A new member of the flavodoxin superfamily from Fusobacterium nucleatum that functions in heme trafficking and reduction of anaerobilin.

Fusobacterium nucleatum is an opportunistic oral pathogen that is associated with various cancers. To fulfill its essential need for iron, this anaerobe will express heme uptake machinery encoded at a single genetic locus. The heme uptake operon includes HmuW, a class C radical SAM-dependent methyltransferase that degrades heme anaerobically to release Fe2+ and a linear tetrapyrrole called anaerobilin. The last gene in the operon, hmuF encodes a member of the flavodoxin superfamily of proteins. We discovered that HmuF and a paralog, FldH, bind tightly to both FMN and heme. The structure of Fe3+-heme-bound FldH (1.6 Å resolution) reveals a helical cap domain appended to the ⍺/ß core of the flavodoxin fold. The cap creates a hydrophobic binding cleft that positions the heme planar to the si-face of the FMN isoalloxazine ring. The ferric heme iron is hexacoordinated to His134 and a solvent molecule. In contrast to flavodoxins, FldH and HmuF do not stabilize the FMN semiquinone but instead cycle between the FMN oxidized and hydroquinone states. We show that heme-loaded HmuF and heme-loaded FldH traffic heme to HmuW for degradation of the protoporphyrin ring. Both FldH and HmuF then catalyze multiple reductions of anaerobilin through hydride transfer from the FMN hydroquinone. The latter activity eliminates the aromaticity of anaerobilin and the electrophilic methylene group that was installed through HmuW turnover. Hence, HmuF provides a protected path for anaerobic heme catabolism, offering F. nucleatum a competitive advantage in the colonization of anoxic sites of the human body.

The Effects of Changes in Cigarette Menthol Content on Acute Nicotine Pharmacology and Smoking Topography.

INTRODUCTION: Menthol influences the appeal and addictiveness of cigarette smoking, however the data regarding menthol's effects on nicotine pharmacokinetics (PK) and smoking topography are inconsistent. This study investigated the impact of different cigarette menthol levels on nicotine pharmacology and smoking topography in current menthol smokers. AIMS AND METHODS: The study was a double-blind, randomized, four-period, crossover study to investigate the effects of smoking cigarettes with varying menthol content (0, 3, 6, and 12 mg menthol) on nicotine PK, smoking topography, and subjective effects in current menthol smokers. Each experimental session consisted of a prescribed use session, followed by 145 min of no smoking and a 1-h ad libitum smoking session. Serial blood samples were collected; smoking topography was recorded using CReSS Lab topography device. RESULTS: There was no significant effect of menthol on nicotine PK after prescribed smoking of cigarettes with varying menthol contents. During ad libitum smoking, there was significantly smaller total puff volume and puff duration in the 12 mg menthol condition compared to other menthol conditions. Subjective and sensory measures indicated significantly higher overall positive ratings for the 3 mg and 6 mg menthol cigarettes compared to the 0 mg menthol cigarette; the 12 mg menthol cigarette was less liked and harsher than the 3 mg condition. CONCLUSIONS: These findings suggest that menthol, at concentrations reflecting the marketplace (3-6 mg), contributes to positive subjective smoking experiences among menthol smokers, but does not have a significant effect on nicotine PK or smoking topography in an acute laboratory setting. IMPLICATIONS: While our data indicate that varying menthol content does not have a significant impact on nicotine's pharmacological effects under acute exposure conditions, these data highlight the contribution of menthol's flavor and sensory effects to product preference and positive smoking experiences, which facilitate repeated experimentation, progression to regular use, and subsequent dependence.

Genes That Escape X Chromosome Inactivation Modulate Sex Differences in Valve Myofibroblasts.

BACKGROUND: Aortic valve stenosis is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain underexplored. METHODS: Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA sequencing was used to define pathways involved in driving sex-dependent activation. Interventions with small molecule inhibitors and siRNA transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors. RESULTS: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation through Rho-associated protein kinase signaling. CONCLUSIONS: Together, in vivo and in vitro results confirm sex dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.

