Organometallic Pillarplexes That Bind DNA 4-Way Holliday Junctions and Forks.

Holliday 4-way junctions are key to important biological DNA processes (insertion, recombination, and repair) and are dynamic structures that adopt either open or closed conformations, the open conformation being the biologically active form. Tetracationic metallo-supramolecular pillarplexes display aryl faces about a cylindrical core, an ideal structure to interact with open DNA junction cavities. Combining experimental studies and MD simulations, we show that an Au pillarplex can bind DNA 4-way (Holliday) junctions in their open form, a binding mode not accessed by synthetic agents before. Pillarplexes can bind 3-way junctions too, but their large size leads them to open up and expand that junction, disrupting the base pairing, which manifests in an increased hydrodynamic size and lower junction thermal stability. At high loading, they rearrange both 4-way and 3-way junctions into Y-shaped forks to increase the available junction-like binding sites. Isostructural Ag pillarplexes show similar DNA junction binding behavior but lower solution stability. This pillarplex binding contrasts with (but complements) that of metallo-supramolecular cylinders, which prefer 3-way junctions and can rearrange 4-way junctions into 3-way junction structures. The pillarplexes' ability to bind open 4-way junctions creates exciting possibilities to modulate and switch such structures in biology, as well as in synthetic nucleic acid nanostructures. In human cells, the pillarplexes do reach the nucleus, with antiproliferative activity at levels similar to those of cisplatin. The findings provide a new roadmap for targeting higher-order junction structures using a metallo-supramolecular approach, as well as expanding the toolbox available to design bioactive junction binders into organometallic chemistry.

Supramolecular Cylinders Target Bulge Structures in the 5' UTR of the RNA Genome of SARS-CoV-2 and Inhibit Viral Replication*.

The untranslated regions (UTRs) of viral genomes contain a variety of conserved yet dynamic structures crucial for viral replication, providing drug targets for the development of broad spectrum anti-virals. We combine in vitro RNA analysis with molecular dynamics simulations to build the first 3D models of the structure and dynamics of key regions of the 5' UTR of the SARS-CoV-2 genome. Furthermore, we determine the binding of metallo-supramolecular helicates (cylinders) to this RNA structure. These nano-size agents are uniquely able to thread through RNA junctions and we identify their binding to a 3-base bulge and the central cross 4-way junction located in stem loop 5. Finally, we show these RNA-binding cylinders suppress SARS-CoV-2 replication, highlighting their potential as novel anti-viral agents.

Rotaxanating Metallo-supramolecular Nano-cylinder Helicates to Switch DNA Junction Binding.

A class of rotaxane is created, not by encapsulating a conventional linear thread, but rather by wrapping a large cucurbit[10]uril macrocycle about a three-dimensional, cylindrical, nanosized, self-assembled supramolecular helicate as the axle. The resulting pseudo-rotaxane is readily converted into a proper interlocked rotaxane by adding branch points to the helicate strands that form the surface of the cylinder (like branches and roots on a tree trunk). The supramolecular cylinder that forms the axle is itself a member of a unique and remarkable class of helicate metallo-drugs that bind Y-shaped DNA junction structures and induce cell death. While pseudo-rotaxanation does not modify the DNA-binding properties, proper, mechanically-interlocked rotaxanation transforms the DNA-binding and biological activity of the cylinder. The ability of the cylinder to de-thread from the rotaxane (and thus to bind DNA junction structures) is controlled by the extent of branching: fully-branched cylinders are locked inside the cucurbit[10]uril macrocycle, while cylinders with incomplete branch points can de-thread from the rotaxane in response to competitor guests. The number of branch points can thus afford kinetic control over the drug de-threading and release.

Iridium Nanoparticles for Multichannel Luminescence Lifetime Imaging, Mapping Localization in Live Cancer Cells.

