Component Of Pulmonary Surfactant Research Articles

Untargeted lipidomics of bronchopulmonary dysplasia induced by hyperoxia exposure in rats.

Bronchopulmonary dysplasia (BPD), characterized by impaired lung development, remains a leading cause of morbidity and mortality in premature infants. The synthesis and metabolism of lipids play a critical role in normal lung development, such as dipalmitoylphosphatidylcholine, a key component of pulmonary surfactant (PS). Therefore, we conducted a lipidomics study of rat lung tissue to explore the changes of pulmonary lipid composition in the progression of BPD disease. In this study, we exposed neonatal Sprague-Dawley (SD) rats to hyperoxia for 14 days. After hyperoxia exposure, the lung tissues of rats were analyzed pathologically, and untargeted lipidomics was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Hematoxylin-eosin (H&E) staining showed that the alveoli enlarged, the number of alveoli decreased and the pulmonary surfactant-associated protein D (SFTPD) decreased in hyperoxia-exposed rats. A total of 620 pulmonary lipids were detected by LC-MS/MS, covering 27 lipid categories. The most common lipids were triacylglycerol (TAG), followed by phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Compared with those rats exposed to normoxic conditions, the lipid levels in the lungs of rats exposed to hyperoxia for 14 days generally decreased, with the levels of TAG and PC decreasing most significantly. In short, our results provide a clue for finding therapeutic targets and biomarkers of a BPD rat model lung liposome.

Effect of lipid-polymer hybrid nanoparticles on the biophysical function and lateral structure of pulmonary surfactant: Mechanistic in vitro studies

The interaction between inhaled drug-loaded nanoparticles and pulmonary surfactant (PS) is critical for the efficacy and safety of inhaled nanomedicines. Here, we investigated the effect of small interfering RNA (siRNA)-loaded lipid-polymer hybrid nanoparticles (LPNs), which are designed for treatment of lung inflammation, on the physiological function of PS. By using biophysical in vitro methods we show that siRNA-loaded LPNs affect the biophysical function and lateral structure of PS. We used the Langmuir monolayer technique to demonstrate that LPNs display intrinsic surface activity by forming interfacial films that collapse at 49 mN/m, and they competitively inhibit the adsorption and spreading of PS components at the air–liquid interface. However, LPNs are excluded from the interface into the aqueous subphase at surface pressures above 49 mN/m, and hence they overcome the PS monolayer film barrier. Epifluorescence microscopy data revealed that LPNs influence the lateral structure of PS by: (i) affecting the nucleation, shape, and growth of compression-driven segregated condensed PS domains, and (ii) facilitating intermixing of liquid-expanded and tilted-condensed domains. However, the total surface area occupied by a highly condensed phase, presumably enriched in the highly surface tension-reducing dipalmitoylphosphatidylcholine, remained constant upon exposure to LPNs. These results suggest that surface-active LPNs influence the lateral structure of PS during translocation from the interface into the subphase, but LPNs do apparently not affect the biophysical function of PS under physiologically relevant conditions.

LPCAT1 levels in the placenta, the maternal plasma and the fetal plasma do not predict fetal lung responses to glucocorticoids in a sheep model of pregnancy

