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The preimplantation embryo is sensitive to culture conditions in vitro and poor maternal diet in vivo. Such environmental perturbations can have long-lasting detrimental consequences for offspring health and physiology. However, early embryo susceptibility to other aspects of maternal health and their potential long-term influence into adulthood is relatively unexplored. In this study, we established an in vivo mouse model of maternal periconceptional systemic inflammation by intraperitoneal lipopolysaccharide (LPS) administration on the day of zygote formation and investigated the consequences into adulthood.
In the short term, maternal LPS challenge induced a transient and typical maternal sickness response (elevated serum proinflammatory cytokines and hypoactive behaviour). Maternal LPS challenge altered preimplantation embryo morphogenesis and cell lineage allocation, resulting in reduced blastocyst inner cell mass (ICM) cell number and a reduced ICM:trophectoderm cell ratio. In the long term, diverse aspects of offspring physiology were affected by maternal LPS treatment. Whilst birthweight, growth and adult blood pressure were unaltered, reduced activity in an open-field behaviour test, increased fat pad:body weight ratio and increased body mass index were observed in male, but not female, offspring. Most importantly, the maternal LPS challenge caused corticosterone-independent blunting of the serum proinflammatory cytokine response to innate immune challenge in both male and female offspring. The suppressed state of innate immunity in challenged offspring was dose-dependent with respect to the maternal LPS concentration administered.
These results demonstrate for the first time that the preimplantation embryo in vivo is sensitive to maternal systemic inflammation, with effects on blastocyst cell lineage allocation and consequences for behaviour, adiposity and innate immune response in adult offspring. Critically, we identify a novel mechanism mediated through maternal-embryonic interactions that confers plasticity in the development of the innate immune system, which is potentially important in setting postnatal tolerance to environmental pathogens. Our study extends the concept of developmental programming of health and disease to include maternal health at the time of conception.
Substantial evidence supports the concept that adult health and physiology are influenced by environmental factors during the gestational and infant stages of development [1-3]. The developmental origins of health and disease (DOHaD) hypothesis proposes that such programming may be contributing to the increasing prevalence of hypertension, diabetes and other noncommunicable diseases in recent decades [1]. Programming can be initiated even during the periconceptional period: the preimplantation embryo has proven sensitive to environmental perturbation mediated through maternal diet or in vitro culture, with concomitant postnatal dysfunction of cardiovascular, metabolic and behavioural systems [2-6]. Whether other aspects of periconceptional maternal health (for example, infection, inflammation) can induce long-term programming has not yet been explored in detail.
Inflammation is associated with the release of potent cellular mediators (for example, cytokines, glucocorticoids (GCs)) which communicate at autocrine and paracrine levels. Such mediators alter and regulate homeostasis and may modify the developmental environment. Maternal infection and inflammation during mid- to late gestation affects offspring behaviour [7,8], immunity [9-11], metabolic status [12] and hypothalamic-pituitary-adrenal (HPA) axis functioning [13]. Little is known about the postnatal sequelae of maternal inflammation during the periconceptional period, although an association with an increased risk of schizophrenia has been reported [14,15]. In this study, we established an in vivo mouse model of maternal periconceptional systemic inflammation (MPSI) by lipopolysaccharide (LPS) administration on the day of zygote formation.
We found that the maternal LPS challenge induces a typical and transient sickness response and, critically, altered developmental programming with abnormal blastocyst cell lineage allocation and led to postnatal changes in behaviour, adiposity and, following innate immune challenge, in cytokine release. Our study therefore establishes that maternal sickness at the time of conception, or immune response conditions associated with it, can have lifelong consequences on offspring health, thereby extending the concept of DOHaD. Moreover, our data show for the first time that the responsiveness of the innate immune system in an adult mammal is subject to environmental conditions originating at the time of conception.
Maternal inflammatory response
Intraperitoneal administration of LPS (10, 50 or 150 μg/kg dose vs. control) into dams at gestational day (GD) 0.5 (zygote stage) induced a typical sickness response, including associated metabolic and behavioural changes (Figure 1). One hour after treatment LPS-injected mice displayed reduced open-field activity, including a 90% to 95% reduction in jumping activity (P < 0.001 for all doses) and increased time spent resting (P < 0.05 at 50 and 150 μg/kg LPS doses) (Figure 1A). At 24 hours post-LPS injection, food consumption was reduced at all doses (10% to 12% reduction; P < 0.001) (Figure 1B), as was water consumption at the 50 and 150 μg/kg LPS doses.
Figure 1. Maternal inflammatory response following treatment with saline or LPS doses. LPS treatment (particularly 50 and 150 μg/kg) (A) reduced jumping activity and increased time spent resting in an open-field test at one hour posttreatment, (B) reduced food and water consumption during the first 24 hours posttreatment, (C) reduced body weight at 24 and 48 hours posttreatment (dashed line = preinjection body weight) and (D) increased serum concentration (pg/mL) of inflammatory cytokines posttreatment. Maternal body weight at each 24-hour interval posttreatment is expressed as a percentage of pretreatment body weight, as were food and water consumption during each 24-hour period posttreatment. All values represent means ± standard error of the mean (SEM). *P ≤ 0.05 vs. control (n = 5 to 7 mothers per treatment).
(P ≤ 0.006) (Figure 1B). Thereafter food and water consumption were unchanged between treatment groups (Figure 1B). Body weight at 24 hours was reduced by 3.8% to 5.7% in an LPS dose-dependent manner (P < 0.001 at 50 and 150 μg/kg and P < 0.01 at 10 μg/kg LPS doses) (Figure 1C). Body weight was similarly reduced at 48 hours (4.2% to 4.5% reduction; P ≤ 0.003) (Figure 1C) but returned to control levels by 72 hours (Figure 1C). A selection of the 23 cytokines detected by multiplex assay in maternal serum at 3 to 72 hours posttreatment is shown in Figure 1D, and the remaining cytokines are shown in Figure S1 in Additional file 1. Serum concentrations of most cytokines peaked at three hours post-LPS treatment, with the 150 μg/kg LPS dose resulting in a three- to fivefold increase in IL-1β, IL-3, IL-10, eotaxin, granulocyte-macrophage colony-stimulating factor (GM-CSF) and macrophage inflammatory protein 1α (MIP-1α; CCL3) (P < 0.05). Greater increases were seen at three hours in IL-12 p40 (48-fold; P = 0.001), monocyte chemoattractant protein 1 (MCP-1; CCL2) (58-fold; P = 0.004) and regulated upon activation, normal T-cell expressed and secreted (RANTES; CCL5) (52-fold; P = 0.044). At 48 hours, most cytokine levels from LPS-treated groups were similar to those of controls; however, some levels remained elevated (IL-6, IL-12 p70, RANTES and TNF-α; P ≤ 0.05), whilst others were decreased (IL-2, IL-10 and IL-13; P ≤ 0.05) (Figure 1D and Additional file 1, Figure S1).
Additional file 1. Figure S1. Maternal serum cytokine concentrations (pg/mL) 3 to 72 hours following treatment with saline or LPS. Bars: maternal LPS treatment; white: saline control; dark: 10 μg/kg; light: 50 μg/kg; hatched: 150 μg/kg. All values represent means ± SEM. *P ≤ 0.05 compared with control (n = 5 to 7 mothers per treatment). The multiplex assay allowed us to detect and quantify the full concentration range of every cytokine at every time point examined, except for G-CSF, KC and IL-6 at three hours postinjection. The data presented here for G-CSF, KC and IL-6 at three hours postinjection are underrepresentative of actual concentrations, particularly for the LPS-treated groups (indicated by ! on graphs).
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Embryo recovery and blastocyst cell number
The effect of maternal LPS administration on preimplantation embryo development was assessed at GD 3.5. Maternal LPS had no effect on total number of embryos recovered or the proportion that were blastocysts (Table S1 in Additional file 2). However, blastocysts from all LPS treatments had reduced inner cell mass (ICM) cell numbers (14% reduced vs. control in the 10 μg/kg LPS group, P = 0.009; 33% reduced in the 50 and 150 μg/kg LPS groups, P < 0.001) (Figure 2A). Trophectoderm (TE) cell numbers were unaffected, except in the 10 μg/kg LPS group (21% increased, P < 0.001) (Figure 2A). Total blastocyst cell numbers were increased in the 10 μg/kg LPS group (P = 0.017) and reduced in the 150 μg/kg LPS group (P = 0.004) (Figure 2A). Such alterations in cell numbers resulted in reduced ICM:TE cell ratios in blastocysts from all LPS treatments (24% to 42% reduced, P ≤ 0.001) (Figure 2B).
Additional file 2. Table S1. Embryos flushed from control and LPS-treated mothers at GD 3.5. Values represent means ± SEM (n = 4 to 8 mothers per treatment).
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Figure 2. GD 3.5 blastocysts from LPS-treated dams have fewer ICM cells and reduced ICM:TE cell ratios vs. controls. (A) Mean total, TE and ICM cell numbers and (B) ICM:TE cell ratios. All values represent means ± SEM. *P ≤ 0.05 vs. control (n = 33 to 50 blastocysts per treatment; n = 4 to 8 mothers per treatment).
Gestation, litter size and offspring growth
Gestation length, male:female ratio, mean birthweight and litter size were all unaffected, except for increased litter size in the 10 μg/kg LPS group (P = 0.046) (Table 1). The mean weekly body weights of male and female offspring from birth to 30 weeks were unaffected (Figure S2 in Additional file 3).
Table 1. Gestation and birth data for each maternal treatment groupa
Additional file 3. Figure S2. No difference in growth of female or male offspring from birth to 30 weeks. Values represent means ± SEM independent of maternal origin and litter size (n = 6 mothers per treatment; n = 18 male and 18 female offspring per treatment).
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Offspring behaviour
Integration of all weekly data (nine repeated analyses over weeks 4 to 23) into mean lifetime open-field activities showed that 150 μg/kg LPS males displayed reduced numbers of jumps and rears (reduced by 40% vs. control, P = 0.015, and reduced by 67% vs. control, P = 0.027, respectively) (Figure 3A), but no differences in distance travelled or time spent resting (Figure S3 in Additional file 4). Mean lifetime open-field activities of female offspring were unaffected (Figure 3A and Additional file 4, Figure S3). When open-field activities were examined on a weekly basis (Figure S4 in Additional file 5), male offspring from 150 μg/kg LPS-treated dams reared less than controls at weeks 4, 6, 11, 14 and 20 (all P < 0.05), while female offspring from 150 μg/kg LPS-treated dams reared less than controls only at week 8 (P = 0.001). In addition, male offspring from 150 μg/kg LPS-treated dams jumped less than controls at weeks 6 and 17 (P < 0.05), and rested longer at week 6 (P = 0.040). No differences between treatments were observed for females in number of jumps or time spent resting, and distance travelled was not different between treatments for males or females (Figure S4 in Additional file 5). Differences were independent of maternal origin and gestational litter size.
Figure 3. MPSI caused dose-dependent, gender-specific abnormalities in adult offspring behaviour and adiposity, but no abnormalities in systolic blood pressure (SBP). (A) Reduced jumping and rearing open-field activity in male, but not female, offspring from 150 μg/kg LPS dams. (B) Increased SBP in male offspring from 10 μg/kg LPS dams. (C) Increased fat pad to body weight ratio (%) and BMI in male offspring from 150 μg/kg LPS dams. All values represent means ± SEM. *P ≤ 0.05 vs. control independent of maternal origin and litter size (n = 6 mothers per treatment; n = 18 male and 18 female offspring per treatment).
Additional file 4. Figure S3. Lifetime mean distance travelled and time spent resting by adult offspring in the open-field behavioural test. Bars: maternal LPS treatment; white: saline control; dark: 10 μg/kg; light: 50 μg/kg; hatched: 150 μg/kg. Values represent means ± SEM independent of maternal origin and litter size (n = 6 mothers per treatment; n = 18 male and 18 female offspring per treatment).
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Additional file 5. Figure S4. Individual weekly open-field activity of adult male and female offspring. Values represent means ± SEM. *P ≤ 0.05 vs. control independent of maternal origin and litter size (n = 6 mothers per treatment; n = 18 male and 18 female offspring per treatment).
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Offspring systolic blood pressure
Integration of weekly measurements (three repeated analyses over weeks 16 to 28) into a 'lifetime' mean showed that systolic blood pressure (SBP) was not different, except in male offspring from 10 μg/kg LPS-treated dams, in which mean lifetime SPB was increased compared with controls (P = 0.029) (Figure 3B). On a weekly basis, male offspring from 10 μg/kg LPS-treated dams displayed increased SBP at weeks 16 and 28 compared with controls (P = 0.035 and P = 0.040, respectively) (Figure S5 in Additional file 6).
Additional file 6. Figure S5. Individual weekly systolic blood pressure (SBP) of adult male and female offspring. Values represent means ± SEM. *P ≤ 0.05 vs. control independent of maternal origin and litter size (n = 6 mothers per treatment; n = 18 male and 18 female offspring per treatment).
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Offspring body composition
Analysis of tissue:body weight ratios and body mass index (BMI) showed that compared with controls, male offspring from 150 μg/kg LPS-treated dams had a 28% increase in inguinal fat:body weight ratio (P = 0.031) (Figure 3C), a 29% increase in retroperitoneal fat:body weight ratio (P = 0.013) (Figure 3C) and a 7% increase in BMI (P = 0.033) (Figure 3C), but no differences in any organ:body weight ratio (Figures S6A and S6D in Additional file 7). In contrast, females showed no treatment group differences in fat pad:body weight ratio, BMI (Figure S6B in Additional file 7) or any organ:body weight ratio (Figure S6C in Additional file 7), except for liver:body weight ratio, which decreased by 12% in the 50 μg/kg LPS-treated dam group (P = 0.033) (Figure S6D in Additional file 7). All differences were independent of maternal origin and gestational litter size.
Additional file 7. Figure S6. Body composition of adult offspring. (A) Organs to body weight (%) of males. (B) Fat pad to body weight ratio (%) and BMI of females. (C) Organs to body weight ratio (%) of females. (D) Liver to body weight ratio (%) in males and females. All values represent means ± SEM. *P ≤ 0.05 vs. control independent of maternal origin and litter size (n = 6 mothers per treatment; n = 18 male and 18 female offspring per treatment).
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Offspring serum cytokine profile
Offspring response to an innate immune challenge was assessed by measuring serum cytokine concentrations in LPS-injected (50 μg/kg) adult offspring of dams that were injected with saline or 10, 50 or 150 μg/kg LPS at the zygote stage. A selection of the 23 cytokines detected by the multiplex assay is shown in Figure 4. The remaining cytokines are shown in Figure S7 in Additional file 8. At 3.5 hours post-LPS injection, male and female offspring of LPS-treated dams had severely blunted serum levels of several cytokines compared with controls. Notably, the cytokine response of challenged offspring was either blunted in a maternal LPS dose-dependent fashion or not altered; in no case did maternal LPS treatment enhance offspring response. Challenged males from 10 μg/kg LPS-treated dams (10 LPS) had blunted levels of IL-1α (31% lower, P = 0.03) and granulocyte colony-stimulating factor (G-CSF) (99% lower, P = 0.042), while those from 50 μg/kg LPS-treated dams (50 LPS) had blunted levels of MCP-1 (91% lower, P = 0.003), IL-1α, IL-9, IL-10, G-CSF, MIP-1β (CCL4) and RANTES (26% to 91% lower, all P < 0.05) and those from 150 μg/kg LPS-treated dams (150 LPS) had blunted levels of IL-1α, IL-9 (44% to 60% lower, all P < 0.01), IL-1β, IL-10, MCP-1, MIP-1β and RANTES (31% to 75% lower, all P < 0.05). Challenged females from 10 μg/kg LPS-treated dams had a serum cytokine profile similar to that of controls. In contrast, challenged females from 50 μg/kg LPS-treated dams had blunted levels of IL-1β, IL-12 p40, G-CSF, keratinocyte-derived chemokine (KC; CXCL1), MCP-1, MIP-1α, MIP-1β, RANTES and TNF-α (42% to 99% lower, all P < 0.01) and those from 150 μg/kg LPS-treated dams had blunted levels of IL-1β, IL-12 p40, G-CSF, KC, MCP-1, MIP-1α (40% to 99% lower, all P < 0.01), IL-1α, IL-2, RANTES and TNF-α (51% to 59% lower, all P < 0.05).
Figure 4. Serum cytokine concentrations (pg/mL) of adult offspring from each maternal treatment group 3.5 hours after LPS challenge. MPSI caused blunted release of predominantly innate-type cytokines in challenged offspring. A selection is shown here; the remaining cytokines are shown in Figure S3 in Additional file 4. All values represent means ± SEM. *P ≤ 0.05 vs. control independent of maternal origin and litter size (n = 6 mothers per treatment). Maternal treatments: Sal = saline; 10 LPS = 10 μg/kg LPS; 50 LPS = 50 μg/kg LPS; 150 LPS = 150 μg/kg LPS.
Additional file 8. Figure S7. Serum cytokine concentrations (pg/mL) of adult offspring from each maternal treatment group 3.5 hours after LPS challenge. Values represent means ± SEM. *P ≤ 0.05 vs. control independent of maternal origin and litter size (n = 6 mothers per treatment). Maternal treatments: Sal = saline; 10 LPS = 10 μg/kg LPS; 50 LPS = 50 μg/kg LPS; 150 LPS = 150 μg/kg LPS.
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Offspring serum corticosterone
In addition to serum cytokines, serum corticosterone concentration was quantified in LPS-injected offspring from each maternal treatment group (the same groups described above in 'Offspring serum cytokine profile'). Serum corticosterone concentration of LPS-injected offspring was unaffected by maternal treatment (Figure S8 in Additional file 9).
Additional file 9. Figure S8. Serum corticosterone concentration (ng/mL) of adult offspring from each maternal treatment. Values represent means ± SEM (n = 6 mothers per treatment). Maternal treatments: Sal = saline; 10 LPS = 10 μg/kg LPS; 50 LPS = 50 μg/kg LPS; 150 LPS = 150 μg/kg LPS.
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We have established an in vivo mouse model of MPSI by intraperitoneal administration of increasing doses of LPS on the day of zygote formation (GD 0.5) and found developmental programming to be significantly altered. We report that MPSI changes blastocyst cellular organisation and induces a complex postnatal phenotype affecting adult behaviour, adiposity and response to an innate immune challenge in a gender-specific manner that is dependent upon the originating maternal LPS dose. These findings provide new insight into both the breadth of environmental cues that early embryos are sensitive to and the range of long-term adult physiological systems that are modulated as a consequence.
Maternal response to LPS
Initially, we confirmed induction of an inflammatory response in LPS-injected dams. LPS treatment caused metabolic and behavioural changes typically associated with an inflammatory response, including reduced locomotor and exploratory activity, reduced food consumption and body weight, and increased circulating cytokine and chemokine levels. Long-term changes to offspring phenotype are known to result from a periconceptional maternal low-protein diet [2,16,17]. It could therefore be argued that the indirect effect of reduced food consumption might be the critical inductive event changing the adult phenotype. We believe this not to be the case, since (1) maternal food consumption was transiently reduced by an equivalent amount in all LPS groups, whilst changes in offspring phenotype were LPS dose-dependent; (2) reduction in maternal food consumption was temporally equivalent in all LPS groups, that is, maternal food consumption in all LPS groups returned to control levels at an equivalent rate; (3) maternal serum cytokine levels, a more direct measure of the inflammatory response, were LPS dose-dependent, corresponding with the resultant changes in offspring phenotype; and (4) maternal diet-induced programming is distinct from that reported here and affects particularly postnatal growth and cardiovascular function [16].
Maternal LPS effect on blastocysts
Administration of LPS, including the low dose, on the day of zygote formation caused a reduction in GD 3.5 blastocyst ICM cell numbers and ICM:TE cell ratios. Reduced rodent blastocyst cell numbers have been induced by maternal protein undernutrition and in vitro culture and are associated with disease-related changes in postnatal physiology [2,6]. Similarly, mouse blastocyst cell numbers have been shown to be altered in response to maternal nutrition over a wider range [18]. Our current findings thus support the concept that altered blastocyst proliferation and lineage allocation may be useful early markers for diverse forms of developmental programming whetherlater effects on foetal and postnatal growth are induced (maternal protein restriction [16]) or not (current study). We also observed increased TE cell numbers coinciding with increased litter size only in the 10 μg/kg LPS group. This may reflect a 'trophic' effect mediated by a moderate release of inflammatory mediators, since cytokines and related signalling molecules are also important factors that are active during blastocyst development and implantation [19]. A greater degree of intrauterine growth restriction may have been exerted by the larger litter size in this low-dose LPS group and may explain the raised SBP (male offspring) seen here.
Maternal LPS effects on offspring phenotype
Our most important finding is that MPSI results in long-term, gender-specific changes to offspring phenotype that are associated with a clear maternal LPS dose dependency. Male offspring from dams treated with the highest LPS dose displayed reduced locomotor and exploratory open-field behaviour, increased mass of specific fat pads, increased BMI and a blunted serum cytokine response to innate (LPS) immune challenge. Male offspring of the dams injected with the lower LPS doses (10 and 50 μg/kg) were much less affected, as were female offspring, which only showed the blunted serum cytokine response to LPS challenge. Notably, but not statistically significantly, the locomotor and exploratory behaviour of female offspring followed the same pattern as that of the males, suggesting that female behaviour is affected by MPSI, but to a lesser extent, such that programming of offspring behaviour may be graded rather than dictated by gender. Gender-specific differences in offspring phenotype have commonly been observed in DOHaD-related studies [2,6,16,17,20], but the underlying mechanisms are not understood yet. Interestingly, gender differences are evident in the preimplantation embryo from conception. For example, male embryos develop more rapidly than female embryos [21,22] and consequently may have different sensitivities to distinct environmental conditions that could thus contribute to the breadth of the postnatal outcomes discussed in this section}.
