Development of multiplexed orthogonal base editor (MOBE) systems.

Base editors (BEs) enable efficient, programmable installation of point mutations while avoiding the use of double-strand breaks. Simultaneous application of two or more different BEs, such as an adenine BE (which converts A·T base pairs to G·C) and a cytosine BE (which converts C·G base pairs to T·A), is not feasible because guide RNA crosstalk results in non-orthogonal editing, with all BEs modifying all target loci. Here we engineer both adenine BEs and cytosine BEs that can be orthogonally multiplexed by using RNA aptamer-coat protein systems to recruit the DNA-modifying enzymes directly to the guide RNAs. We generate four multiplexed orthogonal BE systems that enable rates of precise co-occurring edits of up to 7.1% in the same DNA strand without enrichment or selection strategies. The addition of a fluorescent enrichment strategy increases co-occurring edit rates up to 24.8% in human cells. These systems are compatible with expanded protospacer adjacent motif and high-fidelity Cas9 variants, function well in multiple cell types, have equivalent or reduced off-target propensities compared with their parental systems and can model disease-relevant point mutation combinations.

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.

Genomic Selection Signals in Andean Highlanders Reveal Adaptive Placental Metabolic Phenotypes That Are Disrupted in Preeclampsia.

BACKGROUND: The chronic hypoxia of high-altitude residence poses challenges for tissue oxygen supply and metabolism. Exposure to high altitude during pregnancy increases the incidence of hypertensive disorders of pregnancy and fetal growth restriction and alters placental metabolism. High-altitude ancestry protects against altitude-associated fetal growth restriction, indicating hypoxia tolerance that is genetic in nature. Yet, not all babies are protected and placental pathologies associated with fetal growth restriction occur in some Andean highlanders. METHODS: We examined placental metabolic function in 79 Andeans (18-45 years; 39 preeclamptic and 40 normotensive) living in La Paz, Bolivia (3600-4100 m) delivered by unlabored Cesarean section. Using a selection-nominated approach, we examined links between putatively adaptive genetic variation and phenotypes related to oxygen delivery or placental metabolism. RESULTS: Mitochondrial oxidative capacity was associated with fetal oxygen delivery in normotensive but not preeclamptic placenta and was also suppressed in term preeclamptic pregnancy. Maternal haplotypes in or within 200 kb of selection-nominated genes were associated with lower placental mitochondrial respiratory capacity (PTPRD [protein tyrosine phosphatase receptor-δ]), lower maternal plasma erythropoietin (CPT2 [carnitine palmitoyl transferase 2], proopiomelanocortin, and DNMT3 [DNA methyltransferase 3]), and lower VEGF (vascular endothelial growth factor) in umbilical venous plasma (TBX5 [T-box transcription factor 5]). A fetal haplotype within 200 kb of CPT2 was associated with increased placental mitochondrial complex II capacity, placental nitrotyrosine, and GLUT4 (glucose transporter type 4) protein expression. CONCLUSIONS: Our findings reveal novel associations between putatively adaptive gene regions and phenotypes linked to oxygen delivery and placental metabolic function in highland Andeans, suggesting that such effects may be of genetic origin. Our findings also demonstrate maladaptive metabolic mechanisms in the context of preeclampsia, including dysregulation of placental oxygen consumption.

Inflammation biomarkers in OSA, chronic obstructive pulmonary disease, and chronic obstructive pulmonary disease/OSA overlap syndrome.

