Constitutive activation of nitrate reductase in tobacco alters flowering time and plant biomass.

Pyridine alkaloids produced in tobacco can react with nitrosating agents such as nitrite to form tobacco-specific nitrosamines (TSNA), which are among the most notable toxicants present in tobacco smoke. The market type known as burley tobacco is particularly susceptible to TSNA formation because its corresponding cultivars exhibit a nitrogen-use-deficiency phenotype which results in high accumulation of nitrate, which, in turn, is converted to nitrite by leaf surface microbes. We have previously shown that expression of a constitutively activated nitrate reductase (NR) enzyme dramatically decreases leaf nitrate levels in burley tobacco, resulting in substantial TSNA reductions without altering the alkaloid profile. Here, we show that plants expressing a constitutively active NR construct, designated 35S:S523D-NR, display an early-flowering phenotype that is also associated with a substantial reduction in plant biomass. We hypothesized that crossing 35S:S523D-NR tobaccos with burley cultivars that flower later than normal would help mitigate the undesirable early-flowering/reduced-biomass traits while maintaining the desirable low-nitrate/TSNA phenotype. To test this, 35S:S523D-NR plants were crossed with two late-flowering cultivars, NC 775 and NC 645WZ. In both cases, the plant biomass at harvest was restored to levels similar to those in the original cultivar used for transformation while the low-nitrate/TSNA trait was maintained. Interestingly, the mechanism by which yield was restored differed markedly between the two crosses. Biomass restoration in F1 hybrids using NC 645WZ as a parent was associated with delayed flowering, as originally hypothesized. Unexpectedly, however, crosses with NC 775 displayed enhanced biomass despite maintaining the early-flowering trait of the 35S:S523D-NR parent.

OMARC: An online multimedia application for training health care providers in the assessment of respiratory conditions.

OBJECTIVES: OMARC, a multimedia application designed to support the training of health care providers for the identification of common lung sounds heard in a patient's thorax as part of a health assessment, is described and its positive contribution to user learning is assessed. The main goal of OMARC is to effectively help health-care students become familiar with lung sounds as part of the assessment of respiratory conditions. In addition, the application must be easy to use and accessible to students and practitioners over the internet. SYSTEM DESCRIPTION: OMARC was developed using an online platform to facilitate access to users in remote locations. OMARC's unique contribution as an educational software tool is that it presents a narrative about normal and abnormal lung sounds using interactive multimedia and sample case studies designed by professional health-care providers and educators. Its interface consists of two distinct components: a sounds glossary and a rich multimedia interface which presents clinical case studies and provides access to lung sounds placed on a model of a human torso. OMARC's contents can be extended through the addition of sounds and case studies designed by health-care educators and professionals. VALIDATION AND RESULTS: To validate OMARC and determine its efficacy in improving learning and capture user perceptions about it, we performed a pilot study with ten nursing students. Participants' performance was measured through an evaluation of their ability to identify several normal and adventitious/abnormal sounds prior and after exposure to OMARC. Results indicate that participants are able to better identify different lung sounds, going from an average of 63% (S.D. 18.3%) in the pre-test evaluation to an average of 90% (S.D. of 11.5%) after practising with OMARC. Furthermore, participants indicated in a user satisfaction questionnaire that they found the application helpful, easy to use and that they would recommend it to other persons in their field. CONCLUSIONS: OMARC is an online multimedia application for training health care students in the assessment of respiratory conditions. The software integrates multimedia technology and health-care education concepts to facilitate learning, while being useful and easy to use. Results from a pilot study indicate that OMARC significantly helps to improve the capacity of the users to correctly identify lung sounds for different respiratory conditions. In addition, participants' opinions about OMARC were quite positive: users were likely to recommend the application to other persons in their field and found the application easy to use and helpful to better identify lung sounds.

Transgenic and mutation-based suppression of a berberine bridge enzyme-like (BBL) gene family reduces alkaloid content in field-grown tobacco.

