Triply Selective & Sequential Diversification at Csp 3: Expansion of Alkyl Germane Reactivity for C-C & C-Heteroatom Bond Formation.

We report the triply selective and sequential diversification of a single Csp 3 carbon carrying Cl, Bpin and GeEt3 for the modular and programmable construction of sp3-rich molecules. Various functionalizations of Csp 3-Cl and Csp 3-BPin (e.g. alkylation, arylation, homologation, amination, hydroxylation) were tolerated by the Csp 3-GeEt3 group. Moreover, the methodological repertoire of alkyl germane functionalization was significantly expanded beyond the hitherto known Giese addition and arylation to alkynylation, alkenylation, cyanation, halogenation, azidation, C-S bond formation as well as the first demonstration of stereo-selective functionalization of a Csp 3-[Ge] bond.

Synthesis of biolabile thioalkyl-protected phosphates from an easily accessible phosphotriester precursor.

Robust methods for the synthesis of mixed phosphotriesters are essential to accelerate the development of novel phosphate-containing bioactive molecules. To enable efficient cellular uptake, phosphate groups are commonly masked with biolabile protecting groups, such as S-acyl-2-thioethyl (SATE) esters, that are removed once the molecule is inside the cell. Typically, bis-SATE-protected phosphates are synthesised through phosphoramidite chemistry. This approach, however, suffers from issues with hazardous reagents and can give unreliable yields, especially when applied to the synthesis of sugar-1-phosphate derivatives as tools for metabolic oligosaccharide engineering. Here, we report the development of an alternative approach that gives access to bis-SATE phosphotriesters in two steps from an easy to synthesise tri(2-bromoethyl)phosphotriester precursor. We demonstrate the viability of this strategy using glucose as a model substrate, onto which a bis-SATE-protected phosphate is introduced either at the anomeric position or at C6. We show compability with various protecting groups and further explore the scope and limitations of the methodology on different substrates, including N-acetylhexosamine and amino acid derivatives. The new approach facilitates the synthesis of bis-SATE-protected phosphoprobes and prodrugs and provides a platform that can boost further studies aimed at exploring the unique potential of sugar phosphates as research tools.

A Multiplexing Activity-Based Protein-Profiling Platform for Dissection of a Native Bacterial Xyloglucan-Degrading System.

Bacteria and yeasts grow on biomass polysaccharides by expressing and excreting a complex array of glycoside hydrolase (GH) enzymes. Identification and annotation of such GH pools, which are valuable commodities for sustainable energy and chemistries, by conventional means (genomics, proteomics) are complicated, as primary sequence or secondary structure alignment with known active enzymes is not always predictive for new ones. Here we report a "low-tech", easy-to-use, and sensitive multiplexing activity-based protein-profiling platform to characterize the xyloglucan-degrading GH system excreted by the soil saprophyte, Cellvibrio japonicus, when grown on xyloglucan. A suite of activity-based probes bearing orthogonal fluorophores allows for the visualization of accessory exo-acting glycosidases, which are then identified using biotin-bearing probes. Substrate specificity of xyloglucanases is directly revealed by imbuing xyloglucan structural elements into bespoke activity-based probes. Our ABPP platform provides a highly useful tool to dissect xyloglucan-degrading systems from various sources and to rapidly select potentially useful ones. The observed specificity of the probes moreover bodes well for the study of other biomass polysaccharide-degrading systems, by modeling probe structures to those of desired substrates.

Structure-Uptake Relationship Studies of Oxazolidinones in Gram-Negative ESKAPE Pathogens.

The clinical success of linezolid for treating Gram-positive infections paired with the high conservation of bacterial ribosomes predicts that if oxazolidinones were engineered to accumulate in Gram-negative bacteria, then this pharmacological class would find broad utility in eradicating infections. Here, we report an investigative study of a strategically designed library of oxazolidinones to determine the effects of molecular structure on accumulation and biological activity. Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa strains with varying degrees of compromise (in efflux and outer membrane) were used to identify motifs that hinder permeation across the outer membrane and/or enhance efflux susceptibility broadly and specifically between species. The results illustrate that small changes in molecular structure are enough to overcome the efflux and/or permeation issues of this scaffold. Three oxazolidinone analogues (3e, 8d, and 8o) were identified that exhibit activity against all three pathogens assessed, a biological profile not observed for linezolid.

