Increased Elastase and Matrix Metalloproteinase Levels in the Pulmonary Arteries of Infants With Congenital Diaphragmatic Hernia.

BACKGROUND: Pulmonary vascular disease (PVD) complicated with pulmonary hypertension (PH) is a leading cause of mortality in congenital diaphragmatic hernia (CDH). Unfortunately, CDH patients are often resistant to PH therapy. Using the nitrogen CDH rat model, we previously demonstrated that CDH-associated PVD involves an induction of elastase and matrix metalloproteinase (MMP) activities, increased osteopontin and epidermal growth factor (EGF) levels, and enhanced smooth muscle cell (SMC) proliferation. Here, we aimed to determine whether the levels of the key members of this proteinase-induced pathway are also elevated in the pulmonary arteries (PAs) of CDH patients. METHODS: Neutrophil elastase (NE), matrix metalloproteinase-2 (MMP-2), epidermal growth factor (EGF), tenascin-C, and osteopontin levels were assessed by immunohistochemistry in the PAs from the lungs of 11 CDH patients and 5 normal age-matched controls. Markers of proliferation (proliferating cell nuclear antigen (PCNA)) and apoptosis (cleaved (active) caspase-3) were also used. RESULTS: While expressed by both control and CDH lungs, the levels of NE, MMP-2, EGF, as well as tenascin-C and osteopontin were significantly increased in the PAs from CDH patients. The percentage of PCNA-positive PA SMCs were also enhanced, while those positive for caspase-3 were slightly decreased. CONCLUSIONS: These results suggest that increased elastase and MMPs, together with elevated tenascin-C and osteopontin levels in an EGF-rich environment may contribute to the PVD in CDH infants. The next step of this study is to expand our analysis to a larger cohort, and determine the potential of targeting this pathway for the treatment of CDH-associated PVD and PH. TYPE OF STUDY: Therapeutic. LEVEL OF EVIDENCE: LEVEL III.

Decreased ß-catenin Protein in Lungs From Human Congenital Diaphragmatic Hernia Archival Pathology Specimens: A Case-control Study.

BACKGROUND: Lung hypoplasia contributes to congenital diaphragmatic hernia (CDH) associated morbidity and mortality. Changes in lung wingless-type MMTV integration site family member (Wnt)-signalling and its downstream effector beta-catenin (CTNNB1), which acts as a transcription coactivator, exist in animal CDH models but are not well characterized in humans. We aim to identify changes to Wnt-signalling gene expression in human CDH lungs and hypothesize that pathway expression will be lower than controls. METHODS: We identified 51 CDH cases and 10 non-CDH controls with archival formalin-fixed paraffin-embedded (FFPE) autopsy lung tissue from 2012 to 2022. 11 liveborn CDH cases and an additional two anterior diaphragmatic hernias were excluded from the study, leaving 38 CDH cases. Messenger ribonucleic acid (mRNA) expression of Wnt-signalling effectors WNT2B and CTNNB1 was determined for 19 CDH cases and 9 controls. A subset of CDH cases and controls lung sections were immunostained for ß-catenin. Clinical variables were obtained from autopsy reports. RESULTS: Median gestational age was 21 weeks. 81% (n = 31) of hernias were left-sided. 47% (n = 18) were posterolateral. Liver position was up in 81% (n = 31) of cases. Defect size was Type C or D in 58% (n = 22) of cases based on autopsy photos, and indeterminable in 42% (n = 16) of cases. WNT2B and CTNNB1 mRNA expression did not differ between CDH and non-CDH lungs. CDH lungs had fewer interstitial cells expressing ß-catenin protein than non-CDH lungs (13.2% vs 42.4%; p = 0.006). CONCLUSION: There appear to be differences in the abundance and/or localization of ß-catenin proteins between CDH and non-CDH lungs. LEVEL OF EVIDENCE: Level III. TYPE OF STUDY: Case-Control Study.

