3D bioprinting of collagen to rebuild components of the human heart.

Collagen is the primary component of the extracellular matrix in the human body. It has proved challenging to fabricate collagen scaffolds capable of replicating the structure and function of tissues and organs. We present a method to 3D-bioprint collagen using freeform reversible embedding of suspended hydrogels (FRESH) to engineer components of the human heart at various scales, from capillaries to the full organ. Control of pH-driven gelation provides 20-micrometer filament resolution, a porous microstructure that enables rapid cellular infiltration and microvascularization, and mechanical strength for fabrication and perfusion of multiscale vasculature and tri-leaflet valves. We found that FRESH 3D-bioprinted hearts accurately reproduce patient-specific anatomical structure as determined by micro-computed tomography. Cardiac ventricles printed with human cardiomyocytes showed synchronized contractions, directional action potential propagation, and wall thickening up to 14% during peak systole.

Immunohistochemical expression of the nicotinic acetylcholine receptor (nAChR) subunits in the human placenta, and effects of cigarette smoking and preeclampsia.

INTRODUCTION: Cigarette smoking (CS) and preeclampsia (PE), regulate the expression of nicotinic acetylcholine receptor (nAChR) subunits in the placenta, yet no data exist at the histological level. METHODS: Using immunohistochemistry of formalin fixed and paraffin embedded placental tissue, this study quantified the expression of nine nAChR subunits (α2, α3, α4, α5, α7, α9, ß1, ß2, δ) and compared the expression amongst four groups of non-smoker non-PE (controls, n = 8), smokers (n = 8), PE (n = 8), and those who were smokers with PE (smoke + PE, n = 4). Quantification was of the percentage of villi with positive cells stained (% villi with +ve), percentage of positive stained cells per villous (% +ve cells/villous), percentage of positive cells in the decidua (%+ve Decidua), and intensity of staining in the outer villous trophoblast layer. RESULTS: Changes were restricted to the villi (as opposed to the decidua), and were specific to the α9 (smoke + PE), ß1 (smokers), and ß2 (PE) subunits when compared to controls. CS seemed to have a protective effect for the ß2 subunit and an additive effect for the α9 and ß1 subunits within the villous core/stroma cells and not the trophoblast layer. DISCUSSION: These findings support that both CS and PE affect nAChRs in the placenta, but that this is restricted to the villi.

The IUR Forum: Worldwide Harmonisation of Networks to Support Integration of Scientific Knowledge and Consensus Development in Radioecology.

During the past decades, many specialised networks have formed to meet specific radioecological objectives, whether regional or sectorial (purpose-oriented). Regional networks deal with an array of radioecological issues related to their territories. Examples include the South Pacific network of radioecologists, and the European network of excellence in radioecology. The latter is now part of the European platform for radiation protection. Sectorial networks are more problem-oriented, often with wider international representativeness, but restricted to one specific issue, (e.g. radioactive waste, low-level atmospheric contamination, modelling). All such networks, while often working in relative isolation, contribute to a flow of scientific information which, through United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR's) efforts of synthesis, feeds into the radiation protection frameworks of protecting humans and the environment. The IUR has therefore prompted a co-construction process aimed at improving worldwide harmonisation of radioecology networks. An initiative based on an initial set of 15 networks, now called the IUR FORUM, was launched in June 2014. The IUR Forum agreed to build a framework for improved coordination of scientific knowledge, integration and consensus development relative to environmental radioactivity. Three objectives have been collectively assigned to the IUR FORUM: (1) coordination, (2) global integration and construction of consensus and (3) maintenance of expertise. One particular achievement of the FORUM was an improved description and common understanding of the respective roles and functions of the various networks within the overall scene of radioecology R&D. It clarifies how the various networks assembled within the IUR FORUM interface with UNSCEAR and other international regulatory bodies (IAEA, ICRP), and how consensus on the assessment of risk is constructed. All these agencies interact with regional networks covering different geographical areas, and with other networks which address specific topics within radiation protection. After holding its first Consensus Symposium in 2015, examining the possible ecological impact of radiation from environmental contamination, the IUR FORUM continues its work towards improved radiation protection of humans and the environment. We welcome new members.

