New insights into the natural history of bronchopulmonary dysplasia from proteomics and multiplexed immunohistochemistry.

Bronchopulmonary dysplasia (BPD) is a disease of prematurity related to the arrest of normal lung development. The objective of this study was to better understand how proteome modulation and cell-type shifts are noted in BPD pathology. Pediatric human donors aged 1-3 yr were classified based on history of prematurity and histopathology consistent with "healed" BPD (hBPD, n = 3) and "established" BPD (eBPD, n = 3) compared with respective full-term born (n = 6) age-matched term controls. Proteins were quantified by tandem mass spectroscopy with selected Western blot validations. Multiplexed immunofluorescence (MxIF) microscopy was performed on lung sections to enumerate cell types. Protein abundances and MxIF cell frequencies were compared among groups using ANOVA. Cell type and ontology enrichment were performed using an in-house tool and/or EnrichR. Proteomics detected 5,746 unique proteins, 186 upregulated and 534 downregulated, in eBPD versus control with fewer proteins differentially abundant in hBPD as compared with age-matched term controls. Cell-type enrichment suggested a loss of alveolar type I, alveolar type II, endothelial/capillary, and lymphatics, and an increase in smooth muscle and fibroblasts consistent with MxIF. Histochemistry and Western analysis also supported predictions of upregulated ferroptosis in eBPD versus control. Finally, several extracellular matrix components mapping to angiogenesis signaling pathways were altered in eBPD. Despite clear parsing by protein abundance, comparative MxIF analysis confirms phenotypic variability in BPD. This work provides the first demonstration of tandem mass spectrometry and multiplexed molecular analysis of human lung tissue for critical elucidation of BPD trajectory-defining factors into early childhood.NEW & NOTEWORTHY We provide new insights into the natural history of bronchopulmonary dysplasia in donor human lungs after the neonatal intensive care unit hospitalization. This study provides new insights into how the proteome and histopathology of BPD changes in early childhood, uncovering novel pathways for future study.

Low-dose hyperoxia primes airways for fibrosis in mice after influenza A infection.

It is well known that supplemental oxygen used to treat preterm infants in respiratory distress is associated with permanently disrupting lung development and the host response to influenza A virus (IAV). However, many infants who go home with normally functioning lungs are also at risk for hyperreactivity after a respiratory viral infection. We recently reported a new, low-dose hyperoxia mouse model (40% for 8 days; 40×8) that causes a transient change in lung function that resolves, rendering 40×8 adult animals functionally indistinguishable from room air controls. Here we report that when infected with IAV, 40×8 mice display an early transient activation of TGFß signaling and later airway hyperreactivity associated with peribronchial inflammation (profibrotic macrophages) and fibrosis compared with infected room air controls, suggesting neonatal oxygen induced hidden molecular changes that prime the lung for hyperreactive airways disease. Although searching for potential activators of TGFß signaling, we discovered that thrombospondin-1 (TSP-1) is elevated in naïve 40×8 mice compared with controls and localized to lung megakaryocytes and platelets before and during IAV infection. Elevated TSP-1 was also identified in human autopsy samples of former preterm infants with bronchopulmonary dysplasia. These findings reveal how low doses of oxygen that do not durably change lung function may prime it for hyperreactive airways disease by changing expression of genes, such as TSP-1, thus helping to explain why former preterm infants who have normal lung function are susceptible to airway obstruction and increased morbidity after viral infection.

Collagen VI Deficiency Results in Structural Abnormalities in the Mouse Lung.

Collagen VI (COL6) is known for its role in a spectrum of congenital muscular dystrophies, which are often accompanied by respiratory dysfunction. However, little is known regarding the function of COL6 in the lung. We confirmed the presence of COL6 throughout the basement membrane region of mouse lung tissue. Lung structure and organization were studied in a previously described Col6a1-/- mouse, which does not produce detectable COL6 in the lung. The Col6a1-/- mouse displayed histopathologic alveolar and airway abnormalities. The airspaces of Col6a1-/- lungs appeared simplified, with larger (29%; P < 0.01) and fewer (31%; P < 0.001) alveoli. These airspace abnormalities included reduced isolectin B4+ alveolar capillaries and surfactant protein C-positive alveolar epithelial type-II cells. Alterations in lung function consistent with these histopathologic changes were evident. Col6a1-/- mice also displayed multiple airway changes, including increased branching (59%; P < 0.001), increased mucosal thickness (34%; P < 0.001), and increased epithelial cell density (13%; P < 0.001). Comprehensive transcriptome analysis revealed that the loss of COL6 is associated with reductions in integrin-paxillin-phosphatidylinositol 3-kinase signaling in vivo. In vitro, COL6 promoted steady-state phosphorylated paxillin levels and reduced cell density (16% to 28%; P < 0.05) at confluence. Inhibition of phosphatidylinositol 3-kinase, or its downstream effectors, resulted in increased cell density to a level similar to that seen on matrices lacking COL6.

