Evolutionary selection across the nuclear hormone receptor superfamily with a focus on the NR1I subfamily (vitamin D, pregnane X, and constitutive androstane receptors)

BACKGROUND: The nuclear hormone receptor (NR) superfamily complement in humans is composed of 48 genes with diverse roles in metabolic homeostasis, development, and detoxification. In general, NRs are strongly conserved between vertebrate species, and few examples of molecular adaptation (positive selection) within this superfamily have been demonstrated. Previous studies utilizing two-species comparisons reveal strong purifying (negative) selection of most NR genes, with two possible exceptions being the ligand-binding domains (LBDs) of the pregnane X receptor (PXR, NR1I2) and the constitutive androstane receptor (CAR, NR1I3), two proteins involved in the regulation of toxic compound metabolism and elimination. The aim of this study was to apply detailed phylogenetic analysis using maximum likelihood methods to the entire complement of genes in the vertebrate NR superfamily. Analyses were carried out both across all vertebrates and limited to mammals and also separately for the two major domains of NRs, the DNA-binding domain (DBD) and LBD, in addition to the full-length sequences. Additional functional data is also reported for activation of PXR and the vitamin D receptor (VDR; NR1I1) to gain further insight into the evolution of the NR1I subfamily. RESULTS: The NR genes appear to be subject to strong purifying selection, particularly in the DBDs. Estimates of the ratio of the non-synonymous to synonymous nucleotide substitution rates (the ω ratio) revealed that only the PXR LBD had a sub-population of codons with an estimated ω ratio greater than 1. CAR was also unusual in showing high relative ω ratios in both the DBD and LBD, a finding that may relate to the recent appearance of the CAR gene (presumably by duplication of a pre-mammalian PXR gene) just prior to the evolution of mammals. Functional analyses of the NR1I subfamily show that human and zebrafish PXRs show similar activation by steroid hormones and early bile salts, properties not shared by sea lamprey, mouse, or human VDRs, or by Xenopus laevis PXRs. CONCLUSION: NR genes generally show strong sequence conservation and little evidence for positive selection. The main exceptions are PXR and CAR, genes that may have adapted to cross-species differences in toxic compound exposure

Evolutionary diversity of bile salts in reptiles and mammals, including analysis of ancient human and extinct giant ground sloth coprolites

<p>Abstract</p> <p>Background</p> <p>Bile salts are the major end-metabolites of cholesterol and are also important in lipid and protein digestion and in influencing the intestinal microflora. We greatly extend prior surveys of bile salt diversity in both reptiles and mammals, including analysis of 8,000 year old human coprolites and coprolites from the extinct Shasta ground sloth (<it>Nothrotherium shastense</it>).</p> <p>Results</p> <p>While there is significant variation of bile salts across species, bile salt profiles are generally stable within families and often within orders of reptiles and mammals, and do not directly correlate with differences in diet. The variation of bile salts generally accords with current molecular phylogenies of reptiles and mammals, including more recent groupings of squamate reptiles. For mammals, the most unusual finding was that the Paenungulates (elephants, manatees, and the rock hyrax) have a very different bile salt profile from the Rufous sengi and South American aardvark, two other mammals classified with Paenungulates in the cohort Afrotheria in molecular phylogenies. Analyses of the approximately 8,000 year old human coprolites yielded a bile salt profile very similar to that found in modern human feces. Analysis of the Shasta ground sloth coprolites (approximately 12,000 years old) showed the predominant presence of glycine-conjugated bile acids, similar to analyses of bile and feces of living sloths, in addition to a complex mixture of plant sterols and stanols expected from an herbivorous diet.</p> <p>Conclusions</p> <p>The bile salt synthetic pathway has become longer and more complex throughout vertebrate evolution, with some bile salt modifications only found within single groups such as marsupials. Analysis of the evolution of bile salt structures in different species provides a potentially rich model system for the evolution of a complex biochemical pathway in vertebrates. Our results also demonstrate the stability of bile salts in coprolites preserved in arid climates, suggesting that bile salt analysis may have utility in selected paleontological research.</p