The combined postnatal phenotype, observed especially in male offspring of mothers that were injected with the high LPS dose, of reduced locomotor activity, increased adiposity and blunted cytokine response to LPS challenge may represent diverse manifestations from one or more causative pathways. The open-field behavioural assay has been effective in identifying broader conditions of ill health in mice, including cardiovascular and metabolic dysfunction [16,17], but provides symptomatic information rather than mechanistic insight. The adiposity and BMI outcomes are similar to those found in an earlier study where 790 μg/kg LPS administration to rat dams at midgestation caused central obesity, metabolic syndrome, hyperphagia and altered HPA functionality, but no change in blood pressure or lean body mass, in predominantly male offspring [12]. A similar offspring phenotype can result from administration of key inflammatory (IL-6 and TNF-α) or anti-inflammatory (dexamethasone) mediators during midgestation [23].
Maternal LPS effect on offspring innate immunity
A novel finding of our study is that MPSI resulted in adult offspring with a severely blunted serum cytokine response to innate (LPS) immune challenge. The blunting was most pronounced in offspring of dams treated with the highest LPS doses, and, unlike most phenotypic changes in this study, it affected both male and female offspring. This blunted response was prominent in cytokines and chemokines (for example, IL-1β and MCP-1) typically produced by cells of the monocyte lineage (for example, macrophages), whereas cytokines of the lymphocyte lineage (for example, IL-3 and IL-5) were less affected. Interestingly, Shanks et al. [24] showed that exposure of neonatal rats to low-dose LPS protected them from adjuvant-induced arthritis in later life, suggesting a blunted response to infection and inflammation, in accordance with the findings of the present study.
Physiologically, the inflammatory response is controlled at a number of levels in a context- and cell-specific manner [25]; however, major mechanisms include immunosuppressive signalling via GC pathways, peroxisome proliferator-activated receptors (PPARs), regulatory T cells (Tregs), alterations in Toll-like receptor (TLR) sensing and/or signalling pathways and epigenetic modifications [25-27]. Interestingly, altered expression of GC receptors (GRs) and PPARs has been found in offspring born to mothers fed a protein-restricted diet [28,29], and increased Tregs have been found in cord blood from neonates born to farm-exposed mothers [30]. Thus, several pathways with the potential to regulate inflammation are known to be susceptible to programming influences. In the present study, production of the major rodent GC, corticosterone, was unaffected by MPSI, indicating that the blunted offspring cytokine response was not due to altered GC production. Furthermore, GCs possess broad immunosuppressive activity, whilst the blunting observed in this study predominantly affects innate cytokines. Taken together, these data support a mechanism that affects innate immune cells specifically, for example, altered TLR signalling and/or epigenetic modifications to cytokine promoters. Other aspects of offspring immune status await investigation.
The blunted cytokine response to LPS challenge is reminiscent of the endotoxin tolerance (ET) phenomenon, wherein transient hyporesponsiveness to endotoxins (LPS) follows an initial sublethal exposure [31]. However, the long-lasting intergenerational phenotype of MPSI is distinct from ET. Crucially, ET is not a state of total unresponsiveness, because LPS signalling in endotoxin-tolerant cells induces antimicrobial effector functions without concomitant expression of inflammatory cytokines [25]. In this way, the host is protected from a deleterious and potentially fatal cytokine storm (increased tolerance) whilst maintaining antipathogen defences (sustained resistance). In animals, host tolerance has been studied far less extensively than resistance; however, the importance of tolerance strategies for host survival and host-pathogen interactions is becoming increasingly known [32-34]. At a physiological level, we therefore view the current findings as evidence for a novel form of adaptive response [1] whereby maternal experience can lead to an increased level of postnatal tolerance which, within an anticipated pathogen-rich environment, would protect the offspring from the cytotoxic effects of their own frequent inflammatory responses. Cytokine hyporesponsiveness in ET is thought to be regulated at least in part by epigenetic mechanisms [31], just as metabolic responses are evident in diet-mediated developmental programming [28,29]. The molecular mechanisms underlying the blunted cytokine response in the current intergenerational model will be the subject of future research.
In summary, we have identified for the first time that a maternal periconceptional inflammatory response acts as a mediator of developmental programming to preimplantation embryos. This environmental cue not only alters the cellular organisation of the blastocyst but also leads to permanent, gender-specific changes in adult offspring behaviour, adiposity and innate immune reactivity. Adult offspring are hyporesponsive to inflammatory stimuli which may confer host protection from inflammation-associated tissue damage. These findings in an animal model extend the repertoire of environmental conditions that preimplantation embryos are sensitive to and substantiate the need for broader analysis of underlying molecular mechanisms and applicability across species, including humans.
Animal treatments
With UK Home Office licence and local ethics committee approval, MF1 mice were bred in-house at the University of Southampton Biomedical Research Facility and kept on a 0700 to 1900 hours light cycle with ad libitum access to standard rodent chow. Virgin females (7 to 8.5 weeks old and 27 to 33 g in weight) were naturally mated overnight with MF1 studs. The following morning plug-positive females were housed individually and randomly assigned to receive either sterile saline (0.85%) or one of the following three LPS (Salmonella enterica serovar Enteritidis; Sigma, St Louis, MO, USA) dosages: 10, 50 or 150 μg/kg body weight (freshly prepared on each occasion). Saline or LPS was administered by intraperitoneal injection of 100-μL volumes between 1400 and 1600 hours on the day of plug (that is, at the zygote stage, GD 0.5). Mice were returned to their cages following treatment and were housed individually until required for experiments.
Maternal inflammatory response
A group of mated females were assessed to characterise maternal response to saline (control) or LPS dosages. A subset from each treatment group were assessed for sickness-related behaviour and body weight. At one hour posttreatment, mice underwent an open-field test (see below in Offspring Analyses: Behaviour) to assess locomotor activity. Treated females and their chow and water bottles were weighed at 0, 24, 48 and 72 hours posttreatment to assess body weight and consumption of food and water. Another subset from each treatment group were killed by cervical dislocation at 3, 24, 48 and 72 hours posttreatment, maternal blood was collected by cardiac puncture and serum was aliquoted and stored at -80°C. Maternal serum cytokine profiles were assessed by using the Bio-Plex Pro Mouse Cytokine 23-plex panel (Bio-Rad Laboratories, Hercules, CA, USA).
Embryo recovery and differential nuclear labelling
A group of females from each treatment group were killed as described above in Maternal inflammatory response) at GD 3.5, and embryos were flushed from uteri using prewarmed (37°C) H6 medium with 4 mg/mL BSA. Freshly collected blastocysts were analysed for cell numbers within the TE and ICM lineages by differential nuclear labelling as described previously [35] with modifications [2]. Blastocysts were viewed and imaged using a Zeiss Axiovert 200 fluorescence microscope (Carl Zeiss Ltd, Welwyn Garden City, Hertfordshire, UK) and MetaMorph version 6.2r6 software (Universal Imaging Corp.).
Offspring analyses
For assessment of postnatal phenotype, females from each treatment group were bred to term. At birth, litters were standardised to six (three median weight offspring of each gender) and each offspring was assigned a neutral code that did not reflect the treatment group to which it belonged. Mice were identified by their codes throughout the study, and the results were not decoded until data analysis was performed at the end of the study. Offspring were weaned at three weeks with littermates of the same gender housed together.
Offspring were weighed weekly from birth to 30 weeks of age.
Open-field tests were conducted at 4, 5, 6, 8, 11, 14, 17, 20 and 23 weeks. Open-field tests were conducted in the afternoon in a normally lit room, and, prior to the test, mice were habituated to the behaviour room for 30 minutes. The open-field arena consisted of a white acrylic base enclosed on four sides with transparent 0.7-cm-thick acrylic (dimensions 27.5 × 27.5 × 20 cm, model ENV-520; Med Associates Inc., Georgia, VT, USA). Laser beams crossing the open-field arena allowed animal movement to be detected and recorded automatically. Each open-field arena was surrounded by an aluminium shield that prevented the mouse under investigation from seeing other rodents. Data were acquired by using Activity Monitor version 4.0 software (Med Associates Inc.) for a three-minute period which started as soon as a mouse was detected in the arena [36]. Each mouse was individually placed in the centre of the open-field arena, and the distance travelled, time spent resting, number of jumps and number of rears was recorded by the computer. After each three-minute recording period, the mouse was returned to its home cage and the open-field arena was cleared of any droppings and wiped out with disinfectant solution and paper towels.
Systolic blood pressure
SBP was measured at 16, 22 and 28 weeks of age by tail cuff plethysmography [6] using an ITC model 229 blood pressure monitor (Linton Instruments, Norfolk, UK). SBP was taken at room temperatures of 27°C to 30°C, to which we allowed the mice to acclimatise themselves for 60 minutes prior to measurement. This practice encourages vasodilatation, thus facilitating detection of SBP. Mice were restrained in a ventilated acrylic tube, and their tails were threaded through the tail cuff. Each mouse was allowed to become acclimatised to the apparatus for five minutes prior to readings' being taken. At each time point, five sequential SBP readings were taken for each mouse, and the mean value of the three median readings was calculated and recorded.
Terminal treatments, blood collection and body composition
Offspring were killed by cervical dislocation at 40 weeks of age. Three and one-half hours before they were killed, a subset of male and female littermates were injected with 50 μg/kg LPS (as described above in Animal treatments). Blood was collected by cardiac puncture, then major organs (heart, lungs, liver, brain and left and right kidneys) and fat pads (interscapular, retroperitoneal, mesenteric, gonadal and inguinal) were dissected and weighed. Serum was extracted from blood samples and stored at -80°C.
Serum cytokine profiles
Offspring (and maternal, see above in Maternal inflammatory response{) serum cytokine profiles were assessed using the Bio-Plex Pro Mouse Cytokine 23-plex panel (Bio-Rad Laboratories) as per the manufacturer's instructions. Before analysis, thawed serum samples were diluted 1:4 using Bio-Plex mouse serum diluent (Bio-Rad Laboratories) as recommended by the manufacturer. Prepared plates were run on a Luminex 100 instrument (Luminex Corp., Austin, TX, USA) equipped with STarStation version 2.0 software (Applied Cytometry, Sheffield, Yorkshire, UK). Unknown sample cytokine concentrations were automatically calculated by the STarStation software based on a nine-point standard curve (in duplicate) fitted with a four- or five-parameter logistic regression algorithm to allow a wide range of quantification, and R2 values were routinely > 0.95. Of the 23 cytokines analysed by using this kit, most had a lower limit of detection that was < 3 pg/mL and an upper limit of detection that was well in excess of 15,000 pg/mL. Intra-assay coefficients of variation were routinely < 15%, indicating a good level of precision. Please refer to Bio-Rad Bulletin 3156 for a detailed summary of the performance characteristics of the Bio-Plex Pro Mouse Cytokine 23-plex panel that we used. The profile of the cytokines analysed was IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 p40, IL-12 p70, IL-13, IL-17, eotaxin (CCL11), G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1α, MIP-1β, RANTES and TNF-α.
Serum corticosterone concentrations
Offspring serum corticosterone levels were measured using a quantitative competitive ELISA kit (AssayMax Corticosterone ELISA Kit; AssayPro, St. Charles, MO, USA).
Statistical analysis
All statistics were calculated using SPSS version 14 software (SPSS Inc., Chicago, IL, USA). One-way analysis of variance with the Bonferroni correction was used to analyse all maternal, birth and blastocyst cell number data. All postnatal data were analysed using a multilevel random effects regression model without the Bonferroni correction, which enabled accurate consideration of the hierarchical nature of the data set with between-mother and within-mother variation and incorporation of gestational litter size [16,37]. Thus, differences identified between treatment groups are independent of maternal origin of litter and litter size.
BMI: body mass index; BSA: bovine serum albumin; DOHaD: developmental origins of health and disease; ELISA: enzyme-linked immunosorbent assay; ET: endotoxin tolerance; GD: gestational day; GR: glucocorticoid receptor; HPA: hypothalamic-pituitary-adrenal; ICM: inner cell mass; IFN: interferon; IL: interleukin; LPS: lipopolysaccharide; MPSI: maternal periconceptional systemic inflammation; PPAR: peroxisome proliferator-activated receptor; SBP: systolic blood pressure; TE: trophectoderm; TLR: Toll-like receptor; TNF: tumour necrosis factor.
Authors' contributions
CLW designed the research, performed the experiments and wrote the paper. JLT analysed part of the data and revised the paper. VHP designed the research and revised the paper. TPF designed the research and wrote the paper. All authors read and approved the final manuscript.
This work was funded by a Biotechnology and Biological Sciences Research Council (BBSRC) Strategic Research Postgraduate Studentship awarded to TPF for CLW, with additional funding from the Medical Research Council, the National Institutes of Health and the BBSRC (to TPF). We are grateful to Ferdousi Chowdhury (University of Southampton) for her assistance with the Luminex 100 instrument.
Gluckman PD, Hanson MA, Pinal C: The developmental origins of adult disease.
Matern Child Nutr 2005, 1:130-141. PubMed Abstract | Publisher Full Text
Kwong WY, Wild AE, Roberts P, Willis AC, Fleming TP: Maternal undernutrition during the preimplantation period of rat development causes blastocyst abnormalities and programming of postnatal hypertension.
Development 2000, 127:4195-4202. PubMed Abstract | Publisher Full Text
Watkins AJ, Fleming TP: Blastocyst environment and its influence on offspring cardiovascular health: the heart of the matter.
J Anat 2009, 215:52-59. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ecker DJ, Stein P, Xu Z, Williams CJ, Kopf GS, Bilker WB, Abel T, Schultz RM: Long-term effects of culture of preimplantation mouse embryos on behavior.
Proc Natl Acad Sci USA 2004, 101:1595-1600. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Fernández-Gonzalez R, Moreira P, Bilbao A, Jiménez A, Pérez-Crespo M, Ramírez MA, Rodríguez De Fonesca F, Pintado B, Gutiérrez-Adán A: Long-term effect of in vitro culture of mouse embryos with serum on mRNA expression of imprinting genes, development, and behavior.
Proc Natl Acad Sci USA 2004, 101:5880-5885. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Watkins AJ, Platt D, Papenbrock T, Wilkins A, Eckert JJ, Kwong WY, Osmond C, Hanson M, Fleming TP: Mouse embryo culture induces changes in postnatal phenotype including raised systolic blood pressure.
Proc Natl Acad Sci USA 2007, 104:5449-5454. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Limosin F, Rouillon F, Payan C, Cohen JM, Strub N: Prenatal exposure to influenza as a risk factor for adult schizophrenia.
Acta Psychiatr Scand 2003, 107:331-335. PubMed Abstract | Publisher Full Text
Shi L, Fatemi SH, Sidwell RW, Patterson PH: Maternal influenza infection causes marked behavioral and pharmacological changes in the offspring.
J Neurosci 2003, 23:297-302. PubMed Abstract | Publisher Full Text
Hodyl NA, Krivanek KM, Lawrence E, Clifton VL, Hodgson DM: Prenatal exposure to a pro-inflammatory stimulus causes delays in the development of the innate immune response to LPS in the offspring.
J Neuroimmunol 2007, 190:61-71. PubMed Abstract | Publisher Full Text
Reiterova K, Tomasovicova O, Dubinsky P: Influence of Toxocara canis infection during pregnancy on offspring resistance towards re-infection.
Parasitology 2006, 132:625-633. PubMed Abstract | Publisher Full Text
Yamaguchi N, Shimizu S, Hara A, Saito T: The effect of maternal antigenic stimulation upon the active immune responsiveness of their offspring.
Immunology 1983, 50:229-238. PubMed Abstract | PubMed Central Full Text
Nilsson C, Larsson BM, Jennische E, Eriksson E, Björntorp P, York DA, Holmäng A: Maternal endotoxemia results in obesity and insulin resistance in adult male offspring.
Endocrinology 2001, 142:2622-2630. PubMed Abstract | Publisher Full Text
Reul JM, Stec I, Wiegers GJ, Labeur MS, Linthorst AC, Arzt E, Holsboer F: Prenatal immune challenge alters the hypothalamic-pituitary-adrenocortical axis in adult rats.
J Clin Invest 1994, 93:2600-2607. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Babulas V, Factor-Litvak P, Goetz R, Schaefer CA, Brown AS: Prenatal exposure to maternal genital and reproductive infections and adult schizophrenia.
Am J Psychiatry 2006, 163:927-929. PubMed Abstract | Publisher Full Text
Brown AS, Begg MD, Gravenstein S, Schaefer CA, Wyatt RJ, Bresnahan M, Babulas VP, Susser ES: Serologic evidence of prenatal influenza in the etiology of schizophrenia.
Arch Gen Psychiatry 2004, 61:774-780. PubMed Abstract | Publisher Full Text
Watkins AJ, Ursell E, Panton R, Papenbrock T, Hollis L, Cunningham C, Wilkins A, Perry VH, Sheth B, Kwong WY, Eckert JJ, Wild AE, Hanson MA, Osmond C, Fleming TP: Adaptive responses by mouse early embryos to maternal diet protect fetal growth but predispose to adult onset disease.
Biol Reprod 2008, 78:299-306. PubMed Abstract | Publisher Full Text
Watkins AJ, Wilkins A, Cunningham C, Perry VH, Seet MJ, Osmond C, Eckert JJ, Torrens C, Cagampang FR, Cleal J, Gray WP, Hanson MA, Fleming TP: Low protein diet fed exclusively during mouse oocyte maturation leads to behavioural and cardiovascular abnormalities in offspring.
J Physiol 2008, 586:2231-2244. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Mitchell M, Schulz SL, Armstrong DT, Lane M: Metabolic and mitochondrial dysfunction in early mouse embryos following maternal dietary protein intervention.
Biol Reprod 2009, 80:622-630. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Dimitriadis E, White CA, Jones RL, Salamonsen LA: Cytokines, chemokines and growth factors in endometrium related to implantation.
Hum Reprod Update 2005, 11:613-630. PubMed Abstract | Publisher Full Text
Poore KR, Boullin JP, Cleal JK, Newman JP, Noakes DE, Hanson MA, Green LR: Sex- and age-specific effects of nutrition in early gestation and early postnatal life on hypothalamo-pituitary-adrenal axis and sympathoadrenal function in adult sheep.
J Physiol 2010, 588:2219-2237. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Erickson RP: Does sex determination start at conception?
Bioessays 1997, 19:1027-1032. PubMed Abstract | Publisher Full Text
Mittwoch U: Blastocysts prepare for the race to be male.
Hum Reprod 1993, 8:1550-1555. PubMed Abstract | Publisher Full Text
Dahlgren J, Nilsson C, Jennische E, Ho HP, Eriksson E, Niklasson A, Björntorp P, Albertsson Wikland K, Holmäng A: Prenatal cytokine exposure results in obesity and gender-specific programming.
Am J Physiol Endocrinol Metab 2001, 281:E326-E334. PubMed Abstract | Publisher Full Text
Shanks N, Windle RJ, Perks PA, Harbuz MS, Jessop DS, Ingram CD, Lightman SL: Early-life exposure to endotoxin alters hypothalamic-pituitary-adrenal function and predisposition to inflammation.
Proc Natl Acad Sci USA 2000, 97:5645-5650. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Medzhitov R, Horng T: Transcriptional control of the inflammatory response.
Nat Rev Immunol 2009, 9:692-703. PubMed Abstract | Publisher Full Text
Maloy KJ, Salaun L, Cahill R, Dougan G, Saunders NJ, Powrie F: CD4+CD25+ TR cells suppress innate immune pathology through cytokine-dependent mechanisms.
J Exp Med 2003, 197:111-119. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Ogawa S, Lozach J, Benner C, Pascual G, Tangirala RK, Westin S, Hoffmann A, Subramaniam S, David M, Rosenfeld MG, Glass CK: Molecular determinants of crosstalk between nuclear receptors and toll-like receptors.
Cell 2005, 122:707-721. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lillycrop KA, Slater-Jefferies JL, Hanson MA, Godfrey KM, Jackson AA, Burdge GC: Induction of altered epigenetic regulation of the hepatic glucocorticoid receptor in the offspring of rats fed a protein-restricted diet during pregnancy suggests that reduced DNA methyltransferase-1 expression is involved in impaired DNA methylation and changes in histone modifications.
Br J Nutr 2007, 97:1064-1073. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Lillycrop KA, Phillips ES, Torrens C, Hanson MA, Jackson AA, Burdge GC: Feeding pregnant rats a protein-restricted diet persistently alters the methylation of specific cytosines in the hepatic PPAR alpha promoter of the offspring.
Br J Nutr 2008, 100:278-282. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Schaub B, Liu J, Höppler S, Schleich I, Huehn J, Olek S, Wieczorek G, Illi S, von Mutius E: Maternal farm exposure modulates neonatal immune mechanisms through regulatory T cells.
J Allergy Clin Immunol 2009, 123:774-782.
e5PubMed Abstract | Publisher Full Text
Biswas SK, López-Collazo E: Endotoxin tolerance: new mechanisms, molecules and clinical significance.
Trends Immunol 2009, 30:475-487. PubMed Abstract | Publisher Full Text
Ayres JS, Schneider DS: The role of anorexia in resistance and tolerance to infections in Drosophila.
PLoS Biol 2009, 7:e1000150. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Raberg L, Sim D, Read AF: Disentangling genetic variation for resistance and tolerance to infectious diseases in animals.
Science 2007, 318:812-814. PubMed Abstract | Publisher Full Text
Seixas E, Gozzelino R, Chora A, Ferreira A, Silva G, Larsen R, Rebelo S, Penido C, Smith NR, Coutinho A, Soares MP: Heme oxygenase-1 affords protection against noncerebral forms of severe malaria.
Proc Natl Acad Sci USA 2009, 106:15837-15842. PubMed Abstract | Publisher Full Text | PubMed Central Full Text
Handyside AH, Hunter S: A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using polynucleotide-specific fluorochromes.
J Exp Zool 1984, 231:429-434. PubMed Abstract | Publisher Full Text
Cunningham C, Campion S, Teeling J, Felton L, Perry VH: The sickness behaviour and CNS inflammatory mediator profile induced by systemic challenge of mice with synthetic double-stranded RNA (poly I:C).
Brain Behav Immun 2007, 21:490-502. PubMed Abstract | Publisher Full Text
Kwong WY, Osmond C, Fleming TP: Support for Barker hypothesis upheld in rat model of maternal undernutrition during the preimplantation period: application of integrated 'random effects' statistical model.
Reprod Biomed Online 2004, 8:574-576. PubMed Abstract | Publisher Full Text