Cigarette smoking during pregnancy regulates the expression of specific nicotinic acetylcholine receptor (nAChR) subunits in the human placenta.

Smoking during pregnancy is associated with low birth weight, premature delivery, and neonatal morbidity and mortality. Nicotine, a major pathogenic compound of cigarette smoke, binds to the nicotinic acetylcholine receptors (nAChRs). A total of 16 nAChR subunits have been identified in mammals (9 α, 4 ß, and 1 δ, γ and ε subunits). The effect of cigarette smoking on the expression of these subunits in the placenta has not yet been determined, thus constituting the aim of this study. Using RT-qPCR and western blotting, this study investigated all 16 mammalian nAChR subunits in the normal healthy human placenta, and compared mRNA and protein expressions in the placentas from smokers (n = 8) to controls (n = 8). Our data show that all 16 subunit mRNAs are expressed in the normal, non-diseased human placenta and that the expression of α2, α3, α4, α9, ß2 and ß4 subunits is greater than the other subunits. For mRNA, cigarette smoke exposure was associated with increased expression of the α9 subunit, and decreased expression of the δ subunit. At the protein level, expression of both α9 and δ was increased. Thus, cigarette smoking in pregnancy is sufficient to regulate nAChR subunits in the placenta, specifically α9 and δ subunits, and could contribute to the adverse effects of vasoconstriction and decreased re-epithelialisation (α9), and increased calcification and apoptosis (δ), seen in the placentas of smoking women.

DNA damage caused by chronic transgenerational exposure to low dose gamma radiation in Medaka fish ( Oryzias latipes ).

The effect of transgenerational exposure to low dose rate (2.4 and 21 mGy/day) gamma irradiation on the yield of DNA double-strand breaks and oxidized guanine (8-hydroxyguanine) has been studied in the muscle and liver tissue of a model organism, the Japanese medaka fish. We found the level of unrepaired 8-hydroxyguanine in muscle tissue increased nonlinearly over four generations and the pattern of this change depended on the radiation dose rate, suggesting that our treatment protocols initiated genomic instability and an adaptive response as the generations progressed. The yield of unrepaired double-strand breaks did not vary significantly among successive generations in muscle tissue in contrast to liver tissue in which it varied in a nonlinear manner. The 8-hydroxyguanine and DSB radiation yields were significantly higher at 2.4 mGy/day than at 21 mGy/day in both muscle and liver tissue in all generations. These data are consistent with the hypothesis of a threshold for radiation-induced activation of DNA repair systems below which tissue levels of DNA repair enzymes remain unchanged, leading to the accumulation of unrepaired damage at very low doses and dose rates.

Are radiosensitivity data derived from natural field conditions consistent with data from controlled exposures? A case study of Chernobyl wildlife chronically exposed to low dose rates.

The discrepancy between laboratory or controlled conditions ecotoxicity tests and field data on wildlife chronically exposed to ionising radiation is presented for the first time. We reviewed the available chronic radiotoxicity data acquired in contaminated fields and used a statistical methodology to support the comparison with knowledge on inter-species variation of sensitivity to controlled external γ irradiation. We focus on the Chernobyl Exclusion Zone and effects data on terrestrial wildlife reported in the literature corresponding to chronic dose rate exposure situations (from background ~100 nGy/h up to ~10 mGy/h). When needed, we reconstructed the dose rate to organisms and obtained consistent unbiased data sets necessary to establish the dose rate-effect relationship for a number of different species and endpoints. Then, we compared the range of variation of radiosensitivity of species from the Chernobyl-Exclusion Zone with the statistical distribution established for terrestrial species chronically exposed to purely gamma external irradiation (or chronic Species radioSensitivity Distribution - SSD). We found that the best estimate of the median value (HDR50) of the distribution established for field conditions at Chernobyl (about 100 µGy/h) was eight times lower than the one from controlled experiments (about 850 µGy/h), suggesting that organisms in their natural environmental were more sensitive to radiation. This first comparison highlights the lack of mechanistic understanding and the potential confusion coming from sampling strategies in the field. To confirm the apparent higher sensitive of wildlife in the Chernobyl Exclusion Zone, we call for more a robust strategy in field, with adequate design to deal with confounding factors.

