± 100.9 to 109.8 ± 104.8 minutes) and those with cardiovascular disease (n = 15; 117.6 ± 40.6 to 79.7 ± 33.3 minutes; P < .01 for both groups). The effects of A11 on fibrinolysis were maintained when clots were made from whole blood samples. Mechanistic studies demonstrated that A11 did not affect clot structure or interfere with the incorporation of α2AP into fibrin networks but significantly enhanced plasmin activity and accelerated plasmin generation. Affimer A11 reduced α2AP binding to plasmin(ogen), whereas molecular modeling demonstrated interactions with α2AP in an area responsible for binding to plasminogen, explaining the effects on both plasmin activity and generation. Affimer A11, at 0.15 to 0.60 mg/mL, had the ability to bind 70% to 90% of plasma α2AP. In conclusion, we demonstrate that Affimers are viable tools for inhibiting α2AP function and facilitating fibrinolysis, making them potential future therapeutic agents to reduce thrombosis risk., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Introduction: Cannabis is increasingly available to individuals in the United States, with the majority of states legalizing medical or recreational cannabis use. Cannabis expectancies (e.g., positive expectations from cannabis use) are related to more frequent cannabis use and associated with greater odds of lapse following a cessation attempt., Methods: To evaluate the relationship between expectancies and cannabis- and health-related outcomes, we surveyed adults who used medical cannabis for chronic pain (n = 633; 52.2 % female; 82.1 % white) over a two-year period. Then, we performed a principal components analysis (PCA) on the Cannabis Expectancies Questionnaire to derive components of cannabis expectancies. We performed k-means clustering to identify subgroups with similar expectancies. The relationship between subgroup assignment and cannabis use outcomes (frequency, quantity), and physical and mental health functioning were evaluated., Results: The PCA identified a three-component solution and k-means clustering identified four subgroups: low expectancies, negative expectancies, amotivation expectancies, and positive expectancies. Participants' cannabis expectancies were dynamic over time. After adjusting for demographics, expectancy subgroup was able to predict six-month cannabis use frequency (χ2(3) = 11.244; p = 0.010) and mental health functioning (χ2(3) = 24.926; p < 0.001), but not quantity (χ2(3) = 2.741, p = 0.433) or physical health functioning (χ2(3) = 5.110, p = 0.164)., Conclusion: In a large cohort of individuals who use medical cannabis, four subgroups with differing cannabis expectancies emerged: low expectancies, negative expectancies, amotivation expectancies, and positive expectancies. These subgroups predicted subsequent outcomes and may be important for targeted intervention approaches to reduce cannabis use and improve functioning., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Introduction: Recent addiction and obesity-related research suggests that episodic future thinking (EFT) can serve as a promising intervention to promote healthy decision-making. We used data from a pilot study to investigate the acute neural effects of EFT in alcohol use disorder (AUD). Because of the limitations of those data, we additionally used data from a previously published functional MRI (fMRI) study in which participants had not received any intervention for their AUD. Methods: In an out-of-scanner, guided interview, participants ( n = 24; median age = 37.3 years; median AUDIT = 22.5) generated scenarios and cues about their future (EFT intervention, n = 15) or recent past (control episodic thinking [CET] control intervention, n = 9). Then, they performed both resting-state and task-based (delay discounting [DD]) fMRI. We used nodes from the default mode network and salience networks as well as the hippocampus to perform seed-based analyses of the resting-state data. The results then guided psychophysiological interaction analyses in the DD task. In addition, we used data from a larger, previously reported study as a "no intervention" group of AUD participants ( n = 50; median age = 43.3; median Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV) alcohol dependence score = 7) to reproduce and aid in interpreting our key findings. Results: EFT, but not CET, participants showed statistically improved DD rates-a behavioral marker for addiction. Resting-state analyses of the left hippocampus revealed connectivity differences in the frontal poles. The directionality of this difference suggested that EFT may reduce a hypo-connectivity relationship between these regions in AUD. We also found resting-state connectivity differences between the salience network and the right dorsolateral prefrontal cortex (R DLPFC), which then led us to discover R-to-L DLPFC psychophysiological interaction differences during DD. Moreover, the resting-state salience-to-DLPFC functional connectivity showed an inverse relationship to DD rate while hyperconnectivity between left and right DLPFC reflected slower reaction times during DD trials. Discussion: These findings suggest that previously noted benefits of EFT such as the improved DD replicated here might coincide with changes in neural connectivity patterns in AUD. The alterations in connectivity highlight potential mechanisms underlying the effectiveness of EFT in improving decision-making in AUD. Understanding these neural effects may contribute to the further development of targeted interventions for AUD and related disorders.

Despite SH2 domains, being pivotal in protein interactions linked to various diseases like cancer, we lack specific research tools for intracellular assays. Understanding SH2-mediated interactions and creating effective inhibitors requires tools which target individual protein domains. Affimer reagents exhibit promise, yet their potential against the extensive SH2 domain family remains largely unexplored. Our study aimed to bridge this gap by identifying Affimer reagents that selectively bind to 22 out of 41 SH2 domains. These reagents enabled a medium-throughput screening approach resembling siRNA studies, shedding light on their functionality. Notably, select Affimers demonstrated the ability to curtail the nuclear translocation of pERK, with Grb2 being a prominent target. Further analyses revealed that these Grb2-specific Affimer reagents displayed competitive inhibition with impressive metrics: IC50s ranging from 270.9 nM to 1.22 µM, together with low nanomolar binding affinities. Moreover, they exhibited the ability to pull down endogenous Grb2 from cell lysates, illustrating their efficacy in binding the Grb2 SH2 domain. This comprehensive assessment underscores the potential of Affimer reagents as domain-specific inhibitors. Their viability for medium/high-throughput phenotypic screening presents a promising avenue via which to identify and characterize potential drug targets within the SH2 domain family., Competing Interests: Declarations Competing interests MJ is an employee of Avacta Life Sciences and holds shares in the company, the Affimer technology is licensed to Avacta Life Sciences by the University of Leeds. The royalties from the license are managed by ULIP at the University of Leeds and disseminated to the inventors DCT and MJM. All other authors do not have a competing interest., (© 2024. The Author(s).)