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Originally written on Fox Sports Florida | Last updated 12/12/12
ORLANDO, Fla. (AP) -- Josh Smith and Jeff Teague scored 16 points apiece, Al Horford added 13 points and 13 rebounds, and the Atlanta Hawks held off a late charge by the Orlando Magic for an 86-80 victory Wednesday night. Atlanta extended its regular-season win streak against Orlando to eight with its second win over the Magic this season. Arron Afflalo scored 16 points and J.J. Redick added 13 for the Magic, who were able to dig out of a huge first-half hole, but couldn't overcome 16 turnovers that led to 24 points. Orlando also struggled from the field, connecting on only 31 of 81 shots, in dropping its fourth straight home game. The Magic get a chance to end the skid Friday against Golden State. Wednesday was the Magic's first home game since Nov. 30 following a five-game trip, but it hardly was a welcome return. Atlanta led by as many as 19 in the third quarter before a late Orlando run trimmed it to 12 entering the fourth. The Magic pulled within six late, but costly turno...
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JSU guard Dundrecous Nelson (5) is averaging a team-leading 17.7 points.
- Filed Under
Tevester Anderson has long been a staunch proponent for scheduling a demanding non-conference schedule.
But as Jackson State hosts bitter conference rival Alcorn State in what will be the Tigers' first home game of the season, he admits he might have overdone it.
JSU played nine straight road games to open the year then traveled twice more to open SWAC play at Alabama State and Alabama A&M. The results haven't been pretty, as evidenced by the Tigers' 1-10 record. After dropping its conference opener at Alabama State 70-66, JSU fell 88-87 in double overtime two nights later at Alabama A&M. ...


