One of the key tenets of life-history theory is that reproduction and survival are linked and that they trade-off with each other. When dietary resources are limited, reduced reproduction with a concomitant increase in survival is commonly observed. It is often hypothesized that this dietary restriction effect results from strategically reduced investment in reproduction in favor of somatic maintenance to survive starvation periods until resources become plentiful again. We used experimental evolution to test this "waiting-for-the-good-times" hypothesis, which predicts that selection under sustained dietary restriction will favor increased investment in reproduction at the cost of survival because "good-times" never come. We assayed fecundity and survival of female Drosophila melanogaster fruit flies that had evolved for 50 generations on three different diets varying in protein content-low (classic dietary restriction diet), standard, and high-in a full-factorial design. High-diet females evolved overall increased fecundity but showed reduced survival on low and standard diets. Low-diet females evolved reduced survival on low diet without corresponding increase in reproduction. In general, there was little correspondence between the evolution of survival and fecundity across all dietary regimes. Our results contradict the hypothesis that resource reallocation between fecundity and somatic maintenance underpins life span extension under dietary restriction.

Individual variation in coping with environmental challenges is a well-known phenomenon across vertebrates, including teleost fish. Dopamine is the major transmitter in the brain reward networks, and important for motivational processes and stress coping. Functions of the endogenous opioid system are not well studied in teleosts. However, in mammals the activity in the brain reward networks is regulated by the endogenous opioid system. This study aimed at investigating if there was a correlation between risk-taking behavior and the expression of dopamine and opioid receptors in the zebrafish (Danio rerio) brain. Risk-taking behavior was assessed in a novel tank diving test, and the most extreme high risk taking, i.e. bold, and low risk taking, i.e. shy, fish were sampled for qPCR analysis of whole brain gene expression. The expression analysis showed a significantly higher expression of the dopamine D2 receptors (drd2a and drd2b) and the delta opioid receptor (DOR; oprd1b) in bold compared to shy fish. Besides reward and reinforcing properties, DORs are also involved in emotional responses. Dopamine D2 receptors are believed to be important for active stress coping in rodents, and taken together the results of the current study suggest similar functions in zebrafish. However, additional experiments are required to clarify how dopamine and opioid receptor activation affect behavior and stress coping in this species.

Autosomal recessive congenital ichthyosis (ARCI) is a group of monogenic skin disorders caused by mutations in any of at least 12 different genes, many of which are involved in the epidermal synthesis of ω‐O‐acylceramides (acylCer). AcylCer are essential precursors of the corneocyte lipid envelope crosslinked by transglutaminase‐1 (TGm‐1), or a yet unidentified enzyme, for normal skin barrier formation. We hypothesized that inactivating TGM1 mutations will lead to a compensatory overexpression of the transcripts involved in skin barrier repair, including many other ARCI‐causing genes. Using microarray, we examined the global mRNA expression profile in skin biopsies from five ARCI patients with TGM1 mutations and four healthy controls. There were a total of 599 significantly differentially expressed genes (adjusted P < 0.05), out of which 272 showed more than 1.5 log2fold‐change (FC) up‐ or down‐regulation. Functional classification of the latter group of transcripts showed enrichment of mRNA encoding proteins mainly associated with biological pathways involved in keratinocyte differentiation and immune response. Moreover, the expression of seven out of twelve ARCI‐causing genes was significantly increased (FC = 0.98‐2.05). Also, many of the genes involved in keratinocyte differentiation (cornified envelope formation) and immune response (antimicrobial peptides and proinflammatory cytokines) were upregulated. The results from the microarray analysis were also verified for selected genes at the mRNA level by qPCR and at the protein level by semi‐quantitative immunofluorescence. The upregulation of these genes might reflect a compensatory induction of acylCer biosynthesis as a part of a global barrier repair response in the patient′s epidermis.

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Immunofluorescence (IF) and in situ proximity ligation assay (isPLA) are techniques that are used for in situ protein expression and colocalisation analysis, respectively.However, an efficient quantitative method to analyse both IF and isPLA staining on skin sections is lacking. Therefore, we developed a new method for semi-automatic quantitative layer-by-layer measurement of protein expression and colocalisation in skin sections using the free open-source software CellProfiler. As a proof of principle, IF and isPLA of ichthyosis-related proteins TGm-1 and SDR9C7 were examined. The results indicate that this new method can be used for protein expression and colocalisation analysis in skin sections. [1] isPLA signals, each representing one colocalisation, are seen as fluorescent spots under the microscope.[1]Both IF and isPLA have already been applied in subjective and semi-quantitative analysis of protein expression and colocalisation in skin sections. [2,3] However, an efficient quantitative image analysis method for IF and isPLA is lacking. A previous report described advanced automatic segmentation of the epidermal part of the images using the commercial scripting tool Matlab. [4] Another study used the open-source software ImageJ to quantify colorimetric staining of melanin in skin without segmentation of the epidermis. [5] CellProfiler is also a free and open-source software and does not require any programming skills, which makes it easy to handle. | QUESTION ADDRESSEDWe developed a new, efficient method for objective quantitative image analysis of protein expression and colocalisation in different epidermal layers of skin sections. | EXPERIMENTAL DESIGN | Human skin samplesThis study was conducted in conformity with Declaration of Helsinki Principles and approved by Regional Ethical Review Board at Uppsala University. After informed and written consent and local anaesthesia, punch biopsies (3 mm) were obtained from healthy skin donors, fixed in 4% buffered formalin and embedded in paraffin. | 197 METHODS LETTERS TO THE EDITOR | Immunofluorescence (IF) and in situ proximity ligation assay (isPLA)IF staining was performed as described in Supporting Information. The isPLA was performed according to the manufacturer's instructions (Sigma-Aldrich, St. Louis, MO, USA). Microscopy images of IF and isPLA staining were acquired as described in Supporting Information.Images for IF were collected in single focal plane, whereas isPLA images were collected as z-stacks to enable detection of signals present at different depths in the skin sections. | Quantitative analysis of IF staining and isPLA signalsA detailed instruction on how to use the CellProfiler pipelines, Pipeline I projection, Pipeline II region-of-interest (ROI) and Pipeline III measurement, can be found in the Supporting Information. The CellProfiler software and the pipelines used in this study will be available for downloading at www.cellprofiler.org.