Hydrogel cultures reveal Transient Receptor Potential Vanilloid 4 regulation of myofibroblast activation and proliferation in valvular interstitial cells.

As aortic valve stenosis develops, valve tissue becomes stiffer. In response to this change in environmental mechanical stiffness, valvular interstitial cells (VICs) activate into myofibroblasts. We aimed to investigate the role of mechanosensitive calcium channel Transient Receptor Potential Vanilloid type 4 (TRPV4) in stiffness induced myofibroblast activation. We verified TRPV4 functionality in VICs using live calcium imaging during application of small molecule modulators of TRPV4 activity. We designed hydrogel biomaterials that mimic mechanical features of healthy or diseased valve tissue microenvironments, respectively, to investigate the role of TRPV4 in myofibroblast activation and proliferation. Our results show that TRPV4 regulates VIC proliferation in a microenvironment stiffness-independent manner. While there was a trend toward inhibiting myofibroblast activation on soft microenvironments during TRPV4 inhibition, we observed near complete deactivation of myofibroblasts on stiff microenvironments. We further identified Yes-activated protein (YAP) as a downstream target for TRPV4 activity on stiff microenvironments. Mechanosensitive TRPV4 channels regulate VIC myofibroblast activation, whereas proliferation regulation is independent of the microenvironmental stiffness. Collectively, the data suggests differential regulation of stiffness-induced proliferation and myofibroblast activation. Our data further suggest a regulatory role for TRPV4 regarding YAP nuclear localization. TRPV4 is an important regulator for VIC myofibroblast activation, which is linked to the initiation of valve fibrosis. Although more validation studies are necessary, we suggest TRPV4 as a promising pharmaceutical target to slow aortic valve stenosis progression.

A Method to Amend Cigarettes With Menthol for Use in Research.

INTRODUCTION: Studies have evaluated the role of menthol cigarettes on various addiction-related outcomes; however, the effect of varying menthol content on these outcomes has not been evaluated. We developed a method to amend non-menthol SPECTRUM Research Cigarettes to contain menthol at four different levels. AIMS AND METHODS: SPECTRUM Research Cigarettes, NRC 600 (0.8 mg nicotine; 10 mg tar), were modified to contain target menthol amounts at 3, 6, and 12 mg/cigarette by injecting 25 µL ethanol/triacetin/menthol solutions of varying concentrations (120 mg menthol/mL, 240 mg/mL, and 480 mg/mL) into four distinct locations in the filter and tobacco rod. Menthol content was tested in triplicate in the whole cigarette and in the tobacco rod and filter at 1, 24, 48, and 72 hours for each target menthol level using an extraction solution of quinoline in methyl-tert-butyl ether and measured using gas chromatography with flame ionization detection. RESULTS: Injections into the filter and tobacco rod (12.5 µL each) yielded equal menthol distribution up to 72 hours. However, total menthol content decreased from an average of 90.3% of the target menthol concentration at 1 hour to 80.7% at 72 hours in cigarettes stored individually in glass tubes at room temperature. Analysis of urinary menthol glucuronide confirmed that amended cigarettes used within 24 hours of injection delivered dose-related menthol levels to participants in a clinical laboratory setting. CONCLUSION: This method can be used to modify cigarettes with a range of reliable menthol levels in both filter and tobacco rod for use in laboratory and clinical research. IMPLICATIONS: This study presents a technique for modifying cigarettes with different levels of menthol that can reliably deliver dose-related menthol levels to participants when smoked in a clinical study. The technique can be used to quickly amend cigarettes to examine the independent effects of varying flavor and additive levels on smoking behavior, nicotine pharmacokinetics, mainstream smoke emissions, and other laboratory or clinical research outcomes.

National longitudinal tobacco product discontinuation rates among US youth from the PATH Study: 2013-2019 (waves 1-5).