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Originally written on 60 Max Power O | Last updated 11/11/12
The (7-1) Chicago Bears face (7-1) Houston Texans in what should be a defensive battle. Here is the injury report: Chicago Bears Pos Player Injury Status DE Israel Idonije ANKLE PROBABLE WR Alshon Jeffery HAND DOUBTFUL DT Henry Melton BACK PROBABLE DT Matt Toeaina CALF DOUBTFUL Houston Texans LB Bryan Braman NECK PROBABLE RB James Casey ILLNESS PROBABLE NT Shaun Cody RIBS OUT TE Owen Daniels BACK QUESTIONABLE S Quintin Demps THUMB, FOREARM PROBABLE WR Lestar Jean KNEE PROBABLE CB Johnathan Joseph QUADRICEPS PROBABLE S Shiloh Keo BACK PROBABLE CB Brice McCain KNEE PROBABLE NT Earl Mitchell ANKLE PROBABLE LB Jesse Nading FOOT, NECK PROBABLE DE Antonio Smith ANKLE PROBABLE G Wade Smith KNEE PROBABLE RB Ben Tate HAMSTRING OUT WR Kevin Walter GROIN PROBABLE QB T.J. Yates RIGHT ELBOW PROBABLE var switchTo5x=true; stLight.options({publisher:''});
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Smack in the middle of Jonathan Mahler's best-selling Ladies and Gentlemen, the Bronx Is Burning there unfolds an unforgettable account of the 1977 New York City blackout. Personal narratives, drawn from interviews and documentary sources, of the politicians, technicians, looters, and police who experienced the blackout are all stitched together in Mahler's accelerated and visceral montage. After this, any historian attempting to convey the same events must have a fair amount of chutzpah, but sadly, David Nye's social history of blackouts, When the Lights Went Out, lacks the cinematic flair of Mahler's narrative. Nye's subject is broader, "not simply power outages, but different social constructions of artificial darkness," and his book scholarly. He is working his way toward a theory of the blackout as social and
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Shutdown sets back Willamette Superfund work even further
Portland officials working with businesses on the Willamette Superfund site cleanup say the federal government shutdown has already had a material effect on business' efforts.
Specifically, Dennis McLerran, the Environmental Protection Agency Region 10's administrator, was scheduled to visit with Portland Mayor Charlie Hales, Portland City Commissioner Nick Fish and business representatives whose companies operate along the Willamette River's north reaches, near the Superfund site. The meeting in Portland, which is part of Region 10, was set to take place Oct. 2.
The meeting, at which the sides were set to begin finalizing such matters as costs to businesses as well as clean-up strategies, was outright canceled.
Fish said the cancellation is a tangible example of what the shut-down has meant for Portland.
"These are not easy meetings to schedule," Fish said. "We're worried it will cause further delays in what we're trying to do. We'd been admonished that the city needs to speak with one voice, and we're reaching out to as many potentially responsible parties as possible, but it's hard when you have a government shutdown. Everything gets put on ice.
"We have a sense of urgency on this. A lot of money and a lot of jobs are at stake, and as soon as we figure out the city's cost, the sooner we can figure out how to pay for it."
The EPA is drafting strategies to clean the Willamette River's Superfund site. The clean-up is projected to cost as much as $2.2 billion.
The agency continues to meet with stakeholders along the river to determine culpability. It's a complicated issue because many of the biggest polluters no longer operate businesses along the river or are even functional business entities.
Delays in decision-making thanks to the federal government shutdown make it more difficult for businesses to make decision about spending on such matters as capital equipment or hiring, Fish said.
Plus, "When the city itself has budgeting issues to think about, until that cloud is removed or resolved somewhat, a lot of things are on hold. The sooner we have clarity about the path forward, the sooner we can think about not just maintaining those jobs but expanding."
Businesses and the city are at the point where they want to ascertain what type of credits the businesses will receive for work already performed within the Superfund site. McLerran's visit was expected to address some of those questions.
At the same time, city staffers are reviewing a cleanup feasibility study and want speedy feedback on some of the study's proposals.
The newspaper Willamette Week reported earlier this month that the EPA agreed to suspend a $125,000 penalty against the Lower Willamette Group, which consists of 12 waterfront companies, for delays in issuing business-backed clean-up studies. The fine was waived pending the Group's ability to complete its studies.