IntroductionLysophosphatidylcholine acyltransferase 1 (LPCAT1) is important for saturated phosphatidylcholine (Sat-PC) production in the lung. Sat-PC is a critical component of pulmonary surfactant, which maintains low alveolar surface tension, facilitating respiration. Previous studies have reported an association between maternal and fetal LPCAT1 levels and neonatal lung function. Using a sheep model of pregnancy, we investigated a potential correlation between glucocorticoid-induced lung maturation and LPCAT1 mRNA and/or protein levels in the fetal lung, the placenta, the fetal plasma, and the maternal plasma. MethodsEighty seven single pregnant ewes received maternal intramuscular injections of betamethasone. A sub-group of five animals had both maternal and fetal catheters installed to allow for sequential sampling from both plasma compartments. Lambs were surgically delivered under terminal anaesthesia between 2 and 8 days after initial ANS treatment, at a gestational age of 121–123 days. Lambs were ventilated for 30 min to determine functional lung maturation before being euthanized for necropsy and sample collection. Fetal lung, placenta, and fetal and maternal plasma samples were used to analyse LPCAT1 gene expression and protein levels. ResultsThe expression of LPCAT1 mRNA in the fetal lung was significantly corelated to Sat-PC levels at 8 days (R2 = 0.23, p < 0.001) and lung maturation status overall (gas exchange efficiency as determined by measurements of lamb PaCO2 during ventilation, R2 = 0.20, p < 0.001). Similarly, fetal lung LPCAT1 mRNA was also significantly correlated with the individual durability of ANS effects on fetal lung maturation (R2 = 0.20, p < 0.001). Although ANS therapy altered LPCAT1 mRNA expression in the placenta, observed changes were independent of fetal lung maturation outcomes. Neither maternal nor fetal plasma LPCAT1 levels were changed by ANS therapy over the period, including in analysis of serial maternal and fetal samples from chronically catheterised animals. DiscussionLPCAT1 expression in the fetal lung was associated with the durability of glucocorticoid effects on fetal lung maturation. However, LPCAT1 expression in the placenta, the fetal plasma, and the maternal plasma was neither associated with, nor predictive of fetal lung maturation after glucocorticoid treatment in a sheep model of pregnancy.

Gram-negative bacteria act as a reservoir for aminoglycoside antibiotics that interact with host factors to enhance bacterial killing in a mouse model of pneumonia.

In vitro exposure of multiple Gram-negative bacteria to an aminoglycoside (AG) antibiotic has previously been demonstrated to result in bacterial alterations that interact with host factors to suppress Gram-negative pneumonia. However, the mechanisms resulting in suppression are not known. Here, the hypothesis that Gram-negative bacteria bind and retain AGs, which are introduced into the lung and interact with host defenses to affect bacterial killing, was tested. Following in vitro exposure of one of several, pathogenic Gram-negative bacteria to the AG antibiotics kanamycin or gentamicin, AGs were detected in bacterial cell pellets (up to 208 μg/mL). Using inhibitors of AG binding and internalization, the bacterial outer membrane was implicated as the predominant kanamycin and gentamicin reservoir. Following intranasal administration of gentamicin-bound bacteria or gentamicin solution at the time of infection with live, AG-naïve bacteria, gentamicin was detected in the lungs of infected mice (up to 8 μg/g). Co-inoculation with gentamicin-bound bacteria resulted in killing of AG-naïve bacteria by up to 3-log10, mirroring the effects of intranasal gentamicin treatment. In vitro killing of AG-naïve bacteria mediated by kanamycin-bound bacteria required the presence of detergents or pulmonary surfactant, suggesting that increased bacterial killing inside the murine lung is facilitated by the detergent component of pulmonary surfactant. These findings demonstrate that Gram-negative bacteria bind and retain AGs that can interact with host-derived pulmonary surfactant to enhance bacterial killing in the lung. This may help explain why AGs appear to have unique efficacy in the lung and might expand their clinical utility.

Insights Gained Into the Treatment of COVID19 by Pulmonary Surfactant and Its Components.

Pulmonary surfactant constitutes an important barrier that pathogens must cross to gain access to the rest of the organism via the respiratory surface. The presence of pulmonary surfactant prevents the dissemination of pathogens, modulates immune responses, and optimizes lung biophysical activity. Thus, the application of pulmonary surfactant for the treatment of respiratory diseases provides an effective strategy. Currently, several clinical trials are investigating the use of surfactant preparations to treat patients with coronavirus disease 2019 (COVID-19). Some factors have been considered in the application of pulmonary surfactant for the treatment COVID-19, such as mechanical ventilation strategy, timing of treatment, dose delivered, method of delivery, and preparation utilized. This review supplements this list with two additional factors: accurate measurement of surfactants in patients and proper selection of pulmonary surfactant components. This review provides a reference for ongoing exogenous surfactant trials involving patients with COVID-19 and provides insight for the development of surfactant preparations for the treatment of viral respiratory infections.

