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Objective Variants in the SLCO1B1 gene, encoding a hepatic methotrexate (MTX) transporter, affect clearance of high‐dose MTX. We tested whether in the *14 and *15 alleles of SLCO1B1 influenced the response to low‐dose MTX in juvenile idiopathic arthritis (JIA) patients. Methods The study included 310 JIA patients genotyped for three single nucleotide polymorphisms (SNPs) in SLCO1B1 (rs4149056, rs2306283, and rs11045819). A patient's SLCO1B1 diplotype was determined by combining the SNPs into the *1a, *1b, *4, *5, *14, and *15 alleles. Number of active joints at follow‐up (visit closest to 6 months of treatment and prior to starting a tumor necrosis factor inhibitor) was used as the dependent variable in a negative binomial regression model that included active joint count at baseline as a covariate. Results The SLCO1B1*14 allele was associated with less response to MTX (P = 0.024) and the *15 allele was not associated with response to MTX (P = 0.392). Conclusion SLCO1B1 alleles may be associated with poor response to MTX in JIA patients. The *14 allele has been associated with fast clearance (low exposure) after high‐dose MTX in patients with leukemia. Thus, the SLCO1B1 gene may be informative for precision dosing of MTX in JIA patients. Patients carrying the *14 allele may require a higher dose than noncarriers to achieve a similar response to MTX.

Amid increasing rates of electronic cigarette (e-cigarette) use in the United States, there is an urgent need to monitor patterns of use at the population level in order to inform practice, policy and regulation. This article examines how patterns of e-cigarette use among adults differ between users and nonusers of cigarettes using the most current national data. We analyzed data from the 2014 National Health Interview Survey. We estimated prevalence of ever, current, and daily e-cigarette use and examined how use patterns differed by demographic subgroups and measures of cigarette smoking status that accounted for the recent availability of e-cigarettes in the US marketplace. Current e-cigarette use is extremely low among never cigarette smokers (0.4%) and former smokers who quit cigarettes 4 or more years ago (0.8%). Although e-cigarette experimentation is most common among current cigarette smokers and young adults, daily use is highest among former smokers who quit in the past year (13.0%) and older adults. Compared to daily cigarette smokers, recently quit smokers were more than four times as likely to be daily users of e-cigarettes (AOR: 4.33 [95% CI: 3.08-6.09]). Extremely low e-cigarette use among never-smokers and longer term former smokers suggest that e-cigarettes neither promote widespread initiation nor relapse among adults. Recognition of the heterogeneity of smokers, including the time since quitting, is critical to draw accurate conclusions about patterns of e-cigarette use at the population level and its potential for public health benefit or harm. Data from 2014 National Health Interview Survey indicate that e-cigarettes have not been attracting adult non-smokers or promoting relapse in longer term former smokers. Moreover, the data are suggestive that some recent quitters may have done so with the assistance of e-cigarettes. Creating measures of smoking status that treat former smokers as a homogenous group is insufficient to assess the epidemiology of e-cigarette use and the potential impact on public health.

Electronic cigarettes (ECIGs) aerosolize a liquid that usually contains propylene glycol and/or vegetable glycerin, flavorants, and the dependence-producing drug nicotine in various concentrations. This study examined the extent to which ECIG liquid nicotine concentration is related to user plasma nicotine concentration in ECIG-naïve tobacco cigarette smokers. Sixteen ECIG-naïve cigarette smokers completed four laboratory sessions that differed by the nicotine concentration of the liquid (0, 8, 18, or 36 mg/ml) that was placed into a 1.5 Ohm, dual coil \"cartomizer\" powered by a 3.3V battery. In each session, participants completed two, 10-puff ECIG use bouts with a 30-second inter-puff interval; bouts were separated by 60 minutes. Venous blood was sampled before and after bouts for later analysis of plasma nicotine concentration; puff duration, volume, and average flow rate were measured during each bout. In bout 1, relative to the 0mg/ml nicotine condition (mean = 3.8 ng/ml, SD = 3.3), plasma nicotine concentration increased significantly immediately after the bout for the 8 (mean = 8.8 ng/ml, SD = 6.3), 18 (mean = 13.2 ng/ml, SD = 13.2), and 36 mg/ml (mean = 17.0 ng/ml, SD = 17.9) liquid concentration. A similar pattern was observed after bout 2. Average puff duration in the 36 mg/ml condition was significantly shorter compared to the 0mg/ml nicotine condition. Puff volume increased during the second bout for 8 and 18 mg/ml conditions. For a given ECIG device, nicotine delivery may be directly related to liquid concentration. ECIG-naïve cigarette smokers can, from their first use bout, attain cigarette-like nicotine delivery profiles with some currently available ECIG products. Liquid nicotine concentration can influence plasma nicotine concentration in ECIG-naïve cigarette smokers, and, at some concentrations, the nicotine delivery profile of a 3.3V ECIG with a dual coil, 1.5-Ohm cartomizer approaches that of a combustible tobacco cigarette in this population. Finding a product that delivers nicotine as effectively as a tobacco cigarette, as we report here, may be essential for smokers who want to replace completely their combustible tobacco cigarettes with ECIGs.

