Structural Basis of Transcription Inhibition by Fidaxomicin (Lipiarmycin A3).

Fidaxomicin is an antibacterial drug in clinical use for treatment of Clostridium difficile diarrhea. The active ingredient of fidaxomicin, lipiarmycin A3 (Lpm), functions by inhibiting bacterial RNA polymerase (RNAP). Here we report a cryo-EM structure of Mycobacterium tuberculosis RNAP holoenzyme in complex with Lpm at 3.5-Å resolution. The structure shows that Lpm binds at the base of the RNAP "clamp." The structure exhibits an open conformation of the RNAP clamp, suggesting that Lpm traps an open-clamp state. Single-molecule fluorescence resonance energy transfer experiments confirm that Lpm traps an open-clamp state and define effects of Lpm on clamp dynamics. We suggest that Lpm inhibits transcription by trapping an open-clamp state, preventing simultaneous interaction with promoter -10 and -35 elements. The results account for the absence of cross-resistance between Lpm and other RNAP inhibitors, account for structure-activity relationships of Lpm derivatives, and enable structure-based design of improved Lpm derivatives.

Biologics (mepolizumab and omalizumab) induced remission in severe asthma patients.

BACKGROUND: Asthma remission has emerged as a potential treatment goal. This study evaluated the effectiveness of two biologics (mepolizumab/omalizumab) in achieving asthma remission. METHODS: This observational study included 453 severe asthma patients (41% male; mean age ± SD 55.7 ± 14.7 years) from two real-world drug registries: the Australian Mepolizumab Registry and the Australian Xolair Registry. The composite outcome clinical remission was defined as zero exacerbations and zero oral corticosteroids during the previous 6 months assessed at 12 months and 5-item Asthma Control Questionnaire (ACQ-5) ≤1 at 12 months. We also assessed clinical remission plus optimization (post-bronchodilator FEV1 ≥80%) or stabilization (post-bronchodilator FEV1 not greater than 5% decline from baseline) of lung function at 12 months. Sensitivity analyses explored various cut-offs of ACQ-5/FEV1 scores. The predictors of clinical remission were identified. RESULTS: 29.3% (73/249) of AMR and 22.8% (37/162) of AXR cohort met the criteria for clinical remission. When lung function criteria were added, the remission rates were reduced to 25.2% and 19.1%, respectively. Sensitivity analyses identified that the remission rate ranged between 18.1% and 34.9% in the AMR cohort and 10.6% and 27.2% in the AXR cohort. Better lung function, lower body mass index, mild disease and absence of comorbidities such as obesity, depression and osteoporosis predicted the odds of achieving clinical remission. CONCLUSION: Biologic treatment with mepolizumab or omalizumab for severe asthma-induced asthma remission in a subgroup of patients. Remission on treatment may be an achievable treatment target and future studies should consider remission as an outcome measure.

2D 1H sLASER Long-TE and 3D 31P Chemical Shift Imaging at 3 T for Monitoring Fasting-Induced Changes in Brain Tumor Tissue.

BACKGROUND: Emerging evidence suggests that fasting could play a key role in cancer treatment. Its metabolic effects on gliomas require further investigation. PURPOSE: To design a multi-voxel 1H/31P MR-spectroscopic imaging (MRSI) protocol for noninvasive metabolic monitoring of cerebral, fasting-induced changes on an individual patient/tumor level, and to assess its technical reliability/reproducibility. STUDY TYPE: Prospective. POPULATION: MRS phantom. Twenty-two patients (mean age = 61, 6 female) with suspected WHO grade II-IV glioma examined before and after 72-hour-fasting prior to biopsy/resection. FIELD STRENGTH/SEQUENCE: 3-T, 1H decoupled 3D 31P MRSI, 2D 1H sLASER MRSI at an echo time of 144 msec, 2D 1H MRSI (as water reference), T1-weighted, T1-weighted contrast-enhanced, T2-weighted, and FLAIR. sLASER and PRESS sequences were used for phantom measurements. ASSESSMENT: Phantom measurements and spectral simulations were performed with various echo-times for protocol optimization. In vivo spectral analyses were conducted using LCModel and AMARES, obtaining quality/fitting parameters (linewidth, signal-to-noise-ratio, and uncertainty measures of fitting) and metabolite intensities. The volume of glioma sub-regions was calculated and correlated with MRS findings. Ex-vivo spectra of necrotic tumor tissues were obtained using high-resolution magic-angle spinning (HR-MAS) technique. STATISTICAL TESTS: Wilcoxon signed-rank test, Bland-Altman plots, and coefficient of variation were used for repeatability analysis of quality/fitting parameters and metabolite concentrations. Spearman ρ correlation for the concentration of ketone bodies with volumes of glioma sub-regions was determined. A P-value <0.05 was considered statistically significant. RESULTS: 1H and 31P repeatability measures were highly consistent between the two sessions. ß-hydroxybutyrate and acetoacetate were detectable (fitting-uncertainty <50%) in glioma sub-regions of all patients who completed the 72-hour-fasting cycle. ß-hydroxybutyrate accumulation was significantly correlated with the necrotic/non-enhancing tumor core volume (ρ = 0.81) and validated using ex-vivo 1H HR-MAS. DATA CONCLUSION: We propose a comprehensive MRS protocol that may be used for monitoring cerebral, fasting-induced changes in patients with glioma. EVIDENCE LEVEL: 1 TECHNICAL EFFICACY: Stage 4.