Iron oxide nanoparticles oxidize transformed RAW 264.7 macrophages into foam cells: Impact of pulmonary surfactant component dipalmitoylphosphatidylcholine

Iron oxide nanoparticles (IONPs) are one of the most important components in airborne particulate matter that originally generated from traffic emission, iron ore mining, coal combustion and melting of engine fragments. Once IONPs entered respiratory tract and deposit in the alveoli, they may interact with pulmonary surfactant (PS) that distributed in the alveolar lining. Thereafter, it is necessary to investigate the interaction of inhaled IONPs and PS, which helps the understanding of health risk of respiratory health induced by IONPs. Using dipalmitoyl phosphatidylcholine (DPPC), the major components of PS, as a lipid model, we explored the interaction of DPPC with typical IONPs, Fe3O4 NPs and amino-functionalized analogue (Fe3O4–NH2 NPs). DPPC was readily adsorbed on the surface of both IONPs. Although DPPC corona depressed the cellular uptake of IONPs, IONPs@DPPC complexes caused higher cytotoxicity toward RAW 264.7 macrophages, compared to pristine IONPs. Mechanistic studies have shown that IONPs react with intracellular hydrogen peroxide, which promotes the Fenton reaction, to generate hydroxyl radicals. Iron ions could oxidize lipids to form lipid peroxides, and lipid hydroperoxides will decompose to generate hydroxyl radicals, which further promote cellular oxidative stress, lipid accumulation, foam cell formation, and the release of inflammatory factors. These findings demonstrated the phenomenon of coronal component oxidation, which contributed to IONPs-induced cytotoxicity. This study offered a brand-new toxicological mechanism of IONPs at the molecular level, which is helpful for further understanding the adverse effects of IONPs.

Gene Therapy Potential for Genetic Disorders of Surfactant Dysfunction.

Pulmonary surfactant is critically important to prevent atelectasis by lowering the surface tension of the alveolar lining liquid. While respiratory distress syndrome (RDS) is common in premature infants, severe RDS in term and late preterm infants suggests an underlying genetic etiology. Pathogenic variants in the genes encoding key components of pulmonary surfactant including surfactant protein B (SP-B, SFTPB gene), surfactant protein C (SP-C, SFTPC gene), and the ATP-Binding Cassette transporter A3 (ABCA3, ABCA3 gene) result in severe neonatal RDS or childhood interstitial lung disease (chILD). These proteins play essential roles in pulmonary surfactant biogenesis and are expressed in alveolar epithelial type II cells (AEC2), the progenitor cell of the alveolar epithelium. SP-B deficiency most commonly presents in the neonatal period with severe RDS and requires lung transplantation for survival. SFTPC mutations act in an autosomal dominant fashion and more commonly presents with chILD or idiopathic pulmonary fibrosis than neonatal RDS. ABCA3 deficiency often presents as neonatal RDS or chILD. Gene therapy is a promising option to treat monogenic lung diseases. Successes and challenges in developing gene therapies for genetic disorders of surfactant dysfunction include viral vector design and tropism for target cell types. In this review, we explore adeno-associated virus (AAV), lentiviral, and adenoviral (Ad)-based vectors as delivery vehicles. Both gene addition and gene editing strategies are compared to best design treatments for lung diseases resulting from pathogenic variants in the SFTPB, SFTPC, and ABCA3 genes.

