Developmental stage-dependent effects of perceived predation risk on nestling tree swallows (Tachycineta bicolor).

The risk of predation directly affects the physiology, behavior, and fitness of wild birds. Strong social connections with conspecifics could help individuals recover from a stressful experience such as a predation event; however, competitive interactions also have the potential to exacerbate stress. Few studies have investigated the interaction between environmental stressors and the social landscape in wild bird populations. In 2 years of field studies, we experimentally simulated predation attempts on breeding female tree swallows (Tachicyneta bicolor). At the same time, we manipulated female breast plumage color, a key social signal. Simulated predation events on tree swallows early in the nestling period reduced young nestlings' mass by approximately 20% and shortened telomere lengths. Ultimately, only 31% of nestlings in the predation group fledged compared with 70% of control nestlings. However, the effects of experimental manipulations were timing dependent: the following year when we swapped the order of the experimental manipulations and simulated predation during incubation, there were no significant effects of predation on nestling condition or fledging success. Contrary to our expectations, manipulation of the social environment did not affect the response of tree swallows to simulated predation. However, manipulating female plumage during the nestling period did reduce nestling skeletal size and mass, although the effects depended on original plumage brightness. Our data demonstrate that transient stressors on female birds can have carry-over effects on their nestlings if they occur during critical periods in the breeding season.

Experimentally elevated corticosterone does not affect bacteria killing ability of breeding female tree swallows (Tachycineta bicolor).

The immune system can be modulated when organisms are exposed to acute or chronic stressors. Glucocorticoids (GCs), the primary hormonal mediators of the physiological stress response, are suspected to play a crucial role in immune modulation. However, most evidence of stress-associated immunomodulation does not separate the effects of glucocorticoid-dependent pathways from those of glucocorticoid-independent mechanisms on immune function. In this study, we experimentally elevated circulating corticosterone, the main avian glucocorticoid, in free-living female tree swallows (Tachycineta bicolor) for one to two weeks to test its effects on immune modulation. Natural variation in bacteria killing ability (BKA), a measure of innate constitutive immunity, was predicted by the interaction between timing of breeding and corticosterone levels. However, experimental elevation of corticosterone had no effect on BKA. Therefore, even when BKA is correlated with natural variation in glucocorticoid levels, this relationship may not be causal. Experiments are necessary to uncover the causal mechanisms of immunomodulation and the consequences of acute and chronic stress on disease vulnerability. Findings in other species indicate that acute increases in GCs can suppress BKA; but our results support the hypothesis that this effect does not persist over longer timescales, during chronic elevations in GCs. Direct comparisons of the effects of acute vs. chronic elevation of GCs on BKA will be important for testing this hypothesis.

Social signal manipulation and environmental challenges have independent effects on physiology, internal microbiome, and reproductive performance in tree swallows (Tachycineta bicolor).

The social environment that individuals experience appears to be a particularly salient mediator of stress resilience, as the nature and valence of social interactions are often related to subsequent health, physiology, microbiota, and overall stress resilience. Relatively few studies have simultaneously manipulated the social environment and ecological challenges under natural conditions. Here, we report the results of experiments in wild tree swallows (Tachycineta bicolor) in which we manipulated both ecological challenges (predator encounters and flight efficiency reduction) and social interactions (by experimental dulling of a social signal). In two experiments conducted in separate years, we reversed the order of these treatments so that females experienced either an altered social signal followed by a challenge or vice-versa. Before, during, and after treatments were applied, we tracked breeding success, morphology and physiology (mass, corticosterone, and glucose), nest box visits via an RFID sensor network, cloacal microbiome diversity, and fledging success. Overall, we found that predator exposure during the nestling period reduced the likelihood of fledging and that signal manipulation sometimes altered nest box visitation patterns, but little evidence that the two categories of treatment interacted with each other. We discuss the implications of our results for understanding what types of challenges and what conditions are most likely to result in interactions between the social environment and ecological challenges.

Cold temperatures induce priming of the glucose stress response in tree swallows.

