Long lasting anxiety following early life stress is dependent on glucocorticoid signaling in zebrafish.

Chronic adversity in early childhood is associated with increased anxiety and a propensity for substance abuse later in adulthood, yet the effects of early life stress (ELS) on brain development remain poorly understood. The zebrafish, Danio rerio, is a powerful model for studying neurodevelopment and stress. Here, we describe a zebrafish model of ELS and identify a role for glucocorticoid signaling during a critical window in development that leads to long-term changes in brain function. Larval fish subjected to chronic stress in early development exhibited increased anxiety-like behavior and elevated glucocorticoid levels later in life. Increased stress-like behavior was only observed when fish were subjected to ELS within a precise time window in early development, revealing a temporal critical window of sensitivity. Moreover, enhanced anxiety-like behavior only emerges after two months post-ELS, revealing a developmentally specified delay in the effects of ELS. ELS leads to increased levels of baseline cortisol, and resulted in a dysregulation of cortisol receptors' mRNA expression, suggesting long-term effects on cortisol signaling. Together, these findings reveal a 'critical window' for ELS to affect developmental reprogramming of the glucocorticoid receptor pathway, resulting in chronic elevated stress.

Repression precedes independent evolutionary gains of a highly specific gene expression pattern.

Highly specific expression patterns can be caused by the overlapping activities of activator and repressor sequences in enhancers. However, few studies illuminate how these sequences evolve in the origin of new enhancers. Here, we show that expression of the bond gene in the semicircular wall epithelium (swe) of the Drosophila melanogaster male ejaculatory bulb (EB) is controlled by an enhancer consisting of an activator region that requires Abdominal-B driving expression in the entire EB and a repressor region that restricts this expression to the EB swe. Although this expression pattern is independently gained in the distantly related Scaptodrosophila lebanonensis and does not require Abdominal-B, we show that functionally similar repressor sequences are present in Scaptodrosophila and also in species that do not express bond in the EB. We suggest that during enhancer evolution, repressor sequences can precede the evolution of activator sequences and may lead to similar but independently evolved expression patterns.

Analysis of stress responses in Astyanax larvae reveals heterogeneity among different populations.

Stress responses are conserved physiological and behavioral outcomes as a result of facing potentially harmful stimuli, yet in pathological states, stress becomes debilitating. Stress responses vary considerably throughout the animal kingdom, but how these responses are shaped evolutionarily is unknown. The Mexican cavefish has emerged as a powerful system for examining genetic principles underlying behavioral evolution. Here, we demonstrate that cave Astyanax have reduced behavioral and physiological measures of stress when examined at larval stages. We also find increased expression of the glucocorticoid receptor, a repressible element of the neuroendocrine stress pathway. Additionally, we examine stress in three different cave populations, and find that some, but not all, show reduced stress measures. Together, these results reveal a mechanistic system by which cave-dwelling fish reduced stress, presumably to compensate for a predator poor environment.

Behavioral Approaches to Studying Innate Stress in Zebrafish.

Responding appropriately to stressful stimuli is essential for survival of an organism. Extensive research has been done on a wide spectrum of stress-related diseases and psychiatric disorders, yet further studies into the genetic and neuronal regulation of stress are still required to develop better therapeutics. The zebrafish provides a powerful genetic model to investigate the neural underpinnings of stress, as there exists a large collection of mutant and transgenic lines. Moreover, pharmacology can easily be applied to zebrafish, as most drugs can be added directly to water. We describe here the use of the 'novel tank test' as a method to study innate stress responses in zebrafish, and demonstrate how potential anxiolytic drugs can be validated using the assay. The method can easily be coupled with zebrafish lines harboring genetic mutations, or those in which transgenic approaches for manipulating precise neural circuits are used. The assay can also be used in other fish models. Together, the described protocol should facilitate the adoption of this simple assay to other laboratories.

Manipulation of Gene Function in Mexican Cavefish.

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.

Justice and global care chains: Lessons from Singapore.

Growing demand for direct care workers to assist care-dependent elderly people has created an opening for migrant workers from low- income nations to sell their services to middle and high-income nations. Using Singapore as a case example, we draw on capability theory to make the case that receiving nations that import direct care workers should be held to global justice standards that protect workers' floor level human capabilities. Specifically, we (1) show that Singapore and other receiving nations fail to protect human capabilities at a threshold level required by dignity; (2) identify specific human capabilities placed at risk; and (3) recommend standards for receiving nations that support central capabilities. As populations in both developed and developing nations are rapidly aging, these concerns could not be timelier.

Convergence on reduced stress behavior in the Mexican blind cavefish.

