Functional EPAS1/HIF2A missense variant is associated with hematocrit in Andean highlanders.

Hypoxia-inducible factor pathway genes are linked to adaptation in both human and nonhuman highland species. EPAS1, a notable target of hypoxia adaptation, is associated with relatively lower hemoglobin concentration in Tibetans. We provide evidence for an association between an adaptive EPAS1 variant (rs570553380) and the same phenotype of relatively low hematocrit in Andean highlanders. This Andean-specific missense variant is present at a modest frequency in Andeans and absent in other human populations and vertebrate species except the coelacanth. CRISPR-base-edited human cells with this variant exhibit shifts in hypoxia-regulated gene expression, while metabolomic analyses reveal both genotype and phenotype associations and validation in a lowland population. Although this genocopy of relatively lower hematocrit in Andean highlanders parallels well-replicated findings in Tibetans, it likely involves distinct pathway responses based on a protein-coding versus noncoding variants, respectively. These findings illuminate how unique variants at EPAS1 contribute to the same phenotype in Tibetans and a subset of Andean highlanders despite distinct evolutionary trajectories.

The bidirectional relationship between head injuries and conduct problems: longitudinal modelling of a population-based birth cohort study.

Childhood head injuries and conduct problems increase the risk of aggression and criminality and are well-known correlates. However, the direction and timing of their association and the role of their demographic risk factors remain unclear. This study investigates the bidirectional links between both from 3 to 17 years while revealing common and unique demographic risks. A total of 8,603 participants (50.2% female; 83% White ethnicity) from the Millennium Cohort Study were analysed at 6 timepoints from age 3 to 17. Conduct problems were parent-reported for ages 3 to 17 using the Strengths and Difficulties Questionnaire (SDQ) and head injuries at ages 3 to 14. A cross-lagged path model estimated the longitudinal bidirectional effects between the two whilst salient demographic risks were modelled cumulatively at three ecological levels (child, mother, and household). Conduct problems at age 5 promoted head injuries between 5 and 7 (Z = 0.07; SE = 0.03; 95% CI, 0.02-0.13), and head injuries at ages 7 to 11 promoted conduct problems at age 14 (ß = .0.06; SE = .0.03; 95% CI, 0.01-0.12). Head injuries were associated with direct child-level risk at age 3, whereas conduct problems were associated with direct risks from all ecological levels until 17 years. The findings suggest a sensitive period at 5-11 years for the bidirectional relationship shared between head injuries and conduct problems. They suggest that demographic risks for increased head injuries play an earlier role than they do for conduct problems. Both findings have implications for intervention timing.

Adolescent delinquency following co-occurring childhood head injuries and conduct problem symptoms: findings from a UK longitudinal birth cohort.

Childhood conduct problems and head injuries share a bidirectional association, but how this affects the risk of adolescent delinquency is unknown. Due to their similar underlying mechanisms (i.e. increased impulsivity), this study aims to identify whether their co-occurrence increases the risk of adolescent delinquency. Data was obtained from 11,272 children at age 14 and 10,244 at age 17 years enrolled in the UK Millennium Cohort Study. Conduct problem symptoms (via the Strengths and Difficulties Questionnaire) and head injuries were parent reported from ages 3 to 14 years. Delinquency was self-reported at ages 14 and 17 including substance use, criminality, and antisocial behaviour. Incident rate ratios (IRR) were estimated for delinquency at ages 14 and 17 by childhood conduct problem and head injury status. Co-occurring head injuries and high conduct problem symptoms presented the greatest risk for overall delinquency and substance use at age 14 compared to those with the presence of one or neither (IRRs from 1.20 to 1.60). At age 17, conduct problems (with or without co-occurring head injuries) presented the greatest risk for overall delinquency, substance use, and antisocial behaviour. There was no evidence for an increased risk of delinquency at ages 14 or 17 following a head injury only. Whilst these findings suggest childhood head injuries alone do not increase the risk of adolescent delinquency, when co-occurring alongside high conduct problem symptoms there is a heightened earlier risk. These results provide further insight into adolescent delinquency and the outcomes of co-occurring childhood head injury and conduct problem symptoms.

Linked head injury and conduct problem symptom pathways from early childhood to adolescence and their associated risks: Evidence from the millennium cohort study.

