A Rural Community Pauses to Gauge Attitudes on the Road to an Alternative Economic Strategy

This community-based research uses Q methodology to examine perceptions of tourism and downtown development in a lower Midwest town that has experienced continual economic downturn and decreasing population. With memories of the thriving ranching and oil heyday, some community members are approaching tourism as the new black gold. At the same time, there is a perception of a fractious community characterized by a heritage of racism, cultural tension, and an aging, burned-out volunteer base. One of the goals of this study was to help community planners make sense out of what they perceived as a jumble of viewpoints. The results of a Q study can help planners determine in a systematic way the significant, persistent attitudes surrounding the issue of tourism development from a broad community perspective. The Chamber of Commerce board members, community leaders, and volunteers used this study as a building block for their strategic plan in tourism

A Rural Community Pauses to Gauge Attitudes on the Road to an Alternative Economic Strategy

This is the publisher's official version, also available electronically from: http://dx.doi.org/10.4148/ojrrp.v5i3.201This community-based research uses Q methodology to examine perceptions of tourism and downtown development in a lower Midwest town that has experienced continual economic downturn and decreasing population. With memories of the thriving ranching and oil heyday, some community members are approaching tourism as the new black gold. At the same time, there is a perception of a fractious community characterized by a heritage of racism, cultural tension, and an aging, burned-out volunteer base. One of the goals of this study was to help community planners make sense out of what they perceived as a jumble of viewpoints. The results of a Q study can help planners determine in a systematic way the significant, persistent attitudes surrounding the issue of tourism development from a broad community perspective. The Chamber of Commerce board members, community leaders, and volunteers used this study as a building block for their strategic plan in tourism

Are Anti-Spit Tobacco Campaigns Striking Out? A Survey of Iowa and Nebraska College Baseball Players

This is the publisher's version, also available electronically from: http://digitalcommons.unl.edu/greatplainsresearch/842Anti-spit tobacco information is replete with fear appeals, including firsthand accounts of death and debilitation, to make users aware of the health risks and dangers. Those dangers, however, are well known by baseball players whose association with spit tobacco is historic. A survey of 217 Iowa and Nebraska college players showed that despite their awareness of spit tobacco's dangers, the players use spit tobacco to relax and focus on the field. This study supports other research showing that fear appeals may not be the most appropriate approach for anti-tobacco advertising campaigns. The study suggests that campaigns should promote relaxation and stress reduction techniques as alternatives to spit tobacco

Tup1-Ssn6 and Swi-Snf remodelling activities influence long-range chromatin organization upstream of the yeast SUC2 gene

The traditional model for chromatin remodelling during transcription has focused upon the remodelling of nucleosomes at gene promoters. However, in this study, we have determined that Tup1-Ssn6 and Swi-Snf chromatin remodelling activities extend far upstream of the SUC2 gene promoter into the intergenic region of the Saccharomyces cerevisiae chromosome. We mapped the nucleosomal array over a 7.5 kb region that encompassed the SUC2 gene promoter and upstream region but was devoid of other transcriptionally active genes. Nucleosome positioning over this region was determined under conditions of glucose repression and derepression, and in snf2, ssn6 and snf2 ssn6 mutant strains. A map detailing remodelling events extending as much as 5 kb upstream of the SUC2 gene promoter underlines the roles of the Tup1-Ssn6 and Swi-Snf complexes in respectively organizing and disrupting nucleosome arrays. The gene specificity of these events suggests a role in gene regulation. We propose that long-range chromatin remodelling activities of Swi-Snf and Tup1-Ssn6 may ultimately influence whether the chromosomal state of the SUC2 gene is proficient for transcription. These data raise the possibility that remodelling of extensive chromatin domains may be a general property of the Swi-Snf and Tup1-Ssn6 complexes

Quantitative differential proteomics of yeast extracellular matrix: there is more to it than meets the eye

