Oxidation of caspase-8 by hypothiocyanous acid enables TNF-mediated necroptosis.

Necroptosis is a form of regulated cell death triggered by various host and pathogen-derived molecules during infection and inflammation. The essential step leading to necroptosis is phosphorylation of the mixed lineage kinase domain-like protein by receptor-interacting protein kinase 3. Caspase-8 cleaves receptor-interacting protein kinases to block necroptosis, so synthetic caspase inhibitors are required to study this process in experimental models. However, it is unclear how caspase-8 activity is regulated in a physiological setting. The active site cysteine of caspases is sensitive to oxidative inactivation, so we hypothesized that oxidants generated at sites of inflammation can inhibit caspase-8 and promote necroptosis. Here, we discovered that hypothiocyanous acid (HOSCN), an oxidant generated in vivo by heme peroxidases including myeloperoxidase and lactoperoxidase, is a potent caspase-8 inhibitor. We found HOSCN was able to promote necroptosis in mouse fibroblasts treated with tumor necrosis factor. We also demonstrate purified caspase-8 was inactivated by low concentrations of HOSCN, with the predominant product being a disulfide-linked dimer between Cys360 and Cys409 of the large and small catalytic subunits. We show oxidation still occurred in the presence of reducing agents, and reduction of the dimer was slow, consistent with HOSCN being a powerful physiological caspase inhibitor. While the initial oxidation product is a dimer, further modification also occurred in cells treated with HOSCN, leading to higher molecular weight caspase-8 species. Taken together, these findings indicate major disruption of caspase-8 function and suggest a novel mechanism for the promotion of necroptosis at sites of inflammation.

Coping and Conformity Motives Mediate the Joint Effects of the Behavioral Inhibition and Approach Systems on Alcohol Problems in Young Adults.

Background: Gray's original Reinforcement Sensitivity Theory (RST) posits that an oversensitive behavioral inhibition system (BIS) may increase risk for negative-reinforcement-motivated drinking, given its role in anxiety. However, existing data provides mixed support for the BIS-alcohol use association. The inconsistent evidence is not surprising, as the revised RST predicts that the behavioral approach system (BAS) should moderate the effect of the BIS on alcohol use. A strong BAS is thought to bring attention to the negatively reinforcing effects of alcohol, leading to problem drinking among those with a strong BIS. While emerging results support this interaction, we still have much to learn about the mechanisms underlying this effect on alcohol use. Accordingly, we examined motives for alcohol use as mediators of the joint associations of the BIS and the BAS on drinking behaviors. Specifically, our central hypothesis was that individuals with a strong BIS and a strong BAS would endorse increased negative reinforcement motives for drinking (coping and conformity motives), which in turn would predict heavy drinking and alcohol problems. Method: Participants (N=346; 195 women) completed study measures as part of the baseline assessment for a larger study. Results: Overall, results partially supported the hypotheses. Mediated moderation analyses showed that the indirect effect of the BIS on alcohol problems, through coping and conformity motives, was strongest at high levels of the BAS. This effect was not supported for alcohol use. Conclusions: Our findings suggest that clinical interventions should target coping and conformity reasons for drinking among anxious, reward responsive, young adults.

A Direct Comparison, and Prioritisation, of the Immunotherapeutic Targets Expressed by Adult and Paediatric Acute Myeloid Leukaemia Cells: A Systematic Review and Meta-Analysis.

Acute myeloid leukaemia (AML) is characterized by impaired myeloid differentiation resulting in an accumulation of immature blasts in the bone marrow and peripheral blood. Although AML can occur at any age, the incidence peaks at age 65. The pathobiology of AML also varies with age with associated differences in incidence, as well as the frequency of cytogenetic change and somatic mutations. In addition, 5-year survival rates in paediatrics are 60-75% but fall to 5-15% in older AML patients. This systematic review aimed to determine whether the altered genes in AML affect the same molecular pathways, indifferent of patient age, and, therefore, whether patients could benefit from the repurposing drugs or the use of the same immunotherapeutic strategies across age boundaries to prevent relapse. Using a PICO framework and PRISMA-P checklist, relevant publications were identified using five literature databases and assessed against an inclusion criteria, leaving 36 articles, and 71 targets for therapy, for further analysis. QUADAS-2 was used to determine the risk of bias and perform a quality control step. We then priority-ranked the list of cancer antigens based on predefined and pre-weighted objective criteria as part of an analytical hierarchy process used for dealing with complex decisions. This organized the antigens according to their potential to act as targets for the immunotherapy of AML, a treatment that offers an opportunity to remove residual leukaemia cells at first remission and improve survival rates. It was found that 80% of the top 20 antigens identified in paediatric AML were also within the 20 highest scoring immunotherapy targets in adult AML. To analyse the relationships between the targets and their link to different molecular pathways, PANTHER and STRING analyses were performed on the 20 highest scoring immunotherapy targets for both adult and paediatric AML. There were many similarities in the PANTHER and STRING results, including the most prominent pathways being angiogenesis and inflammation mediated by chemokine and cytokine signalling pathways. The coincidence of targets suggests that the repurposing of immunotherapy drugs across age boundaries could benefit AML patients, especially when used in combination with conventional therapies. However, due to cost implications, we would recommend that efforts are focused on ways to target the highest scoring antigens, such as WT1, NRAS, IDH1 and TP53, although in the future other candidates may prove successful.