Prenatal treprostinil improves pulmonary arteriolar hypermuscularization in the rabbit model of congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a congenital malformation characterized by pulmonary hypoplasia, pulmonary hypertension, and cardiac dysfunction. Pulmonary hypertension represents the major cause of neonatal mortality and morbidity. Prenatal diagnosis allows assessment of severity and selection of foetal surgery candidates. We have shown that treprostinil, a prostacyclin analogue with an anti-remodelling effect, attenuates the relative hypermuscularization of the pulmonary vasculature in rats with nitrofen-induced CDH. Here we confirm these observations in a large animal model of surgically-created CDH. In the rabbit model, subcutaneous maternal administration of treprostinil at 150 ng/kg/min consistently reached target foetal concentrations without demonstrable detrimental foetal or maternal adverse effects. In pups with CDH, prenatal treprostinil reduced pulmonary arteriolar proportional medial wall thickness and downregulated inflammation and myogenesis pathways. No effect on alveolar morphometry or lung mechanics was observed. These findings provide further support towards clinical translation of prenatal treprostinil for CDH.

Bifenox compromises porcine trophectoderm and luminal epithelial cells in early pregnancy by arresting cell cycle progression and impairing mitochondrial and calcium homeostasis.

Bifenox is a widely used herbicide that contains a diphenyl ether group. However its global usage, the cell physiological effects that induce toxicity have not been elucidated. In this study, the effect of bifenox was examined in porcine trophectoderm and uterine epithelial cells to investigate the potential toxicity of the implantation process. To uncover the toxic effects of bifenox, cell viability and apoptosis following treatment with bifenox were evaluated. To investigate the underlying cellular mechanisms, mitochondrial and calcium homeostasis were investigated in both cell lines. In addition, the dysregulation of cell signal transduction and transcriptional alterations were also demonstrated. Bifenox reduced cell viability and significantly increased the number of cells arrested at the sub-G1 stage. Moreover, bifenox depolarized the mitochondrial membrane and upregulated the calcium flux into the mitochondria in both cell lines. Cytosolic calcium flux increased in porcine trophectoderm (pTr) cells and decreased in porcine luminal epithelium (pLE) cells. In addition, bifenox activated the mitogen-activated protein kinase and phosphoinositide 3-kinase signaling pathways. Furthermore, bifenox inhibited the expression of retinoid receptor genes, such as RXRA, RXRB, and RXRG. Chemokine CCL8 was also downregulated at the mRNA level, whereas CCL5 expression remained unchanged. Overall, the results of this study suggest that bifenox deteriorates cell viability by arresting cell cycle progression, damaging mitochondria, and controlling calcium levels in pTr and pLE cells. The present study indicates the toxic potential of bifenox in the trophectoderm and luminal epithelial cells, which can lead to implantation disorders in early pregnancy.

Discovery of diphenyl ether-degrading Streptomyces strains by direct screening based on ether bond-cleaving activity.

Diphenyl ethers (DEs), which are widely used in the agricultural and chemical industries, have become hazardous contaminants in the environment. Although several DE-degrading bacteria have been reported, discovering new types of such microorganisms could enhance understanding of the degradation mechanism in the environment. In this study, we used a direct screening method based on detection of ether bond-cleaving activity to screen for microorganisms that degrade 4,4'-dihydroxydiphenyl ether (DHDE) as a model DE. Microorganisms isolated from soil samples were incubated with DHDE, and strains producing hydroquinone via ether bond cleavage were selected using hydroquinone-sensitive Rhodanine reagent. This screening procedure resulted in the isolation of 3 bacteria and 2 fungi that transform DHDE. Interestingly, all of the isolated bacteria belonged to one genus, Streptomyces. To our knowledge, these are the first microorganisms of the genus Streptomyces shown to degrade a DE. Streptomyces sp. TUS-ST3 exhibited high and stable DHDE-degrading activity. HPLC, LC-MS, and GC-MS analyses revealed that strain TUS-ST3 converts DHDE to its hydroxylated analogue and generates hydroquinone as an ether bond-cleavage product. Strain TUS-ST3 also transformed DEs other than DHDE. In addition, glucose-grown TUS-ST3 cells began to transform DHDE after incubation with this compound for 12 h, and produced 75 µM hydroquinone in 72 h. These activities of streptomycetes may play an important role in DE degradation in the environment. We also report the whole genome sequence of strain TUS-ST3.

