Induction of heat shock proteins in cold- adapted and coldacclimated Fishes

Thesis (M.S.) University of Alaska Fairbanks, 2014I examined the effects of oxidative stress and changes in temperature on heat shock protein (Hsp) levels in cold-adapted and cold-acclimated fishes. Adaptation of Antarctic notothenioids to cold temperature is correlated with high levels of Hsps, thought to minimize cold-induced protein denaturation. Hsp70 levels were measured in red- and white-blooded Antarctic notothenioid fishes exposed to their critical thermal maximum (CTMax), 4°C warm acclimated, and notothenioids from different latitudes. I determined the effect of cold acclimation on Hsp levels and the role of sirtuins in regulating Hsp expression and changes in metabolism in threespine stickleback, Gasterosteus aculeatus, cold-acclimated to 8°C. Levels of Hsps do not increase in Antarctic notothenioids exposed to their CTMax, and warm acclimation reduced levels of Hsp70. Hsp70 levels were higher in Antarctic notothenioids compared to a temperate notothenioid and higher in white-blooded notothenioids compared to red-blooded notothenioids, despite higher oxidative stress levels in red-blooded fish, suggesting Hsp70 does not mitigate oxidative stress. Cold acclimation of stickleback resulted in tissue-specific increases in some Hsps and sirtuins. My research indicates that cold acclimation increases Hsp levels, and moderate increases in temperature reduce Hsp levels in cold-adapted fishes. Together, these data lend support to the hypothesis that cold denatures proteins

FOOD2GATHER: What is migrants’ food all about in Europe? A media discourse analysis through the lens of controversies

This report is part of the HERANET funded project FOOD2GATHER. The project aims at understanding the question of integration/exclusion of migrants through foodscapes. An important step in this direction is to analyse the contextual framework within which food-related practices, norms and values are embedded in European societies. Food controversies that have raised and have been reported in the media since the “2015 migrants’ crisis” across Europe can reveal important aspects related to such norms and values and indicate possible tensions and compromises. This report presents and discusses relevant food controversies that occurred in the six countries participating in the study (Belgium, France, Germany, Italy, Norway, and the Netherlands). This will generate a contextual overview of the integration/exclusion of migrants through foodscapes. Controversy has been used as a tool and a scanner. Each of the six FOOD2GATHER teams provided two relevant controversies that have reached media attention in the last ten years. One of the two had to be related to halal food. The analysis of the controversies has been conducted by identifying issues they tackled, agents they involved, (public) spaces and situations in which controversies took place and what they produced. A comparative analysis of relevant variables related to migrations, such as the geopolitical position of the countries, organization of reception and food provision, has been conducted as well. The six countries included in the study have different traditions related to migration and have been exposed to the “migrants’ crisis” in different ways. These differences are reflected in the proposed controversies. However, some common traits tend to emerge and reveal power relationships within societies that are different or shared by the countries involved in the project. We show that these power relationships particularly deal with the right to food, citizens’ commitment, identity, the place of religion, animal welfare and political issues. Our study indicates that analysing controversies adds an important dimension to the study of foodscapes. Food controversies that reach the media attention are seldom something migrants have brought up themselves. The migrants’ representation in the media based on food controversies indicated that migrants are given little opportunity to negotiating values and practices, as norms about “the right” quantity and quality of food tend to reproduce the food model of the country they migrate to, also when there is a “positive” focus on ethnic business. To better understand these dynamics, we propose the concept of “food encounters” and illustrate how the type of food encounters can play a role in how foodscapes could evolve or even emerge.Consumption Research Norway (SIFO), OsloMe

Ca\u3csup\u3e2+\u3c/sup\u3e Dependency of Limb Muscle Fiber Contractile Mechanics in Young and Older Adults

Age-induced declines in skeletal muscle contractile function have been attributed to multiple cellular factors, including lower peak force (Po), decreased Ca2+ sensitivity, and reduced shortening velocity (Vo). However, changes in these cellular properties with aging remain unresolved, especially in older women, and the effect of submaximal Ca2+ on contractile function is unknown. Thus, we compared contractile properties of muscle fibers from 19 young (24 ± 3 yr; 8 women) and 21 older adults (77 ± 7 yr; 7 women) under maximal and submaximal Ca2+ and assessed the abundance of three proteins thought to influence Ca2+ sensitivity. Fast fiber cross-sectional area was ~44% larger in young (6,479 ± 2,487 µm2) compared with older adults (4,503 ± 2,071 µm2, P \u3c 0.001), which corresponded with a greater absolute Po (young = 1.12 ± 0.43 mN; old = 0.79 ± 0.33 mN, P \u3c 0.001). There were no differences in fast fiber size-specific Po, indicating the age-related decline in force was explained by differences in fiber size. Except for fast fiber size and absolute Po, no age or sex differences were observed in Ca2+ sensitivity, rate of force development (ktr), or Vo in either slow or fast fibers. Submaximal Ca2+ depressed ktr and Vo, but the effects were not altered by age in either sex. Contrary to rodent studies, regulatory light chain (RLC) and myosin binding protein-C abundance and RLC phosphorylation were unaltered by age or sex. These data suggest the age-associated reductions in contractile function are primarily due to the atrophy of fast fibers and that caution is warranted when extending results from rodent studies to humans

FOOD2GATHER: What is migrants’ food all about in Europe? A media discourse analysis through the lens of controversies

