Vertebrate endocrine disruptors induce sex-reversal in blue mussels.

Mollusks are the second most diverse animal phylum, yet little is known about their endocrinology or how they respond to endocrine disrupting compound (EDC) pollution. Characteristic effects of endocrine disruption are reproductive impairment, skewed sex ratios, development of opposite sex characteristics, and population decline. However, whether classical vertebrate EDCs, such as steroid hormone-like chemicals and inhibitors of steroidogenesis, exert effects on mollusks is controversial. In the blue mussel, Mytilus edulis, EDC exposure is correlated with feminized sex ratios in wild and laboratory mussels, but sex reversal has not been confirmed. Here, we describe a non-destructive qPCR assay to identify the sex of M. edulis allowing identification of males and females prior to experimentation. We exposed male mussels to 17α-ethinylestradiol and female mussels to ketoconazole, EDCs that mimic vertebrate steroid hormones or inhibit their biosynthesis. Both chemicals changed the sex of individual mussels, interfered with gonadal development, and disrupted gene expression of the sex differentiation pathway. Impacts from ketoconazole treatment, including changes in steroid levels, confirmed a role for steroidogenesis and steroid-like hormones in mollusk endocrinology. The present study expands the possibilities for laboratory and field monitoring of mollusk species and provides key insights into endocrine disruption and sexual differentiation in bivalves.

Structure-Function Analysis of the Essential Mycobacterium tuberculosis P450 Drug Target, CYP121A1.

Cytochrome P450 CYP121A1 is a well-known drug target against Mycobacterium tuberculosis, the human pathogen that causes the deadly disease tuberculosis (TB). CYP121A1 is a unique P450 enzyme because it uses classical and non-classical P450 catalytic processes and has distinct structural features among P450s. However, a detailed investigation of CYP121A1 protein structures in terms of active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 53 CYP121A1 crystal structures were investigated in this study. Critical amino acids required for CYP121A1's overall activity were identified and highlighted this enzyme's rigid architecture and substrate selectivity. The CYP121A1-fluconazole crystal structure revealed a novel azole drug-P450 binding mode in which azole heme coordination was facilitated by a water molecule. Fragment-based inhibitor approaches revealed that CYP121A1 can be inhibited by molecules that block the substrate channel or by directly interacting with the P450 heme. This study serves as a reference for the precise understanding of CYP121A1 interactions with different ligands and the structure-function analysis of P450 enzymes in general. Our findings provide critical information for the synthesis of more specific CYP121A1 inhibitors and their development as novel anti-TB drugs.

Identification of Potent and Selective Inhibitors of Acanthamoeba: Structural Insights into Sterol 14α-Demethylase as a Key Drug Target.

Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC50 0.45 µM), VT1598 (0.25 µM), and VT1161 (0.20 µM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.

Biosynthesis of a new skyllamycin in Streptomyces nodosus: a cytochrome P450 forms an epoxide in the cinnamoyl chain.

Activation of a silent gene cluster in Streptomyces nodosus leads to synthesis of a cinnamoyl-containing non-ribosomal peptide (CCNP) that is related to skyllamycins. This novel CCNP was isolated and its structure was interrogated using mass spectrometry and nuclear magnetic resonance spectroscopy. The isolated compound is an oxidised skyllamycin A in which an additional oxygen atom is incorporated in the cinnamoyl side-chain in the form of an epoxide. The gene for the epoxide-forming cytochrome P450 was identified by targeted disruption. The enzyme was overproduced in Escherichia coli and a 1.43 Å high-resolution crystal structure was determined. This is the first crystal structure for a P450 that forms an epoxide in a substituted cinnamoyl chain of a lipopeptide. These results confirm the proposed functions of P450s encoded by biosynthetic gene clusters for other epoxidized CCNPs and will assist investigation of how epoxide stereochemistry is determined in these natural products.

Structure-Function Analysis of the Biotechnologically Important Cytochrome P450 107 (CYP107) Enzyme Family.