Effect of pulmonary surfactant on the dispersion of carbon nanoparticles

Carbon nanoparticles (CNs) are widely used and there has been a great concern about their potential adverse effects on human health. CNs can directly interact with the pulmonary surfactant (PS) lining of alveoli, but the dispersion state of CNs at PS film has not yet been explored. Accumulating evidences indicated that the degree of dispersion of nanoparticles has a strong influence on their biological activities. This study investigated the effect of PS and its major components on the dispersion state of three representative CNs (fullerene C60, nano carbon powder, graphene oxide). The results demonstrated that suspended concentrations of all three CNs were lower in the presence of PS and its lipid component dipalmitoylphosphatidylcholine (DPPC) than in the presence of bovine serum album (BSA). Compared with PS and DPPC, BSA notably reduced the hydrodynamic diameters of CNs. In addition, the absolute zeta potentials of CNs in the presence of BSA was higher than those in the presence of PS or DPPC, and this result corresponds to the increase of surface oxygen contents of CNs, indicating that relatively high surface oxygen content is beneficial for the dispersion of CNs in alveolus. π-A isotherms demonstrated that the surface tension and phase behavior of PS were obviously altered in the presence of CNs. This study revealed the role of PS and different active components in the dispersion of CNs, which is helpful for deeply understanding CNs alveolar transport and the associated potential hazards to lung health. • Interaction of PS with carbon nanoparticles (CNs) leads to the aggregation of CNs. • Lipids and protein components of PS exert different effects on CNs dispersion. • Hydrodynamic diameter/zeta potential impact the dispersion of CNs with PS component. • Surface oxygen content of CNs governs the dispersion of CNs. • CNs exposure leads to the alternation of phase behavior of PS film.

Potential hazards associated with interactions between diesel exhaust particulate matter and pulmonary surfactant

Long term exposure to diesel exhaust particulate matter (DEPM) can induce numerous adverse health effects to the respiratory system. Understanding the interaction between DEPM and pulmonary surfactant (PS) can be an essential step toward preliminary evaluation of the impact of DEPM on pulmonary health. Herein, DEPM was explored for its interaction with 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC), the major component of PS. The results indicated that the surface pressure-area (π-A) isotherms of DPPC monolayers shifted toward lower molecular areas and the compression modulus (CS−1) reduced in the presence of DEPM. Atomic force microscopy image showed that DEPM can disrupt the ultrastructure of DPPC monolayers along with the direction of lateral compression. In addition, DPPC can in turn condition the surface properties of DEPM, permitting its agglomeration in aqueous media, which was attributed to the adsorption of DEPM to DPPC. Furthermore, the particle-bound polycyclic aromatic hydrocarbons (PAHs) could be desorbed from DEPM by the solubilization of DPPC and it was positively correlated with the hydrophobicity of PAHs. These findings revealed the toxicity of DEPM-associated PAHs and the role of DPPC in facilitating the removal of the inhaled particles, which can provide a new insight into the potential hazards of airborne particles on lung health.

LAMP3 deficiency affects surfactant homeostasis in mice.

Lysosome-associated membrane glycoprotein 3 (LAMP3) is a type I transmembrane protein of the LAMP protein family with a cell-type-specific expression in alveolar type II cells in mice and hitherto unknown function. In type II pneumocytes, LAMP3 is localized in lamellar bodies, secretory organelles releasing pulmonary surfactant into the extracellular space to lower surface tension at the air/liquid interface. The physiological function of LAMP3, however, remains enigmatic. We generated Lamp3 knockout mice by CRISPR/Cas9. LAMP3 deficient mice are viable with an average life span and display regular lung function under basal conditions. The levels of a major hydrophobic protein component of pulmonary surfactant, SP-C, are strongly increased in the lung of Lamp3 knockout mice, and the lipid composition of the bronchoalveolar lavage shows mild but significant changes, resulting in alterations in surfactant functionality. In ovalbumin-induced experimental allergic asthma, the changes in lipid composition are aggravated, and LAMP3-deficient mice exert an increased airway resistance. Our data suggest a critical role of LAMP3 in the regulation of pulmonary surfactant homeostasis and normal lung function.

