Simultaneous production of biosurfactant and extracellular unspecific peroxygenases by Moesziomyces aphidis XM01 enables an efficient strategy for crude oil degradation.

Crude oil is a hazardous pollutant that poses significant and lasting harm to human health and ecosystems. In this study, Moesziomyces aphidis XM01, a biosurfactant mannosylerythritol lipids (MELs)-producing yeast, was utilized for crude oil degradation. Unlike most microorganisms relying on cytochrome P450, XM01 employed two extracellular unspecific peroxygenases, MaUPO.1 and MaUPO.2, with preference for polycyclic aromatic hydrocarbons (PAHs) and n-alkanes respectively, thus facilitating efficient crude oil degradation. The MELs produced by XM01 exhibited a significant emulsification activity of 65.9% for crude oil and were consequently supplemented in an "exogenous MELs addition" strategy to boost crude oil degradation, resulting in an optimal degradation ratio of 72.3%. Furthermore, a new and simple "pre-MELs production" strategy was implemented, achieving a maximum degradation ratio of 95.9%. During this process, the synergistic up-regulation of MaUPO.1, MaUPO.1 and the key MELs synthesis genes contributed to the efficient degradation of crude oil. Additionally, the phylogenetic and geographic distribution analysis of MaUPO.1 and MaUPO.1 revealed their wide occurrence among fungi in Basidiomycota and Ascomycota, with high transcription levels across global ocean, highlighting their important role in biodegradation of crude oil. In conclusion, M. aphidis XM01 emerges as a novel yeast for efficient and eco-friendly crude oil degradation.

Production, characterization, and application of Pseudoxanthomonas taiwanensis biosurfactant: a green chemical for microbial enhanced oil recovery (MEOR).

Biosurfactants, as microbial bioproducts, have significant potential in the field of microbial enhanced oil recovery (MEOR). Biosurfactants are microbial bioproducts with the potential to reduce the interfacial tension (IFT) between crude oil and water, thus enhancing oil recovery. This study aims to investigate the production and characterization of biosurfactants and evaluate their effectiveness in increasing oil recovery. Pseudoxanthomonas taiwanensis was cultured on SMSS medium to produce biosurfactants. Crude oil was found to be the most effective carbon source for biosurfactant production. The biosurfactants exhibited comparable activity to sodium dodecyl sulfate (SDS) at a concentration of 400 ppm in reducing IFT. It was characterized as glycolipids, showing stability in emulsions at high temperatures (up to 120 °C), pH levels ranging from 3 to 9, and NaCl concentrations up to 10% (w/v). Response surface methodology revealed the optimized conditions for the most stable biosurfactants (pH 7, temperature of 40 °C, and salinity of 2%), resulting in an EI24 value of 64.45%. Experimental evaluations included sand pack column and core flooding studies, which demonstrated additional oil recovery of 36.04% and 12.92%, respectively. These results indicate the potential application of P. taiwanensis biosurfactants as sustainable and environmentally friendly approaches to enhance oil recovery in MEOR processes.

A Rare Mono-Rhamnolipid Congener Efficiently Produced by Recombinant Pseudomonas aeruginosa YM4 via the Expression of Global Transcriptional Regulator irrE.

Rhamnolipids (RLs) are widely used biosurfactants produced mainly by Pseudomonas aeruginosa and Burkholderia spp. in the form of mixtures of diverse congeners. The global transcriptional regulator gene irrE from radiation-tolerant extremophiles has been widely used as a stress-resistant element to construct robust producer strains and improve their production performance. A PrhlA-irrE cassette was constructed to express irrE genes in the Pseudomonas aeruginosa YM4 of the rhamnolipids producer strain. We found that the expression of irrE of Deinococcus radiodurans in the YM4 strain not only enhanced rhamnolipid production and the strain's tolerance to environmental stresses, but also changed the composition of the rhamnolipid products. The synthesized rhamnolipids reached a maximum titer of 26 g/L, about 17.9% higher than the original, at 48 h. The rhamnolipid production of the recombinant strain was determined to be mono-rhamnolipids congener Rha-C10-C12, accounting for 94.1% of total products. The critical micelle concentration (CMC) value of the Rha-C10-C12 products was 62.5 mg/L and the air-water surface tension decreased to 25.5 mN/m. The Rha-C10-C12 products showed better emulsifying activity on diesel oil than the original products. This is the first report on the efficient production of the rare mono-rhamnolipids congener Rha-C10-C12 and the first report that the global regulator irrE can change the components of rhamnolipid products in Pseudomonas aeruginosa.