Antibacterial diphenyl ether production induced by co-culture of Aspergillus nidulans and Aspergillus fumigatus.

Fungi are a rich source of secondary metabolites with potent biological activities. Co-culturing a fungus with another microorganism has drawn much attention as a practical method for stimulating fungal secondary metabolism. However, in most cases, the molecular mechanisms underlying the activation of secondary metabolite production in co-culture are poorly understood. To elucidate such a mechanism, in this study, we established a model fungal-fungal co-culture system, composed of Aspergillus nidulans and Aspergillus fumigatus. In the co-culture of A. nidulans and A. fumigatus, production of antibacterial diphenyl ethers was enhanced. Transcriptome analysis by RNA-sequencing showed that the co-culture activated expression of siderophore biosynthesis genes in A. fumigatus and two polyketide biosynthetic gene clusters (the ors and cic clusters) in A. nidulans. Gene disruption experiments revealed that the ors cluster is responsible for diphenyl ether production in the co-culture. Interestingly, the ors cluster was previously reported to be upregulated by co-culture of A. nidulans with the bacterium Streptomyces rapamycinicus; orsellinic acid was the main product of the cluster in that co-culture. In other words, the main product of the ors cluster was different in fungal-fungal and bacterial-fungal co-culture. The genes responsible for biosynthesis of the bacterial- and fungal-induced polyketides were deduced using a heterologous expression system in Aspergillus oryzae. The molecular genetic mechanisms that trigger biosynthesis of two different types of compounds in A. nidulans in response to the fungus and the bacterium were demonstrated, which provides an insight into complex secondary metabolic response of fungi to microorganisms. KEY POINTS: • Co-culture of two fungal species triggered antibiotic diphenyl ether production. • The co-culture affected expression levels of several genes for secondary metabolism. • Gene cluster essential for induction of the antibiotics production was determined.

Pharmacological Targeting of Executioner Proteins: Controlling Life and Death.

Small-molecule mediated modulation of protein interactions of Bcl-2 (B-cell lymphoma-2) family proteins was clinically validated in 2015 when Venetoclax, a selective inhibitor of the antiapoptotic protein BCL-2, achieved breakthrough status designation by the FDA for treatment of lymphoid malignancies. Since then, substantial progress has been made in identifying inhibitors of other interactions of antiapoptosis proteins. However, targeting their pro-apoptotic counterparts, the "executioners" BAX, BAK, and BOK that both initiate and commit the cell to dying, has lagged behind. However, recent publications demonstrate that these proteins can be positively or negatively regulated using small molecule tool compounds. The results obtained with these molecules suggest that pharmaceutical regulation of apoptosis will have broad implications that extend beyond activating cell death in cancer. We review recent advances in identifying compounds and their utility in the exogenous control of life and death by regulating executioner proteins, with emphasis on the prototype BAX.

Synthesis, Biological Evaluation and Molecular Dynamics Simulation Studies of Novel Diphenyl Ethers.