In Vivo Measurement of Rat Brain Water Content at 9.4 T MR Using Super-Resolution Reconstruction: Validation With Ex Vivo Experiments.

BACKGROUND: Given that changes in brain water content are often correlated with disease, investigating water content non-invasively and in vivo could lead to a better understanding of the pathogenesis of several neurologic diseases. PURPOSE: To adapt a super-resolution-based technique, previously developed for humans, to the rat brain and report in vivo high-resolution (HR) water content maps in comparison with ex vivo wet/dry methods. STUDY TYPE: Prospective. ANIMAL MODEL: Eight healthy male Wistar rats. FIELD STRENGTH/SEQUENCE: 9.4-T, multi-echo gradient-echo (mGRE) sequence. ASSESSMENT: Using super-resolution reconstruction (SRR), a HR mGRE image (200 µm isotropic) was reconstructed from three low-resolution (LR) orthogonal whole-brain images in each animal, which was followed by water content mapping in vivo. The animals were subsequently sacrificed, the brains excised and divided into five regions (front left, front right, middle left, middle right, and cerebellum-brainstem regions), and the water content was measured ex vivo using wet/dry measurements as the reference standard. The water content values of the in vivo and ex vivo methods were then compared for the whole brain and also for the different regions separately. STATISTICAL TESTS: Friedman's non-parametric test was used to test difference between the five regions, and Pearson's correlation coefficient was used for correlation between in vivo and ex vivo measurements. A P-value <0.05 was considered statistically significant. RESULTS: Water content values derived from in vivo MR measurements showed strong correlations with water content measured ex vivo at a regional level (r = 0.902). Different brain regions showed significantly different water content values. Water content values were highest in the frontal brain, followed by the midbrain, and lowest in the cerebellum and brainstem regions. DATA CONCLUSION: An in vivo technique to achieve HR isotropic water content maps in the rat brain using SRR was adopted in this study. The MRI-derived water content values obtained using the technique showed strong correlations with water content values obtained using ex vivo wet/dry methods. LEVEL OF EVIDENCE: 1 TECHNICAL EFFICACY: Stage 1.

Treatable traits, combination inhaler therapy and the future of asthma management.

The landscape of asthma has considerably changed in the last decade. Effective medications and inhaler devices have been developed and integrated into the asthma pharmacopoeia, but unfortunately, the proportion of uncontrolled patients remains unacceptably high. This is now recognized to be mainly due to the inappropriate use of medications or inhaler devices, heterogeneity of the disease or other factors contributing to the disease. Currently, inhaled corticosteroids (ICS), with or without long-acting beta agonists (LABA), are the cornerstone of asthma management, and recently international guidelines recognized the importance of combination inhaler therapy (ICS/LABA) even in mild asthma. In future, ultra-long-acting personalized medications and smart inhalers will complement combination inhaler therapy in order to effectively addresses issues such as adherence, inhaler technique and polypharmacy (both of drugs and devices). Asthma is now acknowledged as a multifaceted cluster of disorders and the treatment model has evolved from one-size-fits-all to precision medicine approaches such as treatable traits (TTs, defined as measurable and treatable clinically important factors) which encourages the quality use of medications and identification and management of all underlying behavioural and biological treatable risk factors. TT requires research and validation in a clinical context and the implementation strategies and efficacy in various settings (primary/secondary/tertiary care, low-middle income countries) and populations (mild/moderate/severe asthma) are currently evolving. Combination inhaler therapy and the TTs approach are complementary treatment approaches. This review examines the current status of personalized medicine and combination inhaler therapy, and describes futuristic views for these two strategies.