STUDY OBJECTIVES: The coexistence of obstructive sleep apnea (OSA) and chronic obstructive pulmonary disease (COPD) in a single individual, also known as overlap syndrome (OVS), is associated with higher cardiovascular risk and mortality than either OSA or COPD alone. However, the underlying mechanisms remain unclear. We hypothesized that patients with OVS have elevated systemic inflammatory biomarkers relative to patients with either disease alone, which could explain greater cardiovascular risk observed in OVS. METHODS: We included 255 participants in the study, 55 with COPD alone, 100 with OSA alone, 50 with OVS, and 50 healthy controls. All participants underwent a home sleep study, spirometry, and a blood draw for high-sensitivity C-reactive protein and total blood count analysis. In a randomly selected subset of 186 participants, inflammatory protein profiling was performed using Bio-Rad Bio-Plex Pro Human Cytokine 27-Plex Assays. Biomarker level differences across groups were identified using a mixed linear model. RESULTS: Levels of interleukin 6 (IL-6), high-sensitivity C-reactive protein (hs-CRP), and granulocyte colony stimulating factor (G-CSF) were higher in participants with OVS and COPD compared with healthy controls and participants with OSA. Furthermore, participants with OVS had higher circulating levels of leukocytes and neutrophils than those with COPD, OSA, and controls. CONCLUSIONS: COPD and OVS are associated with higher systemic inflammation relative to OSA and healthy controls. This work proposes the potential utilization of interleukin 6, granulocyte colony stimulating factor, and high-sensitivity C-reactive protein as screening biomarkers for COPD in patients with OSA. Inflammatory pathways may not fully explain the higher cardiovascular risk observed in OVS, indicating the need for further investigation. CITATION: Sanchez-Azofra A, Gu W, Masso-Silva JA, et al. Inflammation biomarkers in OSA, chronic obstructive pulmonary disease, and chronic obstructive pulmonary disease/OSA overlap syndrome. J Clin Sleep Med. 2023;19(8):1447-1456.

Preserved peak exercise capacity in Andean highlanders with excessive erythrocytosis both before and after isovolumic hemodilution.

In chronic mountain sickness (CMS), increased blood oxygen (O2)-carrying capacity due to excessive erythrocytosis (EE, [Hb] ≥ 21 g/dL) could be offset, especially during exercise by both impaired cardiac output (Q̇t) and O2 diffusion limitation in lungs and muscle. We hypothesized that EE results in reduced peak V̇o2 despite increased blood O2-carrying capacity, and that isovolumic hemodilution (IVHD) improves exercise capacity. In 14 male residents of Cerro de Pasco, Peru (4,340 m), six with and eight without EE, we measured peak cycle-exercise capacity, V̇o2, Q̇t, arterial blood gas parameters, and (resting) blood volume. This was repeated for participants with EE after IVHD, reducing hematocrit by 20% (from 67% to 53%). From these data, we quantified the major O2 transport pathway components (ventilation, pulmonary alveolar-capillary diffusion, Q̇t, and blood-muscle mitochondria diffusion). Participants with EE had similar peak V̇o2, systemic O2 delivery, and O2 extraction as non-EE controls, however, with lower Q̇t and higher arterial [O2]. After IVHD, peak V̇o2 was preserved (but not enhanced), with lower O2 delivery (despite higher Q̇t) balanced by greater O2 extraction. The considerable variance in exercise capacity across the 14 individuals was explained essentially completely by differences in both pulmonary and muscle O2 diffusional conductances and not by any differences in ventilation, [Hb], nor Q̇t. In conclusion, EE does not result in lower peak V̇o2 in Andean males, and IVHD maintains, but does not enhance, exercise capacity.NEW & NOTEWORTHY Male Andean highlanders with and without excessive erythrocytosis (EE) have similar peak V̇o2 at 4,340 m, with higher arterial [O2] in EE and lower cardiac output (Q̇t), thus maintaining similar O2 delivery. Peak V̇o2 in participants with EE was unaffected by isovolumic hemodilution (hematocrit reduced from 67% to 53%), with lower O2 delivery balanced by slightly increased Q̇t and greater O2 extraction. Differences in lung and muscle diffusing capacity, and not hematocrit variation, accounted for essentially all interindividual variance in peak V̇o2.

Very Low Driving-Pressure Ventilation in Patients With COVID-19 Acute Respiratory Distress Syndrome on Extracorporeal Membrane Oxygenation: A Physiologic Study.