Conduct problems and head injuries increase the risk of delinquency and share a bidirectional association. However, how they link across development is unknown. The present study aimed to identify their linked developmental pathways and associated risk factors. Latent class analysis was modeled from Millennium Cohort Study data (n = 8,600) to identify linked pathways of conduct problem symptoms and head injuries. Head injuries were parent-reported from ages 3 to 14 and conduct problems from ages 3 to 17 using the Strengths and Difficulties Questionnaire (SDQ). Multinomial logistic regression then identified various risk factors associated with pathway membership. Four distinct pathways were identified. Most participants displayed low-level conduct problem symptoms and head injuries (n = 6,422; 74.7%). Three groups were characterized by clinically relevant levels of conduct problem symptoms and high-risk head injuries in childhood (n = 1,422; 16.5%), adolescence (n = 567; 6.6%), or persistent across development (n = 189; 2.2%). These clinically relevant pathways were associated with negative maternal parenting styles. These findings demonstrate how pathways of conduct problem symptoms are uniquely linked with distinct head injury pathways. Suggestions for general preventative intervention targets include early maternal negative parenting styles. Pathway-specific interventions are also required targeting cumulative risk at different ecological levels.

Time Domains of Hypoxia Responses and -Omics Insights.

The ability to respond rapidly to changes in oxygen tension is critical for many forms of life. Challenges to oxygen homeostasis, specifically in the contexts of evolutionary biology and biomedicine, provide important insights into mechanisms of hypoxia adaptation and tolerance. Here we synthesize findings across varying time domains of hypoxia in terms of oxygen delivery, ranging from early animal to modern human evolution and examine the potential impacts of environmental and clinical challenges through emerging multi-omics approaches. We discuss how diverse animal species have adapted to hypoxic environments, how humans vary in their responses to hypoxia (i.e., in the context of high-altitude exposure, cardiopulmonary disease, and sleep apnea), and how findings from each of these fields inform the other and lead to promising new directions in basic and clinical hypoxia research.

In vivo macromolecular crowding is differentially modulated by aquaporin 0 in zebrafish lens: Insights from a nanoenvironment sensor and spectral imaging.

Macromolecular crowding is crucial for cellular homeostasis. In vivo studies of macromolecular crowding and water dynamics are needed to understand their roles in cellular physiology and fate determination. Macromolecular crowding in the lens is essential for normal optics, and an understanding of its regulation will help prevent cataract and presbyopia. Here, we combine the use of the nanoenvironmental sensor [6-acetyl-2-dimethylaminonaphthalene (ACDAN)] to visualize lens macromolecular crowding with in vivo studies of aquaporin 0 zebrafish mutants that disrupt its regulation. Spectral phasor analysis of ACDAN fluorescence reveals water dipolar relaxation and demonstrates that mutations in two zebrafish aquaporin 0s, Aqp0a and Aqp0b, alter water state and macromolecular crowding in living lenses. Our results provide in vivo evidence that Aqp0a promotes fluid influx in the deeper lens cortex, whereas Aqp0b facilitates fluid efflux. This evidence reveals previously unidentified spatial regulation of macromolecular crowding and spatially distinct roles for Aqp0 in the lens.

Differences in a Single Extracellular Residue Underlie Adhesive Functions of Two Zebrafish Aqp0s.

Aquaporin 0 (AQP0) is the most abundant lens membrane protein, and loss of function in human and animal models leads to cataract formation. AQP0 has several functions in the lens including water transport and adhesion. Since lens optics rely on strict tissue architecture achieved by compact cell-to-cell adhesion between lens fiber cells, understanding how AQP0 contributes to adhesion would shed light on normal lens physiology and pathophysiology. We show in an in vitro adhesion assay that one of two closely related zebrafish Aqp0s, Aqp0b, has strong auto-adhesive properties while Aqp0a does not. The difference appears to be largely due to a single amino acid difference at residue 110 in the extracellular C-loop, which is T in Aqp0a and N in Aqp0b. Similarly, P110 is the key residue required for adhesion in mammalian AQP0, highlighting the importance of residue 110 in AQP0 cell-to-cell adhesion in vertebrate lenses as well as the divergence of adhesive and water permeability functions in zebrafish duplicates.

Cross-Species Insights Into Genomic Adaptations to Hypoxia.