Responding appropriately to stress is essential for survival, yet in pathological states, these responses can develop into debilitating conditions such as post-traumatic stress disorder and generalized anxiety. While genetic models have provided insight into the neurochemical and neuroanatomical pathways that underlie stress, little is known about how evolutionary processes and naturally occurring variation contribute to the diverse responses to stressful stimuli observed in the animal kingdom. The Mexican cavefish is a powerful system to address how altered genetic and neuronal systems can give rise to altered behaviors. When introduced into a novel tank, surface fish and cavefish display a stereotypic stress response, characterized by reduced exploratory behavior and increased immobility, akin to "freezing". The stress response in cave and surface forms is reduced by pharmacological treatment with the anxiolytic drug, buspirone, fortifying the notion that behavior in the assay represents a conserved stress state. We find that cave populations display reduced behavioral measures of stress compared to surface conspecifics, including increased time in the top half of the tank and fewer periods of immobility. Further, reduced stress responses are observed in multiple independently derived cavefish populations, suggesting convergence on loss of behavioral stress responses in the novel tank assay. These findings provide evidence of a naturally occurring species with two drastically different forms in which a shift in predator-rich ecology to one with few predators corresponds to a reduction in stress behavior.

Management of Patients Who Receive an Organ Transplant Abroad and Return Home for Follow-up Care: Recommendations From the Declaration of Istanbul Custodian Group.

Eradicating transplant tourism depends on complex solutions that include efforts to progress towards self-sufficiency in transplantation. Meanwhile, professionals and authorities are faced with medical, legal, and ethical problems raised by patients who return home after receiving an organ transplant abroad, particularly when the organ has been obtained through illegitimate means. In 2016, the Declaration of Istanbul Custodian Group convened an international, multidisciplinary workshop in Madrid, Spain, to address these challenges and provide recommendations for the management of these patients, which are presented in this paper. The core recommendations are grounded in the belief that principles of transparency, traceability, and continuity of care applied to patients who receive an organ domestically should also apply to patients who receive an organ abroad. Governments and professionals are urged to ensure that, upon return, patients are promptly referred to a transplant center for evaluation and care, not cover the costs of transplants resulting from organ or human trafficking, register standardized information at official registries on patients who travel for transplantation, promote international exchange of data for traceability, and develop a framework for the notification of identified or suspected cases of transnational transplant-related crimes by health professionals to law enforcement agencies.

Hormonal Modulation of Pheromone Detection Enhances Male Courtship Success.

During the lifespans of most animals, reproductive maturity and mating activity are highly coordinated. In Drosophila melanogaster, for instance, male fertility increases with age, and older males are known to have a copulation advantage over young ones. The molecular and neural basis of this age-related disparity in mating behavior is unknown. Here, we show that the Or47b odorant receptor is required for the copulation advantage of older males. Notably, the sensitivity of Or47b neurons to a stimulatory pheromone, palmitoleic acid, is low in young males but high in older ones, which accounts for older males' higher courtship intensity. Mechanistically, this age-related sensitization of Or47b neurons requires a reproductive hormone, juvenile hormone, as well as its binding protein Methoprene-tolerant in Or47b neurons. Together, our study identifies a direct neural substrate for juvenile hormone that permits coordination of courtship activity with reproductive maturity to maximize male reproductive fitness.

Clarifying the best interests standard: the elaborative and enumerative strategies in public policy-making.

One recurring criticism of the best interests standard concerns its vagueness, and thus the inadequate guidance it offers to care providers. The lack of an agreed definition of 'best interests', together with the fact that several suggested considerations adopted in legislation or professional guidelines for doctors do not obviously apply across different groups of persons, result in decisions being made in murky waters. In response, bioethicists have attempted to specify the best interests standard, to reduce the indeterminacy surrounding medical decisions. In this paper, we discuss the bioethicists' response in relation to the state's possible role in clarifying the best interests standard. We identify and characterise two clarificatory strategies employed by bioethicists -elaborative and enumerative-and argue that the state should adopt the latter. Beyond the practical difficulties of the former strategy, a state adoption of it would inevitably be prejudicial in a pluralistic society. Given the gravity of best interests decisions, and the delicate task of respecting citizens with different understandings of best interests, only the enumerative strategy is viable. We argue that this does not commit the state to silence in providing guidance to and supporting healthcare providers, nor does it facilitate the abuse of the vulnerable. Finally, we address two methodological worries about adopting this approach at the state level. The adoption of the enumerative strategy is not defeatist in attitude, nor does it eventually collapse into (a form of) the elaborative strategy.

Sequential Collision- and Ozone-Induced Dissociation Enables Assignment of Relative Acyl Chain Position in Triacylglycerols.

Unambiguous identification of isomeric lipids by mass spectrometry represents a significant analytical challenge in contemporary lipidomics. Herein, the combination of collision-induced dissociation (CID) with ozone-induced dissociation (OzID) on an ion-trap mass spectrometer is applied to the identification of triacylglycerol (TG) isomers that vary only by the substitution pattern of fatty acyl (FA) chains esterified to the glycerol backbone. Isolated product ions attributed to loss of a single FA arising from CID of [TG + Na](+) ions react rapidly with ozone within the ion trap. The resulting CID/OzID spectra exhibit abundant ions that unequivocally reveal the relative position of FAs along the backbone. Isomeric TGs containing two or three different FA substituents are readily differentiated by diagnostic ions present in their CID/OzID spectra. Compatibility of this method with chromatographic separations enables the characterization of unusual TGs containing multiple short-chain FAs present in Drosophila.

The fatty acid elongase Bond is essential for Drosophila sex pheromone synthesis and male fertility.