A longitudinal approach to understanding risk factors for problem alcohol use during the COVID-19 pandemic.

BACKGROUND: We conducted a longitudinal study to examine person-centered heterogeneity in problem drinking risk during the 2019 Coronavirus disease (COVID-19) pandemic. We aimed to differentiate high- from low-risk subgroups of drinkers during the pandemic, to report on the longitudinal follow-up of the baseline sample reported in Wardell et al. (Alcohol Clin Exp Res, 44, 2020, 2073), and to examine how subgroups of drinkers differed on coping-related and pre-pandemic alcohol vulnerability factors. METHODS: Canadian alcohol users (N = 364) were recruited for the study. Participants completed surveys at four waves (spaced 3 months apart), with the first being 7 to 8 weeks after the COVID-19 state of emergency began in Canada. The data were analyzed using a parallel process latent growth class analysis followed by general linear mixed models analysis. RESULTS: We found evidence for three latent classes: individuals who increased drinking (class 1; n = 23), low-risk drinkers (class 2; n = 311), and individuals who decreased drinking (class 3; n = 30). Participants who increased (vs. those who decreased) problem drinking during the pandemic struggled with increasing levels of social disconnection and were also increasingly more likely to report drinking to cope with these issues. Those in the increasing class (relative to low-risk drinkers) reported increasing levels of depression during the study. Relative to low-risk drinkers, participants in the increasing class had higher pre-pandemic AUDIT scores, greater frequency of solitary drinking, and higher alcohol demand. Interestingly, participants in the decreasing class had the highest pre-pandemic AUDIT scores. CONCLUSIONS: We examined longitudinal data to identify subgroups of drinkers during the pandemic and to identify factors that may have contributed to increased problem drinking. Findings suggest that while most of the sample did not change their alcohol use, a small portion of individuals escalated use, while a small portion decreased their drinking. Identifying the vulnerability factors associated with increased drinking could aid in the development of preventative strategies and intervention approaches.

Is talk cheap? Correspondence between self-attributions about changes in drinking and longitudinal changes in drinking during the 2019 coronavirus pandemic.

BACKGROUND: There are concerns that the coronavirus disease 2019 (COVID-19) pandemic may increase drinking, but most accounts to date are cross-sectional studies of self-attributions about alcohol-related impacts and the accuracy of those perceptions has not been investigated. The current study examined the correspondence between self-attributions of pandemic-related changes in drinking and longitudinally-measured changes in drinking and alcohol-related consequences in a sample of emerging adults. METHODS: In an existing ongoing longitudinal study on alcohol misuse (≥1 heavy episodic drinking day/month) in emerging adults, 473 individuals (Mage  = 23.8; 41.7% male) received a supplemental assessment from June 17th to July 1st, 2020, during public health restrictions in Ontario, Canada. These intrapandemic data were matched to the most recent assessment prior to the pandemic (~8 months earlier). Self-attributions about changes in drinking were assessed globally (i.e., increases/decreases/no change) and with higher resolution questions clarifying the magnitude of changes. RESULTS: Global self-attributions about changes in drinking substantively paralleled longitudinal changes in weekly drinking days (DD). In the longitudinal data, individuals' who self-reported increases in drinking exhibited significant increases; individuals' who self-reported decreases exhibited significant decreases; and individuals who self-reported no change exhibited nonsignificant changes. Higher resolution items likewise revealed longitudinal patterns of weekly drinking that were substantively consistent with self-attributions. Heavy DD and alcohol-related consequences exhibited similar patterns, but only individuals who self-reported large increases in drinking exhibited increases on these outcomes. Individuals who reported large increases in drinking also exhibited significant increases in depression and posttraumatic stress disorder symptoms. CONCLUSIONS: Self-attributions about drinking closely corresponded to longitudinal changes in drinking, supporting the validity of self-attributions in population-level surveys, particularly in young adults. Notably, a subgroup was identified that exhibited pronounced increases for all alcohol-related outcomes and concurrent increases in internalizing psychopathology.