Motivation exists to develop tobacco cultivars with reduced nicotine content for the purpose of facilitating compliance with expected tobacco product regulations that could mandate the lowering of nicotine levels per se, or the reduction of carcinogenic alkaloid-derived tobacco specific nitrosamines (TSNAs). A berberine bridge enzyme-like (BBL) gene family was recently characterized for N. tabacum and found to catalyze one of the final steps in pyridine alkaloid synthesis for this species. Because this gene family acts downstream in the nicotine biosynthetic pathway, it may represent an attractive target for genetic strategies with the objective of reducing alkaloid content in field-grown tobacco. In this research, we produced transgenic doubled haploid lines of tobacco cultivar K326 carrying an RNAi construct designed to reduce expression of the BBL gene family. Field-grown transgenic lines carrying functional RNAi constructs exhibited average cured leaf nicotine levels of 0.684%, in comparison to 2.454% for the untransformed control. Since numerous barriers would need to be overcome to commercialize transgenic tobacco cultivars, we subsequently pursued a mutation breeding approach to identify EMS-induced mutations in the three most highly expressed isoforms of the BBL gene family. Field evaluation of individuals possessing different homozygous combinations of truncation mutations in BBLa, BBLb, and BBLc indicated that a range of alkaloid phenotypes could be produced, with the triple homozygous knockout genotype exhibiting greater than a 13-fold reduction in percent total alkaloids. The novel source of genetic variability described here may be useful in future tobacco breeding for varied alkaloid levels.

Structural factors that promote stigmatization of drug users with hepatitis C in hospital emergency departments.

BACKGROUND: Interventions to mediate the stigmatization of people affected with HCV, particularly those who use illicit drugs, have been largely focused on changing health care practitioners' attitudes and knowledge regarding Hepatitis C and illicit drug use and these have had disappointing results. There is a need for research that examines factors beyond individual practitioners that explains why and how stigmatization of the population occurs within health care and informs interventions to mitigate these factors. METHODS: The research was intended to identify structural factors that contribute to the structural stigmatization of people within hospital Emergency Departments who are current users of illicit drugs and are HCV positive. The research had an interpretive description design and occurred in Nova Scotia, Canada. The year-long qualitative study entailed individual interviews of 50 service providers in hospital EDs or community organizations that served this population. RESULTS: The research findings generated a model of structural stigmatization that greatly expands the current understanding of stigmatization beyond individual practitioners' attitudes and knowledge and internal structures to incorporate structures external to hospitals, such as physician shortages within the community and the mandate of EDs to reduce wait times. CONCLUSIONS: The research reported herein has conceptualized stigmatization beyond an individualistic approach to incorporate the multifaceted ways that such stigmatization is fostered and supported by internal and external structures.

Impact of alleles at the Yellow Burley (Yb) loci and nitrogen fertilization rate on nitrogen utilization efficiency and tobacco-specific nitrosamine (TSNA) formation in air-cured tobacco.

Tobacco-specific nitrosamine (TSNA) formation in tobacco is influenced by alkaloid levels and the availability of nitrosating agents. Tobacco types differ in their potential for TSNA accumulation due to genetic, agronomic, and curing factors. Highest TSNA concentrations are typically measured in burley tobaccos. One of the main genetic differences between burley and all other tobacco types is that this tobacco type is homozygous for recessive mutant alleles at the Yellow Burley 1 (Yb(1)) and Yellow Burley 2 (Yb(2)) loci. In addition, burley tobacco is typically fertilized at higher nitrogen (N) rates than most other tobacco types. This study utilized nearly isogenic lines (NILs) differing for the presence of dominant or recessive alleles at the Yb(1) and Yb(2) loci to investigate the potential influence of genes at these loci on TSNA accumulation. Three pairs of NILs were evaluated at three different nitrogen fertilization rates for alkaloid levels, nitrogen physiology measures, and TSNA accumulation after air-curing. As previously observed by others, positive correlations were observed between N application rates and TSNA accumulation. Recessive alleles at Yb(1) and Yb(2) were associated with increased alkaloid levels, reduced nitrogen use efficiency, reduced nitrogen utilization efficiency, and increased leaf nitrate nitrogen (NO(3)-N). Acting together, these factors contributed to significantly greater TSNA levels in genotypes possessing the recessive alleles at these two loci relative to those carrying the dominant alleles. The chlorophyll-deficient phenotype conferred by the recessive yb(1) and yb(2) alleles probably contributes in a substantial way to increase available NO(3)-N during curing and, consequently, increased potential for TSNA formation.