Surface Properties of Pulmonary Surfactant Sampled by Bronchoalveolar Lavage and by Electrostatic Exhaled Aerosol Trapping

The development of efficient methods for non-invasive collection of alveolar lining fluid (ALF) samples containing pulmonary surfactant (PS) components and the study of the surface activity of the obtained native material is relevant for the diagnosis of inflammatory pneumopathies of the lungs. The paper presents an electrostatic aerosol trapping (ESAT) mobile complex for capturing droplets of ALF contained in an exhaled air. Passing the exhaled air through the corona discharge area results in the aerosol droplets charging and their further transferring by electrostatic force into a water surface, where they accumulate forming an adsorbed layer. Additionally, ALF samples were collected using a bronchoalveolar lavage (BAL). The surface properties of the PS obtained by both methods have been examined using the capillary wave method, which was previously modified by the authors specifically for biomedical applications. Significant difference was found in the results obtained with ESAT and BAL in the group of healthy subjects, which can be explained by different origin of the samples obtained by these techniques. Furthermore, significant difference in surface properties was established in the samples collected from healthy volunteers and patient with disseminated tuberculosis, while we did not find significant differences in the limited inflammatory process. The results presented in the paper demonstrate high potential of the proposed non-invasive technique for clinical usage.

Carrier Solvents of Electronic Nicotine Delivery Systems Alter Pulmonary Surfactant.

In late 2019, hundreds of users of electronic products that aerosolize a liquid for inhalation were hospitalized with a variety of respiratory and gastrointestinal symptoms. While some investigations have attributed the disease to the presence of vitamin E acetate in liquids that also contained tetrahydrocannabinol, some evidence suggests that chronic inhalation of two common solvents used in electronic nicotine delivery systems (ENDS), propylene glycol (PG) and vegetable glycerin (VG), can interfere with the lipid components of pulmonary surfactant and cause or exacerbate pulmonary injury. The interaction between PG, VG, and lung surfactant is not yet understood. This study presents an examination of the molecular interactions of PG and VG with lung surfactant mimicked by 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). The interaction of DPPC and PG-VG is studied by attenuated total reflectance fourier transform infrared spectroscopy. The results showed that PG and VG altered the molecular alignment of the DPPC surfactant. The orientation of the surfactant at the surface of the lung affects the surface tension at the air–water interface, thereby influencing breathing. These findings suggest that chronic aerosolization of the primary solvents in ENDS might alter the function of pulmonary surfactant.

Surfactant Protein B Promotes Cytosolic SiRNA Delivery by Adopting a Virus-like Mechanism of Action.

RNA therapeutics are poised to revolutionize medicine. To unlock the full potential of RNA drugs, safe and efficient (nano)formulations to deliver them inside target cells are required. Endosomal sequestration of nanocarriers represents a major bottleneck in nucleic acid delivery. Gaining more detailed information on the intracellular behavior of RNA nanocarriers is crucial to rationally develop delivery systems with improved therapeutic efficiency. Surfactant protein B (SP-B) is a key component of pulmonary surfactant (PS), essential for mammalian breathing. In contrast to the general belief that PS should be regarded as a barrier for inhaled nanomedicines, we recently discovered the ability of SP-B to promote gene silencing by siRNA-loaded and lipid-coated nanogels. However, the mechanisms governing this process are poorly understood. The major objective of this work was to obtain mechanistic insights into the SP-B-mediated cellular delivery of siRNA. To this end, we combined siRNA knockdown experiments, confocal microscopy, and focused ion beam scanning electron microscopy imaging in an in vitro non-small-cell lung carcinoma model with lipid mixing assays on vesicles that mimic the composition of (intra)cellular membranes. Our work highlights a strong correlation between SP-B-mediated fusion with anionic endosomal membranes and cytosolic siRNA delivery, a mode of action resembling that of certain viruses and virus-derived cell-penetrating peptides. Building on these gained insights, we optimized the SP-B proteolipid composition, which dramatically improved delivery efficiency. Altogether, our work provides a mechanistic understanding of SP-B-induced perturbation of intracellular membranes, offering opportunities to fuel the rational design of SP-B-inspired RNA nanoformulations for inhalation therapy.

