Transcriptomic and developmental effects of persistent organic pollutants in sentinel fishes collected near an arctic formerly used defense site.

Alaska contains over 600 formerly used defense (FUD) sites, many of which serve as point sources of pollution. These sites are often co-located with rural communities that depend upon traditional subsistence foods, especially lipid-rich animals that bioaccumulate and biomagnify persistent organic pollutants (POPs). Many POPs are carcinogenic and endocrine-disrupting compounds that are associated with adverse health outcomes. Therefore, elevated exposure to POPs from point sources of pollution may contribute to disproportionate incidence of disease in arctic communities. We investigated PCB concentrations and the health implications of POP exposure in sentinel fishes collected near the Northeast Cape FUD site on Sivuqaq (St. Lawrence Island), Alaska. Sivuqaq residents are almost exclusively Yupik and rely on subsistence foods. At the request of the Sivuqaq community, we examined differential gene expression and developmental pathologies associated with exposure to POPs originating at the Northeast Cape FUD site. We found significantly higher levels of PCBs in Alaska blackfish (Dallia pectoralis) collected from contaminated sites downstream of the FUD site compared to fish collected from upstream reference sites. We compared transcriptomic profiles and histopathologies of these same blackfish. Blackfish from contaminated sites overexpressed genes involved in ribosomal and FoxO signaling pathways compared to blackfish from reference sites. Contaminated blackfish also had significantly fewer thyroid follicles and smaller pigmented macrophage aggregates. Conversely, we found that ninespine stickleback (Pungitius pungitius) from contaminated sites exhibited thyroid follicle hyperplasia. Despite our previous research reporting transcriptomic and endocrine differences in stickleback from contaminated vs. reference sites, we did not find significant differences in kidney or gonadal histomorphologies. Our results demonstrate that contaminants from the Northeast Cape FUD site are associated with altered gene expression and thyroid development in native fishes. These results are consistent with our prior work demonstrating disruption of the thyroid hormone axis in Sivuqaq residents.

A maternal-to-zygotic-transition gene block on the zebrafish sex chromosome.

Wild zebrafish (Danio rerio) have a ZZ/ZW chromosomal sex-determination system with the major sex locus on the right arm of chromosome-4 (Chr4R) near the largest heterochromatic block in the genome, suggesting that Chr4R transcriptomics might differ from the rest of the genome. To test this hypothesis, we conducted an RNA-seq analysis of adult ZW ovaries and ZZ testes in the Nadia strain and identified 4 regions of Chr4 with different gene expression profiles. Unique in the genome, protein-coding genes in a 41.7 Mb section (Region-2) were expressed in testis but silent in ovary. The AB lab strain, which lacks sex chromosomes, verified this result, showing that testis-biased gene expression in Region-2 depends on gonad biology, not on sex-determining mechanism. RNA-seq analyses in female and male brains and livers validated reduced transcripts from Region-2 in somatic cells, but without sex specificity. Region-2 corresponds to the heterochromatic portion of Chr4R and its content of genes and repetitive elements distinguishes it from the rest of the genome. Region-2 lacks protein-coding genes with human orthologs; has zinc finger genes expressed early in zygotic genome activation; has maternal 5S rRNA genes, maternal spliceosome genes, a concentration of tRNA genes, and a distinct set of repetitive elements. The colocalization of (1) genes silenced in ovaries but not in testes that are (2) expressed in embryos briefly at the onset of zygotic genome activation; (3) maternal-specific genes for translation machinery; (4) maternal-specific spliceosome components; and (5) adjacent genes encoding miR-430, which mediates maternal transcript degradation, suggest that this is a maternal-to-zygotic-transition gene regulatory block.

Direct male development in chromosomally ZZ zebrafish.

