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Abstract Background Non-invasive delivery of nebulized surfactant has been a neonatology long-pursued goal. Nevertheless, the clinical efficacy of nebulized surfactant remains inconclusive, in part, due to the great technical challenges of depositing nebulized drugs in the lungs of preterm infants. The aim of this study was to investigate the feasibility of delivering nebulized surfactant (poractant alfa) in vitro and in vivo with an adapted, neonate-tailored aerosol delivery strategy. Methods Particle size distribution of undiluted poractant alfa aerosols generated by a customized eFlow-Neos nebulizer system was determined by laser diffraction. The theoretical nebulized surfactant lung dose was estimated in vitro in a clinical setting replica including a neonatal continuous positive airway pressure (CPAP) circuit, a cast of the upper airways of a preterm neonate, and a breath simulator programmed with the tidal breathing pattern of an infant with mild respiratory distress syndrome (RDS). A dose-response study with nebulized surfactant covering the 100–600 mg/kg nominal dose-range was conducted in RDS-modelling, lung-lavaged spontaneously-breathing rabbits managed with nasal CPAP. The effects of nebulized poractant alfa on arterial gas exchange and lung mechanics were assessed. Exogenous alveolar disaturated-phosphatidylcholine (DSPC) in the lungs was measured as a proxy of surfactant deposition efficacy. Results Laser diffraction studies demonstrated suitable aerosol characteristics for inhalation (mass median diameter, MMD = 3 μm). The mean surfactant lung dose determined in vitro was 13.7% ± 4.0 of the 200 mg/kg nominal dose. Nebulized surfactant delivered to spontaneously-breathing rabbits during nasal CPAP significantly improved arterial oxygenation compared to animals receiving CPAP only. Particularly, the groups of animals treated with 200 mg/kg and 400 mg/kg of nebulized poractant alfa achieved an equivalent pulmonary response in terms of oxygenation and lung mechanics as the group of animals treated with instilled surfactant (200 mg/kg). Conclusions The customized eFlow-Neos vibrating-membrane nebulizer system efficiently generated respirable aerosols of undiluted poractant alfa. Nebulized surfactant delivered at doses of 200 mg/kg and 400 mg/kg elicited a pulmonary response equivalent to that observed after treatment with an intratracheal surfactant bolus of 200 mg/kg. This bench-characterized nebulized surfactant delivery strategy is now under evaluation in Phase II clinical trial (EUDRACT No.:2016–004547-36).

Background and Purpose: Therapeutic hypothermia (TH) has become the standard care to reduce morbidity and mortality in neonates affected by moderate-to-severe hypoxic-ischaemic encephalopathy (HIE). Despite the use of TH for HIE, the incidence of mortality and disabilities remains high., Experimental Approach: Nerve growth factor (NGF) is a potent neurotrophin, but clinical use is limited by its pain eliciting effects. CHF6467 is a recombinant modified form of human NGF devoid of algogenic activity (painless NGF)., Key Results: In rodent hippocampal slices exposed to oxygen and glucose deprivation, CHF6467 protected neurons from death and reverted neurotransmission impairment when combined with hypothermia. In a model of rat neonatal HIE, intranasal CHF6467 (20 μg kg -1 ) significantly reduced brain infarct volume versus vehicle when delivered 10 min or 3 h after the insult. CHF6467 (20 and 40 μg kg -1 , i.n.), significantly decreased brain infarct volume to a similar extent to TH and when combined, showed a synergistic neuroprotective effect. CHF6467 (20 μg kg -1 , i.n.) per se and in combination with hypothermia reversed locomotor coordination impairment (Rotarod test) and memory deficits (Y-maze and novel object recognition test) in the neonatal HIE rat model. Intranasal administration of CHF6467 resulted in meaningful concentrations in the brain, blunted HIE-induced mRNA elevation of brain neuroinflammatory markers and, when combined to TH, significantly counteracted the increase in plasma levels of neurofilament light chain, a peripheral marker of neuroaxonal damage., Conclusion and Implications: CHF6467 administered intranasally is a promising therapy, in combination with TH, for the treatment of HIE., (© 2024 British Pharmacological Society.)

Background: Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory condition. Given patients with COPD continue to experience exacerbations despite the availability of effective therapies, anti-inflammatory treatments targeting novel pathways are needed. Kinases, notably the phosphoinositide 3-kinases (PI3K), are thought to be involved in chronic airway inflammation, with this pathway proposed as a critical regulator of inflammation and oxidative stress response in COPD. CHF6523 is an inhaled PI3Kδ inhibitor that has shown positive preclinical results. This manuscript reports the results of a study of CHF6523 in patients with stable COPD (chronic bronchitis phenotype), and who had evidence of type-2 inflammation., Methods: This randomised, double-blind, placebo-controlled, two-way crossover study comprised two 28-day treatment periods separated by a 28-day washout. Patients (N = 44) inhaled CHF6523 in one period, and placebo in the other, both twice daily. The primary objective was to assess the safety and tolerability of CHF6523; the secondary objective was to assess CHF6523 pharmacokinetics. Exploratory endpoints included target engagement (the relative reduction in phosphatidylinositol (3,4,5)-trisphosphate [PIP 3 ]), pharmacodynamic evaluations such as airflow obstruction, and hyperinflation, and to identify biomarker(s) of drug response using proteomics and transcriptomics., Results: CHF6523 plasma pharmacokinetics were characterised by an early maximum concentration (C max ), reached 15 and 10 min after dosing on Days 1 and 28, respectively, followed by a rapid decline. Systemic exposure on Day 28 showed limited accumulation, with ratios < 1.6 for C max and area under the curve from 0 to 12 h post-dose, and with steady state achieved on Day 20. Target engagement was confirmed by a significant 29.7% reduction from baseline in induced sputum PIP 3 (29.5% reduction vs. placebo; adjusted ratio 0.705 [0.580, 0.856]; p = 0.001), but this did not translate into an anti-inflammatory pharmacodynamic effect, as assessed through measures including biomarkers and multi-omics. Additionally, although CHF6523 was generally well-tolerated, 95.2% of patients reported cough as an adverse event, most mild to moderate and resolving within one-hour post-dose., Conclusions: These data, together with those from other PI3K inhibitors, suggest that PI3Kδ is not a suitable pathway for the management of COPD, as the achieved target engagement did not translate into any pharmacodynamic anti-inflammatory effect., Trial Registration: ClinicalTrials.gov (NCT04032535); posted 23rd July 2019., (© 2024. The Author(s).)

