Atopic dermatitis (AD) is associated with microbial dysbiosis and impaired skin barrier function. Topical therapies, such as moisturisers and antimicrobial fragrance compounds, may modulate the skin microbiome and support disease management. The objective was to evaluate how a moisturiser and a fragrance compound (farnesol) influence skin microbiome composition in individuals with AD and healthy controls. In a randomised, controlled, operator-blinded study, 15 AD patients and 15 healthy controls applied a moisturiser, farnesol, moisturiser + farnesol, or no treatment to defined skin areas over 7 days. Microbiome composition, alpha/beta diversity, and core taxa were analysed using shotgun metagenomics. At baseline, AD patients exhibited distinct microbial profiles, including elevated Staphylococcus aureus and Micrococcus luteus. Neither moisturiser nor farnesol significantly altered richness, beta diversity, or core taxa in either AD patients or controls. However, moisturiser use in healthy individuals modestly increased Shannon diversity, reflecting improved microbial evenness. Despite clear microbiome differences between AD and healthy skin, short-term topical treatment did not markedly shift microbial composition. The observed stability underscores the resilience of the skin microbiome and suggests that longer interventions or more targeted formulations may be necessary to influence microbial dysbiosis in AD.

Introduction: It is an international standard to recommend patients with atopic dermatitis (AD) to use moisturizers; however, little is known about their effect on lipids in the stratum corneum (SC). Methods: In this randomized clinical experiment of 30 Caucasian participants (15 with AD and 15 healthy controls), the superficial SC lipid profile was assessed through tape stripping non-lesional skin following treatment thrice daily for seven days with a moisturizer, and subsequently compared with untreated skin. Results: No discernible disparity in superficial SC lipid quantity was evident between the AD group and the control group. However, the SC lipid composition diverged significantly, with the AD group exhibiting diminished levels of long-chain EO CERs (p = 0.024) and elevated levels of short-chain C34 CERs (p = 0.025) compared to healthy skin. Moisturizer application significantly reduced the total SC lipids and all lipid subgroups in both groups. Within the AD group, a non-significant inclination towards an augmentation in EO CERs (p = 0.053) and reduction in C34 CERs (p = 0.073) was observed. Conclusion: The recent identification of distinctions in SC lipid composition between AD and healthy skin was substantiated by our findings. Topical moisturizer application, despite reducing overall total lipids, indicated a potential tendency towards a healthier lipid constitution in AD skin.

Supplementary figure 1: Effects of cisplatin treatment on expression of ΔNErbB2 and NHE1, and subcellular localization of cytochrome c. MCF-7 vector (V) and ΔNErbB2 (ΔN) cells were washed free of tetracycline 48 h before the experiment, and exposed to cisplatin (25 μM) or control conditions for 18 h. Cells were subsequently processed for immunoblotting (A), immunofluorescence analysis (B) or cell surface biotinylation (C-D). A. ΔNErbB2, top panel shows representative immunoblots, lower panel shows ΔNErbB2 protein level relative to that in vector cells under control conditions. B. Subcellular localization of cytochrome c. Nuclei are stained with DAPI. C. Representative immunoblots of NHE1 in total lysate fractions (TLF), intracellular fractions (ITF) and biotinylated fractions (BF) in MCF-7 vector (V) and ΔNErbB2 (ΔN) cells. F. Top panel shows representative immunoblots of NHE1 in TLF and BF in native MCF-7 cells, lower panel shows NHE1 protein level relative to that in native MCF-7 cells under control conditions. Data are representative of 3-5 independent experiments for each condition. Western blot data are shown as means with SEM error bars. * indicates comparison of the value in cisplatin treated to that in the respective cell line control, # indicate comparison of values in vector- and ΔNErbB2 cell lines under same treatment. */#, **/##, ***/### and ****/#### indicate p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively (C, one-way ANOVA with Bonferroni post-test) (A, two-way ANOVA with Bonferroni post-test).