The Combined Effects of Azoxystrobin and the Biosurfactant-Producing Bacillus sp. Kol B3 against the Phytopathogenic Fungus Fusarium sambucinum IM 6525.

This study aimed to evaluate how the combined presence of the synthetic fungicide azoxystrobin (AZ) and the biosurfactant-producing Bacillus sp. Kol B3 influences the growth of the phytopathogenic fungus Fusarium sambucinum IM 6525. The results showed a noticeable increase in antifungal effectiveness when biotic and abiotic agents were combined. This effect manifested across diverse parameters, including fungal growth inhibition, changes in hyphae morphology, fungal membrane permeability and levels of intracellular reactive oxygen species (ROS). In response to the presence of Fusarium and AZ in the culture, the bacteria changed the proportions of biosurfactants (surfactin and iturin) produced. The presence of both AZ and/or Fusarium resulted in an increase in iturin biosynthesis. Only in 72 h old bacterial-fungal co-culture a 20% removal of AZ was noted. In the fungal cultures (with and without the addition of the bacteria), the presence of an AZ metabolite named azoxystrobin free acid was detected in the 48th and 72nd hours of the process. The possible involvement of increased iturin and ROS content in antifungal activity of Bacillus sp. and AZ when used together are also discussed. Biosurfactants were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Microscopy techniques and biochemical assays were also used.

Modular metabolic engineering of Bacillus amyloliquefaciens for high-level production of green biosurfactant iturin A.

As a kind of biosurfactants, iturin A has attracted people's wide attentions due to their features of biodegradability, environmentally friendly, etc.; however, high production cost limited its extensive application, and the aim of this research wants to improve iturin A production in Bacillus amyloliquefaciens. Firstly, dual promoter was applied to strengthen iturin A synthetase expression, and its yield was increased to 1.25 g/L. Subsequently, original 5'-UTRs of downstream genes (ituA, ituB, and ituC) in iturin A synthetase cluster were optimized, which significantly increased mRNA secondary stability, and iturin A yield produced by resultant strain HZ-T3 reached 2.32 g/L. Secondly, synthetic pathway of α-glucosidase inhibitor 1-deoxynojirimycin was blocked to improve substrate corn starch utilization, and iturin A yield was increased by 34.91% to 3.13 g/L. Thirdly, efficient precursor (fatty acids, Ser, and Pro) supplies were proven as the critical role in iturin A synthesis, and 5.52 g/L iturin A was attained by resultant strain, through overexpressing yngH, serC, and introducing ocD. Meanwhile, genes responsible for poly-γ-glutamic acid, extracellular polysaccharide, and surfactin syntheses were deleted, which led to a 30.98% increase of iturin A yield. Finally, lipopeptide transporters were screened, and iturin A yield was increased by 17.98% in SwrC overexpression strain, reached 8.53 g/L, which is the highest yield of iturin A ever reported. This study laid a foundation for industrial production and application development of iturin A, and provided the guidance of metabolic engineering breeding for efficient production of other metabolites synthesized by non-ribosomal peptide synthetase. KEY POINTS: • Optimizing 5'-UTR is an effective tactics to regulate synthetase cluster expression. • Blocking 1-DNJ synthesis benefited corn starch utilization and iturin A production. • The iturin A yield attained in this work was the highest yield reported so far.