We report the characterization of potent and selective ROCK inhibitors identified through a core-hopping strategy. A virtual screening workflow, combining ligand- and structure-based methods, was applied on a known series of ROCK inhibitors bearing an acetamido-thiazole scaffold. The most promising replacement of the central core was represented by a benzoazepinone ring, which was selected as a starting point for a subsequent chemical exploration. The overall design approach exploited previous SARs available for congeneric series and crystallographic information to optimize the hinge-binding group as well as the terminal aromatic moiety interacting with the glycine-rich loop. The introduction of elongated and flexible charged groups led to compound 15, which exhibited sub-nanomolar potencies in biochemical and cellular assays, as well as a remarkable selectivity over PKA. HDX studies not only supported the postulated binding mode of compound 15 but also confirmed the crucial role of specific ROCK peptide segments in driving ligand selectivity., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: Premature birth, perinatal inflammation, and life-saving therapies such as postnatal oxygen and mechanical ventilation are strongly associated with the development of bronchopulmonary dysplasia (BPD); these risk factors, alone or combined, cause lung inflammation and alter programmed molecular patterns of normal lung development. The current knowledge on the molecular regulation of lung development mainly derives from mechanistic studies conducted in newborn rodents exposed to postnatal hyperoxia, which have been proven useful but have some limitations., Methods: Here, we used the rabbit model of BPD as a cost-effective alternative model that mirrors human lung development and, in addition, enables investigating the impact of premature birth per se on the pathophysiology of BPD without further perinatal insults (e.g., hyperoxia, LPS-induced inflammation). First, we characterized the rabbit's normal lung development along the distinct stages (i.e., pseudoglandular, canalicular, saccular, and alveolar phases) using histological, transcriptomic and proteomic analyses. Then, the impact of premature birth was investigated, comparing the sequential transcriptomic profiles of preterm rabbits obtained at different time intervals during their first week of postnatal life with those from age-matched term pups., Results: Histological findings showed stage-specific morphological features of the developing rabbit's lung and validated the selected time intervals for the transcriptomic profiling. Cell cycle and embryo development, oxidative phosphorylation, and WNT signaling, among others, showed high gene expression in the pseudoglandular phase. Autophagy, epithelial morphogenesis, response to transforming growth factor β, angiogenesis, epithelium/endothelial cells development, and epithelium/endothelial cells migration pathways appeared upregulated from the 28th day of gestation (early saccular phase), which represents the starting point of the premature rabbit model. Premature birth caused a significant dysregulation of the inflammatory response. TNF-responsive, NF-κB regulated genes were significantly upregulated at premature delivery and triggered downstream inflammatory pathways such as leukocyte activation and cytokine signaling, which persisted upregulated during the first week of life. Preterm birth also dysregulated relevant pathways for normal lung development, such as blood vessel morphogenesis and epithelial-mesenchymal transition., Conclusion: These findings establish the 28-day gestation premature rabbit as a suitable model for mechanistic and pharmacological studies in the context of BPD., (© 2023. The Author(s).)

Pulmonary surfactant (PS) has been proposed as an efficient drug delivery vehicle for inhaled therapies. Its ability to adsorb and spread interfacially and transport different drugs associated with it has been studied mainly by different surface balance designs, typically interconnecting various compartments by interfacial paper bridges, mimicking in vitro the respiratory air-liquid interface. It has been demonstrated that only a monomolecular surface layer of PS/drug is able to cross this bridge. However, surfactant films are typically organized as multi-layered structures associated with the interface. The aim of this work was to explore the contribution of surface-associated structures to the spreading of PS and the transport of drugs. We have designed a novel vehiculization balance in which donor and recipient compartments are connected by a whole three-dimensional layer of liquid and not only by an interfacial bridge. By combining different surfactant formulations and liposomes with a fluorescent lipid dye and a model hydrophobic drug, budesonide (BUD), we observed that the use of the bridge significantly reduced the transfer of lipids and drug through the air-liquid interface in comparison to what can be spread through a fully open interfacial liquid layer. We conclude that three-dimensional structures connected to the surfactant interfacial film can provide an important additional contribution to interfacial delivery, as they are able to transport significant amounts of lipids and drugs during surfactant spreading.