OBJECTIVE: Determine longitudinal tobacco product discontinuation rates among youth (ages 12-17 years) in the USA between 2013 and 2019. METHODS: The Population Assessment of Tobacco and Health Study, a nationally representative, longitudinal cohort study, was used to determine annual/biennial rates of tobacco product discontinuation behaviours among youth across 2013-2019: (1) discontinuing product use (transition from past 30-day use to no past 30-day use), (2) attempting to quit product use and (3) discontinuing product use among those who attempted to quit. Discontinuing use was evaluated separately for cigarettes, electronic nicotine delivery systems (ENDS), cigars, hookah, smokeless tobacco and any tobacco. Attempting to quit and discontinuing use among those who attempted were each evaluated for cigarettes and ENDS. Generalised estimating equations were used to evaluate linear and non-linear trends in rates across the study period. RESULTS: Between 2013 and 2019, biennial rates of discontinuing tobacco product use among youth increased for cigarettes from 29% to 40%, increased for smokeless tobacco from 39% to 60%, and decreased for ENDS from 53% to 27%. By 2018/2019, rates of discontinuing use among attempters were 30% for those who used ENDS and 30% for those who smoked cigarettes. CONCLUSIONS: Findings show decreasing rates of discontinuing ENDS use among youth in the USA alongside the changing ENDS marketplace and increasing rates of discontinuing cigarette smoking and smokeless tobacco use. Findings will serve as benchmarks against which future tobacco product discontinuation rates can be compared with evaluating impacts of subsequent tobacco regulatory policies, ENDS product development and public education campaigns.

Tumor necrosis factor-α promotes and exacerbates calcification in heart valve myofibroblast populations.

Pro-inflammatory cytokines play critical roles in regulating valvular interstitial cell (VIC) phenotypic changes that can cause heart valve fibrosis and calcification. Tumor necrosis factor alpha (TNF-α) is a cytokine known to influence VIC behavior and has been reported at high levels in calcified valves ex vivo. We sought to understand the specific effects of TNF-α on VIC phenotypes (eg, fibroblast, profibrotic activated myofibroblasts) and its link with heart valve disorders. We characterize human aortic valve tissue from patients with valve disorders and identify a high variability of fibrotic and calcific markers between tissues. These results motivated in vitro studies to explore the effects of TNF-α on defined VIC fibroblasts and profibrotic activated myofibroblasts, induced via FGF-2 and TGF-ß1 treatment. Using 3D hydrogels to culture VICs, we measure the effect of TNF-α (0.1-10 ng/mL) on key markers of fibrosis (eg, αSMA, COL1A1) and calcification (eg, RUNX2, BMP2, and calcium deposits). We observe calcification in TNF-α-treated VIC activated myofibroblasts and identify the MAPK/ERK signaling cascade as a potential pathway for TNF-α mediated calcification. Conversely, VIC fibroblasts respond to TNF-α with decreased calcification. Treatment of VIC profibrotic activated myofibroblast populations with TNF-α leads to increased calcification. Our in vitro findings correlate with findings in diseased human valves and highlight the importance of understanding the effect of cytokines and signaling pathways on specific VIC phenotypes. Finally, we reveal MAPK/ERK as a potential pathway involved in VIC-mediated matrix calcification with TNF-α treatment, suggesting this pathway as a potential pharmaceutical target for aortic valve disease.

Validation of the Wave 1 and Wave 2 Population Assessment of Tobacco and Health (PATH) Study Indicators of Tobacco Dependence Using Biomarkers of Nicotine Exposure Across Tobacco Products.