A key factor in biomineralization is the use of organic molecules to direct the formation of inorganic materials. However, identification of molecules that can selectively produce the calcium carbonate polymorphs calcite or aragonite has proven extremely challenging. Here, we use a phage display approach to identify proteins - rather than the short peptides typically identified using this method - that can direct calcium carbonate formation. A 1.3 × 10 10 library of Affimer proteins was displayed on modified M13 phage, where an Affimer is a ≈13 kDa protein scaffold that displays two variable regions of 9-13 residues. The phage displaying the Affimer library were then screened in binding assays against calcite and aragonite at pH 7.4, and four different strongly-binding proteins were identified. The two aragonite-binding proteins generated aragonite when calcium and magnesium ions were present at a 1 : 1 ratio, while the calcite-binding proteins produce magnesium-calcite under the same conditions. Calcite alone formed in the presence of all four proteins in the absence of magnesium ions. In combination with molecular dynamics simulations to evaluate the conformations of the proteins in solution, this work demonstrates the importance of conformation in polymorph control, and highlights the importance of magnesium ions, which are abundant in seawater, to reduce the energetic barriers associated with aragonite formation.

Significance: Tobacco product design features, including flavors and cigarette filter ventilation, are subject to regulation. This study examined the effects of cigarette and e-cigarette flavors on demand and substitution by preferred cigarette flavor and ventilation in the Experimental Tobacco Marketplace (ETM)., Methods: In a mixed between-group (usual cigarette flavor and ventilation)/within-subject design (policy conditions), individuals who use tobacco (n=176 cigarette (60.5 % female); n=91 multiple products (26.7 % female)) were recruited using Ipsos and InnovateMR, completed purchasing trials with increasing cigarette prices in the ETM. Participants were exposed to four conditions in a 2×2 factorial design with cigarette flavors restricted or unrestricted and e-cigarette flavors restricted or unrestricted., Results: In individuals who exclusively smoke cigarettes: 1) Cigarette menthol restrictions decreased willingness to purchase cigarettes (OR: 0.001; 95 % CI: 0.00002, 0.015) for individuals that prefer menthol cigarettes and increased willingness to purchase NRT (OR: 4.02; 95 % CI: 1.47, 11.0), 2) cigarette menthol restrictions reduced demand for cigarettes in individuals who smoke menthol cigarettes (p<0.001), 3) e-cigarette flavor restrictions reduced the degree of e-cigarette substitution in individuals who smoke menthol cigarettes (p=0.028), and 4) preference for higher cigarette filter ventilation was associated with higher demand for cigarettes (p=0.003) and lowered substitution of smokeless tobacco products (p=0.028). In individuals who use multiple tobacco products, restrictions did not impact product purchasing., Conclusion: Strategies to reduce flavored product sales and increase cessation resource accessibility may improve population health by reducing smoking and increasing NRT use in individuals who use menthol cigarettes., Competing Interests: Declaration of Competing Interest Although the following activities/relationships do not create a conflict of interest pertaining to this manuscript, in the interest of full disclosure, Dr. Bickel would like to report the following: W. K. Bickel is a principal of HealthSim, LLC; BEAM Diagnostics, Inc.; and Red 5 Group, LLC. In addition, he serves on the scientific advisory board for Ria Health; and serves as a consultant for Lumanity and AlphaSights. Dr. Tegge works on a project supported by Indivior, Inc. The other authors report no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Src homology 3 (SH3) domains play a critical role in mediating protein-protein interactions (PPIs) involved in cell proliferation, migration, and the cytoskeleton. Despite their abundance in the human proteome, the functions and molecular interactions of many SH3 domains remain unknown, and this is in part due to the lack of SH3-domain-specific reagents available for their study. Affimer proteins have been developed as affinity reagents targeting a diverse range of targets, including those involved in PPIs. In this study, Affimer proteins were isolated against both the N- and C-terminal SH3 domains (NSH3 and CSH3) of growth-factor-receptor-bound protein 2 (Grb2), an adapter protein that provides a critical link between cell surface receptors and Ras signalling pathways. Targeting the CSH3 alone for the inhibition of PPIs appeared sufficient for curtailing Ras signalling in mammalian cell lines stimulated with human epidermal growth factor (EGF), which conflicts with the notion that the predominant interactions with Ras activating Son of sevenless (SOS) occur via the NSH3 domain. This result supports a model in which allosteric mechanisms involved in Grb2-SOS1 interaction modulate Ras activation.

