"I Get the Flavors and It Makes Me Love Vaping More": How and Why Youth Users Modify Electronic Nicotine Delivery Systems.

INTRODUCTION: Youth in the United States are using electronic nicotine delivery systems (ENDS) at a high rate. Modifications to ENDS by youth can introduce additional health hazards which have not been previously considered. To better understand these risks, we need more information on what these modifications are, the motivations behind them, and the sources of information on modifications. AIMS AND METHODS: Utilizing a trained moderator, in 2020-2021, we conducted one-on-one interviews with 19 youth ENDS users aged 16-17 living in the United States and analyzed their responses using a qualitative description approach. RESULTS: The most prominent modification was to the e-liquid; youth indicated they mixed e-juices to create new flavors and added substances not intended for vaping, including illicit drugs such as cannabis and cocaine. Few youths from our sample were interested in achieving a specific nicotine level to vape, and modifications to the battery, coil and wick were less frequently mentioned. Some of these modifications were motivated by a desire to achieve specific experiences with their device. At other times, modifications were made due to necessity because of limited access to ENDS devices and supplies. YouTube and peers were the main sources of information about modifying. CONCLUSIONS: Youth are making modifications that are both intended and unintended by the manufacturer. Adding illicit drugs and other substances not made for vaping is of particular concern. Understanding how youth modify ENDS and how that changes their use is important to guide regulatory policy intended to reduce harm to youth from ENDS use. IMPLICATIONS: Youth from our study indicated that they make modifications to the ENDS devices, specifically to the e-liquid. These modifications are both intended by the manufacturer, such as changing the e-liquid and replacing coils, and unintended, such as adding substances not meant for vaping. Future policies aimed at reducing youth ENDS use should consider mandating better safeguards against modifications that appeal to youth.

Support for nicotine reduction in cigarettes: findings from the 2016 and 2020 ITC Four Country Smoking and Vaping Surveys.

INTRODUCTION: The USA and New Zealand have sought to establish a product standard to set a maximum nicotine level for cigarettes to reduce their addictiveness. This study examined support for very low nicotine cigarettes (VLNCs) in Australia, Canada, England and the USA between 2016 and 2020. METHODS: Repeated cross-sectional data were analysed from participants who currently smoke, formerly smoked or vaped and/or currently vape in the 2016 (n=11 150) and/or 2020 (n=5432) International Tobacco Control (ITC) Four Country Smoking and Vaping Survey. Respondents were asked if they would support a law that reduces the amount of nicotine in cigarettes to make them less addictive. Adjusted and weighted logistic regression analyses estimated the prevalence and predictors of support, such as country, age, sex, education, income, race and smoking/vaping status for VLNCs (support vs oppose/do not know). RESULTS: A majority of respondents supported a VLNC law, with support highest in Canada (69%; 2016 and 2020 combined), followed by England (61%), Australia (60%) and the USA (58%). Overall, support decreased from 62% in 2016 to 59% in 2020 (p=0.004), which did not differ by country. Levels of support differed by smoking/vaping status, where those who exclusively smoked daily showed the lowest level of support (59%) and those who exclusively vaped non-daily had the highest level of support (72%). CONCLUSION: More than half of respondents in all four countries-including those who smoked daily-supported a hypothetical VLNC standard to render cigarettes less addictive. It is important to examine if support is sustained after policies are implemented.

Roles of singleton tryptophan motifs in COPI coat stability and vesicle tethering.

Coat protein I (COPI)-coated vesicles mediate retrograde transport from the Golgi to the endoplasmic reticulum (ER), as well as transport within the Golgi. Major progress has been made in defining the structure of COPI coats, in vitro and in vivo, at resolutions as high as 9 Å. Nevertheless, important questions remain unanswered, including what specific interactions stabilize COPI coats, how COPI vesicles recognize their target membranes, and how coat disassembly is coordinated with vesicle fusion and cargo delivery. Here, we use X-ray crystallography to identify a conserved site on the COPI subunit α-COP that binds to flexible, acidic sequences containing a single tryptophan residue. One such sequence, found within α-COP itself, mediates α-COP homo-oligomerization. Another such sequence is contained within the lasso of the ER-resident Dsl1 complex, where it helps mediate the tethering of Golgi-derived COPI vesicles at the ER membrane. Together, our findings suggest that α-COP homo-oligomerization plays a key role in COPI coat stability, with potential implications for the coordination of vesicle tethering, uncoating, and fusion.