This report is part of the HERANET funded project FOOD2GATHER. The project aims at understanding the question of integration/exclusion of migrants through foodscapes. An important step in this direction is to analyse the contextual framework within which food-related practices, norms and values are embedded in European societies. Food controversies that have raised and have been reported in the media since the “2015 migrants’ crisis” across Europe can reveal important aspects related to such norms and values and indicate possible tensions and compromises. This report presents and discusses relevant food controversies that occurred in the six countries participating in the study (Belgium, France, Germany, Italy, Norway, and the Netherlands). This will generate a contextual overview of the integration/exclusion of migrants through foodscapes. Controversy has been used as a tool and a scanner. Each of the six FOOD2GATHER teams provided two relevant controversies that have reached media attention in the last ten years. One of the two had to be related to halal food. The analysis of the controversies has been conducted by identifying issues they tackled, agents they involved, (public) spaces and situations in which controversies took place and what they produced. A comparative analysis of relevant variables related to migrations, such as the geopolitical position of the countries, organization of reception and food provision, has been conducted as well. The six countries included in the study have different traditions related to migration and have been exposed to the “migrants’ crisis” in different ways. These differences are reflected in the proposed controversies. However, some common traits tend to emerge and reveal power relationships within societies that are different or shared by the countries involved in the project. We show that these power relationships particularly deal with the right to food, citizens’ commitment, identity, the place of religion, animal welfare and political issues. Our study indicates that analysing controversies adds an important dimension to the study of foodscapes. Food controversies that reach the media attention are seldom something migrants have brought up themselves. The migrants’ representation in the media based on food controversies indicated that migrants are given little opportunity to negotiating values and practices, as norms about “the right” quantity and quality of food tend to reproduce the food model of the country they migrate to, also when there is a “positive” focus on ethnic business. To better understand these dynamics, we propose the concept of “food encounters” and illustrate how the type of food encounters can play a role in how foodscapes could evolve or even emerge

Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses.

Enrichment methodologies enable the analysis of minor members in multi-species transcriptomic data. We compared the standard enrichment of bacterial and eukaryotic mRNA to a targeted enrichment using an Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with their unenriched counterparts (r2 = 0.559-0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r2 = 0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote enrichment from a single RNA sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase in multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries compared to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using AgSS targeted enrichment on poly(A)-enriched libraries for eukaryotic captures, and total RNA libraries for prokaryotic captures, to increase the robustness of multi-species transcriptomic studies

Targeted enrichment outperforms other enrichment techniques and enables more multi-species RNA-Seq analyses.

Enrichment methodologies enable the analysis of minor members in multi-species transcriptomic data. We compared the standard enrichment of bacterial and eukaryotic mRNA to a targeted enrichment using an Agilent SureSelect (AgSS) capture for Brugia malayi, Aspergillus fumigatus, and the Wolbachia endosymbiont of B. malayi (wBm). Without introducing significant systematic bias, the AgSS quantitatively enriched samples, resulting in more reads mapping to the target organism. The AgSS-enriched libraries consistently had a positive linear correlation with their unenriched counterparts (r2 = 0.559-0.867). Up to a 2,242-fold enrichment of RNA from the target organism was obtained following a power law (r2 = 0.90), with the greatest fold enrichment achieved in samples with the largest ratio difference between the major and minor members. While using a single total library for prokaryote and eukaryote enrichment from a single RNA sample could be beneficial for samples where RNA is limiting, we observed a decrease in reads mapping to protein coding genes and an increase in multi-mapping reads to rRNAs in AgSS enrichments from eukaryotic total RNA libraries compared to eukaryotic poly(A)-enriched libraries. Our results support a recommendation of using AgSS targeted enrichment on poly(A)-enriched libraries for eukaryotic captures, and total RNA libraries for prokaryotic captures, to increase the robustness of multi-species transcriptomic studies

A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics

Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious, requiring 2 h of mass spectrometry time per single muscle fiber; 50 fibers would take approximately 4 days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 min total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 h. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Ninety-four proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, oxidative phosphorylation, and muscle structure and contractile function. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput

A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics

Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious, requiring 2 h of mass spectrometry time per single muscle fiber; 50 fibers would take approximately 4 days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 min total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 h. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Ninety-four proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, oxidative phosphorylation, and muscle structure and contractile function. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput

A Complete Workflow for High Throughput Human Single Skeletal Muscle Fiber Proteomics

Skeletal muscle is a major regulatory tissue of whole-body metabolism and is composed of a diverse mixture of cell (fiber) types. Aging and several diseases differentially affect the various fiber types, and therefore, investigating the changes in the proteome in a fiber-type specific manner is essential. Recent breakthroughs in isolated single muscle fiber proteomics have started to reveal heterogeneity among fibers. However, existing procedures are slow and laborious, requiring 2 h of mass spectrometry time per single muscle fiber; 50 fibers would take approximately 4 days to analyze. Thus, to capture the high variability in fibers both within and between individuals requires advancements in high throughput single muscle fiber proteomics. Here we use a single cell proteomics method to enable quantification of single muscle fiber proteomes in 15 min total instrument time. As proof of concept, we present data from 53 isolated skeletal muscle fibers obtained from two healthy individuals analyzed in 13.25 h. Adapting single cell data analysis techniques to integrate the data, we can reliably separate type 1 and 2A fibers. Ninety-four proteins were statistically different between clusters indicating alteration of proteins involved in fatty acid oxidation, oxidative phosphorylation, and muscle structure and contractile function. Our results indicate that this method is significantly faster than prior single fiber methods in both data collection and sample preparation while maintaining sufficient proteome depth. We anticipate this assay will enable future studies of single muscle fibers across hundreds of individuals, which has not been possible previously due to limitations in throughput