Engineering volatile thiol formation in yeast.

AIMS: Volatile thiols are very potent aroma molecules that contribute to the aroma of many beverages. The characteristic thiols of certain wine varieties such as Sauvignon blanc are partly released during the yeast-based fermentation from plant-synthesized glutathione- or cysteine-conjugated and dipeptic precursors present in the must. In this work, we aimed at the construction and characterization of yeast strains with the ability to synthesize volatile thiols from respective precursors. METHODS AND RESULTS: Besides genome integration of the Escherichia coli gene tnaA, which encodes an enzyme with high ß-lyase activity, a glutathione synthetase and glutathione-S-transferases were overexpressed. Up to 8.9 µg L-1 3-mercaptohexan-1-ol could be formed with the strain from externally added trans-2-hexen-1-ol. Well-characterized thiols such as 2-methyl-2-butanethiol, 3-mercapto-3-methylbutan-1-ol, and 8-mercapto-p-menthan-3-one, as well as several so far undescribed thiol compounds could be synthesized. CONCLUSION: Volatile thiols could be produced by feeding alcohol, alkenol, aldehyde, or ketone precursors like trans-2-hexenal, trans-2-hexen-1-ol, cis-2-hexen-1-ol, 3-methyl-2-buten-1-ol, 3-buten-2-one, and pulegone to the optimized yeast cells.

Investigation of non-Saccharomyces yeasts with intracellular ß-glycosidase activity for wine aroma modification.

Since high proportions of aroma-relevant molecules in plant-derived juices are present in glycosylated forms, the introduction of glycosidase activity during processing is an important tool to modify the aroma composition of the product. During winemaking, the addition of ß-glycosidase enzyme or microorganisms with ß-glycosidase activity is an established technology. However, low stability under acidic conditions and low selectivity for hydrolysis of different glycosides are still drawbacks, which limit application possibilities. Here, we report the identification and characterization of non-Saccharomyces yeast strains with relatively high ß-glycosidase activity in their cultures. We found strong indications for intracellular localization of the enzymes, which is in line with the pH robustness found in experiments with whole cells. Furthermore, we compared the selectivity of aroma compound release from glycoside mixtures using whole cells or cell extracts. The results showed strong differences for the released aroma patterns, which indicates the transport of glycosides and intracellular hydrolysis. Our work demonstrates the application potential of yeasts with intracellular ß-glycosidase activities as catalysts with high pH robustness and selective aroma release properties. PRACTICAL APPLICATION: The yeast strains identified and characterized within this work can be applied in wine processing but also in other processes to release aroma molecules from their glycosylated precursors provided by the plants. The strains show relatively high activity of the relevant enzyme, ß-glycosidase, also at low pH, which is essential in many processes. In contrast to most other approaches, the enzyme is inside the cells, which can lead to a specific release of certain aroma compounds.

Long term betaine supplementation regulates genes involved in lipid and cholesterol metabolism of two muscles from an obese pig breed.

This study evaluates the effects of betaine supplementation (1gkg-1 for 20weeks) on the regulation of genes involved in lipid and cholesterol metabolism of Longissimus lumborum and Biceps femoris from obese Alentejano pigs. Betaine supplementation led to an increase in total cholesterol in both muscles, complementing results previously published indicating a significant increase on the intramuscular lipid content. The expression of twelve genes involved in lipogenesis, lipolysis/FA oxidation, FA transport, and cholesterol metabolism, as well as two transcription factors were also evaluated. Genes related to lipid and cholesterol synthesis plus FA transport were consistently up-regulated in both muscles of betaine fed pigs. On the other hand, genes related to lipolysis/FA oxidation were not affected or down-regulated by betaine supplementation. Our data suggest that the underlying mechanism regulating IMF and cholesterol accumulation in Alentejano pigs supplemented with betaine is associated with the up-regulation of genes involved in lipid synthesis, FA transport, and cholesterol synthesis.

