Carotenoid pigmentation in salmon: variation in expression at BCO2-l locus controls a key fitness trait affecting red coloration.

Carotenoids are primarily responsible for the characteristic red flesh coloration of salmon. Flesh coloration is an economically and evolutionarily significant trait that varies inter- and intra-specifically, yet the underlying genetic mechanism is unknown. Chinook salmon (Oncorhynchus tshawytscha) represents an ideal system to study carotenoid variation as, unlike other salmonids, they exhibit extreme differences in carotenoid utilization due to genetic polymorphisms. Here, we crossed populations of Chinook salmon with fixed differences in flesh coloration (red versus white) for a genome-wide association study to identify loci associated with pigmentation. Here, the beta-carotene oxygenase 2-like (BCO2-l) gene was significantly associated with flesh colour, with the most significant single nucleotide polymorphism explaining 66% of the variation in colour. BCO2 gene disruption is linked to carotenoid accumulation in other taxa, therefore we hypothesize that an ancestral mutation partially disrupting BCO2-l activity (i.e. hypomorphic mutation) allowed the deposition and accumulation of carotenoids within Salmonidae. Indeed, we found elevated transcript levels of BCO2-l in white Chinook salmon relative to red. The long-standing mystery of why salmon are red, while no other fishes are, is thus probably explained by a hypomorphic mutation in the proto-salmonid at the time of divergence of red-fleshed salmonid genera (approx. 30 Ma).

Assessing the capacity for compensatory growth in growth-hormone transgenic coho salmon Oncorhynchus kisutch.

The effect of feed cycling (consisting of periods of starvation followed by periods of refeeding to satiation) on compensatory growth was evaluated in growth hormone transgenic and non-transgenic wild-type coho salmon Oncorhynchus kisutch. The specific growth rate (GSR ) of feed-restricted non-transgenic O. kisutch was not significantly different from the GSR of fully-fed non-transgenic O. kisutch during two refeeding periods, whereas the GSR of feed-restricted transgenic O. kisutch was significantly higher in relation to the GSR of fully-fed transgenic O. kisutch during the second refeeding period, but not during the first, indicating that growth compensation mechanisms are different between non-transgenic and growth-hormone (GH)-transgenic O. kisutch and may depend on life history (i.e. previous starvation). Despite the non-significant growth rate compensation in non-transgenic O. kisutch, these fish showed a level of body mass catch-up growth not displayed by transgenic O. kisutch.

Multispectral Chiral Imaging with a Metalens.

The vast majority of biologically active compounds, ranging from amino acids to essential nutrients such as glucose, possess intrinsic handedness. This in turn gives rise to chiral optical properties that provide a basis for detecting and quantifying enantio-specific concentrations of these molecules. However, traditional chiroptical spectroscopy and imaging techniques require cascading of multiple optical components in sophisticated setups. Here, we present a planar lens with an engineered dispersive response, which simultaneously forms two images with opposite helicity of an object within the same field-of-view. In this way, chiroptical properties can be probed across the visible spectrum using only the lens and a camera without the addition of polarizers or dispersive optical devices. We map the circular dichroism of the exoskeleton of a chiral beetle, Chrysina gloriosa, which is known to exhibit high reflectivity of left-circularly polarized light, with high spatial resolution limited by the numerical aperture of the planar lens. Our results demonstrate the potential of metasurfaces in realizing a compact and multifunctional device with unprecedented imaging capabilities.

Polarization-Insensitive Metalenses at Visible Wavelengths.

In this Letter, we demonstrate highly efficient, polarization-insensitive planar lenses (metalenses) at red, green, and blue wavelengths (λ = 660, 532, and 405 nm). Metalenses with numerical apertures (NA) of 0.85 and 0.6 and corresponding efficiencies as high as 60% and 90% are achieved. These metalenses are less than 600 nm-thick and can focus incident light down to diffraction-limited spots as small as ∼0.64λ and provide high-resolution imaging. In addition, the focal spots are very symmetric with high Strehl ratios. The single step lithography and compatibility with large-scale fabrication processes make metalenses highly promising for widespread applications in imaging and spectroscopy.