Hitching a Ride in the Phyllosphere: Surfactant Production of Pseudomonas spp. Causes Co-swarming of Pantoea eucalypti 299R.

Here, we demonstrate the beneficial effect of surfactant-producing pseudomonads on Pantoea eucalypti 299R. We conducted a series of experiments in environments of increasing complexity. P. eucalypti 299R (Pe299R), and Pseudomonas sp. FF1 (Pff1) or Pe299R and surfactant-production deficient Pseudomonas sp. FF1::ΔviscB (Pff1ΔviscB) were co-inoculated in broth, on swarming agar plates, and on plants. In broth, there were no differences in the growth dynamics of Pe299R when growing in the presence of Pff1 or Pff1ΔviscB. By contrast, on swarming agar plates, Pe299R was able to co-swarm with Pff1 which led to a significant increase in Pe299R biomass compared to Pe299R growing with Pff1ΔviscB or in monoculture. Finally in planta, and using the single-cell bioreporter for reproductive success (CUSPER), we found a temporally distinct beneficial effect of Pff1 on co-inoculated Pe299R subpopulations that did not occur in the presence of Pff1ΔviscB. We tested three additional surfactant-producing pseudomonads and their respective surfactant knockout mutants on PE299R on swarming agar showing similar results. This led us to propose a model for the positive effect of surfactant production during leaf colonization. Our results indicate that co-motility might be common during leaf colonization and adds yet another facet to the already manyfold roles of surfactants.

PAH bioremediation with Rhodococcus rhodochrous ATCC 21198: Impact of cell immobilization and surfactant use on PAH treatment and post-remediation toxicity.

Polycyclic aromatic hydrocarbons (PAHs) are prevalent environmental contaminants that are harmful to ecological and human health. Bioremediation is a promising technique for remediating PAHs in the environment, however bioremediation often results in the accumulation of toxic PAH metabolites. The objectives of this research were to demonstrate the cometabolic treatment of a mixture of PAHs by a pure bacterial culture, Rhodococcus rhodochrous ATCC 21198, and investigate PAH metabolites and toxicity. Additionally, the surfactant Tween ® 80 and cell immobilization techniques were used to enhance bioremediation. Total PAH removal ranged from 70-95% for fluorene, 44-89% for phenanthrene, 86-97% for anthracene, and 6.5-78% for pyrene. Maximum removal was achieved with immobilized cells in the presence of Tween ® 80. Investigation of PAH metabolites produced by 21198 revealed a complex mixture of hydroxylated compounds, quinones, and ring-fission products. Toxicity appeared to increase after bioremediation, manifesting as mortality and developmental effects in embryonic zebrafish. 21198's ability to rapidly transform PAHs of a variety of molecular structures and sizes suggests that 21198 can be a valuable microorganism for catalyzing PAH remediation. However, implementing further treatment processes to address toxic PAH metabolites should be pursued to help lower post-remediation toxicity in future studies.

Development of an in vitro human alveolar epithelial air-liquid interface model using a small molecule inhibitor cocktail.

BACKGROUND: The alveolar epithelium is exposed to numerous stimuli, such as chemicals, viruses, and bacteria that cause a variety of pulmonary diseases through inhalation. Alveolar epithelial cells (AECs) cultured in vitro are a valuable tool for studying the impacts of these stimuli and developing therapies for associated diseases. However, maintaining the proliferative capacity of AECs in vitro is challenging. In this study, we used a cocktail of three small molecule inhibitors to cultivate AECs: Y-27632, A-83-01, and CHIR99021 (YAC). These inhibitors reportedly maintain the proliferative capacity of several types of stem/progenitor cells. RESULTS: Primary human AECs cultured in medium containing YAC proliferated for more than 50 days (over nine passages) under submerged conditions. YAC-treated AECs were subsequently cultured at the air-liquid interface (ALI) to promote differentiation. YAC-treated AECs on ALI day 7 formed a monolayer of epithelial tissue with strong expression of the surfactant protein-encoding genes SFTPA1, SFTPB, SFTPC, and SFTPD, which are markers for type II AECs (AECIIs). Immunohistochemical analysis revealed that paraffin sections of YAC-treated AECs on ALI day 7 were mainly composed of cells expressing surfactant protein B and prosurfactant protein C. CONCLUSIONS: Our results indicate that YAC-containing medium could be useful for expansion of AECIIs, which are recognized as local stem/progenitor cells, in the alveoli.