Thiazolidinediones (TZDs) are potent PPARγ agonists that have been shown to attenuate alveolar simplification after prolonged hyperoxia in term rodent models of bronchopulmonary dysplasia. However, the pulmonary outcomes of postnatal TZDs have not been investigated in preterm animal models. Here, we first investigated the PPARγ selectivity, epithelial permeability, and lung tissue binding of three types of TZDs in vitro (rosiglitazone (RGZ), pioglitazone, and DRF-2546), followed by an in vivo study in preterm rabbits exposed to hyperoxia (95% oxygen) to investigate the pharmacokinetics and the pulmonary outcomes of daily RGZ administration. In addition, blood lipids and a comparative lung proteomics analysis were also performed on Day 7. All TZDs showed high epithelial permeability through Caco-2 monolayers and high plasma and lung tissue binding; however, RGZ showed the highest affinity for PPARγ. The pharmacokinetic profiling of RGZ (1 mg/kg) revealed an equivalent biodistribution after either intratracheal or intraperitoneal administration, with detectable levels in lungs and plasma after 24 h. However, daily RGZ doses of 1 mg/kg did not improve lung function in preterm rabbits exposed to hyperoxia, and daily 10 mg/kg doses were even associated with a significant lung function worsening, which could be partially explained by the upregulation of lung inflammation and lipid metabolism pathways revealed by the proteomic analysis. Notably, daily postnatal RGZ produced an aberrant modulation of serum lipids, particularly in rabbit pups treated with the 10 mg/kg dose. In conclusion, daily postnatal RGZ did not improve lung function and caused dyslipidemia in preterm rabbits exposed to hyperoxia.

Pulmonary surfactant is a complex lipoprotein mixture secreted into the alveolar lumen by type 2 pneumocytes, which is composed by tens of different lipids (approximately 90% of its entire mass) and surfactant proteins (approximately 10% of the mass). It is crucially involved in maintaining lung homeostasis by reducing the values of alveolar liquid surface tension close to zero at end-expiration, thereby avoiding the alveolar collapse, and assembling a chemical and physical barrier against inhaled pathogens. A deficient amount of surfactant or its functional inactivation is directly linked to a wide range of lung pathologies, including the neonatal respiratory distress syndrome. This paper reviews the main biophysical concepts of surfactant activity and its inactivation mechanisms, and describes the past, present and future roles of surfactant replacement therapy, focusing on the exogenous surfactant preparations marketed worldwide and new formulations under development. The closing section describes the pulmonary surfactant in the context of drug delivery. Thanks to its peculiar composition, biocompatibility, and alveolar spreading capability, the surfactant may work not only as a shuttle to the branched anatomy of the lung for other drugs but also as a modulator for their release, leading to innovative therapeutic avenues for the treatment of several respiratory diseases., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Corticosteroids as budesonide can be effective in reducing topic inflammation processes in different organs. Therapeutic use of budesonide in respiratory diseases, like asthma, chronic obstructive pulmonary disease, and allergic rhinitis is well known. However, the pulmonary distribution of budesonide is not well understood, mainly due to the difficulties in tracing the molecule in lung samples without the addition of a label. In this paper, we present a matrix-assisted laser desorption/ionization mass spectrometry imaging protocol that can be used to visualize the pulmonary distribution of budesonide administered to a surfactant-depleted adult rabbit. Considering that budesonide is not easily ionized by MALDI, we developed an on-tissue derivatization method with Girard's reagent P followed by ferulic acid deposition as MALDI matrix. Interestingly, this sample preparation protocol results as a very effective strategy to raise the sensitivity towards not only budesonide but also other corticosteroids, allowing us to track its distribution and quantify the drug inside lung samples.

Direct lung administration of budesonide in combination with surfactant reduces the incidence of bronchopulmonary dysplasia. Although the therapy is currently undergoing clinical development, the lung distribution of budesonide throughout the premature neonatal lung has not yet been investigated. Here, we applied mass spectrometry imaging (MSI) to investigate the surfactant-assisted distal lung distribution of budesonide. Unlabeled budesonide was either delivered using saline as a vehicle ( n = 5) or in combination with a standard dose of the porcine surfactant Poractant alfa ( n = 5). These lambs were ventilated for one minute, and then the lungs were extracted for MSI analysis. Another group of lambs ( n = 5) received the combination of budesonide and Poractant alfa, followed by two hours of mechanical ventilation. MSI enabled the label-free detection and visualization of both budesonide and the essential constituent of Poractant alfa, the porcine surfactant protein C (SP-C). 2D ion intensity images revealed a non-uniform distribution of budesonide with saline, which appeared clustered in clumps. In contrast, the combination therapy showed a more homogeneous distribution of budesonide throughout the sample, with more budesonide distributed towards the lung periphery. We found similar distribution patterns for the SP-C and budesonide in consecutive lung tissue sections, indicating that budesonide was transported across the lungs associated with the exogenous surfactant. After two hours of mechanical ventilation, the budesonide intensity signal in the 2D ion intensity maps dropped dramatically, suggesting a rapid lung clearance and highlighting the relevance of achieving a uniform surfactant-assisted lung distribution of budesonide early after delivery to maximize the anti-inflammatory and maturational effects throughout the lung.