INTRODUCTION: This study examined the predictive relationships between biomarkers of nicotine exposure and 16-item self-reported level of tobacco dependence (TD) and subsequent tobacco use outcomes. AIMS AND METHODS: The Population Assessment of Tobacco and Health (PATH) Study surveyed adult current established tobacco users who provided urine biospecimens at Wave 1 (September 2013-December 2014) and completed the Wave 2 (October 2014-October 2015) interview (n = 6872). Mutually exclusive user groups at Wave 1 included: Cigarette Only, E-cigarette Only, Cigar Only, Hookah Only, Smokeless Tobacco Only, Cigarette Plus E-cigarette, multiple tobacco product users who smoked cigarettes, and multiple tobacco product users who did not smoke cigarettes. Total Nicotine Equivalents (TNE-2) and TD were measured at Wave 1. Approximate one-year outcomes included frequency/quantity used, quitting, and adding/switching to different tobacco products. RESULTS: For Cigarette Only smokers and multiple tobacco product users who smoked cigarettes, higher TD and TNE-2 were associated with: a tendency to smoke more, smoking more frequently over time, decreased likelihood of switching away from cigarettes, and decreased probability of quitting after one year. For other product user groups, Wave 1 TD and/or TNE-2 were less consistently related to changes in quantity and frequency of product use, or for adding or switching products, but higher TNE-2 was more consistently predictive of decreased probability of quitting. CONCLUSIONS: Self-reported TD and nicotine exposure assess common and independent aspects of dependence in relation to tobacco use behaviors for cigarette smokers. For other product user groups, nicotine exposure is a more consistent predictor of quitting than self-reported TD. IMPLICATIONS: This study suggests that smoking cigarettes leads to the most coherent pattern of associations consistent with a syndrome of TD. Because cigarettes continue to be prevalent and harmful, efforts to decrease their use may be accelerated via conventional means (eg, smoking cessation interventions and treatments), but also perhaps by decreasing their dependence potential. The implications for noncombustible tobacco products are less clear as the stability of tobacco use patterns that include products such as e-cigarettes continue to evolve. TD, nicotine exposure measures, and consumption could be used in studies that attempt to understand and predict product-specific tobacco use behavioral outcomes.

National longitudinal tobacco product cessation rates among US adults from the PATH Study: 2013-2019 (waves 1-5).

OBJECTIVE: To report on longitudinal tobacco product cessation rates, by product type, among adults (ages 18+ years) in the USA between 2013 and 2019. METHODS: The Population Assessment of Tobacco and Health Study, a nationally representative, longitudinal cohort study was used to report on annual and biennial rates of the following three cessation behaviours across 2013-2019: (1) discontinuing tobacco product use (ie, transition from past 30-day use to no past 30-day use), (2) attempting to quit tobacco product use and (3) quitting tobacco product use among those who attempted to quit. Each cessation behaviour was evaluated separately for cigarettes, electronic nicotine delivery systems (ENDS), cigars, hookah and smokeless tobacco. Generalised estimating equations were used to evaluate linear and nonlinear trends in cessation rates across the study period. RESULTS: Between 2013 and 2019, rates of discontinuing cigarette smoking among adults in the USA statistically increased from 16% to 18%, though these were consistently lower than rates of discontinuing use of other tobacco products. Similarly, quit attempt rates and rates of quitting among attempters increased for cigarette smokers. However, rates of discontinuing ENDS use sharply declined across the study period, from 62% to 44%. CONCLUSIONS: Findings show that tobacco product cessation rates have been changing in recent years in the USA alongside the changing tobacco product marketplace and regulatory environment, though rates of discontinuing cigarette smoking remain relatively low. Findings can serve as a benchmark against which future cessation rates can be compared with evaluate the impacts of future tobacco regulatory policies.

Investigation of SARS-CoV-2 infection and associated lesions in exotic and companion animals.

Documented natural infections with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in exotic and companion animals following human exposures are uncommon. Those documented in animals are typically mild and self-limiting, and infected animals have only infrequently died or been euthanized. Through a coordinated One Health initiative, necropsies were conducted on 5 animals from different premises that were exposed to humans with laboratory-confirmed SARS-CoV-2 infection. The combination of epidemiologic evidence of exposure and confirmatory real-time reverse transcriptase-polymerase chain reaction testing confirmed infection in 3 cats and a tiger. A dog was a suspect case based on epidemiologic evidence of exposure but tested negative for SARS-CoV-2. Four animals had respiratory clinical signs that developed 2 to 12 days after exposure. The dog had bronchointerstitial pneumonia and the tiger had bronchopneumonia; both had syncytial-like cells with no detection of SARS-CoV-2. Individual findings in the 3 cats included metastatic mammary carcinoma, congenital renal disease, and myocardial disease. Based on the necropsy findings and a standardized algorithm, SARS-CoV-2 infection was not considered the cause of death in any of the cases. Continued surveillance and necropsy examination of animals with fatal outcomes will further our understanding of natural SARS-CoV-2 infection in animals and the potential role of the virus in development of lesions.