Continuous vs. single-shot adductor canal block for pain management following primary total knee arthroplasty: A systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: Adductor canal block (ACB) provides effective analgesia following total knee arthroplasty (TKA). This systematic review aimed to compare continuous and single-shot ACB for pain management and functional recovery following TKA. METHODS: MEDLINE, Embase, Web of Science and CENTRAL were searched up to January 5th, 2021. Included studies were randomized controlled trials comparing continuous to single-shot ACB for postoperative pain management after primary TKA. Primary outcome was opioid consumption and secondary outcomes were pain intensity, quadriceps strength, mobility, complications, and length of hospital stay. Meta-analyses were performed using random-effects method. RESULTS: Eleven studies (910 patients) were included in this systematic review. Continuous ACB did not significantly decrease opioid consumption (8 studies; 642 patients; MD=-5.67; 95% CI: -13.87 to 2.54; I2=13%) but significantly decreased 48hours pain scores (10 studies; 852 patients; MD=-0,73; 95% CI: -0.93 to -0.54; I2=54%). Continuous ACB improved quadriceps strength (4 studies; 250 patients; SMD=0.59; 95% CI: 0.16 to 1.03; I2=63%) but not Timed Up and Go test performance (5 studies; 524 patients; MD=3.99; 95% CI: -8.98 to 1.01; I2=89%). Type of ACB did not affect nausea and vomiting (5 studies; 357 patients; RR=1.23; 95% CI: 0.65 to 2.34; I2=0%) nor length of hospital stay (8 studies; 655 patients; MD=-0.13; 95% CI: -0.28 to 0.01; I2=36%). CONCLUSION: Continuous ACB did not reduce opioid consumption following TKA. Larger trials are required.

Return to Baseline Physical Activity After Bone-Patellar Tendon-Bone Versus Hamstring Tendon Autografts for Anterior Cruciate Ligament Reconstruction: A Systematic Review and Meta-analysis of Randomized Controlled Trials.

BACKGROUND: Bone-patellar tendon-bone (BPTB) and hamstring tendon (HT) autografts are the most utilized grafts for primary anterior cruciate ligament (ACL) reconstruction. The ability of a patient to return to a preinjury level of physical activity is a key consideration in choice of graft; the influence of graft choice on this metric lacks consensus in the literature. PURPOSE: To assess the effects of autograft choice (BPTB vs HT) for primary ACL reconstruction on return to baseline level of physical activity and/or sports participation. STUDY DESIGN: Meta-analysis; Level of evidence, 1. METHODS: A systematic review of randomized controlled trials comparing the use of BPTB and HT autografts for primary ACL reconstruction was conducted. The electronic databases EMBASE, MEDLINE, Cochrane CENTRAL, and Web of Science were comprehensively queried through September 23, 2019. The primary outcome was return to preinjury level of activity/sports. Secondary outcomes included knee stability testing (Lachman, KT-1000 arthrometer, and pivot-shift tests) and clinical subjective knee scores (Tegner, Cincinnati, International Knee Documentation Committee, and Lysholm). Two independent reviewers were involved in the screening of titles and abstracts, data extraction, and the assessment of risk of bias. Meta-analyses were performed respecting the Cochrane Handbook for Systematic Reviews of Intervention. RESULTS: A total of 29 studies (N = 3099 patients) were eligible for this review, of which 13 (n = 1029 patients) reported on return to baseline level of sports as an endpoint. The risk ratio (RR) of using BPTB vs HT on return to baseline sport level was 1.03 (0.91-1.17; P = .63). Absence of a positive pivot-shift test was the only secondary outcome, with a statistically significant RR of 0.66 (95% CI, 0.50-0.86) in favor of BPTB autografts (P = .002). CONCLUSION: In reviewing the current literature, no recommendation can be made on the optimal graft choice when using a return to baseline level of physical activity and/or sports participation as a primary metric.

Perchlorate exposure does not induce obesity or non-alcoholic fatty liver disease in zebrafish.