The development of long-lived luminescent nanoparticles for lifetime imaging is of wide interest as luminescence lifetime is environmentally sensitive detection independent of probe concentration. We report novel iridium-coated gold nanoparticles as probes for multiphoton lifetime imaging with characteristic long luminescent lifetimes based on iridium luminescence in the range of hundreds of nanoseconds and a short signal on the scale of picoseconds based on gold allowing multichannel detection. The tailor-made IrC6 complex forms stable, water-soluble gold nanoparticles (AuNPs) of 13, 25, and 100 nm, bearing 1400, 3200, and 22 000 IrC6 complexes per AuNP, respectively. The sensitivity of the iridium signal on the environment of the cell is evidenced with an observed variation of lifetimes. Clusters of iridium nanoparticles show lifetimes from 450 to 590 ns while lifetimes of 660 and 740 ns are an average of different points in the cytoplasm and nucleus. Independent luminescence lifetime studies of the nanoparticles in different media and under aggregation conditions postulate that the unusual long lifetimes observed can be attributed to interaction with proteins rather than nanoparticle aggregation. Total internal reflection fluorescence microscopy (TIRF), confocal microscopy studies and 3D luminescence lifetime stacks confirm the presence of bright, nonaggregated nanoparticles inside the cell. Inductively coupled plasma mass spectrometry (ICPMS) analysis further supports the presence of the nanoparticles in cells. The iridium-coated nanoparticles provide new nanoprobes for lifetime detection with dual channel monitoring. The combination of the sensitivity of the iridium signal to the cell environment together with the nanoscaffold to guide delivery offer opportunities for iridium nanoparticles for targeting and tracking in in vivo models.

Partial meniscectomy is associated with increased risk of incident radiographic osteoarthritis and worsening cartilage damage in the following year.

OBJECTIVES: To assess whether partial meniscectomy is associated with increased risk of radiographic osteoarthritis (ROA) and worsening cartilage damage in the following year. METHODS: We studied 355 knees from the Osteoarthritis Initiative that developed ROA (Kellgren-Lawrence grade ≥ 2), which were matched with control knees. The MR images were assessed using the semi-quantitative MOAKS system. Conditional logistic regression was applied to estimate risk of incident ROA. Logistic regression was used to assess the risk of worsening cartilage damage in knees with partial meniscectomy that developed ROA. RESULTS: In the group with incident ROA, 4.4 % underwent partial meniscectomy during the year prior to the case-defining visit, compared with none of the knees that did not develop ROA. All (n = 31) knees that had partial meniscectomy and 58.9 % (n = 165) of the knees with prevalent meniscal damage developed ROA (OR = 2.51, 95 % CI [1.73, 3.64]). In knees that developed ROA, partial meniscectomy was associated with an increased risk of worsening cartilage damage (OR = 4.51, 95 % CI [1.53, 13.33]). CONCLUSIONS: The probability of having had partial meniscectomy was higher in knees that developed ROA. When looking only at knees that developed ROA, partial meniscectomy was associated with greater risk of worsening cartilage damage. KEY POINTS: • Partial meniscectomy is a controversial treatment option for degenerative meniscal tears. • Partial meniscectomy is strongly associated with incident osteoarthritis within 1 year. • Partial meniscectomy is associated with increased risk of worsening cartilage damage.

Testing a Positive Psychological Intervention for Osteoarthritis.

OBJECTIVE: Osteoarthritis is a leading cause of disability for which there is no cure. Psychosocial-oriented treatments are underexplored. We developed and tested an intervention to build positive psychological skills (e.g., gratitude) to reduce osteoarthritis symptom severity, including pain and functioning, and to improve psychosocial well-being in patients with knee or hip osteoarthritis. DESIGN: Two-arm randomized design with six-month follow-up. SETTING: An academic Veterans Affairs Medical Center. SUBJECTS: Patients aged 50 years or older with knee or hip osteoarthritis and pain ratings of 4 or higher. METHODS: Patients (N = 42) were randomized to a six-week program containing positive skill-building activities or neutral control activities tailored to the patient population. Adherence was assessed by telephone each week. We assessed osteoarthritis symptom severity (WOMAC Osteoarthritis Index) and measures of well-being (positive affect, negative affect, and life satisfaction) at baseline and by telephone one, three, and six months after the program ended. We used linear mixed models to examine changes over time. RESULTS: The majority (64%) of patients completed more than 80% of their weekly activities. Patients in the positive (vs neutral) program reported significantly more improvement over time in osteoarthritis symptom severity (P = 0.02, Cohen's d = 0.86), negative affect (P = 0.03, Cohen's d = 0.50), and life satisfaction (P = 0.02, Cohen's d = 0.36). CONCLUSIONS: The study successfully engaged patients with knee or hip osteoarthritis in a six-week intervention to build positive psychological skills. Improving osteoarthritis symptom severity and measures of psychosocial well-being, the intervention shows promise as a tool for chronic pain management.

