Amyloid-like aggregates sequester numerous metastable proteins with essential cellular functions.

Protein aggregation is linked with neurodegeneration and numerous other diseases by mechanisms that are not well understood. Here, we have analyzed the gain-of-function toxicity of artificial ß sheet proteins that were designed to form amyloid-like fibrils. Using quantitative proteomics, we found that the toxicity of these proteins in human cells correlates with the capacity of their aggregates to promote aberrant protein interactions and to deregulate the cytosolic stress response. The endogenous proteins that are sequestered by the aggregates share distinct physicochemical properties: They are relatively large in size and significantly enriched in predicted unstructured regions, features that are strongly linked with multifunctionality. Many of the interacting proteins occupy essential hub positions in cellular protein networks, with key roles in chromatin organization, transcription, translation, maintenance of cell architecture and protein quality control. We suggest that amyloidogenic aggregation targets a metastable subproteome, thereby causing multifactorial toxicity and, eventually, the collapse of essential cellular functions.

Crystal structure and activity of a de novo enzyme, ferric enterobactin esterase Syn-F4.

Producing novel enzymes that are catalytically active in vitro and biologically functional in vivo is a key goal of synthetic biology. Previously, we reported Syn-F4, the first de novo protein that meets both criteria. Syn-F4 hydrolyzed the siderophore ferric enterobactin, and expression of Syn-F4 allowed an inviable strain of Escherichia coli (Δfes) to grow in iron-limited medium. Here, we describe the crystal structure of Syn-F4. Syn-F4 forms a dimeric 4-helix bundle. Each monomer comprises two long α-helices, and the loops of the Syn-F4 dimer are on the same end of the bundle (syn topology). Interestingly, there is a penetrated hole in the central region of the Syn-F4 structure. Extensive mutagenesis experiments in a previous study showed that five residues (Glu26, His74, Arg77, Lys78, and Arg85) were essential for enzymatic activity in vivo. All these residues are located around the hole in the central region of the Syn-F4 structure, suggesting a putative active site with a catalytic dyad (Glu26-His74). The complete inactivity of purified proteins with mutations at the five residues supports the putative active site and reaction mechanism. Molecular dynamics and docking simulations of the ferric enterobactin siderophore binding to the Syn-F4 structure demonstrate the dynamic property of the putative active site. The structure and active site of Syn-F4 are completely different from native enterobactin esterase enzymes, thereby demonstrating that proteins designed de novo can provide life-sustaining catalytic activities using structures and mechanisms dramatically different from those that arose in nature.

A de novo protein catalyzes the synthesis of semiconductor quantum dots.

De novo proteins constructed from novel amino acid sequences are distinct from proteins that evolved in nature. Construct K (ConK) is a binary-patterned de novo designed protein that rescues Escherichia coli from otherwise toxic concentrations of copper. ConK was recently found to bind the cofactor PLP (pyridoxal phosphate, the active form of vitamin B6). Here, we show that ConK catalyzes the desulfurization of cysteine to H2S, which can be used to synthesize CdS nanocrystals in solution. The CdS nanocrystals are approximately 3 nm, as measured by transmission electron microscope, with optical properties similar to those seen in chemically synthesized quantum dots. The CdS nanocrystals synthesized using ConK have slower growth rates and a different growth mechanism than those synthesized using natural biomineralization pathways. The slower growth rate yields CdS nanocrystals with two desirable properties not observed during biomineralization using natural proteins. First, CdS nanocrystals are predominantly of the zinc blende crystal phase; this is in stark contrast to natural biomineralization routes that produce a mixture of zinc blende and wurtzite phase CdS. Second, in contrast to the growth and eventual precipitation observed in natural biomineralization systems, the CdS nanocrystals produced by ConK stabilize at a final size. Future optimization of CdS nanocrystal growth using ConK-or other de novo proteins-may help to overcome the limits on nanocrystal quality typically observed from natural biomineralization by enabling the synthesis of more stable, high-quality quantum dots at room temperature.

Engagement with Adolescent Substance Prevention Messages: Impact and Dissemination of REAL Media.

This article reviews the development and testing of a youth substance prevention program, REAL media. The contributions of this body of research include theoretical development, measurement, and dissemination of an efficacious independently evaluated program. Special attention is given to the impact of the program through collaborations with multiple community groups and multiple phases of development and testing.