A degenerate pair of primers for simultaneous detection of four alpha- and betanecroviruses.

The high infection levels due to Olive latent virus 1 (OLV-1), Olive mild mosaic virus (OMMV) (alphanecrovirus) and Tobacco necrosis virus D (TNV-D) (betanecrovirus) in Portuguese olive orchards prompted us to develop a rapid PCR-based assay for the simultaneous detection of these viruses aimed at the sanitary selection and marketing of plant material in compliance with European Union regulations. A pair of degenerate oligonucleotide primers, parRdRp5' and parCoat3' was designed based on conserved regions located in the RNA-dependent RNA polymerase (RdRp) and coat protein (CP) genes of these viruses and one other alphanecrovirus, Tobacco necrosis virus A. Its use in RT-PCR assays generated a product of ca. 2000 bp for the 4 viral species tested. These primers were compared with virus specific primers in multiplex RT-PCR, and identical results were obtained. Its application to dsRNA extracted from 54 olive field growing trees originated the expected ca. 2000 bp amplicon in 17 trees. The virus identity was determined by sequencing the cloned RT-PCR products. No TNV-A was found. The RT-PCR assay using the degenerate primers described in this study were shown to be reliable in detecting any of the above-mentioned alpha- and betanecroviruses, and it is as sensitive as that which uses virus specific primers in multiplex assays. Therefore, this assay is well suited for the rapid screen of virus-free plant material in selection and improvement crop programmes. Additionally, it has the potential to reveal virus diversity and the presence of new viruses, provided the RT-PCR generated amplicon is further sequenced.

Genetic diversity of the coat protein of olive latent virus 1 isolates.

The CP gene variability among 21 olive latent virus 1 (OLV-1) isolates obtained from different hosts and locations and at different times was assessed. Amplicons obtained by RT-PCR were cloned, and at least 10 sequences from each isolate were analyzed and compared. OLV-1 sequences available in GenBank were included. The encoded CPs consisted of 270 amino acids, except those of isolates G1S and C7 (269 aa) and G6 (271 aa). Comparison of CP genomic sequences of the isolates under study showed very low values of nucleotide diversity, 0.02, and maximum nucleotide distances between (0.087) or within isolates (0.001). Although very few nucleotide sequence differences were observed among the isolates, olive isolates exhibited lower diversity (0.012). In addition, at position 158 (157 in C7 and G1S and 159 in G6) of the deduced aa sequences, an alanine residue was found to be conserved among the olive isolates. In citrus and tulip isolates, a threonine residue was present at position 158, whereas a valine was present at this same position in tomato isolates. Phylogenetic analysis indicated that OLV-1 isolates clustered in five groups according to original host. However, G6, originally recovered from olive but repeatedly inoculated and maintained in N. benthamiana plants for 8 years in our laboratory, was separated from other isolates. This may be attributable to adaptation to the experimental host over time. There was no correlation of phylogenetic grouping of isolates based on geographical location or year of collection. Strong negative selection may have contributed to the low diversity among the OLV-1 CP isolates.

Viral properties, primary structure and phylogenetic analysis of the coat protein of an Olive latent virus 1 isolate from Olea europaea L.

An Olive latent virus 1 isolate designated GM6, obtained from a Portuguese olive tree, was characterized and the coat protein gene sequenced and analysed. The purified virus particles showed to be isometric with ca. 30 nm in diameter and contained a single-stranded RNA species with ca. 3.7 kb. The dsRNA profile obtained from infected tissues showed three major species with ca. 3.7, 1.5 and 1.3 kbp. SDS-PAGE analysis revealed a major peptide with an apparent molecular mass of 32 kDa identified as the coat protein. A viral genome region containing the coat protein gene was amplified by RT-PCR and the cDNA was cloned and sequenced. The coat protein gene revealed to be 813 nucleotides long and encode a peptide with 270 amino acid residues and an estimated Mr of 29,851. Alignment of the deduced amino acid sequence with that of other necroviruses showed a higher identity with OLV-1 tulip isolate (97.7%) than with OLV-1 citrus isolate (87.7%). The consensus pattern of the coat protein 'S' domain is conserved in GM6 isolate coat protein sequence, except in amino acid 151, leucine. This is the first report on the coat protein sequence of an OLV-1 olive isolate.