BACKGROUND: The well-known antibacterial agent Triclosan (TCL) that targets bacterial enoylacyl protein reductase has been described to inhibit human fatty acid synthase (FASN) via the enoylacyl reductase domain. A Literature survey indicates that TCL is selectively toxic to cancer cells and furthermore might indeed reduce cancer incidence in vivo. A recent study found that TCL inhibits FASN by acting as an allosteric protein-protein interface (PPI) inhibitor. It induces dimer orientation changes that effect in a downstream reorientation of catalytic residues in the NADPH binding site proposing TCL as a viable scaffold to design a superior molecule that might have more inhibitory potential. This unveils tons of potential interaction space to take advantage of future inhibitor design. OBJECTIVES: Synthesis of TCL mimicking novel diphenyl ether derivatives, biological evaluation as potential antiproliferative agents and molecular docking and molecular dynamics simulation studies. METHODS: A series of novel N-(1-(3-hydroxy-4-phenoxyphenyl)-3-oxo-3-phenylpropyl)acetamides (3a-n) and N-(3(3-hydroxy-4phenoxyphenyl)-3-oxo-1-phenylpropyl) acetamides (6a-n) were designed, synthesized, characterized and evaluated against HepG2, A-549, MCF-7 and Vero cell lines. The induction of antiproliferative activity of selected compounds (3d and 6c) was done by AO/EB (acridine orange/ethidium bromide) nuclear staining method, DNA fragmentation study, and cell cycle analysis was performed by flow cytometry. Molecular docking and dynamics simulation study was also performed. RESULTS: Among the tested compounds, compound 3d was most active (IC50 13.76 ± 0.43 µM) against A-549 cell line. Compounds 3d and 3g were found to be moderately active with IC50 30.56 ± 1.1 µM and 25.05 ± 0.8 µM respectively against MCF-7 cell line. Morphological analysis of A-549 cells treated with 3d and 6c clearly demonstrated the reduction of cell viability and induction of apoptosis. DNA fragmentation was observed as a characteristic of apoptosis in treated cells. Further, cell cycle analysis by flow cytometry confirmed that compounds 3d and 6c significantly arrested the cell cycle at the G0/G1 phase. Molecular docking study demonstrated that these compounds exhibit high affinity for the human fatty acid synthase (hFASN) target. Molecular dynamics simulation study of the most active compound 3d was performed for calculating binding free energies using Molecular Mechanics-Generalized Born Surface Area (MM/GBSA). CONCLUSION: Compound 3d (IC50 13.76 ± 0.43 µM) has been identified as a potential lead molecule for anticancer activity against A-549 cells followed by 3l, 6c, and 3g. Thus, the design of diphenyl ether derivatives with enhanced affinity to the binding site of hER may lead to the discovery of potential anticancer agents.

Enhancement Effect of Poly-L-ornithine on the Nasal Absorption of Water-Soluble Macromolecules in Rats.

The transnasal route for the delivery of water-soluble macromolecules, such as bioactive peptides and proteins, has attracted interest, although the use of permeation enhancers is required due to the poor permeabilities of these macromolecules across the nasal mucosa. With polycationic compounds, such as poly-L-arginine and chitosan, the nasal absorption of hydrophilic macromolecules is molecular weight- and concentration-dependently enhanced without causing cytotoxicity. In the present study, we evaluated the effect of various molecular weights and concentrations of poly-L-ornithine (PLO), a polycationic compound, on the nasal absorption and the damage to the nasal mucosa in vivo. PLO enhanced the nasal absorption of fluorescein isothiocyanate-dextran (FD-4), used as a model drug, and the bioavailability of FD-4 increased with the concentration of PLO. The enhancement effect was also dependent on the molecular weight. The administration of PLO at a concentration that sufficed for enhancing the nasal absorption had no effect on the activity of lactic dehydrogenase and the protein leakage in the nasal fluid, as indices of nasal mucosa damage. These findings suggest that a transnasal delivery system using PLO is a useful strategy for improving the nasal absorption of water-soluble macromolecules without toxicity to the nasal mucosa.

Ligand binding to human prostaglandin E receptor EP4 at the lipid-bilayer interface.

Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family.

Design, synthesis, and evaluation of novel diphenyl ether derivatives against drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis.