An invitation to contribute to a strategic research agenda in radioecology.

With intentions of integrating a portion of their respective research efforts into a trans-national programme that will enhance radioecology, eight European organisations recently formed the European Radioecology ALLIANCE (www.er-alliance.org). The ALLIANCE is an Association open to other organisations throughout the world with similar interests in promoting radioecology. The ALLIANCE members recognised that their shared radioecological research could be enhanced by efficiently pooling resources among its partner organizations and prioritising group efforts along common themes of mutual interest. A major step in this prioritisation process was to develop a Strategic Research Agenda (SRA). An EC-funded Network of Excellence in Radioecology, called STAR (Strategy for Allied Radioecology), was formed, in part, to develop the SRA. This document is the first published draft of the SRA. The SRA outlines a suggested prioritisation of research topics in radioecology, with the goal of improving research efficiency and more rapidly advancing the science. It responds to the question: "What topics, if critically addressed over the next 20 years, would significantly advance radioecology?" The three Scientific Challenges presented within the SRA, with their 15 associated research lines, are a strategic vision of what radioecology can achieve in the future. Meeting these challenges will require a directed effort and collaboration with many organisations the world over. Addressing these challenges is important to the advancement of radioecology and in providing scientific knowledge to decision makers. Although the development of the draft SRA has largely been a European effort, the hope is that it will initiate an open dialogue within the international radioecology community and its stakeholders. This is an abbreviated document with the intention of introducing the SRA and inviting contributions from interested stakeholders. Critique and input for improving the SRA are welcomed via a link on the STAR website (www.star-radioecology.org).

Effects of chronic exposure in populations of Koeleria gracilis Pers. from the Semipalatinsk nuclear test site, Kazakhstan.

Morphological and cytogenetic abnormalities were examined in crested hairgrass (Koeleria gracilis Pers.) populations inhabiting the Semipalatinsk nuclear test site (STS), Kazakhstan. Sampling of biological material and soil was carried out during 3 years (2005-2007) at 4 sites within the STS. Activity concentrations of 10 radionuclides and 8 heavy metals content in soils were measured. Doses absorbed by plants were estimated and varied, depending on the plot, from 4 up to 265 mGy/y. The frequency of cytogenetic alterations in apical meristem of germinated seeds from the highly contaminated plot significantly exceeded the level observed at other plots with lower levels of radioactive contamination during all three years of the study. A significant excess of chromosome aberrations, typical for radiation exposure, as well as a dependence of the frequency of these types of mutations on dose absorbed by plants were revealed. The results indicate the role radioactive contamination plays in the occurrence of cytogenetic effects. However, no radiation-dependent morphological alterations were detected in the progeny of the exposed populations. Given that the crested hairgrass populations have occupied the radioactively contaminated plots for some 50 years, adaptation to the radiation stress was not evident. The findings obtained were in agreement with the benchmark values proposed in the FASSET and ERICA projects to restrict radiation impacts on biota.

Investigating the embryo/larval toxic and genotoxic effects of γ irradiation on zebrafish eggs.

Eggs/larval of freshwater fish (Danio rerio) were exposed to low dose rates of external gamma radiation (from 1 to 1000 mGy d(-1)) over a 20-day period, with the objective of testing the appropriateness of the 10 mGy d(-1) guideline suggested by the IAEA. The present study examines different endpoints, mortality and hatching time and success of embryos as well as the genotoxicity of γ-irradiations (after 48 h). The 20-day embryo-larval bioassay showed an enhanced larval resistance to starvation after chronic exposure to γ irradiation (from low 1 mGy d(-1) to high dose rate 1000 mGy d(-1)) and an acceleration in hatching time. Gamma irradiation led to increased genotoxic damage Ito zebrafish egg (40-50% DNA in tail in Comet assay) from the lowest dose rate (1 mGy d(-1)). Possible mechanisms of γ radiotoxicity and implications for radioprotection are discussed.