Does Narrative Quality Matter During Implementation of a School-Based Prevention Intervention? A Test of Narrative Engagement Theory.

Narratives play a powerful role in sharing meaning and making sense of experiences. Specifically, health narratives convey storylines, characters, and messages about health-related behaviors and provide audiences with models for healthy behaviors, prompting audiences' health-related reflections and decision-making. Narrative engagement theory (NET) explains how personal narratives can be integrated into interventions to promote health. This study utilizes NET to test direct and indirect effects of teachers' narrative quality on adolescent outcomes during a school-based substance use prevention intervention that includes narrative pedagogy and an implementation strategy. Observational coding of teacher narratives in video-recorded lessons along with self-report student surveys (N = 1,683) were subjected to path analysis. Findings showed significant direct effects of narrative quality on student engagement, norms (i.e. personal, best-friend injunctive, and descriptive norms), and substance use behavior. The analysis also yielded support for indirect effects of narrative quality on adolescent substance use behavior via student engagement, personal norms, and descriptive norms. Findings highlight important issues related to teacher-student interaction during implementation and contributes implications for adolescent substance use prevention research.

Developing the Opioid Rapid Response System™ for Lay Citizen Response to the Opioid Overdose Crisis: a Randomized Controlled Trial.

Emergency responders face challenges in arriving timely to administer naloxone in opioid overdoses. Therefore, interest in having lay citizens administer naloxone nasal spray has emerged. These citizens, however, must be recruited and trained, and be in proximity to the overdose. This study aimed to develop the Opioid Rapid Response System (ORRS)tm to meet this need by developing a system to recruit and train citizen responders and evaluate outcomes in a randomized clinical trial. ORRS recruitment messages and training platform were developed iteratively and then outcomes for each were evaluated in a randomized, unblinded two-arm waitlist-controlled trial. ORRS was field tested in 5 Indiana counties, recruiting adult citizen responders (age 18 or older) who did not self-identity as a certified first responder. Participants were recruited using either personal or communal messages and then randomly assigned to online naloxone training and waitlisted-control conditions. Pre- and post-surveys were administered online to measure the exposure to recruitment messages and training effects on knowledge of opioid overdose, confidence responding, concerns about responding, and intent to respond. Of the 220 randomized participants (114 training, 106 waitlisted-control), 140 were analyzed (59 training, 81 waitlisted-control). Recruited participants more frequently identified with communal appeal than with the personal appeal (chi-square = 53.5; p < 0.0001). Between-group differences for intervention effects were significant for knowledge of overdose signs (Cohen's d = 1.17), knowledge of overdose management (d = 1.72), self-efficacy (d = 1.39), and concerns (d = 1.31), but not for intent (d = 0.17), which suffered from a ceiling effect. ORRS provides stronger support for efficacy than that reported for other training interventions and the digital modality eases rapid dissemination.Trial Registration: NCT04589676.

The Student Athlete Wellness Portal: Translating Student Athletes' Prescription Opioid Use Narratives into a Targeted Public Health Intervention.

Background and Objectives: The opioid epidemic has permeated all strata of society over the last two decades, especially within the adolescent student athletic environment, a group particularly at risk and presenting their own challenges for science and practice. This paper (a) describes the development of a web-based intervention called the Student Athlete Wellness Portal that models effective opioid misuse resistance strategies and (b) details the findings of a single-group design to test its effectiveness. Materials and Methods: Formative research included 35 student athletes residing in the United States, ages 14 to 21, who had been injured in their school-based sport. They participated in in-depth qualitative interviews to explore narratives relating to their injuries and pain management plans. Inductive analyses of interview transcripts revealed themes of the challenges of being a student athlete, manageable vs. unmanageable pain, and ways to stay healthy. These themes were translated into prevention messages for the portal, which was then tested in a single-group design. Results: Users of the portal (n = 102) showed significant decreases in their willingness to misuse opioids and increases in their perceptions of opioid risks. Conclusions: This manuscript illuminates the processes involved in translating basic research knowledge into intervention scripts and reveals the promising effects of a technology-based wellness portal.

The Role of Engagement in Effective, Digital Prevention Interventions: the Function of Engagement in the REAL Media Substance Use Prevention Curriculum.