Which smokers enroll in a hospital based smoking cessation trial? Survey of smoking related behaviors, quit attempts, and motivation to quit.

BACKGROUND: Understanding smoking behaviors in hospital patients who smoke may improve inpatient cessation treatments. This study aimed to describe smoking-related behaviors, past-quit attempts, and self-reported difficulties experienced in quitting among those who enrolled in a smoking cessation trial of varenicline. METHODS: Baseline data were obtained from adult hospitalized smokers (average ≥ 10 cigarettes/day in 4-weeks prior to hospitalization) who enrolled in a randomized, placebo-controlled trial of varenicline ± nicotine lozenges at five Australian public hospitals. A logistic regression model tested the association between participant characteristics and quitting in the previous 12 months. RESULTS: Participants' (n = 320; 57% male, 52.5 ± 12.1 years old) motivation and confidence in quitting were high. A total of 120 participants (37.5%) had attempted quitting in the previous 12-months. Prior hospitalization (P = .008) and employment status (P = .015) were significantly associated with past quit attempts. No statistically significant differences were noted in the reason for hospitalization or the level of nicotine dependence between participants who attempted quitting in the previous 12 months and their counterparts. Smoking cessation pharmacotherapy was used by 55% of those attempting to quit; nicotine replacement therapy (65.2%) and varenicline (16.7%) most common. Stress or anxiety, urges to smoke and a lack of motivation were the difficulties experienced in past quit attempts. CONCLUSIONS: Those who had a prior hospitalization and were unemployed had significantly greater odds of reporting past quit attempts. Further research is needed to investigate the degree of adherence among inpatient smokers with the smoke-free hospital policies and the frequency of NRT provision and uptake on admission.

Asthma remission: what is it and how can it be achieved?

Asthma treatment goals currently focus on symptom and exacerbation control rather than remission. Remission is not identical to cure, but is a step closer. This review considers the current definitions of remission in asthma, the prevalence and predictors, the pathophysiology of remission, the possibility of achieving it using the available treatment options, and the future research directions. Asthma remission is characterised by a high level of disease control, including the absence of symptoms and exacerbations, and normalisation or optimisation of lung function with or without ongoing treatment. Even in those who develop a symptomatic remission of asthma, persistent pathological abnormalities are common, leading to a risk of subsequent relapse at any time. Complete remission requires normalisation or stabilisation of any underlying pathology in addition to symptomatic remission. Remission is possible as part of the natural history of asthma, and the prevalence of remission in the adult asthma population varies between 2% and 52%. The factors associated with remission include mild asthma, better lung function, better asthma control, younger age, early-onset asthma, shorter duration of asthma, milder bronchial hyperresponsiveness, fewer comorbidities and smoking cessation or never smoking. Although previous studies have not targeted treatment-induced remission, there is some evidence to show that the current long-term add-on therapies such as biologics and azithromycin can achieve some criteria for asthma remission on treatment, at least in a subgroup of patients. However, more research is required. Long-term remission could be included as a therapeutic goal in studies of asthma treatments.

Whole-Brain Water Content Mapping Using Super-Resolution Reconstruction with MRI Acquisition in 3 Orthogonal Orientations.

PURPOSE: Brain water content provides rich tissue contrast comparable to that of longitudinal relaxation time T1 , but mapping is usually performed at modest resolution. In particular, the slice thickness in 2D mapping methods is limited. Here, we combine super-resolution reconstruction techniques with a fast water content mapping method to acquire high and isotropic resolution (0.75 mm) water content maps at 3 Tesla. METHODS: A high-resolution multi-echo gradient echo image is super-resolution-reconstructed from 3 low-resolution, orthogonal multi-echo gradient echo image acquisitions, followed by water content mapping. The mapping accuracy and SNR of the proposed method are assessed using numerical simulations, phantom studies, and in vivo data acquired from 6 healthy volunteers at 3 Tesla. A high-resolution acquisition with an established mapping method is used as a reference. RESULTS: Whole-brain water content maps with 0.75 mm isotropic resolution are demonstrated. No bias in the water content values was seen following super-resolution reconstruction. In the in vivo experiments, a lower SD of the mean water content values was observed with the proposed method compared to the reference method. CONCLUSIONS: Super-resolution reconstruction of multi-echo gradient echo data is demonstrated, enabling whole-brain water content mapping with high and isotropic resolution. The accuracy of the proposed method is shown using phantoms and 6 healthy volunteers and was found to be unchanged compared to the conventional acquisition. The proposed method could increase the sensitivity of water content mapping sufficiently to enable the detection of very small lesions, such as cortical lesions in multiple sclerosis.