Behaviour of growth hormone transgenic coho salmon Oncorhynchus kisutch in marine mesocosms assessed by acoustic tag telemetry.

Underwater acoustic tag telemetry was used to assess behavioural differences between juvenile wild-type (i.e. non-transgenic, NT) and growth hormone (GH) transgenic (T) coho salmon Oncorhynchus kisutch in a contained simulated ocean environment. T O. kisutch were found across days to maintain higher baseline swimming speeds than NT O. kisutch and differences in response to feeding were detected between T and NT genotypes. This is the first study to assess behaviour of GH transgenic salmonids in a marine environment and has relevance for assessing whether behavioural effects of GH overexpression seen in freshwater environments can be extrapolated to oceanic phases of the life cycle.

Gene--environment interactions influence feeding and anti-predator behavior in wild and transgenic coho salmon.

Environmental conditions are known to affect phenotypic development in many organisms, making the characteristics of an animal reared under one set of conditions not always representative of animals reared under a different set of conditions. Previous results show that such plasticity can also affect the phenotypes and ecological interactions of different genotypes, including animals anthropogenically generated by genetic modification. To understand how plastic development can affect behavior in animals of different genotypes, we examined the feeding and risk-taking behavior in growth-enhanced transgenic coho salmon (with two- to threefold enhanced daily growth rates compared to wild type) under a range of conditions. When compared to wild-type siblings, we found clear effects of the rearing environment on feeding and risk-taking in transgenic animals and noted that in some cases, this environmental effect was stronger than the effects of the genetic modification. Generally, transgenic fish, regardless of rearing conditions, behaved similar to wild-type fish reared under natural-like conditions. Instead, the more unusual phenotype was associated with wild-type fish reared under hatchery conditions, which possessed an extreme risk averse phenotype compared to the same strain reared in naturalized conditions. Thus, the relative performance of genotypes from one environment (e.g., laboratory) may not always accurately reflect ecological interactions as would occur in a different environment (e.g., nature). Further, when assessing risks of genetically modified organisms, it is important to understand how the environment affects phenotypic development, which in turn may variably influence consequences to ecosystem components across different conditions found in the complexity of nature.

Accuracy of nonmolecular identification of growth-hormone- transgenic coho salmon after simulated escape.

Concerns with transgenic animals include the potential ecological risks associated with release or escape to the natural environment, and a critical requirement for assessment of ecological effects is the ability to distinguish transgenic animals from wild type. Here, we explore geometric morphometrics (GeoM) and human expertise to distinguish growth-hormone-transgenic coho salmon (Oncorhynchus kisutch) specimens from wild type. First, we simulated an escape of 3-month-old hatchery-reared wild-type and transgenic fish to an artificial stream, and recaptured them at the time of seaward migration at an age of 13 months. Second, we reared fish in the stream from first-feeding fry until an age of 13 months, thereby simulating fish arising from a successful spawn in the wild of an escaped hatchery-reared transgenic fish. All fish were then assessed from 'photographs by visual identification (VID) by local staff and by GeoM based on 13 morphological landmarks. A leave-one-out discriminant analysis of GeoM data had on average 86% (72-100% for individual groups) accuracy in assigning the correct genotypes, whereas the human experts were correct, on average, in only 49% of cases (range of 18-100% for individual fish groups). However, serious errors (i.e., classifying transgenic specimens as wild type) occurred for 7% (GeoM) and 67% (VID) of transgenic fish, and all of these incorrect assignments arose with fish reared in the stream from the first-feeding stage. The results show that we presently lack the skills of visually distinguishing transgenic coho salmon from wild type with a high level of accuracy, but that further development-of GeoM methods could be useful in identifying second-generation,fish from nature as a nonmolecular approach.