CysE and CysK, the last two enzymes of the cysteine biosynthetic pathway, engage in a bienzyme complex, cysteine synthase, with yet incompletely characterized three-dimensional structure and regulatory function. Being absent in mammals, the two enzymes and their complex are attractive targets for antibacterial drugs. We have used hydrogen/deuterium exchange MS to unveil how complex formation affects the conformational dynamics of CysK and CysE. Our results support a model where CysE is present in solution as a dimer of trimers, and each trimer can bind one CysK homodimer. When CysK binds to one CysE monomer, intratrimer allosteric communication ensures conformational and dynamic symmetry within the trimer. Furthermore, a long-range allosteric signal propagates through CysE to induce stabilization of the interface between the two CysE trimers, preparing the second trimer for binding the second CysK with a nonrandom orientation. These results provide new molecular insights into the allosteric formation of the cysteine synthase complex and could help guide antibacterial drug design., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Poractant alfa and Calsurf are two natural surfactants widely used in China for the treatment of neonatal respiratory distress syndrome, which are extracted from porcine and calf lungs, respectively. The purpose of this experimental study was to compare their in vitro characteristics and in vivo effects in the improvement of pulmonary function and protection of lung injury. The biophysical properties, ultrastructure, and lipid composition of both surfactant preparations were respectively analysed in vitro by means of Langmuir-Blodgett trough (LBT), atomic force microscopy (AFM), and liquid-chromatography mass-spectrometry (LC-MS). Then, as core pharmacological activity, both head-to-head (100 and 200 mg/kg for both surfactants) and licensed dose comparisons (70 mg/kg Calsurf vs. 200 mg/kg Poractant alfa) between the two surfactants were conducted as prophylaxis in preterm rabbits with primary surfactant deficiency, assessing survival time and rate and dynamic compliance of the respiratory system (Cdyn). Intrapulmonary surfactant pools, morphometric volume density as alveolar expansion (Vv), and lung injury scores were determined post mortem. AFM and LC-MS analysis revealed qualitative differences in the ultrastructure as well as in the lipid composition of both preparations. Calsurf showed a longer plateau region of the LBT isotherm and lower film compressibility. In vivo, both surfactant preparations improved Cdyn at any dose, although maximum benefits in terms of Vv and intrapulmonary surfactant pools were seen with the 200 mg/kg dose in both surfactants. The group of animals treated with 200 mg/kg of Poractant alfa showed a prolonged survival time and rate compared to untreated but ventilated controls, and significantly ameliorated lung injury compared to Calsurf at any dose, including 200 mg/kg. The overall outcomes suggest the pulmonary effects to be dose dependent for both preparations. The group of animals treated with 200 mg/kg of Poractant alfa showed a significant reduction of mortality. Compared to Calsurf, Poractant alfa exerted better effects if licensed doses were compared, which requires further investigation., Competing Interests: The study was supported by Chiesi Farmaceutici S.p.A, which owns the marketing rights for Poractant alfa. The company contributed to the study design but had neither influence on the performance, analysis, and interpretation of experimental data nor in writing the manuscript. BP, SC, and FS are employees of Chiesi Farmaceutici S.p.A. XM served as consultant for Chiesi in this study. YD is a recipient of research grants from the National Natural Science Foundation (No. 81501288) and Shanghai Municipal Commission of Health (Project Young Physician Investigator). This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Background: The amount of surfactant deposited in the lungs and its overall pulmonary distribution determine the therapeutic outcome of surfactant replacement therapy. Most of the currently available methods to determine the intrapulmonary distribution of surfactant are time-consuming and require surfactant labelling. Our aim was to assess the potential of Mass Spectrometry Imaging (MSI) as a label-free technique to qualitatively and quantitatively evaluate the distribution of surfactant to the premature lamb., Methods: Twelve preterm lambs (gestational age 126-127d, term ~150d) were allocated in two experimental groups. Seven lambs were treated with an intratracheal bolus of the synthetic surfactant CHF5633 (200 mg/kg) and 5 lambs were managed with mechanical ventilation for 120 min, as controls. The right lung lobes of all lambs were gradually frozen while inflated to 20 cmH 2 O pressure for lung cryo-sections for MSI analysis. The intensity signals of SP-C analog and SP-B analog, the two synthetic peptides contained in the CHF5633 surfactant, were used to locate, map and quantify the intrapulmonary exogenous surfactant., Results: Surfactant treatment was associated with a significant improvement of the mean arterial oxygenation and lung compliance (p < 0.05). Nevertheless, the physiological response to surfactant treatment was not uniform across all animals. SP-C analog and SP-B analog were successfully imaged and quantified by means of MSI in the peripheral lungs of all surfactant-treated animals. The intensity of the signal was remarkably low in untreated lambs, corresponding to background noise. The signal intensity of SP-B analog in each surfactant-treated animal, which represents the surfactant distributed to the peripheral right lung, correlated well with the physiologic response as assessed by the area under the curves of the individual arterial partial oxygen pressure and dynamic lung compliance curves of the lambs., Conclusions: Applying MSI, we were able to detect, locate and quantify the amount of exogenous surfactant distributed to the lower right lung of surfactant-treated lambs. The distribution pattern of SP-B analog correlated well with the pulmonary physiological outcomes of the animals. MSI is a valuable label-free technique which is able to simultaneously evaluate qualitative and quantitative drug distribution in the lung.