Perchlorate is a water-soluble contaminant found throughout the United States and many other countries. Perchlorate competitively inhibits iodide uptake at the sodium/iodide symporter, reducing thyroid hormone synthesis, which can lead to hypothyroidism and metabolic syndromes. Chronic perchlorate exposure induces hepatic steatosis and non-alcoholic fatty liver disease (NAFLD) in developing threespine stickleback (Gasterosteus aculeatus). We hypothesized that perchlorate would also induce zebrafish (Danio rerio) to develop phenotypes consistent with NAFLD and to accumulate lipids throughout the body. We exposed zebrafish embryos to four concentrations of perchlorate treated water (10µg/L, 10mg/L, 30mg/L, and 100mg/L) and a control (0mg/L) over the course of 133 days. Adult zebrafish were euthanized, sectioned, H&E and Oil Red-O stained, and analyzed for liver morphology and whole body lipid accumulation. In a representative section of the liver, we counted the number of lipid droplets and measured the area of each droplet and the total lipid area. For whole body analysis, we calculated the ratio of lipid area to body area within a section. We found that zebrafish exposed to perchlorate did not differ in any measured liver variables or whole body lipid area when compared to controls. In comparison to stickleback, we see a trend that control stickleback accumulate more lipids in their liver than do control zebrafish. Differences between the species indicate that obesogenic effects due to perchlorate exposure are not uniform across fish species, and likely are mediated by evolutionary differences related to geographic location. For example, high latitude fishes such as stickleback evolved to deposit lipid stores for over-winter survival, which may lead to more pronounced obesogenic effects than seen in tropical fish such as zebrafish.

Multimodal Imaging Mass Spectrometry to Identify Markers of Pulmonary Arterial Hypertension in Human Lung Tissue Using MALDI-ToF, ToF-SIMS, and Hybrid SIMS.

Pulmonary arterial hypertension (PAH) is a rare and deadly disease affecting roughly 15-60 people per million in Europe with a poorly understood pathology. There are currently no diagnostic tools for early detection nor does a curative treatment exist. The lipid composition of arteries in lung tissue samples from human PAH and control patients were investigated using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) combined with time-of-flight secondary ion mass spectrometry (TOF-SIMS) imaging. Using random forests as an IMS data analysis technique, it was possible to identify the ion at m/z 885.6 as a marker of PAH in human lung tissue. The m/z 885.6 ion intensity was shown to be significantly higher around diseased arteries and was confirmed to be a diacylglycerophosphoinositol PI(C18:0/C20:4) via MS/MS using a novel hybrid SIMS instrument. The discovery of a potential biomarker opens up new research avenues which may finally lead to a better understanding of the PAH pathology and highlights the vital role IMS can play in modern biomedical research.

Characterizing the selectivity of ER α-glucosidase inhibitors.

The endoplasmic reticulum (ER) contains both α-glucosidases and α-mannosidases which process the N-linked oligosaccharides of newly synthesized glycoproteins and thereby facilitate polypeptide folding and glycoprotein quality control. By acting as structural mimetics, iminosugars can selectively inhibit these ER localized α-glycosidases, preventing N-glycan trimming and providing a molecular basis for their therapeutic applications. In this study, we investigate the effects of a panel of nine iminosugars on the actions of ER luminal α-glucosidase I and α-glucosidase II. Using ER microsomes to recapitulate authentic protein N-glycosylation and oligosaccharide processing, we identify five iminosugars that selectively inhibit N-glycan trimming. Comparison of their inhibitory activities in ER microsomes against their effects on purified ER α-glucosidase II, suggests that 3,7a-diepi-alexine acts as a selective inhibitor of ER α-glucosidase I. The other active iminosugars all inhibit α-glucosidase II and, having identified 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) as the most effective of these compounds, we use in silico modeling to understand the molecular basis for this enhanced activity. Taken together, our work identifies the C-3 substituted pyrrolizidines casuarine and 3,7a-diepi-alexine as promising "second-generation" iminosugar inhibitors.

Abiotic synthesis of amino acids in the recesses of the oceanic lithosphere.

