Social environment and anogenital distance length phenotype interact to explain testosterone levels in a communally rearing rodent: Part 1: The male side.

In vertebrates, male testosterone levels vary across the year being generally higher during the mating season relative to the offspring rearing season. However, male testosterone levels may also be associated with male anogenital distance (AGD) length (a proxy of prenatal androgen exposition), and influenced by the social group environment. In social species, it has been proposed that high levels of testosterone could be incompatible with the development of an amicable social environment. Thus, in these species, it is predicted that males have relatively low levels of testosterone. Our goal was to examine the potential association between male serum testosterone levels, season, male AGD length, and the social environment in the rodent Octodon degus under natural conditions. We quantified male serum testosterone levels during the mating and offspring rearing seasons, and we determined the number of females and males in each social group, as well as the composition of groups, in terms of the AGD length of the female and male group mates, from 2009 to 2019. Our results revealed that male testosterone levels covary with season, being highest during the offspring rearing season. Additionally, male testosterone levels vary with male AGD length, and female and male social group environments. More importantly, male degus exhibit low levels of testosterone that are indistinguishable from female levels during offspring rearing season. Similar to other highly social mammals, where males and females live together year-round, male amicable behavior could be the best male mating strategy, thus leading to a reduction in circulating testosterone levels.

Social environment and anogenital distance length phenotype interact to explain testosterone levels in a communally rearing rodent: Part 2: The female side.

Testosterone is known as a "male" hormone; however, females also synthetize testosterone, which influences female sexual and aggressive behavior. In female vertebrates, as in males, testosterone levels can vary seasonally. However, female testosterone levels may also be related with female anogenital distance (AGD) length phenotype (a proxy of prenatal androgen exposure), and the social group environment. We used data from a long-term rodent study (2009-2019) in a natural population of degus (Octodon degus) to examine the potential associations between female serum testosterone levels, season, female AGD phenotype, and social group composition. We quantified female serum testosterone levels during the mating and offspring rearing seasons, and we determined the number of females and males in social groups, as well the composition of groups, in terms of the AGD of the female and male group mates. Our results indicate that female testosterone levels vary with season, being highest during the offspring rearing season. Additionally, female testosterone levels were associated with the number of male group-members and the AGD of male group-members but were not associated with female social environment and focal female AGD phenotype. Together, our results suggest that female testosterone levels are sensitive to intersexual interactions. Our results also reveal that female and male testosterone levels do not differ between the sexes, a finding previously reported only in rock hyraxes. We discuss how the complex social system of degus could be driving this physiological similarity between the sexes.

Physico-Chemical and Nutritional Properties of Chia Seeds from Latin American Countries.

In the last few decades, chia (Salvia hispanica L.) cultivation has expanded around the world, and the seeds have become well known due to their rich composition of nutrients and bioactive compounds. The aim of this work was to evaluate the physical, chemical, and nutritional profile of eight types of chia seeds grown in different Latin-American countries (Argentina, Bolivia, Chile, Ecuador, Mexico, Paraguay, and Peru). The results showed that several nutritional parameters of the seeds, such as the protein content and amino acid profile, dietary fiber content, lipid content, mineral composition, and presence of phytate, depend on the location in which they were grown. Other parameters, such as ash content, fatty acid profile, or various physical parameters, were uniform across locations (except for color parameters). The results support the notion that the nutritional characteristics of seeds are determined by the seeds' origin, and further analysis is needed to determine the exact mechanisms that control the changes in the seed nutritional properties of chia seeds.

Spectroscopy, molecular structure, and electropolymerization of Ni(II) and Cu(II) complexes containing a thiophene-appending fluorinated Schiff base ligand.