Capricious environments often present wild animals with challenges that coincide or occur in sequence. Conceptual models of the stress response predict that one threat may prime or dampen the response to another. Although evidence has supported this for glucocorticoid responses, much less is known about the effects of previous challenges on energy mobilization. Food limitation may have a particularly important effect, by altering the ability to mobilize energy when faced with a subsequent challenge. We tested the prediction that challenging weather conditions, which reduce food availability, alter the energetic response to a subsequent acute challenge (capture and restraint). Using a three-year dataset from female tree swallows measured during three substages of breeding, we used a model comparison approach to test if weather (temperature, wind speed, and precipitation) over 3- or 72-hour timescales predicted baseline and post-restraint glucose levels, and if so which environmental factors were the strongest predictors. Contrary to our predictions, weather conditions did not affect baseline glucose; however, birds that had experienced lower temperatures over the preceding 72 h tended to have higher stress-induced glucose when faced with an acute stressor. We also saw some support for an effect of rainfall on stress-induced glucose: around the time that eggs hatched, birds that had experienced more rainfall over the preceding 72 h mounted lower responses. Overall, we find support in a wild animal for the idea that the glucose stress response may be primed by exposure to prior challenges.

Timing of Breeding Reveals a Trade-Off between Immune Investment and Life History in Tree Swallows.

The allocation of limited resources among life history traits creates trade-offs that constrain the range of possible phenotypes of organisms. In animals, the cost of maintaining an effective immune response may reduce the ability to invest in reproduction, resulting in altered susceptibility to disease. However, not all members of a population face identical constraints because differences in an individual's environmental context or physiological state can influence the degree to which traits are negatively associated. Here, we evaluated how variation in timing of breeding, a correlate of fitness, may result in different patterns of trait associations between immunity and reproduction. We measured constitutive immunity in breeding female tree swallows (Tachycineta bicolor) using a bacteria killing assay with blood plasma to assess the relationships between bacteria killing ability (BKA), reproductive effort, and reproductive success. We found that timing of breeding can influence the association between BKA and reproductive effort, but its effects are not homogeneous among all traits. Late-breeding tree swallows with stronger BKA laid smaller clutches, a pattern that was not apparent in early breeders. Regardless of the timing of breeding, birds with stronger BKA fed their nestlings less. Despite a negative association with reproductive effort, we found no association between immunity and reproductive success. We provide evidence that individual tree swallows do not experience some trade-offs equally, and that timing of breeding likely plays a role in how costs of immunity are weighed. To understand how investment in immunity can limit life history traits, we must consider how a variation among individuals influences the relative costs of immunity.

Individual variation in natural or manipulated corticosterone does not covary with circulating glucose in a wild bird.

Animals respond to sudden challenges with a coordinated set of physiological and behavioral responses that enhance the ability to cope with stressors. While general characteristics of the vertebrate stress response are well described, it is not as clear how individual components covary between or within individuals. A rapid increase in glucocorticoids coordinates the stress response and one of the primary downstream results is an increase in glucose availability via reduced glucose utilization. Here, we asked whether between- and within-individual variation in corticosterone directly predict variation in glucose. We collected 2673 paired glucose and corticosterone measures from 776 tree swallows (Tachycineta bicolor) from four populations spanning the species range. In adults, glucose and corticosterone both increased during a standardized restraint protocol in all four populations. Moreover, in one population experimentally increasing a precursor that stimulates corticosterone release resulted in a further increase in both measures. In contrast, nestlings did not show a robust glucose response to handling or manipulation. Despite this group-level variation, there was very little evidence in any population that between-individual variation in corticosterone predicted between-individual variation in glucose regulation. Glucose was moderately repeatable within individuals, but within-individual variation in glucose and corticosterone were unrelated. Our results highlight the fact that a strong response in one aspect of the coordinated acute stress response (corticosterone) does not necessarily indicate that specific downstream components, such as glucose, will show similarly strong responses. These results have implications for understanding the evolution of integrated stress response systems.

Live imaging of intra-lysosome pH in cell lines and primary neuronal culture using a novel genetically encoded biosensor.