Educational interventions for health professionals managing chronic obstructive pulmonary disease in primary care.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a common, preventable and treatable health condition. COPD is associated with substantial burden on morbidity, mortality and healthcare resources. OBJECTIVES: To review existing evidence for educational interventions delivered to health professionals managing COPD in the primary care setting. SEARCH METHODS: We searched the Cochrane Airways Trials Register from inception to May 2021. The Register includes records from the Cochrane Central Register of Controlled Trials (CENTRAL), MEDLINE, Embase, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Allied and Complementary Medicine Database (AMED) and PsycINFO. We also searched online trial registries and reference lists of included studies. SELECTION CRITERIA: We included randomised controlled trials (RCTs) and cluster-RCTs. Eligible studies tested educational interventions aimed at any health professionals involved in the management of COPD in primary care. Educational interventions were defined as interventions aimed at upskilling, improving or refreshing existing knowledge of health professionals in the diagnosis and management of COPD. DATA COLLECTION AND ANALYSIS: Two review authors independently reviewed abstracts and full texts of eligible studies, extracted data and assessed the risk of bias of included studies. We conducted meta-analyses where possible and used random-effects models to yield summary estimates of effect (mean differences (MDs) with 95% confidence intervals (CIs)). We performed narrative synthesis when meta-analysis was not possible. We assessed the overall certainty of evidence for each outcome using Grades of Recommendation, Assessment, Development and Evaluation (GRADE). Primary outcomes were: 1) proportion of COPD diagnoses confirmed with spirometry; 2) proportion of patients with COPD referred to, participating in or completing pulmonary rehabilitation; and 3) proportion of patients with COPD prescribed respiratory medication consistent with guideline recommendations. MAIN RESULTS: We identified 38 studies(22 cluster-RCTs and 16 RCTs) involving 4936 health professionals (reported in 19/38 studies) and 71,085 patient participants (reported in 25/38 studies). Thirty-six included studies evaluated interventions versus usual care; seven studies also reported a comparison between two or more interventions as part of a three- to five-arm RCT design. A range of simple to complex interventions were used across the studies, with common intervention features including education provided to health professionals via training sessions, workshops or online modules (31 studies), provision of practice support tools, tool kits and/or algorithms (10 studies), provision of guidelines (nine studies) and training on spirometry (five studies). Health professionals targeted by the interventions were most commonly general practitioners alone (20 studies) or in combination with nurses or allied health professionals (eight studies), and the majority of studies were conducted in general practice clinics. We identified performance bias as high risk for 33 studies. We also noted risk of selection, detection, attrition and reporting biases, although to a varying extent across studies. The evidence of efficacy was equivocal for all the three primary endpoints evaluated: 1) proportion of COPD diagnoses confirmed with spirometry (of the four studies that reported this outcome, two supported the intervention); 2) proportion of patients with COPD who are referred to, participate in or complete pulmonary rehabilitation (of the four studies that reported this outcome, two supported the intervention); and 3) proportion of patients with COPD prescribed respiratory medications consistent with guideline recommendations (12 studies reported this outcome, the majority evaluated multiple drug classes and reported a mixed effect). Additionally, the low quality of evidence and potential risk of bias make the interpretation more difficult. Moderate-quality evidence (downgraded due to risk of bias concerns) suggests that educational interventions for health professionals probably improve the proportion of patients with COPD vaccinated against influenza (three studies) and probably have little impact on the proportion of patients vaccinated against pneumococcal infection (two studies). Low-quality evidence suggests that educational interventions for health professionals may have little or no impact on the frequency of COPD exacerbations (10 studies). There was a high degree of heterogeneity in the reporting of health-related quality of life (HRQoL). Low-quality evidence suggests that educational interventions for health professionals may have little or no impact on HRQoL overall, and when using the COPD-specific HRQoL instrument, the St George's Respiratory Questionnaire (at six months MD 0.87, 95% CI -2.51 to 4.26; 2 studies, 406 participants, and at 12 months MD -0.43, 95% CI -1.52 to 0.67, 4 studies, 1646 participants; reduction in score indicates better health). Moderate-quality evidence suggests that educational interventions for health professionals may improve patient satisfaction with care (one study). We identified no studies that reported adverse outcomes. AUTHORS' CONCLUSIONS: The evidence of efficacy was equivocal for educational interventions for health professionals in primary care on the proportion of COPD diagnoses confirmed with spirometry, the proportion of patients with COPD who participate in pulmonary rehabilitation, and the proportion of patients prescribed guideline-recommended COPD respiratory medications. Educational interventions for health professionals may improve influenza vaccination rates among patients with COPD and patient satisfaction with care. The quality of evidence for most outcomes was low or very low due to heterogeneity and methodological limitations of the studies included in the review, which means that there is uncertainty about the benefits of any currently published educational interventions for healthcare professionals to improve COPD management in primary care. Further well-designed RCTs are needed to investigate the effects of educational interventions delivered to health professionals managing COPD in the primary care setting.