OBJECTIVES: To determine in patients with acute respiratory distress syndrome (ARDS) on venovenous extracorporeal membrane oxygenation (VV ECMO) whether reducing driving pressure (ΔP) would decrease plasma biomarkers of inflammation and lung injury (interleukin-6 [IL-6], IL-8, and the soluble receptor for advanced glycation end-products sRAGE). DESIGN: A single-center prospective physiologic study. SETTING: At a single university medical center. PARTICIPANTS: Adult patients with severe COVID-19 ARDS on VV ECMO. INTERVENTIONS: Participants on VV ECMO had the following biomarkers measured: (1) pre-ECMO with low-tidal-volume ventilation (LTVV), (2) post-ECMO with LTVV, (3) during low-driving-pressure ventilation (LDPV), (4) after 2 hours of very low driving-pressure ventilation (V-LDPV, main intervention ΔP = 1 cmH2O), and (5) 2 hours after returning to LDPV. MAIN MEASUREMENTS AND RESULTS: Twenty-six participants were enrolled; 21 underwent V-LDPV. There was no significant change in IL-6, IL-8, and sRAGE from LDPV to V-LDPV and from V-LDPV to LDPV. Only participants (9 of 21) with nonspontaneous breaths had significant change (p < 0.001) in their tidal volumes (Vt) (mean ± SD), 1.9 ± 0.5, 0.1 ± 0.2, and 2.0 ± 0.7 mL/kg predicted body weight (PBW). Participants with spontaneous breathing, Vt were unchanged-4.5 ± 3.1, 4.7 ± 3.1, and 5.6 ± 2.9 mL/kg PBW (p = 0.481 and p = 0.065, respectively). There was no relationship found when accounting for Vt changes and biomarkers. CONCLUSIONS: Biomarkers did not significantly change with decreased ΔPs or Vt changes during the first 24 hours post-ECMO. Despite deep sedation, reductions in Vt during V-LDPV were not reliably achieved due to spontaneous breaths. Thus, patients on VV ECMO for ARDS may have higher Vt (ie, transpulmonary pressure) than desired despite low ΔPs or Vt.

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.

Isovolemic hemodilution in chronic mountain sickness acutely worsens nocturnal oxygenation and sleep apnea severity.

STUDY OBJECTIVES: Chronic mountain sickness (CMS) is commonly observed among Andean and other highland populations. Sleep-disordered breathing (SDB) is highly prevalent at high altitude, and SDB and nocturnal hypoxemia have been observed in CMS. Phlebotomy is commonly performed to treat CMS, but it is unknown whether reducing hematocrit improves SDB. We hypothesized that isovolemic hemodilution (IVHD) in CMS would reduce SBD severity and improve sleep efficiency. METHODS: Six participants with CMS and 8 without CMS, all residents of Cerro de Pasco, Peru (altitude 4340 m), completed baseline nocturnal sleep studies. CMS participants then underwent IVHD, and nocturnal sleep studies were repeated 24-48 hours after IVHD. We analyzed sleep apnea severity, nocturnal oxygenation, and sleep quality in those with CMS relative to those without CMS, and the effects of IVHD in CMS participants. RESULTS: Participants with CMS did not have altered sleep architecture, sleep apnea severity, or nocturnal oxygenation relative to non-CMS participants. However, IVHD in CMS increased apnea-hypopnea index (40.9 ± 6.9 events/h to 61.5 ± 7.7 events/h, P = .009). IVHD increased oxyhemoglobin desaturation index (P = .008) and the percentage of sleep time spent with oxyhemoglobin saturation at or below 80% (P = .012). There was no effect of IVHD on sleep efficiency, arousal index, or sleep staging. CONCLUSIONS: In this cohort, CMS was not associated with worsened SDB or changes in sleep architecture. IVHD, a putative therapeutic option for participants with CMS, appears to worsen nocturnal oxygenation and SDB within 48 hours post-IVHD. CITATION: Sanchez-Azofra A, Villafuerte FC, DeYoung PN, et al. Isovolemic hemodilution in chronic mountain sickness acutely worsens nocturnal oxygenation and sleep apnea severity. J Clin Sleep Med. 2022;18(10):2423-2432.