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Digital Copy
STLE's 2014 Report on Emerging Issues and Trends, is the culmination of a multi-phase research effort to evaluate current trends and to predict future developments impacting tribology and lubrication engineering. The research encompasses the global opinions and experiences of nearly 1,000 tribologists and lubrication engineers who participated in the project as advisors, interview and focus group participants, and survey respondents. The 80-page report is divided into nine major areas: the outlook on the field, energy, manufacturing, transportation, atomistic processes, other tribology topics, government regulation, safety and the environment, and basic human needs.
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A new 20-page report examines the forces likely to influence markets and business practices in the tribology and lubricants sector during the next three years. Readers will gain insights into global trends in the six areas of business and economics, government, supply chains, finance, workforces and culture.
This is a joint project by STLE and Association Laboratory Inc. (ALI), a Chicago based consultancy. It combines information from a recent STLE membership needs study (also conducted by ALI) with a comprehensive research project involving business leaders from 54 different industries and professions.
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Author Philip Yancey discusses growing up in a toxic church and, after leaving the church, how he later found his way back to faith.
Religious liberty champion James Dunn dies July 4 BJC
Fugitive African warlord Joseph Kony is said to be sickly AP
Boyd Packer, head of Mormon Church top governing body, has died RNS
Evangelicals create 'sin-free' Facebook CNET
Was America founded as a Christian nation? CNN