Role of Glycosylation in Lipid-Interacting Properties of Pulmosaposin A, from the Pulmonary Surfactant Protein B Precursor

Pulmonary surfactant, the secretory product of type II pneumocytes, is a complex with a unique protein-lipid composition that lines the alveoli surface. At the air-liquid interface, surfactant reduces surface tension and avoids lung collapse during breathing. Surfactant Protein B (SP-B), an essential component of pulmonary surfactant, is synthesized as a long precursor to prevent from protein aggregation due to its hydrophobicity. The N-terminus of this precursor contains the Pulmosaposin A (or SP-BN), the murine version being originally described as a defensive protein with some bactericidal functions under particular conditions. Moreover, this precursor domain contains, in humans, a glycosylation site apparently related to susceptibility to certain pulmonary pathologies. However, none of these properties have been deeply characterized from a molecular point of view. Here we describe the production of the recombinant human Pulmosaposin A (hSP-BN) in the yeast Pichia pastoris, the isolation of the glycosylated and deglycosylated forms as well as a non-glycosylated mutant. The purified proteins conserved the saposin fold rich in helical content. Toxicity against bacteria could not be observed, a discrepancy with previous results obtained with the murine version expressed in E. coli. Nevertheless, these pulmosaposin variants showed lipid-interacting properties, especially under acidic conditions, which is consistent with their saposin fold as well as with their potential role within the precursor during SP-B maturation and secretion. Results are discussed in terms of the specific role of this lipid-interacting activity as well as the involvement of glycosylation and its relation with pulmonary dysfunction and disease.

In silico study reveals binding potential of rotenone at multiple sites of pulmonary surfactant proteins: A matter of concern

Rotenone is a broad-spectrum pesticide employed in various agricultural practices all over the world. Human beings are exposed to this chemical through oral, nasal, and dermal routes. Inhalation of rotenone exposes bio-molecular components of lungs to this chemical. Biophysical activity of lungs is precisely regulated by pulmonary surfactant to facilitate gaseous exchange. Surfactant proteins (SPs) are the fundamental components of pulmonary surfactant. SPs like SP-A and SP-D have antimicrobial activities providing a crucial first line of defense against infections in lungs whereas SP-B and SP-C are mainly involved in respiratory cycle and reduction of surface tension at air–water interface. In this study, molecular docking analysis using AutoDock Vina has been conducted to investigate binding potential of rotenone with the four SPs. Results indicate that, rotenone can bind with carbohydrate recognition domain (CRD) of SP-A, N-, and C- terminal peptide of SP-B, SP-C, and CRD of SP-D at multiples sites via several interaction mediators such as H bonds, C–H bonds, alkyl bonds, pi-pi stacked, Van der Waals interaction, and other. Such interactions of rotenone with SPs can disrupt biophysical and anti-microbial functions of SPs in lungs that may invite respiratory ailments and pathogenic infections.

A 20-Mer Peptide Derived from the Lectin Domain of SP-A2 Decreases Tumor Necrosis Factor Alpha Production during Mycoplasma pneumoniae Infection.