Supplementary figure 2: Inhibition of ATM and knockdown of noxa, caspase-9, μ-calpain and cathepsin B. MCF-7 vector (V) and ΔNErbB2 (ΔN) cells were washed free of tetracycline 72 h before the experiment. Cells were exposed to KU-55933 (10 μM) for 19 h, cisplatin (25 μM) or control conditions for 18 h and subsequently processed for immunoblotting (A-B). Top panels show representative immunoblots, lower panels show protein level relative to that in vector cells under control conditions. A. p-ATM B. p-p53. ΔNErbB2 cells were washed and transfected with siRNA (200nM) 72 h before the experiment. Cells were exposed to cisplatin (25 μM), and subsequently processed for immunoblotting (C-F). Top panels show representative immunoblots, lower panels show protein level relative to that in cells transfected with mock control. C. Noxa D. Caspase-9. E. μ-calpain. F. Cathepsin B. Data are representative of 3-4 independent experiments for each condition. Western blot data are shown as means with SEM error bars. * indicates comparison of the value in cisplatin treated to that in the respective cell line control, # indicate comparison of values in vector- and ΔNErbB2 cell lines under same treatment. */#, **/##, ***/### and ****/#### indicate p < 0.05, p < 0.01, p < 0.001 and p < 0.0001, respectively (C-F), one-way ANOVA with Bonferroni post-test) (A-B, two-way ANOVA with Bonferroni post-test).

Supplementary figure 3: Working model. Pathways involved in cisplatin-induced death in MCF-7 breast cancer cells, and their relation to ΔNErbB2 expression.

Skeletal metastatic disease excludes many cancer patients from participating in exercise and physical activity due to safety concerns. Empirical evidence from high-quality trials is warranted to guide clinicians and patients. To evaluate the safety and potential benefits of high-impact aerobic exercise in patients with prostate cancer with skeletal metastases. Exploratory subgroup analysis of a pragmatic, multicentre, parallel randomised controlled trial. The trial recruited 214 patients from five hospital urological departments in Denmark. Patients with prostate cancer with skeletal metastases (n = 41). Six months of football training twice weekly at a local club or usual care. Both groups received brief information on physical activity recommendations at the time of randomisation. Safety, defined as falls, fractures and hospital admissions. Effects were evaluated on the primary outcome (prostate cancer-specific quality of life) and secondary outcomes (lean body mass, fat mass, hip and spine bone mineral density, and general physical and mental health). The original trial comprised 214 participants, 41 of whom had skeletal metastases at enrolment. Of these, 22 were allocated to football and 19 to usual care. The trial retention rate was 95% at 12 weeks and 88% at 6 months. Football participants attended 13 sessions on average at 12 weeks and 23 at 6 months. There were two falls, one in each group after 6 months, and no fractures. There were four unplanned hospital admissions in the study period, all four in the usual care group. Statistically significant between-group difference was observed in the primary outcome change in prostate cancer-specific quality of life at 12 weeks (7.6 points [95% CI 0.5 to 15.0]; P = 0.038). No statistical changes were found in the secondary outcomes. The analysis showed that football training was safe in patients with skeletal metastatic prostate cancer and significantly improved quality of life. Larger analyses and/or trials are warranted to confirm the safety of exercise more broadly in cancer patients with skeletal metastatic disease. ClinicalTrials.gov, NCT02430792 . Date of registration 30 April 2015

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 Staphylococcus phages, 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.

