Reference proteins (RP) or the total protein (TP) loaded is used to correct for uneven loading and/or transfer in Western blotting. However, the signal sensitivity and the influence of physiological conditions may question the normalization methods. Therefore, three widely used reference proteins [β-actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and α-tubulin], as well as TP loaded measured by Stain-Free technology (SF) as normalization tool were tested. This was done using skeletal muscle samples from men subjected to physiological conditions often investigated in applied physiology where the intervention has been suggested to impede normalization (ageing, muscle atrophy, and different muscle fiber type composition). The linearity of signal and the methodological variation coefficient was obtained. Furthermore, the inter- and intraindividual variation in signals obtained from SF and RP was measured in relation to ageing, muscle atrophy, and different muscle fiber type composition, respectively. A stronger linearity of SF and β-actin compared with GAPDH and α-tubulin was observed. The methodological variation was relatively low in all four methods (4–11%). Protein level of β-actin and GAPDH was lower in older men compared with young men. In conclusion, β-actin, GAPDH, and α-tubulin may not be used for normalization in studies that include subjects with a large age difference. In contrast, the RPs may not be affected in studies that include muscle wasting and differences in muscle fiber type. The novel SF technology adds lower variation to the results compared with the existing methods for correcting for loading inaccuracy in Western blotting of human skeletal muscle in applied physiology.

The human skin is colonized by a wide array of microorganisms playing a role in skin disorders. Studying the skin microbiome provides unique obstacles such as low microbial biomass. The objective of this study was to establish methodology for skin microbiome analyses, focusing on sampling technique and DNA extraction. Skin swabs and scrapes were collected from 9 healthy adult subjects, and DNA extracted using 12 commercial kits. All 165 samples were sequenced using the 16S rRNA gene. Comparing the populations captured by eSwabs and scrapes, 99.3% of sequences overlapped. Using eSwabs yielded higher consistency. The success rate of library preparation applying different DNA extraction kits ranged from 39% to 100%. Some kits had higher Shannon alpha-diversity. Metagenomic shotgun analyses were performed on a subset of samples (N = 12). These data indicate that a reduction of human DNA from 90% to 57% is feasible without lowering the success of 16S rRNA library preparation and without introducing taxonomic bias. Using swabs is a reliable technique to investigate the skin microbiome. DNA extraction methodology is crucial for success of sequencing and adds a substantial amount of variation in microbiome analyses. Reduction of host DNA is recommended for interventional studies applying metagenomics.

Background: Microbial dysbiosis with increased Staphylococcus aureus (S. aureus) colonization on the skin is a hallmark of atopic dermatitis (AD), however most microbiome studies focus on bacteria in the flexures and the microbial composition at other body sites have not been studied systematically. Objectives: The aim of the study is to characterize the skin microbiome, including bacteria, fungi and virus, at different body sites in relation to AD, lesional state, and S. aureus colonization, and to test whether the nares could be a reservoir for S. aureus strain colonization. Methods: Using shotgun metagenomics we characterized microbial compositions from 14 well defined skin sites from 10 patients with AD and 5 healthy controls. Results: We found clear differences in microbial composition between AD and controls at multiple skin sites, most pronounced on the flexures and neck. The flexures exhibited lower alpha-diversity and were colonized by S. aureus, accompanied by S. epidermidis in lesions. Malassezia species were absent on the neck in AD. Virus mostly constituted Propionibacterium and Staphylococcusphages, with increased abundance of Propionibacterium phages PHL041 and PHL092 and Staphylococcus epidermidis phages CNPH82 and PH15 in AD. In lesional samples, both the genus Staphylococcus and Staphylococcus phages were more abundant. S. aureus abundance was higher across all skin sites except from the feet. In samples where S. aureus was highly abundant, lower abundances of S. hominis and Cutibacterium acnes were observed. M. osloensis and M. luteus were more abundant in AD. By single nucleotide variant analysis of S. aureus we found strains to be subject specific. On skin sites some S. aureus strains were similar and some dissimilar to the ones in the nares. Conclusions: Our data indicate a global and site-specific dysbiosis in AD, involving both bacteria, fungus and virus. When defining targeted treatment clinicians should both consider the individual and skin site and future research into potential crosstalk between microbiota in AD yields high potential.