Which tobacco control policies do smokers support? Findings from the International Tobacco Control Four Country Smoking and Vaping Survey.

As governments consider policy action to reduce smoking, a key factor in creating political will is the level of public support, particularly among smokers who are most affected by the policies. The goal of this paper is to assess and compare the level of support in Canada, the United States, England, and Australia for five smoking control policies: 1) banning menthol in cigarettes, 2) banning cigarette additives, 3) reducing nicotine in cigarettes to make them less addictive, 4) raising the minimum age to purchase cigarettes to 21 years and older, and 5) requiring pictorial warning labels on cigarette packs (examined in the US only). Data for these analyses come from 8165 daily cigarette smokers who responded to the 2016 International Tobacco Control Four Country Smoking and Vaping Survey. In all countries, the highest level of support was for raising the legal age for purchase to 21 years and older (62-70%) and reducing the nicotine content of cigarettes to make them less addictive (57-70%). Smokers who were less dependent on cigarettes and those expressing interest in quitting were more likely to support all policies. When asked how they would respond to a nicotine reduction policy, the most common response given was to try the non-nicotine cigarettes to see how they liked them (42-48%), with the next most common response being to quit smoking entirely (16-24%). The high level of support for these proposed policies among daily smokers provides important evidence for policymakers to counteract claims that such policies would be unpopular.

Exploring the Point-of-Sale Among Vape Shops Across the United States: Audits Integrating a Mystery Shopper Approach.

INTRODUCTION: Vape shops represent prominent, unique retailers, subject to Food and Drug Administration (FDA) regulation in the United States. AIMS AND METHODS: This study assessed compliance of US vape shop retail marketing strategies with new regulations (eg, required age verification, prohibited free samples) and pre-implementation conditions for other regulations (eg, health warning labels on all nicotine products, required disclosures of e-liquid contents). RESULTS: 95.0% of shops displayed minimum-age signage; however, mystery shoppers were asked for age verification at 35.6% upon entry and at 23.4% upon purchase. Although 85.5% of shops had some evidence of implementing FDA health warnings, 29.1% had signage indicating prohibited health claims, 16.3% offered free e-liquid samples, 27.4% had signage with cartoon imagery, and 33.3% were within two blocks of schools. All shops sold open-system devices, 64.8% sold closed-system devices, 68.2% sold their own brand of e-liquids, 42.5% sold e-liquids containing cannabidiol, 83.2% offered price promotions of some kind, and 89.9% had signage for product and price promotions. CONCLUSIONS: Results indicated that most shops complied with some implementation of FDA health warnings and with free sampling bans and minimum-age signage. Other findings indicated concerns related to underage access, health claims, promotional strategies, and cannabidiol product offerings, which call for further FDA and state regulatory/enforcement efforts.

Uncovering protein-protein interactions through a team-based undergraduate biochemistry course.

How can we provide fertile ground for students to simultaneously explore a breadth of foundational knowledge, develop cross-disciplinary problem-solving skills, gain resiliency, and learn to work as a member of a team? One way is to integrate original research in the context of an undergraduate biochemistry course. In this Community Page, we discuss the development and execution of an interdisciplinary and cross-departmental undergraduate biochemistry laboratory course. We present a template for how a similar course can be replicated at other institutions and provide pedagogical and research results from a sample module in which we challenged our students to study the binding interface between 2 important biosynthetic proteins. Finally, we address the community and invite others to join us in making a larger impact on undergraduate education and the field of biochemistry by coordinating efforts to integrate research and teaching across campuses.

Structure, Regulation, and Inhibition of the Quorum-Sensing Signal Integrator LuxO.