Disorders of lysosomal physiology have increasingly been found to underlie the pathology of a rapidly growing cast of neurodevelopmental disorders and sporadic diseases of aging. One cardinal aspect of lysosomal (dys)function is lysosomal acidification in which defects trigger lysosomal stress signaling and defects in proteolytic capacity. We have developed a genetically encoded ratiometric probe to measure lysosomal pH coupled with a purification tag to efficiently purify lysosomes for both proteomic and in vitro evaluation of their function. Using our probe, we showed that lysosomal pH is remarkably stable over a period of days in a variety of cell types. Additionally, this probe can be used to determine that lysosomal stress signaling via TFEB is uncoupled from gross changes in lysosomal pH. Finally, we demonstrated that while overexpression of ARL8B GTPase causes striking alkalinization of peripheral lysosomes in HEK293 T cells, peripheral lysosomes per se are no less acidic than juxtanuclear lysosomes in our cell lines.Abbreviations: ARL8B: ADP ribosylation factor like GTPase 8B; ATP: adenosine triphosphate; ATP5F1B/ATPB: ATP synthase F1 subunit beta; ATP6V1A: ATPase H+ transporting V1 subunit A; Baf: bafilomycin A1; BLOC-1: biogenesis of lysosome-related organelles complex 1; BSA: bovine serum albumin; Cos7: African green monkey kidney fibroblast-like cell line; CQ: chloroquine; CTSB: cathepsin B; CYCS: cytochrome c, somatic; DAPI: 4',6-diamidino -2- phenylindole; DIC: differential interference contrast; DIV: days in vitro; DMEM: Dulbecco's modified Eagle's medium; E8: embryonic day 8; EEA1: early endosome antigen 1; EGTA: ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid; ER: endoplasmic reticulum; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; GABARAPL2: GABA type A receptor associated protein like 2; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GOLGA2/GM130: golgin A2; GTP: guanosine triphosphate; HEK293T: human embryonic kidney 293 cells, that expresses a mutant version of the SV40 large T antigen; HeLa: Henrietta Lacks-derived cell; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; HRP: horseradish peroxidase; IGF2R/ciM6PR: insulin like growth factor 2 receptor; LAMP1/2: lysosomal associated membrane protein 1/2; LMAN2/VIP36: lectin, mannose binding 2; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTORC1: mechanistic target of rapamycin kinase complex 1; PCR: polymerase chain reaction; PDL: poly-d-lysine; PGK1p: promotor from human phosphoglycerate kinase 1; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PPT1/CLN1: palmitoyl-protein thioesterase 1; RPS6KB1/p70: ribosomal protein S6 kinase B1; STAT3: signal transducer and activator of transcription 3; TAX1BP1: Tax1 binding protein 1; TFEB: transcription factor EB; TGN: trans-Golgi network; TGOLN2/TGN46: trans-Golgi network protein 2; TIRF: total internal reflection fluorescence; TMEM106B: transmembrane protein 106B; TOR: target of rapamycin; TRPM2: transient receptor potential cation channel subfamily M member 2; V-ATPase: vacuolar-type proton-translocating ATPase; VPS35: VPS35 retromer complex component.

Tollip coordinates Parkin-dependent trafficking of mitochondrial-derived vesicles.

Multiple mitochondrial quality control pathways exist to maintain the health of mitochondria and ensure cell homeostasis. Here, we investigate the role of the endosomal adaptor Tollip during the mitochondrial stress response and identify its interaction and colocalisation with the Parkinson's disease-associated E3 ubiquitin ligase Parkin. The interaction between Tollip and Parkin is dependent on the ubiquitin-binding CUE domain of Tollip, but independent of Tom1 and mitophagy. Interestingly, this interaction is independent of Parkin mitochondrial recruitment and ligase activity but requires an intact ubiquitin-like (UBL) domain. Importantly, Tollip regulates Parkin-dependent endosomal trafficking of a discrete subset of mitochondrial-derived vesicles (MDVs) to facilitate delivery to lysosomes. Retromer function and an interaction with Tom1 allow Tollip to facilitate late endosome/lysosome trafficking in response to mitochondrial stress. We find that upregulation of TOM20-positive MDVs upon mitochondrial stress requires Tollip interaction with ubiquitin, endosomal membranes and Tom1 to ensure their trafficking to the lysosomes. Thus, we conclude that Tollip, via an association with Parkin, is an essential coordinator to sort damaged mitochondrial-derived cargo to the lysosomes.