The complete genome sequence of a new necrovirus isolated from Olea europaea L.

The complete nucleotide sequence of a virus isolated from Olea europaea L. (GP isolate), previously identified as an isolate of Tobacco necrosis virus D (TNV-D) based on its coat protein sequence, was determined. The viral RNA genome consists of 3683 nucleotides and contains five open reading frames. The putative RNA-dependent RNA polymerase shows 91.2% amino acid identity with that of an isolate of Olive latent virus 1 (OLV-1) and the coat protein reveals highest sequence identity with that of TNV-D. Based on the deduced genome organization and phylogenetic analysis of predicted functional translation products with that of other necroviruses, the GP isolate genome appears to represent an example of a new virus arisen by gene exchange and is proposed to be a new necrovirus, provisionally named Olive mild mosaic virus.

A Tobacco necrosis virus D isolate from Olea europaea L.: viral characterization and coat protein sequence analysis.

A virus isolated from Olea europaea L. grown in Portugal, was identified as a member of the species Tobacco necrosis virus D (TNV-D, genus Necrovirus, family Tombusviridae), based on the molecular and serological properties of the purified virus particles. The genomic region encoding the coat protein (CP) of this isolate (named GP isolate) was amplified by RT-PCR and the cDNA was cloned and sequenced. The CP gene encodes a predicted protein of 269 amino acids showing high identity (86.2%) to TNV-D coat protein sequence. Phylogenetic analysis based on necroviruses CP sequences, confirmed GP as a TNV-D isolate. The alignment with homologous TNV-D CP sequences revealed four conserved amino acids involved in Ca(2+) binding as well as the plant virus icosahedral capsid protein "S' signature. Based on the determined nucleotide sequence, specific primers were designed and successfully used in RT-PCR for virus diagnosis in naturally infected olive trees.

The SF-12 as a population health measure: an exploratory examination of potential for application.

OBJECTIVE: To describe the relationships among functional health status measures (SF-12 physical and mental components summary scores), traditional measures of community health status, and social determinants of health among respondents to community health status surveys conducted in nine different communities. DATA SOURCES/STUDY SETTING: Data collected as part of comprehensive community health status assessments conducted in each of nine communities (in seven states) between 1992 and 1997. The purpose of each assessment was to gather data to plan and evaluate population health improvement initiatives. STUDY DESIGN AND DATA COLLECTION: This is an opportunistic study drawing on the universe of community health survey data collected by the authors to support local health improvement initiatives. Both community-level as well as an aggregate of individual-level measures are used in the analysis. Within each locality, survey respondents were randomly selected using a telephone-facilitated, mailed survey methodology. PRINCIPAL FINDINGS: The key variables reported here are functional health status measures (SF-12) and social determinants of health variables. SF-12 physical and mental component scales correlated with two of four traditional measures of community health status. At the aggregate level of analysis, significant relationships were found for seven of nine social determinants of health measures when compared with SF-12 component summary scores. Relationships between social determinants measures and PCS-12 and MCS-12 scores suggest both application possibilities and the need for additional analysis in order to understand the nature of those relationships. CONCLUSIONS: Physical and mental health functioning summary scores as measured by the SF-12 are useful in describing overall community health status when compared with traditional measures such as total deaths, age-adjusted mortality, or physician to population ratio. The SF-12 can also be used to measure the relationship between physical and mental health functioning (as proxies for community health status) and the social determinants of health. This analysis can help to refine our understanding of how social determinants and health status interact in a community or population as a precursor to the development of models of community or population health.