Declining Sex Ratio in a First Nation Community

Members of the Aamjiwnaang First Nation community near Sarnia, Ontario, Canada, voiced concerns that there appeared to be fewer male children in their community in recent years. In response to these concerns, we assessed the sex ratio (proportion of male births) of the Aamjiwnaang First Nation over the period 1984–2003 as part of a community-based participatory research project. The trend in the proportion of male live births of the Aamjiwnaang First Nation has been declining continuously from the early 1990s to 2003, from an apparently stable sex ratio prior to this time. The proportion of male births (m) showed a statistically significant decline over the most recent 10-year period (1994–2003) (m = 0.412, p = 0.008) with the most pronounced decrease observed during the most recent 5 years (1999–2003) (m = 0.348, p = 0.006). Numerous factors have been associated with a decrease in the proportion of male births in a population, including a number of environmental and occupational chemical exposures. This community is located within the Great Lakes St. Clair River Area of Concern and is situated immediately adjacent to several large petrochemical, polymer, and chemical industrial plants. Although there are several potential factors that could be contributing to the observed decrease in sex ratio of the Aamjiwnaang First Nation, the close proximity of this community to a large aggregation of industries and potential exposures to compounds that may influence sex ratios warrants further assessment into the types of chemical exposures for this population. A community health survey is currently under way to gather more information about the health of the Aamjiwnaang community and to provide additional information about the factors that could be contributing to the observed decrease in the proportion of male births in recent years

The evolution of farnesoid X, vitamin D, and pregnane X receptors: insights from the green-spotted pufferfish (Tetraodon nigriviridis) and other non-mammalian species

<p>Abstract</p> <p>Background</p> <p>The farnesoid X receptor (FXR), pregnane X receptor (PXR), and vitamin D receptor (VDR) are three closely related nuclear hormone receptors in the NR1H and 1I subfamilies that share the property of being activated by bile salts. Bile salts vary significantly in structure across vertebrate species, suggesting that receptors binding these molecules may show adaptive evolutionary changes in response. We have previously shown that FXRs from the sea lamprey (<it>Petromyzon marinus</it>) and zebrafish (<it>Danio rerio</it>) are activated by planar bile alcohols found in these two species. In this report, we characterize FXR, PXR, and VDR from the green-spotted pufferfish (<it>Tetraodon nigriviridis</it>), an actinopterygian fish that unlike the zebrafish has a bile salt profile similar to humans. We utilize homology modelling, docking, and pharmacophore studies to understand the structural features of the <it>Tetraodon </it>receptors.</p> <p>Results</p> <p><it>Tetraodon </it>FXR has a ligand selectivity profile very similar to human FXR, with strong activation by the synthetic ligand GW4064 and by the primary bile acid chenodeoxycholic acid. Homology modelling and docking studies suggest a ligand-binding pocket architecture more similar to human and rat FXRs than to lamprey or zebrafish FXRs. <it>Tetraodon </it>PXR was activated by a variety of bile acids and steroids, although not by the larger synthetic ligands that activate human PXR such as rifampicin. Homology modelling predicts a larger ligand-binding cavity than zebrafish PXR. We also demonstrate that VDRs from the pufferfish and Japanese medaka were activated by small secondary bile acids such as lithocholic acid, whereas the African clawed frog VDR was not.</p> <p>Conclusions</p> <p>Our studies provide further evidence of the relationship between both FXR, PXR, and VDR ligand selectivity and cross-species variation in bile salt profiles. Zebrafish and green-spotted pufferfish provide a clear contrast in having markedly different primary bile salt profiles (planar bile alcohols for zebrafish and sterically bent bile acids for the pufferfish) and receptor selectivity that matches these differences in endogenous ligands. Our observations to date present an integrated picture of the co-evolution of bile salt structure and changes in the binding pockets of three nuclear hormone receptors across the species studied.</p