The genetics of sex determination varies across taxa, sometimes even within a species. Major domesticated strains of zebrafish (Danio rerio), including AB and TU, lack a strong genetic sex determining locus, but strains more recently derived from nature, like Nadia (NA), possess a ZZ male/ZW female chromosomal sex-determination system. AB fish pass through a juvenile ovary stage, forming oocytes that survive in fish that become females but die in fish that become males. To understand mechanisms of gonad development in NA zebrafish, we studied histology and single cell transcriptomics in developing ZZ and ZW fish. ZW fish developed oocytes by 22 days post-fertilization (dpf) but ZZ fish directly formed testes, avoiding a juvenile ovary phase. Gonads of some ZW and WW fish, however, developed oocytes that died as the gonad became a testis, mimicking AB fish, suggesting that the gynogenetically derived AB strain is chromosomally WW. Single-cell RNA-seq of 19dpf gonads showed similar cell types in ZZ and ZW fish, including germ cells, precursors of gonadal support cells, steroidogenic cells, interstitial/stromal cells, and immune cells, consistent with a bipotential juvenile gonad. In contrast, scRNA-seq of 30dpf gonads revealed that cells in ZZ gonads had transcriptomes characteristic of testicular Sertoli, Leydig, and germ cells while ZW gonads had granulosa cells, theca cells, and developing oocytes. Hematopoietic and vascular cells were similar in both sex genotypes. These results show that juvenile NA zebrafish initially develop a bipotential gonad; that a factor on the NA W chromosome, or fewer than two Z chromosomes, is essential to initiate oocyte development; and without the W factor, or with two Z doses, NA gonads develop directly into testes without passing through the juvenile ovary stage. Sex determination in AB and TU strains mimics NA ZW and WW zebrafish, suggesting loss of the Z chromosome during domestication. Genetic analysis of the NA strain will facilitate our understanding of the evolution of sex determination mechanisms.

Differential gene expression and developmental pathologies associated with persistent organic pollutants in sentinel fish in Troutman Lake, Sivuqaq, Alaska.

Persistent organic pollutants (POPs) are lipophilic compounds that bioaccumulate in animals and biomagnify within food webs. Many POPs are endocrine disrupting compounds that impact vertebrate development. POPs accumulate in the Arctic via global distillation and thereby impact high trophic level vertebrates as well as people who live a subsistence lifestyle. The Arctic also contains thousands of point sources of pollution, such as formerly used defense (FUD) sites. Sivuqaq (St. Lawrence Island), Alaska was used by the U.S. military during the Cold War and FUD sites on the island remain point sources of POP contamination. We examined the effects of POP exposure on ninespine stickleback (Pungitius pungitius) collected from Troutman Lake in the village of Gambell as a model for human exposure and disease. During the Cold War, Troutman Lake was used as a dump site by the U.S. military. We found that PCB concentrations in stickleback exceeded the U.S. Environmental Protection Agency's guideline for unlimited consumption despite these fish being low trophic level organisms. We examined effects at three levels of biological organization: gene expression, endocrinology, and histomorphology. We found that ninespine stickleback from Troutman Lake exhibited suppressed gonadal development compared to threespine stickleback (Gasterosteus aculeatus) studied elsewhere. Troutman Lake stickleback also displayed two distinct hepatic phenotypes, one with lipid accumulation and one with glycogen-type vacuolation. We compared the transcriptomic profiles of these liver phenotypes using RNA sequencing and found significant upregulation of genes involved in ribosomal and metabolic pathways in the lipid accumulation group. Additionally, stickleback displaying liver lipid accumulation had significantly fewer thyroid follicles than the vacuolated phenotype. Our study and previous work highlight health concerns for people and wildlife due to pollution hotspots in the Arctic, and the need for health-protective remediation.

Colour as a behavioural guide for fish near hydrokinetic turbines.

Hydropower is a traditional and widespread form of renewable energy and vertical axis turbines are an emerging technology suitable for low to medium velocity water bodies such as rivers. Such devices can provide renewable power to remote communities but may also contribute to fragmenting already poorly connected riverine habitats and the impact could be particularly pronounced for migratory diadromous aquatic species such as salmonids by limiting their ability to pass the turbines. Optimising the design of such turbines is therefore essential to mitigate their impact on aquatic fauna. One easily altered property that does not impact turbine performance is blade colour. Here, juvenile rainbow trout (Oncorhynchus mykiss) free swimming within a flume were monitored in the presence of a vertical axis turbine that was either stationary or rotating, and coloured white or orange. The orange colour of the turbine affected behaviour by increasing turbine avoidance and decreasing the number of potentially harmful interactions with the turbine when it was rotating, whilst not affecting passage or mobility of the trout compared to the white turbine. Visibility is therefore a potentially useful tool in mitigating the environmental impact of hydrokinetic turbines.

A maternal-to-zygotic-transition gene block on the zebrafish sex chromosome.