Survivin' Acute Myeloid Leukaemia-A Personalised Target for inv(16) Patients.

Despite recent advances in therapies including immunotherapy, patients with acute myeloid leukaemia (AML) still experience relatively poor survival rates. The Inhibition of Apoptosis (IAP) family member, survivin, also known by its gene and protein name, Baculoviral IAP Repeat Containing 5 (BIRC5), remains one of the most frequently expressed antigens across AML subtypes. To better understand its potential to act as a target for immunotherapy and a biomarker for AML survival, we examined the protein and pathways that BIRC5 interacts with using the Kyoto Encyclopedia of Genes and Genomes (KEGG), search tool for recurring instances of neighbouring genes (STRING), WEB-based Gene Set Analysis Toolkit, Bloodspot and performed a comprehensive literature review. We then analysed data from gene expression studies. These included 312 AML samples in the Microarray Innovations In Leukemia (MILE) dataset. We found a trend between above median levels of BIRC5 being associated with improved overall survival (OS) but this did not reach statistical significance (p = 0.077, Log-Rank). There was some evidence of a beneficial effect in adjusted analyses where above median levels of BIRC5 were shown to be associated with improved OS (p = 0.001) including in Core Binding Factor (CBF) patients (p = 0.03). Above median levels of BIRC5 transcript were associated with improved relapse free survival (p < 0.0001). Utilisation of a second large cDNA microarray dataset including 306 AML cases, again showed no correlation between BIRC5 levels and OS, but high expression levels of BIRC5 correlated with worse survival in inv(16) patients (p = 0.077) which was highly significant when datasets A and B were combined (p = 0.001). In addition, decreased BIRC5 expression was associated with better clinical outcome (p = 0.004) in AML patients exhibiting CBF mainly due to patients with inv(16) (p = 0.007). This study has shown that BIRC5 expression plays a role in the survival of AML patients, this association is not apparent when we examine CBF patients as a cohort, but when those with inv(16) independently indicating that those patients with inv(16) would provide interesting candidates for immunotherapies that target BIRC5.

Physiologically Important Electrolytes as Regulators of TDP-43 Aggregation and Droplet-Phase Behavior.

Intraneuronal aggregation of TDP-43 is seen in 97% of all amyotrophic lateral sclerosis cases and occurs by a poorly understood mechanism. We developed a simple in vitro model system for the study of full-length TDP-43 aggregation in solution and in protein droplets. We found that soluble, YFP-tagged full-length TDP-43 (yTDP-43) dimers can be produced by refolding in low-salt HEPES buffer; these solutions are stable for several weeks. We found that physiological electrolytes induced reversible aggregation of yTDP-43 into 10-50 nm tufted particles, without amyloid characteristics. The order of aggregation induction potency was K+ < Na+ < Mg2+ < Ca2+, which is the reverse of the Hofmeister series. The kinetics of aggregation were fit to a single-step model, and the apparent rate of aggregation was affected by yTDP-43 and NaCl concentrations. While yTDP-43 alone did not form stable liquid droplets, it partitioned into preformed Ddx4N1 droplets, showing dynamic diffusion behavior consistent with liquid-liquid phase transition, but then aggregated over time. Aggregation of yTDP-43 in droplets also occurred rapidly in response to changes in electrolyte concentrations, mirroring solution behavior. This was accompanied by changes to droplet localization and solvent exchange. Exposure to extracellular-like electrolyte conditions caused rapid aggregation at the droplet periphery. The aggregation behavior of yTDP-43 is controlled by ion-specific effects that occur at physiological concentrations, suggesting a mechanistic role for local electrolyte concentrations in TDP-43 proteinopathies.

Associations Between Drinking and Cortical Thickness in Younger Adult Drinkers: Findings From the Human Connectome Project.