In our efforts to develop druggable diphenyl ethers as potential antitubercular agents, a series of novel diphenyl ether derivatives (5a-f, 6a-f) were designed and synthesized. The representative compounds showed promising in vitro activity against drug-susceptible, isoniazid-resistant, and multidrug-resistant strains of Mycobacterium tuberculosis with MIC values of 1.56 µg/ml (6b), 6.25 µg/ml (6a-d), and 3.125 µg/ml (6b-c), respectively. All the synthesized compounds exhibited satisfactory safety profile (CC50  > 300 µg/ml) against Vero and HepG2 cells. Reverse phase HPLC method was used to probe the physicochemical properties of the synthesized compounds. This series of compounds demonstrated comparatively low logP values. pKa values of representative compounds indicated that they were weak acids. Additionally, in vitro human liver microsomal stability assay confirmed that the synthesized compounds possessed acceptable stability under study conditions. The present study thus establishes compound 6b as the most promising antitubercular agent with acceptable drug-likeness.

Effect of overnight smoking abstinence on a marker for microglial activation: a [11C]DAA1106 positron emission tomography study.

RATIONALE: Microglia are the main immune cells in the central nervous system and participate in neuroinflammation. When activated, microglia express increased levels of the translocator protein 18 kDa (TSPO), thereby making TSPO availability a marker for neuroinflammation. Using positron emission tomography (PET) scanning, our group recently demonstrated that smokers in the satiated state had 16.8% less binding of the radiotracer [11C]DAA1106 (a radioligand for TSPO) in the brain than nonsmokers. OBJECTIVES: We sought to determine the effect of overnight smoking abstinence on [11C]DAA1106 binding in the brain. METHODS: Forty participants (22 smokers and 18 nonsmokers) completed the study (at one of two sites) and had usable data, which included images from a dynamic [11C]DAA1106 PET scanning session (with smokers having been abstinent for 17.9 ± 2.3 h) and a blood sample for TSPO genotyping. Whole brain standardized uptake values (SUVs) were determined, and analysis of variance was performed, with group (overnight abstinent smoker vs. nonsmoker), site, and TSPO genotype as factors, thereby controlling for site and genotype. RESULTS: Overnight abstinent smokers had lower whole brain SUVs (by 15.5 and 17.0% for the two study sites) than nonsmokers (ANCOVA, P = 0.004). The groups did not significantly differ in injected radiotracer dose or body weight, which were used to calculate SUV. CONCLUSIONS: These results in overnight abstinent smokers are similar to those in satiated smokers, indicating that chronic cigarette smoking leads to global impairment of microglial activation which persists into early abstinence. Other explanations for study results, such as smoking leading to reduced numbers of microglia or smokers having more rapid metabolism of the radiotracer than nonsmokers, are also possible.

Isolation of Phytotoxic Phenols and Characterization of a New 5-Hydroxymethyl-2-isopropoxyphenol from Dothiorella vidmadera, a Causal Agent of Grapevine Trunk Disease.

Polyphenols were characterized from Dothiorella vidmadera (DAR78993), which was isolated from a grapevine in Australia. In total, six polyphenols were isolated including a new polyphenol characterized by a spectroscopic method (essentially NMR and HR ESIMS) as 5-hydroxymethyl-2-isopropoxyphenol. Tyrosol, benzene-1,2,4-triol, resorcinol, 3-(hydroxymethyl)phenol, and protocatechuic alcohol, the latter being the main metabolite, were also isolated. Although these are already known as naturally occurring compounds in microorganisms and plants, this is the first time they have been isolated from fungal organisms involved in grapevine trunk disease. When assayed on tomato seedlings, all the compounds show similar phytotoxic effects. However, when assayed on grapevine leaves (Vitis vinifera cv Shiraz), resorcinol was the most toxic compound, followed by protocatechuic alcohol and 5-hydroxymethyl-2-isopropoxyphenol.

