Breath analysis by ultra-sensitive broadband laser spectroscopy detects SARS-CoV-2 infection

Rapid testing is essential to fighting pandemics such as COVID-19, the disease caused by the SARS-CoV-2 virus. Exhaled human breath contains multiple volatile molecules providing powerful potential for non-invasive diagnosis of diverse medical conditions. We investigated breath detection of SARS-CoV-2 infection using cavity-enhanced direct frequency comb spectroscopy (CE-DFCS), a state-of-the-art laser spectroscopic technique capable of a real-time massive collection of broadband molecular absorption features at ro-vibrational quantum state resolution and at parts-per-trillion volume detection sensitivity. Using a total of 170 individual breath samples (83 positive and 87 negative with SARS-CoV-2 based on Reverse Transcription Polymerase Chain Reaction tests), we report excellent discrimination capability for SARS-CoV-2 infection with an area under the Receiver-Operating-Characteristics curve of 0.849(4). Our results support the development of CE-DFCS as an alternative, rapid, non-invasive test for COVID-19 and highlight its remarkable potential for optical diagnoses of diverse biological conditions and disease states

HIF1A-Dependent Induction of Alveolar Epithelial PFKFB3 Dampens Acute Lung Injury

Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us toward a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxP/loxP SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid instillation. Studies in genetic models linked Pfkfb3 expression and function to Hif1a. Not only did intratracheal pyruvate instillation reconstitute Pfkfb3loxP/loxP or Hif1aloxP/loxP SPC-ER-Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies demonstrated increased PFKFB3 staining in injured lungs and colocalized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counterbalancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI

DUSP5-mediated inhibition of smooth muscle cell proliferation suppresses pulmonary hypertension and right ventricular hypertrophy

Pulmonary hypertension (PH) is associated with structural remodeling of pulmonary arteries (PAs) because of excessive proliferation of fibroblasts, endothelial cells, and smooth muscle cells (SMCs). The peptide hormone angiotensin II (ANG II) contributes to pulmonary vascular remodeling, in part, through its ability to trigger extracellular signal-regulated kinase (ERK1/2) activation. Here, we demonstrate that the ERK1/2 phosphatase, dual-specificity phosphatase 5 (DUSP5), functions as a negative regulator of ANG II-mediated SMC proliferation and PH. In contrast to wild-type controls, Dusp5 null mice infused with ANG II developed PH and right ventricular (RV) hypertrophy. PH in Dusp5 null mice was associated with thickening of the medial layer of small PAs, suggesting an in vivo role for DUSP5 as a negative regulator of ANG II-dependent SMC proliferation. Consistent with this, overexpression of DUSP5 blocked ANG II-mediated proliferation of cultured human pulmonary artery SMCs (hPASMCs) derived from patients with idiopathic PH or from failed donor controls. Collectively, the data support a role for DUSP5 as a feedback inhibitor of ANG II-mediated ERK signaling and PASMC proliferation and suggest that disruption of this circuit leads to adverse cardiopulmonary remodeling. NEW & NOTEWORTHY Dual-specificity phosphatases (DUSPs) serve critical roles in the regulation of mitogen-activated protein kinases, but their functions in the cardiovascular system remain poorly defined. Here, we provide evidence that DUSP5, which resides in the nucleus and specifically dephosphorylates extracellular signal-regulated kinase (ERK1/2), blocks pulmonary vascular smooth muscle cell proliferation. In response to angiotensin II infusion, mice lacking DUSP5 develop pulmonary hypertension and right ventricular cardiac hypertrophy. These findings illustrate DUSP5-mediated suppression of ERK signaling in the lungs as a protective mechanism

A Protocol for Fabrication and on-Chip Cell Culture to Recreate PAH-Afflicted Pulmonary Artery on a Microfluidic Device

Pulmonary arterial hypertension (PAH) is a rare pulmonary vascular disease that affects people of all ethnic origins and age groups including newborns. In PAH, pulmonary arteries and arterioles undergo a series of pathological changes including remodeling of the entire pulmonary vasculatures and extracellular matrices, mis-localized growth of pulmonary arterial cells, and development of glomeruloid-like lesions called plexiform lesions. Traditionally, various animal and cellular models have been used to understand PAH pathophysiology, investigate sex-disparity in PAH and monitor therapeutic efficacy of PAH medications. However, traditional models can only partially capture various pathological features of PAH, and they are not adaptable to combinatorial study design for deciphering intricately intertwined complex cellular processes implicated in PAH pathogenesis. While many microfluidic chip-based models are currently available for major diseases, no such disease-on-a-device model is available for PAH, an under investigated disease. In the absence of any chip-based models of PAH, we recently proposed a five-channel polydimethylsiloxane (PDMS)-based microfluidic device that can emulate major pathological features of PAH. However, our proposed model can make a bigger impact on the PAH field only when the larger scientific community engaged in PAH research can fabricate the device and develop the model in their laboratory settings. With this goal in mind, in this study, we have described the detailed methodologies for fabrication and development of the PAH chip model including a thorough explanation of scientific principles for various steps for chip fabrication, a detailed list of reagents, tools and equipment along with their source and catalogue numbers, description of laboratory setup, and cautionary notes. Finally, we explained the methodologies for on-chip cell seeding and application of this model for studying PAH pathophysiology. We believe investigators with little or no training in microfluidic chip fabrication can fabricate this eminently novel PAH-on-a-chip model. As such, this study will have a far-reaching impact on understanding PAH pathophysiology, unravelling the biological mystery associated with sexual dimorphism in PAH, and developing PAH therapy based on patient sex and age

Neonatal Selenium Deficiency Decreases Selenoproteins in the Lung and Impairs Pulmonary Alveolar Development

Decreased selenium (Se) levels during childhood and infancy are associated with worse respiratory health. Se is biologically active after incorporation into Se-containing antioxidant enzymes (AOE) and proteins. It is unknown how decreased maternal Se during pregnancy and lactation impacts neonatal pulmonary selenoproteins, growth, and lung development. Using a model of neonatal Se deficiency that limits Se intake to the dam during pregnancy and lactation, we evaluated which neonatal pulmonary selenoproteins are decreased in both the saccular (postnatal day 0, P0) and early alveolar (postnatal day 7, P7) stages of lung development. We found that Se deficient (SeD) pups weigh less and exhibit impaired alveolar development compared to Se sufficient (SeS) pups at P7. The activity levels of glutathione peroxidase (GPx) and thioredoxin reductase (Txnrd) were decreased at P0 and P7 in SeD lungs compared to SeS lungs. Protein content of GPx1, GPx3 and Txnrd1 were decreased in SeD lungs at P0 and P7, whereas Txnrd2 content was unaltered compared to SeS controls. The expression of NRF-2 dependent genes and several non-Se containing AOE were similar between SeS and SeD lungs. SeD lungs exhibited a decrease in selenoprotein N, an endoplasmic reticulum protein implicated in alveolar development, at both time points. We conclude that exposure to Se deficiency during pregnancy and lactation impairs weight gain and lung growth in offspring. Our data identify multiple selenoproteins in the neonatal lung that are vulnerable to decreased Se intake, which may impact oxidative stress and cell signaling under physiologic conditions as well as after oxidative stressors