Abstract: Glycoprotein VI (GPVI) plays a key role in collagen-induced platelet aggregation. Affimers are engineered binding protein alternatives to antibodies. We screened and characterized GPVI-binding Affimers as novel tools to probe GPVI function. Among the positive clones, M17, D22, and D18 bound GPVI with the highest affinities (dissociation constant (KD) in the nanomolar range). These Affimers inhibited GPVI-collagen-related peptide (CRP)-XL/collagen interactions, CRP-XL/collagen-induced platelet aggregation, and D22 also inhibited in vitro thrombus formation on a collagen surface under flow. D18 bound GPVI dimer but not monomer. GPVI binding was increased for D18 but not M17/D22 upon platelet activation by CRP-XL and adenosine 5'-diphosphate. D22 but not M17/D18 displaced nanobody 2 (Nb2) binding to GPVI, indicating similar epitopes for D22 with Nb2 but not for M17/D18. Mapping of binding sites revealed that D22 binds a site that overlaps with Nb2 on the D1 domain, whereas M17 targets a site on the D2 domain, overlapping in part with the glenzocimab binding site, a humanized GPVI antibody fragment antigen-binding fragment. D18 targets a new region on the D2 domain. We found that D18 is a stable noncovalent dimer and forms a stable complex with dimeric GPVI with 1:1 stoichiometry. Taken together, our data demonstrate that Affimers modulate GPVI-ligand interactions and bind different sites on GPVI D1/D2 domains. D18 is dimer-specific and could be used as a tool to detect GPVI dimerization or clustering in platelets. A dimeric epitope regulating ligand binding was identified on the GPVI D2 domain, which could be used for the development of novel bivalent antithrombotic agents selectively targeting GPVI dimer on platelets., (© 2024 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Monoclonal antibodies have revolutionized therapies, but non-immunoglobulin scaffolds are becoming compelling alternatives owing to their adaptability. Their ability to be labeled with imaging or cytotoxic compounds and to create multimeric proteins is an attractive strategy for therapeutics. Focusing on HER2, a frequently overexpressed receptor in breast cancer, this study addresses some limitations of conventional targeting moieties by harnessing the potential of these scaffolds. HER2-binding Affimers were isolated and characterized, demonstrating potency as binding reagents and efficient internalization by HER2-overexpressing cells. Affimers conjugated with cytotoxic agent achieved dose-dependent reductions in cell viability within HER2-overexpressing cell lines. Bispecific Affimers, targeting HER2 and virus-like particles, facilitated efficient internalization of virus-like particles carrying enhanced green fluorescent protein (eGFP)-encoding RNA, leading to protein expression. Anti-HER2 affibody or designed ankyrin repeat protein (DARPin) fusion constructs with the anti-VLP Affimer further underscore the adaptability of this approach. This study demonstrates the versatility of scaffolds for precise delivery of cargos into cells, advancing biotechnology and therapeutic research., Competing Interests: The authors declare a potential conflict of interest. A patent application has been filed related to the work described in this manuscript. [Application number: LU505126, Inventors: George P. Lomonossoff, Sachin Shah, Keith Saunders, Darren C Tomlinson, Christian Tiede, Heather Martin, Ross Overman]. D.C.T. also is a named inventor on patent “scaffold protein derived from plant cystatins” Europe 14705565.1, US 14/768,032, Australia 2014217628. G.P.L. also sits as a chair on the advisory board for Leaf Expression Systems., (© 2024 The Authors. Published by Elsevier Inc.)

Introduction: Cigarette smoking accounts for >30% of the socioeconomic gap in life expectancy. Flavored restrictions claim to promote equity; however, no previous studies have compared the effect of cigarette and e-cigarette flavor restrictions among individuals who smoke with lower and higher socioeconomic status (SES)., Aims and Methods: In a between-group within-subject design, individuals with lower (n = 155) and higher (n = 125) SES completed hypothetical purchasing trials in the experimental tobacco marketplace (ETM). Conditions were presented in a 2 × 2 factorial design (cigarette flavors restricted or unrestricted and e-cigarette flavors restricted or unrestricted) with increasing cigarette prices across trials., Results: Results show (1) SES differences in cigarette, e-cigarette, and NRT purchases under unrestricted policies, with lower SES showing higher cigarette demand and lower e-cigarette and NRT substitution than higher SES, (2) cigarette restrictions decreased cigarette and increased NRT purchases among lower SES, but no significant changes among higher SES, (3) decreased SES differences in cigarette demand under cigarette restrictions, but persistence under e-cigarette restrictions or their combination, (4) persistence of SES differences in e-cigarette purchases when all restrictions were enforced, and (5) waning of SES differences in NRT purchasing under all restrictions., Conclusions: Flavor restrictions differentially affected individuals based on SES. Within-group comparisons demonstrated restrictions significantly impacted lower SES, but not higher SES. Between-group comparisons showed SES differences in cigarette purchasing decreased under cigarette restrictions, but persisted under e-cigarette-restrictions or their combination. Additionally, SES differences in NRT substitution decreased under flavor restrictions. These findings highlight the utility of the ETM to investigate SES disparities., Implications: With increasing trends of socioeconomic differences in smoking prevalence and cessation rates, smoking-related health disparities are expected to continue to widen. Restricting menthol flavor in cigarettes while enhancing the availability and affordability of NRT have the potential to alleviate SES disparities in tobacco use, therefore, positively impacting health equity. However, this effect may depend on flavor availability in other tobacco products., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Research on Nicotine and Tobacco.)