Comparison of radiographic joint space width and magnetic resonance imaging for prediction of knee replacement: A longitudinal case-control study from the Osteoarthritis Initiative.

OBJECTIVE: To evaluate whether change in fixed-location measures of radiographic joint space width (JSW) and cartilage thickness by MRI predict knee replacement. METHODS: Knees replaced between 36 and 60 months' follow-up in the Osteoarthritis Initiative were each matched with one control by age, sex and radiographic status. Radiographic JSW was determined from fixed flexion radiographs and subregional femorotibial cartilage thickness from 3 T MRI. Changes between the annual visit before replacement (T0) and 2 years before T0 (T-2) were compared using conditional logistic regression. RESULTS: One hundred and nineteen knees from 102 participants (55.5 % women; age 64.2 ± 8.7 [mean ± SD] years) were studied. Fixed-location JSW change at 22.5 % from medial to lateral differed more between replaced and control knees (case-control [cc] OR = 1.57; 95 % CI: 1.23-2.01) than minimum medial JSW change (ccOR = 1.38; 95 % CI: 1.11-1.71). Medial femorotibial cartilage loss displayed discrimination similar to minimum JSW, and central tibial cartilage loss similar to fixed-location JSW. Location-independent thinning and thickening scores were elevated prior to knee replacement. CONCLUSIONS: Discrimination of structural progression between knee pre-placement cases versus controls was stronger for fixed-location than minimum radiographic JSW. MRI displayed similar discrimination to radiography and suggested greater simultaneous cartilage thickening and loss prior to knee replacement. KEY POINTS: • Fixed-location JSW predicts surgical knee replacement more strongly than minimum JSW. • MRI predicts knee replacement with similar accuracy to radiographic JSW. • MRI reveals greater cartilage thinning and thickening prior to knee replacement.

Can structural joint damage measured with MR imaging be used to predict knee replacement in the following year?

PURPOSE: To assess whether magnetic resonance (MR) imaging-based cross-sectional measures of structural joint damage can be used to predict knee replacement during the following year. MATERIALS AND METHODS: Participants were drawn from the Osteoarthritis Initiative, a longitudinal observational study that includes 4796 participants who have knee osteoarthritis or are at risk. The HIPAA-compliant protocol was approved by the institutional review boards of all participating centers, and written informed consent was obtained from all participants. During the 5 years of follow-up, 199 knees underwent knee replacement and were matched with 199 control knees that did not undergo knee replacement. Knees were matched according to radiographic disease stage and patient sex and age. All knees that underwent knee replacement and had MR images available from the year before surgery were included. MR images were assessed for cartilage damage, bone marrow lesions, meniscal damage, meniscal extrusion, synovitis, and effusion prior to reported knee replacement. Conditional logistic regression was applied to assess the risk of knee replacement. Analyses were performed on a compartmental and knee level. RESULTS: Participants had a mean age ± standard deviation of 64.2 years ± 8.4 (range, 47-82 years) and were predominantly women (232 of 398 participants, 58.3%). Risk for knee replacement was significantly increased for knees that exhibited two or more subregions with severe cartilage loss (odds ratio [OR], 16.5; 95% confidence interval [CI]: 3.96, 68.76), more than two subregions with bone marrow lesions (OR, 4.00; 95% CI: 1.75, 9.16), medial meniscal maceration (OR, 1.84; 95% CI: 1.13, 2.99), effusion (OR, 4.75; 95% CI: 2.55, 8.85), or synovitis (OR, 2.17; 95% CI: 1.33, 3.56), but not extrusion (OR, 1.00; 95% CI: 0.60,1.67), when compared with knees that did not exhibit these features as the reference standard. CONCLUSION: Apart from meniscal extrusion, all features of tissue abnormalities at MR imaging were related to clinical prognosis and could be used to predict knee replacement in the following year.

