High Sox2 expression predicts taste lineage competency of lingual progenitors in vitro.

Taste buds on the tongue contain taste receptor cells (TRCs) that detect sweet, sour, salty, umami and bitter stimuli. Like non-taste lingual epithelium, TRCs are renewed from basal keratinocytes, many of which express the transcription factor SOX2. Genetic lineage tracing has shown that SOX2+ lingual progenitors give rise to both taste and non-taste lingual epithelium in the posterior circumvallate taste papilla (CVP) of mice. However, SOX2 is variably expressed among CVP epithelial cells, suggesting that their progenitor potential may vary. Using transcriptome analysis and organoid technology, we show that cells expressing SOX2 at higher levels are taste-competent progenitors that give rise to organoids comprising both TRCs and lingual epithelium. Conversely, organoids derived from progenitors that express SOX2 at lower levels are composed entirely of non-taste cells. Hedgehog and WNT/ß-catenin are required for taste homeostasis in adult mice. However, manipulation of hedgehog signaling in organoids has no impact on TRC differentiation or progenitor proliferation. By contrast, WNT/ß-catenin promotes TRC differentiation in vitro in organoids derived from higher but not low SOX2+ expressing progenitors.

Structural snapshots from the oxidative half-reaction of a copper amine oxidase: implications for O2 activation.

The mechanism of molecular oxygen activation is the subject of controversy in the copper amine oxidase family. At their active sites, copper amine oxidases contain both a mononuclear copper ion and a protein-derived quinone cofactor. Proposals have been made for the activation of molecular oxygen via both a Cu(II)-aminoquinol catalytic intermediate and a Cu(I)-semiquinone intermediate. Using protein crystallographic freeze-trapping methods under low oxygen conditions combined with single-crystal microspectrophotometry, we have determined structures corresponding to the iminoquinone and semiquinone forms of the enzyme. Methylamine reduction at acidic or neutral pH has revealed protonated and deprotonated forms of the iminoquinone that are accompanied by a bound oxygen species that is likely hydrogen peroxide. However, methylamine reduction at pH 8.5 has revealed a copper-ligated cofactor proposed to be the semiquinone form. A copper-ligated orientation, be it the sole identity of the semiquinone or not, blocks the oxygen-binding site, suggesting that accessibility of Cu(I) may be the basis of partitioning O2 activation between the aminoquinol and Cu(I).

Generation of purified nitric oxide from liquid N2O4 for the treatment of pulmonary hypertension in hypoxemic swine.

Inhaled nitric oxide (NO) selectively dilates pulmonary blood vessels, reduces pulmonary vascular resistance (PVR), and enhances ventilation-perfusion matching. However, existing modes of delivery for the treatment of chronic pulmonary hypertension are limited due to the bulk and heft of large tanks of compressed gas. We present a novel system for the generation of inhaled NO that is based on the initial heat-induced evaporation of liquid N2O4 into gas phase NO2 followed by the room temperature reduction to NO by an antioxidant, ascorbic acid cartridge just prior to inhalation. The biologic effects of NO generated from liquid N2O4 were compared with the effects of NO gas, on increased mean pulmonary artery pressure (mPAP) and PVR in a hypoxemic (FiO2 15%) swine model of pulmonary hypertension. We showed that NO concentration varied directly with the fixed cross sectional flow of the outflow aperture when studied at temperatures of 45, 47.5 and 50°C and was independent of the rate of heating. Liquid N2O4-sourced NO at 1, 5, and 20 ppm significantly reduced the elevated mPAP and PVR induced by experimental hypoxemia and was biologically indistinguishable from gas source NO in this model. These experiments show that it is feasible to generate highly purified NO gas from small volumes of liquid N2O4 at concentrations sufficient to lower mPAP and PVR in hypoxemic swine, and suggest that a miniaturized ambulatory system designed to generate biologically active NO from liquid N2O4 is achievable.

Respiratory viral infection promotes the awakening and outgrowth of dormant metastatic breast cancer cells in lungs.