Functional evolution of the vitamin D and pregnane X receptors

<p>Abstract</p> <p>Background</p> <p>The vitamin D receptor (VDR) and pregnane X receptor (PXR) are nuclear hormone receptors of the NR1I subfamily that show contrasting patterns of cross-species variation. VDR and PXR are thought to have arisen from duplication of an ancestral gene, evident now as a single gene in the genome of the chordate invertebrate <it>Ciona intestinalis </it>(sea squirt). VDR genes have been detected in a wide range of vertebrates including jawless fish. To date, PXR genes have not been found in cartilaginous fish. In this study, the ligand selectivities of VDRs were compared in detail across a range of vertebrate species and compared with those of the <it>Ciona </it>VDR/PXR. In addition, several assays were used to search for evidence of PXR-mediated hepatic effects in three model non-mammalian species: sea lamprey (<it>Petromyzon marinus</it>), zebrafish (<it>Danio rerio</it>), and African clawed frog (<it>Xenopus laevis</it>).</p> <p>Results</p> <p>Human, mouse, frog, zebrafish, and lamprey VDRs were found to have similar ligand selectivities for vitamin D derivatives. In contrast, using cultured primary hepatocytes, only zebrafish showed evidence of PXR-mediated induction of enzyme expression, with increases in testosterone 6β-hydroxylation activity (a measure of cytochrome P450 3A activity in other species) and flurbiprofen 4-hydroxylation activity (measure of cytochrome P450 2C activity) following exposure to known PXR activators. A separate assay in vivo using zebrafish demonstrated increased hepatic transcription of another PXR target, multidrug resistance gene (ABCB5), following injection of the major zebrafish bile salt, 5α-cyprinol 27-sulfate. The PXR target function, testosterone hydroxylation, was detected in frog and sea lamprey primary hepatocytes, but was not inducible in these two species by a wide range of PXR activators in other animals. Analysis of the sea lamprey draft genome also did not show evidence of a PXR gene.</p> <p>Conclusion</p> <p>Our results show tight conservation of ligand selectivity of VDRs across vertebrate species from Agnatha to mammals. Using a functional approach, we demonstrate classic PXR-mediated effects in zebrafish, but not in sea lamprey or African clawed frog liver cells. Using a genomic approach, we failed to find evidence of a PXR gene in lamprey, suggesting that VDR may be the original NR1I gene.</p

Pbx homeodomain proteins pattern both the zebrafish retina and tectum

<p>Abstract</p> <p>Background</p> <p><it>Pbx </it>genes encode TALE class homeodomain transcription factors that pattern the developing neural tube, pancreas, and blood. Within the hindbrain, Pbx cooperates with Hox proteins to regulate rhombomere segment identity. Pbx cooperates with Eng to regulate midbrain-hindbrain boundary maintenance, and with MyoD to control fast muscle cell differentiation. Although previous results have demonstrated that Pbx is required for proper eye size, functions in regulating retinal cell identity and patterning have not yet been examined.</p> <p>Results</p> <p>Analysis of retinal ganglion cell axon pathfinding and outgrowth in <it>pbx2/4 </it>null embryos demonstrated a key role for <it>pbx </it>genes in regulating neural cell behavior. To identify Pbx-dependent genes involved in regulating retino-tectal pathfinding, we conducted a microarray screen for Pbx-dependent transcripts in zebrafish, and detected genes that are specifically expressed in the eye and tectum. A subset of Pbx-dependent retinal transcripts delineate specific domains in the dorso-temporal lobe of the developing retina. Furthermore, we determined that some Pbx-dependent transcripts also require Meis1 and Gdf6a function. Since <it>gdf6a </it>expression is also dependent on Pbx, we propose a model in which Pbx proteins regulate expression of the growth factor <it>gdf6a</it>, which in turn regulates patterning of the dorso-temporal lobe of the retina. This, in concert with aberrant tectal patterning in <it>pbx2/4 </it>null embryos, may lead to the observed defects in RGC outgrowth.</p> <p>Conclusion</p> <p>These data define a novel role for Pbx in patterning the vertebrate retina and tectum in a manner required for proper retinal ganglion cell axon outgrowth.</p