Upper thermal tolerance of wild-type, domesticated and growth hormone-transgenic coho salmon Oncorhynchus kisutch.

In coho salmon Oncorhynchus kisutch, no significant differences in critical thermal maximum (c. 26·9° C, CTmax ) were observed among size-matched wild-type, domesticated, growth hormone (GH)-transgenic fish fed to satiation, and GH-transgenic fish on a ration-restricted diet. Instead, GH-transgenic fish fed to satiation had significantly higher maximum heart rate and Arrhenius breakpoint temperature (mean ± s.e. = 17·3 ± 0·1° C, TAB ). These results provide insight into effects of modified growth rate on temperature tolerance in salmonids, and can be used to assess the potential ecological consequences of GH-transgenic fishes should they enter natural environments with temperatures near their thermal tolerance limits.

Rationed and satiated growth hormone transgenic Coho Salmon (Oncorhynchus kisutch) show tissue specific differences in energy stores.

Growth hormone transgenic coho salmon experience increased growth rates, driven primarily through elevated feed intake and feed conversion. However, neuropeptides that signal appetite stimulation have been shown to exhibit variable responses across fed states, suggesting a more complex system mediating growth in these fish. Studies have proposed that growth hormone may have a modulatory role on the energy reserves of fish, possibly through AMP-activated protein kinase (AMPK) activation. AMPK, an energy sensor in cells, has previously been shown to be upregulated in growth hormone transgenic salmon when compared to wild type, however, whether this effect is seen across fed states is unknown. Here, we tested the hypothesis that growth hormone induces an energetic deficit in metabolic tissues, leading to constitutive AMPK activation in growth hormone transgenic salmon. This study compared AMPK activity, ATP, and glycogen, of the liver, heart, and muscle of wild-type, and growth hormone transgenic salmon either fed to satiation or a wild-type ration. The results suggest that white muscle ATP levels in growth hormone salmon are elevated in satiation and rationed conditions. In the liver, growth hormone transgenic salmon fed a rationed wild-type diet experience reductions in ATP level and glycogen. In none of the tissues examined, did AMPK activity change. Taken together, these results indicate that growth hormone transgenic salmon experience metabolic duress when not fed to satiation.

Growth and endocrine effects of recombinant bovine growth hormone treatment in non-transgenic and growth hormone transgenic coho salmon.

To examine the relative growth, endocrine, and gene expression effects of growth hormone (GH) transgenesis vs. GH protein treatment, wild-type non-transgenic and GH transgenic coho salmon were treated with a sustained-release formulation of recombinant bovine GH (bGH; Posilac). Fish size, specific growth rate (SGR), and condition factor (CF) were monitored for 14 weeks, after which endocrine parameters were measured. Transgenic fish had much higher growth, SGR and CF than non-transgenic fish, and bGH injection significantly increased weight and SGR in non-transgenic but not transgenic fish. Plasma salmon GH concentrations decreased with bGH treatment in non-transgenic but not in transgenic fish where levels were similar to controls. Higher GH mRNA levels were detected in transgenic muscle and liver but no differences were observed in GH receptor (GHR) mRNA levels. In non-transgenic pituitary, GH and GHR mRNA levels per mg pituitary decreased with bGH dose to levels seen in transgenic salmon. Plasma IGF-I was elevated with bGH dose only in non-transgenic fish, while transgenic fish maintained an elevated level of IGF-I with or without bGH treatment. A similar trend was seen for liver IGF-I mRNA levels. Thus, bGH treatment increased fish growth and influenced feedback on endocrine parameters in non-transgenic but not in transgenic fish. A lack of further growth stimulation of GH transgenic fish suggests that these fish are experiencing maximal growth stimulation via GH pathways.

Inside the heads of David and Goliath: environmental effects on brain morphology among wild and growth-enhanced coho salmon Oncorhynchus kisutch.