Glucose phosphorylation by hexokinase (HK) is a rate-limiting step in glucose metabolism. Regulation of HK includes feedback inhibition by its product glucose-6-phosphate (G6P) and mitochondria binding. HK affinity for G6P is difficult to measure because its natural product (G6P) inhibits enzyme activity. HK phosphorylates several hexoses, and we have taken advantage of the fact that 2-deoxyglucose (2-DG)-6-phosphate does not inhibit HK activity. By this, we have developed a new method for rapid radiochemical analysis of HK activity with 2-DG as a substrate, which allows control of the concentrations of G6P to investigate HK affinity for inhibition by G6P. We verified that 2-DG serves as a substrate for the HK reaction with linear time and concentration dependency as well as expected maximal velocity and KM. This is the first simple assay that evaluates feedback inhibition of HK by its product G6P and provides a unique technique for future research evaluating the regulation of glucose phosphorylation under various physiological conditions.NEW & NOTEWORTHY Traditionally, hexokinase activity has been analyzed spectrophotometrically in which the product formation of glucose-6-phosphate (G6P) is analyzed by an indirect reaction coupled to NADPH formation during conversion of G6P to 6-P gluconolactone. By nature, this assay prevents measurements of hexokinase (HK) affinity for inhibition by G6P. We have developed a rapid radiochemical filter paper assay to study HK affinity for G6P by use of radiolabeled 2-deoxyglucose as substrate to study physiological regulation of HK affinity for G6P-induced inhibition.

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.