Taking the pulse of the community.

In recent years, the technology and methodology have been developed to help you identify, collect, analyze, and apply health status information about the community you serve. Called a population-based community health status assessment, this tool is not intended to help you identify "needs"--you probably already know what many of them are in your community. Rather, it allows you to develop precise indicators of the health status of the community in general as well as of specific subgroups with particular health problems and risk factors. The assessment is useful both for the data and insights it provides and for organizing, implementing, and evaluating community health improvement interventions. Seven steps for the successful design and implementation of a population-based community health status assessment follow.

Age and sex differences in health habits and beliefs of schoolchildren.

All children in Grades 3 through 12 of one school system completed a survey about health habits and beliefs including smoking and eating habits, perceptions of exercise, weight, and parental involvement in health. The surveys were factor-analyzed within grade and sex, and the overall factors that emerged were Smoking Habits, Family Discussion of Health, Family Thinking About Health, Nutritional Habits, and Health Locus of Control. Analysis of variance of each factor revealed that girls generally reported healthier food habits than did boys. However, girls reported more smoking and less exercise. There are also changes in habits and belief with age; junior high school is a particularly important time for the development of several habits. The findings are discussed in relation to theories of child development and the implications for the content and timing of future health education intervention programs with children.

Weight loss competitions at the worksite: how they work and how well.

Three studies were conducted to evaluate the effectiveness of competition as a method of weight loss at the worksite. Study 1 found team competitions more effective than either cooperation or individual competition for men, and more effective than individual competition for women. Study 2 replicated the effectiveness of team competitions at 10 worksites. The influence of four variables [gender, age, type of employment (blue-collar vs white-collar), and method of assignment to teams] upon four outcomes (recruitment, attrition, weight loss, and cost/effectiveness), was assessed. This study showed that the results of competition were robust and widely generalizable. Recruitment was high, attrition very low, weight losses large, and cost/effectiveness favorable. Study 3, however, found only limited maintenance of weight loss following competitions.

The role of parents and older peers in school-based cardiovascular prevention programs: implications for program development.

This article describes a set of studies which compare the effectiveness of innovative interventions led by older peers and which included a parent component with teacher-led interventions for nutrition, blood pressure, and smoking prevention. Information about the agreement between parents' and children's (grades six through eight) perception of the children's health behavior and family interaction was also found, by surveying parents and children in 1051 households. Both teacher-led and older peer-led interventions were successful in increasing behavioral capabilities for nutrition and blood pressure, measured one year after the interventions. Results of the parent-child survey showed reasonable agreement between parents and children for reports of the child's exercise, dieting, and fast food consumption, but poorer agreement for smoking and perceptions of family interaction. Results are discussed in relation to the planning of future programs designed to address the importance of peer and parental role models.

Experimentation with smokeless tobacco and cigarettes by children and adolescents: relationship to beliefs, peer use, and parental use.

Experimentation with both cigarettes and smokeless tobacco by children in grades 3-12 in a school district in Pennsylvania was related to peer experimentation, parental use, and personal beliefs about the harm of the product for both males and females. Experimentation with both products had begun as early as the third grade and increased with age, with a major increase in experimentation during junior high school. Nearly half the males in grades 7-12 did not believe smokeless tobacco was harmful.

Comparison of three worksite weight-loss competitions.

Competition is an effective means of promoting weight loss in worksite programs. This study was designed to determine the most effective structure of competition through a comparison of two kinds of competition within one worksite. Two competitions were between teams and one was among individuals. While all three competitions produced significant weight loss and lower attrition than previous worksite programs, the team competitions produced significantly lower attrition rates and significantly greater weight loss than the individual competition. Patterns of loss over time and the distribution of losses for groups also differed, with a smaller proportion of successful participants in the individual competition. The results suggest that team support as well as competition is a necessary component of effective worksite weight loss competitions.