Cytochrome P450 monooxygenases (CYPs; P450s) are a superfamily of heme-containing enzymes that are recognized for their vast substrate range and oxidative multifunctionality. CYP107 family members perform hydroxylation and epoxidation processes, producing a variety of biotechnologically useful secondary metabolites. Despite their biotechnological importance, a thorough examination of CYP107 protein structures regarding active site cavity dynamics and key amino acids interacting with bound ligands has yet to be undertaken. To address this research knowledge gap, 44 CYP107 crystal structures were investigated in this study. We demonstrate that the CYP107 active site cavity is very flexible, with ligand binding reducing the volume of the active site in some situations and increasing volume size in other instances. Polar interactions between the substrate and active site residues result in crucial salt bridges and the formation of proton shuttling pathways. Hydrophobic interactions, however, anchor the substrate within the active site. The amino acid residues within the binding pocket influence substrate orientation and anchoring, determining the position of the hydroxylation site and hence direct CYP107's catalytic activity. Additionally, the amino acid dynamics within and around the binding pocket determine CYP107's multifunctionality. This study serves as a reference for understanding the structure-function analysis of CYP107 family members precisely and the structure-function analysis of P450 enzymes in general. Finally, this work will aid in the genetic engineering of CYP107 enzymes to produce novel molecules of biotechnological interest.

Structural modeling of cytochrome P450 51 from a deep-sea fish points to a novel structural feature in other CYP51s.

Cytochromes P450 (CYP), enzymes involved in the metabolism of endogenous and xenobiotic substrates, provide an excellent model system to study how membrane proteins with unique functions have catalytically adapted through evolution. Molecular adaptation of deep-sea proteins to high hydrostatic pressure remains poorly understood. Herein, we have characterized recombinant cytochrome P450 sterol 14α-demethylase (CYP51), an essential enzyme of cholesterol biosynthesis, from an abyssal fish species, Coryphaenoides armatus. C. armatus CYP51 was heterologously expressed in Escherichia coli following N-terminal truncation and purified to homogeneity. Recombinant C. armatus CYP51 bound its sterol substrate lanosterol giving a Type I binding spectra (KD 15 µM) and catalyzed lanosterol 14α-demethylation turnover at 5.8 nmol/min/nmol P450. C. armatus CYP51 also bound the azole antifungals ketoconazole (KD 0.12 µM) and propiconazole (KD 0.54 µM) as determined by Type II absorbance spectra. Comparison of C. armatus CYP51 primary sequence and modeled structures with other CYP51s identified amino acid substitutions that may confer an ability to function under pressures of the deep sea and revealed heretofore undescribed internal cavities in human and other non-deep sea CYP51s. The functional significance of these cavities is not known. PROLOGUE: This paper is dedicated in memory of Michael Waterman and Tsuneo Omura, who as good friends and colleagues enriched our lives. They continue to inspire us.

Unravelling the role of transient redox partner complexes in P450 electron transfer mechanics.

The molecular evolution of cytochromes P450 and associated redox-driven oxidative catalysis remains a mystery in biology. It is widely believed that sterol 14α-demethylase (CYP51), an essential enzyme of sterol biosynthesis, is the ancestor of the whole P450 superfamily given its conservation across species in different biological kingdoms. Herein we have utilized X-ray crystallography, molecular dynamics simulations, phylogenetics and electron transfer measurements to interrogate the nature of P450-redox partner binding using the naturally occurring fusion protein, CYP51-ferredoxin found in the sterol-producing bacterium Methylococcus capsulatus. Our data advocates that the electron transfer mechanics in the M. capsulatus CYP51-ferredoxin fusion protein involves an ensemble of ferredoxin molecules in various orientations and the interactions are transient. Close proximity of ferredoxin, however, is required to complete the substrate-induced large-scale structural switch in the P450 domain that enables proton-coupled electron transfer and subsequent oxygen scission and catalysis. These results have fundamental implications regarding the early evolution of electron transfer proteins and for the redox reactions in the early steps of sterol biosynthesis. They also shed new light on redox protein mechanics and the subsequent diversification of the P450 electron transfer machinery in nature.

Characterization of a Virally Encoded Flavodoxin That Can Drive Bacterial Cytochrome P450 Monooxygenase Activity.

Flavodoxins are small electron transport proteins that are involved in a myriad of photosynthetic and non-photosynthetic metabolic pathways in Bacteria (including cyanobacteria), Archaea and some algae. The sequenced genome of 0305φ8-36, a large bacteriophage that infects the soil bacterium Bacillus thuringiensis, was predicted to encode a putative flavodoxin redox protein. Here we confirm that 0305φ8-36 phage encodes a FMN-containing flavodoxin polypeptide and we report the expression, purification and enzymatic characterization of the recombinant protein. Purified 0305φ8-36 flavodoxin has near-identical spectral properties to control, purified Escherichia coli flavodoxin. Using in vitro assays we show that 0305φ8-36 flavodoxin can be reconstituted with E. coli flavodoxin reductase and support regio- and stereospecific cytochrome P450 CYP170A1 allyl-oxidation of epi-isozizaene to the sesquiterpene antibiotic product albaflavenone, found in the soil bacterium Streptomyces coelicolor. In vivo, 0305φ8-36 flavodoxin is predicted to mediate the 2-electron reduction of the ß subunit of phage-encoded ribonucleotide reductase to catalyse the conversion of ribonucleotides to deoxyribonucleotides during viral replication. Our results demonstrate that this phage flavodoxin has the potential to manipulate and drive bacterial P450 cellular metabolism, which may affect both the host biological fitness and the communal microbiome. Such a scenario may also be applicable in other viral-host symbiotic/parasitic relationships.