Breast cancer is the second most common cancer globally. Most deaths from breast cancer are due to metastatic disease which often follows long periods of clinical dormancy1. Understanding the mechanisms that disrupt the quiescence of dormant disseminated cancer cells (DCC) is crucial for addressing metastatic progression. Infection with respiratory viruses (e.g. influenza or SARS-CoV-2) is common and triggers an inflammatory response locally and systemically2,3. Here we show that influenza virus infection leads to loss of the pro-dormancy mesenchymal phenotype in breast DCC in the lung, causing DCC proliferation within days of infection, and a greater than 100-fold expansion of carcinoma cells into metastatic lesions within two weeks. Such DCC phenotypic change and expansion is interleukin-6 (IL-6)-dependent. We further show that CD4 T cells are required for the maintenance of pulmonary metastatic burden post-influenza virus infection, in part through attenuation of CD8 cell responses in the lungs. Single-cell RNA-seq analyses reveal DCC-dependent impairment of T-cell activation in the lungs of infected mice. SARS-CoV-2 infected mice also showed increased breast DCC expansion in lungs post-infection. Expanding our findings to human observational data, we observed that cancer survivors contracting a SARS-CoV-2 infection have substantially increased risks of lung metastatic progression and cancer-related death compared to cancer survivors who did not. These discoveries underscore the significant impact of respiratory viral infections on the resurgence of metastatic cancer, offering novel insights into the interconnection between infectious diseases and cancer metastasis.

The precursor form of Hansenula polymorpha copper amine oxidase 1 in complex with CuI and CoII.

Copper amine oxidases (CAOs) catalyze the oxidative deamination of primary amines to their corresponding aldehydes, with the concomitant reduction of O(2) to H(2)O(2). Catalysis requires two cofactors: a mononuclear copper center and the cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ). TPQ is synthesized through the post-translational modification of an endogenous tyrosine residue and requires only oxygen and copper to proceed. TPQ biogenesis in CAO can be supported by alternate metals, albeit at decreased rates. A variety of factors are thought to contribute to the degree to which a metal can support TPQ biogenesis, including Lewis acidity, redox potential and electrostatic stabilization capability. The crystal structure has been solved of one of two characterized CAOs from the yeast Hansenula polymorpha (HPAO-1) in its metal-free (apo) form, which contains an unmodified precursor tyrosine residue instead of fully processed TPQ (HPAO-1 was denoted HPAO in the literature prior to 2010). Structures of apoHPAO-1 in complex with Cu(I) and Co(II) have also been solved, providing structural insight into metal binding prior to biogenesis.

Use of ultra pure nitric oxide generated by the reduction of nitrogen dioxide to reverse pulmonary hypertension in hypoxemic swine.

Inhaled nitric oxide (NO) has the capacity to selectively dilate pulmonary blood vessels, and thus enhance the matching of ventilation and perfusion, improve oxygenation and decrease pulmonary hypertension. However, existing approaches for the administration of inhaled NO are associated with the co-delivery of potentially toxic concentrations of nitrogen dioxide (NO2) due to the oxidation of NO in oxygen rich environments. We tested the ability of a novel methodology for generating highly purified NO through the reduction of NO2 by ascorbic acid to reverse pulmonary hypertension. In vitro testing demonstrated that the NO output of the novel device is ultrapure and free of NO2. An in vivo hypoxemic swine model of pulmonary hypertension was used to examine the dose response to NO in terms of pulmonary pressures and pulmonary vascular resistance. Pulmonary hypertension was induced by lowering inspired oxygen to 15% prior to treatment with inhaled ultra purified NO (1, 5, 20, and 80PPM). Hypoxemia increased mean pulmonary artery pressures and pulmonary vascular resistance. Inhaled ultra purified NO doses (down to 1PPM) show a marked reduction of hypoxemia-induced pulmonary vascular resistance. These experiments demonstrate a simple and robust method to generate purified inhaled NO that is devoid of NO2 and capable of reversing hypoxemia induced pulmonary hypertension.

Kinetic and structural analysis of substrate specificity in two copper amine oxidases from Hansenula polymorpha.