Abiotic hydrocarbons and carboxylic acids are known to be formed on Earth, notably during the hydrothermal alteration of mantle rocks. Although the abiotic formation of amino acids has been predicted both from experimental studies and thermodynamic calculations, its occurrence has not been demonstrated in terrestrial settings. Here, using a multimodal approach that combines high-resolution imaging techniques, we obtain evidence for the occurrence of aromatic amino acids formed abiotically and subsequently preserved at depth beneath the Atlantis Massif (Mid-Atlantic Ridge). These aromatic amino acids may have been formed through Friedel-Crafts reactions catalysed by an iron-rich saponite clay during a late alteration stage of the massif serpentinites. Demonstrating the potential of fluid-rock interactions in the oceanic lithosphere to generate amino acids abiotically gives credence to the hydrothermal theory for the origin of life, and may shed light on ancient metabolisms and the functioning of the present-day deep biosphere.

An Integrative Approach to Decipher the Chemical Antagonism between the Competing Endophytes Paraconiothyrium variabile and Bacillus subtilis.

An integrative approach combining traditional natural products chemistry, molecular networking, and mass spectrometry imaging has been undertaken to decipher the molecular dialogue between the fungus Paraconiothyrium variabile and the bacterium Bacillus subtilis, which were isolated as endophytes from the conifer Cephalotaxus harringtonia and are characterized by a strong and mutual antibiosis. From this study, we highlight that bacterial surfactins and a fungal tetronic acid are involved in such competition and that the fungus is able to hydrolyze surfactins to fight against the bacterial partner.

Human hypoxic pulmonary vasoconstriction is unaltered by 8 h of preceding isocapnic hyperoxia.

Exposure to sustained hypoxia of 8 h duration increases the sensitivity of the pulmonary vasculature to acute hypoxia, but it is not known whether exposure to sustained hyperoxia affects human pulmonary vascular control. We hypothesized that exposure to 8 h of hyperoxia would diminish the hypoxic pulmonary vasoconstriction (HPV) that occurs in response to a brief exposure to hypoxia. Eleven healthy volunteers were studied in a crossover protocol with randomization of order. Each volunteer was exposed to acute isocapnic hypoxia (end-tidal PO2 = 50 mmHg for 10 min) before and after 8 h of hyperoxia (end-tidal PO2 = 420 mmHg) or euoxia (end-tidal PO2 = 100 mmHg). After at least 3 days, each volunteer returned and was exposed to the other condition. Systolic pulmonary artery pressure (an index of HPV) and cardiac output were measured, using Doppler echocardiography. Eight hours of hyperoxia had no effect on HPV or the response of cardiac output to acute hypoxia.

Analysis of Chemotherapeutic Drug Delivery at the Single Cell Level Using 3D-MSI-TOF-SIMS.

In this work, we show the advantages of label-free, tridimensional mass spectrometry imaging using dual beam analysis (25 keV Bi3+) and depth profiling (20 keV with a distribution centered at Ar1500+) coupled to time of flight secondary ion mass spectrometry (3D-MSI-TOF-SIMS) for the study of A-172 human glioblastoma cell line treated with B-cell lymphoma 2 (Bcl-2) inhibitor ABT-737. The high spatial (~250 nm) and high mass resolution (m/Δm ~10,000) of TOF-SIMS permitted the localization and identification of the intact, unlabeled drug molecular ion (m/z 811.26 C42H44ClN6O5S2- [M - H]-) as well as characteristic fragment ions. We propose a novel approach based on the inspection of the drug secondary ion yield, which showed a good correlation with the drug concentration during cell treatment at therapeutic dosages (0-200 µM with 4 h incubation). Chemical maps using endogenous molecular markers showed that the ABT-737 is mainly localized in subsurface regions and absent in the nucleus. A semiquantitative workflow is proposed to account for the biological cell diversity based on the spatial distribution of endogenous molecular markers (e.g., nuclei and cytoplasm) and secondary ion confirmation based on the ratio of drug-specific fragments to molecular ion as a function of the therapeutic dosage. Graphical Abstract ᅟ.

Mapping Dicorynia guianensis Amsh. wood constituents by submicron resolution cluster-TOF-SIMS imaging.

The preparation of tropical wood surface sections for time-of-flight secondary ion mass spectrometry imaging is described, and the use of delayed extraction of secondary ions and its interest for the analysis of vegetal surface are shown. The method has been applied to the study by time-of-flight secondary ion mass spectrometry imaging with a resolution of less than one micron of a tropical wood species, Dicorynia guianensis, which is one of the most exploited wood in French Guiana for its durable heartwood. The heartwood of this species exhibits an economical importance, but its production is not controlled in forestry. Results show an increase of tryptamine from the transition zone and a concomitant decrease of inorganic ions and starch fragment ions. These experiments lead to a better understanding of the heartwood formation and the origin of the natural durability of D. guianensis. Copyright © 2016 John Wiley & Sons, Ltd.