In this contribution, we describe the preparation, characterization, and electrochemical behavior of a series of four new mononuclear M(II) complexes featuring a symmetric substituted N2O2-tetradentate Schiff base ligand, bearing either trifluoromethyl and p-bromophenyl (M = Ni, 3; Cu, 4) or trifluoromethyl and the π-extended p-(2-thienyl)phenylene (M = Ni, 5; Cu, 6) substituents. Complexes 3 and 4 were readily synthesized by reacting the diprotic fluorinated Schiff base proligand 2 with the appropriate hydrated metal(II) acetates, whereas 5 and 6 were obtained upon Stille cross-coupling reaction of 3 and 4 with 2-(tributylstannyl)-thiophene, respectively. Compounds 3-6 were isolated as neutral, air, and thermally stable-coloured solids, with yields ranging from 60 to 80%. The four complexes, the diimine precursor 1 and its trifluoroacetylated derivative 2, were identified using analytical (EA, ESI-MS), spectroscopic (IR, 1H, 13C, and 19F NMR), and X-ray crystallographic methods. X-ray crystal structure determination of complexes 3-5 revealed that both four-coordinate Ni(II) and Cu(II) metal ions adopt a square planar geometry. The magnetic properties of powdered samples of the Cu(II) derivatives 4 and 6 have been investigated (2-300 K) and found consistent in both cases with a single isolated copper(II) ion (s = 1/2). DFT calculations were used to examine the optimal geometries of complexes 5 and 6, allowing for a consistent perspective of their structure and characteristics. The primary aspects of the UV-vis spectra were interpreted using TD-DFT computations. Finally, electrochemical data indicate that complexes 5 and 6 polymerize at high anodic potentials in acetonitrile (greater than 2.0 V vs. Ag/AgCl). Cyclic voltammetry, scanning electron microscopy, and energy-dispersive X-ray spectroscopy (SEM-EDS) analyses were used to characterize the obtained films poly-5 and poly-6.

Can the genetic variability of Blastocystis sp. be associated with the climatic region of its human carriers?

Blastocystis sp. is a widespread microorganism that colonizes the intestinal tract of several animals, including human beings, while its pathogenic role in humans is still under debate. The objective of the present study was to describe the frequency of Blastocystis sp. subtypes (STs) and their genetic variation within and among samples recovered from scholars inhabiting two rural villages with tropical climates and compare this information with previously documented data from arid and temperate zones in Mexico. Blastocystis sp. positive samples and ST identification were achieved by coprological analysis screening and Polymerase Chain Reaction-sequencing, respectively. Classical population genetics indexes (nucleotide diversity (π), haplotype polymorphism (θ), gene flow (Nm), genetic differentiation (ST), and Tajima's D) were calculated by comparing the sequences here obtained (n = 42) and those from previous studies from the arid (n = 80) and temperate (n = 61) climates from Mexico. Although Blastocystis sp. was the parasite most frequently found between 33% and 26% in both communities, only STs 1-3 were found. Haplotype network inference of Blastocystis sp. STs showed different haplotype profiles among STs vs. climate zones, although no specific haplotypes were identified for any particular climatic zone. Population genetics indexes showed different values within STs and climate zones (π and θ values ranged from 0.004 to 0.147; Nm > 4 and ST from 0.006 to 0.12). Our results show that Blastocystis sp. subtypes exhibit a different genetic variability profile according to the climate zone, suggesting a balancing process between the genetic variability within the Blastocystis sp. subtype and the number of haplotypes identified in each climate.

Basil Seeds as a Novel Food, Source of Nutrients and Functional Ingredients with Beneficial Properties: A Review.

Basil (Ocimum basilicum L.) is found worldwide and is used in the food, pharmaceutical, and cosmetic industries; however, the nutritional and functional properties of the seeds are scarcely known. Basil seeds contain high concentrations of proteins (11.4-22.5 g/100 g), with all the essential amino acids except S-containing types and tryptophan; dietary fiber (soluble and insoluble) ranging from 7.11 to 26.2 g/100 g lipids, with linoleic (12-85.6 g/100 g) and linolenic fatty acids (0.3-75 g/100 g) comprising the highest proportions; minerals, such as calcium, potassium, and magnesium, in high amounts; and phenolic compounds, such as orientine, vicentine, and rosmarinic acid. In addition, their consumption is associated with several health benefits, such as the prevention of type-2 diabetes, cardio-protection, antioxidant and antimicrobial effects, and anti-inflammatory, antiulcer, anticoagulant, and anti-depressant properties, among others. The focus of this systematic review was to study the current state of knowledge and explore the enormous potential of basil seeds as a functional food and source of functional ingredients to be incorporated into foods.

