We show that double strand breaks (DSBs) induced in chromatin of low as well as high density by exposure of human cells to gamma-rays are repaired in low-density chromatin. Extensive chromatin decondensation manifested in the vicinity of DSBs by decreased intensity of chromatin labelling, increased H4K5 acetylation, and decreased H3K9 dimethylation was observed already 15 min after irradiation. Only slight movement of sporadic DSB loci for short distances was noticed in living cells associated with chromatin decondensation around DSBs. This frequently resulted in their protrusion into the low-density chromatin domains. In these regions, the clustering (contact or fusion) of DSB foci was seen in vivo, and in situ after cell fixation. The majority of these clustered foci were repaired within 240 min, but some of them persisted in the nucleus for several days after irradiation, indicating damage that is not easily repaired. We propose that the repair of DSB in clustered foci might lead to misjoining of ends and, consequently, to exchange aberrations. On the other hand, the foci that persist for several days without being repaired could lead instead to cell death.

Researchers interested in ecological immunology face substantial methodological problems: 1) most immunological approaches are diffi cult to perform in free-living animals, 2) in some of the applicable methods the immunological background of the test remains unclear. Th e latter is also true for the phytohaemagglutinin (PHA) skin-swelling test, a trait of cell-mediated immunity commonly measured in ecology. A lack of direct evidence documenting the immunological processes in the tissue limits our understanding of the mechanism triggering the response to PHA. Understanding of this mechanism is, nonetheless, crucial for us to uncover the nature of ecological costs and benefi ts of investments into the response. As knowledge of cytokine signalling in the tissue may clarify the response mechanism, in our study we investigated the association between the PHA-induced skin-swelling and tissue cytokine expression in males of grey partridge Perdix perdix . In PHA-challenged birds we assessed expression of nine cytokines (IL-1 β , IL-2, IL-4, IL-6, IL-10, IL-12, IL-17, TGF-β , IFN-γ ) in wing-web skin during an early stage of the immune response. We examined the relationship between the magnitude of tissue swelling and cytokine expression. Contrary to some earlier expectations we did not fi nd any diff erential expression of T-cell growth factor, IL-2, in the tissue. Hence, T-cell proliferation at the time of the swelling measurement is unlikely. We detected diff erential expression in Th 17 pro-infl ammatory (IL-1 β , IL-6) and anti-infl ammatory (TGF-β ) cytokines. Th e PHA-induced swelling response was only weakly linked to the expression of TGF-β . We also found relationships between the PHA-induced swelling response and phenotypic traits of the birds; the PHA swelling was positively associated with the extent of melanin-based breast ornamentation and negatively related to body size. Our results might suggest that variation in swelling is infl uenced by total numbers of responding cells rather than by diff erences in signalling. Moreover, we revealed signifi cant correlations in expression of IL-1 β , IL-6 and TGF-β . Th ese fi ndings are the fi rst to show on the molecular level that the PHA skin-swelling test actually measures infl ammation process which is part of innate immune defence and not the adaptive immune response (as assumed if the test was the refl ection of T-cell proliferation).

Several recent hypotheses consider oxidative stress to be a primary constraint ensuring honesty of condition-dependent carotenoid-based signalling. The key testable difference between these hypotheses is the assumed importance of carotenoids for redox homeostasis, with carotenoids being either antioxidant, pro-oxidant or unimportant. We tested the role of carotenoids in redox balance and sexual signalling by exposing adult male zebra finches (Taeniopygia guttata) to oxidative challenge (diquat dibromide) and manipulating carotenoid intake. As the current controversy over the importance of carotenoids as antioxidants could stem from the hydrophilic basis of commonly-used antioxidant assays, we used the novel measure of in vivo lipophilic antioxidant capacity. Oxidative challenge reduced beak pigmentation but elicited an increase in antioxidant capacity suggesting resource reallocation from signalling to redox homeostasis. Carotenoids counteracted the effect of oxidative challenge on lipophilic (but not hydrophilic) antioxidant capacity, thereby supporting carotenoid antioxidant function in vivo. This is inconsistent with hypotheses proposing that signalling honesty is maintained through either ROS-induced carotenoid degradation or the pro-oxidant effect of high levels of carotenoid-cleavage products acting as a physiological handicap. Our data further suggest that assessment of lipophilic antioxidant capacity is necessary to fully understand the role of redox processes in ecology and evolution.

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