Over millions of years, vertebrate species populated vast environments spanning the globe. Among the most challenging habitats encountered were those with limited availability of oxygen, yet many animal and human populations inhabit and perform life cycle functions and/or daily activities in varying degrees of hypoxia today. Of particular interest are species that inhabit high-altitude niches, which experience chronic hypobaric hypoxia throughout their lives. Physiological and molecular aspects of adaptation to hypoxia have long been the focus of high-altitude populations and, within the past decade, genomic information has become increasingly accessible. These data provide an opportunity to search for common genetic signatures of selection across uniquely informative populations and thereby augment our understanding of the mechanisms underlying adaptations to hypoxia. In this review, we synthesize the available genomic findings across hypoxia-tolerant species to provide a comprehensive view of putatively hypoxia-adaptive genes and pathways. In many cases, adaptive signatures across species converge on the same genetic pathways or on genes themselves [i.e., the hypoxia inducible factor (HIF) pathway). However, specific variants thought to underlie function are distinct between species and populations, and, in most cases, the precise functional role of these genomic differences remains unknown. Efforts to standardize these findings and explore relationships between genotype and phenotype will provide important clues into the evolutionary and mechanistic bases of physiological adaptations to environmental hypoxia.

Seq-ing Higher Ground: Functional Investigation of Adaptive Variation Associated With High-Altitude Adaptation.

Human populations at high altitude exhibit both unique physiological responses and strong genetic signatures of selection thought to compensate for the decreased availability of oxygen in each breath of air. With the increased availability of genomic information from Tibetans, Andeans, and Ethiopians, much progress has been made to elucidate genetic adaptations to chronic hypoxia that have occurred throughout hundreds of generations in these populations. In this perspectives piece, we discuss specific hypoxia-pathway variants that have been identified in high-altitude populations and methods for functional investigation, which may be used to determine the underlying causal factors that afford adaptation to high altitude.

Optical development in the zebrafish eye lens.

The optics of the eye is the key to a functioning visual system. The exact nature of the correlation between ocular optics and eye development is not known because of the paucity of knowledge about the growth of a key optical element, the eye lens. The sophisticated optics of the lens and its gradient of refractive index provide the superior optical quality that the eye needs and which, it is thought, has a major influence on the development of proper visual function. The nature of a gradient refractive index lens, however, renders accurate measurements of its development difficult to make and has been the reason why the influence of lens growth on visual function remains largely unknown. Novel imaging techniques have made it possible to investigate growth of the eye lens in the zebrafish. This study shows measurements using X-ray Talbot interferometry of three-dimensional gradient index profiles in eye lenses of zebrafish from late larval to adult stages. The zebrafish lens shows evidence of a gradient of refractive index from the earliest stages measured and its growth suggests an apparent coincidence between periods of rapid increase in refractive index in the lens nucleus and increased expression of a particular crystallin protein group.

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity.

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for generation of transgenics for in vivo imaging. These unique capabilities make the zebrafish an ideal platform to study ocular lens development and physiology. Our recent findings that an Aquaporin-0, Aqp0a, is required for stability of the anterior lens suture, as well as for the shift of the lens nucleus to the lens center with age led us to develop tools especially suited to analyzing the properties of zebrafish lenses. Here we outline detailed methods for lens dissection that can be applied to both larval and adult lenses, to prepare them for histological analysis, immunohistochemistry and imaging. We focus on analysis of lens suture integrity and cortical cell morphology and compare data generated from dissected lenses with data obtained from in vivo imaging of lens morphology made possible by a novel transgenic zebrafish line with a genetically encoded fluorescent marker. Analysis of dissected lenses perpendicular to their optical axis allows quantification of the relative position of the lens nucleus along the anterior-posterior axis. Movement of the lens nucleus from an initial anterior position to the center is required for normal lens optics in adult zebrafish. Thus, a quantitative measure of lens nuclear position directly correlates with its optical properties.

Experimental and Simulation Studies of Aquaporin 0 Water Permeability and Regulation.

We begin with the history of aquaporin zero (AQP0), the most prevalent membrane protein in the eye lens, from the early days when AQP0 was a protein of unknown function known as Major Intrinsic Protein 26. We progress through its joining the aquaporin family as a water channel in its own right and discuss how regulation of its water permeability by pH and calcium came to be discovered experimentally and linked to lens homeostasis and development. We review the development of molecular dynamics (MD) simulations of lipid bilayers and membrane proteins, including aquaporins, with an emphasis on simulation studies that have elucidated the mechanisms of water conduction, selectivity, and proton exclusion by aquaporins in general. We also review experimental and theoretical progress toward understanding why mammalian AQP0 has a lower water permeability than other aquaporins and the evolution of our present understanding of how its water permeability is regulated by pH and calcium. Finally, we discuss how MD simulations have elucidated the nature of lipid interactions with AQP0.