BACKGROUND: Previous neuroimaging studies examining relations between alcohol misuse and cortical thickness have revealed that increased drinking quantity and alcohol-related problems are associated with thinner cortex. Although conflicting regional effects are often observed, associations are generally localized to frontal regions (e.g., dorsolateral prefrontal cortex [DLPFC], inferior frontal gyrus [IFG], and anterior cingulate cortex). Inconsistent findings may be attributed to methodological differences, modest sample sizes, and limited consideration of sex differences. METHODS: This study examined neuroanatomical correlates of drinking quantity and heavy episodic drinking in a large sample of younger adults (N = 706; Mage  = 28.8; 51% female) using magnetic resonance imaging data from the Human Connectome Project. Exploratory analyses examined neuroanatomical correlates of executive function (flanker task) and working memory (list sorting). RESULTS: Hierarchical linear regression models (controlling for age, sex, education, income, smoking, drug use, twin status, and intracranial volume) revealed significant inverse associations between drinks in past week and frequency of heavy drinking and cortical thickness in a majority of regions examined. The largest effect sizes were found for frontal regions (DLPFC, IFG, and the precentral gyrus). Follow-up regression models revealed that the left DLPFC was uniquely associated with both drinking variables. Sex differences were also observed, with significant effects largely specific to men. CONCLUSIONS: This study adds to the understanding of brain correlates of alcohol use in a large, gender-balanced sample of younger adults. Although the cross-sectional methodology precludes causal inferences, these findings provide a foundation for rigorous hypothesis testing in future longitudinal investigations.

Technological advances in the assessment of impulse control in offenders: A systematic review.

Deficits in impulse control have been linked to criminal offending, risk of recidivism, and other maladaptive behaviours relevant to the criminal justice system (e.g. substance use). Impulse control can be conceptualized as encompassing the broad domains of response inhibition and impulsive/risky decision-making. Advancements in technology have led to the development of computerized behavioural measures to assess performance in these domains, such as go/no-go and delay discounting tasks. Despite a relatively large literature examining these tasks in offenders, findings are not universally consistent. This systematic review aims to synthesize the literature using computerized neurocognitive tasks to assess two domains of impulse control in offenders: response inhibition and impulsive/risky decision-making. The review included 28 studies from diverse geographic locations, settings, and offender populations. The results largely support the general conclusion that offenders exhibit deficits in impulse control compared with non-offenders, with studies of response inhibition more consistently reporting differences than studies using impulsive and risky decision-making tasks. Findings are discussed in the context of contemporary neuroimaging research emphasizing dysfunction in prefrontal cortex as a key contributor to impulse control deficits in offenders.

The role of IL-12/23 in T cell-related chronic inflammation: implications of immunodeficiency and therapeutic blockade.

In this review we discuss the divergent role of two closely related cytokines, IL-12 and IL-23, in shaping immune responses. In light of current therapeutic developments using biologic agents to block these two pathways, a better understanding of the immunological function of these cytokines is pivotal.

Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS.

The hydrophobin EAS from the fungus Neurospora crassa forms functional amyloid fibrils called rodlets that facilitate spore formation and dispersal. Self-assembly of EAS into fibrillar rodlets occurs spontaneously at hydrophobic:hydrophilic interfaces and the rodlets further associate laterally to form amphipathic monolayers. We have used site-directed mutagenesis and peptide experiments to identify the region of EAS that drives intermolecular association and formation of the cross-ß rodlet structure. Transplanting this region into a nonamyloidogenic hydrophobin enables it to form rodlets. We have also determined the structure and dynamics of an EAS variant with reduced rodlet-forming ability. Taken together, these data allow us to pinpoint the conformational changes that take place when hydrophobins self-assemble at an interface and to propose a model for the amphipathic EAS rodlet structure.

Solid-state NMR structure determination from diagonal-compensated, sparsely nonuniform-sampled 4D proton-proton restraints.

We report acquisition of diagonal-compensated protein structural restraints from four-dimensional solid-state NMR spectra on extensively deuterated and (1)H back-exchanged proteins. To achieve this, we use homonuclear (1)H-(1)H correlations with diagonal suppression and nonuniform sampling (NUS). Suppression of the diagonal allows the accurate identification of cross-peaks which are otherwise obscured by the strong autocorrelation or whose intensity is biased due to partial overlap with the diagonal. The approach results in unambiguous spectral interpretation and relatively few but reliable restraints for structure calculation. In addition, the diagonal suppression produces a spectrum with low dynamic range for which ultrasparse NUS data sets can be readily reconstructed, allowing straightforward application of NUS with only 2% sampling density with the advantage of more heavily sampling time-domain regions of high signal intensity. The method is demonstrated here for two proteins, α-spectrin SH3 microcrystals and hydrophobin functional amyloids. For the case of SH3, suppression of the diagonal results in facilitated identification of unambiguous restraints and improvement of the quality of the calculated structural ensemble compared to nondiagonal-suppressed 4D spectra. For the only partly assigned hydrophobin rodlets, the structure is yet unknown. Applied to this protein of biological significance with large inhomogeneous broadening, the method allows identification of unambiguous crosspeaks that are otherwise obscured by the diagonal.