Summary Pseudomonas species have become reliable platforms for bioproduction due to their capability to tolerate harsh conditions imposed by large‐scale bioprocesses and their remarkable resistance to diverse physicochemical stresses. The last few years have brought forth a variety of synthetic biology tools for the genetic manipulation of pseudomonads, but most of them are either applicable only to obtain certain types of mutations, lack efficiency, or are not easily accessible to be used in different Pseudomonas species (e.g. natural isolates). In this work, we describe a versatile, robust and user‐friendly procedure that facilitates virtually any kind of genomic manipulation in Pseudomonas species in 3–5 days. The protocol presented here is based on DNA recombination forced by double‐stranded DNA cuts (through the activity of the I‐SceI homing meganuclease from yeast) followed by highly efficient counterselection of mutants (aided by a synthetic CRISPR‐Cas9 device). The individual parts of the genome engineering toolbox, tailored for knocking genes in and out, have been standardized to enable portability and easy exchange of functional gene modules as needed. The applicability of the procedure is illustrated both by eliminating selected genomic regions in the platform strain P. putida KT2440 (including difficult‐to‐delete genes) and by integrating different reporter genes (comprising novel variants of fluorescent proteins) into a defined landing site in the target chromosome. The present protocol describes a streamlined method for precise genome engineering of Pseudomonas species. By using a combination of fluorescent markers, coupled with the activity of the I‐SceI meganuclease or CRISPR/Cas9‐mediated counterselection, virtually any genomic manipulation can be performed in 3–5 days.

Summary Microbial conversion offers a promising strategy for overcoming the intrinsic heterogeneity of the plant biopolymer, lignin. Soil microbes that natively harbour aromatic‐catabolic pathways are natural choices for chassis strains, and Pseudomonas putida KT2440 has emerged as a viable whole‐cell biocatalyst for funnelling lignin‐derived compounds to value‐added products, including its native carbon storage product, medium‐chain‐length polyhydroxyalkanoates (mcl‐PHA). In this work, a series of metabolic engineering targets to improve mcl‐PHA production are combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene phaZ was knocked out, and the genes involved in β‐oxidation (fadBA1 and fadBA2) were deleted. Additionally, to increase carbon flux into mcl‐PHA biosynthesis, phaG, alkK, phaC1 and phaC2 were overexpressed. The best performing strain – which contains all the genetic modifications detailed above – demonstrated a 53% and 200% increase in mcl‐PHA titre (g l−1) and a 20% and 100% increase in yield (g mcl‐PHA per g cell dry weight) from p‐coumaric acid and lignin, respectively, compared with the wild type strain. Overall, these results present a promising strain to be employed in further process development for enhancing mcl‐PHA production from aromatic compounds and lignin. In this work, a series of metabolic engineering targets to improve mcl‐PHA production were combined in the P. putida chromosome and evaluated in strains growing in a model aromatic compound, p‐coumaric acid, and in lignin streams. Specifically, the PHA depolymerase gene (phaZ) and genes involved in β‐oxidation (fadBA1 and fadBA2) were deleted, while phaG, alkK, phaC1, and phaC2 were overexpressed to increase carbon flux from fatty acid biosynthesis to mcl‐PHA production. This strain demonstrated an increase in mcl‐PHA titer (g l−1) and yield (g mcl‐PHA per g cell dry weight) from p‐coumaric acid and from lignin compared to the wild‐type strain, resulting in a promising strain to be employed in further process development for enhancing mcl‐PHA production from aromatic compounds and lignin.