Parental care in male degus (Octodon degus) is flexible and contingent upon female care.

Parents in many animal species provide care to their offspring as a mechanism to enhance their own fitness. In mammals, this behavior is expressed mostly by the females, but also by males of some species. Proximally, rates of paternal offspring care have been linked to organizational and activational effects of testosterone. Specifically, intrauterine position of male fetuses is associated with differential exposure to testosterone, leading to development of males with different levels of masculinization (assessed through differences in the length of the anogenital distance (AGD). The relative roles played by organizational and activational effects of testosterone on male parental care remain unclear. In this study, we aimed to determine if male sex-biased uterine environment and testosterone levels across the breeding period explain variation in paternal care in the social rodent, Octodon degus. Neither quantity (time with the offspring) nor quality (frequency of grooming and retrieving) of paternal care was affected by male sex-biased uterine environment, nor did paternal care significantly differ across the different stages of male reproduction. In contrast, paternal care was associated with maternal care. Quantity of male care decreased with increasing quantity of maternal care, and quality of male care increased with increasing quality of maternal care. While serum testosterone did not differ between males with different sex-biased uterine environment, male testosterone tended to increase during mating and decrease when pregnant females or offspring were present.

Socially unstable conditions experienced during development prime female Octodon degus to shape the phenotype of their own offspring.

Because residents and immigrants from group living species may experience fitness costs associated with permanent changes in group membership, we examined the hypothesis that females experiencing socially unstable or socially stable conditions during development compensate these costs by shaping the phenotype of their own offspring differently. Groups of adult females experiencing either socially stable or unstable conditions in the early social environment were assigned to either socially stable or unstable conditions in the social environment as adults. We quantified affiliative and agonistic interactions among the females during pregnancy and lactation of the focal female, maternal and allomaternal care, hypothalamic-anterior pituitary-adrenal axis (HPA) acute stress response, and early offspring growth. Social instability during breeding enhanced agonistic interactions among adult females, and offspring that experienced socially unstable conditions exhibited enhanced offspring care, regardless of adult environments. Neither social behavior, offspring care, acute stress physiology, nor early growth was influenced by early or adult social stability conditions. These findings imply that socially unstable conditions prime developing females to shape the phenotype of their offspring to prevent negative effects of socially unstable environments.

Intake of soluble fibre from chia seed reduces bioaccessibility of lipids, cholesterol and glucose in the dynamic gastrointestinal model simgi®.

The role of soluble fibres on hypoglycemic and hypocholesterolemic effects has been widely documented, but the effect on glucose and cholesterol binding capacity of soluble fibre extracted from chia seed mucilage has not been studied until now. In the present research, dynamic gastrointestinal model simgi® combined with absorption static techniques have been used to explore the effect of chia seed mucilage at 0.75 and 0.95% w/w on the bioaccessibility of glucose, dietary lipids and cholesterol along the gastrointestinal tract. Glucose bioaccessibility was reduced when 0.95% of chia mucilage was present in sugar food models. The total reduction of glucose bioaccessibility reached a maximum of 66.7% while glucose dialysis retardation index presented its maximum of 53.4% at the end of small intestine digestion. The in vitro studies with lipid food models, showed that the presence of both, 0.75 and 0.95% of chia seed mucilage caused substantial reductions on the bioaccessibility of free fatty acids (16.8 and 56.1%), cholesterol (18.2 and 37.2% respectively) and bile salts (4.8 and 64.6%), revealing a clear dependence on fibre concentration. These innovative results highlight the potential functionality of the soluble fibre extracted from chia seeds to improve lipid and glycemic profiles and suggest the dietary health benefits of this new soluble fibre source as an ingredient in functional foods designed to reduce the risk of certain non-communicable diseases.

Stress, sleep, and sex: A review of endocrinological research in Octodon degus.