Background: Regulatory flexibility (RF) involves three distinct components of self-regulation: context sensitivity, repertoire, and feedback responsiveness. Subgroups based on differences in RF have been identified in a general sample and are differentially associated with symptoms of anxiety and depression. However, potential RF profiles have not been examined in individuals with substance use disorders. This study examined RF subtypes in individuals with alcohol use disorder (AUD) and their associations with psychosocial outcomes (i.e., depression, anxiety, and stress) and delay discounting (a core feature of addiction)., Methods: Individuals (n = 200) with an Alcohol Use Disorders Identification Test score of >16 (mean = 24.12 (±6.92)) were recruited from Amazon Mechanical Turk (mean = 37.26 years old (±11.41); 94 (47%) women). Participants completed the Context Sensitivity Index, the Flexible Regulation of Emotional Expression Scale, and the Coping Flexibility Scale to assess RF. Participants also completed an Adjusting Amount Delay Discounting Task and the Depression, Anxiety, and Stress Scale (DASS-21). Latent profile analyses (LPA) were used to identify patterns in RF deficits. Kruskal-Wallis and Dunn's tests were performed to examine differences in discounting rates and symptoms of depression, anxiety, and stress across RF profiles., Results: The LPA revealed a 2-profile characterization, including (1) context sensitive regulators (CSR; n = 39) and (2) moderate flexibility regulators (MFR; n = 161). CSR demonstrated significantly lower symptoms of depression (p = 0.004), anxiety (p < 0.001), and stress (p < 0.001) than MFR. CSR also displayed significantly lower AUDIT scores (p = 0.031)., Conclusions: Findings illustrate that among individuals with moderate-severe AUD, those high in context sensitivity coupled with moderate abilities in repertoire and feedback responsiveness have fewer symptoms of depression, anxiety, and stress. Together, context sensitivity may be an important and protective component of RF among individuals with AUD., (© 2023 Research Society on Alcohol.)

Introduction: Delay discounting (DD) and self-regulation are important predictors of substance use disorder (SUD) outcomes. Further, regulatory flexibility (RF; i.e., selecting, monitoring, and adapting coping techniques based on contextual demands) is related to psychological resilience. However, studies have yet to examine associations among DD, RF, and remission from SUDs among individuals in recovery., Methods: Individuals (N = 148) in SUD recovery completed the Context Sensitivity Index (CSI), the Flexible Regulation of Emotional Expression (FREE) Scale, and the Perceived Ability to Cope with Trauma (PACT) Scale to assess RF and, an $1000 hypothetical reward Adjusting Amount Delay Discounting Task. The study considered individuals to be in remission from SUD if they did not endorse any SUD DSM-5 symptom other than craving (except tobacco use disorder) in the past three months. The study team used t-tests to examine differences in RF and DD by remission status. Univariate linear regressions were used to examine the relationship between RF and DD. Finally, mediation models examined the dynamic relationship among DD, RF, and remission status., Results: Remitted individuals (n = 82) had significantly lower DD (i.e., greater preference for larger, later rewards) rates (p < .001) and higher context sensitivity (p < .001) and coping flexibility (p < .001). The study found significant negative associations between DD and context sensitivity (p = .008), coping flexibility (p = .002), and emotion regulation flexibility (p < .001). Finally, context sensitivity (p = .023) and coping flexibility (p = .009) mediated the relationship between DD and SUD remission., Conclusions: Results suggest that individuals in recovery with broader temporal windows can better identify contextual demands and flexibly cope, contributing to improved SUD recovery outcomes., Competing Interests: Declaration of competing interest Although the following activities/relationships do not create a conflict of interest pertaining to this manuscript, in the interest of full disclosure, W.K. Bickel would like to report the following: W. K. Bickel is a principal of HealthSim, LLC; BEAM Diagnostics, Inc.; and Red 5 Group, LLC. In addition, he serves on the scientific advisory board for Sober Grid, Inc.; and Ria Health; serves as a consultant for Boehringer Ingelheim International; and works on a project supported by Indivior, Inc. Dr. Tegge would like to report the following: works on a project supported by Indivior, Inc. None of the other authors report any potential conflicts of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Tobacco regulations may increase demand for illegal cigarettes. We use the Experimental Tobacco Marketplace to estimate the impact of banning menthol cigarettes (Experiment 1) and decreasing allowable cigarette filter ventilation levels (Experiment 2)., Methods: Crowdsourced participants were randomized into one of four groups (2×2 factorial design). Experiment 1 included menthol availability (yes/no) by purchasing option (legal only vs illegal available). Experiment 2 included filter-vented cigarettes availability (yes/no) by purchasing option (legal only vs illegal available). Participants were given an individualized budget to purchase tobacco. Percent budget spent was the outcome measure., Results: Experiment 1, with a legal marketplace only, non-menthol cigarette purchasing was lower (p=0.010) and electronic-cigarette purchasing was higher (p=0.016), when cigarettes were banned compared to when they were available. With an illegal marketplace, switching to legal non-menthol cigarettes was less likely (p<0.001) and purchasing illegal menthol cigarettes was higher (p<0.001), when cigarettes were banned compared to when they were available. Experiment 2, with a legal marketplace only, cigarette purchasing was lower (p=0.010), when the participant's filtered vented cigarettes were banned compared to when they were available. With an illegal marketplace, purchasing the legal low-ventilated cigarette option was lower (p<0.001) and significant differences in illegal filter-vented cigarette purchasing were not observed, when their filter-vented cigarettes were banned compared to when they were available legally., Conclusions: Without an illegal option, both restrictions decreased cigarette purchasing, but the menthol ban increased e-cigarette purchasing. With an illegal option, a menthol ban increased illegal cigarette purchasing, but decreasing filter ventilation did not., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Although the following activities/relationships do not create a conflict of interest pertaining to this manuscript, in the interest of full disclosure, Dr. Bickel would like to report the following: W. K. Bickel is a principal of HealthSim, LLC; BEAM Diagnostics, Inc.; and Red 5 Group, LLC. In addition, he serves on the scientific advisory board for Sober Grid, Inc.; and Ria Health; serves as a consultant for Boehringer Ingelheim International and Lumanity; and works on a project supported by Indivior, Inc. Dr. Tegge reports work on a project supported by Indivior, Inc. Dr. Cummings and Shields report that they have and continue to serve as a paid expert witness in litigation filed against cigarette manufacturers. The other authors report no conflict of interest., (Copyright © 2023. Published by Elsevier B.V.)