Can Increased Locomotor Task Difficulty Differentiate Knee Muscle Forces After Anterior Cruciate Ligament Reconstruction?

Changes in knee mechanics following anterior cruciate ligament (ACL) reconstruction are known to be magnified during more difficult locomotor tasks, such as when descending stairs. However, it is unclear if increased task difficulty could distinguish differences in forces generated by the muscles surrounding the knee. This study examined how knee muscle forces differ between individuals with ACL reconstruction with different graft types (hamstring tendon and patellar tendon autograft) and "healthy" controls when performing tasks with increasing difficulty. Dynamic simulations were used to identify knee muscle forces in 15 participants when walking overground and descending stairs. The analysis was restricted to the stance phase (foot contact through toe-off), yielding 162 separate simulations of locomotion in increasing difficulty: overground walking, step-to-floor stair descent, and step-to-step stair descent. Results indicated that knee muscle forces were significantly reduced after ACL reconstruction, and stair descent tasks better discriminated changes in the quadriceps and gastrocnemii muscle forces in the reconstructed knees. Changes in quadriceps forces after a patellar tendon graft and changes in gastrocnemii forces after a hamstring tendon graft were only revealed during stair descent. These results emphasize the importance of incorporating sufficiently difficult tasks to detect residual deficits in muscle forces after ACL reconstruction.

Matters of the heart: Cellular sex differences.

Nearly all cardiovascular diseases show sexual dimorphisms in prevalence, presentation, and outcomes. Until recently, most clinical trials were carried out in males, and many animal studies either failed to identify the sex of the animals or combined data obtained from males and females. Cellular sex in the heart is relatively understudied and many studies fail to report the sex of the cells used for in vitro experiments. Moreover, in the small number of studies in which sex is reported, most of those studies use male cells. The observation that cells from males and females are inherently different is becoming increasingly clear - either due to acquired differences from hormones and other factors or due to intrinsic differences in genotype (XX or XY). Because of the likely contribution of cellular sex differences in cardiac health and disease, here, we explore differences in mammalian male and female cells in the heart, including the less-studied non-myocyte cell populations. We discuss how the heart's microenvironment impacts male and female cellular phenotypes and vice versa, including how secretory profiles are dependent on cellular sex, and how hormones contribute to sexually dimorphic phenotypes and cellular functions. Intracellular mechanisms that contribute to sex differences, including gene expression and epigenetic remodeling, are also described. Recent single-cell sequencing studies have revealed unexpected sex differences in the composition of cell types in the heart which we discuss. Finally, future recommendations for considering cellular sex differences in the design of bioengineered in vitro disease models of the heart are provided.

Secreted Factors From Proinflammatory Macrophages Promote an Osteoblast-Like Phenotype in Valvular Interstitial Cells.