Cooperativity and allostery in aquaporin 0 regulation by Ca2.

Aquaporin 0 (AQP0) is essential for eye lens homeostasis as is regulation of its water permeability by Ca2+, which occurs through interactions with calmodulin (CaM), but the underlying molecular mechanisms are not well understood. Here, we use molecular dynamics (MD) simulations on the microsecond timescale under an osmotic gradient to explicitly model water permeation through the AQP0 channel. To identify any structural features that are specific to water permeation through AQP0, we also performed simulations of aquaporin 1 (AQP1) and a pure mixed lipid bilayer under the same conditions. The relative single-channel water osmotic permeability coefficients (pf) calculated from all of our simulations are in reasonable agreement with experiment. Our simulations allowed us to characterize the dynamics of the key structural elements that modulate the diffusion of water single-files through the AQP0 and AQP1 pores. We find that CaM binding influences the collective dynamics of the whole AQP0 tetramer, promoting the closing of both the extracellular and intracellular gates by inducing cooperativity between neighboring subunits.

BFSP1 C-terminal domains released by post-translational processing events can alter significantly the calcium regulation of AQP0 water permeability.

BFSP1 (beaded filament structural protein 1, filensin) is a cytoskeletal protein expressed in the eye lens. It binds AQP0 in vitro and its C-terminal sequences have been suggested to regulate the water channel activity of AQP0. A myristoylated fragment from the C-terminus of BFSP1 was found in AQP0 enriched fractions. Here we identify BFSP1 as a substrate for caspase-mediated cleavage at several C-terminal sites including D433. Cleavage at D433 exposes a cryptic myristoylation sequence (434-440). We confirm that this sequence is an excellent substrate for both NMT1 and 2 (N-myristoyl transferase). Thus caspase cleavage may promote formation of myristoylated fragments derived from the BFSP1 C-terminus (G434-S665). Myristoylation at G434 is not required for membrane association. Biochemical fractionation and immunogold labeling confirmed that C-terminal BFSP1 fragments containing the myristoylation sequence colocalized with AQP0 in the same plasma membrane compartments of lens fibre cells. To determine the functional significance of the association of BFSP1 G434-S665 sequences with AQP0, we measured AQP0 water permeability in Xenopus oocytes co-transfected with transcripts expressing both AQP0 and various C-terminal domain fragments of BFSP1 generated by caspase cleavage. We found that different fragments dramatically alter the response of AQP0 to different concentrations of Ca2+. The complete C-terminal fragment (G434-S665) eliminates calcium regulation altogether. Shorter fragments can enhance regulation by elevated calcium or reverse the response, indicative of the regulatory potential of BFSP1 with respect to AQP0. In particular, elimination of the myristoylation site by the mutation G434A reverses the order of water permeability sensitivity to different Ca2+ concentrations.

Aqp0a Regulates Suture Stability in the Zebrafish Lens.

Purpose: To investigate the roles of Aquaporin 0a (Aqp0a) and Aqp0b in zebrafish lens development and transparency. Methods: CRISPR/Cas9 gene editing was used to generate loss-of-function deletions in zebrafish aqp0a and/or aqp0b. Wild type (WT), single mutant, and double mutant lenses were analyzed from embryonic to adult stages. Lens transparency, morphology, and growth were assessed. Immunohistochemistry was used to map protein localization as well as to assess tissue organization and distribution of cell nuclei. Results: aqp0a-/- and/or aqp0b-/- cause embryonic cataracts with variable penetrance. While lenses of single mutants of either gene recover transparency in juveniles, double mutants consistently form dense cataracts that persist in adults, indicating partially redundant functions. Double mutants also reveal redundant Aqp0 functions in lens growth. The nucleus of WT lenses moves from the anterior pole to the lens center with age. In aqp0a-/- mutants, the nucleus fails to centralize as it does in WT or aqp0b-/- lenses, and in double mutant lenses there is no consistent lens nuclear position. In addition, the anterior sutures of aqp0a-/-, but not aqp0b-/- mutants, are unstable resulting in failure of suture maintenance at older stages and anterior polar opacity. Conclusions. Zebrafish Aqp0s have partially redundant functions, but only Aqp0a promotes suture stability, which directs the lens nucleus to centralize, failure of which results in anterior polar opacity. These studies support the hypothesis that the two Aqp0s subfunctionalized during fish evolution and that Aqp0-dependent maintenance of the anterior suture is essential for lens transparency.