Pulmonary mechanics and structural lung development after neonatal hyperoxia in mice.

BACKGROUND: Supplemental oxygen exposure administered to premature infants is associated with chronic lung disease and abnormal pulmonary function. This study used mild (40%), moderate (60%), and severe (80%) oxygen to determine how hyperoxia-induced changes in lung structure impact pulmonary mechanics in mice. METHODS: C57BL/6J mice were exposed to room air or hyperoxia from birth through postnatal day 8. Baseline pulmonary function and methacholine challenge was assessed at 4 and 8 weeks of age, accompanied by immunohistochemical assessments of both airway (smooth muscle, tethering) and alveolar (simplification, elastin deposition) structure. RESULTS: Mild/moderate hyperoxia increased baseline airway resistance (40% only) and airway hyperreactivity (40 and 60%) at 4 weeks accompanied by increased airway smooth muscle deposition, which resolved at 8 weeks. Severe hyperoxia increased baseline compliance, baseline resistance, and total elastin/surface area ratio without increasing airway hyperreactivity, and was accompanied by increased alveolar simplification, decreased airway tethering, and changes in elastin distribution at both time points. CONCLUSIONS: Mild to moderate hyperoxia causes changes in airway function and airway hyperreactivity with minimal parenchymal response. Severe hyperoxia drives its functional changes through alveolar simplification, airway tethering, and elastin redistribution. These differential responses can be leveraged to further develop hyperoxia mouse models.

Transglutaminase 2 knockout mice are protected from bleomycin-induced lung fibrosis with preserved lung function and reduced metabolic derangements.

Pulmonary fibrosis is an interstitial scarring disease of the lung characterized by poor prognosis and limited treatment options. Tissue transglutaminase 2 (TG2) is believed to promote lung fibrosis by crosslinking extracellular matrix components and activating latent TGFß. This study assessed physiologic pulmonary function and metabolic alterations in the mouse bleomycin model with TG2 genetic deletion. TG2-deficient mice demonstrated attenuated the fibrosis and preservation of lung function, with significant reduction in elastance and increases in compliance and inspiratory capacity compared to control mice treated with bleomycin. Bleomycin induced metabolic changes in the mouse lung that were consistent with increased aerobic glycolysis, including increased expression of lactate dehydrogenase A and increased production of lactate, as well as increased glutamine, glutamate, and aspartate. TG2-deficient mice treated with bleomycin exhibited similar metabolic changes but with reduced magnitude. Our results demonstrate that TG2 is required for a typical fibrosis response to injury. In the absence of TG2, the fibrotic response is biochemically similar to wild-type, but lesions are smaller and lung function is preserved. We also show for the first time that profibrotic pathways of tissue stiffening and metabolic reprogramming are interconnected, and that metabolic disruptions in fibrosis go beyond glycolysis.

Neonatal hyperoxia enhances age-dependent expression of SARS-CoV-2 receptors in mice.

The severity of COVID-19 lung disease is higher in the elderly and people with pre-existing co-morbidities. People who were born preterm may be at greater risk for COVID-19 because their early exposure to oxygen (hyperoxia) at birth increases the severity of respiratory viral infections. Hyperoxia at birth increases the severity of influenza A virus infections in adult mice by reducing the number of alveolar epithelial type 2 (AT2) cells. Since AT2 cells express the SARS-CoV-2 receptors angiotensin converting enzyme (ACE2) and transmembrane protease/serine subfamily member 2 (TMPRSS2), their expression should decline as AT2 cells are depleted by hyperoxia. Instead, ACE2 was detected in airway Club cells and endothelial cells at birth, and then AT2 cells at one year of age. Neonatal hyperoxia stimulated expression of ACE2 in Club cells and in AT2 cells by 2 months of age. It also stimulated expression of TMPRSS2 in the lung. Increased expression of SARS-CoV-2 receptors was blocked by mitoTEMPO, a mitochondrial superoxide scavenger that reduced oxidative stress and DNA damage seen in oxygen-exposed mice. Our finding that hyperoxia enhances the age-dependent expression of SARS-CoV-2 receptors in mice helps explain why COVID-19 lung disease is greater in the elderly and people with pre-existing co-morbidities.

Neonatal hyperoxia enhances age-dependent expression of SARS-CoV-2 receptors in mice.