Cesium accumulation by aquatic organisms at different trophic levels following an experimental release into a small reservoir.

The rates of accumulation and subsequent loss of stable cesium (¹³³Cs) by organisms at different trophic levels within plankton-based and periphyton-based food chains were measured following the addition of ¹³³Cs into a small reservoir near Aiken, South Carolina, USA. An uptake parameter u (L kg⁻¹ d⁻¹ dry mass) and a loss rate parameter k (d⁻¹) were estimated for each organism using time-series measurements of ¹³³Cs concentrations in water and biota, and these parameters were used to estimate maximum concentrations, times to maximum concentrations, and concentration ratios (C(r)). The maximum ¹³³Cs concentrations for plankton, periphyton, the insect larva Chaoborus punctipennis, which feeds on plankton, and the snail Helisoma trivolvis, which feeds on periphyton, occurred within the first 14 days following the addition, whereas the maximum concentrations for the fish species Lepomis macrochirus and Micropterus salmoides occurred after 170 days. The C(r) based on dry mass for plankton and C. punctipennis were 1220 L kg⁻¹ and 5570 L kg⁻¹, respectively, and were less than the C(r) of 8630 L kg⁻¹ for periphyton and 47,700 L kg⁻¹ for H. trivolvis. Although the C(r) differed between plankton-based and periphyton-based food chains, they displayed similar levels of biomagnification. Biomagnification was also indicated for fish where the C(r) for the mostly nonpiscivorous L. macrochirus of 22,600 L kg⁻¹ was three times less than that for mostly piscivorous M. salmoides of 71,500 L kg⁻¹. Although the C(r) for M. salmoides was greater than those for periphyton and H. trivolvis, the maximum ¹³³Cs concentrations for periphyton and H. trivolvis were greater than that for M. salmoides.

Differential genetic responses to ionizing irradiation in individual families of Japanese medaka, Oryzias latipes.

Although no statistically significant hereditary effects have yet been detected in the children of survivors from the atomic bombings in Hiroshima and Nagasaki, recent animal studies have found that exposure to ionizing radiation can cause genomic and epigenomic instability in the exposed individuals, as well as their offspring, and therefore, may have much larger genetic effects than predicted by earlier studies. When individuals are exposed to various environmental insults, including radiation, individual sensitivity to the insults often varies. Variance in germ-line response to radiation among individuals has been widely recognized, but it is difficult to address due to the use of inbred strains and the limited number of offspring that can be produced by a pair of mice, the common model used to study genetic effects of radiation. Herein is the first study to examine individual family responses to ionizing radiation using a parent-pedigree approach in an outbred strain of a vertebrate model, the Japanese medaka fish. Changes in frequencies of radiation-induced germline mutations at nine microsatellite loci were examined in the same families before and after exposure to one of four acute doses of ionizing radiation (0.1, 0.5, 2.5, 5Gy, plus sham-exposed controls). Families varied significantly in pre-exposure mutation frequencies and responses to irradiation, but germline mutations were elevated in at least one family after 0.1, 0.5, and 5Gy exposures. Variance among individuals in sensitivity to radiation is well documented for many endpoints, and our work now extends these endpoints to include germ-line mutations. Further studies are needed to elucidate dose response, effects at varying stages of spermatogenesis, and the mechanisms underlying the variance in these individual responses to radiation.

Contrasting cesium dynamics in neighboring deep and shallow warm-water reservoirs.