Stress Resilience and the Dynamic Regulation of Glucocorticoids.

Vertebrates respond to a diversity of stressors by rapidly elevating glucocorticoid (GC) levels. The changes in physiology and behavior triggered by this response can be crucial for surviving a variety of challenges. Yet the same process that is invaluable in coping with immediate threats can also impose substantial damage over time. In addition to the pathological effects of long-term exposure to stress hormones, even relatively brief elevations can impair the expression of a variety of behaviors and physiological processes central to fitness, including sexual behavior, parental behavior, and immune function. Therefore, the ability to rapidly and effectively terminate the short-term response to stress may be fundamental to surviving and reproducing in dynamic environments. Here we review the evidence that variation in the ability to terminate the stress response through negative feedback is an important component of stress coping capacity. We suggest that coping capacity may also be influenced by variation in the dynamic regulation of GCs-specifically, the ability to rapidly turn on and off the stress response. Most tests of the fitness effects of these traits to date have focused on organisms experiencing severe or prolonged stressors. Here we use data collected from a long-term study of tree swallows (Tachycineta bicolor) to test whether variation in negative feedback, or other measures of GC regulation, predict components of fitness in non-chronically stressed populations. We find relatively consistent, but generally weak relationships between different fitness components and the strength of negative feedback. Reproductive success was highest in individuals that both mounted a robust stress response and had strong negative feedback. We did not see consistent evidence of a relationship between negative feedback and adult or nestling survival: negative feedback was retained in the best supported models of nestling and adult survival, but in two of three survival-related analyses the intercept-only model received only slightly less support. Both negative feedback and stress-induced GC levels-but not baseline GCs-were individually repeatable. These measures of GC activity did not consistently covary across ages and life history stages, indicating that they are independently regulated. Overall, the patterns seen here are consistent with the predictions that negative feedback-and the dynamic regulation of GCs-are important components of stress coping capacity, but that the fitness benefits of having strong negative feedback during the reproductive period are likely to manifest primarily in individuals exposed to chronic or repeated stressors.

A MAPK/c-Jun-mediated switch regulates the initial adaptive and cell death responses to mitochondrial damage in a neuronal cell model.

Parkinson's disease (PD) is defined by the progressive loss of dopaminergic neurons. Mitochondrial dysfunction and oxidative stress are associated with PD although it is not fully understood how neurons respond to these stresses. How adaptive and apoptotic neuronal stress response pathways are regulated and the thresholds at which they are activated remains ambiguous. Utilising SH-SY5Y neuroblastoma cells, we show that MAPK/AP-1 pathways are critical in regulating the response to mitochondrial uncoupling. Here we found the AP-1 transcription factor c-Jun can act in either a pro- or anti-apoptotic manner, depending on the level of stress. JNK-mediated cell death in differentiated cells only occurred once a threshold of stress was surpassed. We also identified a novel feedback loop between Parkin activity and the c-Jun response, suggesting defective mitophagy may initiate MAPK/c-Jun-mediated neuronal loss observed in PD. Our data supports the hypothesis that blocking cell death pathways upstream of c-Jun as a therapeutic target in PD may not be appropriate due to crossover of the pro- and anti-apoptotic responses. Boosting adaptive responses or targeting specific aspects of the neuronal death response may therefore represent more viable therapeutic strategies.

Characterization of Primary Cilia in Normal Fallopian Tube Epithelium and Serous Tubal Intraepithelial Carcinoma.