A new lysine biosynthetic enzyme from a bacterial endosymbiont shaped by genetic drift and genome reduction.

The effect of population bottlenecks and genome reduction on enzyme function is poorly understood. Candidatus Liberibacter solanacearum is a bacterium with a reduced genome that is transmitted vertically to the egg of an infected psyllid-a population bottleneck that imposes genetic drift and is predicted to affect protein structure and function. Here, we define the function of Ca. L. solanacearum dihydrodipicolinate synthase (CLsoDHDPS), which catalyzes the committed branchpoint reaction in diaminopimelate and lysine biosynthesis. We demonstrate that CLsoDHDPS is expressed in Ca. L. solanacearum and expression is increased ~2-fold in the insect host compared to in planta. CLsoDHDPS has decreased thermal stability and increased aggregation propensity, implying mutations have destabilized the enzyme but are compensated for through elevated chaperone expression and a stabilized oligomeric state. CLsoDHDPS uses a ternary-complex kinetic mechanism, which is to date unique among DHDPS enzymes, has unusually low catalytic ability, but an unusually high substrate affinity. Structural studies demonstrate that the active site is more open, and the structure of CLsoDHDPS with both pyruvate and the substrate analogue succinic-semialdehyde reveals that the product is both structurally and energetically different and therefore evolution has in this case fashioned a new enzyme. Our study suggests the effects of genome reduction and genetic drift on the function of essential enzymes and provides insights on bacteria-host co-evolutionary associations. We propose that bacteria with endosymbiotic lifestyles present a rich vein of interesting enzymes useful for understanding enzyme function and/or informing protein engineering efforts.

Amyloid formation and depolymerization of tumor suppressor p16INK4a are regulated by a thiol-dependent redox mechanism.

The conversion of a soluble protein into polymeric amyloid structures is a process that is poorly understood. Here, we describe a fully redox-regulated amyloid system in which cysteine oxidation of the tumor suppressor protein p16INK4a leads to rapid amyloid formation. We identify a partially-structured disulfide-bonded dimeric intermediate species that subsequently assembles into fibrils. The stable amyloid structures disassemble when the disulfide bond is reduced. p16INK4a is frequently mutated in cancers and is considered highly vulnerable to single-point mutations. We find that multiple cancer-related mutations show increased amyloid formation propensity whereas mutations stabilizing the fold prevent transition into amyloid. The complex transition into amyloids and their structural stability is therefore strictly governed by redox reactions and a single regulatory disulfide bond.

Surface functionalization of carbon nanomaterials by self-assembling hydrophobin proteins.

Class I fungal hydrophobins are small surface-active proteins that self-assemble to form amphipathic monolayers composed of amyloid-like rodlets. The monolayers are extremely robust and can adsorb onto both hydrophobic and hydrophilic surfaces to reverse their wettability. This adherence is particularly strong for hydrophobic materials. In this report, we show that the class I hydrophobins EAS and HYD3 can self-assemble to form a single-molecule thick coating on a range of nanomaterials, including single-walled carbon nanotubes (SWCNTs), graphene sheets, highly oriented pyrolytic graphite, and mica. Moreover, coating by class I hydrophobin results in a stable, dispersed preparation of SWCNTs in aqueous solutions. No cytotoxicity is detected when hydrophobin or hydrophobin-coated SWCNTs are incubated with Caco-2 cells in vitro. In addition, we are able to specifically introduce covalently linked chemical moieties to the hydrophilic side of the rodlet monolayer. Hence, class I hydrophobins provide a simple and effective strategy for controlling the surfaces of a range of materials at a molecular level and exhibit strong potential for biomedical applications.

Fast-track pathway for early diagnosis and management of giant cell arteritis: the combined role of vascular ultrasonography and [18F]-fluorodeoxyglucose PET-computed tomography imaging.