Structure of the Bacterial Cell Envelope and Interactions with Antimicrobials: Insights from Molecular Dynamics Simulations.

Bacteria have evolved over 3 billion years, shaping our intrinsic and symbiotic coexistence with these single-celled organisms. With rising populations of drug-resistant strains, the search for novel antimicrobials is an ongoing area of research. Advances in high-performance computing platforms have led to a variety of molecular dynamics simulation strategies to study the interactions of antimicrobial molecules with different compartments of the bacterial cell envelope of both Gram-positive and Gram-negative species. In this review, we begin with a detailed description of the structural aspects of the bacterial cell envelope. Simulations concerned with the transport and associated free energy of small molecules and ions through the outer membrane, peptidoglycan, inner membrane and outer membrane porins are discussed. Since surfactants are widely used as antimicrobials, a section is devoted to the interactions of surfactants with the cell wall and inner membranes. The review ends with a discussion on antimicrobial peptides and the insights gained from the molecular simulations on the free energy of translocation. Challenges involved in developing accurate molecular models and coarse-grained strategies that provide a trade-off between atomic details with a gain in sampling time are highlighted. The need for efficient sampling strategies to obtain accurate free energies of translocation is also discussed. Molecular dynamics simulations have evolved as a powerful tool that can potentially be used to design and develop novel antimicrobials and strategies to effectively treat bacterial infections.

Regulation mechanism and bioactivity characteristic of surfactin homologues with C14 and C15 fatty acid chains.

BACKGROUND: Surfactin, a green lipopeptide bio-surfactant, exhibits excellent surface, hemolytic, antibacterial, and emulsifying activities. However, a lack of clear understanding of the synthesis regulation mechanism of surfactin homologue components has hindered the customized production of surfactin products with different biological activities. RESULTS: In this study, exogenous valine and 2-methylbutyric acid supplementation significantly facilitated the production of C14-C15 surfactin proportions (up to 75% or more), with a positive correlation between the homologue proportion and fortified concentration. Subsequently, the branched-chain amino acid degradation pathway and the glutamate synthesis pathway are identified as critical pathways in regulating C14-C15 surfactin synthesis by transcriptome analysis. Overexpression of genes bkdAB and glnA resulted in a 1.4-fold and 1.3-fold increase in C14 surfactin, respectively. Finally, the C14-rich surfactin was observed to significantly enhance emulsification activity, achieving an EI24 exceeding 60% against hexadecane, while simultaneously reducing hemolytic activity. Conversely, the C15-rich surfactin demonstrated an increase in both hemolytic and antibacterial activities. CONCLUSION: This study presents the first evidence of a potential connection between surfactin homologue synthesis and the conversion of glutamate and glutamine, providing a theoretical basis for targeting the synthesis regulation and structure-activity relationships of surfactin and other lipopeptide compounds.

The expression of the surfactant proteins SP-A and SP-B during postnatal alveolarization of the rat lung.