To measure the long term retention and seasonal dynamics of an initial 4 kg addition of (133)Cs into an 11.4-ha, 157,000 m(3) reservoir (Pond 4, near Aiken, South Carolina, USA), the concentrations and inventories of (133)Cs in the water column were measured at periodical intervals for 522 days following the 1 August, 1999 release. After rapid declines in concentrations and inventories during the first 90 days, the (133)Cs concentrations in the water column declined at an average proportional rate of 0.004 d(-1). However, there were periods of less rapid and more rapid rates of declines, and these were correlated with periods of increasing and decreasing K concentrations in the water column. The decline rates were less and the K concentrations greater in the winter than in the summer. In the deeper, neighboring monomictic reservoirs of Par Pond and Pond B, a yearly cycle of increasing and decreasing (137)Cs concentrations in the water column is driven by anoxic remobilization of Cs from the sediments into a persistent summer hypolimnion. In Pond 4, whose mean depth of 1.6 m is too shallow to support a persistent anoxic hypolimnion, the pattern of yearly dynamics for K and Cs appear to be related to the accumulation and release of these elements from the extensive, seasonal macrophyte communities. The contrasting results between Pond 4 and Pond B suggest that a full appreciation of the relative importance of 1) anoxic remobilization and 2) accumulation and release by macrophytes in these systems remains to be established.

Communication of radiation-induced signals in vivo between DNA repair deficient and proficient medaka (Oryzias latipes).

Radiation-induced bystander effects are established consequences of exposure to ionizing radiation. The operation of this mechanism has been seen in vitro and also between fish, mammals, and plants in vive where stress signals from treated organisms induce responses in neighbors. In vitro research shows that DNA repair deficient cells produce more toxic bystander responses. To test this in vivo two strains of Japanese medaka were tested. One is a mutant, repair deficient strain (ric2) and the other, the wildtype repair proficient strain (CAB). Irradiated fish swam with unirradiated partners in a strain mix and match protocol. The data suggest that medaka produce signals, when exposed to radiation, that induce unirradiated fish ofthe same strain swimming with them to produce an altered response to that seen in bystanders to sham irradiated fish. More apoptosis was seen in bystanders to repair deficient fish. When the strains are mixed, the bystanders of either strain respond like the donor strain. Measurements of Bcl-2 and cmyc proteins in the explants confirmed these observations. A possible role for p53 was also identified in that the use of reporters with mutant p53 demonstrated that CAB signals killed all the reporter cells by apoptosis. Use of a similar but p53 wildtype cell line had no such effect. The data add to the body of knowledge showing that bystander signals operate at hierarchical levels of organization greater than the individual and may therefore have relevance in radioecology and (eco)systems biology.

Cesium accumulation by fish following acute input to lakes: a comparison of experimental and Chernobyl-impacted systems.

An uptake parameter u (Lkg(-1)d(-1)) and a loss rate parameter k (d(-1)) were estimated for the patterns of accumulation and loss of (133)Cs by three fish species following an experimental (133)Cs addition into a pond in South Carolina, USA. These u and k parameters were compared to similar estimates for fish from other experimental ponds and from lakes that received (137)Cs deposition from Chernobyl. Estimates of u from ponds and lakes declined with increasing potassium concentrations in the water column. Although loss rates were greater in the experimental ponds, the times required to reach maximum Cs concentrations in fish were similar between ponds and lakes, because ponds and lakes had similar retentions of Cs in the water column. The maximum Cs concentrations in fish were largely determined by initial Cs concentrations in the water column. These maximum concentrations in fish and the times required to reach these maxima are potentially useful indicators for assessments of risks to humans from fish consumption.

Sex-differences and stress: effects on regional high and low affinity [3H]GABA binding.

Sex-differences are observed in the GABAergic neurotransmitter system both at rest and following acute stress, yet the brain regions and functional implications of these differences are unknown. We examined sex-differences in the number of low- and high-affinity [3H]GABA binding sites in various brain regions of male and female mice and the effect of stress on such sex-differences. Male (n=6) and female (n=6) QS mice were exposed to a brief swim stress (3 min at 32+/-1 degrees C) either individually or with cage-mates whilst control males (n=6) and females (n=6) remained undisturbed in the home cage. Using quantitative receptor autoradiography, sections of mouse brain were labelled with either 30 or 1000 nM [3H]GABA to label high or low affinity binding sites, respectively. Results indicated that males had more low affinity [3H]GABA binding sites in various forebrain cortical regions but less high affinity binding sites in many of these regions compared with females. Forced swim stress-induced rapid changes in forebrain GABA binding sites in females and group stressed males, suggesting a mechanism for rapid GABAergic adaptations. However the number of functional binding sites for GABA in certain forebrain regions was altered by stress in opposite directions in males and females, such that baseline sex-differences were removed following stress. These results exemplify sex-differences in brain chemical function and stress responses, and are of potential importance for understanding sex-differences in response to GABAergic compounds and disorders with sex and stress as predisposing factors.