The structural underpinnings of enzyme substrate specificity are investigated in a pair of copper amine oxidases (CAOs) from Hansenula polymorpha (HPAO-1 and HPAO-2). The X-ray crystal structure (to 2.0 A resolution) and steady state kinetic data of the second copper amine oxidase (HPAO-2) are presented for comparison to those of HPAO-1. Despite 34% sequence identity and superimposable active site residues implicated in catalysis, the enzymes vary considerably in their substrate entry channel. The previously studied CAO, HPAO-1, has a narrow substrate channel. In contrast, HPAO-2 has a wide funnel-shaped substrate channel, which also contains a side chamber. In addition, there are a number of amino acid changes within the channels of HPAO-2 and HPAO-1 that may sterically impact the ability of substrates to form covalent Schiff base catalytic intermediates and to initiate chemistry. These differences can partially explain the greatly different substrate specificities as characterized by k(cat)/K(m) value differences. In HPAO-1, the k(cat)/K(m) for methylamine is 330-fold greater than for benzylamine, whereas in HPAO-2, it is benzylamine that is the better substrate by 750-fold. In HPAO-2, an inflated (D)k(cat)/K(m)(methylamine) in relation to (D)k(cat)/K(m)(benzylamine) indicates that proton abstraction has been impeded more than substrate release. In HPAO-1, (D)k(cat)/K(m)(S) changes little with the slow substrate and indicates a similar increase in the energy barriers that control both substrate binding and subsequent catalysis. In neither case is k(cat)/K(m) for the second substrate, O(2), significantly altered. These results reinforce the modular nature of the active sites of CAOs and show that multiple factors contribute to substrate specificity and catalytic efficiency. In HPAO-1, the enzyme with the smaller substrate binding pocket, both initial substrate binding and proton loss are affected by an increase in substrate size, while in HPAO-2, the enzyme with the larger substrate binding pocket, the rate of proton loss is differentially affected when a phenyl substituent in the substrate is reduced to the size of a methyl group.

Ozonesonde Quality Assurance: The JOSIE-SHADOZ (2017) Experience.

The ozonesonde is a small balloon-borne instrument that is attached to a standard radiosonde to measure profiles of ozone from the surface to 35 km with ~100-m vertical resolution. Ozonesonde data constitute a mainstay of satellite calibration and are used for climatologies and analysis of trends, especially in the lower stratosphere where satellites are most uncertain. The electrochemical-concentration cell (ECC) ozonesonde has been deployed at ~100 stations worldwide since the 1960s, with changes over time in manufacture and procedures, including details of the cell chemical solution and data processing. As a consequence, there are biases among different stations and discontinuities in profile time-series from individual site records. For 22 years the Jülich [Germany] Ozone Sonde Intercomparison Experiment (JOSIE) has periodically tested ozonesondes in a simulation chamber designated the World Calibration Centre for Ozonesondes (WCCOS) by WMO. In October-November 2017 a JOSIE campaign evaluated the sondes and procedures used in SHADOZ (Southern Hemisphere Additional Ozonesondes), a 14-station sonde network operating in the tropics and subtropics. A distinctive feature of the 2017 JOSIE was that the tests were conducted by operators from eight SHADOZ stations. Experimental protocols for the SHADOZ sonde configurations, which represent most of those in use today, are described, along with preliminary results. SHADOZ stations that follow WMO-recommended protocols record total ozone within 3% of the JOSIE reference instrument. These results and prior JOSIEs demonstrate that regular testing is essential to maintain best practices in ozonesonde operations and to ensure high-quality data for the satellite and ozone assessment communities.

Tropospheric ozonesonde profiles at long-term U.S. monitoring sites: 2. Links between Trinidad Head, CA, profile clusters and inland surface ozone measurements.