OBJECTIVE: Surfactant-specific proteins (SP) are responsible for the functional and structural integrity as well as for the stabilization of the intra-alveolar surfactant. Morphological lung maturation starts in rat lungs after birth. The aim of this study was to investigate whether the expression of the hydrophilic SP-A and the hydrophobic SP-B is associated with characteristic postnatal changes characterizing morphological lung maturation. METHODS: Stereological methods were performed on the light microscope. Using immunohistochemical and molecular biological methods (Western Blot, RT-qPCR), the SP-A and SP-B of adult rat lungs and of those with different postnatal developmental stages (3, 7, 14 and 21 days after birth) were characterized. RESULTS: As signs of alveolarization the total septal surface and volume increased and the septal thickness decreased. The significantly highest relative surface fraction of SP-A labeled alveolar epithelial cells type II (AEII) was found together with the highest relative SP-A gene expression before the alveolarization (3th postnatal day). With the downregulation of SP-A gene expression during and after alveolarization (between postnatal days 7 and 14), the surface fraction of the SP-A labeled AEII also decreased, so they are lowest in adult animals. The surface fraction of SP-B labeled AEII and the SP-B gene expression showed the significantly highest levels in adults, the protein expression increased also significantly at the end of morphological lung maturation. There were no alterations in the SP-B expression before and during alveolarization until postnatal day 14. The protein expression as well as the gene expression of SP-A and SP-B correlated very well with the total surface of alveolar septa independent of the postnatal age. CONCLUSION: The expression of SP-A and SP-B is differentially associated with morphological lung maturation and correlates with increased septation of alveoli as indirect clue for alveolarization.

Effect of salinity stress and surfactant treatment with zinc and boron on morpho-physiological and biochemical indices of fenugreek (Trigonella foenum-graecum).

Micronutrient application has a crucial role in mitigating salinity stress in crop plants. This study was carried out to investigate the effect of zinc (Zn) and boron (B) as foliar applications on fenugreek growth and physiology under salt stress (0 and 120 mM). After 35 days of salt treatments, three levels of zinc (0, 50, and 100 ppm) and two levels of boron (0 and 2 ppm) were applied as a foliar application. Salinity significantly reduced root length (72.7%) and shoot length (33.9%), plant height (36%), leaf area (37%), root fresh weight (48%) and shoot fresh weight (75%), root dry weight (80%) and shoot dry weight (67%), photosynthetic pigments (78%), number of branches (50%), and seeds per pod (56%). Fenugreek's growth and physiology were improved by foliar spray of zinc and boron, which increased the length of the shoot (6%) and root length (2%), fresh root weight (18%), and dry root weight (8%), and chlorophyll a (1%), chlorophyll b (25%), total soluble protein content (3%), shoot calcium (9%) and potassium (5%) contents by significantly decreasing sodium ion (11%) content. Moreover, 100 ppm of Zn and 2 ppm of B enhanced the growth and physiology of fenugreek by reducing the effect of salt stress. Overall, boron and zinc foliar spray is suggested for improvement in fenugreek growth under salinity stress.

Effects of sodium dodecylbenzene sulfonate (SDBS) on zebrafish (Danio rerio) gills and blood.

Sodium dodecylbenzene sulfonate (SDBS) is an important surfactant used as a cleaning agent and industrial additive to remove unwanted chemicals which have been detected in the aquatic environment. The aim of this study was to examine the toxicological potential of SDBS on the gills of adult male zebrafish (Danio rerio) exposed to this chemical. For the 96 hr acute exposure, fish were divided into three groups: control, 0.25 mg/L, and 0.5 mg/L of SDBS. After the experiment, morphophysiological analyses (gill histopathology and histochemistry), oxidative stress (determination of gill activities of superoxide dismutase (SOD) and catalase (CAT)), and hematological analyses (leukocyte differentiation) were conducted. Data demonstrated that SDBS at both tested concentrations altered the histopathological index and initiated circulatory disturbances, as well as adverse, progressive, and immunological changes in the gills. In the 0.5 mg/L group, SOD activity decreased significantly, but CAT activity was not altered. Prominent blood changes observed in this group were neutrophilia and lymphocytosis. The number of mucous and chloride cells increased significantly in both groups. Taken together, our findings demonstrated that exposure of D. rerio to SDBS, even for 96 hr, produced adverse morphological and hematological effects associated with a reduction in SOD activity. Our findings indicate that exposure of aquatic species to the anionic surfactant SDBS may lead to adverse consequences associated with oxidative stress. Therefore, this study highlights the risks that this substance may pose to aquatic ecosystems and emphasizes the need for further investigations and strict regulations on its disposal.