Accessible Synthetic Probes for Staining Actin inside Platelets and Megakaryocytes by Employing Lifeact Peptide.

Lifeact is a 17-residue peptide that can be employed in cell microscopy as a probe for F-actin when fused to fluorescent proteins, but therefore is not suitable for all cell types. We have conjugated fluorescently labelled Lifeact to three different cell-penetrating systems (a myristoylated carrier (myr), the pH low insertion peptide (pHLIP) and the cationic peptide TAT) as a strategy to deliver Lifeact into cells and developed new tools for actin staining with improved synthetic accessibility and low toxicity, focusing on their suitability in platelets and megakaryocytes. Using confocal microscopy, we characterised the cell distribution of the new hybrids in fixed cells, and found that both myr- and pHLIP-Lifeact conjugates provide efficient actin staining upon cleavage of Lifeact from the carriers, without affecting cell spreading. This new approach could facilitate the design of new tools for actin visualisation.

Iron(II) supramolecular helicates condense plasmid DNA and inhibit vital DNA-related enzymatic activities.

The dinuclear iron(II) supramolecular helicates [Fe2 L3 ]Cl4 (L=C25 H20 N4 ) bind to DNA through noncovalent (i.e., hydrogen-bonding, electrostatic) interactions and exhibit antimicrobial and anticancer effects. In this study, we show that the helicates condense plasmid DNA with a much higher potency than conventional DNA-condensing agents. Notably, molecules of DNA in the presence of the M enantiomer of [Fe2 L3 ]Cl4 do not form intermolecular aggregates typically formed by other condensing agents, such as spermidine or spermine. The helicates inhibit the activity of several DNA-processing enzymes, such as RNA polymerase, DNA topoisomerase I, deoxyribonuclease I, and site-specific restriction endonucleases. However, the results also indicate that the DNA condensation induced by the helicates does not play a crucial role in these inhibition reactions. The mechanisms for the inhibitory effects of [Fe2 L3 ]Cl4 helicates on DNA-related enzymatic activities have been proposed.

Racial differences in treatment preferences among lupus patients: a two-site study.

OBJECTIVES: To identify the demographic, clinical and psychosocial characteristics associated with racial differences in willingness to receive cyclophosphamide (CYC) or participate in a research clinical trial (RCT) among patients with systemic lupus erythematosus (SLE). METHODS: Data from 163 African-American (AA) and 180 white (WH) SLE patients were evaluated. Structured interviews and chart reviews were conducted to determine treatment preferences in hypothetical situations and identify variables that may affect preferences. Logistic regression models were performed to evaluate the relationship between patient preferences and race, adjusted for patient characteristics. RESULTS: Among patients who had never received CYC (n=293), 62.9% AAs compared to 87.6% WHs were willing to receive the medication (p<0.001). This difference persisted (OR 0.37 [95% CI, 0.16-0.87]) after adjusting for socio-demographics, clinical characteristics, and perceptions about CYC and physicians. Income and higher perception of CYC effectiveness were other determinants of willingness to receive CYC. Among patients who had never participated in an RCT (n=326), 64.9% AAs compared to 84.3% WHs were willing to do so (p<0.001). This difference persisted (OR 0.41 [95% CI, 0.20-0.83]) after adjusting for socio-demographics, clinical context and patients' perceptions of physicians. SLE damage score, number of immunosuppressive medications and higher trust in physicians were also independently associated with willingness to participate in an RCT. CONCLUSIONS: Race remains an independent determinant of treatment preferences after adjustment for income, medications, medication efficacy expectations and trust in physicians. While some factors related to racial differences in preferences are relatively fixed, others that may alleviate these differences also exist, including medication beliefs and provider trust.