Prevention curricula rely on audience engagement to effectively communicate their messages. However, to date, measurement of engagement has primarily focused on self-report that is often an indicator of liking or satisfaction. Emerging technologies for intervention delivery hold promise not only for additional engagement indicators but also for dissemination outside of traditional vehicles such as classroom delivery. The present study, grounded in the theory of active involvement (Greene 2013), explores the role of engagement (as measured by self-report, program analytics, and observation) with short-term substance use prevention outcomes such as self-efficacy to counter-argue and descriptive and injunctive norms. The study tracks 4-H youth (N = 310) engaged with a media literacy focused e-learning substance prevention curriculum, REAL media. Results indicate that self-reports of engagement predicted self-efficacy to counter-argue, but a program-analytic indicator of dosage predicted lower injunctive and descriptive norms, all at 3 months. The observational indicator was correlated with self-efficacy to counter-argue but not significant in the predictive models. The implications and directions for future research regarding how engagement is measured in prevention and included in studying program effects are discussed. Clinical trial: NCT03157700, May 2017.

A Completely De Novo ATPase from Combinatorial Protein Design.

Our understanding of biological chemistry is shaped by the observation that all life comes from other life-as Pasteur put it, omne vivum ex vivo. A key step in expanding our biochemical vocabulary is to recapitulate biogenic catalysis using non-natural sequences that did not arise from common ancestry. Here we describe an enzyme designed completely de novo that hydrolyzes ATP. This protein was designed to lack ß-sheet structure and is competitively inhibited by magnesium, two traits that are unlike natural ATPases.

A de novo enzyme catalyzes a life-sustaining reaction in Escherichia coli.

Producing novel enzymes that are catalytically active in vitro and biologically functional in vivo is a key goal of synthetic biology. Here we describe Syn-F4, the first de novo protein that meets both criteria. Purified Syn-F4 hydrolyzes the siderophore ferric enterobactin, and expression of Syn-F4 allows an inviable strain of Escherichia coli to grow in iron-limited medium. These findings demonstrate that entirely new sequences can provide life-sustaining enzymatic functions in living organisms.

Design of a Fe4 S4 cluster into the core of a de novo four-helix bundle.

We explore the capacity of the de novo protein, S824, to incorporate a multinuclear iron-sulfur cluster within the core of a single-chain four-helix bundle. This topology has a high intrinsic designability because sequences are constrained largely by the pattern of hydrophobic and hydrophilic amino acids, thereby allowing for the extensive substitution of individual side chains. Libraries of novel proteins based on these constraints have surprising functional potential and have been shown to complement the deletion of essential genes in E. coli. Our structure-based design of four first-shell cysteine ligands, one per helix, in S824 resulted in successful incorporation of a cubane Fe4 S4 cluster into the protein core. A number of challenges were encountered during the design and characterization process, including nonspecific metal-induced aggregation and the presence of competing metal-cluster stoichiometries. The introduction of buried iron-sulfur clusters into the helical bundle is an initial step toward converting libraries of designed structures into functional de novo proteins with catalytic or electron-transfer functionalities.

Exploring Indicators of Engagement in Online Learning as Applied to Adolescent Health Prevention: A Pilot Study of REAL media.

Engagement is central to the effectiveness of online health messages and the related educational programs that aim to deliver these messages to the intended audience (Li, Won, Yang et al. 2019: Lin, Hung, Kinshuk et al. 2019). Drawing from health communication and social learning theories, the Theory of Active Involvement (TAI) (Greene, 2013) posits that an online prevention program's impact depends on how engaged participants are. In practice, measuring engagement in this context has relied primarily on self-report measures (e.g., Hamutoglu, Gemikonakli, Duman et al. 2019). However, the emergence and growth of online learning platforms to deliver health-specific information offers other options for assessing engagement. This includes program analytics that capture interaction with content and facilitate examination of patterns via multiple indicators such as responses to interactive questions and time spent in the program (Herodotou, Rienties, Boroowa, et al. 2019; Li, Wong, Yang et al. 2019; van Leeuwen, 2019). However, little is known about the relationships between these different indicators of engagement as it applies to health curricula. This study uses self-report, observational, and program analytic data collected on a small (N = 38) sample using REAL media, an online substance use prevention program, to examine relationships among various indicators of engagement. Findings suggest a cluster of indicators across the three modalities that provide a useful way of measuring engagement. A cluster centered around complexity suggests a separate factor to be considered when designing engaging interventions.