Hypothetical purchase tasks (HPTs) are effective tools for evaluating participants' demand for substances. The present study evaluated the effect of task presentation on producing unsystematic data and purchasing behavior in a sample of individuals who smoke cigarettes. Participants ( n = 365) were recruited from Amazon Mechanical Turk and randomly assigned to complete two of three HPT presentations: List (prices on one page in an increasing order), Ascending (one price per page in an increasing order), or Random (one price per page in a random order). We evaluated outcomes using a mixed model regression with a random effect for participants. We observed a significant effect of task presentation on passing the criterion assessing consistency in effects of contiguous prices (i.e., Bounce; X ²(2) = 13.31, p = .001). A significant effect of task presentation on Trend or Reversals from Zero was not observed. For purchasing behavior, we observed a significant effect of task presentation on R ², X ²(2) = 17.89, p < .001; BP₁ , X ²(2) = 13.64, p = .001; ln(α), X ²(2) = 332.94, p < .001; and ln( O max ), X ²(2) = 20.26, p < .001; we did not observe a significant effect of task presentation on ln( Q ₀) or ln( P max ). We recommend against using the Random HPT presentation to avoid unsystematic data. While the List and Ascending presentations do not differ across unsystematic criteria or purchasing behavior, the List presentation may be preferred due to participant experience. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Concentration-therapeutic efficacy relationships have been observed for several therapeutic monoclonal antibodies (TmAb), where low circulating levels can result in ineffective treatment and high concentrations can cause adverse reactions. Rapid therapeutic drug monitoring (TDM) of TmAb drugs would provide the opportunity to adjust an individual patient's dosing regimen to improve treatment results. However, TDM for immunotherapies is currently limited to centralised testing methods with long sample-collection to result timeframes. Here, we show four point-of-care (PoC) TmAb biosensors by combining anti-idiotypic Affimer proteins and NanoBiT split luciferase technology at a molecular level to provide a platform for rapid quantification (<10 minutes) for four clinically relevant TmAb (rituximab, adalimumab, ipilimumab and trastuzumab). The rituximab sensor performed best with 4 pM limit of detection (LoD) and a quantifiable range between 8 pM-2 nM with neglectable matrix effects in serum up to 1%. After dilution of serum samples, the resulting quantifiable range for all four sensors falls within the clinically relevant range and compares favourably with the sensitivity and/or time-to-result of current ELISA standards. Further development of these sensors into a PoC test may improve treatment outcome and quality of life for patients receiving immunotherapy., Competing Interests: The authors declare the following competing financial interest(s): the Affimer reagents used in this report are owned by the University of Leeds (UoL) but licensed to Avacta Life Sciences. The UoL received royalties from Avacta Life Sciences as part of the license agreement, which is managed by the commercialization team. The authors declare no competing financial interest., (This journal is © The Royal Society of Chemistry.)