Wild zebrafish (Danio rerio) have a ZZ/ZW chromosomal sex determination system with the major sex locus on the right arm of chromosome-4 (Chr4R) near the largest heterochromatic block in the genome, suggesting the hypothesis that the Chr4R transcriptome might be different from the rest of the genome. We conducted an RNA-seq analysis of adult ZW ovaries and ZZ testes and identified four regions of Chr4 with different gene expression profiles. Unique in the genome, protein-coding genes in a 41.7 Mb section (Region-2) were expressed in testis but silent in ovary. The AB lab strain, which lacks sex chromosomes, verified this result, showing that testis-biased gene expression in Region-2 depends on gonad biology, not on sex-determining mechanism. RNA-seq analyses in female and male brain and liver validated few transcripts from Region-2 in somatic cells, but without sex-specificity. Region-2 corresponds to the heterochromatic portion of Chr4R and its content of genes and repetitive elements distinguishes it from the rest of the genome. In Region-2, protein-coding genes lack human orthologs; it has zinc finger genes expressed early in zygotic genome activation; it has maternal 5S rRNA genes, maternal spliceosome genes, a concentration of tRNA genes, and an distinct set of repetitive elements. The colocalization of 1) genes silenced in ovaries but not in testes that are 2) expressed in embryos briefly at the onset of zygotic genome activation; 3) maternal-specific genes for translation machinery; 4) maternal-specific spliceosome components; and 4) adjacent genes encoding miR-430, which mediates maternal transcript degradation, suggest that this is a Maternal-to-Zygotic-Transition Gene Regulatory Block.

Fish response to the presence of hydrokinetic turbines as a sustainable energy solution.

Hydrokinetic turbines such as vertical axis turbines (VATs) may provide decentralised, clean, sustainable energy for remote communities that lack access to the main energy grid or renewable resources. As traditional hydropower adversely alters aquatic ecosystems, it is essential to evaluate the environmental consequences of deploying VATs in riverine ecosystems to meet current and future energy needs. This study explores the implications of VATs on fish movement by observing fish swimming behaviour under two discharges, turbine operation states, and cross-sections confinements using scaled laboratory experiments. Our findings reveal that for cross-sectional confined conditions neither discharge, turbine presence, nor device operation, prevented fish from passing around and through the turbine both in the up- and downstream directions. However, fish spent the least time near the turbine vicinity and within the turbine's turbulent, low-velocity wake, indicating avoidance behaviour. Swimming in a less confined test section further reduced the time spent within the turbine's vicinity and wake, increasing the distance fish kept away from the device. Our results contribute to an understanding of VATs as low-risk hazards for fish swimming behaviour, advancing the potential of deploying VATs in rivers, estuaries or sea as a renewable energy solution for remote communities.

Direct Male Development in Chromosomally ZZ Zebrafish.

The genetics of sex determination varies across taxa, sometimes even within a species. Major domesticated strains of zebrafish ( Danio rerio ), including AB and TU, lack a strong genetic sex determining locus, but strains more recently derived from nature, like Nadia (NA), possess a ZZ male/ZW female chromosomal sex-determination system. AB strain fish pass through a juvenile ovary stage, forming oocytes that survive in fish that become females but die in fish that become males. To understand mechanisms of gonad development in NA zebrafish, we studied histology and single cell transcriptomics in developing ZZ and ZW fish. ZW fish developed oocytes by 22 days post-fertilization (dpf) but ZZ fish directly formed testes, avoiding a juvenile ovary phase. Gonads of some ZW and WW fish, however, developed oocytes that died as the gonad became a testis, mimicking AB fish, suggesting that the gynogenetically derived AB strain is chromosomally WW. Single-cell RNA-seq of 19dpf gonads showed similar cell types in ZZ and ZW fish, including germ cells, precursors of gonadal support cells, steroidogenic cells, interstitial/stromal cells, and immune cells, consistent with a bipotential juvenile gonad. In contrast, scRNA-seq of 30dpf gonads revealed that cells in ZZ gonads had transcriptomes characteristic of testicular Sertoli, Leydig, and germ cells while ZW gonads had granulosa cells, theca cells, and developing oocytes. Hematopoietic and vascular cells were similar in both sex genotypes. These results show that juvenile NA zebrafish initially develop a bipotential gonad; that a factor on the NA W chromosome or fewer than two Z chromosomes is essential to initiate oocyte development; and without the W factor or with two Z doses, NA gonads develop directly into testes without passing through the juvenile ovary stage. Sex determination in AB and TU strains mimics NA ZW and WW zebrafish, suggesting loss of the Z chromosome during domestication. Genetic analysis of the NA strain will facilitate our understanding of the evolution of sex determination mechanisms.