Determinants of ventilation and pulmonary artery pressure during early acclimatization to hypoxia in humans.

Pulmonary ventilation and pulmonary arterial pressure both rise progressively during the first few hours of human acclimatization to hypoxia. These responses are highly variable between individuals, but the origin of this variability is unknown. Here, we sought to determine whether the variabilities between different measures of response to sustained hypoxia were related, which would suggest a common source of variability. Eighty volunteers individually underwent an 8-h isocapnic exposure to hypoxia (end-tidal P(O2)=55 Torr) in a purpose-built chamber. Measurements of ventilation and pulmonary artery systolic pressure (PASP) assessed by Doppler echocardiography were made during the exposure. Before and after the exposure, measurements were made of the ventilatory sensitivities to acute isocapnic hypoxia (G(pO2)) and hyperoxic hypercapnia, the latter divided into peripheral (G(pCO2)) and central (G(cCO2)) components. Substantial acclimatization was observed in both ventilation and PASP, the latter being 40% greater in women than men. No correlation was found between the magnitudes of pulmonary ventilatory and pulmonary vascular responses. For G(pO2), G(pCO2) and G(cC O2), but not the sensitivity of PASP to acute hypoxia, the magnitude of the increase during acclimatization was proportional to the pre-acclimatization value. Additionally, the change in G(pO2) during acclimatization to hypoxia correlated well with most other measures of ventilatory acclimatization. Of the initial measurements prior to sustained hypoxia, only G(pCO2) predicted the subsequent rise in ventilation and change in G(pO2) during acclimatization. We conclude that the magnitudes of the ventilatory and pulmonary vascular responses to sustained hypoxia are predominantly determined by different factors and that the initial G(pCO2) is a modest predictor of ventilatory acclimatization.

Time-of-flight secondary ion mass spectrometry imaging of biological samples with delayed extraction for high mass and high spatial resolutions.

RATIONALE: In Time-of-Flight Secondary Ion Mass Spectrometry (TOF-SIMS), pulsed and focused primary ion beams enable mass spectrometry imaging, a method which is particularly useful to map various small molecules such as lipids at the surface of biological samples. When using TOF-SIMS instruments, the focusing modes of the primary ion beam delivered by liquid metal ion guns can provide either a mass resolution of several thousand or a sub-µm lateral resolution, but the combination of both is generally not possible. METHODS: With a TOF-SIMS setup, a delayed extraction applied to secondary ions has been studied extensively on rat cerebellum sections in order to compensate for the effect of long primary ion bunches. RESULTS: The use of a delayed extraction has been proven to be an efficient solution leading to unique features, i.e. a mass resolution up to 10000 at m/z 385.4 combined with a lateral resolution of about 400 nm. Simulations of ion trajectories confirm the experimental determination of optimal delayed extraction and allow understanding of the behavior of ions as a function of their mass-to-charge ratio. CONCLUSIONS: Although the use of a delayed extraction has been well known for many years and is very popular in MALDI, it is much less used in TOF-SIMS. Its full characterization now enables secondary ion images to be recorded in a single run with a submicron spatial resolution and with a mass resolution of several thousand. This improvement is very useful when analyzing lipids on tissue sections, or rare, precious, or very small size samples.

Contrasting effects of ascorbate and iron on the pulmonary vascular response to hypoxia in humans.