Effects of mango and mint pod-based e-cigarette aerosol inhalation on inflammatory states of the brain, lung, heart, and colon in mice.

While health effects of conventional tobacco are well defined, data on vaping devices, including one of the most popular e-cigarettes which have high nicotine levels, are less established. Prior acute e-cigarette studies have demonstrated inflammatory and cardiopulmonary physiology changes while chronic studies have demonstrated extra-pulmonary effects, including neurotransmitter alterations in reward pathways. In this study we investigated the impact of inhalation of aerosols produced from pod-based, flavored e-cigarettes (JUUL) aerosols three times daily for 3 months on inflammatory markers in the brain, lung, heart, and colon. JUUL aerosol exposure induced upregulation of cytokine and chemokine gene expression and increased HMGB1 and RAGE in the nucleus accumbens in the central nervous system. Inflammatory gene expression increased in the colon, while gene expression was more broadly altered by e-cigarette aerosol inhalation in the lung. Cardiopulmonary inflammatory responses to acute lung injury with lipopolysaccharide were exacerbated in the heart. Flavor-specific findings were detected across these studies. Our findings suggest that daily e-cigarette use may cause neuroinflammation, which may contribute to behavioral changes and mood disorders. In addition, e-cigarette use may cause gut inflammation, which has been tied to poor systemic health, and cardiac inflammation, which leads to cardiovascular disease.

The use of e-cigarettes or 'vaping' has become widespread, particularly among young people and smokers trying to quit. One of the most popular e-cigarette brands is JUUL, which offers appealing flavors and a discrete design. Many e-cigarette users believe these products are healthier than traditional tobacco products. And while the harms of conventional tobacco products have been extensively researched, the short- and long-term health effects of e-cigarettes have not been well studied. There is even less information about the health impacts of newer products like JUUL. E-cigarettes made by JUUL are different relative to prior generations of e-cigarettes. The JUUL device uses disposable pods filled with nicotinic salts instead of nicotine. One JUUL pod contains as much nicotine as an entire pack of cigarettes (41.3 mg). These differences make studying the health effects of this product particularly important. Moshensky, Brand, Alhaddad et al. show that daily exposure to JUUL aerosols increases the expression of genes encoding inflammatory molecules in the brain, lung, heart and colon of mice. In the experiments, mice were exposed to JUUL mint and JUUL mango flavored aerosols for 20 minutes, 3 times a day, and for 4 and 12 weeks. The changes in inflammatory gene expression varied depending on the flavor. This suggests that the flavorings themselves contribute to the observed changes. The findings suggest that daily use of pod-based e-cigarettes or e-cigarettes containing high levels of nicotinic salts over months to years, may cause inflammation in various organs, increasing the risk of disease and poor health. This information may help individuals, clinicians and policymakers make more informed decisions about e-cigarettes. Further studies assessing the impact of these changes on long-term physical and mental health in humans are desperately needed. These should assess health effects across different e-cigarette types, flavors and duration of use.

Notch Signaling and Cross-Talk in Hypoxia: A Candidate Pathway for High-Altitude Adaptation.