Surface-active antibiotic production as a multifunctional adaptation for postfire microorganisms.

Wildfires affect soils in multiple ways, leading to numerous challenges for colonizing microorganisms. Although it is thought that fire-adapted microorganisms lie at the forefront of postfire ecosystem recovery, the specific strategies that these organisms use to thrive in burned soils remain largely unknown. Through bioactivity screening of bacterial isolates from burned soils, we discovered that several Paraburkholderia spp. isolates produced a set of unusual rhamnolipid surfactants with a natural methyl ester modification. These rhamnolipid methyl esters (RLMEs) exhibited enhanced antimicrobial activity against other postfire microbial isolates, including pyrophilous Pyronema fungi and Amycolatopsis bacteria, compared to the typical rhamnolipids made by organisms such as Pseudomonas spp. RLMEs also showed enhanced surfactant properties and facilitated bacterial motility on agar surfaces. In vitro assays further demonstrated that RLMEs improved aqueous solubilization of polycyclic aromatic hydrocarbons, which are potential carbon sources found in char. Identification of the rhamnolipid biosynthesis genes in the postfire isolate, Paraburkholderia kirstenboschensis str. F3, led to the discovery of rhlM, whose gene product is responsible for the unique methylation of rhamnolipid substrates. RhlM is the first characterized bacterial representative of a large class of integral membrane methyltransferases that are widespread in bacteria. These results indicate multiple roles for RLMEs in the postfire lifestyle of Paraburkholderia isolates, including enhanced dispersal, solubilization of potential nutrients, and inhibition of competitors. Our findings shed new light on the chemical adaptations that bacteria employ to navigate, grow, and outcompete other soil community members in postfire environments.

Advances in the co-production of biosurfactant and other biomolecules: statistical approaches for process optimization.

An efficient microbial conversion for simultaneous synthesis of multiple high-value compounds, such as biosurfactants and enzymes, is one of the most promising aspects for an economical bioprocess leading to a marked reduction in production cost. Although biosurfactant and enzyme production separately have been much explored, there are limited reports on the predictions and optimization studies on simultaneous production of biosurfactants and other industrially important enzymes, including lipase, protease, and amylase. Enzymes are suited for an integrated production process with biosurfactants as multiple common industrial processes and applications are catalysed by these molecules. However, the complexity in microbial metabolism complicates the production process. This study details the work done on biosurfactant and enzyme co-production and explores the application and scope of various statistical tools and methodologies in this area of research. The use of advanced computational tools is yet to be explored for the optimization of downstream strategies in the co-production process. Given the complexity of the co-production process and with various new methodologies based on artificial intelligence (AI) being invented, the scope of AI in shaping the biosurfactant-enzyme co-production process is immense and would lead to not only efficient and rapid optimization, but economical extraction of multiple biomolecules as well.

In utero ventilation induces lung parenchymal and vascular alterations in extremely preterm fetal sheep.