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease., Competing Interests: Conflicts of interest M. J. M. and D. C. T. are named inventors of the Affimer technology and this is filed under U.S. patent number #10,844,370 assigned to the University of Leeds on “Scaffold proteins derived from plant cystatins”., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible., Competing Interests: Declaration of interests A.L.T. owns personal shares in Avacta Life Sciences. D.C.T. is a named inventor on the “artificial binding protein” patent, which is held be the University of Leeds and covers the Affimer reagents used in the study., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Therapeutic monoclonal antibodies (TmAb) have emerged as effective treatments for a number of cancers and autoimmune diseases. However, large interpatient disparities in the pharmacokinetics of TmAb treatment requires close therapeutic drug monitoring (TDM) to optimise dosage for individual patients. Here we demonstrate an approach for achieving rapid, sensitive quantification of two monoclonal antibody therapies using a previously described enzyme switch sensor platform. The enzyme switch sensor consists of a β-lactamase - β-lactamase inhibitor protein (BLA-BLIP) complex with two anti-idiotype binding proteins (Affimer proteins) as recognition elements. The BLA-BLIP sensor was engineered to detect two TmAbs (trastuzumab and ipilimumab) by developing constructs incorporating novel synthetic binding reagents to each of these mAbs. Trastuzumab and ipilimumab were successfully monitored with sub nM sensitivity in up to 1% serum, thus covering the relevant therapeutic range. Despite the modular design, the BLA-BLIP sensor was unsuccessful in detecting two further TmAbs (rituximab and adalimumab), an explanation for which was explored. In conclusion, the BLA-BLIP sensors provide a rapid biosensor for TDM of trastuzumab and ipilimumab with the potential to improve therapy. The sensitivity of this platform alongside its rapid action would be suitable for bedside monitoring in a point-of-care (PoC) setting., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The Affimer reagents used in this report are owned by the University of Leeds (UoL) but licensed to Avacta Life Sciences. The UoL receive royalties from Avacta Life Sciences as part of the license agreement, which is managed by the commercialisation team. The authors declare no competing financial interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Despite being a major threat to health, vaccine hesitancy (i.e., refusal or reluctance to vaccinate despite vaccine availability) is on the rise. Using a longitudinal cohort of young adults (N = 1260) from Los Angeles County, California we investigated the neurobehavioral mechanisms underlying COVID-19 vaccine hesitancy. Data were collected at two time points: during adolescence (12th grade; fall 2016; average age = 16.96 (±0.42)) and during young adulthood (spring 2021; average age = 21.33 (±0.49)). Main outcomes and measures were delay discounting (DD; fall 2016) and tendency to act rashly when experiencing positive and negative emotions (UPPS-P; fall 2016); self-reported vaccine hesitancy and vaccine beliefs/knowledge (spring 2021). A principal components analysis determined four COVID-19 vaccine beliefs/knowledge themes: Collective Responsibility, Confidence and Risk Calculation, Complacency, and Convenience . Significant relationships were found between themes, COVID-19 vaccine hesitancy, and DD. Collective Responsibility (β = -1.158[-1.213,-1.102]) and Convenience (β = -0.132[-0.185,-0.078]) scores were negatively associated, while Confidence and Risk Calculation (β = 0.283[0.230,0.337]) and Complacency (β = 0.412[0.358,0.466]) scores were positively associated with COVID-19 vaccine hesitancy. Additionally, Collective Responsibility (β = -0.060[-0.101,-0.018]) was negatively associated, and Complacency (β = -0.063[0.021,0.105]) was positively associated with DD from fall 2016. Mediation analysis revealed immediacy bias during adolescence, measured by DD, predicted vaccine hesitancy 4 years later while being mediated by two types of vaccine beliefs/knowledge: Collective Responsibility (β = 0.069[0.022,0.116]) and Complacency (β = 0.026[0.008,0.044]). These findings provide a further understanding of individual vaccine-related decision-making among young adults and inform public health messaging to increase vaccination acceptance., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Although the following activities/relationships do not create a conflict of interest pertaining to this manuscript, in the interest of full disclosure, Dr. Bickel would like to report the following:  W. K. Bickel is a principal of HealthSim, LLC; BEAM Diagnostics, Inc.; and Red 5 Group, LLC.  In addition, he serves on the scientific advisory board for Sober Grid, Inc.; and Ria Health; serves as a consultant for Boehringer Ingelheim International and Lumanity; and works on a project supported by Indivior, Inc. Dr Tegge would like to report work on a project supported by Indivior, Inc. The other authors report no conflict of interest., (© 2023 The Authors.)

This systematic review aims to characterize the utility of machine learning to identify the predictors of smoking cessation outcomes and identify the machine learning methods applied in this area. In the current study, multiple searches occurred through December 9, 2022 in MEDLINE, Science Citation Index, Social Science Citation Index, EMBASE, CINAHL Plus, APA PsycINFO, PubMed, Cochrane Central Register of Controlled Trials, and the IEEE Xplore were performed. Inclusion criteria included various machine learning techniques, studies reporting cigarette smoking cessation outcomes (smoking status and the number of cigarettes), and various experimental designs (e.g., cross-sectional and longitudinal). Predictors of smoking cessation outcomes were assessed, including behavioral markers, biomarkers, and other predictors. Our systematic review identified 12 papers fitting our inclusion criteria. In this review, we identified gaps in knowledge and innovation opportunities for machine learning research in the field of smoking cessation., Competing Interests: Although the following activities/relationships do not create a conflict of interest pertaining to this manuscript, in the interest of full disclosure, Dr. Bickel would like to report the following: W. K. Bickel is a principal of HealthSim, LLC; BEAM Diagnostics, Inc.; and Red 5 Group, LLC. In addition, he serves on the scientific advisory board for Sober Grid, Inc., and Ria Health, is a consultant for Alkermes, Inc., and works on a project supported by Indivior, Inc. The other authors have no conflicts to report.