In a process called quorum sensing, bacteria communicate with chemical signal molecules called autoinducers to control collective behaviors. In pathogenic vibrios, including Vibrio cholerae, the accumulation of autoinducers triggers repression of genes responsible for virulence factor production and biofilm formation. The vibrio autoinducer molecules bind to transmembrane receptors of the two-component histidine sensor kinase family. Autoinducer binding inactivates the receptors' kinase activities, leading to dephosphorylation and inhibition of the downstream response regulator LuxO. Here, we report the X-ray structure of LuxO in its unphosphorylated, autoinhibited state. Our structure reveals that LuxO, a bacterial enhancer-binding protein of the AAA+ ATPase superfamily, is inhibited by an unprecedented mechanism in which a linker that connects the catalytic and regulatory receiver domains occupies the ATPase active site. The conformational change that accompanies receiver domain phosphorylation likely disrupts this interaction, providing a mechanistic rationale for LuxO activation. We also determined the crystal structure of the LuxO catalytic domain bound to a broad-spectrum inhibitor. The inhibitor binds in the ATPase active site and recapitulates elements of the natural regulatory mechanism. Remarkably, a single inhibitor molecule may be capable of inhibiting an entire LuxO oligomer.

Sedimentation Velocity Analysis with Fluorescence Detection of Mutant Huntingtin Exon 1 Aggregation in Drosophila melanogaster and Caenorhabditis elegans.

At least nine neurodegenerative diseases that are caused by the aggregation induced by long tracts of glutamine sequences have been identified. One such polyglutamine-containing protein is huntingtin, which is the primary factor responsible for Huntington's disease. Sedimentation velocity with fluorescence detection is applied to perform a comparative study of the aggregation of the huntingtin exon 1 protein fragment upon transgenic expression in Drosophila melanogaster and Caenorhabditis elegans. This approach allows the detection of aggregation in complex mixtures under physiologically relevant conditions. Complementary methods used to support this biophysical approach included fluorescence microscopy and semidenaturing detergent agarose gel electrophoresis, as a point of comparison with earlier studies. New analysis tools developed for the analytical ultracentrifuge have made it possible to readily identify a wide range of aggregating species, including the monomer, a set of intermediate aggregates, and insoluble inclusion bodies. Differences in aggregation in the two animal model systems are noted, possibly because of differences in levels of expression of glutamine-rich sequences. An increased level of aggregation is shown to correlate with increased toxicity for both animal models. Co-expression of the human Hsp70 in D. melanogaster showed some mitigation of aggregation and toxicity, correlating best with inclusion body formation. The comparative study emphasizes the value of the analytical ultracentrifuge equipped with fluorescence detection as a useful and rigorous tool for in situ aggregation analysis to assess commonalities in aggregation across animal model systems.

Self-association motifs in the enteroaggregative Escherichia coli heat-resistant agglutinin 1.

The heat-resistant agglutinin 1 (Hra1) is an integral outer membrane protein found in strains of Escherichia coli that are exceptional colonizers. Hra1 from enteroaggregative E. coli strain 042 is sufficient to confer adherence to human epithelial cells and to cause bacterial autoaggregation. Hra1 is closely related to the Tia invasin, which also confers adherence, but not autoaggregation. Here, we have demonstrated that Hra1 mediates autoaggregation by self-association and we hypothesize that at least some surface-exposed amino acid sequences that are present in Hra1, but absent in Tia, represent autoaggregation motifs. We inserted FLAG tags along the length of Hra1 and used immune-dot blots to verify that four in silico-predicted outer loops were indeed surface exposed. In Hra1 we swapped nine candidate motifs in three of these loops, ranging from one to ten amino acids in length, to the corresponding sequences in Tia. Three of the motifs were required for Hra1-mediated autoaggregation. The database was searched for other surface proteins containing these motifs; the GGXWRDDXK motif was also present in a surface-exposed region of Rck, a Salmonella enterica serotype Typhimurium complement resistance protein. Cloning and site-specific mutagenesis demonstrated that Rck can confer weak, GGXWRDDXK-dependent autoaggregation by self-association. Hra1 and Rck appear to form heterologous associations and GGXWRDDXK is required on both molecules for Hra1-Rck association. However, a GGYWRDDLKE peptide was not sufficient to interfere with Hra1-mediated autoaggregation. In the present study, three autoaggregation motifs in an integral outer membrane protein have been identified and it was demonstrated that at least one of them works in the context of a different cell surface.

Probing the selectivity of ß-hydroxylation reactions in non-ribosomal peptide synthesis using analytical ultracentrifugation.