CHF5633 (Chiesi Farmaceutici, Italy) is a synthetic surfactant developed for respiratory distress syndrome replacement therapy in pre-term newborn infants. CHF5633 contains two phospholipids (dipalmitoylphosphatidylcholine and 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoglycerol sodium salt), and peptide analogues of surfactant protein C (SP-C analogue) and surfactant protein B (SP-B analogue). Both proteins are fundamental for an optimal surfactant activity in vivo and SP-B genetic deficiency causes lethal respiratory failure after birth. Fluorescence emission of the only tryptophan residue present in SP-B analogue (SP-C analogue has none) could in principle be exploited to probe SP-B analogue conformation, localization and interaction with other components of the pharmaceutical formulation. However, the high light scattering activity of the multi-lamellar vesicles suspension characterizing the pharmaceutical surfactant formulation represents a challenge for such studies. We show here that quenching of tryptophan fluorescence and Singular Value Decomposition analysis can be used to accurately calculate and subtract background scattering. The results indicate, with respect to Trp microenvironment, a conformationally homogeneous population of SP-B. Trp is highly accessible to the water phase, suggesting a surficial localization on the membrane of phospholipid vesicles, similarly to what observed for full length SP-B in natural lung surfactant, and supporting an analogous role in protein anchoring to the lipid phase., Competing Interests: The following authors are Chiesi Farmaceutici full-time employees: Barbara Pioselli, Fabrizio Salomone and Silvia Catinella. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

BackgroundThe intratracheal (IT) administration of budesonide using surfactant as a vehicle has been shown to reduce the incidence of bronchopulmonary dysplasia (BPD) in preterm infants. The objective of this study was to characterize the in vitro characteristics and in vivo safety and efficacy of the extemporaneous combination of budesonide and poractant alfa.MethodsThe stability, minimum surface tension, and viscosity of the preparation were evaluated by means of high-performance liquid chromatography (HPLC), Wilhelmy balance, and Rheometer, respectively. The safety and efficacy of the IT administration of the mixture were tested in two respiratory distress syndrome (RDS) animal models: twenty-seventh day gestational age premature rabbits and surfactant-depleted adult rabbits.ResultsA pre-formulation trial identified a suitable procedure to ensure the homogeneity and stability of the formulation. Wilhelmy Balance tests clarified that budesonide supplementation has no detrimental effect on poractant alfa surface tension activity. The addition of budesonide to poractant alfa did not affect the physiological response to surfactant treatment in both RDS animal models, and was associated to a significant reduction of lung inflammation in surfactant-depleted rabbits.ConclusionOur in vitro and in vivo analysis suggests that the IT administration of a characterized extemporaneous combination of poractant alfa and budesonide is a safe and efficacious procedure in the context of RDS.

Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT*) that is pH insensitive, stabilized and hypersoluble compared to wild-type NT. NT*-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT* enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT* also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.

Nasal continuous positive airway pressure (nCPAP) is a widely accepted technique of non-invasive respiratory support in spontaneously-breathing premature infants with respiratory distress syndrome (RDS). Surfactant administration techniques compatible with nCPAP ventilation strategy are actively investigated. Our aim is to set up and validate a respiratory distress animal model that can be managed on nCPAP suitable for surfactant administration techniques studies. Surfactant depletion was induced by bronchoalveolar lavages (BALs) on 18 adult rabbits. Full depletion was assessed by surfactant component analysis on the BALs samples. Animals were randomized into two groups: Control group (nCPAP only) and InSurE group, consisting of a bolus of surfactant (Poractant alfa, 200 mg/kg) followed by nCPAP. Arterial blood gases were monitored until animal sacrifice, 3 hours post treatment. Lung mechanics were evaluated just before and after BALs, at the time of treatment, and at the end of the procedure. Surfactant phospholipids and protein analysis as well as surface tension measurements on sequential BALs confirmed the efficacy of the surfactant depletion procedure. The InSurE group showed a significant improvement of blood oxygenation and lung mechanics. On the contrary, no signs of recovery were appreciated in animals treated with just nCPAP. The surfactant-depleted adult rabbit RDS model proved to be a valuable and efficient preclinical tool for mimicking the clinical scenario of preterm infants affected by mild/moderate RDS who spontaneously breathe and do not require mechanical ventilation. This population is of particular interest as potential target for the non-invasive administration of surfactant., Competing Interests: Ricci F, Catozzi C, Bianco F, Catinella S, Villetti G, Civelli M, Pioselli B, Salomone F are employees of Chiesi Farmaceutici S.p.A. Murgia X, Lorenzini L, Dani C served as consultant for Chiesi Farmaceutici S.p.A. in the present work. Commercial affiliations of authors FR, CC, BR, LL, FB, SC, GV, BP, FS does not alter their adherence to PLOS ONE policies on sharing data and materials.

Background: The development of synthetic surfactants for the treatment of lung pulmonary diseases has been going on for many years., Objectives: To investigate the effects of phospholipid mixtures combined with SP-B and SP-C analogues on lung functions in an animal model of respiratory distress syndrome., Methods: Natural and synthetic phospholipid mixtures with/without SP-B and/or SP-C analogues were instilled in ventilated premature newborn rabbits. Lung functions were evaluated., Results: Treatment with Curosurf or phospholipids from Curosurf combined with SP-B and SP-C analogues gave similar results. Treatment with phospholipids from adult rabbit lungs or liver combined with dipalmitoylphosphatidylcholine (DPPC) and palmitoyloleoylphosphatidylglycerol (POPG) gave tidal volumes (VT) well above physiological levels, but alveolar stability at end-expiration was only achieved when these phospholipids were combined with analogues of SP-B and SP-C. Treatment with egg yolk-PC mixed with DPPC with and without POPG gave small VT, but after addition of both analogues VT was only somewhat lower and lung gas volumes (LGV) similar to those obtained with Curosurf. Substitution of egg yolk-PC (≥99% PC) with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, and combining them with DPPC, POPG and 2% each of the SP-B and SP-C analogue gave a completely synthetic surfactant with similar effects on VT and LGV as Curosurf., Conclusions: Phospholipid composition is important for VT while the SP-B and SP-C analogues increase alveolar stability at end-expiration. Synthetic surfactant consisting of unsaturated and saturated phosphatidylcholines, POPG and the analogues of SP-B and SP-C has similar activity as Curosurf regarding VT and LGV in an animal model using preterm newborn rabbits ventilated without positive end-expiratory pressure., (© 2016 S. Karger AG, Basel.)