The Common Degu (Octodon degus) is a small rodent endemic to central Chile. It has become an important model for comparative vertebrate endocrinology because of several uncommon life-history features - it is diurnal, shows a high degree of sociality, practices plural breeding with multiple females sharing natal burrows, practices communal parental care, and can easily be studied in the laboratory and the field. Many studies have exploited these features to make contributions to comparative endocrinology. This review summarizes contributions in four major areas. First are studies on degu stress responses, focusing on seasonal changes in glucocorticoid (GC) release, impacts of parental care on offspring GC responses, and fitness consequences of individual variations of GC responses. These studies have helped confirm the ecological relevance of stress responses. Second are studies exploring diurnal circadian rhythms of melatonin and sex steroids. These studies have formed important work translating circadian biology from nocturnal laboratory rodents to diurnal humans. Third are studies that exploit the open nature of degu natural habitat, combined with laboratory studies, to explore the impact of testosterone on agonistic behavior. Studies have focused primarily on male:male, female:female, male:female, and parental behaviors. Fourth, are contributions to the study of female masculinization from male siblings in the uterus. These studies have focused on both the behavioral consequences of masculinization and the impact of those behaviors on fitness. Taken together, the studies reviewed here have formed a strong foundation for further studies in the degu so that future studies can address how endocrinological components underlie new mechanistic connections to the ecological effects on behavior and fitness.

Evaluation of protective effect of different dietary fibers on polyphenolic profile stability of maqui berry (Aristotelia chilensis (Molina) Stuntz) during in vitro gastrointestinal digestion.

The aim of this work was to determine the protective effect of different dietary fibers on (i) the recovery and bioaccessibility indexes, and (ii) the stability of polyphenolic compounds (phenolic acids, flavonoids and anthocyanins) of maqui berry powder subjected to in vitro gastrointestinal digestion (GID). The extracts obtained in each phase (oral, gastric and intestinal) of GID were used to analyze the stability of polyphenolic compounds by HPLC, and the bioaccessibility of these compounds was also determined. At the end of the GID process, the mixture of maqui berry with the different fibers increased the bioaccessibility index of the phenolic and flavonoid compounds in all cases. The results obtained suggest that the anthocyanins and phenolic acids and flavonoid compounds present in maqui are stabilized through dietary fiber interactions, which might provide sufficient levels for absorption during gastrointestinal digestion. The gums sodium carboxymethyl cellulose, xanthan gum and guar gum provided the best protective effect.

Masculinized females produce heavier offspring in a group living rodent.

Alternative morphotypes have been reported less frequently in females than in males. An exception to this rule is the gradient of phenotypical masculinization reported in some female mammals, in which feminized and masculinized females represent two opposite ends along this gradient. These phenotypical differences originate during prenatal development as the consequence of maternal effects. Feminized and masculinized females differ in several traits, including morphological, physiological, behavioural and reproductive traits. Differences previously reported in reproductive traits between feminized and masculinized females come mostly from mechanistic studies performed in the laboratory, and not necessarily on social species. As a result, it is unclear to what extent these reported differences between female alternative morphotypes materialize in wild, natural populations. We quantified the effect of female alternative morphotype on female reproductive traits in a natural population of Octodon degus, a highly social rodent. We assessed female alternative morphotype through a continuous gradient of anogenital distance. Thus, feminized females were close to the short end of anogenital distance, while masculinized females were close to the long end of this gradient. We also tested the hypothesis that the social environment interacts with female morphotype to influence female reproductive traits. In female degus, only body weight affected litter size, where heavier females weaned more offspring. Masculinized females delivered male-biased litters and weaned heavier offspring. Lastly, masculinized females gave birth later in the breeding season compared to feminized females. Contrary to previous claims, our findings do not support that masculinized females are less fertile than feminized females. Moreover, masculinized females produced heavier, potentially higher quality offspring compared with feminized females.

The modulating role of group stability on fitness effects of group size is different in females and males of a communally rearing rodent.