Identification of storage conditions stabilizing extracellular vesicles pre

Extracellular vesicles (EVs) play a key role in many physiological and pathophysiological processes and hold great potential for therapeutic and diagnostic use. Despite significant advances within the last decade, the key issue of EV storage stability remains unresolved and under investigated. Here, we aimed to identify storage conditions stabilizing EVs and comprehensively compared the impact of various storage buffer formulations at different temperatures on EVs derived from different cellular sources for up to 2 years. EV features including concentration, diameter, surface protein profile and nucleic acid contents were assessed by complementary methods, and engineered EVs containing fluorophores or functionalized surface proteins were utilized to compare cellular uptake and ligand binding. We show that storing EVs in PBS over time leads to drastically reduced recovery particularly for pure EV samples at all temperatures tested, starting already within days. We further report that using PBS as diluent was found to result in severely reduced EV recovery rates already within minutes. Several of the tested new buffer conditions largely prevented the observed effects, the lead candidate being PBS supplemented with human albumin and trehalose (PBS-HAT). We report that PBS-HAT buffer facilitates clearly improved short-term and long-term EV preservation for samples stored at -80°C, stability throughout several freeze-thaw cycles, and drastically improved EV recovery when using a diluent for EV samples for downstream applications

Programmable antivirals targeting critical conserved viral RNA secondary structures from influenza A virus and SARS-CoV-2

Influenza A virus’s (IAV’s) frequent genetic changes challenge vaccine strategies and engender resistance to current drugs. We sought to identify conserved and essential RNA secondary structures within IAV’s genome that are predicted to have greater constraints on mutation in response to therapeutic targeting. We identified and genetically validated an RNA structure (packaging stem–loop 2 (PSL2)) that mediates in vitro packaging and in vivo disease and is conserved across all known IAV isolates. A PSL2-targeting locked nucleic acid (LNA), administered 3 d after, or 14 d before, a lethal IAV inoculum provided 100% survival in mice, led to the development of strong immunity to rechallenge with a tenfold lethal inoculum, evaded attempts to select for resistance and retained full potency against neuraminidase inhibitor-resistant virus. Use of an analogous approach to target SARS-CoV-2, prophylactic administration of LNAs specific for highly conserved RNA structures in the viral genome, protected hamsters from efficient transmission of the SARS-CoV-2 USA_WA1/2020 variant. These findings highlight the potential applicability of this approach to any virus of interest via a process we term ‘programmable antivirals’, with implications for antiviral prophylaxis and post-exposure therapy

Characterizing a World Within the Hot-Neptune Desert: Transit Observations of LTT 9779 b with the Hubble Space Telescope/WFC3

We present an atmospheric analysis of LTT 9779 b, a rare planet situated in the hot-Neptune desert, that has been observed with Hubble Space Telescope (HST)/WFC3 with G102 and G141. The combined transmission spectrum, which covers 0.8–1.6 μm, shows a gradual increase in transit depth with wavelength. Our preferred atmospheric model shows evidence for H2O, CO2, and FeH with a significance of 3.1σ, 2.4σ, and 2.1σ, respectively. In an attempt to constrain the rate of atmospheric escape for this planet, we search for the 1.083 μm helium line in the G102 data but find no evidence of excess absorption that would indicate an escaping atmosphere using this tracer. We refine the orbital ephemerides of LTT 9779 b using our HST data and observations from TESS, searching for evidence of orbital decay or apsidal precession, which are not found. The phase-curve observation of LTT 9779 b with JWST NIRISS should provide deeper insights into the atmosphere of this planet and the expected atmospheric escape might be detected with further observations concentrated on other tracers such as Lyα