Patterns of azithromycin use in obstructive airway diseases: a real-world observational study.

BACKGROUND: Low-dose long-term azithromycin is recommended in clinical practice guidelines for obstructive airway diseases (OAD); however, an optimal therapeutic regimen is not yet established. AIM: To understand the patterns of azithromycin use in OAD, characterise the patients who received it and evaluate its safety and efficacy using real-world data. METHODS: We audited 91 patients who had received azithromycin for at least 4 weeks for the management of asthma, chronic obstructive pulmonary disease (COPD) or non-cystic fibrosis bronchiectasis. RESULTS: The mean age was 65 ± 18 years, 60% were female and 48% were ex-smokers. The majority had asthma (75%), either alone (50%) or in combination with COPD (12%) or bronchiectasis (13%). Most (64%) reported cough or sputum at baseline. The most common treatment regimen was azithromycin 250 mg daily (73%) for more than 1 year (57%), with only seven adverse events. There was a significant reduction in the proportions of patients requiring emergency department visits (48% vs 32%; P < 0.001) and hospital admissions (35% vs 31%; P < 0.001) after starting azithromycin. In 88% of cases, physicians favoured the use of azithromycin. CONCLUSION: Physicians are currently using low-dose azithromycin for a long duration of more than 1 year for the management of OAD. The typical case definition is an older non-smoking adult with persistent asthma, often in combination with another OAD and presenting with bothersome cough or sputum. Azithromycin was well tolerated and led to reduced healthcare utilisation. Further research is required to establish an optimal dosage regimen of azithromycin in OAD.

High-resolution cryo-EM structures of outbreak strain human norovirus shells reveal size variations.

Noroviruses are a leading cause of foodborne illnesses worldwide. Although GII.4 strains have been responsible for most norovirus outbreaks, the assembled virus shell structures have been available in detail for only a single strain (GI.1). We present high-resolution (2.6- to 4.1-Å) cryoelectron microscopy (cryo-EM) structures of GII.4, GII.2, GI.7, and GI.1 human norovirus outbreak strain virus-like particles (VLPs). Although norovirus VLPs have been thought to exist in a single-sized assembly, our structures reveal polymorphism between and within genogroups, with small, medium, and large particle sizes observed. Using asymmetric reconstruction, we were able to resolve a Zn2+ metal ion adjacent to the coreceptor binding site, which affected the structural stability of the shell. Our structures serve as valuable templates for facilitating vaccine formulations.

Pterocarpan synthase (PTS) structures suggest a common quinone methide-stabilizing function in dirigent proteins and proteins with dirigent-like domains.

The biochemical activities of dirigent proteins (DPs) give rise to distinct complex classes of plant phenolics. DPs apparently began to emerge during the aquatic-to-land transition, with phylogenetic analyses revealing the presence of numerous DP subfamilies in the plant kingdom. The vast majority (>95%) of DPs in these large multigene families still await discovery of their biochemical functions. Here, we elucidated the 3D structures of two pterocarpan-forming proteins with dirigent-like domains. Both proteins stereospecifically convert distinct diastereomeric chiral isoflavonoid precursors to the chiral pterocarpans, (-)- and (+)-medicarpin, respectively. Their 3D structures enabled comparisons with stereoselective lignan- and aromatic terpenoid-forming DP orthologs. Each protein provides entry into diverse plant natural products classes, and our experiments suggest a common biochemical mechanism in binding and stabilizing distinct plant phenol-derived mono- and bis-quinone methide intermediates during different C-C and C-O bond-forming processes. These observations provide key insights into both their appearance and functional diversification of DPs during land plant evolution/adaptation. The proposed biochemical mechanisms based on our findings provide important clues to how additional physiological roles for DPs and proteins harboring dirigent-like domains can now be rationally and systematically identified.