A protein constructed de novo enables cell growth by altering gene regulation.

Recent advances in protein design rely on rational and computational approaches to create novel sequences that fold and function. In contrast, natural systems selected functional proteins without any design a priori. In an attempt to mimic nature, we used large libraries of novel sequences and selected for functional proteins that rescue Escherichia coli cells in which a conditionally essential gene has been deleted. In this way, the de novo protein SynSerB3 was selected as a rescuer of cells in which serB, which encodes phosphoserine phosphatase, an enzyme essential for serine biosynthesis, was deleted. However, SynSerB3 does not rescue the deleted activity by catalyzing hydrolysis of phosphoserine. Instead, SynSerB3 up-regulates hisB, a gene encoding histidinol phosphate phosphatase. This endogenous E. coli phosphatase has promiscuous activity that, when overexpressed, compensates for the deletion of phosphoserine phosphatase. Thus, the de novo protein SynSerB3 rescues the deletion of serB by altering the natural regulation of the His operon.

"Choose Today, Live Tomorrow": A Content Analysis of Anti-Substance Use Messages Produced by Adolescents.

Adolescent-produced anti-substance use messaging is an increasingly popular and effective prevention strategy. However, little is known about the content of these messages and the production elements adolescents use to bring that content to life. In this article, we present a content analysis of 95 anti-substance use messages developed by 4-H club members across nine U.S. states as part of their participation in the media literacy program REAL media. Posters and videos were content-analyzed for target substance, prevention goal, message form, message content, persuasion strategies, and production elements. Results of the content analysis revealed that combustible tobacco (smoking) was the most popular target substance in the sample among the choices of alcohol, marijuana, e-cigarettes, and chewing tobacco. More youth developed messages with the goal of preventing substance use, rather than stopping current use. Slogans were used in the majority of messages, and nearly all messages took an informational form, rather than narrative or statistical form. Persuasion strategies covered in the curriculum, including fun with the group, unexpected, style, and endorsement were scantily used. Finally, results showed that production value was high in this sample, reflected by the extensive use of color and variety of fonts and font sizes. Implications for future media literacy interventions and research are discussed.

Differential Effects of Parental "drug talk" Styles and Family Communication Environments on Adolescent Substance Use.

The current study examines the relationships among adolescent reports of parent-adolescent drug talk styles, family communication environments (e.g., expressiveness, structural traditionalism, and conflict avoidance), and adolescent substance use. ANCOVAs revealed that the 9th grade adolescents (N = 718) engaged in four styles of "drug talks" with parents (e.g., situated direct, ongoing direct, situated indirect, and ongoing indirect style) and these styles differed in their effect on adolescent substance use. Multiple regression analyses showed that expressiveness and structural traditionalism were negatively related to adolescent substance use, whereas conflict avoidance was positively associated with substance use. When controlling for family communication environments and gender, adolescents with an ongoing indirect style reported the lowest use of substance. The findings suggest implications and future directions for theory and practice.

Trends of Parent-Adolescent Drug Talk Styles in Early Adolescence.

The present study seeks to understand how parents as prevention agents approach substance use prevention messages during the period of early adolescence. Students (N = 410) in a drug prevention trial completed surveys from 7th to 9th grade. Using longitudinal data, a series of latent transition analyses was conducted to identify major trends of parent-adolescent drug talk styles (i.e., never talked, situated direct, ongoing direct, situated indirect, and ongoing indirect) in control and treatment conditions. Findings demonstrate a developmental trend in drug talk styles toward a situated style of talk as youth transitioned from 7th grade to 9th grade. Findings also show that even though the drug prevention trial did not specifically target parental communication, parents in the treatment condition provide more ongoing substance use prevention messages to their adolescent children than do parents in the control condition. The present study discusses relevant developmental issues, potential intervention effects, and future research directions for communication research in substance use prevention.

Artificial Gene Amplification in Escherichia coli Reveals Numerous Determinants for Resistance to Metal Toxicity.