Group size may influence fitness benefits and costs that emerge from cooperative and competitive interactions in social species. However, evidence from plural breeding mammals indicates that group size is insufficient to explain variation in direct fitness, implying other attributes of social groups were overlooked. We studied the natural population of a social rodent during 5 years to test the hypothesis that social stability - in terms of group composition - modulates the effects of increasing number of breeding females (a proxy of communal rearing) and males on the number of offspring weaned (sired) and on the number of offspring weaned (sired) surviving to breeding age (two proxies of direct fitness). We quantified the effects of social stability (measured as changes in female or male group members between mating and the onset of lactation) on these fitness measures. We used live trapping, telemetry and DNA markers to determine social and fitness measures. Social stability in degus was variable in terms of the number of changes in group composition across groups. Low stability was mostly due to mortality and emigration of group members. Results supported a modulating role of social stability on the relationship between group size and the number of offspring weaned (sired). Stability in female and male group composition were both modulators of fitness to females and males. The modulatory role of stability was sex specific, where high social stability was often fitness beneficial to the females. Instead, low social stability was fitness enhancing to the males.

Determination of polyphenolic profile, antioxidant activity and antibacterial properties of maqui [Aristotelia chilensis (Molina) Stuntz] a Chilean blackberry.

BACKGROUND: The aim of the present study was to determine (1) the polyphenolic profile (phenolic acids, flavonoids and anthocyanins), (2) the antioxidant using four different methodologies (DPPH, ABTS, FRAP and FIC) and (3) the antibacterial properties of maqui berry [Aristotelia chilensis (Molina) Stuntz] (MB) grown in Chile. RESULTS: The HPLC analysis of MB showed a total of 19 polyphenolic compounds identified as anthocyanins (eight compounds), flavonols (10 compounds) and ellagic acid. Delphinidin derivatives were the predominant anthocyanins while quercetin derivatives were the predominant flavonols. MB showed an antioxidant activity measured with DPPH, ABTS, FRAP and FIC methods of 28.18, 18.66, 25.22 g Trolox equivalent kg(-1) and 0.12 g ethylenediaminetetraacetic acid equivalent kg(-1) , respectively. With regard to the antibacterial activity, all strains tested were affected by MB. Aeromonas hydrophila and Listeria innocua showed the highest sensitivity to maqui berry extracts with MIC values of 40 and a 50 mg mL(-1) , respectively. CONCLUSIONS: The results suggest that maqui berry has a great potential to be employed in the food industry as potential food ingredient to functional food development or as bio-preservative. © 2016 Society of Chemical Industry.

Electrosynthesis and characterization of nanostructured polyquinone for use in detection and quantification of naturally occurring dsDNA.

The detection of naturally occurring desoxyribonucleic acid (DNA) has become a subject of study by the projections that would generate to be able to sense the genetic material for the detection of future diseases. Bearing this in mind, to provide new measuring strategies, in the current work the preparation of a low-cost electrode, modified with poly(1-amino-9,10-anthraquinone) nanowires using a SiO2 template, is carried out; the assembly is next modified by covalently attaching ssDNA strands. It must be noted that all this is accomplished by using solely electrochemical techniques, according to methodology developed for this purpose. SEM images of the modified surface show high order and homogeneity in the distribution of modified nanowires over the electrode surface. In turn, after the hybridization with its complementary strand, the voltammetric responses enable corroborating the linear relationship between hybridization at different DNA concentrations and normalized current response, obtaining a limit of detection (LOD) 5.7·10(-12)gL(-1) and limit of quantification (LOQ) 1.9·10(-11)gL(-1). The working dynamic range is between 1.4·10(-7) and 8.5·10(-9)gL(-1) with a correlation coefficient 0.9998. The successful obtaining of the modified electrode allows concluding that the high order reached by the nanostructures, guides the subsequent single strand of DNA (ssDNA) covalent attachment, which after hybridization with its complementary strand brings about a considerable current increase. This result allows foreseeing a guaranteed breakthrough with regard to the use of the biosensor in real samples.

A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration.