Transgenic and wild-type individual coho salmon Oncorhynchus kisutch were reared in hatchery and near-natural stream conditions and their brain and structure sizes were determined. Animals reared in the hatchery grew larger and developed larger brains, both absolutely and when controlling for body size. In both environments, transgenics developed relatively smaller brains than wild types. Further, the volume of the optic tectum of both genotypes was larger in the hatchery animals and the cerebellum of transgenics was smaller when reared in near-natural streams. Finally, wild types developed a markedly smaller telencephalon under hatchery conditions. It is concluded that, apart from the environment, genetic factors that modulate somatic growth rate also have a strong influence on brain size and structure.

Mixed lymphocyte reactivity against normal cells by splenic lymphocytes from tumor-bearing mice : I. Studies of vigorous immune responsiveness induced in f(1) mice by parental strain tumor cells.

The establishment of an intimate connection between autoimmunity and neoplasia would require the demonstration of an experimentally induced, tumor-dependent autoimmune process. For this reason, we have studied cellular immune reactions of mice bearing a transplantable leukemia (L1210). Spleen cells from hybrid BDF(1) mice bearing the L1210 tumor (BDFt) reacted vigorously in mixed lymphocyte culture with mitomycin-treated, normal spleen cells from mice of the parental strain from which the L1210 tumor was derived (DBA/2). Spleen cells from nontumor-bearing BDF(1) mice reacted only weakly with these parental cells. The BDFt cells likewise did not respond when cultured with mitomycin-treated spleen cells from the other parental strain (C57B1/6). The vigorous mixed lymphocyte reaction (MLR) by BDFt cells against normal parental cells of the same strain as the tumor was not due to a double exposure of the reacting cells to histocompatibility antigens shared by tumor cells and normal parental cells. The response of cells from tumor-bearing F(1) mice against normal parental cells seen in these experiments suggests the possibility of the induction of an autoimmune-like process against host lymphocytes by spleen cells from leukemic mice. Theoretically such a phenomenon would considerably reduce an animal's ability to mount an immune attack against malignant cells.

Mixed lymphocyte reactivity against normal cells by splenic lymphocytes from tumor-bearing mice : ii. Studies of autoimmune-like activity in completely syngeneic and semisyngeneic systems.

A possible consequence of an antilymphocytic autoimmune process would be serious impairment of an animal's ability to destroy tumor cells. One measure of autoimmune reactivity of this type would be the demonstration of cellular immune responsiveness by cells from tumor-bearing mice against syngeneic normal cells. These experiments demonstrate that spleen cells from mice bearing a lymphocytic leukemia of identical histocompatability type as the host mounted a vigorous immune response against normal syngeneic cells in a mixed lymphocyte reaction (MLR). Moreover, ascitic cells from leukemic mice responded significantly to normal syngeneic spleen cells in MLR's. The former reactions are usually much more vigorous than the responses of normal to malignant cells. These results are discussed in terms of the relationship between autoimmunity and neoplasia. Alternative explanations necessitated by the dangers involved in the interpretation of the immunology of transplantable tumors are considered.

Effects of temperature and growth hormone on individual growth trajectories of wild-type and transgenic coho salmon Oncorhynchus kisutch.

In this study, individual growth patterns of wild-type and growth-enhanced coho salmon Oncorhynchus kisutch at 8, 12 and 16 degrees C water temperature were followed. Despite large differences among individuals in growth rates, there was generally little variation in the shape of the growth curves among O. kisutch individuals of both genotypes and at all temperatures. Typically, individuals that were relatively large initially were also relatively large at the end of the growth period. The limitation in variation was more pronounced in the growth-enhanced O. kisutch than in the wild type, where the relative size of some individuals reared at 12 and 8 degrees C changed by the end of the trial. As a warmer temperature seems to decrease the plasticity of growth trajectories in wild-type fish, it is possible that global warming will influence the ability of wild fish to adapt their growth to changing conditions.

Team-based primary care practice and physician's services: Evidence from Family Health Teams in Ontario, Canada.