OBJECTIVE: Resident valvular interstitial cells (VICs) activate to myofibroblasts during aortic valve stenosis progression, which further promotes fibrosis or even differentiate into osteoblast-like cells that can lead to calcification of valve tissue. Inflammation is a hallmark of aortic valve stenosis, so we aimed to determine proinflammatory cytokines secreted from M1 macrophages that give rise to a transient VIC phenotype that leads to calcification of valve tissue. Approach and Results: We designed hydrogel biomaterials as valve extracellular matrix mimics enabling the culture of VICs in either their quiescent fibroblast or activated myofibroblast phenotype in response to the local matrix stiffness. When VIC fibroblasts and myofibroblasts were treated with conditioned media from THP-1-derived M1 macrophages, we observed robust reduction of αSMA (alpha smooth muscle actin) expression, reduced stress fiber formation, and increased proliferation, suggesting a potent antifibrotic effect. We further identified TNF (tumor necrosis factor)-α and IL (interleukin)-1ß as 2 cytokines in M1 media that cause the observed antifibrotic effect. After 7 days of culture in M1 conditioned media, VICs began differentiating into osteoblast-like cells, as measured by increased expression of RUNX2 (runt-related transcription factor 2) and osteopontin. We also identified and validated IL-6 as a critical mediator of the observed pro-osteogenic effect. CONCLUSIONS: Proinflammatory cytokines in M1 conditioned media inhibit myofibroblast activation in VICs (eg, TNF-α and IL-1ß) and promote their osteogenic differentiation (eg, IL-6). Together, our work suggests inflammatory M1 macrophages may drive a myofibroblast-to-osteogenic intermediate VIC phenotype, which may mediate the switch from fibrosis to calcification during aortic valve stenosis progression.

Nicotine Content and Physical Properties of Large Cigars and Cigarillos in the United States.

Introduction: Cigars are combusted tobacco products consisting of filler, binder, and wrapper, which are derived from tobacco. Despite the abundance of literature on the composition of traditional combusted cigarettes, research is limited on the physical and chemical properties of cigars. Therefore, research on cigar properties may be useful to better understand their health impact. Methods: In this study, twenty large cigar and cigarillo products were characterized for physical properties (ie, weight, length, and diameter), filler nicotine content, and tobacco pH. Tobacco pH was used to calculate free nicotine content, free nicotine concentration, and percent free nicotine for all cigars using the Henderson-Hasselbach equation. An additional analysis was performed on a second batch of two large cigar and two cigarillo brands to determine within-brand consistency. All analyses were performed in triplicate. Results: The initial analysis of the twenty cigars showed that cigars exhibited wide variation in product size and nicotine content, although tobacco pH was similar across cigars. Furthermore, in the two large cigar and cigarillo brands analyzed a second time, there was considerable within-brand variance in nicotine content and concentration between the first and second analyses. Conclusions: While only a small sample of commercially-available cigars was analyzed, our data suggest there is wide variability in nicotine content and some physical properties in the domestic cigar market. The data may help to inform potential future regulatory decisions related to these products. Implications: This study reveals some of the challenges to experimental cigar research and illustrates the need to characterize cigar products (eg, nicotine and tobacco content) before use in clinical studies. Additional studies and characterization of the physical and chemical properties of cigars may be useful to further understand these products' toxicity, abuse potential, and public health impact.

Dependence symptoms and cessation intentions among US adult daily cigarette, cigar, and e-cigarette users, 2012-2013.

BACKGROUND: Cigar and e-cigarette use is becoming increasingly common among US tobacco users and the Food and Drug Administration recently asserted regulatory jurisdiction over these products, among others, in May 2016. Research on tobacco dependence among users of these products is limited, however. We therefore examined several symptoms of dependence and cessation intentions among adult cigarette, cigar, and/or e-cigarette users in a nationally representative sample. METHODS: We used nationally representative data from more than 60,000 participants in the US National Adult Tobacco Survey (NATS) from 2012 to 2013 to analyze dependence symptoms and cessation intentions for users of cigarettes, cigars, and/or e-cigarettes but not other tobacco products. RESULTS: Among daily tobacco users, dual cigarette and cigar users on average smoked more cigarettes per day (17.3, 95 % CI = 16.1, 18.6 vs. 15.8, 95 % CI = 15.4, 16.2), had shorter times to first tobacco use after waking (21.4 min, 95 % CI = 16.6, 24.9 vs. 25.9 min, 95 % CI = 25.3, 26.5), and were more likely to report withdrawal and craving symptoms than exclusive cigarette smokers. Dual cigarette and e-cigarette users were more likely than exclusive cigarette smokers to report withdrawal and craving symptoms and cessation intentions. Exclusive cigar and e-cigarette users were less likely to report withdrawal and craving symptoms than users of other products, but even so, more than a third of exclusive cigar (38.8 %, 95 % CI = 27.4 %, 51.6 %) and e-cigarette (46.1 %, 95 % CI = 35.1 %, 57.4 %) users reported experiencing a strong craving for a tobacco product in the past 30 days. CONCLUSIONS: Dual cigarette and cigar users show evidence of greater dependence symptoms and dual cigarette and e-cigarette users show evidence of greater dependence symptoms and cessation intentions compared with exclusive cigarette smokers. A sizeable number of users of all of the tobacco products report dependence symptoms such as craving for tobacco.