Laccase-mediated functionalization of chitosan with 4-hexyloxyphenol enhances antioxidant and hydrophobic properties of copolymer.

An effective method to functionalize chitosan with 4-hexyloxyphenol (HP) under homogeneous reaction conditions was developed using laccase as the catalyst. The resulting copolymer was characterized for chemical structure, grafted-HP content, surface morphology, thermal stability, antioxidant capacity, hydrophobic properties and tensile strength. Solid-state 13C NMR spectrum confirmed the incorporation of HP onto chitosan. X-ray diffraction (XRD) showed a decrease in the degree of crystallinity for laccase/HP treated chitosan compared to pure chitosan. The grafted-HP content in laccase/HP-treated chitosan first increased and then declined with increase of the initial HP/chitosan ratio. A heterogeneous surface with spherical particles on the laccase/HP treated chitosan was observed by environmental scanning electron microscopy (ESEM) and scanning probe microscopy (SPM). The laccase/HP treatment of chitosan improved the thermal stability of copolymer. More significantly, the HP functionalized chitosan showed greatly improved ABTS+ and DPPH radicals scavenging capacity, compared with pure chitosan. The hydrophobicity property of the HP functionalized chitosan also significantly increased although its tensile strength decreased. This new type of composite with double functionalities (i.e., antioxidant and hydrophobic) could potentially be used as food packaging materials or coating agents.

Phosphorogenic sensors for biothiols derived from cyclometalated iridium(III) polypyridine complexes containing a dinitrophenyl ether moiety.

We report the synthesis and characterization of three cyclometalated iridium(III) polypyridine complexes containing a 2,4-dinitrophenyl ether moiety [Ir(pq)2(N^N)](PF6) (Hpq=2-phenylquinoline; N^N=4-(N-(4-(2,4-dinitrophenoxy)benzyloxy)carbonyl)aminomethyl-4'-methyl-2,2'-bipyridine (bpy-dinitro-1) (1a), 4-(2,4-dinitrophenoxy)methyl-4'-methyl-2,2'-bipyridine (bpy-dinitro-2) (2a), 4-(4-(2,4-dinitrophenoxy)phenyl)-2,2'-bipyridine (bpy-dinitro-3) (3a)) as intracellular sensors for biothiols. Due to the quenching effect of the dinitroaromatic moiety, these complexes were extremely weakly emissive. Upon the reaction with biothiols, however, the emission was turned on as a consequence of the departure of the quenching unit. The results from a range of experiments demonstrated that complex 1a was noncytotoxic under the conditions used for confocal imaging, showed facile cellular uptake, and can serve as a phosphorogenic intracellular sensor for biothiols including glutathione (GSH) and hydrogen sulfide.

Bioresolution of racemic phenyl glycidyl ether by a putative recombinant epoxide hydrolase from Streptomyces griseus NBRC 13350.

In order to produce enantiomerically pure epoxides for the synthesis of value-added chemicals, a novel putative epoxide hydrolase (EH) sgeh was cloned and overexpressed in pET28a/Escherichia coli BL21(DE3). The 1047 bp sgeh gene was mined from Streptomyces griseus NBRC 13350 genome sequence. The recombinant hexahistidyl-tagged SGEH was purified (16.6-fold) by immobilized metal-affinity chromatography, with 90% yield as a homodimer of 100 kDa. The recombinant E. coli whole cells overexpressing SGEH could kinetically resolve racemic phenyl glycidyl ether (PGE) into (R)-PGE with 98% ee, 40% yield, and enantiomeric ratio (E) of 20. This was achieved under the optimized reaction conditions i.e. cell/substrate ratio of 20:1 (w/w) at pH 7.5 and 20 °C in 10% (v/v) dimethylformamide (DMF) in a 10 h reaction. 99% enantiopure (R)-PGE was obtained when the reaction time was prolonged to 12 h with a yield of 34%. In conclusion, an economically viable and environment friendly green process for the production of enantiopure (R)-PGE was developed by using wet cells of E. coli expressing recombinant SGEH.