Hypoxia triggers complex inter- and intracellular signals that regulate tissue oxygen (O2) homeostasis, adjusting convective O2 delivery and utilization (i.e., metabolism). Human populations have been exposed to high-altitude hypoxia for thousands of years and, in doing so, have undergone natural selection of multiple gene regions supporting adaptive traits. Some of the strongest selection signals identified in highland populations emanate from hypoxia-inducible factor (HIF) pathway genes. The HIF pathway is a master regulator of the cellular hypoxic response, but it is not the only regulatory pathway under positive selection. For instance, regions linked to the highly conserved Notch signaling pathway are also top targets, and this pathway is likely to play essential roles that confer hypoxia tolerance. Here, we explored the importance of the Notch pathway in mediating the cellular hypoxic response. We assessed transcriptional regulation of the Notch pathway, including close cross-talk with HIF signaling, and its involvement in the mediation of angiogenesis, cellular metabolism, inflammation, and oxidative stress, relating these functions to generational hypoxia adaptation.

Diagnostic Accuracy of the Progressive Collapsing Foot Deformity (PCFD) Classification.

BACKGROUND: A consensus group recently proposed the term progressive collapsing foot deformity (PCFD) and a new classification with 2 stages plus 5 classes to describe the complex array of flatfoot deformities. This study aimed to validate different diagnostic accuracy rates of the PCFD classification. METHODS: This was a survey-based study distributed among 13 foot and ankle fellowship programs for 3 groups of participants with varied experience in practice (group 1: fellows in training, group 2: surgeons in practice for 1-4 years, and group 3: surgeons in practice for ≥5 years). Each participant was asked to assign 20 different cases of flatfoot deformity to the appropriate classes and stages using the PCFD classification. The overall diagnostic accuracy, class, and stage diagnostic accuracy rates for the 20 cases were calculated first for the entire cohort and then compared among the 3 groups. The misdiagnosis rate for each class of deformity (the sum of overdiagnosis and underdiagnosis rates) of the entire cohort was calculated and compared with the other classes. Mean and standard evidence were used to describe numerical data. One-way analysis of variance was used to compare values among the 3 groups and the 5 classes. P <.05 was considered statistically significant. RESULTS: For the whole cohort, the overall diagnostic accuracy, class diagnostic accuracy, and stage diagnostic accuracy rates were 71.0%, 78.3%, and 81.7%, respectively There was a statistically significant difference between group 1 and 2, and group 1 and 3, in overall diagnostic accuracy and class diagnostic accuracy, with no significant difference among the 3 groups regarding stage diagnostic accuracy. Class B had a significantly higher overdiagnosis rate than the rest of the classes, whereas class D was significantly underdiagnosed than others. The misdiagnosis rates for classes A to E were 3.3%, 17.5%, 11.1%, 26.0%, and 3.7%, respectively. CONCLUSION: The PCFD classification showed overall fair diagnostic accuracy rates. The highest diagnostic accuracy was for "hindfoot valgus deformity" and "ankle instability." Further content validation of the PCFD classification is needed to examine the terminology and interpretation of those classes with low diagnostic accuracy including "midfoot/forefoot abduction deformity," "forefoot varus deformity/medial column instability," and "peritalar subluxation/dislocation."Level of Evidence: Level II, prospective comparative study.

High-Altitude Erythrocytosis: Mechanisms of Adaptive and Maladaptive Responses.

Erythrocytosis, or increased production of red blood cells, is one of the most well-documented physiological traits that varies within and among in high-altitude populations. Although a modest increase in blood O2-carrying capacity may be beneficial for life in highland environments, erythrocytosis can also become excessive and lead to maladaptive syndromes such as chronic mountain sickness (CMS).

Combined intermittent and sustained hypoxia is a novel and deleterious cardio-metabolic phenotype.