3D cell culture models of cancer are currently being developed to recapitulate in vivo physiological conditions and to assess therapeutic responses. However, most models failed to incorporate the biochemical and biophysical stimuli from fluid flow. In this study, a three-dimensional scaffold, SeedEZ was applied within the PerfusionPal perfused culture system to investigate how perfusion, and blood-like oxygen delivery influenced breast cancer cell growth and their responses to a commonly used breast cancer drug tamoxifen. Our results showed that breast cancer cells could be maintained over 3 weeks in PerfusionPal with increased cell viability compared to static 3D culture in fully humanised conditions. This platform also supported examining the effect of tamoxifen on breast cancer cell lines and in primary patient-derived breast cancer samples. Future work is warranted to further the adaption for fully humanised assessment of drug effectiveness in a patient personalized approach with the aim to reduce the burden of animal use in cancer research and increase the degree of human pre-clinical data translation to clinic., Competing Interests: PerfusionPal system and SeedEZ scaffold are commercial products sold by Lena Biosciences. JS is the Chief Science Officer at Lena Biosciences and holds stock options of the company. JV is the President and Chief Executive Officer at Lena Biosciences and holds shares of the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Delay discounting (DD), the decrease in reward valuation as a function of delay to receipt, is a key process undergirding alcohol use. Narrative interventions, including episodic future thinking (EFT), have decreased delay discounting and demand for alcohol. Rate dependence, the relationship between a baseline rate and change in that rate after an intervention, has been evidenced as a marker of efficacious substance use treatment, but whether narrative interventions have rate-dependent effects needs to be better understood. We investigated the effects of narrative interventions on delay discounting and hypothetical demand for alcohol in this longitudinal, online study., Methods: Individuals (n = 696) reporting high- or low-risk alcohol use were recruited for a longitudinal 3-week survey via Amazon Mechanical Turk. Delay discounting and alcohol demand breakpoint were assessed at baseline. Individuals returned at weeks 2 and 3 and were randomized into the EFT or scarcity narrative interventions and again completed the delay discounting tasks and alcohol breakpoint task. Oldham's correlation was used to explore the rate-dependent effects of narrative interventions. Study attrition as a function of delay discounting was assessed., Results: Episodic future thinking significantly decreased, while scarcity significantly increased delay discounting relative to baseline. No effects of EFT or scarcity on the alcohol demand breakpoint were observed. Significant rate-dependent effects were observed for both narrative intervention types. Higher delay discounting rates were associated with a greater likelihood of attrition from the study., Conclusion: The evidence of a rate-dependent effect of EFT on delay discounting rates offers a more nuanced, mechanistic understanding of this novel therapeutic intervention and can allow more precise treatment targeting by demonstrating who is likely to receive the most benefit from it., (© 2023 The Authors. Alcohol: Clinical and Experimental Research published by Wiley Periodicals LLC on behalf of Research Society on Alcohol.)

Previous research with individuals with substance use disorder indicated that different narratives have different effects on one's behaviors and decision-making. One question that remains unanswered is whether simulating real-world consequences of illegal purchases using narratives would induce psychological distress levels as expected in real-world situations. The present experiment used a between-group design to study how different narratives regarding penalties impact smokers' affective state and more specifically, psychological distress. Participants ( N = 93) were randomized into one of the three groups: (a) legal tobacco purchases (LTP), (b) illegal tobacco purchases with fines (ITP&#95;F), and (c) illegal tobacco purchases with fines, criminal record, and negative public exposure (ITP&#95;F + CR + NPE) to complete an online survey. The survey contained one narrative randomly assigned to each participant, demographic questions, the heaviness of smoking index, the Positive and Negative Affect Schedule (PANAS), and a single-item psychological distress question. The results showed that (a) narratives about making illegal tobacco purchases significantly increased psychological distress when compared to narratives about making LTP, (b) no differences were observed on psychological distress and negative affective state between a narrative describing a fine and a narrative describing a fine, a criminal record, and negative public exposure, and (c) a narrative about a criminal record had the greatest effect on psychological distress among the different narratives about penalty types. Narratives involving illegal purchases increase psychological distress, which varies according to the hypothetical penalties described. More research regarding the use of narratives on decision-making and purchasing of hypothetical substances is warranted. (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Background : Recovery from substance use disorders (SUDs) requires sustained and purposeful support to maintain long-term remission. Methods : This study investigated the association between assessment of recovery capital, household chaos, delay discounting (DD) and probability discounting (PD), and remission status among individuals in recovery from SUD. Data from 281 participants from the International Quit & Recovery Registry (IQRR), an ongoing online registry that aims to study the recovery process, were included in the analysis. Results : Lower DD rates and higher recovery capital were found among those in remission compared to those not in remission after controlling for demographics. In contrast, the association of household chaos and PD with remission status were insignificant. Overall, DD accounted for 20% of the total effect between the recovery capital and the remission status. Conclusion : This study contributes to the understanding of recovery as a multidimensional process, supports DD as a behavioral marker of addiction, and suggests areas for future research.