Degradation of Diphenyl Ether in Sphingobium phenoxybenzoativorans SC_3 Is Initiated by a Novel Ring Cleavage Dioxygenase.

Sphingobium phenoxybenzoativorans SC_3 degrades and utilizes diphenyl ether (DE) or 2-carboxy-DE as its sole carbon and energy source. In this study, we report the degradation of DE and 2-carboxy-DE initiated by a novel ring cleavage angular dioxygenase (diphenyl ether dioxygenase [Dpe]) in the strain. Dpe functions at the angular carbon and its adjacent carbon (C-1a, C-2) of a benzene ring in DE (or the 2-carboxybenzene ring in 2-carboxy-DE) and cleaves the C-1a-C-2 bond (decarboxylation occurs simultaneously for 2-carboxy-DE), yielding 2,4-hexadienal phenyl ester, which is subsequently hydrolyzed to muconic acid semialdehyde and phenol. Dpe is a type IV Rieske non-heme iron oxygenase (RHO) and consists of three components: a hetero-oligomer oxygenase, a [2Fe-2S]-type ferredoxin, and a glutathione reductase (GR)-type reductase. Genetic analyses revealed that dpeA1A2 plays an essential role in the degradation and utilization of DE and 2-carboxy-DE in S. phenoxybenzoativorans SC_3. Enzymatic study showed that transformation of 1 molecule of DE needs two molecules of oxygen and two molecules of NADH, supporting the assumption that the cleavage of DE catalyzed by Dpe is a continuous two-step dioxygenation process: DE is dioxygenated at C-1a and C-2 to form a hemiacetal-like intermediate, which is further deoxygenated, resulting in the cleavage of the C-1a-C-2 bond to form one molecule of 2,4-hexadienal phenyl ester and two molecules of H2O. This study extends our knowledge of the mode and mechanism of ring cleavage of aromatic compounds.IMPORTANCE Benzene ring cleavage, catalyzed by dioxygenase, is the key and speed-limiting step in the aerobic degradation of aromatic compounds. As previously reported, in the ring cleavage of DEs, the benzene ring needs to be first dihydroxylated at a lateral position and subsequently dehydrogenated and opened through extradiol cleavage. This process requires three enzymes (two dioxygenases and one dehydrogenase). In this study, we identified a novel angular dioxygenase (Dpe) in S. phenoxybenzoativorans SC_3. Under Dpe-mediated catalysis, the benzene ring of DE is dioxygenated at the angular position (C-1a, C-2), resulting in the cleavage of the C-1a-C-2 bond to generate a novel product, 2,4-hexadienal phenyl ester. This process needs only one angular dioxygenase, Dpe. Thus, the ring cleavage catalyzed by Dpe represents a novel mechanism of benzene ring cleavage.

Effect of Cigarette Smoking on a Marker for Neuroinflammation: A [11C]DAA1106 Positron Emission Tomography Study.

In the brain, microglia continuously scan the surrounding extracellular space in order to respond to damage or infection by becoming activated and participating in neuroinflammation. When activated, microglia increase the expression of translocator protein (TSPO) 18 kDa, thereby making the TSPO expression a marker for neuroinflammation. We used the radiotracer [11C]DAA1106 (a ligand for TSPO) and positron emission tomography (PET) to determine the effect of smoking on availability of this marker for neuroinflammation. Forty-five participants (30 smokers and 15 non-smokers) completed the study and had usable data. Participants underwent a dynamic PET scanning session with bolus injection of [11C]DAA1106 (with smokers in the satiated state) and blood draws during PET scanning to determine TSPO affinity genotype and plasma nicotine levels. Whole-brain standardized uptake values (SUVs) were determined, and analysis of variance was performed, with group (smoker vs non-smoker) and genotype as factors, thereby controlling for genotype. Smokers and non-smokers differed in whole-brain SUVs (P=0.006) owing to smokers having 16.8% lower values than non-smokers. The groups did not differ in injected radiotracer dose or body weight, which were used to calculate SUV. An inverse association was found between whole-brain SUV and reported cigarettes per day (P<0.05), but no significant relationship was found for plasma nicotine. Thus, smokers have less [11C]DAA1106 binding globally than non-smokers, indicating less microglial activation. Study findings are consistent with much prior research demonstrating that smokers have impaired inflammatory functioning compared with non-smokers and that constituents of tobacco smoke other than nicotine affect inflammatory processes.