Electronic cigarettes and nicotine clinical pharmacology.

OBJECTIVE: To review the available literature evaluating electronic cigarette (e-cigarette) nicotine clinical pharmacology in order to understand the potential impact of e-cigarettes on individual users, nicotine dependence and public health. METHODS: Literature searches were conducted between 1 October 2012 and 30 September 2013 using key terms in five electronic databases. Studies were included in the review if they were in English and publicly available; non-clinical studies, conference abstracts and studies exclusively measuring nicotine content in e-cigarette cartridges were excluded from the review. RESULTS: Nicotine yields from automated smoking machines suggest that e-cigarettes deliver less nicotine per puff than traditional cigarettes, and clinical studies indicate that e-cigarettes deliver only modest nicotine concentrations to the inexperienced e-cigarette user. However, current e-cigarette smokers are able to achieve systemic nicotine and/or cotinine concentrations similar to those produced from traditional cigarettes. Therefore, user experience is critically important for nicotine exposure, and may contribute to the products' ability to support and maintain nicotine dependence. CONCLUSIONS: Knowledge about e-cigarette nicotine pharmacology remains limited. Because a user's e-cigarette experience may significantly impact nicotine delivery, future nicotine pharmacokinetic and pharmacodynamic studies should be conducted in experienced users to accurately assess the products' impact on public health.

Mutational analysis of the rotavirus NSP4 enterotoxic domain that binds to caveolin-1.

BACKGROUND: Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. Our previous data show a direct interaction between RV NSP4 and the structural protein of caveolae, caveolin-1 (cav-1), in yeast and mammalian cells. The binding site of cav-1 mapped to the NSP4 amphipathic helix, and led us to examine which helical face was responsible for the interaction. METHODS: A panel of NSP4 mutants were prepared and tested for binding to cav-1 by yeast two hybrid and direct binding assays. The charged residues of the NSP4 amphipathic helix were changed to alanine (NSP446-175-ala6); and three residues in the hydrophobic face were altered to charged amino acids (NSP4(46-175)-HydroMut). In total, twelve mutants of NSP4 were generated to define the cav-1 binding site. Synthetic peptides corresponding to the hydrophobic and charged faces of NSP4 were examined for structural changes by circular dichroism (CD) and diarrhea induction by a neonatal mouse study. RESULTS: Mutations of the hydrophilic face (NSP4(46-175)-Ala6) bound cav-1 akin to wild type NSP4. In contrast, disruption of the hydrophobic face (NSP4(46-175)-HydroMut) failed to bind cav-1. These data suggest NSP4 and cav-1 associate via a hydrophobic interaction. Analyses of mutant synthetic peptides in which the hydrophobic residues in the enterotoxic domain of NSP4 were altered suggested a critical hydrophobic residue. Both NSP4HydroMut112-140, that contains three charged amino acids (aa113, 124, 131) changed from the original hydrophobic residues and NSP4AlaAcidic112-140 that contained three alanine residues substituted for negatively charged (aa114, 125, 132) amino acids failed to induce diarrhea. Whereas peptides NSP4wild type 112-140 and NSP4AlaBasic112-140 that contained three alanine substituted for positively charged (aa115, 119, 133) amino acids, induced diarrhea. CONCLUSIONS: These data show that the cav-1 binding domain is within the hydrophobic face of the NSP4 amphipathic helix. The integrity of the helical structure is important for both cav-1 binding and diarrhea induction implying a connection between NSP4 functional and binding activities.