Much attention has been focused on the transport of ozone (O3) to the Western U.S., particularly given the latest revision of the National Ambient Air Quality Standard (NAAQS) to 70 parts per billion by volume (ppbv) of O3. This makes defining a "background" O3 amount essential so that the effects of stratosphere-to-troposphere exchange and pollution transport to this region can be quantified. To evaluate free-tropospheric and surface O3 in the Western U.S., we use self-organizing maps to cluster 18 years of ozonesonde profiles (940 samples) from Trinidad Head, CA. Two of nine O3 mixing ratio profile clusters exhibit thin laminae of high O3 above Trinidad Head. A third, consisting of background (~20 - 40 ppbv) O3, occurs in ~10% of profiles. The high O3 layers are located between 1 and 4 km amsl, and reside above a subsidence inversion associated with a northern location of the semi-permanent Pacific subtropical high. Several ancillary data sets are examined to identify the high O3 sources (reanalyses, trajectories, remotely-sensed carbon monoxide), but distinguishing chemical and stratospheric influences of the elevated O3 is difficult. There is marked and long-lasting impact of the elevated tropospheric O3 on high-altitude surface O3 monitors at Lassen Volcanic and Yosemite National Parks, and Truckee, CA. Days corresponding to the high O3 clusters exhibit hourly surface O3 anomalies of +5 - 10 ppbv compared to a climatology; the anomalies can last up to four days. The profile and surface O3 links demonstrate the importance of regular ozonesonde profiling at Trinidad Head.

Ozone Variability and Anomalies Observed during SENEX and SEAC4RS Campaigns in 2013.

Tropospheric ozone variability occurs because of multiple forcing factors including surface emission of ozone precursors, stratosphere-to-troposphere transport (STT), and meteorological conditions. Analyses of ozonesonde observations made in Huntsville, AL, during the peak ozone season (May to September) in 2013 indicate that ozone in the planetary boundary layer was significantly lower than the climatological average, especially in July and August when the Southeastern United States (SEUS) experienced unusually cool and wet weather. Because of a large influence of the lower stratosphere, however, upper-tropospheric ozone was mostly higher than climatology, especially from May to July. Tropospheric ozone anomalies were strongly anti-correlated (or correlated) with water vapor (or temperature) anomalies with a correlation coefficient mostly about 0.6 throughout the entire troposphere. The regression slopes between ozone and temperature anomalies for surface up to mid-troposphere are within 3.0-4.1 ppbv·K-1. The occurrence rates of tropospheric ozone laminae due to STT are ≥50% in May and June and about 30% in July, August and September suggesting that the stratospheric influence on free-tropospheric ozone could be significant during early summer. These STT laminae have a mean maximum ozone enhancement over the climatology of 52±33% (35±24 ppbv) with a mean minimum relative humidity of 2.3±1.7%.

Quantifying TOLNet Ozone Lidar Accuracy during the 2014 DISCOVER-AQ and FRAPPÉ Campaigns.

The Tropospheric Ozone Lidar Network (TOLNet) is a unique network of lidar systems that measure high-resolution atmospheric profiles of ozone. The accurate characterization of these lidars is necessary to determine the uniformity of cross-instrument calibration. From July to August 2014, three lidars, the TROPospheric OZone (TROPOZ) lidar, the Tunable Optical Profiler for Aerosol and oZone (TOPAZ) lidar, and the Langley Mobile Ozone Lidar (LMOL), of TOLNet participated in the "Deriving Information on Surface conditions from Column and Vertically Resolved Observations Relevant to Air Quality" (DISCOVER-AQ)mission and the "Front Range Air Pollution and Photochemistry Éxperiment" (FRAPPÉ)to measure ozone variations from the boundary layer to the top of the troposphere. This study presents the analysis of the intercomparison between the TROPOZ, TOPAZ, and LMOL lidars, along with comparisons between the lidars and other in situ ozone instruments including ozonesondes and a P-3B airborne chemiluminescence sensor. In terms of the range-resolving capability, the TOLNet lidars measured vertical ozone structures with an accuracy generally better than ±15% within the troposphere. Larger differences occur at some individual altitudes in both the near-field and far-field range of the lidar systems, largely as expected. In terms of column average, the TOLNet lidars measured ozone with an accuracy better than ±5% for both the intercomparison between the lidars and between the lidars and other instruments. These results indicate very good measurement accuracy for these three TOLNet lidars, making them suitable for use in air quality, satellite validation, and ozone modeling efforts.