Multi-lingual "Asthma APP" improves health knowledge of asthma among Australian First Nations carers of children with asthma.

Background: Among Australian First Nations people, asthma is associated with worse morbidity and mortality than non-First Nations people. Improving the delivery of health education that is innovative and culturally relevant to linguistically diverse populations is needed. Digital platforms, such as mobile applications (APP), have the potential to improve evidence-based health education, particularly in settings where access to specialist services is limited and turnover of staff is high, such as in remote Australia. In response to consumer needs, we developed a multi-lingual Asthma APP from our existing asthma flipchart, with a "voice-over" in seven local First Nations languages and English, using a mixture of static and interactive formats. In this study, we evaluated (a) the functionality and usability of the APP with First Nations health professionals with and without asthma and (b) whether the APP improves health knowledge and understanding of asthma among First Nations carers of children with asthma. Methods: In total, 7 First Nations health professionals participated in semi-structured interviews prior to the evaluation with 80 First Nations carers of children with asthma from the Northern Territory and Queensland, Australia. Carers underwent pre- and post-education questionnaires (maximum score = 25), where the post-questionnaire was administered immediately post the APP education session. Results: Health professionals found that APP was easy to navigate and culturally appropriate. Among the 80 carers, most were mothers (86%), aged between 26 and 50 years (75%) and 61% lived in remote settings (>100 km from a tertiary hospital). Most carers chose English audio (76%) with the remainder choosing one of the First Nations languages. Overall, asthma knowledge significantly improved post-education (median scores pre = 21 [interquartile range (IQR), 19-22; post = 24 (IQR 22-24), p = 0.05]. Conclusion: The First Nations-specific multi-lingual Asthma APP was easy to use and acceptable for the use by health professionals that also significantly improved short-term asthma knowledge among First Nations carers of children with asthma. The Asthma APP is an innovative and culturally acceptable method of delivering evidence-based, health education to culturally and linguistically diverse populations among Australian First Nations people.

An inertial mechanism behind dynamic station holding by fish swinging in a vortex street.

Many aquatic and aerial animal species are known to utilise their surrounding flow field and/or the induced flow field of a neighbour to reduce their physical exertion, however, the mechanism by which such benefits are obtained has remained elusive. In this work, we investigate the swimming dynamics of rainbow trout in the wake of a thrust-producing oscillating hydrofoil. Despite the higher flow velocities in the inner region of the vortex street, some fish maintain position in this region, while exhibiting an altered swimming gait. Estimates of energy expenditure indicate a reduction in the propulsive cost when compared to regular swimming. By examining the accelerations of the fish, an explanation of the mechanism by which energy is harvested from the vortices is proposed. Similar to dynamic soaring employed by albatross, the mechanism can be linked to the non-equilibrium hydrodynamic forces produced when fish encounter the cross-flow velocity generated by the vortex street.

Environmentally-induced sex reversal in fish with chromosomal vs. polygenic sex determination.

Sex ratio depends on sex determination mechanisms and is a key demographic parameter determining population viability and resilience to natural and anthropogenic stressors. There is increasing evidence that the environment can alter sex ratio even in genetically sex-determined species (GSD), as elevated temperature can cause female-to-male sex reversal (neomales). Alarmingly, neomales are being discovered in natural populations of several fish, amphibian and reptile species worldwide. Understanding the basis of neomale development is important for conservation biology. Among GSD species, it is unknown whether those with chromosomal sex determination (CSD), the most common system, will better resist the influence of high temperature than those with polygenic sex determination (PSD). Here, we compared the effects of elevated temperature in two wild zebrafish strains, Nadia (NA) and Ekkwill (EKW), which have CSD with a ZZ/ZW system, against the AB laboratory strain, which has PSD. First, we uncovered novel sex genotypes and the results showed that, at control temperature, the masculinization rate roughly doubled with the addition of each Z chromosome, while some ZW and WW fish of the wild strains became neomales. Surprisingly, we found that at elevated temperatures WW fish were just as likely as ZW fish to become neomales and that all strains were equally susceptible to masculinization. These results demonstrate that the Z chromosome is not essential for male development and that the dose of W buffers masculinization at the control temperature but not at elevated temperature. Furthermore, at the elevated temperature the testes of neomales, but not of normal males, contained more spermatozoa than at the control temperature. Our results show in an unprecedented way that, in a global warming scenario, CSD species may not necessarily be better protected against the masculinizing effect of elevated temperature than PSD species, and reveal genotype-by-temperature interactions in male sex determination and spermatogenesis.