STUDY OBJECTIVES: Chronic obstructive pulmonary disease and obstructive sleep apnea overlap syndrome is associated with excess mortality, and outcomes are related to the degree of hypoxemia. People at high altitudes are susceptible to periodic breathing, and hypoxia at altitude is associated with cardio-metabolic dysfunction. Hypoxemia in these scenarios may be described as superimposed sustained hypoxia (SH) plus intermittent hypoxia (IH), or overlap hypoxia (OH), the effects of which have not been investigated. We aimed to characterize the cardio-metabolic consequences of OH in mice. METHODS: C57BL/6J mice were subjected to either SH (FiO2 = 0.10), IH (FiO2 = 0.21 for 12 h, and FiO2 oscillating between 0.21 and 0.06, 60 times/hour, for 12 h), OH (FiO2 = 0.13 for 12 h, and FiO2 oscillating between 0.13 and 0.06, 60 times/hour, for 12 h), or room air (RA), n = 8/group. Blood pressure and intraperitoneal glucose tolerance test were measured serially, and right ventricular systolic pressure (RVSP) was assessed. RESULTS: Systolic blood pressure transiently increased in IH and OH relative to SH and RA. RVSP did not increase in IH, but increased in SH and OH by 52% (p < .001) and 20% (p = .001). Glucose disposal worsened in IH and improved in SH, with no change in OH. Serum low- and very-low-density lipoproteins increased in OH and SH, but not in IH. Hepatic oxidative stress increased in all hypoxic groups, with the highest increase in OH. CONCLUSIONS: OH may represent a unique and deleterious cardio-metabolic stimulus, causing systemic and pulmonary hypertension, and without protective metabolic effects characteristic of SH.

Targeting Mitochondria and Metabolism in Acute Kidney Injury.

Acute kidney injury (AKI) significantly contributes to morbidity and mortality in critically ill patients. AKI is also an independent risk factor for the development and progression of chronic kidney disease. Effective therapeutic strategies for AKI are limited, but emerging evidence indicates a prominent role of mitochondrial dysfunction and altered tubular metabolism in the pathogenesis of AKI. Therefore, a comprehensive, mechanistic understanding of mitochondrial function and renal metabolism in AKI may lead to the development of novel therapies in AKI. In this review, we provide an overview of current state of research on the role of mitochondria and tubular metabolism in AKI from both pre-clinical and clinical studies. We also highlight current therapeutic strategies which target mitochondrial function and metabolic pathways for the treatment of AKI.

Upregulation of Calcium Homeostasis Modulators in Contractile-To-Proliferative Phenotypical Transition of Pulmonary Arterial Smooth Muscle Cells.

Excessive pulmonary artery (PA) smooth muscle cell (PASMC) proliferation and migration are implicated in the development of pathogenic pulmonary vascular remodeling characterized by concentric arterial wall thickening and arteriole muscularization in patients with pulmonary arterial hypertension (PAH). Pulmonary artery smooth muscle cell contractile-to-proliferative phenotypical transition is a process that promotes pulmonary vascular remodeling. A rise in cytosolic Ca2+ concentration [(Ca2+) cyt ] in PASMCs is a trigger for pulmonary vasoconstriction and a stimulus for pulmonary vascular remodeling. Here, we report that the calcium homeostasis modulator (CALHM), a Ca2+ (and ATP) channel that is allosterically regulated by voltage and extracellular Ca2+, is upregulated during the PASMC contractile-to-proliferative phenotypical transition. Protein expression of CALHM1/2 in primary cultured PASMCs in media containing serum and growth factors (proliferative PASMC) was significantly greater than in freshly isolated PA (contractile PASMC) from the same rat. Upregulated CALHM1/2 in proliferative PASMCs were associated with an increased ratio of pAKT/AKT and pmTOR/mTOR and an increased expression of the cell proliferation marker PCNA, whereas serum starvation and rapamycin significantly downregulated CALHM1/2. Furthermore, CALHM1/2 were upregulated in freshly isolated PA from rats with monocrotaline (MCT)-induced PH and in primary cultured PASMC from patients with PAH in comparison to normal controls. Intraperitoneal injection of CGP 37157 (0.6 mg/kg, q8H), a non-selective blocker of CALHM channels, partially reversed established experimental PH. These data suggest that CALHM upregulation is involved in PASMC contractile-to-proliferative phenotypical transition. Ca2+ influx through upregulated CALHM1/2 may play an important role in the transition of sustained vasoconstriction to excessive vascular remodeling in PAH or precapillary PH. Calcium homeostasis modulator could potentially be a target to develop novel therapies for PAH.