Giant cell arteritis (GCA) is a medical emergency, which can lead to irreversible blindness and other ischaemic vascular events if left untreated. Prompt access to specialist assessment, diagnostics in the form of a fast-track pathway (FTP) and access to appropriate treatment are key factors in preventing morbidity associated with this disease. Recent developments in vascular imaging prompted review of our management of GCA patients. Here, we present the newly implemented FTP in GCA at the University College London Hospital, with added vascular imaging in the form of temporal artery ultrasound (TAUS) and [18F]-fluorodeoxyglucose PET-computed tomography ( 18 F-FDG PET-CT) with temporal artery biopsy. The initial pilot data on the FTP showed a significant negative predictive value of the combined TAUS and 18 F-FDG PET-CT, and the vast majority of cases positive on imaging were confirmed by biopsy. Through the new FTP in GCA, the diagnosis was completed within 48-72 h, compared with the conventional pathway time of up to 2-3 weeks awaiting biopsy results. Prompt and accurate diagnosis of GCA enables commencement of corticosteroid (prednisolone) treatment in the appropriate patient population while avoiding unnecessary steroid exposure and toxicity in GCA-negative patients.

Hazardous Drinking Mediates the Relation Between Externalizing Personality and Reduced Adherence to COVID-19 Public Health Guidelines in University Students.

Using a multigroup path analysis, we examined if hazardous alcohol use mediated the relations between elevated externalizing personality traits (i.e., impulsivity or sensation seeking) and reduced adherence to COVID-19 public health guidelines. We hypothesized that those high in externalizing personality traits would demonstrate less adherence to public health guidelines and that hazardous alcohol use would mediate this relationship. First- and second-year undergraduates (N = 1232; ages 18-25) from five Canadian universities participated in a cross-sectional survey between January to April 2021. Individuals with higher levels of impulsive or sensation seeking personality traits demonstrated poorer adherence to COVID-19 public health guidelines and these relations were mediated by hazardous alcohol use. Results suggest that hazardous drinking is an important target for students high in impulsivity and sensation seeking to increase their adherence to public health guidelines and thereby help control viral spread.

Efficacy of a novel online integrated treatment for problem gambling and tobacco smoking: Results of a randomized controlled trial.

Background and aims: Problem gambling and tobacco use are highly comorbid among adults. However, there are few treatment frameworks that target both gambling and tobacco use simultaneously (i.e., an integrated approach), while also being accessible and evidence-based. The aim of this two-arm open label RCT was to examine the efficacy of an integrated online treatment for problem gambling and tobacco use. Methods: A sample of 209 participants (Mage = 37.66, SD = 13.81; 62.2% female) from North America were randomized into one of two treatment conditions (integrated [n = 91] or gambling only [n = 118]) that lasted for eight weeks and consisted of seven online modules. Participants completed assessments at baseline, after treatment completion, and at 24-week follow-up. Results: While a priori planned generalized linear mixed models showed no condition differences on primary (gambling days, money spent, time spent) and secondary outcomes, both conditions did appear to significantly reduce problem gambling and smoking behaviours over time. Post hoc analyses showed that reductions in smoking and gambling craving were correlated with reductions in days spent gambling, as well as with gambling disorder symptoms. Relatively high (versus low) nicotine replacement therapy use was associated with greater reductions in gambling behaviours in the integrated treatment condition. Discussion and conclusions: While our open label RCT does not support a clear benefit of integrated treatment, findings suggest that changes in smoking and gambling were correlated over time, regardless of treatment condition, suggesting that more research on mechanisms of smoking outcomes in the context of gambling treatment may be relevant.

Recruitment of class I hydrophobins to the air:water interface initiates a multi-step process of functional amyloid formation.

Class I fungal hydrophobins form amphipathic monolayers composed of amyloid rodlets. This is a remarkable case of functional amyloid formation in that a hydrophobic:hydrophilic interface is required to trigger the self-assembly of the proteins. The mechanism of rodlet formation and the role of the interface in this process have not been well understood. Here, we have studied the effect of a range of additives, including ionic liquids, alcohols, and detergents, on rodlet formation by two class I hydrophobins, EAS and DewA. Although the conformation of the hydrophobins in these different solutions is not altered, we observe that the rate of rodlet formation is slowed as the surface tension of the solution is decreased, regardless of the nature of the additive. These results suggest that interface properties are of critical importance for the recruitment, alignment, and structural rearrangement of the amphipathic hydrophobin monomers. This work gives insight into the forces that drive macromolecular assembly of this unique family of proteins and allows us to propose a three-stage model for the interface-driven formation of rodlets.