Leaky barriers: leaky enough for fish to pass?

Perceived as environmental-friendly hydraulic structures, leaky barriers used for natural flood management are introduced into rivers, potentially creating migration barriers for fish. Using sustainable, local materials to construct wooden barriers across river channels in upper catchments, these barriers aim to slow down the flow, reduce flood peaks and attenuate the flow reaching downstream communities. Yet little is known about their impact on hydrodynamics and fish passage. Here, we examined two model barrier designs under 100% and 80% bankfull flow conditions in an open channel flume. These barriers included a porous and a non-porous design, with the latter emulating the natural accumulation of brush, sediment and leaf material between logs over time. Flow visualization and velocity measurements recorded with acoustic Doppler velocimetry characterized the flow field upstream and downstream of the barriers. Our fish behavioural studies revealed that juvenile salmon (Salmo salar) movement between downstream and upstream sections of the flume was inhibited by barrier design rather than discharge, influencing upstream fish passage and their spatial preference, indicating the importance of barrier design criteria to facilitate fish movement.

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.

A Search for Sex-Linked Loci in the Agamid Lizard, Calotes versicolor.

The Indian garden lizard, Calotes versicolor, lacks cytologically recognizable sex chromosomes, and its mechanism of sex determination is unclear. We evaluated genotype-to-sex-phenotype association using RAD-seq in wild-caught males and females, 30 of each sex. Of 210,736 unique, 96-nt long RAD-tags, 48% contained polymorphisms, 23% of which were present in at least 40 of 60 individuals. Twenty one RAD-tags neared, but none achieved, the inclusion criteria for sex enrichment, as expected if C. versicolor lacks highly differentiated sex chromosomes. Three RAD-tags with alleles most strongly associated with sex tended to be heterozygous in females and to lack male-specific alleles, suggesting a ZW female/ZZ male system. Putative female alleles, however, were present in some males and lacking from some females, suggesting either recombination between these markers and the sex locus or sex reversal due to environmental or genetic factors. Paired-end, 250-nt reads from 1 male provided a fragmented draft genome assembly. Four sex-associated RAD-tags were identical to portions of 4 unique C. versicolor genomic contigs rather than linked to a single putative sex-linked region. The lack of strongly sex-linked loci coupled with weak evidence for temperature-associated sex determination intensifies the need for further investigation of the puzzling sex determination mechanism in C. versicolor.

Antarctic blackfin icefish genome reveals adaptations to extreme environments.

Icefishes (suborder Notothenioidei; family Channichthyidae) are the only vertebrates that lack functional haemoglobin genes and red blood cells. Here, we report a high-quality genome assembly and linkage map for the Antarctic blackfin icefish Chaenocephalus aceratus, highlighting evolved genomic features for its unique physiology. Phylogenomic analysis revealed that Antarctic fish of the teleost suborder Notothenioidei, including icefishes, diverged from the stickleback lineage about 77 million years ago and subsequently evolved cold-adapted phenotypes as the Southern Ocean cooled to sub-zero temperatures. Our results show that genes involved in protection from ice damage, including genes encoding antifreeze glycoprotein and zona pellucida proteins, are highly expanded in the icefish genome. Furthermore, genes that encode enzymes that help to control cellular redox state, including members of the sod3 and nqo1 gene families, are expanded, probably as evolutionary adaptations to the relatively high concentration of oxygen dissolved in cold Antarctic waters. In contrast, some crucial regulators of circadian homeostasis (cry and per genes) are absent from the icefish genome, suggesting compromised control of biological rhythms in the polar light environment. The availability of the icefish genome sequence will accelerate our understanding of adaptation to extreme Antarctic environments.

Cold Fusion: Massive Karyotype Evolution in the Antarctic Bullhead Notothen Notothenia coriiceps.