Abstract Background The herpes zoster subunit vaccine (HZ/su), consisting of varicella-zoster virus glycoprotein E (gE) and AS01B Adjuvant System, was highly efficacious in preventing herpes zoster in the ZOE-50 and ZOE-70 trials. We present immunogenicity results from those trials. Methods Participants (ZOE-50: ≥50; ZOE-70: ≥70 years of age) received 2 doses of HZ/su or placebo, 2 months apart. Serum anti-gE antibodies and CD4 T cells expressing ≥2 of 4 activation markers assessed (CD42+) after stimulation with gE-peptides were measured in subcohorts for humoral (n = 3293) and cell-mediated (n = 466) immunogenicity. Results After vaccination, 97.8% of HZ/su and 2.0% of placebo recipients showed a humoral response. Geometric mean anti-gE antibody concentrations increased 39.1-fold and 8.3-fold over baseline in HZ/su recipients at 1 and 36 months post-dose 2, respectively. A gE-specific CD42+ T-cell response was shown in 93.3% of HZ/su and 0% of placebo recipients. Median CD42+ T-cell frequencies increased 24.6-fold (1 month) and 7.9-fold (36 months) over baseline in HZ/su recipients and remained ≥5.6-fold above baseline in all age groups at 36 months. The proportion of CD4 T cells expressing all 4 activation markers increased over time in all age groups. Conclusions Most HZ/su recipients developed robust immune responses persisting for 3 years following vaccination. Clinical Trials Registration NCT01165177; NCT01165229. The herpes zoster subunit vaccine, consisting of varicella-zoster virus glycoprotein E and the AS01B Adjuvant System, stimulated specific antibody and CD4 T-cell responses in >90% of recipients which, in most, persisted for the 36-month duration of the study.

ObjectiveTo explore patterns in flavoured e-cigarette sales following Juul Labs’ 2019 removal of mint-flavoured products and the Food and Drug Administration’s (FDA) 2020 e-cigarette flavour guidance which prohibits flavoured cartridge-based sales, but allows for the sale of tobacco-flavoured and menthol-flavoured cartridges, open-system and disposable e-cigarettes.MethodsWe examined Nielsen Retail Scanner data from September 2013 to March 2020. Inflation-adjusted sales dollars for e-liquid-containing products were classified into five flavour categories (fruit, menthol, mint, tobacco and other).ResultsFollowing the Juul Labs 2019 and FDA 2020 actions, total e-cigarette sales declined; however, menthol-flavoured e-cigarette sales dollars increased, while mint-flavoured e-cigarette sales dollars decreased in both instances. Juul Labs’ removal of mint-flavoured products was followed by a 59.4% increase in the market share of menthol-flavoured e-cigarettes over 4 weeks. The FDA’s 2020 guidance was followed by a 54.5% increase in the market share of menthol-flavoured e-cigarettes over 4 weeks and a 82.8% increase over 8 weeks.ConclusionsJuul Labs’ self-regulation and the current FDA flavour guidance were followed by a shift towards menthol-flavoured e-cigarettes. Industry self-regulation and current federal guidance appear insufficient in reversing the youth vaping epidemic. E-cigarettes must be fully regulated as a tobacco product including the removal of flavoured e-cigarettes, including menthol, from the market to reduce youth e-cigarette use.