To the Editor: The prevalence of permanent tattooing is increasing,1Laumann A.E. Derick A.J. Tattoos and body piercings in the United States: a national data set.J Am Acad Dermatol. 2006; 55: 413-421Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar, 2Sagoe D. Pallesen S. Andreassen C.S. Prevalence and correlates of tattooing in Norway: a large-scale cross-sectional study.Scand J Psychol. 2017; 58: 562-570Crossref PubMed Scopus (9) Google Scholar along with its harms. Case studies provide mounting evidence of various tattoo-associated skin problems often ascribed to red and black ink.3Brady B.G. Gold H. Leger E.A. Leger M.C. Self-reported adverse tattoo reactions: a New York City Central Park study.Contact Dermatitis. 2015; 73: 91-99Crossref PubMed Scopus (38) Google Scholar However, as the prevalence of tattoo-related skin reactions in the general population is unknown, the scale of the adverse reactions remains uncertain. Tattooing has been associated with marginalized societal groups,4Pers M. von Herbst T. The demand for removal of tattoos. A plea for regulations against tattooing of minors.Acta Chir Scand. 1966; 131: 201-204PubMed Google Scholar but little is known about the current social characteristics of the tattooed population. Our baseline cohort study from 2006 compromised 3471 randomly selected adult Danes representative of the general population. Five years later, a follow-up was conducted including 2212 individuals (participation rate 63.7%) who answered questions regarding permanent tattoos, adverse skin reactions, and social characteristics. Permanent tattooing was defined as a tattoo pricked into the skin by a needle. We used SPSS version 22 for descriptive statistics and regression models to estimate odds ratios (ORs) with 95% confidence intervals (95% CIs). In total, 14.2% had ≥1 tattoo (Table I). Tattoos were more frequent among the young age groups, with 28.6% for those 24-30 years of age and 27.1% for those 31-39 years of age compared with 10.4% in the 40-76 year age group. More men were tattooed than women, and men were younger (median age 20 years) than women (median age 28 years) when they had their first tattoo (P < .001). Notably, tattooed individuals were more often unmarried, less educated, and cohabiting than individuals without tattoos. Moreover, they had an unhealthy diet, frequently used tanning beds, and rated their social position as in the middle (Table I).Table ICharacteristics of study population from Health2006 cohort and logistic regression analysis with ≥1 tattoo as dependent variable and covariates influencing likelihood of having tattoo as independent variablesExplanatory variablesNo tattoos, % (n)≥1 tattoo, % (n)P value, χ2 testOR (95% CI)General All participants, n = 221285.8 (1899)14.2 (313) SexFemale, n = 119089.0 (1059)11.0 (131)<.001ReferenceMale, n = 102282.2 (840)17.8 (182)1.89 (1.48-2.43)∗Adjusted for age. Age group, y40-7689.6 (1546)10.4 (180)<.001Referenceφ31-3972.9 (283)27.1 (105)3.34 (2.53-4.40)†Adjusted for sex.24-3071.4 (70)28.6 (28)3.65 (2.28-5.84)†Adjusted for sex.Skin health Itchy skin ever48.3 (897/1856)46.5 (140/301).5581.05 (0.81-1.34) Itchy skin within past 12 months56.9 (645/1133)54.9 (100/182).6161.08 (0.78-1.49)Social characteristics and lifestyle Marriage, n = 2204Ever married85.6 (1619/1892)70.5 (220/312)<.001ReferenceUnmarried14.4 (273/1892)29.5 (92/312)1.80 (1.30-2.48) Cohabitation, n = 2162Never cohabited42.2 (783/1856)27.1 (83/306)<.001ReferenceEver cohabited57.8 (1073/1856)72.9 (223/306)1.74 (1.32-2.29) Educational level, n = 2132High59.0 (1082/1833)37.5 (112/299)<.001ReferenceLow, <7-10 y41.0 (751/1833)62.5 (187/299)2.92 (2.23-3.84) Social position, self-assessed, n = 2198High32.9 (620/1886)24.7 (77/312)<.05ReferenceMiddle64.8 (1222/1886)72.8 (227/312)1.74 (1.30-2.32)Low2.3 (44/1886)2.6 (8/312)1.51 (0.67-3.40) Diet, self-assessed, n = 2196Healthy27.0 (508/1884)20.5 (64/312)<.05ReferenceAverage64.9 (1222/1884)66 (206/312)1.20 (0.88-1.63)Unhealthy8.2 (154/1884)13.5 (42/312)1.83 (1.17-2.86)Tanning habits Use of sunscreen during summer63.4 (1194/1882)65.4 (202/309).5130.90 (0.68-1.18) Use of tanning bed8.7 (164/1889)26.9 (84/312)<.0013.89 (2.84-5.34)Bold indicates statistically significant by logistic regression analysis (P < .05).CI, Confidence interval; OR, odds ratio.∗ Adjusted for age.† Adjusted for sex. Open table in a new tab Bold indicates statistically significant by logistic regression analysis (P < .05). CI, Confidence interval; OR, odds ratio. Adverse reactions to tattoos were reported by 5.9% (18 of 306). The reasons for adverse reactions were eczema/rash (2.9%), infection (1.3%), erosions (1.0%), or all symptoms (0.7%). In most cases, the adverse reactions disappeared without any action; 5.6% of reactions disappeared after medical treatment and 11.1% of individuals had the tattoo removed (Table II). Red ink was involved in most adverse reactions.Table IISelf-reported adverse skin reactions in tattooed population (N = 306)Adverse skin reaction in tattoo and characteristicsValue, n (%)Adverse skin reaction in tattoo, self-reported18 (5.9) Eczema/rash9 (2.9) Infection4 (1.3) Erosion3 (1.0) All 32 (0.7)Sex, male9 (50)Age group, y 24-30, n = 281 (3.6) 31-39, n = 1056 (5.7) 40-76, n = 18011 (6.1)Low educational level (<7-10 years), n = 18710 (5.3)Skin health Itchy skin ever, n = 14012 (8.6) Itchy skin within the past 12 months, n = 1008 (8.0)Tanning habits Use of sunscreen, n = 20212 (5.9) Use of tanning bed, n = 844 (4.8)Color affected by adverse reaction (P value = .22, Fisher's exact test) Blue1 Black4 Red4 Green0 Yellow2 Mix∗Blue and red; red, green, yellow, and other; yellow and other; blue, red, green, and yellow; and black, red, green, and yellow.5 Other1 No answer1Handling of adverse reaction (P = .239, Fisher's exact test) Disappeared without any action12 Disappeared after medical treatment1 Tattoo had to be removed2 Other2 No answer1∗ Blue and red; red, green, yellow, and other; yellow and other; blue, red, green, and yellow; and black, red, green, and yellow. Open table in a new tab Our study suggests that tattooing is becoming more mainstream, which is in-line with previous literature.2Sagoe D. Pallesen S. Andreassen C.S. Prevalence and correlates of tattooing in Norway: a large-scale cross-sectional study.Scand J Psychol. 2017; 58: 562-570Crossref PubMed Scopus (9) Google Scholar We show that 5.9% of tattooed individuals report adverse reactions, which is high considering our focus on well-defined clinical symptoms. Potentially this restricted range of reactions from our questionnaire was a study limitation, resulting in an underestimation of the prevalence of tattoo-associated problems. Further, we do not know when the reactions occurred, and whether they were late events or associated with the pricking itself. A low rate of medical consultation and a habit of consulting the tattooist or asking tattooed friends for advice regarding tattoo-related skin problems have been shown elsewhere.5Hutton Carlsen K. Serup J. Photosensitivity and photodynamic events in black, red, and blue tattoos are common: a ‘beach study’.J Eur Acad Dermatol Venereol. 2014; 28: 231-237Crossref PubMed Scopus (61) Google Scholar However, the tattooist is not required to report adverse reactions or to provide general risk information. Face-to-face interviews in tattooed subgroups have shown that 42% of 144 tattooed sunbathers in Denmark experienced complaints5Hutton Carlsen K. Serup J. Photosensitivity and photodynamic events in black, red, and blue tattoos are common: a ‘beach study’.J Eur Acad Dermatol Venereol. 2014; 28: 231-237Crossref PubMed Scopus (61) Google Scholar and 10.3% of 300 tattooed individuals in Central Park, New York, had experienced an adverse reaction.3Brady B.G. Gold H. Leger E.A. Leger M.C. Self-reported adverse tattoo reactions: a New York City Central Park study.Contact Dermatitis. 2015; 73: 91-99Crossref PubMed Scopus (38) Google Scholar The high number of adverse reactions supports the need for systematic surveillance regarding tattoo-related skin problems. Our findings underline the importance of counseling emphasizing risks associated with red ink.