Bacteria and fungi use non-ribosomal peptide synthetases (NRPSs) to produce peptides of broad structural diversity and biological activity, many of which have proven to be of great importance for human health. The impressive diversity of non-ribosomal peptides originates in part from the action of tailoring enzymes that modify the structures of single amino acids and/or the mature peptide. Studying the interplay between tailoring enzymes and the peptidyl carrier proteins (PCPs) that anchor the substrates is challenging owing to the transient and complex nature of the protein-protein interactions. Using sedimentation velocity (SV) methods, we studied the collaboration between the PCPs and cytochrome P450 enzyme that results in the installation of ß-hydroxylated amino acid precursors in the biosynthesis of the depsipeptide skyllamycin. We show that SV methods developed for the analytical ultracentrifuge are ideally suited for a quantitative exploration of PCP-enzyme equilibrium interactions. Our results suggest that the PCP itself and the presence of substrate covalently tethered to the PCP together facilitate productive PCP-P450 interactions, thereby revealing one of nature's intricate strategies for installing interesting functionalities using natural product synthetases.

Effect of helix length on the stability of the Lac repressor antiparallel coiled coil.

The helix length dependence of the stability of antiparallel four-chain coiled coils is investigated using eight synthetic peptides (Lac21-Lac28) whose sequences are derived from the tetramerization domain of the Lac repressor protein. Previous studies using analytical ultracentrifugation sedimentation equilibrium experiments to characterize Lac21 and Lac28 justifies the use of a two state model to describe the unfolding behavior of these two peptides. Using circular dichroism spectropolarimetry as a measure of tetramer assembly, both chemical and thermal denaturation experiments were carried out to determine thermodynamic parameters. We found that the hydrophobic core residues provide the greatest impact on stability and, as a consequence, must reorganize the register of the antiparallel helices to accommodate the burial of the nonpolar amino acids. Addition of noncore residues appears to have only a minor effect on stability, and in some cases, show a slight destabilization.

Role of the coiled-coil structural motif in polyglutamine aggregation.

Polyglutamine repeat motifs are known to induce protein aggregation in various neurodegenerative diseases, and flanking sequences can modulate this behavior. It has been proposed that the 17 N-terminal residues (Htt(NT)) of the polyglutamine-containing huntingtin protein accelerate the kinetics of aggregation due to the formation of helix-rich oligomers through helix-pairing interactions. Several hypotheses that explain the role of helical interactions in modulating aggregation have been proposed. These include (1) an increase in the effective concentration of polyglutamine chains (proximity model), (2) the induction of helical structure within the polyglutamine domain itself (transformation model), and/or (3) interdomain interactions between the flanking sequence and the polyglutamine domain (domain cross-talk model). These hypotheses are tested by studying the kinetics of polyglutamine aggregation using a Q25 sequence fused to a well-defined heterotetrameric coiled-coil model system. Using a combined spectroscopic and dye binding approach, it is shown that stable coiled-coil formation strongly inhibits polyglutamine aggregation, suggesting that the proximity and transformation models are insufficient to explain the enhanced aggregation seen in Htt(NT)-polyglutamine constructs. Consistent with other published work, our data support a model in which domain cross-talk prevents formation of a nonspecific aggregated collapsed polyglutamine state, which can act to inhibit conversion to a fibrillar state. Because our model system has a charged to nonpolar residue ratio much higher than that of the Htt(NT) sequence, domain cross-talk is severely weakened, thus favoring the nonspecific aggregation pathway and significantly inhibiting aggregation through a fibrillar pathway.

Effect of helical flanking sequences on the morphology of polyglutamine-containing fibrils.

A peptide model system has been developed to study the effects of helical flanking sequences on polyglutamine aggregation. In a companion manuscript, the kinetics of aggregation are described, comparing the influence of a well-defined heterotetrameric coiled coil to that of the helix-rich structure found in Htt(NT), a 17-residue flanking sequence found in the huntingtin protein, on polyglutamine aggregation. Here, the morphological characterization of the resultant fibrils that form for a set of peptides is reported, only one of which, KKQ25KK, has been previously studied. A careful analysis of TEM and AFM images of KKQ25KK confirms that it forms bundled fibrils of varying length and reveals, unexpectedly, that they are composed of fully extended cross-ß-strands. Second, it is shown that helical flanking sequences do not disrupt the assembly of a core cross-ß-sheet structure, but such flanking sequences can influence higher order processes, such as inhibiting the bundling of the fibrils.

Testing the role of charge and structure on the stability of peptide-porphyrin complexes.