Willingness and access to joint replacement among African American patients with knee osteoarthritis: a randomized, controlled intervention.

OBJECTIVE: African American patients are significantly less likely to undergo knee replacement for the management of knee osteoarthritis (OA). Racial difference in preference (willingness) has emerged as a key factor. This study was undertaken to examine the efficacy of a patient-centered educational intervention on patient willingness and the likelihood of receiving a referral to an orthopedic clinic. METHODS: A total of 639 African American patients with moderate-to-severe knee OA from 3 Veterans Affairs primary care clinics were enrolled in a randomized, controlled trial with a 2 × 2 factorial design. Patients were shown a knee OA decision-aid video with or without brief counseling. The main outcome measures were change in patient willingness and receipt of a referral to an orthopedic clinic. Also assessed were whether patients discussed knee pain with their primary care provider or saw an orthopedic surgeon within 12 months of the intervention. RESULTS: At baseline, 67% of the participants were definitely/probably willing to consider knee replacement, with no difference among the groups. The intervention increased patient willingness (75%) in all groups at 1 month. For those who received the decision aid intervention alone, the gains were sustained for up to 3 months. By 12 months postintervention, patients who received any intervention were more likely to report engaging their provider in a discussion about knee pain (92% versus 85%), to receive a referral to an orthopedic surgeon (18% versus 13%), and for those with a referral, to attend an orthopedic consult (61% versus 50%). CONCLUSION: An educational intervention significantly increased the willingness of African American patients to consider knee replacement. It also improved the likelihood of patient-provider discussion about knee pain and access to surgical evaluation.

Youth use of electronic nicotine delivery systems (ENDS) in the Population Assessment of Tobacco and Health (PATH) Wave 6: Impact of survey mode.

BACKGROUND: The Population Assessment of Tobacco and Health (PATH) provides annual prevalence data on youth use of electronic nicotine delivery systems (ENDS). However, trends may be complicated by COVID-related changes in survey mode (self-completed vs. telephone interview) across 2019-2021. METHODS: Trends in past 30-day (P30D) ENDS use over PATH waves 5 (2019; 100 % self-completed), 5.5 (2020; 100 % telephone interview), and 6 (2021; 33.5 % self-completed, 66.5 % telephone interview) were examined among continuing youth, overall and within survey mode. Further analyses examined the nature of these changes by examining 1) potential response biases in social contexts of nicotine use, and 2) sources of ENDS over time. RESULTS: Telephone interviewees were less likely to report ENDS use (4.6 % vs. 8.6 % for self-completers), and more likely to report social disapproval of nicotine use, suggesting a reporting bias in telephone interviews. Survey-mode-naïve analyses suggested a large decline in P30D ENDS use prevalence between 2019-20 (10.2-4.6 %) followed by an apparent uptick in 2021 (5.9 %); however, comparing like-to-like survey modes showed a more modest decline (10.2 % in 2019; 8.6 % in 2021; self-completed) with no change between 2020 and 2021 (4.6 % in both; telephone interviews). Analyses suggested that the mode effects were partly, but not wholly, related to social desirability effects. DISCUSSION: Changes in PATH survey mode introduced artifacts into ENDS use prevalence, possibly due in part to social desirability bias suppressing reporting in telephone interviews, rather than a true uptick. It is essential to account for survey mode in PATH surveys.

Quantitative MRI measures of cartilage predict knee replacement: a case-control study from the Osteoarthritis Initiative.