Team-based primary care offers a wide range of health services to patients by using interdisciplinary health care providers committed to delivering comprehensive, coordinated and high-quality care through team collaboration. Ontario's Family Health Team (FHT), the largest team-based practice model in Canada, was introduced to improve access to and effectiveness of primary health care services, and was available primarily for physicians paid under blended capitation models (Family Health Organizations and Family Health Networks). Using health administrative data on physicians practicing under blended capitation models in Ontario between 2006 and 2015, we study the impact of switching from non-FHT to FHTs on the production of capitated comprehensive care services, after-hours services, non-incentivized services, and services provided to non-enrolled patients by family physicians. We find that when in FHTs, physicians increase the production of total services and non-incentivized services by 26% and 5% per annum and reduce capitated comprehensive care services by 3.2% per annum. When in FHTs, physicians also see and enroll more patients relative to those practicing in non-FHTs. We find evidence of improved access to physician's services under team-based primary care, but switching to FHTs has no effect on the production of after-hours services and services provided to non-enrolled patients.

Heterogeneous ozone effects on the DNA methylome of bronchial cells observed in a crossover study.

We used a randomized crossover experiment to estimate the effects of ozone (vs. clean air) exposure on genome-wide DNA methylation of target bronchial epithelial cells, using 17 volunteers, each randomly exposed on two separated occasions to clean air or 0.3-ppm ozone for two hours. Twenty-four hours after exposure, participants underwent bronchoscopy to collect epithelial cells whose DNA methylation was measured using the Illumina 450 K platform. We performed global and regional tests examining the ozone versus clean air effect on the DNA methylome and calculated Fisher-exact p-values for a series of univariate tests. We found little evidence of an overall effect of ozone on the DNA methylome but some suggestive changes in PLSCR1, HCAR1, and LINC00336 DNA methylation after ozone exposure relative to clean air. We observed some participant-to-participant heterogeneity in ozone responses.

Prevalence of community-associated methicillin-resistant Staphylococcus aureus colonization in men who have sex with men.

Outbreaks of skin and soft tissue infections mediated by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are being reported with increasing frequency among men who have sex with men (MSM). However, the potential role of asymptomatic colonization with this organism in perpetuating these infections is unclear. The purpose of this cross-sectional study was to determine the prevalence of colonization with CA-MRSA among a cohort of 500 MSM recruited from two inner city clinics in Toronto, Canada. Following the provision of informed consent, subjects completed a questionnaire capturing demographic and clinical variables, which may be associated with MRSA colonization. A nasal swab for MRSA was collected from each subject, and instructions were provided regarding the self-collection of a rectal swab. Cultured MRSA underwent pulsed-field gel electrophoresis and virulence testing for Panton-Valentine leukocidin gene expression. The prevalence of CA-MRSA colonization was 1.6% (95% CI: 0.5-2.6%).

Endocrine effects of growth hormone overexpression in transgenic coho salmon.

Non-transgenic (wild-type) coho salmon (Oncorhynchus kisutch), growth hormone (GH) transgenic salmon (with highly elevated growth rates), and GH transgenic salmon pair fed a non-transgenic ration level (and thus growing at the non-transgenic rate) were examined for plasma hormone concentrations, and liver, muscle, hypothalamus, telencephalon, and pituitary mRNA levels. GH transgenic salmon exhibited increased plasma GH levels, and enhanced liver, muscle and hypothalamic GH mRNA levels. Insulin-like growth factor-I (IGF-I) in plasma, and growth hormone receptor (GHR) and IGF-I mRNA levels in liver and muscle, were higher in fully fed transgenic than non-transgenic fish. GHR mRNA levels in transgenic fish were unaffected by ration-restriction, whereas plasma GH was increased and plasma IGF-I and liver IGF-I mRNA were decreased to wild-type levels. These data reveal that strong nutritional modulation of IGF-I production remains even in the presence of constitutive ectopic GH expression in these transgenic fish. Liver GHR membrane protein levels were not different from controls, whereas, in muscle, GHR levels were elevated approximately 5-fold in transgenic fish. Paracrine stimulation of IGF-I by ectopic GH production in non-pituitary tissues is suggested by increased basal cartilage sulphation observed in the transgenic salmon. Levels of mRNA for growth hormone-releasing hormone (GHRH) and cholecystokinin (CCK) did not differ between groups. Despite its role in appetite stimulation, neuropeptide Y (NPY) mRNA was not found to be elevated in transgenic groups.