NAD metabolism regulates diverse biological processes, including ageing, circadian rhythm and axon survival. Axons depend on the activity of the central enzyme in NAD biosynthesis, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2), for their maintenance and degenerate rapidly when this activity is lost. However, whether axon survival is regulated by the supply of NAD or by another action of this enzyme remains unclear. Here we show that the nucleotide precursor of NAD, nicotinamide mononucleotide (NMN), accumulates after nerve injury and promotes axon degeneration. Inhibitors of NMN-synthesising enzyme NAMPT confer robust morphological and functional protection of injured axons and synapses despite lowering NAD. Exogenous NMN abolishes this protection, suggesting that NMN accumulation within axons after NMNAT2 degradation could promote degeneration. Ectopic expression of NMN deamidase, a bacterial NMN-scavenging enzyme, prolongs survival of injured axons, providing genetic evidence to support such a mechanism. NMN rises prior to degeneration and both the NAMPT inhibitor FK866 and the axon protective protein Wld(S) prevent this rise. These data indicate that the mechanism by which NMNAT and the related Wld(S) protein promote axon survival is by limiting NMN accumulation. They indicate a novel physiological function for NMN in mammals and reveal an unexpected link between new strategies for cancer chemotherapy and the treatment of axonopathies.

Social structure in a family group of Guanaco (Lama guanicoe, Ungulate): is female hierarchy based on 'prior attributes' or 'social dynamics'?

Social life involves costs and benefits mostly associated with how individuals interact with each other. The formation of hierarchies inside social groups has evolved as a common strategy to avoid high costs stemming from social interactions. Hierarchical relationships seem to be associated with different features such as body size, body condition and/or age, which determine dominance ability ('prior attributes' hypothesis). In contrast, the 'social dynamic' hypothesis suggests that an initial social context is a determinant in the formation of the hierarchy, more so than specific individual attributes. Hierarchical rank places individuals in higher positions, which presumably increases resource accessibility to their benefit, including opportunities for reproduction. We evaluate the maintenance of hierarchy in a family group of guanacos (Lama guanicoe) and evaluate the possible mechanisms involved in the stability of these interactions and their consequences. We estimate the linearity of social hierarchy and their dynamics. We find evidence of the formation of a highly linear hierarchy among females with males positioned at the bottom of the hierarchy. This hierarchy is not affected by physical characteristics or age, suggesting that it is established only through intra-group interactions. Rank is not related with calves' weight gain either; however, subordinated females, with lower rank, exhibit higher rates of allosuckling. We found no evidence of hierarchical structure in calves suggesting that hierarchical relationship in guanacos could be established during the formation of the family group. Hence, our results suggest that hierarchical dynamics could be related more to social dynamics than to prior attributes. We finally discuss the importance of hierarchies established by dominance and their role in minimizing social costs of interactions.

Aquaporin 1, Nox1, and Ask1 mediate oxidant-induced smooth muscle cell hypertrophy.

AIMS: Reactive oxygen species (ROS)-mediated intracellular signalling is well described in the vasculature, yet the precise roles of ROS in paracrine signalling are not known. Studies implicate interstitial ROS hydrogen peroxide (H(2)O(2)) in vascular disease, and plasma H(2)O(2) levels in the micromolar range are detectable in animal models and humans with hypertension. Recently, H(2)O(2) was shown to cross biological membranes of non-vascular cells via aquaporin (Aqp) water channels. Previous findings suggest that H(2)O(2) activates NADPH oxidase (Nox) enzymes in vascular cells and apoptosis signal-regulating kinase 1 (Ask1) in non-vascular cells. We hypothesized that extracellular H(2)O(2) induces smooth muscle cell (SMC) hypertrophy by a mechanism involving Aqp1, Nox1, and Ask1. METHODS AND RESULTS: Treatment of rat aortic SMCs (rASMC) with exogenous H(2)O(2) resulted in a concentration-dependent increase in Nox-derived superoxide (O(2)(•-)), determined by L-012 chemiluminescence, cytochrome c and electron paramagnetic resonance. Nox1 was verified as the source of O(2)(·-) by siRNA. Aqp1 siRNA attenuated H(2)O(2) cellular entry and H(2)O(2)-induced O(2)(•-) production. H(2)O(2) treatment increased Ask1 activation and induced rASMC hypertrophy in a Nox1-dependent mechanism. Adenoviral-dominant-negative Ask1 attenuated H(2)O(2)-induced rASMC hypertrophy and adenoviral overexpression of Ask1 augmented it. CONCLUSION: Our results demonstrate for the first time that extracellular H(2)O(2), at pathophysiological concentrations, stimulates rASMC Nox1-derived O(2)(•-), subsequent Ask1 activation and SMC hypertrophy. The data demonstrate a novel pathway by which H(2)O(2) enters vascular cells via aquaporins and activates Nox, leading to hypertrophy, and provide multiple novel targets for combinatorial therapeutics development targeting hypertrophy and vascular disease.