Exploring the Impacts of Conformer Selection Methods on Ion Mobility Collision Cross Section Predictions.

The prediction of structure dependent molecular properties, such as collision cross sections as measured using ion mobility spectrometry, are crucially dependent on the selection of the correct population of molecular conformers. Here, we report an in-depth evaluation of multiple conformation selection techniques, including simple averaging, Boltzmann weighting, lowest energy selection, low energy threshold reductions, and similarity reduction. Generating 50 000 conformers each for 18 molecules, we used the In Silico Chemical Library Engine (ISiCLE) to calculate the collision cross sections for the entire data set. First, we employed Monte Carlo simulations to understand the variability between conformer structures as generated using simulated annealing. Then we employed Monte Carlo simulations to the aforementioned conformer selection techniques applied on the simulated molecular property: the ion mobility collision cross section. Based on our analyses, we found Boltzmann weighting to be a good trade-off between precision and theoretical accuracy. Combining multiple techniques revealed that energy thresholds and root-mean-squared deviation-based similarity reductions can save considerable computational expense while maintaining property prediction accuracy. Molecular dynamic conformer generation tools like AMBER can continue to generate new lowest energy conformers even after tens of thousands of generations, decreasing precision between runs. This reduced precision can be ameliorated and theoretical accuracy increased by running density functional theory geometry optimization on carefully selected conformers.

De novo sequencing and native mass spectrometry revealed hetero-association of dirigent protein homologs and potential interacting proteins in Forsythia × intermedia.

The discovery of dirigent proteins (DPs) and their functions in plant phenol biochemistry was made over two decades ago with Forsythia × intermedia. Stereo-selective, DP-guided, monolignol-derived radical coupling in vitro was then reported to afford the optically active lignan, (+)-pinoresinol from coniferyl alcohol, provided one-electron oxidase/oxidant capacity was present. It later became evident that DPs have several distinct sub-families, presumably with different functions. Some known DPs require other essential enzymes/proteins (e.g. oxidases) for their functions. However, the lack of a fully sequenced genome for Forsythia × intermedia made it difficult to profile other components co-purified with the (+)-pinoresinol forming DP. Herein, we used an integrated bottom-up, top-down, and native mass spectrometry (MS) approach to de novo sequence the extracted proteins via adaptation of our initial report of DP solubilization and purification. Using publicly available transcriptome and genomic data from closely related species, we identified 14 proteins that were putatively associated with either DP function or the cell wall. Although their co-occurrence after extraction and chromatographic separation is suggestive for potential protein-protein interactions, none were found to form stable protein complexes with DPs in native MS under the specific experimental conditions we have explored. Interestingly, two new DP homologs were found and they formed hetero-trimers. Molecular dynamics simulations suggested that similar hetero-trimers were possible between Arabidopsis DP homologs with comparable sequence similarities. Nevertheless, our integrated mass spectrometry method development helped prepare for future investigations directed to the discovery of novel proteins and protein-protein interactions. These advantages can be highly beneficial for plant and microbial research where fully sequenced genomes may not be readily available.

Effect of Cytisine vs Varenicline on Smoking Cessation: A Randomized Clinical Trial.

Importance: Cytisine is more effective than placebo and nicotine replacement therapy for smoking cessation. However, cytisine has not been tested against the most effective smoking cessation medication, varenicline, which is associated with adverse events known to lead to discontinuation of therapy. Objective: To examine whether standard cytisine treatment (25 days) was at least as effective as standard varenicline treatment (84 days) for smoking cessation. Design, Setting, and Participants: This noninferiority, open-label randomized clinical trial with allocation concealment and blinded outcome assessment was undertaken in Australia from November 2017 through May 2019; follow-up was completed in January 2020. A total of 1452 Australian adult daily smokers willing to make a quit attempt were included. Data collection was conducted primarily by computer-assisted telephone interview, but there was an in-person visit to validate the primary outcome. Interventions: Treatments were provided in accordance with the manufacturers' recommended dosage: cytisine (n = 725), 1.5-mg capsules taken 6 times daily initially then gradually reduced over the 25-day course; varenicline (n = 727), 0.5-mg tablets titrated to 1 mg twice daily for 84 days (12 weeks). All participants were offered referral to standard telephone behavioral support. Main Outcomes and Measures: The primary outcome was 6-month continuous abstinence verified using a carbon monoxide breath test at 7-month follow-up. The noninferiority margin was set at 5% and the 1-sided significance threshold was set at .025. Results: Among 1452 participants who were randomized (mean [SD] age, 42.9 [12.7] years; 742 [51.1%] women), 1108 (76.3%) completed the trial. Verified 6-month continuous abstinence rates were 11.7% for the cytisine group and 13.3% for the varenicline group (risk difference, -1.62% [1-sided 97.5% CI, -5.02% to ∞]; P = .03 for noninferiority). Self-reported adverse events occurred less frequently in the cytisine group (997 events among 482 participants) compared with the varenicline group (1206 events among 510 participants) and the incident rate ratio was 0.88 (95% CI, 0.81 to 0.95; P = .002). Conclusions and Relevance: Among daily smokers willing to quit, cytisine treatment for 25 days, compared with varenicline treatment for 84 days, failed to demonstrate noninferiority regarding smoking cessation. Trial Registration: anzctr.org.au Identifier: ACTRN12616001654448.