OBJECTIVE: Knee osteoarthritis commonly requires joint replacement, substantially reduces quality of life and increases healthcare utilisation and costs. This study aimed to identify whether quantitative measures of articular cartilage structure predict knee replacement, and to establish their utility as outcomes in clinical trials of disease-modifying therapy. METHODS: A nested case-control study was performed in Osteoarthritis Initiative participants, a multicentre observational cohort of 4796 participants with or at risk of knee osteoarthritis. 127 knees were replaced between baseline and 4 years follow-up, and one control knee per case matched for baseline radiographic disease stage (Kellgren-Lawrence grade; KLG), gender and age. Quantitative cartilage measures were obtained from 3 T magnetic resonance images at the exam before knee replacement, and longitudinal change during the previous 12 months when available (n=110). RESULTS: Cartilage thickness loss in the central and total medial femorotibial compartment (primary and secondary predictor variables) was significantly greater in case than control knees (AUC=0.59/0.58). Differences in cartilage loss were greater at earlier than later radiographic disease stages (p<0.01 for interaction with KLG). Cartilage thickness loss in the central tibia was the most predictive longitudinal measure (AUC=0.64). Denuded bone areas in the medial femur were the most predictive and discriminatory cross-sectional measure between case and control knees (AUC=0.66). CONCLUSIONS: This study demonstrates the predictive value of quantitative, MRI-based measures of cartilage for the clinically relevant endpoint of knee replacement, providing support for their utility in clinical trials to evaluate the effectiveness of structure-modifying intervention.

Risk factors for magnetic resonance imaging-detected patellofemoral and tibiofemoral cartilage loss during a six-month period: the joints on glucosamine study.

OBJECTIVE: To assess several baseline risk factors that may predict patellofemoral and tibiofemoral cartilage loss during a 6-month period. METHODS: For 177 subjects with chronic knee pain, 3T magnetic resonance imaging (MRI) of both knees was performed at baseline and followup. Knees were semiquantitatively assessed, evaluating cartilage morphology, subchondral bone marrow lesions, meniscal morphology/extrusion, synovitis, and effusion. Age, sex, and body mass index (BMI), bone marrow lesions, meniscal damage/extrusion, synovitis, effusion, and prevalent cartilage damage in the same subregion were evaluated as possible risk factors for cartilage loss. Logistic regression models were applied to predict cartilage loss. Models were adjusted for age, sex, treatment, and BMI. RESULTS: Seventy-nine subregions (1.6%) showed incident or worsening cartilage damage at followup. None of the demographic risk factors was predictive of future cartilage loss. Predictors of patellofemoral cartilage loss were effusion, with an adjusted odds ratio (OR) of 3.5 (95% confidence interval [95% CI] 1.3-9.4), and prevalent cartilage damage in the same subregion with an adjusted OR of 4.3 (95% CI 1.3-14.1). Risk factors for tibiofemoral cartilage loss were baseline meniscal extrusion (adjusted OR 3.6 [95% CI 1.3-10.1]), prevalent bone marrow lesions (adjusted OR 4.7 [95% CI 1.1-19.5]), and prevalent cartilage damage (adjusted OR 15.3 [95% CI 4.9-47.4]). CONCLUSION: Cartilage loss over 6 months is rare, but may be detected semiquantitatively by 3T MRI and is most commonly observed in knees with Kellgren/Lawrence grade 3. Predictors of patellofemoral cartilage loss were effusion and prevalent cartilage damage in the same subregion. Predictors of tibiofemoral cartilage loss were prevalent cartilage damage, bone marrow lesions, and meniscal extrusion.

Prevalence of MRI-detected mediopatellar plica in subjects with knee pain and the association with MRI-detected patellofemoral cartilage damage and bone marrow lesions: data from the Joints On Glucosamine study.