Half of all vertebrate species share a series of chromosome fusions that preceded the teleost genome duplication (TGD), but we do not understand the causative evolutionary mechanisms. The "Robertsonian-translocation hypothesis" suggests a regular fusion of each ancestral acro- or telocentric chromosome to just one other by centromere fusions, thus halving the karyotype. An alternative "genome-stirring hypothesis" posits haphazard and repeated fusions, inversions, and reciprocal and nonreciprocal translocations. To study large-scale karyotype reduction, we investigated the decrease of chromosome numbers in Antarctic notothenioid fish. Most notothenioids have 24 haploid chromosomes, but bullhead notothen (Notothenia coriiceps) has 11. To understand mechanisms, we made a RAD-tag meiotic map with ∼10,000 polymorphic markers. Comparative genomics aligned about a thousand orthologs of platyfish and stickleback genes along bullhead chromosomes. Results revealed that 9 of 11 bullhead chromosomes arose by fusion of just two ancestral chromosomes and two others by fusion of three ancestral chromosomes. All markers from each ancestral chromosome remained contiguous, implying no inversions across fusion borders. Karyotype comparisons support a history of: (1) Robertsonian fusions of 22 ancestral chromosomes in pairs to yield 11 fused plus two small unfused chromosomes, like N. angustata; (2) fusion of one of the remaining two ancestral chromosomes to a preexisting fused pair, giving 12 chromosomes like N. rossii; and (3) fusion of the remaining ancestral chromosome to another fused pair, giving 11 chromosomes in N. coriiceps These results raise the question of what selective forces promoted the systematic fusion of chromosomes in pairs and the suppression of pericentric inversions in this lineage, and provide a model for chromosome fusions in stem teleosts.

A randomized trial of once-daily fluticasone furoate/vilanterol or vilanterol versus placebo to determine effects on arterial stiffness in COPD.

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is associated with increased cardiovascular morbidity and mortality. Elevated arterial stiffness, measured by aortic pulse wave velocity (aPWV), is a cardiovascular risk surrogate and is potentially modifiable by inhaled corticosteroid/long-acting beta2-agonist combinations in patients with COPD. MATERIALS AND METHODS: The effects of once-daily inhaled fluticasone furoate/vilanterol (FF/VI) 100/25 µg, VI 25 µg, versus placebo on arterial stiffness in patients with COPD and baseline aPWV ≥11.0 m/s were investigated in a 24-week, multicenter, double-blind, randomized, stratified (by COPD exacerbation history), parallel-group, placebo-controlled trial. Eligible patients were ≥40 years old, with ≥10 pack-year smoking history, forced expiratory volume in 1 s (FEV1)/forced vital capacity ≤0.70, and post-bronchodilator FEV1 ≤70% of predicted. Patients with a major cardiovascular event in the previous 6 months/current severe heart failure/uncontrolled hypertension were excluded. Primary endpoint is change from baseline in aPWV after 24 weeks of treatment. Safety analyses included adverse events (AEs). RESULTS: The intent-to-treat population included 430 patients: FF/VI (n=135), VI (n=154), and placebo (n=141). Patients were predominantly male (79%) and Asian or White (each 48%), with a mean age of 68.5 years (standard deviation [SD] =7.9), percentage predicted post-bronchodilator FEV1 50.1% (SD =13.3), and aPWV 13.26 m/s (SD =2.22) at screening. At 24 weeks, mean (standard error [SE]) changes from baseline in aPWV were -1.75 m/s (SE =0.26, FF/VI), -1.95 m/s (SE =0.24, VI), and -1.97 m/s (SE =0.28, placebo). AEs occurred in 57% (FF/VI), 51% (VI), and 41% (placebo) of patients. CONCLUSION: No differences were observed in aPWV-adjusted mean change from baseline for FF/VI 100/25 µg, compared with placebo.

A randomised, phase III trial of once-daily fluticasone furoate/vilanterol 100/25 µg versus once-daily vilanterol 25 µg to evaluate the contribution on lung function of fluticasone furoate in the combination in patients with COPD.