text:
Connected BooksSee More Connected Books
If you like this, Harlequin recommends
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Langston Collingsworth was a man to be reckoned with. Powerful and president of the family oil business, opportunists looked at him and saw money, but he saw the land as his life force. So when the Collingsworth empire was threatened--it was personal.
It happened innocently enough--a young girl needing his help. But her mother was none other than Trish Edwards--Langston's high school sweetheart. Trish and her daughter were trapped in a blackmail and murder investigation and had been on the run since the day Trish gave birth to a Collingsworth heir.
Could Langston trust Trish after the years of deception? He had no choice if he wanted to protect her child--his child--and face down the trouble headed their way.


text:
First post in a while. Worked on this one in the past week to get it ready and out for the USHL Draft that happened this past week in Chicago. A little of it from this original design didn't come through but that's all good. You can see the site live at http://draft.ushl.com
The first slider I've built from scratch, while a little rough I managed to work it with CSS animations and some jQuery fall backs for IE.
Used Futura-PT from Typekit and Zamenhof Inline to replicate Neutraface.
More Joe Barrett
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Cable TV: The Re-Regulation, Re-Wiring, and Re-Education of America
Nelson, Milo, Computers in Libraries
After four years of room and board at the White House and a winning streak of thirty-five successful vetoes, it is interesting that the congressional override of President Bush's thirty-sixth veto concerned the regulation of cable television companies. This had something to do, naturally, with the opening of poll booths in November and something to do with the fact that the 102nd Congress was within a few hours of adjournment. But it also had something to do with the simple non-partisan popularity of the measure to authorize regulation of minimum cable service and seek to stimulate competition. The Senate voted 74 to 25 and the House 308 to 114 in support of the bill to restore regulation. In the Senate, twenty-four Republicans joined fifty Democrats in voting to override the presidential veto, a margin too large to ascribe to political waywardness.
Standing Square
Bush's arguments on the dangers of regulation had little impact on the legislators, causing speculation that he either disliked regulation simply on principle, or that he had small grasp of the implications of the bill. Bush said that the legislation would do little to increase competition in the cable television industry, would cost American jobs, and would discourage investment in telecommunications. He also objected to a provision in the bill that would allow television broadcasters to demand royalties from cable companies for carrying their programs.
Senator Albert Gore (D-TN), a principal author of the bill, said: "Cable consumers are getting ripped off and George Bush is giving the cable monopolies permission to do it." He accused the President of "standing square with the big cable operators, the monopolies that have been raising rates and squeezing out competition."
Senator Joe Lieberman (D-CT) said that the cable companies were engaging in "highway robbery." In the past, he said, "the cable industry has relentlessly raised rates on the American consumer, year after year." Without protective regulation, this will only continue.
Defenders of the bill argued that costs to consumers would rise if the measure were not passed, and critics of the bill declared that its enactment would result in greater costs to users. In addition to providing regulation of rates and seeking to encourage competition, the bill also enables over-theair broadcasters to seek payment from cable companies that carry their broadcasts. The movie industry vigorously opposed the bill because there was no provision for it to share in these revenues.
Let's Not Micromanage
In September on NBC's "Meet the Press," David Broder attempted to extract some insight on the pending legislation from Vice President Dan Quayle. Broder asked Quayle a few questions help clarify the matter.
Q: Explain in simple terms why the cable companies should not be regulated.
A: O.K. Simple terms, here's the choice. Here's the choice in simple terms. Are you going to try to constrain the price increase through regulation or through genuine competition? Our preference is to do it through genuine competition.
Q: But there isn't genuine competition.
A: That's right. Because you have -- but the cities that grant these things can certainly be more competitive and have more openness if they're --
Q: So your suggestion is that they go out and have two or three different companies wire these communities to get competition?
A: I'm not going to get into the micromanagement of the cable industry.
So much for the insight of the Vice President of the United States and, I would think, so much for his much touted Council on Competitiveness.
The product of over three years of political debate, the bill actually provides for only modest rate regulation along with several provisions designed to foster greater competition in the industry. The FCC is empowered to set guidelines that would "approximate" prices levied in the few markets where cable companies actually compete with one another. …


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By Kmartin
from Las Vegas
Comments about Whirlpool 30" Double Electric Wall Oven - Black on Black:
Fan is a bit noisy and I wish the upper and lower had separate lights. Overall nice oven.
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Ease of operation
By Sensei
from Parker, Colorado
I had not ever owned a Whirlpool oven and now I don't think I'd want to own anything else! Very easy to use, even temperatures and fast.
Bottom Line Yes, I would recommend this to a friend
Love This Double Oven
By Kelly
from Spokane, WA
I have had this in my newly remodeled kitchen for about 3 months now. I love how easy it is to operate. So far I have only used some of the basic functions and everything works as expected. The even baking is a treat. The only negative for us is the vent noise during cool down. We have not had a dinner party yet, but seems like it will be annoying when sitting down with friends. The solution we have come up with which works well is to simply leave the oven door ajar just a bit and that seems to allow the oven to cool down sooner and the vent to shut off. Overall I am very happy with this purchase.
Unbelievably Noisey
By Oven Disappointment
from Denver, CO
This purchase was a HUGE disappointment and mistake! When our Whirlpool microwave went out, we decided to buy all new appliances to have a matching set. Our 14-year old Whirlpool wall oven was extremely quiet and never required repair. The fan on our new wall oven is terribly noisy and sounds like the convection fan is running. There is a constant high pitch buzzing noise that makes it almost impossible to be in the kitchen while the oven is running. Consider purchasing another brand of wall oven until Whirlpool addresses this design flaw.
Bottom Line No, I would not recommend this to a friend
Great Oven - good looking & performs really well
By rosettaquarrier
from SLC Utah
Great Oven - good looking in our remodelled kitchen & performs really well
(1 of 1 customers found this review helpful)
Bakes great
By txgirlatlrg
from Kansas City, KS, United States
I have only had this oven for less than a month, but so far this oven has been great! Cookies, muffins, and pizza all have an even browning to them, it keeps temperature well, and bakes within the recommended times. It bakes evenly in both ovens in the bake and convection mode of the top oven. My only complaint is the fan. It runs in both conventional and convection mode and both ovens. It is kind of loud and will be noticed in an open concept home. An improvement would be to have the option to turn off the fan at times or provide some type of baffling similar to those used for dishwashers if that's possible. The look of the ovens is very sleek and it has low profile handles that are not obtrusive.I only gave 4 stars for the quality not because of a problem, but because I have not had the oven long enough to say it's without problems.
Great dbl oven
By Joji P
from Fort Belvoir, VA, United States
I bought this three weeks ago and we are already enjoying the features. All our major appliances are whirlpool and we are about to do another upgrade on our fridge.
Great Value for the Price
By Ed V
from Austin, TX, United States
Great looking, huge interior capacity, excellent viewing windows to see what's going on inside. Fills all the needs of a large family.
By cottonlady
from Dallas, TX
This double oven was installed by the builder when we built our new home. It is the worst quality appliance I have EVER owned. I can't count the number of times I have had meals ruined and had to call for repair because it would not maintain cooking temperature. After numerous repairs for the same issue, the oven was replaced. What a waste because the replaced oven is no better than the original unit. My husband & I have decided we will just toss it to the curb and buy a different brand. After doing some research, it WILL NOT be Whirlpool!!
Love the spacious interior
By Blindy
from Undisclosed
Bought this two months ago. Love the stainless steel. Larger interior than my previous oven. I haven't used the steam clean cycle yet but looking forward to that and not having to wait hours for the cleaning to complete so I can bake. The bake cycle is easy to program as is the timers.
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Helen DeWitt has 1 media appearance.
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Helen DeWitt
To The Best of Our Knowledge, Sunday, May 27, 2012 at 0am
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Attending the Manchester Science Festival was incredible. Not only were there computers, mobiles and televisions from decades gone by, but they were all in full working condition, which meant I could re-live some of the highs (space invaders) and lows (error messages and recovery) of my first experiences with computers. Did you have any of these computers? Did… Continue Reading
Latest in: UX (User Experience)
Have you ever wondered if there's a difference between how men and women shop online? What can you do to persuade each sex to buy from your website? This infographic by Minewhat showcases 18 strategies you can use to persuade both men and women to buy from you.
Yesterday at the London e-commerce expo, Google's Martijn Bertisen, spoke about e-commerce trends for 2015. Mobile is set to be BIG business and this christmas, over 50% of christmas retail enquiries are likely to come from smartphones. 2015 will be: The year that mobile takes over. The year of wearables – watch out for Google… Continue Reading
When I first started life as a user researcher, it was commonplace (and it it still is) to refer to research as user testing or usability testing. I soon observed that when you use the word 'test' it: a) Implies that you're testing the end user (which is wrong, you're testing the interface, you're understanding of… Continue Reading
I've been super busy recently which you can probably tell by the infrequency of my posts. As a business, Keep It Usable, has grown phenomenally this year and we've been working day and night to make sure we do great work for every single one of our clients. We genuinely care about every business we… Continue Reading
I just had to share with you this video on how guys might use Google Glass (or should that be glasses?) in the future. Pretty funny! Maybe we shouldn't laugh too soon though as it may well become the future! People are distracted enough as it is, it's just that the glasses may make the… Continue Reading
In my spare time I love baking (I love eating cakes), I run a local baking club and I absolutely love KitchenAid products. I love the industrial user-centred design and the attention to ease of use. So, for the Keep It Usable blog this month, I decided to interview my friend Brandon Satanek who is… Continue Reading