BACKGROUND: The mediopatellar plica is a synovial fold representing an embryonic remnant from the developmental process of the synovial cavity formation in the knee. We aimed to examine the frequency of MRI-detected mediopatellar plica and its cross-sectional association with MRI-detected cartilage damage and bone marrow lesions (BMLs) in the patellofemoral joint (PFJ) in a cohort of subjects with knee pain. METHODS: 342 knees with chronic frequent knee pain were evaluated for MRI-detected mediopatellar plica (type A, B or C according to the modified Sakakibara classification). Cartilage damage (scored 0 to 6) and BMLs (scored 0 to 3) were semiquantitatively assessed in four subregions of the PFJ on MRI. Hoffa-synovitis and effusion-synovitis were graded 0 to 3. Patellar length ratio (PLR), lateral patellar tilt angle (LPTA), bisect offset (BO), and sulcus angle (SA) were measured on MRI. The presence of mediopatellar plica and its association with cartilage damage and BMLs in the PFJ was assessed using logistic regression after adjusting for age, gender, body mass index, PLR, LPTA, BO, SA, and Hoffa- and effusion-synovitis. RESULTS: 163 (47.7%) knees exhibited mediopatellar plica (76 (22.2%) type A, 69 (20.2%) type B, and 18 (5.3%) type C) on MRI. Significant cross-sectional associations of MRI-detected mediopatellar plica and cartilage damage were observed for the medial patella (adjusted odds ratio (aOR) 2.12, 95% CI 1.23-3.64 for all types combined, and aOR 4.20, 95% CI 1.92-9.19 for type B lesion), but not for the anterior medial femur or the lateral PFJ. No associations were found between the presence of MRI-detected mediopatellar plica and BMLs in any patellofemoral subregion. CONCLUSION: On MRI, types A and B mediopatellar plicae were commonly observed in this cohort of subjects with knee pain. MRI-detected mediopatellar plica was cross-sectionally associated with higher likelihood of the presence of MRI-detected medial patellar cartilage damage after adjustment for confounders.

Binding of a designed anti-cancer drug to the central cavity of an RNA three-way junction.

Getting to the heart of it: Co-crystallization of an RNA three-way junction with a cylindrical di-iron(II)-based anti-cancer drug (green) results in π-stacking interactions between the cylinder and the central base pairs of the RNA structure. The shape, size, and cationic nature of the cylinder were found to be responsible for this perfect fit. Native gel electrophoresis studies confirmed stabilization of the RNA three-way junction by the iron(II) cylinder.

Switching away from smoking at 12 months among adult JUUL users varying in recent history of quit attempts made with and without smoking cessation medication.

Some smokers switch away from smoking using e-cigarettes, but guidelines recommend trying approved medications first. We analyzed switching in adult smokers using JUUL by their recent history of quit attempts and use of smoking cessation medications. Participants were 8511 adult (21+) established smokers (at baseline), in which 50.3% are daily smokers, in a longitudinal observational study who completed a survey 12 months after first purchasing a JUUL Starter Kit. At baseline, participants reported attempts to quit smoking in the prior year and use of pharmacotherapy (nicotine replacement therapy [NRT] or prescription medication) in their most recent attempt. The outcomes were switching (self-reported no past-30-day smoking) and 50%+ reductions in cigarette consumption. Multivariable analyses were adjusted for baseline covariates. Two thirds of the participants had made a quit attempt in the year before purchasing JUUL. Overall, 59% [58%, 60%] had switched at 12 months. Switching was more likely in those who had used NRT and who attempted quitting without medication versus those who used prescription medications or made no quit attempt. In adjusted multivariable analyses, only making a past-year quit attempt (vs. not) was associated with higher odds of switching (OR = 1.15 [1.04, 1.28]). Over 60% of dual users reduced cigarette consumption by ≥50%. These associations were largely similar in daily smokers. Twelve months after purchasing JUUL, almost all smokers reported either switching or reducing their smoking by 50%+, including those who had recently failed to quit smoking with approved pharmacotherapies. E-cigarettes provide an alternative route to abstinence from smoking for smokers with a history of cessation and cessation treatment failure.

Youth patterns of use of electronic nicotine delivery systems (ENDS), Population Assessment of Tobacco and Health (PATH) waves 4-5.5.