This study aims to extend a structural and biophysical understanding of a coiled-coil based peptide model system that serves as a scaffold for the anionic porphyrin, TPPS4. This is part of an ongoing biomaterials effort to create photoelectronically active mesoscale fibrils for surface deposition and characterization of conductivity properties. The goals are two-fold: (1) to explore optimal basic side-chain moieties for tight binding to TPPS4 and (2) to test the binding of various metalated TPPS4 derivatives to our peptide model system. The latter goal is to control the electronic and redox properties of the fibrillar biomaterials. A soluble version of the peptide biomaterial was used in order to probe binding and to extract thermodynamically rigorous equilibrium binding constants. UV-visible spectroscopy and circular dichroism spectropolarimtery are used to measure the effects of binding on the Soret band of the porphyrin and the helical signal of the peptide, respectively. For the first study, it was found that lysine, ornithine, and arginine are equally robust at engaging TPPS4 with low micromolar binding affinity. In the case of the metalated porphyrins, submicromolar binding affinity was observed for Cu(II), Ni(II), and Pd(II). The ability of these metalated porphyrins to bind with high affinity is dependent largely on structural perturbations of the porphyrin molecule, rather than on induced electronic effects.

The Effects of poly-GA and poly-PR C9orf72 Dipeptide Repeats on Sleep Patterns in Drosophila melanogaster.

C9orf72 is the most common familial gene associated with amyotrophic lateral sclerosis (ALS). Dipeptide repeats (DPRs) encoded by an expanded nucleotide repeat sequence in the C9orf72 gene were found in the sleep-related neurons of patients, indicating a role of DPRs in ALS-associated sleep disruptions. Poly-GA or poly-PR DPRs were expressed in male Drosophila melanogaster to study their effect on sleep . Poly-PR expression caused sleep disruptions while poly-GA expression did not. This study validates the use of Drosophila as an in vivo model system for exploring the roles of DPRs in perturbing the underlying molecular mechanisms in sleep regulation.

Vaccination in Kidney Transplant Candidates.

Background: Kidney transplant (KT) candidates have historically low immunization rates against recommended vaccines. A retrospective single-center study of contemporary KT candidates was conducted to assess vaccination rates and vaccine uptake. Methods: All KT candidates ≥18 y evaluated between January 1, 2020, and December 31, 2020, were retrospectively reviewed for history of prior vaccination against tetanus, diphtheria, and pertussis; 13-valent pneumococcal conjugate vaccine; 23-valent pneumococcal polysaccharide vaccine; and recombinant zoster vaccine. Positive hepatitis A IgG total, hepatitis B surface antibody, measles, mumps, rubella, and varicella IgG were assessed as surrogate markers of immunity. Vaccine uptake among vaccine-eligible candidates was also assessed. Results: Among 150 KT candidates, the rate of prior vaccination against tetanus, diphtheria, and pertussis; 13-valent pneumococcal conjugate vaccine; 23-valent pneumococcal polysaccharide vaccine; and recombinant zoster vaccine (latter among patients ≥50 y) was found to be as low as 11%. Hepatitis A IgG total, hepatitis B surface antibody, measles, mumps, rubella, and varicella IgG seropositivity rates were 30%, 66%, 88%, 78%, 90%, and 96%, respectively. Only 7 (5%) of 150 patients had complete immunization or seropositivity. Five (3%) of 143 vaccine-eligible patients declined vaccination. Hepatitis A vaccine declination was relatively common with 15 (16%) of 94 vaccine-eligible patients declining it. Conclusions: KT candidates have low baseline rates of prior immunization/seropositivity against most recommended vaccines. Overall vaccine uptake among eligible candidates was high.

C9orf72 proline-arginine dipeptide repeats disrupt the proteasome and perturb proteolytic activities.