Metabolic arsenal of giant viruses: Host hijack or self-use?

Viruses generally are defined as lacking the fundamental properties of living organisms in that they do not harbor an energy metabolism system or protein synthesis machinery. However, the discovery of giant viruses of amoeba has fundamentally challenged this view because of their exceptional genome properties, particle sizes and encoding of the enzyme machinery for some steps of protein synthesis. Although giant viruses are not able to replicate autonomously and still require a host for their multiplication, numerous metabolic genes involved in energy production have been recently detected in giant virus genomes from many environments. These findings have further blurred the boundaries that separate viruses and living organisms. Herein, we summarize information concerning genes and proteins involved in cellular metabolic pathways and their orthologues that have, surprisingly, been discovered in giant viruses. The remarkable diversity of metabolic genes described in giant viruses include genes encoding enzymes involved in glycolysis, gluconeogenesis, tricarboxylic acid cycle, photosynthesis, and ß-oxidation. These viral genes are thought to have been acquired from diverse biological sources through lateral gene transfer early in the evolution of Nucleo-Cytoplasmic Large DNA Viruses, or in some cases more recently. It was assumed that viruses are capable of hijacking host metabolic networks. But the giant virus auxiliary metabolic genes also may represent another form of host metabolism manipulation, by expanding the catalytic capabilities of the host cells especially in harsh environments, providing the infected host cells with a selective evolutionary advantage compared to non-infected cells and hence favoring the viral replication. However, the mechanism of these genes' functionality remains unclear to date.

Characterization of a flexible AAV-DTR/DT mouse model of acute epithelial lung injury.

Animal models are important to mimic certain pathways or biological aspects of human pathologies including acute and chronic pulmonary diseases. We developed a novel and flexible mouse model of acute epithelial lung injury based on adeno-associated virus (AAV) variant 6.2-mediated expression of the human diphtheria toxin receptor (DTR). Following intratracheal administration of diphtheria toxin (DT), a cell-specific death of bronchial and alveolar epithelial cells can be observed. In contrast to other lung injury models, the here described mouse model provides the possibility of targeted injury using specific tropisms of AAV vectors or cell-type-specific promotors to drive the human DTR expression. Also, generation of cell-specific mouse lines is not required. Detailed characterization of the AAV-DTR/DT mouse model including titration of viral genome (vg) load and administered DT amount revealed increasing cell numbers in bronchoalveolar lavage (BAL; macrophages, neutrophils, and unspecified cells) and elevation of degenerated cells and infiltrated leukocytes in lung tissue, dependent of vg load and DT dose. Cytokine levels in BAL fluid showed different patterns with higher vg load, e.g., IFNγ, TNFα, and IP10 increasing and IL-5 and IL-6 decreasing, whereas lung function was not affected. In addition, laser-capture microdissection (LCM)-based proteomics of bronchial epithelium and alveolar tissue revealed upregulated immune and inflammatory responses in all regions and extracellular matrix deposition in infiltrated alveoli. Overall, our novel AAV-DTR/DT model allows investigation of repair mechanisms following epithelial injury and resembles specific mechanistic aspects of acute and chronic pulmonary diseases.

Structural analysis of P450 AmphL from Streptomyces nodosus provides insights into substrate selectivity of polyene macrolide antibiotic biosynthetic P450s.