INTRODUCTION: Youth use of electronic nicotine delivery systems (ENDS) is a continuing concern, making it important to assess evolving patterns, especially as non-tobacco, non-menthol (NTM) flavors were withdrawn for pod-based (but not disposable) ENDS in February 2020. METHODS: Trends in past-30-day (P30D) ENDS use and smoking prevalence, usual device type, flavor (tobacco, mint/menthol, or fruit/sweet/other), and regular/last-used brand in PATH Waves 4 (2017), 4.5 (2018), 5 (2019), and 5.5 (2020) were examined. Shifts between 2019 and 2020 in flavor use for pods and disposables were examined. RESULTS: P30D ENDS use peaked in 2019 at 8.6 % of all youth, subsequently declining by nearly half to 4.5 % in 2020. Meanwhile, P30D cigarette smoking declined to an all-time low (1.3 %) in 2020. Within this overall decline, consumption shifted to disposable ENDS, which increased nearly 10-fold (from 5.0 % to 49.2 % of P30D ENDS users). Relatedly, use of fruit/sweet/other flavors remained similar overall between 2019 and 2020 (approximately 75-80 % of P30D ENDS users), but the use of these flavors became concentrated in disposable ENDS in 2020 (a 12-fold increase from 4.4 % to 58.4 % of fruit/sweet/other-flavor users). CONCLUSIONS: PATH results show similar trends to other US national surveys in youth ENDS trends. The removal of non-tobacco, non-menthol flavors in pod-based ENDS (while remaining available in disposables) has likely driven youth towards disposable devices, resulting in continued high use of fruit/sweet/other flavors, which are now predominant in users of disposable ENDS. Wave 5.5 is uninformative regarding brand use because common disposable brands were not queried.

A New Questionnaire to Assess Respiratory Symptoms (The Respiratory Symptom Experience Scale): Quantitative Psychometric Assessment and Validation Study.

BACKGROUND: Smokers often experience respiratory symptoms (eg, morning cough), and those who stop smoking, including those who do so by switching completely to electronic nicotine delivery systems (ENDS), may experience reductions in symptoms. Existing respiratory symptom questionnaires may not be suitable for studying these changes, as they are intended for patient populations, such as those with chronic obstructive pulmonary disease (COPD). OBJECTIVE: This study aimed to develop a respiratory symptom questionnaire appropriate for current smokers and for assessing changes when smokers stop smoking. METHODS: The Respiratory Symptom Experience Scale (RSES) was derived from existing instruments and subject matter expert input and refined through cognitive debriefing interviews (n=49). Next, for purposes of the quantitative psychometric evaluation, the RSES was administered to smokers (n=202), former smokers (no tobacco use in >6 months; n=200), and switchers (n=208, smokers who switched to ENDS for >6 months), all of whom had smoked for at least 10 years (mean age 33 years). Participants, who averaged 62 (SD 12) years of age, included 28% (173/610) with respiratory allergy symptoms and 17% (104/610) with COPD. Test-retest reliability was assessed by repeat assessment after 1 week in 128 participants. RESULTS: A generalized partial credit model confirmed that the response options were ordered, and a parallel analysis using principal components confirmed that the scale was unidimensional. With allowance for 2 sets of correlated errors between pairs of items, a 1-factor graded response model fit the data. Discrimination parameters were approximately 1 or greater for all items. Scale reliability was 0.80 or higher across a broad range of severity (standardized scores -0.40 to 3.00). Test-retest reliability (absolute intraclass correlation) was good, at 0.89. RSES convergent validity was supported by substantial differences (Cohen d=0.74) between those with and without a diagnosis of respiratory disease (averaging 0.57 points, indicating that differences of this size or smaller represent meaningful differences). RSES scores also strongly differentiated those with and without COPD (d=1.52). Smokers' RSES scores were significantly higher than former smokers' scores (P<.001). Switchers' RSES scores were significantly lower than smokers' scores (P<.001) and no different from former smokers' scores (P=.34). CONCLUSIONS: The RSES fills an important gap in the existing toolkit of respiratory symptom questionnaires; it is a reliable and valid tool to assess respiratory symptoms in adult current and former smokers, including those who have switched to noncombusted nicotine products. This suggests that the scale is sensitive to respiratory symptoms that develop in smokers and to their remission when smokers quit or switch to noncombusted nicotine products intended to reduce the harm of smoking. The findings also suggest that switching from cigarettes to ENDS may improve respiratory health.