Introduction: Research defines recovery capital as the amount of tangible and intangible resources (e.g., human/personal, physical, social, and cultural) available to initiate and sustain recovery from substance use disorders (SUDs). An individual's amount of recovery capital is dynamic over time and influenced by a number of factors such as baseline amount at initiation of recovery/treatment, length of abstinence, access/availability of resources, and individual factors such as the decision to utilize available resources. Research has been proposed delay discounting (DD), which reflects an individual's relative preference for immediate versus delayed rewards, as a candidate behavioral marker for SUDs but has not yet examined it in the context of recovery capital, and DD may be an important aspect of human capital. Thus, the aim of the current study was to examine associations among recovery capital, DD, and length of abstinence., Methods: The study included in its analysis data from 111 individuals in recovery from SUDs from the International Quit and Recovery Registry, an ongoing data collection program used to further scientific understanding of recovery. The study assessed recovery capital using the Assessment of Recovery Capital (ARC) and assessed discounting rates using an adjusting-delay task. The study team performed univariate linear regression to examine the relationship between total ARC score and demographic variables, length of abstinence, and DD. The research team performed a mediation analysis to understand the role of length of abstinence in mediating the relationship between DD and ARC score., Results: Total ARC score was significantly negatively associated with DD and positively associated with length of abstinence, even after adjusting for covariates. Mediation analysis indicated that length of abstinence significantly partially mediated the relationship between DD and ARC score., Conclusion: These findings support the characterization of DD as an important aspect of human capital and a candidate behavioral marker for SUDs. Future research may wish to investigate whether interventions designed to increase the value of future rewards also increase recovery capital., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Crowdsourcing platforms allow researchers to quickly recruit and collect behavioral economic measures in substance-using populations, such as cigarette smokers. Despite the broad utility and flexibility, data quality issues have been an object of concern. In two separate studies recruiting cigarette smokers, we sought to investigate the association between a practical quality control measure (accuracy on an instruction quiz), on internal consistency of number of cigarettes smoked per day and purchasing patterns of tobacco products in an experimental tobacco marketplace (ETM; Study 1), and in a cigarette purchase task (CPT; Study 2). Participants (N = 312 in Study 1; N = 119 in Study 2) were recruited from Amazon mechanical turk. Both studies included task instructions, a quiz, a purchase task, cigarette usage and dependence questions, and demographics. The results show that participants who answered all instruction items correctly: (a) reported the number of cigarettes per day more consistently (partial η² = 0.11, p < .001, Study 1; partial η² = 0.09, p = .016, Study 2), (b) demonstrated increased model fit among the cigarette demand curves (partial η² = 0.23, p < .001, Study 1; partial η² = 0.08, p = .002, Study 2), and purchased tobacco products in the ETM more consistently with their current usage. We conclude that instruction quizzes before purchase tasks may be useful for researchers evaluating demand data. Instruction quizzes with multiple items may allow researchers to choose the level of data quality appropriate for their studies. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Crowdsourced methods of data collection such as Amazon Mechanical Turk (MTurk) have been widely adopted in addiction science. Recent reports suggest an increase in poor quality data on MTurk, posing a challenge to the validity of findings. However, empirical investigations of data quality in addiction-related samples are lacking. In this study of individuals with alcohol use disorder (AUD), we compared poor quality delay discounting data to randomly generated data. A reanalysis of prior published delay discounting data was conducted comparing included, excluded, and randomly generated data samples. Nonsystematic criteria were implemented as a measure of data quality. The excluded data was statistically different from the included sample but did not differ from randomly generated data on multiple metrics. Moreover, a response bias was identified in the excluded data. This study provides empirical evidence that poor quality delay discounting data in an AUD sample is not statistically different from randomly generated data, suggesting data quality concerns on MTurk persist in addiction samples. These findings support the use of rigorous methods of a priori defined criteria to remove poor quality data post hoc. Additionally, it highlights that the use of nonsystematic delay discounting criteria to remove poor quality data is rigorous and not simply a way of removing data that does not conform to an expected theoretical model. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

C. difficile infection (CDI) is a leading healthcare-associated infection with a high morbidity and mortality and is a financial burden. No current standalone point-of-care test (POCT) is sufficient for the identification of true CDI over a disease-free carriage of C. difficile , so one is urgently required to ensure timely, appropriate treatment. Here, two types of binding proteins, Affimers and nanobodies, targeting two C. difficile biomarkers, glutamate dehydrogenase (GDH) and toxin B (TcdB), are combined in NanoBiT (NanoLuc Binary Technology) split-luciferase assays. The assays were optimized and their performance controlling parameters were examined. The 44 fM limit of detection (LoD), 4-5 log range and 1300-fold signal gain of the TcdB assay in buffer is the best observed for a NanoBiT assay to date. In the stool sample matrix, the GDH and TcdB assay sensitivity (LoD = 4.5 and 2 pM, respectively) and time to result (32 min) are similar to a current, commercial lateral flow POCT, but the NanoBit assay has no wash steps, detects clinically relevant TcdB over TcdA, and is quantitative. Development of the assay into a POCT may drive sensitivity further and offer an urgently needed ultrasensitive TcdB test for the rapid diagnosis of true CDI. The NanoBiTBiP (NanoBiT with Binding Proteins) system offers advantages over NanoBiT assays with antibodies as binding elements in terms of ease of production and assay performance. We expect this methodology and approach to be generally applicable to other biomarkers.

Introduction: Substance use disorders (SUDs) remain challenging maladies to treat in the United States and impose significant societal costs. Despite these challenges, a significant number of individuals endorse being in recovery from SUD. The scientific understanding of SUD recovery has evolved to include not only improvements in substance use but also improvements in personal wellness and psychosocial functioning. The devaluation of future rewards (delay discounting; DD) is broadly associated with SUD inception and outcomes. We sought to investigate the relationship between DD, time in recovery, and recovery progress., Methods: We conducted an online assessment of 127 individuals in recovery from SUD who the study recruited via the International Quit and Recovery Registry (IQRR). The research team obtained measures of recovery progress via the Addiction Recovery Questionnaire (ARQ) and the Treatment Effectiveness Assessment (TEA). Additionally, the study collected measures of DD, time in recovery, and endorsement of abstinence in recovery (i.e., requiring abstinence vs. not). We utilized linear regression to test for associations among these variables and performed a mediation analysis to test the role of DD in mediating the relationship between time in recovery and measures of recovery progress., Results: Time in recovery was positively associated with the ARQ (p < .001) and TEA (p < .001). Furthermore, an individual's delay discounting rate mediated the relationship between time in recovery and ARQ/TEA. Of the participants, 66% endorsed recovery requiring total abstinence from alcohol and drugs. Last, through an exhaustive model selection, the study did not find an individual's endorsement of abstinence in recovery to be a primary predictor of recovery progress., Conclusions: This study presents evidence that, for individuals in recovery, the temporal view (i.e., focus on immediate vs. future rewards) is a significant influence on recovery progress. Additionally, an individual's endorsement of abstinence in recovery was not significantly associated with recovery progress, suggesting the importance of a holistic view of SUD recovery. These findings contribute to the understanding of recovery as a multidimensional process and provide further support for DD as a behavioral marker of addiction., (Copyright © 2021 Elsevier Inc. All rights reserved.)