When organisms are subjected to environmental challenges, including growth inhibitors and toxins, evolution often selects for the duplication of endogenous genes, whose overexpression can provide a selective advantage. Such events occur both in natural environments and in clinical settings. Microbial cells-with their large populations and short generation times-frequently evolve resistance to a range of antimicrobials. While microbial resistance to antibiotic drugs is well documented, less attention has been given to the genetic elements responsible for resistance to metal toxicity. To assess which overexpressed genes can endow gram-negative bacteria with resistance to metal toxicity, we transformed a collection of plasmids overexpressing all E. coli open reading frames (ORFs) into naive cells, and selected for survival in toxic concentrations of six transition metals: Cd, Co, Cu, Ni, Ag, Zn. These selections identified 48 hits. In each of these hits, the overexpression of an endogenous E. coli gene provided a selective advantage in the presence of at least one of the toxic metals. Surprisingly, the majority of these cases (28/48) were not previously known to function in metal resistance or homeostasis. These findings highlight the diverse mechanisms that biological systems can deploy to adapt to environments containing toxic concentrations of metals.

Entertainment-Education Videos as a Persuasive Tool in the Substance Use Prevention Intervention "keepin' it REAL".

Based on social cognitive theory and narrative engagement theory, the current study examined hypothesized indirect effects of engagement with keepin' it REAL (kiR) curriculum entertainment-education (E-E) videos on youth alcohol use via youth drug offer refusal efficacy. Students in 7th grade (N = 1,464) at 25 public schools in two Midwestern states were randomly assigned to one of the two versions of the kiR curriculum, the kiR urban version and the kiR rural version. Each version had their own set of five culturally-grounded E-E videos depicting communicative skills to refuse drug offers. Differential effects for engagement components were expected depending on the degree of cultural matching. Pre/post surveys were administered at the beginning and the end of 7th grade. Structural equation modeling analysis resulted in partial support for the research hypotheses. Rural youth receiving the urban curriculum who reported higher interest in the E-E videos were more likely to report having higher refusal efficacy, and in turn, less likely to use alcohol. Rural youth receiving the rural curriculum who identified with the E-E video main characters were more likely to report having higher refusal efficacy, and in turn, less likely to use alcohol. Implications for E-E health promotion are discussed.

Parental Messages about Substance Use in Early Adolescence: Extending a Model of Drug-Talk Styles.

This study extends a typology of parent-offspring drug talk styles to early adolescents and investigates associations with adolescent substance use. Data come from a self-report survey associated with a school-based, 7th grade drug prevention curriculum. Mixed methods were used to collect data across four measurement occasions spanning 30 months. Findings highlight the frequencies of various drug-talk styles over time (i.e., situated direct, ongoing direct, situated indirect, ongoing indirect, never talked), messages adolescents hear from parents, and comparisons of alcohol, cigarette, and marijuana use by drug-talk style. This study advances an understanding of parent-adolescent communication about substances and holds practical implications for drug prevention efforts.

Designed Cultural Adaptation and Delivery Quality in Rural Substance Use Prevention: an Effectiveness Trial for the Keepin' it REAL Curriculum.

This study examined how cultural adaptation and delivery quality of the school-based intervention keepin' it REAL (kiR) influenced adolescent substance use. The goal of the study was to compare the effectiveness of the multi-cultural, urban (non-adapted) kiR intervention, a re-grounded (adapted) rural version of the kiR intervention and control condition in a new, rural setting. A total of 39 middle schools in rural communities of two states in the USA were randomly assigned to one of three conditions (i.e., control, non-adapted urban kiR, and adapted rural kiR). Data included adolescent self-reported lifetime substance use and observers' ratings of delivery quality from video recordings of lessons. Ratings of delivery quality were used to create four comparison groups (i.e., low/high delivery quality in non-adapted/urban kiR condition and low/high quality in adapted/rural kiR condition). Controlling for substance use in the 7th grade, findings compared 9th graders' (N = 2781) lifetime alcohol, cigarette, marijuana, and chewing tobacco use. Mixed model analyses revealed that rural youth receiving the culturally adapted/rural curriculum reported significantly less cigarette use than rural youth in the control condition regardless of delivery quality. In the non-adapted/urban condition, youth receiving high delivery quality delivery reported less marijuana use than those receiving low delivery quality condition. However, substance use outcomes of youth receiving high and low delivery quality in the non-adapted intervention did not differ significantly from those the control group. Findings support the effectiveness of the culturally adapted/rural keepin' it REAL curriculum for rural youth.