Surfactant protein C (SP-C) is deemed as the surfactant protein most specifically expressed in type II alveolar epithelial cells and plays an important role in surfactant function. SP-C turnover in humans and its meaning in the clinical context have never been approached. In this study, we used mass spectrometry to investigate SP-C turnover in humans. We studied four infants and eight adults requiring mechanical ventilation. All patients had no lung disease. Patients received a 24-h continuous infusion of (13)C-leucine as precursor of SP-C, and serial tracheal aspirates and plasma samples were obtained every 6 h till 48 h. SP-C was isolated from tracheal aspirates by sorbent-phase chromatography. (13)C-leucine SP-C enrichment could be successfully measured in three infant and in four adult samples by using mass spectrometry coupled with a gas chromatographer. Median SP-C fractional synthesis rate, secretion time, and peak time were 15.7 (14.1-27.5)%/day, 6.0 (4.7-11.5) h, and 24 (20-27) h. In conclusion, this study shows that it is feasible to accurately determine SP-C turnover in humans by stable isotopes.

The armory of GFP mutants available to biochemists and molecular biologists is huge. Design and selection of mutants are usually driven by tailored spectroscopic properties, but some key aspects of stability, folding and dynamics of selected GFP variants still need to be elucidated. We have prepared, expressed and characterized three H148 mutants of the highly fluorescent variant GFPmut2. H148 is known to be involved in the H-bonding network surrounding the chromophore, and all the three mutants, H148G, H148R and H148K, show increased pKa values of the chromophore. Only H148G GFPmut2 (Mut2G) gave good expression and purification yields, indicating that position 148 is critical for efficient folding in vivo. The chemical denaturation of Mut2G was monitored by fluorescence emission, absorbance and far-UV circular dichroism spectroscopy. The mutation has little effect on the spectroscopic properties of the protein and on its stability in solution. However, the unfolding kinetics of the protein encapsulated in wet nanoporous silica gels, a system that allows to stabilize conformations that are poorly or only transiently populated in solution, indicate that the unfolding pathway of Mut2G is markedly different from the parent molecule. In particular, encapsulation allowed to identify an unfolding intermediate that retains a native-like secondary structure despite a destructured chromophore environment. Thus, H148 is a critical residue not only for the chromophoric and photodynamic properties, but also for the correct folding of GFP, and its substitution has great impact on expression yields and stability of the mature protein., (Copyright © 2013 Elsevier B.V. All rights reserved.)

During the last decade, significant technological improvements in mass spectrometry have had a great impact on drug discovery. The development of matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has set a new frontier for the study of the distribution of endogenous and exogenous molecules present within a tissue. MALDI-IMS is a surface sampling technique that allows not only the detection of multiple analytes but also gives the spatial distribution of those analytes. Active compounds for pulmonary disease need an optimal and well-studied delivery into the lungs, in order to assure distribution with greater penetration into the peripheral or the alveolar region of the lung to maximize the therapeutic effects. IMS is very useful in the field of drug discovery, showing drug delivery and distribution in the body and organs. In this study, we present a comparison between two different ways of carrying out pulmonary drug administration: inhalation of a nebulized aerosol of aqueous drug solutions and intratracheal administration, which is much simpler, not expensive and commonly used during in vivo screening. Tiotropium bromide is a long-acting anticholinergic medicine used for maintenance treatment of chronic obstructive pulmonary disease. In the present work, tiotropium was administered by nebulization and by intratracheal instillation to guinea pigs at doses able to induce significant anti-bronchoconstrictive activity. Lung samples were dissected, frozen, cryosectioned and coated with matrix (α-hydroxy-cinnamic acid). IMS analyses were performed using a MALDI-LTQ-Orbitrap XL. Using this technique we were able to compare different distributions of the drug depending on the method of administration.

The industrial production of cooked ham from pork meat involves, as initial steps, the injection of brine and a prolonged meat massage. These processes strongly affect the quality of the final product because they determine the breakage of muscle cells and the release of their protein content. The produced dense exudates act as a glue in the final cooked ham. In order to exploit modern tools to direct the technological process, still mainly based on empirical observations and traditional recipes, we have carried out a comprehensive proteomic analysis of the exudates as a function of brine concentration, temperature, and length of meat massage. Each condition was found to generate specific protein patterns. Peptide mass fingerprinting analysis was applied allowing the identification of proteins, whose presence and/or quantity can be defined as biomarkers of meat processing, and, potentially, of final product quality.

Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Polyethylene glycol (PEG)-conjugated hemoglobins, a novel class of blood substitutes, were investigated by a combination of native and denaturing one- and two-dimensional polyacrylamide gel electrophoresis (PAGE) coupled with the microspectrophotometric characterization of single bands and the functional analysis of electrophoretically separated fractions. For these intrinsically heterogeneous products, the molecular mass, the size distribution, and the degree of PEGylation are strictly correlated to their side effects and, therefore, are crucial pieces of information to evaluate their safety and efficacy. The PEGylation pattern was shown to strongly depend on the quaternary conformation of hemoglobin during the reaction, and the degree of conjugation was shown to correlate with the oxygen binding properties of the individual electrophoretically separated fractions. Moreover, small but not negligible fractions of underivatized tetramers, known to be responsible for serious side effects, were detected even in preparations with a high average degree of PEGylation. Overall, this approach might be exploited to characterize other products of protein PEGylation, an increasingly relevant technology for the optimization of the pharmacokinetic properties of protein-based drugs., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Chemically or genetically modified hemoglobins are a therapeutic class indicated for the treatment of a variety of hypo-oxygenation pathologies, severe trauma-related hemorrhages or elective surgery when blood transfusions are refused or not available. Recombinant heterologous hemoglobins offer the possibility of a potentially unlimited production and genetically optimized properties in terms of oxygen affinity, NO reactivity and resistance to autoxidation. Hemoglobin Polytaur is an autopolymerizing human-bovine hybrid mutant, previously obtained as a 500kDa polymer, shown to reduce the infarct volume from focal cerebral ischemia in in vivo animal models. In this work, hemoglobin Polytaur polymerization, carried out under conditions to minimize heme oxidation and modification, resulted in a 180kDa cyclic homogeneous trimer of hemoglobin tetramers. This novel oligomer was characterized by electrophoresis, MALDI-TOF mass spectrometry and gel filtration. The size and the oxygen binding properties were shown to be ideally suited for its use as a blood substitute. Co-expression with the human α hemoglobin-stabilizing protein (AHSP), a chaperone that assists hemoglobin folding in vivo, resulted in an unexpected decrease in yield and in unusual spectroscopic and functional properties, suggesting the formation of strong protein-protein interactions that reduce the expression, hinder the tetramer assembly and prevent purification., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Here we report for the first time the use of species-specific isotope dilution mass spectrometry for the absolute quantification of a metalloprotein using nondenaturing gel electrophoresis laser ablation inductively coupled plasma mass spectrometry (GE-LA-ICP-MS). The concept utilises the intrinsic metals of the metalloprotein for labelling of the isotopically labelled spike ((65)Cu, (68)Zn SOD). The stability of the metal-protein complex under non-denaturing conditions during 1-D PAGE was confirmed and the performance of the method evaluated. Between 4 and 64 microg, SOD was quantified with a recovery rate between 82% and 110% in a standard. The use of the isotopically enriched SOD was utilised to identify the extent of orthogonal diffusion in 1-D gel electrophoresis. Orthogonal diffusion of natural and isotopically enriched SOD in the gel can interfere with the correct determination of the isotope ratios. The matrix effect of a cytosolic liver extract on the non-covalently bound copper and zinc in SOD was evaluated and no significant metal loss from the SOD spike was observed. This study represents the first step necessary for establishing and evaluating the use of a species-specific isotope dilution approach for the absolute quantification of SOD in real samples based on the combination of gel electrophoresis and LA-ICP-MS.

Metals bound to proteins play essential roles in living systems. Elements such as phosphorus, selenium and iodine are commonly covalently linked to proteins while others are non-covalently complexed. Thus, the identification and characterization of the metal-protein complexes require a careful hyphenation of techniques able to separate and detect the intact binding complexes with both high resolution and high sensitivity. This study has investigated for the first time the potential of microsolution isoelectric focussing to separate a mixture of metal-binding protein standards under well-established denaturing conditions and a novel non-denaturing separation protocol has also been developed. SEC-ICP-MS analysis was used to evaluate the ability of the two separation procedures to separate and maintain the integrity of standard metal-protein complexes. Microsolution isoelectric focussing under denaturing conditions separates the metalloprotein mixtures with high resolution, although the stability of the complexes is affected. Microsolution isoelectric focussing under our newly developed non-denaturing conditions shows a lower degree of resolution, although the stability of the metal-protein complexes is preserved. The applicability of the two procedures to a biological metalloproteome has also been evaluated.

Hemoglobin conjugated with poly(ethylene glycol) (PEG) acts as an oxygen carrier free in plasma, substituting red blood cells in supplementing oxygen in hypo-oxygenation pathologies. Given the complexity of oxygen delivery controls, subtle structural and functional differences in PEGylated hemoglobins might be associated with distinct physiological responses and, potentially, adverse effects. We have compared hemoglobin PEGylated under anaerobic conditions, called PEG-Hb(deoxy), with hemoglobin PEGylated under aerobic conditions, called PEG-Hb(oxy), a product that mimics Hemospan, produced by Sangart, Inc. SDS PAGE and MALDI-TOF analyses demonstrated that PEG conjugation yields products characterized by a broad distribution of PEG/hemoglobin ratios. The elution profiles in size-exclusion chromatography indicate that both products exhibit a more homogeneous distribution of molecular weight/hydrodynamic volume under deoxy conditions and at higher concentrations. PEG-Hb(oxy) shows high oxygen affinity, low modulation of allosteric effectors, almost no cooperativity, a fast and monophasic CO binding, and a limited dependence of functional properties on concentration, whereas PEG-Hb(deoxy) exhibits oxygen binding curves that significantly depend on protein concentration, and a slow CO binding, similar to native hemoglobin. PEGylated CO-hemoglobins, probed by flash photolysis, exhibited a lower amplitude for the geminate rebinding phase with respect to native hemoglobin and a negligible T state bimolecular CO rebinding phase. These findings are explained by an increased dissociation of PEGylated hemoglobins into dimers and perturbed T and R states with decreased quaternary transition rates. These features are more pronounced for PEG-Hb(oxy) than PEG-Hb(deoxy). The detected heterogeneity might be a source of adverse effects when PEGylated Hbs are used as blood substitutes.