The hexanucleotide G4C2 repeat expansion in C9orf72 is the most frequent genetic cause of familial amyotrophic lateral sclerosis (ALS). Aberrant translation of this hexanucleotide sequence leads to production of 5 dipeptide repeats (DPRs). One of these DPRs is proline-arginine (polyPR), which is found in C9orf72-expanded ALS (C9ALS) patient brain tissue and is neurotoxic across multiple model systems. PolyPR was previously reported to bind and impair proteasomes in vitro. Nevertheless, the clinical relevance of the polyPR-proteasome interaction and its functional consequences in vivo are yet to be established. Here, we aim to confirm and functionally characterize polyPR-induced impairment of proteolysis in C9ALS patient tissue and an in vivo model system. Confocal microscopy and immunofluorescence studies on both human and Drosophila melanogaster brain tissues revealed sequestration of proteasomes by polyPR into inclusion-like bodies. Co-immunoprecipitation in D. melanogaster showed that polyPR strongly binds to the proteasome. In vivo, functional evidence for proteasome impairment is further shown by the accumulation of ubiquitinated proteins along with lysosomal accumulation and hyper-acidification, which can be rescued by a small-molecule proteasomal enhancer. Together, we provide the first clinical report of polyPR-proteasome interactions and offer in vivo evidence proposing polyPR-induced proteolytic dysfunction as a pathogenic mechanism in C9ALS.

It's addiction at this Point": A qualitative examination of youth E-cigarette use trajectory in the United States.

E-cigarettes (electronic cigarettes) have been the most used tobacco product among US youth since 2014, reaching a plateau during the COVID-19 pandemic. Youth e-cigarette use is associated with negative health consequences such as impaired cognitive functioning. For many, the COVID-19 pandemic altered social interactions, harm perceptions, and product availability. This changed the frequency and locations in which youth use e-cigarettes. To better understand youth e-cigarette use, we need more information on factors that can alter e-cigarette use, specifically, how the pandemic changed e-cigarette use among youth. In 2020-2021, we conducted online, individual interviews with 19 youth (aged 13-17) e-cigarette users living in the US to explore how COVID-19 impacted their e-cigarette use. Youth described a progression of e-cigarette use from initial experimentation, regular social use, and ultimately to nicotine addiction demonstrated by individual use in isolation. Many youth initiated e-cigarette use due to influences by friends or family members. Youth discussed progression to social use, with social interactions as an important reason for use and an avenue for expanding one's knowledge of e-cigarettes. After a period of time, youth began to recognize that the social interactions mattered less, suggesting to them that they had become addicted. This realization became more apparent during COVID-19, which changed how youth used e-cigarettes, especially around where use was occurring, health concerns, and use behavior and frequency. In our interviews, youth trajectory began with an initiation with family and friends, progressed to social use, and eventually developed to addiction, at which point social use was no longer the primary motivation for e-cigarette use. Understanding the trajectory of e-cigarette use will allow for effective interventions that reduce harm to youth from e-cigarette use.

Evaluation of a health system's implementation of a monkeypox care model under the RE-AIM framework.

Objective: Emerging infectious diseases challenge healthcare systems to implement new models of care. We aim to evaluate the rapid implementation of a new care model for monkeypox in our health system. Design: This is a retrospective case series evaluation under the Reach, Effectiveness, Adoption, Implementation, and Maintenance (RE-AIM) framework of implementation of a testing and care model for monkeypox in a large, integrated health system. Methods: Atrium Health implemented education of providers, testing protocols, and management of potential monkeypox cases using electronic health record (EHR) data capabilities, telehealth, and collaboration between multiple disciplines. The first 4 weeks of care model implementation were evaluated under the RE-AIM framework. Results: One hundred fifty-three patients were tested for monkeypox by 117 unique providers at urgent care, emergency departments, and infectious disease clinics in our healthcare system between 18 July 2022 and 14 August 2022. Fifty-eight monkeypox cases were identified, compared with 198 cases in the state during the time period, a disproportionate number compared with the health system service area, and 52 patients were assessed for need for tecovirimat treatment. The number of tests performed and providers sending tests increased during the study period. Conclusion: Implementation of a dedicated care model leveraging EHR data support, telehealth, and cross-disciplinary collaboration led to more effective identification and management of emerging infectious diseases and is important for public health. Plain Language Summary: Impact of care model implementation on monkeypox New infectious diseases challenge health systems to implement new care practices. Our health system responded to this challenge by implementing a care model for education, testing, and clinical care of monkeypox patients. We analyzed results from implementing the model. We were able to identify a disproportionate number of monkeypox cases compared with the rest of our state by using our model to educate medical providers, encourage testing, and ensure patients had access to best disease care. Implementation of care models for testing and management of new diseases will improve patient care and public health.