Human surfactant protein-A2 (hSP-A2) is a component of pulmonary surfactant that plays an important role in the lung's immune system by interacting with viruses, bacteria, and fungi to facilitate pathogen clearance and by downregulating inflammatory responses after an allergic challenge. Genetic variation in SP-A2 at position Gln223Lys is present in up to ∼30% of the population and has been associated with several lung diseases, such as asthma, pulmonary fibrosis, and lung cancer (M. M. Pettigrew, J. F. Gent, Y. Zhu, E. W. Triche, et al., BMC Med Genet 8:15, 2007, https://bmcmedgenet.biomedcentral.com/articles/10.1186/1471-2350-8-15; Y. Wang, P. J. Kuan, C. Zing, J. T. Cronkhite, et al., Am J Hum Genet 84:52-59, 2009, https://www.cell.com/ajhg/fulltext/S0002-9297(08)00595-8). Previous work performed by our group showed differences in levels of SP-A binding to non-live mycoplasma membrane fractions that were dependent on the presence of a lysine (K) or a glutamine (Q) at amino acid position 223 in the carbohydrate region of SP-A2. On the basis of these differences, we have derived 20-amino-acid peptides flanking this region of interest in order to test the ability of each to regulate various immune responses to live Mycoplasma pneumoniae in SP-A knockout mice and RAW 264.7 cells. In both models, the 20-mer containing 223Q significantly decreased both tumor necrosis factor alpha (TNF-α) mRNA levels and protein levels in comparison to the 20-mer containing 223K during M. pneumoniae infection. While neither of the 20-mer peptides (223Q and 223K) had an effect on p38 phosphorylation during M. pneumoniae infection, the 223Q-20mer peptide significantly reduced NF-κB p65 phosphorylation in both models. Taken together, our data suggest that small peptides derived from the lectin domain of SP-A2 that contain the major allelic variant (223Q) maintain activity in reducing TNF-α induction during M. pneumoniae infection.

Electrostatic precipitation of exhaled particles for tensiometric examination of pulmonary surfactant

Collecting exhaled particles that represent small droplets of the alveolar lining fluid shows great promise as a tool for pulmonary surfactant (PS) sampling. In this work we present a setup consisting of two modules, namely, a module for droplet collecting, called an electrostatic aerosol trapping (ESAT) system, and a measurement module for studying PS properties. We also suggest a method to extract numerical values from experimental data. The operating principle of ESAT is based on electrostatic precipitation of exhaled particles. Native material was collected directly on the water surface, where the adsorbed pulmonary surfactant film was investigated by the tensiometric method. The modified capillary wave method proposed in our previous work was adapted for studying small-volume samples. The efficiency of the setup was verified in the experiments involved the participation of healthy subjects. The accumulation of PS components on the water surface in an amount sufficient for tensiometric study was reported. The idea underlying the new concept used in this work may give impetus to the development of point-of-care facilities for collecting PS samples needed to provide their express analysis.

Immunohistochemical expression of Napsin A in normal human fetal lungs at different gestational ages and in acquired and congenital pathological pulmonary conditions.

Surfactant protein B (SP-B) is a key component of pulmonary surfactant. SP-B is processed to a mature, surface-active protein from a pro-peptide by two distinct cleavage events in its N-terminal and C-terminal regions. Napsin A, a protease expressed in type II pneumocytes, is responsible for the N-terminal cleavage event. Here, for the first time, we have evaluated the expression of Napsin A in normal fetal lungs at different gestational ages and in lungs from fetuses and neonates with congenital and acquired pathological pulmonary conditions. Lung samples were collected from fetal and neonatal autopsies at the Department of Medicine and Surgery's Pathology Unit of Parma University (Italy). Immunohistochemical analysis was performed using a primary anti-Napsin A (clone IP64 clone) monoclonal antibody. A section of lung adenocarcinoma was used as an external positive control. Napsin A was expressed early in normal fetal lungs throughout the epithelium of the distal pseudoglandular tracts. In fetuses at 30weeks of gestation and term newborns, Napsin A was already expressed only in isolated cells within the alveolar epithelium, similar to adult subjects. Furthermore, increased expression of Napsin A compared with a control group was observed in lung tissue from fetuses and a newborn with pathological conditions (inflammatory diseases and pulmonary hypoplasia). In conclusion, this study demonstrates that Napsin A is produced early in fetal life, and that its production is increased in many diseases, presumably in an effort to remedy functional pulmonary failure.