OBJECTIVES: The aim of this study was to investigate the distribution of primary cilia on secretory cells in normal fallopian tube (FT) and serous tubal intraepithelial carcinoma (STIC). METHODS: Fallopian tube tissue samples were obtained from 4 females undergoing prophylactic hysterectomies and 6 patients diagnosed with STIC. A mogp-TAg transgenic mouse STIC sample was also compared with a wild-type mouse FT sample. Serous tubal intraepithelial carcinoma was identified by hematoxylin and eosin staining and confirmed by positive Ki-67 and p53 immunohistochemical staining of tissue sections. We assessed the relative distribution of primary cilia on secretory cells and motile cilia on multiple ciliated cells by immunofluorescence and immunohistochemical staining. Ciliary function was assessed by immunofluorescence staining of specific ciliary marker proteins and responsiveness to Sonic Hedgehog signaling. RESULTS: Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of human FT where they may appear to mediate ciliary-mediated Sonic Hedgehog signaling. A statistically significant reduction in the number of primary cilia on secretory cells was observed in human STIC samples compared with normal controls (P < 0.0002, Student t test), supported by similar findings in a mouse STIC sample. Immunohistochemical staining for dynein axonemal heavy chain 5 discriminated multiple motile cilia from primary cilia in human FT. CONCLUSIONS: Primary cilia are widespread on secretory cells in the ampulla, isthmus, and in particular, the fimbriae of the human FT but are significantly reduced in both human and mouse STIC samples. Immunohistochemical staining for ciliary proteins may have clinical utility for early detection of STIC.

Optineurin: A Coordinator of Membrane-Associated Cargo Trafficking and Autophagy.

Optineurin is a multifunctional adaptor protein intimately involved in various vesicular trafficking pathways. Through interactions with an array of proteins, such as myosin VI, huntingtin, Rab8, and Tank-binding kinase 1, as well as via its oligomerisation, optineurin has the ability to act as an adaptor, scaffold, or signal regulator to coordinate many cellular processes associated with the trafficking of membrane-delivered cargo. Due to its diverse interactions and its distinct functions, optineurin is an essential component in a number of homeostatic pathways, such as protein trafficking and organelle maintenance. Through the binding of polyubiquitinated cargoes via its ubiquitin-binding domain, optineurin also serves as a selective autophagic receptor for the removal of a wide range of substrates. Alternatively, it can act in an ubiquitin-independent manner to mediate the clearance of protein aggregates. Regarding its disease associations, mutations in the optineurin gene are associated with glaucoma and have more recently been found to correlate with Paget's disease of bone and amyotrophic lateral sclerosis (ALS). Indeed, ALS-associated mutations in optineurin result in defects in neuronal vesicular localisation, autophagosome-lysosome fusion, and secretory pathway function. More recent molecular and functional analysis has shown that it also plays a role in mitophagy, thus linking it to a number of other neurodegenerative conditions, such as Parkinson's. Here, we review the role of optineurin in intracellular membrane trafficking, with a focus on autophagy, and describe how upstream signalling cascades are critical to its regulation. Current data and contradicting reports would suggest that optineurin is an important and selective autophagy receptor under specific conditions, whereby interplay, synergy, and functional redundancy with other receptors occurs. We will also discuss how dysfunction in optineurin-mediated pathways may lead to perturbation of critical cellular processes, which can drive the pathologies of number of diseases. Therefore, further understanding of optineurin function, its target specificity, and its mechanism of action will be critical in fully delineating its role in human disease.

Role of matrix metalloproteinases 2 and 9, toll-like receptor 4 and platelet-leukocyte aggregate formation in sepsis-associated thrombocytopenia.

BACKGROUND: The development of thrombocytopenia in sepsis is a poor prognostic indicator associated with a significantly increased mortality risk. Mechanisms underlying this phenomenon remain to be clearly elucidated. Matrix metalloproteinases (MMPs) are enzymes that regulate the turnover of the extra-cellular matrix. MMP-2 is recognised as a platelet agonist with MMP-9 proposed as an inhibitor of platelet activation. The existence of MMP-9 in platelets is a subject of debate. There is limited evidence thus far to suggest that toll-like receptor 4 (TLR-4) and platelet-leukocyte aggregate (PLA) formation may be implicated in the development of sepsis-associated thrombocytopenia. OBJECTIVES: To investigate whether MMP -2/-9, toll-like receptor 4 (TLR-4) or platelet-leukocyte aggregate (PLA) formation are implicated in a decline in platelet numbers during septic shock. METHODS: This was an observational study which recruited healthy controls, non-thrombocytopenic septic donors and thrombocytopenic septic donors. MMP-2, MMP-9 and TLR-4 platelet surface expression as well as PLA formation was examined using flow cytometry. In addition MMP-2 and MMP-9 were examined by gelatin zymography and enzyme-linked immunosorbent assay (ELISA) using a 3 compartment model (plasma, intraplatelet and platelet membrane). RESULTS: There was no difference found in MMP-2, MMP-9 or TLR-4 levels between non-thrombocytopenic and thrombocytopenic septic donors. PLA formation was increased in thrombocytopenic patients. MMP-9 was detected in platelets using flow cytometry, gelatin zymography and ELISA techniques. CONCLUSIONS: Platelet consumption into PLAs may account for the development of thrombocytopenia in septic shock. MMP-9 is found in platelets and it is upregulated during septic shock.