Key points This study aimed to provide molecular insight into the differential effects of age and physical inactivity on the regulation of substrate metabolism during moderate-intensity exercise. Using the arteriovenous balance technique, we studied the effect of immobilization of one leg for 2weeks on leg substrate utilization in young and older men during two-legged dynamic knee-extensor moderate-intensity exercise, as well as changes in key proteins in muscle metabolism before and after exercise. Age and immobilization did not affect relative carbohydrate and fat utilization during exercise, but the older men had higher uptake of exogenous fatty acids, whereas the young men relied more on endogenous fatty acids during exercise. Using a combined whole-leg and molecular approach, we provide evidence that both age and physical inactivity result in intramuscular lipid accumulation, but this occurs only in part through the same mechanisms.AbstractAge and inactivity have been associated with intramuscular triglyceride (IMTG) accumulation. Here, we attempt to disentangle these factors by studying the effect of 2weeks of unilateral leg immobilization on substrate utilization across the legs during moderate-intensity exercise in young (n=17; 231years old) and older men (n=15; 681years old), while the contralateral leg served as the control. After immobilization, the participants performed two-legged isolated knee-extensor exercise at 201W (approximate to 50% maximal work capacity) for 45min with catheters inserted in the brachial artery and both femoral veins. Biopsy samples obtained from vastus lateralis muscles of both legs before and after exercise were used for analysis of substrates, protein content and enzyme activities. During exercise, leg substrate utilization (respiratory quotient) did not differ between groups or legs. Leg fatty acid uptake was greater in older than in young men, and although young men demonstrated net leg glycerol release during exercise, older men showed net glycerol uptake. At baseline, IMTG, muscle pyruvate dehydrogenase complex activity and the protein content of adipose triglyceride lipase, acetyl-CoA carboxylase 2 and AMP-activated protein kinase (AMPK)3 were higher in young than in older men. Furthermore, adipose triglyceride lipase, plasma membrane-associated fatty acid binding protein and AMPK3 subunit protein contents were lower and IMTG was higher in the immobilized than the contralateral leg in young and older men. Thus, immobilization and age did not affect substrate choice (respiratory quotient) during moderate exercise, but the whole-leg and molecular differences in fatty acid mobilization could explain the age- and immobilization-induced IMTG accumulation.Key pointsThis study aimed to provide molecular insight into the differential effects of age and physical inactivity on the regulation of substrate metabolism during moderate-intensity exercise. Using the arteriovenous balance technique, we studied the effect of immobilization of one leg for 2weeks on leg substrate utilization in young and older men during two-legged dynamic knee-extensor moderate-intensity exercise, as well as changes in key proteins in muscle metabolism before and after exercise. Age and immobilization did not affect relative carbohydrate and fat utilization during exercise, but the older men had higher uptake of exogenous fatty acids, whereas the young men relied more on endogenous fatty acids during exercise. Using a combined whole-leg and molecular approach, we provide evidence that both age and physical inactivity result in intramuscular lipid accumulation, but this occurs only in part through the same mechanisms.