Block Design with Common Reference Samples Enables Robust Large-Scale Label-Free Quantitative Proteome Profiling.

Label-free quantitative proteomics has become an increasingly popular tool for profiling global protein abundances. However, one major limitation is the potential performance drift of the LC-MS platform over time, which, in turn, limits its utility for analyzing large-scale sample sets. To address this, we introduce an experimental and data analysis scheme based on a block design with common references within each block for enabling large-scale label-free quantification. In this scheme, a large number of samples (e.g., >100 samples) are analyzed in smaller and more manageable blocks, minimizing instrument drift and variability within individual blocks. Each designated block also contains common reference samples (e.g., controls) for normalization across all blocks. We demonstrated the robustness of this approach by profiling the proteome response of human macrophage THP-1 cells to 11 engineered nanomaterials at two different doses. A total of 116 samples were analyzed in six blocks, yielding an average coverage of 4500 proteins per sample. Following a common reference-based correction, 2537 proteins were quantified with high reproducibility without any imputation of missing values from 116 data sets. The data revealed the consistent quantification of proteins across all six blocks, as illustrated by the highly consistent abundances of house-keeping proteins in all samples and the high levels of correlation among samples from different blocks. The data also demonstrated that label-free quantification is robust and accurate enough to quantify even very subtle abundance changes as well as large fold-changes. Our streamlined workflow is easy to implement and can be readily adapted to other large cohort studies for reproducible label-free proteome quantification.

Targeted Intervention to Reduce Smoking among People with Severe Mental Illness: Implementation of a Smoking Cessation Intervention in an Inpatient Mental Health Setting.

Background and Objectives: Smoking and smoking-related harms are highly prevalent among people with severe mental illness. Targeted smoking cessation programs are much needed in this population. This pilot study aimed to assess the effectiveness of implementing smoking cessation system change interventions within an acute inpatient mental health unit. Materials and Methods: Design: Pre-post intervention study. System change interventions for smoking cessation were delivered over a three-month period (05 March 2018-04 June 2018) on an acute inpatient mental health unit. Participants (n = 214) were all individuals receiving care as inpatients during the three-month intervention. Outcomes assessed pre- and post-intervention were: (i) recording of patient smoking status in medical notes, (ii) number of inpatients offered smoking cessation medication, and iii) number of violent incidents reported. Results: Recording of smoking status significantly increased from 1.9% to 11.4% (X2 = 14.80; p ≤ 0.001). The proportion of inpatients offered smoking cessation treatment significantly increased from 11.0% to 26.8% (X2 = 16.01; p ≤ 0.001). The number of violent incidents decreased by half, which was not statistically significant. Conclusion: Evidence-based smoking cessation interventions can be successfully implemented on an inpatient mental health unit. Modest gains were made in routine screening for smoking and in smoking cessation treatment prescription. Future studies should prioritize effective participatory collaboration with staff to optimize effectiveness of interventions and should include additional strategies such as brief intervention training and smoking cessation treatments such as varenicline and buproprion in addition to nicotine replacement therapy (NRT).

ISiCLE: A Quantum Chemistry Pipeline for Establishing in Silico Collision Cross Section Libraries.