BACKGROUND: The contribution of fluticasone furoate (FF) on lung function in the FF/vilanterol (VI) 100/25 µg combination has been demonstrated numerically, but not statistically. METHODS: This multicentre, randomised, double-blind, controlled trial (GlaxoSmithKline study number 200820; clinicaltrials.gov NCT02105974) enrolled ≥40-year-old patients with chronic obstructive pulmonary disease (COPD), a ≥10-pack-year smoking history, a post-bronchodilator forced expiratory volume in 1 s (FEV1) 30-70% of the predicted value, a FEV1/forced vital capacity ratio of ≤0.70, ≥1 COPD exacerbation in the previous 12 months requiring corticosteroids, antibiotics and/or hospitalisation, and current COPD symptoms. Participants received FF/VI 100/25 µg or VI 25 µg once daily. The primary endpoint was the change from baseline in trough FEV1 at day 84. FINDINGS: 1620 patients were randomised and received at least one dose of FF/VI 100/25 µg (n = 806) or VI 25 µg (n = 814). At day 84, the FF/VI 100/25 µg group showed an adjusted mean treatment difference of 34 mL over VI 25 µg in change from baseline trough FEV1 (95% confidence interval [CI] 14-55; p = 0.001). There was no significant difference between the groups in the percentage of rescue medication-free 24-h periods. The FF/VI 100/25 µg group demonstrated a 42% risk reduction compared with the VI 25 µg group in time to first moderate/severe COPD exacerbation (95% CI 22-57; nominal p < 0.001). The incidence of on-treatment adverse events was similar between the groups. INTERPRETATION: The contribution of FF in the FF/VI 100/25 µg combination on lung function in COPD was statistically significant. FUNDING: GlaxoSmithKline.

Variations on a theme: Genomics of sex determination in the cichlid fish Astatotilapia burtoni.

BACKGROUND: Sex chromosomes change more frequently in fish than in mammals or birds. However, certain chromosomes or genes are repeatedly used as sex determinants in different members of the teleostean lineage. East African cichlids are an enigmatic model system in evolutionary biology representing some of the most diverse extant vertebrate adaptive radiations. How sex is determined and if different sex-determining mechanisms contribute to speciation is unknown for almost all of the over 1,500 cichlid species of the Great Lakes. Here, we investigated the genetic basis of sex determination in a cichlid from Lake Tanganyika, Astatotilapia burtoni, a member of the most species-rich cichlid lineage, the haplochromines. RESULTS: We used RAD-sequencing of crosses for two populations of A. burtoni, a lab strain and fish caught at the south of Lake Tanganyika. Using association mapping and comparative genomics, we confirmed male heterogamety in A. burtoni and identified different sex chromosomes (LG5 and LG18) in the two populations of the same species. LG5, the sex chromosome of the lab strain, is a fusion chromosome in A. burtoni. Wnt4 is located on this chromosome, representing the best candidate identified so far for the master sex-determining gene in our lab strain of A. burtoni. CONCLUSIONS: Cichlids exemplify the high turnover rate of sex chromosomes in fish with two different chromosomes, LG5 and LG18, containing major sex-determining loci in the two populations of A. burtoni examined here. However, they also illustrate that particular chromosomes are more likely to be used as sex chromosomes. Chromosome 5 is such a chromosome, which has evolved several times as a sex chromosome, both in haplochromine cichlids from all Great Lakes and also in other teleost fishes.

A Genetic Map for the Only Self-Fertilizing Vertebrate.

The mangrove killifish Kryptolebias marmoratus, and its close relative Kryptolebias hermaphroditus, are the only vertebrate species known to reproduce by self-fertilization due to functional ovotestis development. To improve our understanding of their genomes, we constructed a genetic map. First, a single F1 fish was made by artificial fertilization between K. marmoratus and K. hermaphroditus strains. F2 progeny were then obtained by self-fertilization of the F1 fish. We used RAD-seq to query genomic DNAs from the two parental strains, the F1 individual and 49 F2 progeny. Results identified 9904 polymorphic RAD-tags (DNA markers) that mapped to 24 linkage groups, corresponding to the haploid chromosome number of these species. The total length of the map was 1248 cM, indicating that about one recombination occurred for each of the 24 homologous chromosome pairs in each meiosis. Markers were not evenly distributed along the chromosomes: in all chromosomes, many markers (> 8% of the total markers for each chromosome) mapped to chromosome tips. Centromeres suppress recombination, and this uneven distribution is probably due to the species' acrocentric chromosomes. Mapped marker sequences were compared to genomic sequences of medaka and platyfish, the next most closely related species with sequenced genomes that are anchored to genetic maps. Results showed that each mangrove killifish chromosome corresponds to a single chromosome of both platyfish and medaka, suggesting strong conservation of chromosomes over 100 million years of evolution. Our genetic map provides a framework for the K. marmoratus/K. hermaphroditus genome sequence and an important resource for understanding the biology of hermaphroditism.