Characterization of avian poxvirus in Anna's Hummingbird (Calypte anna) in California, USA.

Avian poxvirus (genus Avipoxvirus, family Poxviridae) is an enveloped double-stranded DNA virus that may be transmitted to birds by arthropod vectors or mucosal membrane contact with infectious particles. We characterized the infection in Anna's Hummingbird (Calypte anna; n = 5 birds, n = 9 lesions) by conducting diagnostic tests on skin lesions that were visually similar to avian poxvirus lesions in other bird species. Skin lesions were single or multiple, dry and firm, pink to yellow, with scabs on the surface, and located at the base of the bill, wings, or legs. Microscopically, the lesions were characterized by epidermal hyperplasia and necrosis with ballooning degeneration, and intracytoplasmic inclusions (Bollinger bodies) in keratinocytes. The 4b core gene sequence of avian poxvirus was detected by PCR in samples prepared from lesions. Nucleotide sequences were 75-94% similar to the sequences of other published avian poxvirus sequences. Phylogenetic analyses showed that the Anna's Hummingbird poxvirus sequence was distinguished as a unique subclade showing similarities with sequences isolated from Ostrich (Struthio camelus), Wild Turkey (Meleagris gallopavo), falcons (Falco spp.), Black-browed Albatross (Diomedea melanophris), Mourning Dove (Zenaida macroura) and White-tailed Eagle (Haliaeetus albicilla). To our knowledge this is the first published report of definitive laboratory diagnosis of avian poxvirus in a hummingbird. Our results advance the science of disease ecology in hummingbirds, providing management information for banders, wildlife rehabilitators, and avian biologists.

Physical and functional interaction between the dopamine transporter and the synaptic vesicle protein synaptogyrin-3.

Uptake through the dopamine transporter (DAT) represents the primary mechanism used to terminate dopaminergic transmission in brain. Although it is well known that dopamine (DA) taken up by the transporter is used to replenish synaptic vesicle stores for subsequent release, the molecular details of this mechanism are not completely understood. Here, we identified the synaptic vesicle protein synaptogyrin-3 as a DAT interacting protein using the split ubiquitin system. This interaction was confirmed through coimmunoprecipitation experiments using heterologous cell lines and mouse brain. DAT and synaptogyrin-3 colocalized at presynaptic terminals from mouse striatum. Using fluorescence resonance energy transfer microscopy, we show that both proteins interact in live neurons. Pull-down assays with GST (glutathione S-transferase) proteins revealed that the cytoplasmic N termini of both DAT and synaptogyrin-3 are sufficient for this interaction. Furthermore, the N terminus of DAT is capable of binding purified synaptic vesicles from brain tissue. Functional assays revealed that synaptogyrin-3 expression correlated with DAT activity in PC12 and MN9D cells, but not in the non-neuronal HEK-293 cells. These changes were not attributed to changes in transporter cell surface levels or to direct effect of the protein-protein interaction. Instead, the synaptogyrin-3 effect on DAT activity was abolished in the presence of the vesicular monoamine transporter-2 (VMAT2) inhibitor reserpine, suggesting a dependence on the vesicular DA storage system. Finally, we provide evidence for a biochemical complex involving DAT, synaptogyrin-3, and VMAT2. Collectively, our data identify a novel interaction between DAT and synaptogyrin-3 and suggest a physical and functional link between DAT and the vesicular DA system.