Annexin II increases osteoclast formation by stimulating the proliferation of osteoclast precursors in human marrow cultures.

Annexin II (AXII), a calcium-dependent phospholipid-binding protein, has been recently found to be an osteoclast (OCL) stimulatory factor that is also secreted by OCLs. In vitro studies showed that AXII induced OCL formation and bone resorption. However, the mechanism of action by which AXII acts as a soluble extracellular protein to induce OCL formation is unknown. In this paper, we demonstrate that AXII gene expression is upregulated by 1,25-dihydroxyvitamin D3 [1, 25-(OH)2D3] and that addition of AXII significantly increased OCL-like multinucleated cell formation. Time-course studies suggested that AXII acted on the proliferative stage of OCL precursors and that AXII increased thymidine incorporation in OCL precursors. Moreover, AXII enhanced the growth of CFU-GM, the earliest identifiable OCL precursor, when bone marrow cultures were treated with low concentrations of GM-CSF. This capacity of AXII to induce OCL precursor proliferation was due to induction of GM-CSF expression, because the addition of neutralizing antibodies to GM-CSF blocked the stimulatory effect of AXII on OCL formation. RT-PCR analysis using RNA from highly purified subpopulations of marrow cells demonstrated that T cells, especially CD4(+) T cells, produced GM-CSF in response to AXII. Furthermore, FACS(R) analysis of T-cell subpopulations treated with fluorescein-labeled AXII suggested that the CD4(+), but not CD8(+), subpopulation of T cells express an AXII receptor. Taken together, these data suggest that AXII stimulates OCL formation by activating T cells through a putative receptor to secrete GM-CSF. GM-CSF then expands the OCL precursor pool to enhance OCL formation.

Cloning and identification of human Sca as a novel inhibitor of osteoclast formation and bone resorption.

Increased osteoclast activity is responsible for the enhanced bone destruction in postmenopausal osteoporosis, Paget's disease, bone metastasis, and hypercalcemia of malignancy. However, the number of known inhibitory factors that block osteoclast formation and bone resorption are limited. Therefore, we used an expression-cloning approach to identify novel factors produced by osteoclasts that inhibit osteoclast activity. A candidate clone was identified and isolated from a human osteoclast-like multinucleated cell (MNC) cDNA library, named osteoclast inhibitory peptide-1 (OIP-1), and the cDNA sequence was determined. This sequence matched that of the recently identified human stem cell antigen, was structurally similar to the mouse Ly-6 gene family, and the sequence predicted it was a glycosyl phosphatidyl inositol (GPI)-anchored protein that had a cleavable COOH-terminal peptide. Western blot analysis of conditioned media from 293 cells transfected with the OIP-1 cDNA clone confirmed that OIP-1 was released into the media as a membrane-bound GPI-linked protein. Interestingly, both recombinant OIP-1 expressed in Escherichia coli (which does not have GPI linker) and OIP-1 expressed by mammalian cells significantly reduced osteoclast-like MNC formation induced by 1,25-dihydroxyvitamin D3 or PTH-related protein in mouse and human bone marrow cultures, and inhibited 45Ca release from prelabeled bone in fetal rat organ cultures. In contrast, recombinant OIP-1 did not inhibit the growth of a variety of other cell types. These data indicate that OIP-1 is a novel, specific inhibitor of osteoclast formation and bone resorption.