Isolation of bacterial strains able to degrade biphenyl, diphenyl ether and the heat transfer fluid used in thermo-solar plants.

Thermo-solar plants use eutectic mixtures of diphenyl ether (DE) and biphenyl (BP) as heat transfer fluid (HTF). Potential losses of HTF may contaminate soils and bioremediation is an attractive tool for its treatment. DE- or BP-degrading bacteria are known, but up to now bacteria able to degrade HTF mixture have not been described. Here, five bacterial strains which are able to grow with HTF or its separate components DE and BP as sole carbon sources have been isolated, either from soils exposed to HTF or from rhizospheric soils of plants growing near a thermo-solar plant. The organisms were identified by 16S rRNA gene sequencing as Achromobacter piechaudii strain BioC1, Pseudomonas plecoglossicida strain 6.1, Pseudomonas aeruginosa strains HBD1 and HBD3, and Pseudomonas oleovorans strain HBD2. Activity of 2,3-dihydroxybiphenyl dioxygenase (BphC), a key enzyme of the biphenyl upper degradation pathway, was detected in all isolates. Pseudomonas strains almost completely degraded 2000ppm HTF after 5-day culture, and even tolerated and grew in the presence of 150,000ppm HTF, being suitable candidates for in situ soil bioremediation. Degradation of both components of HTF is of particular interest since in the DE-degrader Sphingomonas sp. SS3, growth on DE or benzoate was strongly inhibited by addition of BP.

Effect of fungal mycelia on the HPLC-UV and UV-vis spectrophotometric assessment of mycelium-bound epoxide hydrolase using glycidyl phenyl ether.

The use of mycelia as biocatalysts has technical and economic advantages. However, there are several difficulties in obtaining accurate results in mycelium-catalysed reactions. Firstly, sample extraction, indispensable because of the presence of mycelia, can bring into the extract components with a similar structure to that of the analyte of interest; secondly, mycelia can influence the recovery of the analyte. We prepared calibration standards of 3-phenoxy-1,2-propanediol (PPD) in the pure solvent and in the presence of mycelia (spiked before or after extraction) from five fungi (Aspergillus niger, Aspergillus tubingensis, Penicillium aurantiogriseum, Penicillium sp. and Aspergillus terreus). The quantification of PPD was carried out by HPLC-UV and UV-vis spectrophotometry. The manuscript shows that the last method is as accurate as the HPLC method. However, the colorimetric method led to a higher data throughput, which allowed the study of more samples in a shorter time. Matrix effects were evaluated visually from the plotted calibration data and statistically by simultaneously comparing the intercept and slope of calibration curves performed with solvent, post-extraction spiked standards and pre-extraction spiked standards. Significant differences were found between the post- and pre-extraction spiked matrix-matched functions. Pre-extraction spiked matrix-matched functions based on A. tubingensis mycelia, selected as the reference, were validated and used to compensate for low recoveries. These validated functions were successfully applied to the quantification of PPD achieved during the hydrolysis of glycidyl phenyl ether by mycelium-bound epoxide hydrolases and equivalent hydrolysis yields were determined by HPLC-UV and UV-vis spectrophotometry. This study may serve as starting point to implement matrix effects evaluation when mycelium-bound epoxide hydrolases are studied.