The severity of COVID-19 lung disease is higher in the elderly and people with pre-existing co-morbidities. People who were born preterm may be at greater risk for COVID-19 because their early exposure to oxygen at birth increases their risk of being hospitalized when infected with RSV and other respiratory viruses. Our prior studies in mice showed how high levels of oxygen (hyperoxia) between postnatal days 0-4 increases the severity of influenza A virus infections by reducing the number of alveolar epithelial type 2 (AT2) cells. Because AT2 cells express the SARS-CoV-2 receptors angiotensin converting enzyme (ACE2) and transmembrane protease/serine subfamily member 2 (TMPRSS2), we expected their expression would decline as AT2 cells were depleted by hyperoxia. Instead, we made the surprising discovery that expression of Ace2 and Tmprss2 mRNA increases as mice age and is accelerated by exposing mice to neonatal hyperoxia. ACE2 is primarily expressed at birth by airway Club cells and becomes detectable in AT2 cells by one year of life. Neonatal hyperoxia increases ACE2 expression in Club cells and makes it detectable in 2-month-old AT2 cells. This early and increased expression of SARS-CoV-2 receptors was not seen in adult mice who had been administered the mitochondrial superoxide scavenger mitoTEMPO during hyperoxia. Our finding that early life insults such as hyperoxia enhances the age-dependent expression of SARS-CoV-2 receptors in the respiratory epithelium helps explain why COVID-19 lung disease is greater in the elderly and people with pre-existing co-morbidities.

Common mechanisms of DNA translocation motors in bacteria and viruses using one-way revolution mechanism without rotation.

Biomotors were once described into two categories: linear motor and rotation motor. Recently, a third type of biomotor with revolution mechanism without rotation has been discovered. By analogy, rotation resembles the Earth rotating on its axis in a complete cycle every 24h, while revolution resembles the Earth revolving around the Sun one circle per 365 days (see animations http://nanobio.uky.edu/movie.html). The action of revolution that enables a motor free of coiling and torque has solved many puzzles and debates that have occurred throughout the history of viral DNA packaging motor studies. It also settles the discrepancies concerning the structure, stoichiometry, and functioning of DNA translocation motors. This review uses bacteriophages Phi29, HK97, SPP1, P22, T4, and T7 as well as bacterial DNA translocase FtsK and SpoIIIE or the large eukaryotic dsDNA viruses such as mimivirus and vaccinia virus as examples to elucidate the puzzles. These motors use ATPase, some of which have been confirmed to be a hexamer, to revolve around the dsDNA sequentially. ATP binding induces conformational change and possibly an entropy alteration in ATPase to a high affinity toward dsDNA; but ATP hydrolysis triggers another entropic and conformational change in ATPase to a low affinity for DNA, by which dsDNA is pushed toward an adjacent ATPase subunit. The rotation and revolution mechanisms can be distinguished by the size of channel: the channels of rotation motors are equal to or smaller than 2 nm, that is the size of dsDNA, whereas channels of revolution motors are larger than 3 nm. Rotation motors use parallel threads to operate with a right-handed channel, while revolution motors use a left-handed channel to drive the right-handed DNA in an anti-chiral arrangement. Coordination of several vector factors in the same direction makes viral DNA-packaging motors unusually powerful and effective. Revolution mechanism that avoids DNA coiling in translocating the lengthy genomic dsDNA helix could be advantageous for cell replication such as bacterial binary fission and cell mitosis without the need for topoisomerase or helicase to consume additional energy.

Discovery of a new motion mechanism of biomotors similar to the earth revolving around the sun without rotation.

Biomotors have been classified into linear and rotational motors. For 35 years, it has been popularly believed that viral dsDNA-packaging apparatuses are pentameric rotation motors. Recently, a third class of hexameric motor has been found in bacteriophage phi29 that utilizes a mechanism of revolution without rotation, friction, coiling, or torque. This review addresses how packaging motors control dsDNA one-way traffic; how four electropositive layers in the channel interact with the electronegative phosphate backbone to generate four steps in translocating one dsDNA helix; how motors resolve the mismatch between 10.5 bases and 12 connector subunits per cycle of revolution; and how ATP regulates sequential action of motor ATPase. Since motors with all number of subunits can utilize the revolution mechanism, this finding helps resolve puzzles and debates concerning the oligomeric nature of packaging motors in many phage systems. This revolution mechanism helps to solve the undesirable dsDNA supercoiling issue involved in rotation.

Isolation and characterization of bioactive compounds from the bark of Litsea costalis.

The bark of Litsea costalis affords two new compounds named 4,4'-diallyl-5,5'-dimethoxy-[1,1'-biphennyl]-2,2'-diol, biseugenol A (1) and 2,2'-oxybis (4-allyl-1-methoxybenzene), biseugenol B (2) along with two known compounds (3-4), namely 5-methoxy-2-Hydroxy Benzaldehyde (3), and (E)-4-styrylphenol (4). The structures of 1 and 2 were determined using 1D and 2D NMR data. Also, the IR and NMR data were combined with quantum chemical calculations in the DFT approach using the hybrid B3LYP exchange-correlation function to confirm the structures of the compounds. Compounds showed fairly potent anticancer activity against cell lines and antioxidant (DPPH).