Halofuginone, a promising drug for treatment of pulmonary hypertension.

BACKGROUND AND PURPOSE: Halofuginone is a febrifugine derivative originally isolated from Chinese traditional herb Chang Shan that exhibits anti-hypertrophic, anti-fibrotic and anti-proliferative effects. We sought to investigate whether halofuginone induced pulmonary vasodilation and attenuates chronic hypoxia-induced pulmonary hypertension (HPH). EXPERIMENTAL APPROACH: Patch-clamp experiments were conducted to examine the activity of voltage-dependent Ca2+ channels (VDCCs) in pulmonary artery smooth muscle cells (PASMCs). Digital fluorescence microscopy was used to measure intracellular Ca2+ concentration in PASMCs. Isolated perfused and ventilated mouse lungs were used to measure pulmonary artery pressure (PAP). Mice exposed to hypoxia (10% O2 ) for 4 weeks were used as model of HPH for in vivo experiments. KEY RESULTS: Halofuginone increased voltage-gated K+ (Kv ) currents in PASMCs and K+ currents through KCNA5 channels in HEK cells transfected with KCNA5 gene. HF (0.03-1 µM) inhibited receptor-operated Ca2+ entry in HEK cells transfected with calcium-sensing receptor gene and attenuated store-operated Ca2+ entry in PASMCs. Acute (3-5 min) intrapulmonary application of halofuginone significantly and reversibly inhibited alveolar hypoxia-induced pulmonary vasoconstriction dose-dependently (0.1-10 µM). Intraperitoneal administration of halofuginone (0.3 mg·kg-1 , for 2 weeks) partly reversed established PH in mice. CONCLUSION AND IMPLICATIONS: Halofuginone is a potent pulmonary vasodilator by activating Kv channels and blocking VDCC and receptor-operated and store-operated Ca2+ channels in PASMCs. The therapeutic effect of halofuginone on experimental PH is probably due to combination of its vasodilator effects, via inhibition of excitation-contraction coupling and anti-proliferative effects, via inhibition of the PI3K/Akt/mTOR signalling pathway.

Impacts of Changes in Atmospheric O2 on Human Physiology. Is There a Basis for Concern?

Concern is often voiced over the ongoing loss of atmospheric O2. This loss, which is caused by fossil-fuel burning but also influenced by other processes, is likely to continue at least for the next few centuries. We argue that this loss is quite well understood, and the eventual decrease is bounded by the fossil-fuel resource base. Because the atmospheric O2 reservoir is so large, the predicted relative drop in O2 is very small even for extreme scenarios of future fossil-fuel usage which produce increases in atmospheric CO2 sufficient to cause catastrophic climate changes. At sea level, the ultimate drop in oxygen partial pressure will be less than 2.5 mm Hg out of a baseline of 159 mmHg. The drop by year 2300 is likely to be between 0.5 and 1.3 mmHg. The implications for normal human health is negligible because respiratory O2 consumption in healthy individuals is only weakly dependent on ambient partial pressure, especially at sea level. The impacts on top athlete performance, on disease, on reproduction, and on cognition, will also be very small. For people living at higher elevations, the implications of this loss will be even smaller, because of a counteracting increase in barometric pressure at higher elevations due to global warming.

Silent hypoxaemia in COVID-19 patients.

The clinical presentation of COVID-19 due to infection with SARS-CoV-2 is highly variable with the majority of patients having mild symptoms while others develop severe respiratory failure. The reason for this variability is unclear but is in critical need of investigation. Some COVID-19 patients have been labelled with 'happy hypoxia', in which patient complaints of dyspnoea and observable signs of respiratory distress are reported to be absent. Based on ongoing debate, we highlight key respiratory and neurological components that could underlie variation in the presentation of silent hypoxaemia and define priorities for subsequent investigation.