Radiation-induced untargeted germline mutations in Japanese medaka.

Radiation has been shown to increase mutation frequencies at tandem repeat loci by indirect interactions of radiation with DNA. We studied germline mutations in chronically exposed Japanese medaka (Oryzias latipes) using microsatellite loci. After screening 26 randomly selected loci among unirradiated parents and their 200 offspring, we selected seven highly mutable loci (0.5-1.0 x 10(-2) mutants per locus per gamete) and two bonus loci for further study. To determine if radiation exposure increases mutation frequencies in these loci, medaka were chronically irradiated from subadults through maturation at relatively low dose rates of 68 mGy/d. Total doses for males and females were 10.4 and 3 Gy, respectively. The mean number of mutations for the offspring of exposed families (0.149+/-0.044) was significantly higher (P=0.018) than for control families (0.080+/-0.028), indicating induction of germline mutations from chronic irradiation. This increase in the microsatellite mutation rate is greater than expected from direct interaction of radiation with DNA, suggesting indirect, untargeted mechanism(s) for mutations. This study identified microsatellite loci with a high mutational background in medaka, variation among loci and families as important variables, and demonstrated the usefulness of this fish model for studying radiation-induced germline mutations.

Foliar uptake of cesium from the water column by aquatic macrophytes.

The probable occurrence and rate of foliar absorption of stable cesium (133Cs) from the water column by aquatic macrophyte species was analyzed following the addition of 133Cs into a small reservoir near Aiken, South Carolina, USA. An uptake parameter u (10(3)Lkg(-1)d(-1)) and a loss rate parameter k (d(-1)) were estimated for each species using time series of 133Cs concentrations in the water and plant tissues. Foliar uptake, as indicated by rapid increases in plant concentrations following the 133Cs addition, occurred in two floating-leaf species, Brasenia schreberi and Nymphaea odorata, and two submerged species, Myriophyllum spicatum and Utricularia inflata. These species had values of u> or =0.75 x 10(3)Lkg(-1)d(-1). Less evidence for foliar uptake was observed in three emergent species, including Typha latifolia. Ratios of u to k for B. schreberi, M. spicatum, N. odorata and U. inflata can be used to estimate concentration ratios (CR) at equilibrium, and these estimates were generally within a factor of 2 of the CR for 137Cs for these species in the same reservoir. This correspondence suggests that foliar uptake of Cs was the principal absorption mechanism for these species. Assessments of: (1) the prevalence of foliar uptake of potassium, rubidium and Cs isotopes by aquatic macrophytes and (2) the possible importance of foliar uptake of Cs in other lentic systems are made from a review of foliar uptake studies and estimation of comparable u and k values from lake studies involving Cs releases.

The influence of a whole-lake addition of stable cesium on the remobilization of aged 137Cs in a contaminated reservoir.

To document the short-term dynamics of Cs, 4 kg of (133)Cs were introduced into an 11.4-ha, 157 000 m(3) reservoir previously contaminated with (137)Cs from past reactor operations at the US Department of Energy's Savannah River Site near Aiken, South Carolina, USA. The (133)Cs addition resulted in an increase of 6.1 MBq of (137)Cs (1.9 mug (137)Cs) in the water column over the following 260 days. Possible sources for the increased (137)Cs included (1) release from the sediments, (2) release from the approximately 26 000 kg of aquatic macrophytes that occupied 80% of the reservoir, and (3) wash-in from the pond's watershed. Data are presented to indicate that release from the sediments was the principal source of the (137)Cs increase. The fraction of (137)Cs released from the sediments (0.7%) is consistent with laboratory measurements of (137)Cs desorption from neighboring ponds on the Savannah River Site.