Developmental Basis and Consequences of a Key Innovation in Lungless Salamanders

A longstanding goal of evolutionary developmental biology is to document the precise mechanisms that underlie formation of, and differences among, basic body plans, organ systems and physiological functions. Another, less‐studied approach is to reveal how those mechanisms are perturbed to enable, or even mediate, the origin of novel adaptations that are critical for the evolutionary success of individual clades. I will report results of our recent studies of evolutionary lung loss in plethodontid salamanders and of the correlated evolution of cutaneous respiration, which is the principal mode of respiratory gas exchange in all 478 species in this, the largest family of extant urodeles. Surprisingly, plethodontids are not entirely lungless: a lung rudiment forms initially in embryos but is resorbed before hatching. Some embryonic cells express lung‐associated molecular markers, such as the gene Nkx2.1, although at lower levels than in lunged species. Moreover, elevated levels of TGF‐ß signaling, which is known to mediate Nkx2.1 expression during normal lung development, may be part of the genetic mechanism responsible for lung loss. In contrast, we have discovered in salamanders a novel paralog of the gene surfactant‐associated protein C (SFTPC), which is a critical component of pulmonary surfactant expressed exclusively in the lung in other vertebrates. In plethodontids, however, and only in plethodontids, this gene is expressed in novel sites of respiration outside the lungs and at different sites in larvae vs. adults that correlate with gas exchange. These and other changes to the ancestral urodele ontogeny may have contributed to the remarkable evolutionary radiation of the lungless salamanders.

The novel potential biomarkers for multidrug-resistance tuberculosis using UPLC-Q-TOF-MS.

The lack of rapid and efficient diagnostics impedes largely the epidemic control of multidrug-resistant tuberculosis, and might misguide the therapeutic strategies as well. This study aimed to identify novel multidrug-resistant tuberculosis biomarkers to improve the early intervention, symptomatic treatment and control of the prevalence of multidrug-resistant tuberculosis. The serum small molecule metabolites in healthy controls, patients with drug-susceptible tuberculosis, and patients with multidrug-resistant tuberculosis were screened using ultra-high-performance liquid chromatography combined with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The differentially abundant metabolites were filtered out through multidimensional statistical analysis and bioinformatics analysis. Compared with drug-susceptible tuberculosis patients and healthy controls, the levels of 13 metabolites in multidrug-resistant tuberculosis patients altered. Among them, the most significant changes were found in N1-Methyl-2-pyridone-5-carboxamide (N1M2P5C), 1-Myristoyl-sn-glycerol-3-phosphocholine (MG3P), Caprylic acid (CA), and D-Xylulose (DX). And a multidrug-resistant tuberculosis/drug-susceptible tuberculosis differential diagnostic model was built based on these four metabolites, achieved the accuracy, sensitivity, and specificity of 0.928, 86.7%, and 86.7%, respectively. The enrichment analysis of metabolic pathways showed that the phospholipid remodeling of cell membranes was active in multidrug-resistant tuberculosis patients. In addition, in patients with tuberculosis, the metabolites of dipalmitoyl phosphatidylcholine (DPPC), a major component of pulmonary surfactant, were down-regulated. N1M2P5C, MG3P, CA, and DX may have the potential to serve as novel multidrug-resistant tuberculosis biomarkers. This research provides a preliminary experimental basis to further investigate potential multidrug-resistant tuberculosis biomarkers. Impact statement The MDR-TB incidence remains high, making the effective control of TB epidemic yet challenging. Rapid and accurate diagnosis is vitally important for improving the therapeutic efficacy and controlling the prevalence of drug resistance TB. Metabolomics has dramatic potential to distinguish MDR-TB and DS-TB. N1M2P5C, MG3P, CA, and DX that we identified in this study might have potential as novel MDR-TB biomarkers. The phospholipid remodeling of cell membranes was highly active in MDR-TB. The DPPC metabolites in TB were significantly down-regulated. This work aimed to investigate potential MDR-TB biomarkers to enhance the clinical diagnostic efficacy. The metabolic pathway distinctly altered in MDR-TB might provide novel targets to develop new anti-TB drugs.