Insects use a spectacular variety of chemical signals to guide their social behaviours. How such chemical diversity arises is a long-standing problem in evolutionary biology. Here we describe the contribution of the fatty acid elongase Bond to both pheromone diversity and male fertility in Drosophila. Genetic manipulation and mass spectrometry analysis reveal that the loss of bond eliminates the male sex pheromone (3R,11Z,19Z)-3-acetoxy-11,19-octacosadien-1-ol (CH503). Unexpectedly, silencing bond expression severely suppresses male fertility and the fertility of conspecific rivals. These deficits are rescued on ectopic expression of bond in the male reproductive system. A comparative analysis across six Drosophila species shows that the gain of a novel transcription initiation site is correlated with bond expression in the ejaculatory bulb, a primary site of male pheromone production. Taken together, these results indicate that modification of cis-regulatory elements and subsequent changes in gene expression pattern is one mechanism by which pheromone diversity arises.

Where families and healthcare meet.

Recent developments in professional healthcare pose moral problems that standard bioethics cannot even identify as problems, but that are fully visible when redefined as problems in the ethics of families. Here, we add to the growing body of work that began in the 1990 s by demonstrating the need for a distinctive ethics of families. First, we discuss what 'family' means and why families can matter so deeply to the lives of those within them. Then, we briefly sketch how, according to an ethics of families, responsibilities must be negotiated against the backdrop of family relationships, treatment decisions must be made in the light of these negotiated responsibilities and justice must be served, both between families and society more generally and within families themselves.

Increase in cellular triacylglycerol content and emergence of large ER-associated lipid droplets in the absence of CDP-DG synthase function.

Excess fatty acids and sterols are stored as triacylglycerols and sterol esters in specialized cellular organelles, called lipid droplets. Understanding what determines the cellular amount of neutral lipids and their packaging into lipid droplets is of fundamental and applied interest. Using two species of fission yeast, we show that cycling cells deficient in the function of the ER-resident CDP-DG synthase Cds1 exhibit markedly increased triacylglycerol content and assemble large lipid droplets closely associated with the ER membranes. We demonstrate that these unusual structures recruit the triacylglycerol synthesis machinery and grow by expansion rather than by fusion. Our results suggest that interfering with the CDP-DG route of phosphatidic acid utilization rewires cellular metabolism to adopt a triacylglycerol-rich lifestyle reliant on the Kennedy pathway.

Sex-specific triacylglycerides are widely conserved in Drosophila and mediate mating behavior.

Pheromones play an important role in the behavior, ecology, and evolution of many organisms. The structure of many insect pheromones typically consists of a hydrocarbon backbone, occasionally modified with various functional oxygen groups. Here we show that sex-specific triacylclyerides (TAGs) are broadly conserved across the subgenus Drosophila in 11 species and represent a novel class of pheromones that has been largely overlooked. In desert-adapted drosophilids, 13 different TAGs are secreted exclusively by males from the ejaculatory bulb, transferred to females during mating, and function synergistically to inhibit courtship from other males. Sex-specific TAGs are comprised of at least one short branched tiglic acid and a long linear fatty acyl component, an unusual structural motif that has not been reported before in other natural products. The diversification of chemical cues used by desert-adapted Drosophila as pheromones may be related to their specialized diet of fermenting cacti.

Evolving legal responses to dependence on families in New Zealand and Singapore healthcare.

Healthcare decision-making has traditionally focused on individual autonomy, but there is now a change occurring in which the involvement of families is gaining prominence. This appears to stem from an increasing emphasis on relational aspects of autonomy which recognises the individual's connectedness to their family, and also state reliance upon families to share the burdens and costs of caring for elderly and disabled dependents. Such a reorientation calls for similar legal emphasis on patient autonomy as understood in relational terms, and one that offers more adequate conceptions of independence, confidentiality and decision-making authority in the light of this change. This paper outlines how two common law jurisdictions, New Zealand and Singapore, have accommodated, or are responding to, these changes.

Pheromones in the fruit fly.

The identification of pheromones (chemical communication cues) is critical to our understanding of complex social behavior in insects and other animals. In this chapter, we describe analytical methods for the purification of lipid pheromones by thin layer chromatography and the quantification and determination of their elemental composition by mass spectrometry.

miR-124 controls male reproductive success in Drosophila.

Many aspects of social behavior are controlled by sex-specific pheromones. Gender-appropriate production of the sexually dimorphic transcription factors doublesex and fruitless controls sexual differentiation and sexual behavior. miR-124 mutant males exhibited increased male-male courtship and reduced reproductive success with females. Females showed a strong preference for wild-type males over miR-124 mutant males when given a choice of mates. These effects were traced to aberrant pheromone production. We identified the sex-specific splicing factor transformer as a functionally significant target of miR-124 in this context, suggesting a role for miR-124 in the control of male sexual differentiation and behavior, by limiting inappropriate expression of the female form of transformer. miR-124 is required to ensure fidelity of gender-appropriate pheromone production in males. Use of a microRNA provides a secondary means of controlling the cascade of sex-specific splicing events that controls sexual differentiation in Drosophila. DOI:http://dx.doi.org/10.7554/eLife.00640.001.