Extremely preterm infants are often exposed to long durations of mechanical ventilation to facilitate gas exchange, resulting in ventilation-induced lung injury (VILI). New lung protective strategies utilizing noninvasive ventilation or low tidal volumes are now common but have not reduced rates of bronchopulmonary dysplasia. We aimed to determine the effect of 24 h of low tidal volume ventilation on the immature lung by ventilating preterm fetal sheep in utero. Preterm fetal sheep at 110 ± 1(SD) days' gestation underwent sterile surgery for instrumentation with a tracheal loop to enable in utero mechanical ventilation (IUV). At 112 ± 1 days' gestation, fetuses received either in utero mechanical ventilation (IUV, n = 10) targeting 3-5 mL/kg for 24 h, or no ventilation (CONT, n = 9). At necropsy, fetal lungs were collected to assess molecular and histological markers of lung inflammation and injury. IUV significantly increased lung mRNA expression of interleukin (IL)-1ß, IL-6, IL-8, IL-10, and tumor necrosis factor (TNF) compared with CONT, and increased surfactant protein (SP)-A1, SP-B, and SP-C mRNA expression compared with CONT. IUV produced modest structural changes to the airways, including reduced parenchymal collagen and myofibroblast density. IUV increased pulmonary arteriole thickness compared with CONT but did not alter overall elastin or collagen content within the vasculature. In utero ventilation of an extremely preterm lung, even at low tidal volumes, induces lung inflammation and injury to the airways and vasculature. In utero ventilation may be an important model to isolate the confounding mechanisms of VILI to develop effective therapies for preterm infants requiring prolonged respiratory support.NEW & NOTEWORTHY Preterm infants often require prolonged respiratory support, but the relative contribution of ventilation to the development of lung injury is difficult to isolate. In utero mechanical ventilation allows for mechanistic investigations into ventilation-induced lung injury without confounding factors associated with sustaining extremely preterm lambs ex utero. Twenty-four hours of in utero ventilation, even at low tidal volumes, increased lung inflammation and surfactant protein expression and produced structural changes to the lung parenchyma and vasculature.

Bioconcentration Assessment of Three Cationic Surfactants in Permanent Fish Cell Lines.

Cationic surfactants are used in many industrial processes and in consumer products with concurrent release into the aquatic environment, where they may accumulate in aquatic organisms to regulatoryly relevant thresholds. Here, we aimed to better understand the bioconcentration behavior of three selected cationic surfactants, namely N,N-dimethyldecylamine (T10), N-methyldodecylamine (S12), and N,N,N-trimethyltetradecylammonium cation (Q14), in the cells of fish liver (RTL-W1) and gill (RTgill-W1) cell lines. We conducted full mass balances for bioconcentration tests with the cell cultures, in which the medium, the cell surface, the cells themselves, and the plastic compartment were sampled and quantified for each surfactant by HPLC MS/MS. Accumulation in/to cells correlated with the surfactants' alkyl chain lengths and their membrane lipid-water partitioning coefficient, DMLW. Cell-derived bioconcentration factors (BCF) of T10 and S12 were within a factor of 3.5 to in vivo BCF obtained from the literature, while the cell-derived BCF values for Q14 were >100 times higher than the in vivo BCF. From our experiments, rainbow trout cell lines appear as a suitable conservative in vitro screening method for bioconcentration assessment of cationic surfactants and are promising for further testing.

Human pluripotent stem cell modeling of alveolar type 2 cell dysfunction caused by ABCA3 mutations.

Mutations in ATP-binding cassette A3 (ABCA3), a phospholipid transporter critical for surfactant homeostasis in pulmonary alveolar type II epithelial cells (AEC2s), are the most common genetic causes of childhood interstitial lung disease (chILD). Treatments for patients with pathological variants of ABCA3 mutations are limited, in part due to a lack of understanding of disease pathogenesis resulting from an inability to access primary AEC2s from affected children. Here, we report the generation of AEC2s from affected patient induced pluripotent stem cells (iPSCs) carrying homozygous versions of multiple ABCA3 mutations. We generated syngeneic CRISPR/Cas9 gene-corrected and uncorrected iPSCs and ABCA3-mutant knockin ABCA3:GFP fusion reporter lines for in vitro disease modeling. We observed an expected decreased capacity for surfactant secretion in ABCA3-mutant iPSC-derived AEC2s (iAEC2s), but we also found an unexpected epithelial-intrinsic aberrant phenotype in mutant iAEC2s, presenting as diminished progenitor potential, increased NFκB signaling, and the production of pro-inflammatory cytokines. The ABCA3:GFP fusion reporter permitted mutant-specific, quantifiable characterization of lamellar body size and ABCA3 protein trafficking, functional features that are perturbed depending on ABCA3 mutation type. Our disease model provides a platform for understanding ABCA3 mutation-mediated mechanisms of alveolar epithelial cell dysfunction that may trigger chILD pathogenesis.