Background: Saccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation. Methods: S. cerevisiae W303-1A wt strain and gup1Δ mutant were used as previously described to generate biofilmlike mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis. Results: The proteome of S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133.Conclusions: yECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive moonlighting or actual metabolism occurs extracellularly. The gup1Δ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.This work was funded by the Marie Curie Initial Training Network GLYCOPHARM (PITN-GA-2012-317297), and by national funds from FCT I.P. through the strategic funding UID/BIA/04050/2013. Fábio Faria-Oliveira was supported by a PhD scholarship (SFRH/BD/45368/2008) from FCT (Fundação para a Ciência e a Tecnologia). We thank David Caceres and Montserrat MartinezGomariz from the Unidad de Proteómica, Universidad Complutense de Madrid – Parque Científico de Madrid, Spain for excellent technical assistance in the successful implementation of all proteomics procedures including peptide identification, and Joana Tulha from the CBMA, Universidade do Minho, Portugal, for helping with the SDS-PAGE experiments, and the tedious and laborious ECM extraction procedures. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium, via the PRIDE partner repository, with the dataset identifier PXD001133. We would like to thank the PRIDE team for all the help and support during the submission process.info:eu-repo/semantics/publishedVersio

Causes of Proteolytic Degradation of Secreted Recombinant Proteins Produced in ethylotrophic Yeast Pichia pastoris: Case Study With Recombinant Ovine Interferon-T

It was observed that during fermentative production of recombinant ovine interferon-H (r-oIFN-H ) in Pichia pastoris, a secreted recombinant protein, the protein was degraded increasingly after 48 h of induction and the rate of degradation increased towards the end of fermentation at 72 h, when the fermentation was stopped. Proteases, whose primary source was the vacuoles, was found in in-creasing levels in the cytoplasm and in the fermentation broth after 48 h of induction and reached maximal values when the batch was completed at 72 h. Protease levels at various cell fractions as well as in the culture supernatant were lower when glycerol was used as the carbon source instead of methanol. It can be concluded that methanol me-tabolism along with cell lysis towards the end of fermenta-tion contributes to increased proteolytic activity and even-tual degradation of recombinant protei

External Control of the GAL Network in S. cerevisiae: A View from Control Theory

While there is a vast literature on the control systems that cells utilize to regulate their own state, there is little published work on the formal application of control theory to the external regulation of cellular functions. This paper chooses the GAL network in S. cerevisiae as a well understood benchmark example to demonstrate how control theory can be employed to regulate intracellular mRNA levels via extracellular galactose. Based on a mathematical model reduced from the GAL network, we have demonstrated that a galactose dose necessary to drive and maintain the desired GAL genes' mRNA levels can be calculated in an analytic form. And thus, a proportional feedback control can be designed to precisely regulate the level of mRNA. The benefits of the proposed feedback control are extensively investigated in terms of stability and parameter sensitivity. This paper demonstrates that feedback control can both significantly accelerate the process to precisely regulate mRNA levels and enhance the robustness of the overall cellular control system

Regulation of conditional gene expression by coupled transcription repression and RNA degradation

Gene expression is determined by a combination of transcriptional and post-transcriptional regulatory events that were thought to occur independently. This report demonstrates that the genes associated with the Snf3p–Rgt2p glucose-sensing pathway are regulated by interconnected transcription repression and RNA degradation. Deletion of the dsRNA-specific ribonuclease III Rnt1p increased the expression of Snf3p–Rgt2p-associated transcription factors in vivo and the recombinant enzyme degraded their messenger RNA in vitro. Surprisingly, Rnt1ps effect on gene expression in vivo was both RNA and promoter dependent, thus linking RNA degradation to transcription. Strikingly, deletion of RNT1-induced promoter-specific transcription of the glucose sensing genes even in the absence of RNA cleavage signals. Together, the results presented here support a model in which co-transcriptional RNA degradation increases the efficiency of gene repression, thereby allowing an effective cellular response to the continuous changes in nutrient concentrations