BackgroundJUUL is an electronic cigarette that aerosolises a nicotine-containing liquid, while IQOS heats tobacco to produce an aerosol. Both are marketed to smokers, but their effects have seldom been examined in this population.MethodsEighteen cigarette smokers (13 men) with no JUUL or IQOS experience completed a within-subject, laboratory study assessing nicotine delivery and subjective effects after controlled (10 puffs, ~30 s interpuff interval) and ad libitum (90 min) use of JUUL, IQOS or own-brand (OB) cigarettes.ResultsJUUL increased mean plasma nicotine concentration significantly from 2.2 (SD=0.7) ng/mL to 9.8 (4.9) ng/mL after 10 puffs and to 11.5 (9.3) ng/mL after ad libitum use. IQOS increased mean plasma nicotine significantly from 2.1 (0.2) ng/mL to 12.7 (6.2) ng/mL after 10 puffs and to 11.3 (8.0) ng/mL after ad libitum use. OB increased mean plasma nicotine significantly from 2.1 (0.2) ng/mL to 20.4 (11.4) ng/mL after 10 puffs and to 21.0 (10.2) ng/mL after ad libitum use. Mean OB plasma nicotine concentration was significantly higher than JUUL and IQOS. OB increased expired carbon monoxide concentration, but IQOS and JUUL did not. ‘Craving a cigarette/nicotine’ and ‘Urges to smoke’ were reduced significantly for all products following the directed bout.ConclusionsAmong smokers, JUUL and IQOS delivered less nicotine than cigarettes. Also, in this sample, IQOS and OB reduced abstinence symptoms more effectively than JUUL. Additional work with experienced JUUL and IQOS users is needed, as their nicotine delivery profiles and subjective experiences may differ.

Summary Hydrolases acting on polyesters like cutin, polycaprolactone or polyethylene terephthalate (PET) are of interest for several biotechnological applications like waste treatment, biocatalysis and sustainable polymer modifications. Recent studies suggest that a large variety of such enzymes are still to be identified and explored in a variety of microorganisms, including bacteria of the genus Pseudomonas. For activity‐based screening, methods have been established using agar plates which contain nanoparticles of polycaprolactone or PET prepared by solvent precipitation and evaporation. In this protocol article, we describe a straightforward agar plate‐based method using emulsifiable artificial polyesters as substrates, namely Impranil® DLN and liquid polycaprolactone diol (PLD). Thereby, the currently quite narrow set of screening substrates is expanded. We also suggest optional pre‐screening with short‐chain and middle‐chain‐length triglycerides as substrates to identify enzymes with lipolytic activity to be further tested for polyesterase activity. We applied these assays to experimentally demonstrate polyesterase activity in bacteria from the P. pertucinogena lineage originating from contaminated soils and diverse marine habitats. Hydrolases acting on polyesters like cutin, polycaprolactone or polyethylene terephthalate (PET) are of interest for several biotechnological applications like waste treatment, biocatalysis, and sustainable polymer modifications. In this protocol article, we describe a straightforward agar plate based method using emulsifiable artificial polyesters as substrates. We applied these assays to experimentally demonstrate polyesterase activity in bacteria from the Pseudomonas pertucinogena lineage originating from contaminated soils and diverse marine habitats.

Objectives This study aimed to compare the longitudinal change of the psychological distress of healthcare workers (HCWs) with non‐HCWs during the repeated outbreaks of the COVID‐19 in Japan. Methods The data were retrieved from the Employee Cohort Study in the Covid‐19 pandemic in Japan study. An online survey was conducted on March 2020 (T1), on May 2020 (T2), on August 2020 (T3), and on November 2020 (T4). Psychological distress was measured by the Brief Job Stress Questionnaire. A mixed‐model repeated‐measures ANOVA was conducted as an indicator of the group differences. Results A total sample of analysis was n = 996 (HCWs, n = 111; non‐HCWs, n = 885). HCWs consisted of physicians/nurses/midwives and other HCWs (eg, pharmacists, clinical laboratory technicians) in the clinical settings (n = 19; 17% and n = 61; 55%, respectively), and HCWs not working in the clinical settings (n = 31; 28%). Being HCWs were associated with a significant increase in psychological distress from T1 to T2, T3 and T4 (P = .001, P = .002, P < .001; respectively). Conclusions The mental health of HCWs deteriorated through the COVID‐19 outbreaks compared with non‐HCWs. HCWs are continuously the important targets to provide mental health support.