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I'm having trouble getting the NAND Permissions Patch to download from the NUS. It gives me no option to install from SD, and all i get a connect error. Any ideas how to get around this?
I'm having trouble getting the NAND Permissions Patch to download from the NUS. It gives me no option to install from SD, and all i get a connect error. Any ideas how to get around this?
Step three isnt really working for me, in the trucha bug loader and dop_mii, it says getting ios15 from revision 523, and it wont let me check off yes or no on the options listed in step 3. I went ahead and went to restore ios15 using ios36, hopefully this works.
ShadowSonic2, I would just like to say thank you for a very clear and easy guide. I was able to successfully softmod my Wii with your instructions.
Thanks again!
Does this work on a Wii running FW version 4.2U? It said in the article that it works on all 4.2 system, but I do wish to make sure before I do mess up Wii. I tried searching with Live Search but I did not find anything, maybe I was doing something wrong.
Thanks in advance.
It's called 'Softmod for ANY 4.2 Wii!' So if instructions are done properly, it will work.
Just making sure, I did not want to risk it. Great article.
Last edited by quintrex; 04-26-2010 at 04:00 PM. Reason: correction
would i be able to skip part 6b? My wiimote will never connect during that section no matter how many times I tried (gc controller and power buttons dont work). It worked during the other sections.Would skipping this part cause the mod to not work?
This is part 6b if you dont remember:
b) Load up homebrew channel once more, then load up WAD once more, then load up WAD Manager. Load IOS249, SD Slot and NAND Emulation: Disabled, navigate to the wad folder, highlight IOS60patched.wad and press A. Make sure action is set to install and Install the wad. After a successful install, Press Home to go back to homebrew channel.


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I Think I'm Having Triplets
Is it possible that four ultrasounds and a CVS later someone–or everyone–missed extra fetuses in my belly? Could I possibly be having twins? Or gasp, triplets? I swear, I'm only 25 weeks along and I am already waddling.
I was excited a few weeks ago when someone gave up their seat on the subway for me. Woo Hoo! I thought. I'm reaping the benefits of my baby bump. Now I get on, and the whole train practically jumps up to offer me their seats.
I don't look fat, but I swear my stomach is twice as big this time as it was last time at 6 months. Is it because it's already stretched out? Or because I'm carrying a boy? Could he be a giant? I don't know, but I can't imagine what I'm going to be like in 3-4 months. I can barely bend over now and put Fia in her crib. Lord help me!
Okay, I'm off to lie down on the floor –since the movers took all our furniture–and feel like a beached whale.Add a Comment
Tags: baby bump, cvs, genetic testing, pregnancy, pregnant, subway, ultrasounds | Categories: Fearless Feisty Mama, Mom Situations, Must Read
Must Read
by Colleen - Sunrise Learning Lab @F5th on Twitter
On October 18, 2011 at 3:57 pm
Hi Jill! We had been very busy with travel and such so i am just now getting caught up on reading some of my favorite blogs. How exciting about your news!
Was super excited that you cat has lost weight but then, saw the part about you moving to L.A. and then, saw that you are preggers!!! Awesome! Wow, you have had so much going on! So happy for you and hope that you will love living in L.A.
We have been very busy here. My younger brother got married so yet another family wedding up in Erie!
Was very fun as all of the little cousins were there. And if you go the route of looking for a nanny when you get out there, I have some friends from high school who live in L.A. that probably would be able to connect you with some good people for nanny stuff…or even Mom's helper stuff, if that is what you will need with the baby…
Well, all the best to you with your move and your baby-to-be, etc.
Have a super week!
Fantastic news on all accounts!!!
On June 16, 2012 at 1:33 pm
I am having triplets, babys will be here in 4 1/2 more month. I am 14 weeks.


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Villa Dilara A
from £115 /night help Price for guests, Nights
from £115 /night help Price for guests, Nights
Estimated nightly price based on a weekly stay. Excludes fees (if applicable). Enter your dates to see the total cost.
Villa / 4 bedrooms / sleeps 8
Availability Your dates are available
Villa / 4 bedrooms / sleeps 8
|Key Info
Two Detached 4 bed villa each with gated entrance, private gardens and large pool Situated about 4 kms from Kas town up the mountain road the Villa is set. In a quiet level location with beautiful views to sea and mountains.
Fully equipped property with air conditioning and insect screens. All linens provided except beach towels
Lovely mountain breezes ensuring less humidity and pleasant cooler temperatures. .
Villa Dilara A provides a beautiful family orientated and friendly atmosphere for those who want to escape the bustle of town.
Private parking
|Sleeps up to 8, 4 bedrooms
|Nearest beach
|Big Pebble Beach 3 km
|Car advised
|Nearest Amenities
|4 km
|Nearest travel links
|Nearest airport: Dalaman and Antalya 160 km
|Family friendly
|Suitable for children over 5
|No pets allowed, No smoking at this property
Features and Facilities
|Internet access, Sea view
|Private outdoor pool (unheated)
|Air conditioning, TV, CD player, Safe, Wi-Fi available
|Kettle, Toaster, Iron, Hair dryer
|Dishwasher, Cooker, Microwave, Fridge, Freezer, Washing machine
|4 bedrooms, 4 bathrooms of which 1 Family bathrooms and 3 En suites
|Double beds (1), Single beds (6), Dining seats for 8, Lounge seats for 8
|Linen provided, Towels provided
|Private outdoor pool (unheated), Balcony or terrace, Private garden, BBQ
|Secure parking, Not suitable for wheelchair users
The Turkish Mediterranean Coast region
Absolutely charming town of Kas nestling in the foothills of the Taurus mountains in southern Turkey. The amazing coastline boasts the clearest torquoise sea and breathtaking views. Loved by travellers who it is said, once tasted the waters of Kas will always return.
Kas is a small friendly town popular with travellers, walkers, boaters, divers and families wanting a quiet relaxed holiday.
There is plenty to do in Kas the busy working harbour in middle of town offers plenty of different boat trips, paragliding, many diving schools where you can get your diving certificates
Many beaches, lots of restaurants and bars. Quaint alleyways with pretty bouganvilla hanging from Ottoman balconies. Boutique shops offering wonderful selections of leather goods. and beautiful hand painted porcelain, clothes etc.
Friday market offers lovely fresh fruit and vegetables and usual sun glasses, copy designer clothes, spices etc
Plenty of supermarkets and Banks and ATM machines.
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90% Response rate
Calendar last updated:29 Jun 2015
Based in
- English


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Are Adventure Bikes Heading in 2 different directions?
View Single Post
Beastly Adventurer
Joined: Dec 2008
Oddometer: 1,986
"Adventure bike" is a term used to sell mediocre underpowered, undersuspended 10+ year old designs that require minimum tooling changes and maximise profits while providing a perception of "value". (read - Japanese adventure bikes)
The exceptions to the rule fall into 2 camps - under 700cc high tech expensive bikes, or over 700cc high tech expensive bikes. (read - Euro adventure bikes)
Between these categories everyones interests is covered. To some extent.
You are then free to customise to your hearts content.
That is part of the joy of owning one, and the basis of the frustration we all feel as owners.
Why don't they make a bike for what I want to do?
Sure, if they could build a bike that weighed next to nothing, handled rough stuff like a motoX bike, had the power and torque to pull 2 people plus camping gear at GP speeds, was cheap as chips, adaptable, reliable, and could still smash single track like an ISDE bike...we'd all have one.....right?
OUCH.....reality just bitch slapped me.
View Public Profile
Find More Posts by Snowy


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Google Play Music All Access crosses the border to Canada
Google's Play Music All Access subscription service is now streaming in Canada! By signing up to All Access, you can get a healthy cloud library of tunes, as well as your own private collection. Us Canadians are used to these sorts of services taking awhile to get up here, but it sure is welcome. As an early bird promotion, Google is cutting the normal subscription rate from $9.99 to $7.99, and tossing in 30 days of service for free. You'll also get Chromecast support as usual, plus, as of recently, you can stream out to Sonos.
Of course, Google Play isn't the only music service available on Android — be sure to check out our comparison of DoubleTwist, Google Play Music, Poweramp, Shuttle Music Player, and Equalizer+, as well as iMore's fantastic cloud music services show-down. Andrew also had some great stuff to say about All Access, if you're looking for thoughts and impressions. Canadians, are you interested in signing up to Google Play All Access? Which services do you subscribe to now?
Head over to Google Play Music to sign up!
Have something to say about this story? Leave a comment! Need help with something else? Ask in our forums!
Reader comments
Google Play Music All Access crosses the border to Canada
What's missing from the service that would make it a good deal?
I'm tempted to switch from Rdio, but I still don't see the Google Play Music app in the app store, so I'm a bit reluctant. It is supposed to be on mobile, not just web is it now?
Ok, the app is finally available. I tried it, but will be sticking with Rdio.


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Picture: Eric Roberts Special Screening of ' The Expendables' at Planet Hollywood Resort Hotel & Casino Las Vegas, Nevada - 11.08.10
"Sly (Sylvester Stallone) said, 'How is it to be up against your sister?' But she's in a chick flick. I'm in a big action movie. She's a woman, I'm a man. We don't compete for roles. We can't be in competition." Eric Roberts is adamant he didn't worry about seeing his new movie THE EXPENDABLES competing with his sister JULIA's EAT PRAY LOVE at the box office last weekend (14-15Aug10).