Although laser ablation (LA)-ICP-MS has been reported for the determination of metalloproteins separated by gel electrophoretic techniques (GE), systematic studies that define the conditions essential for successful measurements are still scarce. In this paper we present the results of our studies of basic conditions for the effective application of GE-LA-ICP-MS for the separation of metal-binding proteins, focusing on their stability during GE and post-separation gel treatment. The stability of metal-protein complexes (haemoglobin, myoglobin, superoxide dismutase, carbonic anhydrase, transferrin, albumin, cytochrome c) during GE is dependent on the nature of the metal-protein interaction and the principle of separation. We have observed that non-denaturing GE is a suitable separation technique for most metal-protein complexes (e.g. Zn in carbonic anhydrase and Fe in Tf and myoglobin were quantitatively recovered in a spiked liver cytosol), whereas separation by denaturing GE strongly impaired the stability of the complexes. Equally important is the post-separation treatment of the gel to enable successful detection of the metal. LA-ICP-MS requires drying of the gel without loss of protein-bound metal or cracking of the gel. This was successfully achieved using glycerol followed by heating. We demonstrate that staining of the gel prior to LA-ICP-MS using silver or Coomassie blue is not recommended, since most protein-bound metal is lost during the staining procedure. Furthermore it has been shown that only line scanning with a speed of less than 30 microm/s can reliably distinguish between lines 1 mm apart, while raster spot analysis carries the risk of misinterpretation due to contamination in/on inhomogeneous gels.

The development of methods to analyze accurately and precisely individual metalloproteins is of increasing importance. Here we describe for the first time the chemical preparation and characterization of an isotopically enriched metalloenzyme containing two different metal isotopes. Its evaluation as a standard in species-specific isotope dilution analysis by HPLC coupled to inductively coupled plasma mass spectrometry is carefully evaluated. Our model enzyme bovine superoxide dismutase (SOD) contains both Cu and Zn and is remarkably stable at high temperatures and even under denaturing conditions. The enzyme's metal cofactors were removed under a range of different conditions and replaced with isotopically enriched 65Cu and 68Zn. Depending on the conditions, various isotopic ratios differing from the natural Cu and Zn abundances were obtained for the reconstituted enzyme. Both the wild type and isotopically enriched enzyme had the same migration pattern on native 1D-PAGE. Using an enzyme activity test, we showed that the incorporated 65Cu was bound to the right SOD-binding site, since the measured activity correlated directly with the amount of Cu incorporated. Mixing the native and the isotopically enriched enzyme standard with free enriched 65Cu and 68Zn or a metal chelator did not result in any exchange or loss of the metals from the enzyme at neutral pH. This verifies the stability of the enzyme metal center under the chosen conditions. The isotopically enriched enzyme standard was spiked into a wild type SOD solution to evaluate its use for species-specific isotope dilution experiments. To our knowledge, this is the first report of the chemical preparation of a metalloenzyme containing two different isotopically enriched metals. We provide evidence that the incorporated isotopically enriched metals are bound to the right binding site of SOD using an specific enzymatic activity assay.

Biological molecules experience in vivo a highly crowded environment. The investigation of the functional properties of the tryptophan synthase alpha(2)beta(2) complex either entrapped in wet nanoporous silica gels or in the presence of the crowding agents dextran 70 and ficoll 70 indicates that the rates of the conformational transitions associated to catalysis and regulation are reduced, and an open and less catalytically active conformation is stabilized.

The pyridoxal 5'-phosphate-dependent enzymes tyrosine phenol-lyase and tryptophan indole-lyase were encapsulated in wet nanoporous silica gels, a powerful method to selectively stabilize tertiary and quaternary protein conformations and to develop bioreactors and biosensors. A comparison of the enzyme reactivity in silica gels and in solution was carried out by determining equilibrium and kinetic parameters, exploiting the distinct spectral properties of catalytic intermediates and reaction products. The encapsulated enzymes exhibit altered distributions of ketoenamine and enolimine tautomers, increased values of inhibitors dissociation constants, slow attaining of steady-state in the presence of substrate and substrate analogs, modified steady-state distribution of catalytic intermediates, and a sixfold-eightfold decrease of specific activities. This behavior can be rationalized by a reduced conformational flexibility for the encapsulated enzymes and a selective stabilization of either the open (inactive) or the closed (active) form of the enzymes. Despite very similar structures and catalytic mechanisms, the influence of encapsulation is more pronounced for tyrosine phenol-lyase than tryptophan indole-lyase. This finding indicates that subtle structural and dynamic differences can lead to distinct interactions of the protein with the gel matrix.

The pyridoxal 5'-phosphate (PLP)-dependent tryptophan synthase is a alpha(2)beta(2) complex. The alpha-beta subunit interaction plays a critical role both in the reciprocal activation of the individual subunits and in the allosteric regulation. We have investigated whether mutations of alpha loop6 Gly(181) and beta helix6 Ser(178) affect intersubunit communication. The loss of the hydrogen bond between these residues, achieved by proline substitution, does not significantly influence the intersubunit catalytic activation, but completely abolishes ligand-induced intersubunit signaling. The comparison of the crystal structure of the wild type and beta Ser(178)Pro mutant, in the absence and presence of alpha-subunit ligands, indicates that the removal of the interaction between beta Ser(178) and alpha Gly(181) strongly affects the equilibrium between active (closed) and inactive (open) conformations of the alpha-active site, the latter being stabilized in both mutants.