Glutamatergic regulation of brain histamine neurons: In vivo microdialysis and electrophysiology studies in the rat.

The interactions between the glutamatergic and the histaminergic systems in the brain are not fully understood. Here we studied histamine release in the medial prefrontal cortex and the posterior hypothalamus-tuberomamillary nucleus (PH-TMN) using in vivo microdialysis and electrophysiological recordings of histaminergc neurons in the PH-TMN in vivo to further address the mechanistic details of these interactions. We demonstrated that histaminergic activity was regulated by group II metabotropic glutamate receptors (mGluR 2 and 3) using systemic dosing with mGluR 2/3 agonist and antagonists and an mGluR 2 positive allosteric modulator. These interactions likely occur via direct modulation of glutamate release in the PH-TMN. The importance of circadian rhythm for histamine release was also shown using microdialysis studies with mGluR 2/3 compounds under light and dark conditions. Based on histamine release studies with NMDA and ketamine, we propose the existence of two sub-populations of NMDA receptors where one subtype is located on histaminergic cell bodies in the PH-TMN and the second on GABA-ergic neurons projecting to the PH-TMN. These subpopulations could be distinguished based on function, notably opposing actions were seen on histamine release in the medial prefrontal cortex of the rat. In summary, this paper provides evidence that the histaminergic system is closely regulated by glutamate neurons in multiple ways. In addition, this interaction depends to a great extent on the activity state of the subject.

Calix[4]arenes linked to multiple bidentate N-donors: potential ligands for synthetic modeling of multinuclear metalloenzymes.

A series of calix[4]arenes incorporating three or four bidentate diamines or pyridylamines attached at the "upper rim" were synthesized via practical protocols. Proof of structure was obtained in one instance by X-ray crystallography. These molecules are designed for general use as ligands for the preparation of multinuclear active site models of metalloenzymes. [reaction: see text]

Exploring molecular oxygen pathways in Hansenula polymorpha copper-containing amine oxidase.

The accessibility of large substrates to buried enzymatic active sites is dependent upon the utilization of proteinaceous channels. The necessity of these channels in the case of small substrates is questionable because diffusion through the protein matrix is often assumed. Copper amine oxidases contain a buried protein-derived quinone cofactor and a mononuclear copper center that catalyze the conversion of two substrates, primary amines and molecular oxygen, to aldehydes and hydrogen peroxide, respectively. The nature of molecular oxygen migration to the active site in the enzyme from Hansenula polymorpha is explored using a combination of kinetic, x-ray crystallographic, and computational approaches. A crystal structure of H. polymorpha amine oxidase in complex with xenon gas, which serves as an experimental probe for molecular oxygen binding sites, reveals buried regions of the enzyme suitable for transient molecular oxygen occupation. Calculated O(2) free energy maps using copper amine oxidase crystal structures in the absence of xenon correspond well with later experimentally observed xenon sites in these systems, and allow the visualization of O(2) migration routes of differing probabilities within the protein matrix. Site-directed mutagenesis designed to block individual routes has little effect on overall k(cat)/K(m) (O(2)), supporting multiple dynamic pathways for molecular oxygen to reach the active site.

Copper-containing amine oxidases. Biogenesis and catalysis; a structural perspective.

This review will focus on how X-ray crystallographic studies of copper-containing amine oxidases have complemented the solution, kinetic, and spectroscopic research on this ubiquitous class of enzymes. These enzymes not only contain a copper ion at the active site, but also a unique organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), which is absolutely required for catalysis. Structural data have not only shed light on the catalytic mechanism of the enzyme, which converts primary amines, using molecular oxygen, to aldehydes, ammonia, and hydrogen peroxide, but also on biogenesis of the cofactor. The cofactor is derived from a tyrosine in the enzyme amino acid sequence and requires only the addition of copper(II) and molecular oxygen in a self-processing event.