Platelet microaggregation in sepsis examined by quartz crystal microbalance with dissipation technology.

Antiplatelet therapies remain an area of potential interest for the treatment of sepsis; however, studies of platelet aggregation in sepsis have yielded conflicting results. We examined platelet aggregation patterns in patients with septic shock using quartz crystal microbalance with dissipation technology, a microfluidic device capable of measuring platelet microaggregate formation under flow conditions. Platelet aggregation was increased in the washed platelet samples of septic patients. Conversely, these same platelets aggregated less than healthy controls when examined in their plasma.

Differences in sexual development in inbred and outbred zebrafish (Danio rerio) and implications for chemical testing.

Outbred laboratory animal strains used in ecotoxicology are intended to represent wild populations. However, breeding history may vary considerably between strains, driving differences in genetic variation and phenotypes used for assessing effects of chemical exposure. We compared a range of phenotypic endpoints in zebrafish from four different "breeding treatments" comprising a Wild Indian Karyotype (WIK) zebrafish strain and a WIK/Wild strain with three levels of inbreeding (F(IT)=n, n+0.25, n+0.375) in a new Fish Sexual Development Test (FSDT). There were no differences between treatments in terms of egg viability, hatch success or fry survival. However, compared with WIKs, WIK/Wild hybrids were significantly larger in size, with more advanced gonadal (germ cell) development at the end of the test (63 days post fertilisation). Increasing the levels of inbreeding in the related WIK/Wild lines did not affect body size, but there was a significant male-bias (72%) in the most inbred line (F(IT)=n+0.375). Conversely, in the reference WIK strain there was a significant female-bias in the population (80% females). Overall, our results support the use of outbred zebrafish strains in the FSDT, where one of the core endpoints is sex ratio. Despite increased variance (and reduced statistical power) for some endpoints, WIK/Wild outbreds (F(IT)=n) met all acceptance criteria for controls in this test, whereas WIKs failed to comply with tolerance limits for sex ratio (30-70% females). Sexual development was also more advanced in WIK/Wild outbreds (cf. WIKs), providing greater scope for detection of developmental reproductive toxicity following chemical exposure.

Correction of phenotype in a thalassemia mouse model using a nonmyeloablative marrow transplantation regimen.

Gene therapy, the replacement of normal human beta- or gamma-globin genes into the hematopoietic stem cells of patients with homozygous beta-thalassemia, is a promising therapy for the future. High-level lineage-specific stable globin expression in transduced cells reinfused into patients in an autologous transplantation setting could be curative, if successful. Previous studies have shown high-level donor chimerism in nonmyeloablated non-thalassemic hosts. We have now studied the conditions for stable long-term engraftment of normal cells into a thalassemia mouse model that lead to high-level donor chimerism and correction of the abnormal phenotype. Thalassemic female mice treated with 0 to 300 cGy whole-body irradiation received transplantations of donor cells harvested from wild-type males. Engraftment of male cells was quantitated by Y-chromosome polymerase chain reaction analysis of blood and marrow progenitors, and changes in hemoglobin levels, red cell morphology, and spleen size were measured at various times posttransplantation. High-level stable donor cell engraftment was achieved in mice given 200 cGy and receiving transplants of 2 x 10(7) or more donor cells. The anemia, abnormal peripheral blood smears, and splenomegaly improved in the thalassemic mice that had successful engraftment. These studies demonstrate that stable and successful levels of engraftment of normal cells can correct the thalassemic phenotype without fully myeloablating the host. This animal model should allow us to test the amount of cytoreduction required and the level of engraftment and beta-globin expression needed in autologous transplantation of beta-globin gene-transduced cells to correct the abnormal phenotype in thalassemic mice, and it may be relevant to human clinical trials, as well.