Hypoxia causes an increase in pulmonary artery pressure. Gene expression controlled by the hypoxia-inducible factor (HIF) family of transcription factors plays an important role in the underlying pulmonary vascular responses. The hydroxylase enzymes that regulate HIF are highly sensitive to varying iron availability, and iron status modifies the pulmonary vascular response to hypoxia, possibly through its effects on HIF. Ascorbate (vitamin C) affects HIF hydroxylation in a similar manner to iron and may therefore have similar pulmonary effects. This study investigated the possible contribution of ascorbate availability to hypoxic pulmonary vasoconstriction in humans. Seven healthy volunteers undertook a randomized, controlled, double-blind, crossover protocol which studied the effects of high-dose intravenous ascorbic acid (total 6 g) on the pulmonary vascular response to 5 h of sustained hypoxia. Systolic pulmonary artery pressure (SPAP) was assessed during hypoxia by Doppler echocardiography. Results were compared with corresponding data from a similar study investigating the effect of intravenous iron, in which SPAP was measured in seven healthy volunteers during 8 h of sustained hypoxia. Consistent with other studies, iron supplementation profoundly inhibited hypoxic pulmonary vasoconstriction (P < 0.001). In contrast, supraphysiological supplementation of ascorbate did not affect the increase in pulmonary artery pressure induced by several hours of hypoxia (P = 0.61). We conclude that ascorbate does not interact with hypoxia and the pulmonary circulation in the same manner as iron. Whether the effects of iron are HIF-mediated remains unknown, and the extent to which ascorbate contributes to HIF hydroxylation in vivo is also unclear.

Multifunctional structural energy storage composite supercapacitors.

This paper addresses the challenge of producing multifunctional composites that can simultaneously carry mechanical loads whilst storing (and delivering) electrical energy. The embodiment is a structural supercapacitor built around laminated structural carbon fibre (CF) fabrics. Each cell consists of two modified structural CF fabric electrodes, separated by a structural glass fibre fabric or polymer membrane, infused with a multifunctional polymeric electrolyte. Rather than using conventional activated carbon fibres, structural carbon fibres were treated to produce a mechanically robust, high surface area material, using a variety of methods, including direct etching, carbon nanotube sizing, and carbon nanotube in situ growth. One of the most promising approaches is to integrate a porous bicontinuous monolithic carbon aerogel (CAG) throughout the matrix. This nanostructured matrix both provides a dramatic increase in active surface area of the electrodes, and has the potential to address mechanical issues associated with matrix-dominated failures. The effect of the initial reaction mixture composition is assessed for both the CAG modified carbon fibre electrodes and resulting devices. A low temperature CAG modification of carbon fibres was evaluated using poly(3,4-ethylenedioxythiophene) (PEDOT) to enhance the electrochemical performance. For the multifunctional structural electrolyte, simple crosslinked gels have been replaced with bicontinuous structural epoxy-ionic liquid hybrids that offer a much better balance between the conflicting demands of rigidity and molecular motion. The formation of both aerogel precursors and the multifunctional electrolyte are described, including the influence of key components, and the defining characteristics of the products. Working structural supercapacitor composite prototypes have been produced and characterised electrochemically. The effect of introducing the necessary multifunctional resin on the mechanical properties has also been assessed. Larger scale demonstrators have been produced including a full size car boot/trunk lid.

Tibetans living at sea level have a hyporesponsive hypoxia-inducible factor system and blunted physiological responses to hypoxia.

Tibetan natives have lived on the Tibetan plateau (altitude ∼ 4,000 m) for at least 25,000 years, and as such they are adapted to life and reproduction in a hypoxic environment. Recent studies have identified two genetic loci, EGLN1 and EPAS1, that have undergone natural selection in Tibetans, and further demonstrated an association of EGLN1/EPAS1 genotype with hemoglobin concentration. Both genes encode major components of the hypoxia-inducible factor (HIF) transcriptional pathway, which coordinates an organism's response to hypoxia. Patients living at sea level with genetic disease of the HIF pathway have characteristic phenotypes at both the integrative-physiology and cellular level. We sought to test the hypothesis that natural selection to hypoxia within Tibetans results in related phenotypic differences. We compared Tibetans living at sea level with Han Chinese, who are Tibetans' most closely related major ethnic group. We found that Tibetans had a lower hemoglobin concentration, a higher pulmonary ventilation relative to metabolism, and blunted pulmonary vascular responses to both acute (minutes) and sustained (8 h) hypoxia. At the cellular level, the relative expression and hypoxic induction of HIF-regulated genes were significantly lower in peripheral blood lymphocytes from Tibetans compared with Han Chinese. Within the Tibetans, we found a significant correlation between both EPAS1 and EGLN1 genotype and the induction of erythropoietin by hypoxia. In conclusion, this study provides further evidence that Tibetans respond less vigorously to hypoxic challenge. This is evident at sea level and, at least in part, appears to arise from a hyporesponsive HIF transcriptional system.