Late-occurring and Long-circulating Metabolites of GABAAα2,3 Receptor Modulator AZD7325 Involving Metabolic Cyclization and Aromatization: Relevance to MIST Analysis and Application for Patient Compliance.

AZD7325 [4-amino-8-(2-fluoro-6-methoxyphenyl)-N-propylcinnoline-3-carboxamide] is a selective GABAAα2,3 receptor modulator intended for the treatment of anxiety disorders through oral administration. An interesting metabolic cyclization and aromatization pathway led to the tricyclic core of M9, i.e., 2-ethyl-7-(2-fluoro-6-methoxyphenyl)pyrimido[5,4-c]cinnolin-4(3H)-one. Further oxidative metabolism generated M10 via O-demethylation and M42 via hydroxylation. An authentic standard of M9 was synthesized to confirm the novel structure of M9 and that of M10 and M42 by liver microsomal incubation of the M9 standard. Metabolites M9, M10, and M42 were either minor or absent in plasma samples after a single dose; however, all became major metabolites in human and preclinical animal plasma after repeated doses and circulated in humans longer than 48 hours after the end of seven repeated doses. The absence of these long circulating metabolites from selected patients' plasma samples was used to demonstrate patient noncompliance as the cause of unexpected lack of drug exposure in some patients during a Phase IIb outpatient clinical study. The observation of late-occurring and long-circulating metabolites demonstrates the need to collect plasma samples at steady state after repeated doses when conducting metabolite analysis for the safety testing of drug metabolites. All 12 major nonconjugate metabolites of AZD7325 observed in human plasma at steady state were also observed in dog, rat, and mouse plasma samples collected from 3-month safety studies and at higher exposures in the animals than humans. This eliminated concern about human specific or disproportional metabolites.

Calmodulin Gates Aquaporin 0 Permeability through a Positively Charged Cytoplasmic Loop.

Aquaporin 0 (AQP0), the major intrinsic protein of the eye lens, plays a vital role in maintaining lens clarity by facilitating the transport of water across lens fiber cell membranes. AQP0 reduces its osmotic water permeability constant (Pf) in response to increases in the external calcium concentration, an effect that is mediated by an interaction with the calcium-binding messenger protein, calmodulin (CaM), and phosphorylation of the CaM-binding site abolishes calcium sensitivity. Despite recent structural characterization of the AQP0-CaM complex, the mechanism by which CaM modulates AQP0 remains poorly understood. By combining atomistic molecular dynamics simulations and oocyte permeability assays, we conclude that serine phosphorylation of AQP0 does not inhibit CaM binding to the whole AQP0 protein. Instead, AQP0 phosphorylation alters calcium sensitivity by modifying the AQP0-CaM interaction interface, particularly at an arginine-rich loop that connects the fourth and fifth transmembrane helices. This previously unexplored loop, which sits outside of the canonical CaM-binding site on the AQP0 cytosolic face, mechanically couples CaM to the pore-gating residues of the second constriction site. We show that this allosteric loop is vital for CaM regulation of the channels, facilitating cooperativity between adjacent subunits and regulating factors such as serine phosphorylation. Similar allosteric interactions may also mediate CaM modulation of the properties of other CaM-regulated proteins.

Structures and Rotational Barriers of a Diiodobinorsnoutane: Energetic Preference for Gauche Conformation.

The diiodobinorsnoutane, bi(5-iodopentacyclo[4.3.0.0(2,4).0(3,8).0(5,7)]non-4-yl) (5), exists in a sterically hindered gauche conformation rather than an anti or an averaged (freely rotating) C2v structure. Density functional theory (DFT) predictions place the gauche conformation 11 kcal/mol more stable than the anti conformation with a barrier of 17 kcal/mol connecting the minima. These are consistent with variable-temperature NMR (17.1 ± 0.8 kcal/mol) estimates and X-ray analysis. Predictions of the torsional profiles of the yet-unsynthesized bromo-, chloro-, and fluoro- analogues show a progressive lowering of the barriers.