Interspecies comparison of metabolism of two novel prototype PFAS.

As a result of proposed global restrictions and regulations on current-use per-and polyfluoroalkyl substances (PFAS), research on possible alternatives is highly required. In this study, phase I in vitro metabolism of two novel prototype PFAS in human and rat was investigated. These prototype chemicals are intended to be safer-by-design and expected to mineralize completely, and thus be less persistent in the environment compared to the PFAS available on the market. Following incubation with rat liver S9 (RL-S9) fractions, two main metabolites per initial substance were produced, namely an alcohol and a short-chain carboxylic acid. While with human liver S9 (HL-S9) fractions, only the short-chain carboxylic acid was detected. Beyond these major metabolites, two and five additional metabolites were identified at very low levels by non-targeted screening for the ether- and thioether-linked prototype chemicals, respectively. Overall, complete mineralization during the in vitro hepatic metabolism of these novel PFAS by HL-S9 and RL-S9 fractions was not observed. The reaction kinetics of the surfactants was determined by using the metabolite formation, rather than the substrate depletion approach. With rat liver enzymes, the formation rates of primary metabolite alcohols were at least two orders of magnitude higher than those of secondary metabolite carboxylic acids. When incubating with human liver enzymes, the formation rates of single metabolite carboxylic acids, were similar or smaller than those experienced in rat. It also indicates that the overall metabolic rate and clearance of surfactants are significantly higher in rat liver than in human liver. The maximum formation rate of the thioether congener exceeded 10-fold that of the ether in humans but were similar in rats. Overall, the results suggest that metabolism of the prototype chemicals followed a similar trend to those reported in studies of fluorotelomer alcohols.

Preterm birth affects both surfactant synthesis and lung liquid resorption actors in fetal sheep.

INTRODUCTION: After birth, the lungs must resorb the fluid they contain. This process involves multiple actors such as surfactant, aquaporins and ENaC channels. Preterm newborns often exhibit respiratory distress syndrome due to surfactant deficiency, and transitory tachypnea caused by a delay in lung liquid resorption. Our hypothesis is that surfactant, ENaC and aquaporins are involved in respiratory transition to extrauterine life and altered by preterm birth. We compared these candidates in preterm and term fetal sheeps. MATERIALS AND METHODS: We performed cesarean sections in 8 time-dated pregnant ewes (4 at 100 days and 4 at 140 days of gestation, corresponding to 24 and 36 weeks of gestation in humans), and obtained 13 fetal sheeps in each group. We studied surfactant synthesis (SP-A, SP-B, SP-C), lung liquid resorption (ENaC, aquaporins) and corticosteroid regulation (glucocorticoid receptor, mineralocorticoid receptor and 11-betaHSD2) at mRNA and protein levels. RESULTS: The mRNA expression level of SFTPA, SFTPB and SFTPC was higher in the term group. These results were confirmed at the protein level for SP-B on Western Blot analysis and for SP-A, SP-B and SP-C on immunohistochemical analysis. Regarding aquaporins, ENaC and receptors, mRNA expression levels for AQP1, AQP3, AQP5, ENaCα, ENaCß, ENaCγ and 11ßHSD2 mRNA were also higher in the term group. DISCUSSION: Expression of surfactant proteins, aquaporins and ENaC increases between 100 and 140 days of gestation in an ovine model. Further exploring these pathways and their hormonal regulation could highlight some new explanations in the pathophysiology of neonatal respiratory diseases.