High-throughput, comprehensive, and confident identifications of metabolites and other chemicals in biological and environmental samples will revolutionize our understanding of the role these chemically diverse molecules play in biological systems. Despite recent technological advances, metabolomics studies still result in the detection of a disproportionate number of features that cannot be confidently assigned to a chemical structure. This inadequacy is driven by the single most significant limitation in metabolomics, the reliance on reference libraries constructed by analysis of authentic reference materials with limited commercial availability. To this end, we have developed the in silico chemical library engine (ISiCLE), a high-performance computing-friendly cheminformatics workflow for generating libraries of chemical properties. In the instantiation described here, we predict probable three-dimensional molecular conformers (i.e., conformational isomers) using chemical identifiers as input, from which collision cross sections (CCS) are derived. The approach employs first-principles simulation, distinguished by the use of molecular dynamics, quantum chemistry, and ion mobility calculations, to generate structures and chemical property libraries, all without training data. Importantly, optimization of ISiCLE included a refactoring of the popular MOBCAL code for trajectory-based mobility calculations, improving its computational efficiency by over 2 orders of magnitude. Calculated CCS values were validated against 1983 experimentally measured CCS values and compared to previously reported CCS calculation approaches. Average calculated CCS error for the validation set is 3.2% using standard parameters, outperforming other density functional theory (DFT)-based methods and machine learning methods (e.g., MetCCS). An online database is introduced for sharing both calculated and experimental CCS values ( metabolomics.pnnl.gov ), initially including a CCS library with over 1 million entries. Finally, three successful applications of molecule characterization using calculated CCS are described, including providing evidence for the presence of an environmental degradation product, the separation of molecular isomers, and an initial characterization of complex blinded mixtures of exposure chemicals. This work represents a method to address the limitations of small molecule identification and offers an alternative to generating chemical identification libraries experimentally by analyzing authentic reference materials. All code is available at github.com/pnnl .

Evaluation of In Silico Multifeature Libraries for Providing Evidence for the Presence of Small Molecules in Synthetic Blinded Samples.

The current gold standard for unambiguous molecular identification in metabolomics analysis is comparing two or more orthogonal properties from the analysis of authentic reference materials (standards) to experimental data acquired in the same laboratory with the same analytical methods. This represents a significant limitation for comprehensive chemical identification of small molecules in complex samples. The process is time consuming and costly, and the majority of molecules are not yet represented by standards. Thus, there is a need to assemble evidence for the presence of small molecules in complex samples through the use of libraries containing calculated chemical properties. To address this need, we developed a Multi-Attribute Matching Engine (MAME) and a library derived in part from our in silico chemical library engine (ISiCLE). Here, we describe an initial evaluation of these methods in a blinded analysis of synthetic chemical mixtures as part of the U.S. Environmental Protection Agency's (EPA) Non-Targeted Analysis Collaborative Trial (ENTACT, Phase 1). For molecules in all mixtures, the initial blinded false negative rate (FNR), false discovery rate (FDR), and accuracy were 57%, 77%, and 91%, respectively. For high evidence scores, the FDR was 35%. After unblinding of the sample compositions, we optimized the scoring parameters to better exploit the available evidence and increased the accuracy for molecules suspected as present. The final FNR, FDR, and accuracy were 67%, 53%, and 96%, respectively. For high evidence scores, the FDR was 10%. This study demonstrates that multiattribute matching methods in conjunction with in silico libraries may one day enable reduced reliance on experimentally derived libraries for building evidence for the presence of molecules in complex samples.

Isolation of Tryptanthrin and Reassessment of Evidence for Its Isobaric Isostere Wrightiadione in Plants of the Wrightia Genus.

A series of Wrightia hanleyi extracts was screened for activity against Mycobacterium tuberculosis H37Rv. One active fraction contained a compound that initially appeared to be either the isoflavonoid wrightiadione or the alkaloid tryptanthrin, both of which have been previously reported in other Wrightia species. Characterization by NMR and MS, as well as evaluation of the literature describing these compounds, led to the conclusion that wrightiadione (1) was misidentified in the first report of its isolation from W. tomentosa in 1992 and again in 2015 when reported in W. pubescens and W. religiosa. Instead, the molecule described in these reports and in the present work is almost certainly the isobaric (same nominal mass) and isosteric (same number of atoms, valency, and shape) tryptanthrin (2), a well-known quinazolinone alkaloid found in a variety of plants including Wrightia species. Tryptanthrin (2) is also accessible synthetically via several routes and has been thoroughly characterized. Wrightiadione (1) has been synthesized and characterized and may have useful biological activity; however, this compound can no longer be said to be known to exist in Nature. To our knowledge, this misidentification of wrightiadione (1) has heretofore been unrecognized.