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All-New X-Men #6 : Amazing start, was genuinely shocked. This story has been great so far and will be first off the pile next issue. Jean and Kitty have both been portrayed really well and the friend/foe tightrope between Cyclops and Wolverine is back, feeling like it had never left. 5/5
Captain America #3 : Kinda lacking the gruesome disparate feeling that the last 2 had. Was hoping he'd spend more time in the wasteland. The thing that attracted me to this series was the constant struggle and how Cap had adapted to overcome it. Now that he's back on track, he's quickly became too comfortable and back to his old self. 3/5
Punisher: Nightmare #3 : Still loving this, I miss the connection with Jonny and J though. Seeing Punisher play the "good cop" in an investigation really hit home the potential for darkness in Jonny Nightmare. Still one of the best runs of this year so far. 4/5
Ultimate Comics Iron Man #4 : Better than the main Iron Man run, in my opinion. Looking into the foundations of Stark Industries has been fascinating. I can only wonder what it'd be like if they had a stronger artist behind it! 3/5
Batgirl #16 : DotF has got me hooked on Batgirl, to the point that I'll probably keep reading it after the event. 5/5
Batman #16 : Awesome awesome awesome. Scott Snyder's run on Batman is sure to be a future classic. Always surprised, always left guessing. Great artwork by Greg Capullo too; graphic and gory. 5/5
Batman and Robin #16 : One of the weaker DotF's for me...just doesn't seem to mesh well. I feel like the writer is determined to tell his own story away from the tether of DotF, but is sacrificing the book in doing so. 3/5
Superboy #16 : Really liked this, The Justice League members were all given a chance to show what they were great at, and watching them in action and utilizing Superboy was great. Worried that the choices made by Superboy and by Superman (accidentally) in this issue, might slow the event down...will have to wait for the Annual and see! 5/5
The Crow: Skinning the Wolves #2 : I love the original Crow, and to hear James O'Barr was back on the project is what got me onto this book, but it has so far been a little disappointing. It lacks a lot of the ceremony and depth of other Crow incarnations and seems to just wade into the killing without fully explaining the main character. 2/5
Todd the Ugliest Kid On Earth #1 : Thought I'd give this a go, judged a book by it's cover. You can tell this was made with love, but for me, it just rambled on a little too much and I struggled to get into it, great art and refreshing to read something different though. 3/5


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Gear Review
Jeff Guest
SKDY Ditched the volume control and added a mic
I used to have the older version of the Hesh that had the inline volume control and I loved that at work. after 2 of using the Hesh for 8 hours a day 5 days a week one of the speakers died. No problem with the lifetime warranty! I sent them back to Skullcandy and they replaced them with the most updated version! I was a little dissapointed that they got rid of the inline volume control and replaced it with a mic, but the sound is still great and these are still my go to headphones for bumpin' Dubstep at work all day!
can i plug these into my phone?
Jeff Guest
Jordan, Yes, you can still use them with your smarphone with the 3.5mm jack.
View More Details on Skullcandy Hesh 2.0 Headphones


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Senior placekicker Tyler Sievertsen was named the College Football Performance Awards FCS National Placekicker of the Week.
The former Kennedy Cougar went 3 for 3 on field goals against North Dakota State on Saturday. This is the second player of the week honor he has received this season.
Cedar Rapids, Iowa News, Sports, and Weather
Sievertsen Named National Placekicker of the Week
By Josh Christensen, TV9 Sports
Most Popular


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ISRAEL21c takes you to the boardwalk along the Mediterranean coast in Tel Aviv.
Bet you don't know about this southern tourist oasis
Arad lies west of the Dead Sea, south of the Judean Desert and north of the Eastern Negev with its moon-like craters and breathtaking ridges.
The beautiful Banias
It's worth a trip to the Golan Heights to see the tallest waterfall in Israel and the stunning scenery around the Hermon-Banias Nature Reserve.
Postcard from Israel: Port of Tel Aviv
You'll wish you were here, hanging out with family and friends at one of Tel Aviv's best spots for leisure and recreation.
Postcard from Israel: Spring in the Negev
Take a spin with ISRAEL21c through the Red South Festival to see the brilliant wildflowers adding surprising color to the desert.
Business students tour the startup nation
'I enjoyed learning about how direct Israelis are, and the laid-back work environment that helps promote creativity,' says one visiting US student.
Both the old and the new are Jaffa's lure
ISRAEL21c tours the ancient port city to see how the already popular seaside tourist stop is getting more attractive by the day.
Mysterious ancient masks displayed in Israel
Twelve 9,000-year-old stone masks unearthed in the Judean Hills and desert are exhibited together for the first time ever, at the Israel Museum.
The museum that honors a peacemaker and his nation
The Rabin Center and Israeli Museum in Ramat Aviv has an innovative way of illustrating each decade of Yitzhak Rabin's life.
18 cool things to do in Haifa for FREE
Fantastic views, beautiful gardens, holy shrines, world-class art museums – and much of Haifa can be toured for free.
Top 10 places to see Israel's spring flowers
Right now is the peak season for Israel's brilliant wildflowers. Try these spots for some of the best viewing of anemones, cyclamen, irises and more.


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Hostelworld "meet the world" by Lucky Generals
Rating: 5.0
campaignlive.co.uk, Thursday, 17 January 2013 08:00AM
We don't know much about art, but we know what we like. And we're glad to say a small art exhibition at Wieden & Kennedy is on Diary's to-view list. The Shoreditch agency is currently displaying artwork by the famed photographer Sam Baker. As adland can get very tribal sometimes, the subject matter is particularly fitting. The photos (pictured, right) are of tribesmen and tribeswomen taken during Baker's trip to Ethiopia's Omo Valley. The works are on display at W&K over the next month.
This article was first published on campaignlive.co.uk
Before commenting please read our rules for commenting on articles.
If you see a comment you find offensive, you can flag it as inappropriate. In the top right-hand corner of an individual comment, you will see 'flag as inappropriate'. Clicking this prompts us to review the comment. For further information see our rules for commenting on articles.
Rating: 5.0
Agency: Grey London
Rating: 5.0


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You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
21 year old woman | Whiteland, IN, USA | Seeking men 19-23 within 50 miles
I'm a passionate, though slightly out there, writer, who is good and making people laugh and will always listen.
Oh lord, things like this make me nervous. I'm overly passionate about almost anything that catches my attention. I love to read, write, and sing when I'm bored. I'm from a very tight-knit family and they mean everything to me. I also have a collect few friends who might as well be family. I'm looking for someone who can match my levels of passion and make me laugh no matter what. Someone who can handle being dragged to slightly embarrassing family events and even more embarrassing gatherings with friends. I guess to sum it up, I just want someone who's positive.
Book club , Camping , Hobbies and crafts , Movies/Videos , Music and concerts , Nightclubs/Dancing , Playing cards
Dancing, Swimming, Walking / Hiking, Yoga
I exercise 1-2 times per week.
I have Cats
I like Dogs, Exotic Pets, Fish, Horses, Other
The only pet I have is a cat named Lacy. She was a former stray and she was found pregnant. She was the oldest cat up for adoption and they were going to move her to the pound if she didn't get adopted soon....needless to say my bleeding heart won.
Any local park. I like being outside and I tend to find a spot under a tree and either read, write, or people watch.
I love music, movies, and books. I generally like music that either makes me think or is something I can have fun listening to. I am absolutely in love with horror movies and movies that draw me in. As for books, I will read almost anything.
To be completely honest, I can have fun doing almost anything. I had fun wearing a fake mustache on the floor of a factory while sorting parts once.
I just recently finished reading a young adult book called A Great and Terrible Beauty. It's based in the Victorian era in England and focuses mainly on women trying to break free from the chains that bind them.
Z{{Cta}} {{#if HasMoreInfo}} ?{{CtaSecondary}} {{/if}}
|What she is looking for
|5'2" (157cms)
|5'3" (160cms) to 6'2" (187cms)
|Body type:
|Slender, About average, Athletic and toned, A few extra pounds
|No preference
|Dark brown
|No preference
|No Way
|No preference
|Social Drinker
|No preference
|No preference
Other than being a student I'm temporarily working at a factory that makes car parts called Tasus. I do something called rework, which is pretty much fixing everyone's problems. It's not the most fun, but I've met some interesting people.
|I'll tell you later
|No preference
|Never Married
|No preference
|Have kids:
|Wants kids:
|I'll tell you later
|No preference
|White / Caucasian
|No preference
|No preference
See, this is the part that's going to throw people off. I'm Wiccan. Yes, that means I'm a witch. No, that does not mean I worship the devil. Wicca is a nature based religion with a God and Goddess that focuses on finding balance in your life.
|Some college
|High school, Some college
It took me a while to figure out what I wanted to major in, but currently I'm a library science major. I've always wanted a career that I could be happy with and not hate, no matter how much it pays. Happiness matters.


text: Mashup: Shazam Share via FacebookShare via TwitterShare via LinkedinShare via Google+Share via RedditShare via Mail Shazam's auto playlist functionality, exclusive to Rdio, allows Rdio users to tag a song in Shazam then create a new Rdio playlist from the tagged songs. Track this Mashup Submit a mashup Summary Comments Followers Comments (0) Comment * Leave this field blank Post Comment Followers (0) Track this Mashup Specs Related APIs Rdio URL http://www.rdio.com/developers/gallery/ Mashups using the same API (6) /View all Fathom Fathom leverages the Rdio and Last.fm APIs to provide an immersive music service. With each click, Fathom provides new artists and songs that adhere to the users preferences. re/spin Re/Spin leverages the Last.fm, Rdio, and Spotify Metadata API to allow music lovers to import any of their hand crafted playlists into Rdio. Cyborg Karaoke Party A Music Hack Day application using karaoke tracks from Rdio and a text to speech library to convert synchronized lyrics from LyricFind to digital speech. Add Mashup


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- Business & Professional
- Karl-Marx-Str. 141, Berlin, 12043
Angel and Venture Capital Investment Term Sheets
Berlin Startup Culture
Tuesday, August 28, 2012 at 7:30 PM (CEST)
Michael, who has a PhD in venture capital financing will give a presentation on venture capital and angel term sheets explaining the key clauses that founders need to understand. The presentation will cover basic funding, corporate governance and liquidation clauses:
Funding Clauses
Amount to be Invested
Capital Structure
Conversion Rights
Vesting Terms
Transfer Restrictions of Founders' Shares
Dividend Rights
Pre-Emption Rights
Right of First Refusal
Contingent Proxy
Stock Option Pool
Staged Financing
Anti-dilution of Venture Capital Shares
Confidentiality and Non-Disclosure Agreements
Executive Employee Agreement
Affirmative and Negative Covenants
Representations and Warranties
Applicable Law
Legal Fees
Corporate Governance Clauses
Board Composition
Information Rights
Consent Rights
Super-majority Voting Rights
Liquidation Clauses
Liquidation Preference
Liquidation Preference Multiple
Liquidation Participation Rights
Drag-Along Right
Tag-Along Right
Attending this event will give you a good basic understanding of investment term sheets. A Q&A session will follow after the presentation. For the event real examples of terms sheets will be provided. Looking forward to see you. Looking forward to see a lot of people. There will be pizza and drinks for refreshment.
Join millions of people on Eventbrite.
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