Multifunctional polyampholyte hydrogels with fouling resistance and protein conjugation capacity.

Materials that are resistant to nonspecific protein adsorption are critical in the biomedical community. Specifically, nonfouling implantable biomaterials are necessary to reduce the undesirable, but natural foreign body response. The focus of this investigation is to demonstrate that polyampholyte hydrogels prepared with equimolar quantities of positively charged [2-(acryloyloxy)ethyl] trimethylammonium chloride (TMA) and negatively charged 2-carboxyethyl acrylate (CAA) monomers are a viable solution to this problem. TMA/CAA hydrogels were prepared and their physical and chemical properties were characterized. The fouling resistance of the TMA/CAA hydrogels were assessed at varying cross-linker densities using enzyme-linked immunosorbant assays (ELISAs). The results clearly demonstrate that TMA/CAA hydrogels are resistant to nonspecific protein adsorption. A unique advantage of the fouling resistant TMA/CAA system is that bioactive proteins can be covalently attached to these materials using standard conjugation chemistry. This was demonstrated in this study through a combination of ELISA investigations and short-term cell adhesion assays. The multifunctional properties of the TMA/CAA polyampholyte hydrogels shown in this work clearly demonstrate the potential for these materials for use as tissue regeneration scaffolds for many biomedical applications.

Detection of Tritrichomonas foetus by RT-rtPCR in pooled bovine preputial washings.

Trichomonosis is a venereal disease of cattle caused by the protozoan Tritrichomonas foetus. T. foetus infection in cattle herds can be economically costly for cattle producers; therefore, testing is important for detection of the agent. Given that bulls are considered to be subclinical carriers of T. foetus, it is important to detect T. foetus infection prior to movement and/or breeding season. We have described previously the development of an updated set of PCR primers and probes that offer increased sensitivity of T. foetus detection in preputial washings collected in PBS by utilizing reverse-transcription real-time PCR (RT-rtPCR) that targets the 5.8S ribosomal RNA of the T. foetus organism. Here, we report improvements in the updated RT-rtPCR reagents as well as the evaluation of testing of pooled preputial washings. We found that up to 5 preputial washings can be pooled, similar to routine testing practices (InPouch culture), without reducing the sensitivity of detection of T. foetus.

Osteopontin activity modulates sex-specific calcification in engineered valve tissue mimics.

Patients with aortic valve stenosis (AVS) have sexually dimorphic phenotypes in their valve tissue, where male valvular tissue adopts a calcified phenotype and female tissue becomes more fibrotic. The molecular mechanisms that regulate sex-specific calcification in valvular tissue remain poorly understood. Here, we explored the role of osteopontin (OPN), a pro-fibrotic but anti-calcific bone sialoprotein, in regulating the calcification of female aortic valve tissue. Recognizing that OPN mediates calcification processes, we hypothesized that aortic valvular interstitial cells (VICs) in female tissue have reduced expression of osteogenic markers in the presence of elevated OPN relative to male VICs. Human female valve leaflets displayed reduced and smaller microcalcifications, but increased OPN expression relative to male leaflets. To understand how OPN expression contributes to observed sex dimorphisms in valve tissue, we employed enzymatically degradable hydrogels as a 3D cell culture platform to recapitulate male or female VIC interactions with the extracellular matrix. Using this system, we recapitulated sex differences observed in human tissue, specifically demonstrating that female VICs exposed to calcifying medium have smaller mineral deposits within the hydrogel relative to male VICs. We identified a change in OPN dynamics in female VICs in the presence of calcification stimuli, where OPN deposition localized from the extracellular matrix to perinuclear regions. Additionally, exogenously delivered endothelin-1 to encapsulated VICs increased OPN gene expression in male cells, which resulted in reduced calcification. Collectively, our results suggest that increased OPN in female valve tissue may play a sex-specific role in mitigating mineralization during AVS progression.