AbstractDespite the frequent expression of N-terminally truncated ErbB2 (ΔNErbB2/p95HER2) in breast cancer and its association with Herceptin resistance and poor prognosis, it remains poorly understood how ΔNErbB2 affects chemotherapy-induced cell death. Previously it was shown that ΔNErbB2 upregulates acid extrusion from MCF-7 breast cancer cells and that inhibition of the Na+/H+ exchanger (SLC9A1/NHE1) strongly sensitizes ΔNErbB2-expressing MCF-7 cells to cisplatin chemotherapy. The aim of this study was to identify the mechanism through which ΔNErbB2 regulates cisplatin-induced breast cancer cell death, and determine how NHE1 regulates this process. Cisplatin treatment elicited apoptosis, ATM phosphorylation, upregulation of p53, Noxa (PMAIP1), and PUMA (BBC3), and cleavage of caspase-9, -7, fodrin, and PARP-1 in MCF-7 cells. Inducible ΔNErbB2 expression strongly reduced cisplatin-induced ATM- and p53-phosphorylation, augmented Noxa upregulation and caspase-9 and -7 cleavage, doubled p21WAF1/Cip1 (CDKN1A) expression, and nearly abolished Bcl-2 expression. LC3-GFP analysis demonstrated that autophagic flux was reduced by cisplatin in a manner augmented by ΔNErbB2, yet did not contribute to cisplatin-induced death. Using knockdown approaches, it was shown that cisplatin-induced caspase-7 cleavage in ΔNErbB2-MCF-7 cells was Noxa- and caspase-9 dependent. This pathway was augmented by NHE1 inhibition, while the Na+/HCO3− cotransporter (SLC4A7/NBCn1) was internalized following cisplatin exposure.Implications: This work reveals that ΔNErbB2 strongly affects several major pro- and antiapoptotic pathways and provides mechanistic insight into the role of NHE1 in chemotherapy resistance. These findings have relevance for defining therapy regimens in breast cancers with ΔNErbB2 and/or NHE1 overexpression. Mol Cancer Res; 13(1); 63–77. ©2014 AACR.

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.

High‐intensity interval training (HIT) is known to increase mitochondrial content in a similar way as endurance training [60–90% of maximal oxygen uptake (VO2peak)]. Whether HIT increases the mitochondria's ability to oxidize lipids is currently debated. We investigated the effect of HIT on mitochondrial fat oxidation in skeletal muscle and adipose tissue. Mitochondrial oxidative phosphorylation (OXPHOS) capacity, mitochondrial substrate sensitivity (Kmapp), and mitochondrial content were measured in skeletal muscle and adipose tissue in healthy overweight subjects before and after 6 weeks of HIT (three times per week at 298 ± 21 W). HIT significantly increased VO2peak from 2.9 ± 0.2 to 3.1 ± 0.2 L/min. No differences were seen in maximal fat oxidation in either skeletal muscle or adipose tissue. Kmapp for octanoyl carnitine or palmitoyl carnitine were similar after training in skeletal muscle and adipose tissue. Maximal OXPHOS capacity with complex I‐ and II‐linked substrates was increased after training in skeletal muscle but not in adipose tissue. In conclusion, 6 weeks of HIT increased VO2peak. Mitochondrial content and mitochondrial OXPHOS capacity were increased in skeletal muscle, but not in adipose tissue. Furthermore, mitochondrial fat oxidation was not improved in either skeletal muscle or adipose tissue.

Many recent studies of ecological speciation have focused on "magic trait" scenarios, in which divergent selection on viability traits leads inextricably to corresponding divergence in mechanisms, especially mate recognition systems, that facilitate assortative mating. Speciation however may also proceed via other scenarios, such as when populations experience directly selected or random divergence in mate recognition systems. The relative contributions of magic trait versus other scenarios for speciation remain virtually unexplored. The present study aims to test the relative contribution of the magic trait scenario in the divergence of populations of the medium ground finch Geospiza fortis of Santa Cruz Island, Galapagos. First, we assess differences in G. fortis song between a northern population (Borrero Bay) and a southeastern population (El Garrapatero), differences that we propose (along with other within-island geographic song variations) have arisen via scenarios that do not involve a magic trait scenario. Pairwise comparisons of raw and composite (PC) song parameters, as well as discriminant functions analyses, reveal significant patterns of song divergence between sites. Second, we test the ability of territorial males at Borrero Bay to discriminate songs from the two sites. We find that G. fortis males can discriminate within-island song variants, responding more strongly to local than to "foreign" songs, along 3 raw and 1 composite response measures. Third, we compare these findings to prior data sets on song divergence and discrimination in Santa Cruz G. fortis. These comparisons suggest that song divergence and discrimination are shaped less strongly by geographic sources than by morphological (beak-related) sources. We thus argue that interpopulation song divergence and discrimination, fundamental elements of assortative mating in Darwin's finches, can be fostered in early stages of divergence under magic trait as well as alternative scenarios for speciation, but with more emphasis on the magic trait scenario, at least for this species on this island [Current Zoology 59 (1): 8-19, 2013].

We have shown that expression of a truncated constitutively active ErbB2 receptor (ΔNErbB2), which is associated with poor prognosis in breast cancer, greatly increased net acid extrusion in MCF‐7 breast cancer cells in a manner mediated by the Na+/H+ exchanger NHE1 and the Na+,HCO3− cotransporter NBCn1. Here, we explore the roles of NHE1 and NBCn1 in cisplatin‐induced cancer cell death. In both vector‐ and ΔNErbB2 cells, cisplatin treatment (25 μM, 18 h) induced cleavage of caspase‐9,‐7, PARP, and fodrin (a calpain substrate), and cysteine cathepsin release, all in a manner slightly augmented by NHE1 inhibition. Expression of the anti‐apoptotic Bcl‐2 protein was markedly reduced in ΔNErbB2‐ compared to vector cells. Cisplatin treatment increased p53‐ and PUMA levels in both cell types, and reduced the number of autophagic vesicles and increased p62 levels, most markedly in ΔNErbB2 cells. This reduction of autophagic flux was not significantly affected by inhibition of NHE1 or NBCn1. Finally, cisplatin treatment elicited marked perinuclear accumulation of NBCn1, but not of NHE1, and reduced the protein levels of both full length‐ and ΔN‐ErbB2. In conclusion, cisplatin treatment of MCF‐7 cells activates several death mechanisms, some of which are further exacerbated by NHE1 inhibition in agreement with previous findings that NHE1 inhibition sensitizes MCF‐7‐ΔNErbB2 cells to cisplatin‐induced death.Funding: Danish Cancer Society, Danish Research Council