AmphL is a cytochrome P450 enzyme that catalyzes the C8 oxidation of 8-deoxyamphotericin B to the polyene macrolide antibiotic, amphotericin B. To understand this substrate selectivity, we solved the crystal structure of AmphL to a resolution of 2.0 Å in complex with amphotericin B and performed molecular dynamics (MD) simulations. A detailed comparison with the closely related P450, PimD, which catalyzes the epoxidation of 4,5-desepoxypimaricin to the macrolide antibiotic, pimaricin, reveals key catalytic structural features responsible for stereo- and regio-selective oxidation. Both P450s have a similar access channel that runs parallel to the active site I helix over the surface of the heme. Molecular dynamics simulations of substrate binding reveal PimD can "pull" substrates further into the P450 access channel owing to additional electrostatic interactions between the protein and the carboxyl group attached to the hemiketal ring of 4,5-desepoxypimaricin. This substrate interaction is absent in AmphL although the additional substrate -OH groups in 8-deoxyamphotericin B help to correctly position the substrate for C8 oxidation. Simulations of the oxy-complex indicates that these -OH groups may also participate in a proton relay network required for O2 activation as has been suggested for two other macrolide P450s, PimD and P450eryF. These findings provide experimentally testable models that can potentially contribute to a new generation of novel macrolide antibiotics with enhanced antifungal and/or antiprotozoal efficacy.

Incomplete tricarboxylic acid cycle and proton gradient in Pandoravirus massiliensis: is it still a virus?

The discovery of Acanthamoeba polyphaga Mimivirus, the first isolated giant virus of amoeba, challenged the historical hallmarks defining a virus. Giant virion sizes are known to reach up to 2.3 µm, making them visible by optical microscopy. Their large genome sizes of up to 2.5 Mb can encode proteins involved in the translation apparatus. We have investigated possible energy production in Pandoravirus massiliensis. Mitochondrial membrane markers allowed for the detection of a membrane potential in purified virions and this was enhanced by a regulator of the tricarboxylic acid cycle but abolished by the use of a depolarizing agent. Bioinformatics was employed to identify enzymes involved in virion proton gradient generation and this approach revealed that eight putative P. massiliensis proteins exhibited low sequence identities with known cellular enzymes involved in the universal tricarboxylic acid cycle. Further, all eight viral genes were transcribed during replication. The product of one of these genes, ORF132, was cloned and expressed in Escherichia coli, and shown to function as an isocitrate dehydrogenase, a key enzyme of the tricarboxylic acid cycle. Our findings show for the first time that a membrane potential can exist in Pandoraviruses, and this may be related to tricarboxylic acid cycle. The presence of a proton gradient in P. massiliensis makes this virus a form of life for which it is legitimate to ask the question "what is a virus?".

Orphan cytochrome P450 20a1 CRISPR/Cas9 mutants and neurobehavioral phenotypes in zebrafish.

Orphan cytochrome P450 (CYP) enzymes are those for which biological substrates and function(s) are unknown. Cytochrome P450 20A1 (CYP20A1) is the last human orphan P450 enzyme, and orthologs occur as single genes in every vertebrate genome sequenced to date. The occurrence of high levels of CYP20A1 transcripts in human substantia nigra and hippocampus and abundant maternal transcripts in zebrafish eggs strongly suggest roles both in the brain and during early embryonic development. Patients with chromosome 2 microdeletions including CYP20A1 show hyperactivity and bouts of anxiety, among other conditions. Here, we created zebrafish cyp20a1 mutants using CRISPR/Cas9, providing vertebrate models with which to study the role of CYP20A1 in behavior and other neurodevelopmental functions. The homozygous cyp20a1 null mutants exhibited significant behavioral differences from wild-type zebrafish, both in larval and adult animals. Larval cyp20a1-/- mutants exhibited a strong increase in light-simulated movement (i.e., light-dark assay), which was interpreted as hyperactivity. Further, the larvae exhibited mild hypoactivity during the adaptation period of the optomotor assays. Adult cyp20a1 null fish showed a pronounced delay in adapting to new environments, which is consistent with an anxiety paradigm. Taken together with our earlier morpholino cyp20a1 knockdown results, the results described herein suggest that the orphan CYP20A1 has a neurophysiological role.

Molecular Dissection of Pro-Fibrotic IL11 Signaling in Cardiac and Pulmonary Fibroblasts.

In fibroblasts, TGFß1 stimulates IL11 upregulation that leads to an autocrine loop of IL11-dependent pro-fibrotic protein translation. The signaling pathways downstream of IL11, which acts via IL6ST, are contentious with both STAT3 and ERK implicated. Here we dissect IL11 signaling in fibroblasts and study IL11-dependent protein synthesis pathways in the context of approved anti-fibrotic drug mechanisms of action. We show that IL11-induced ERK activation drives fibrogenesis and while STAT3 phosphorylation (pSTAT3) is also seen, this appears unrelated to fibroblast activation. Ironically, recombinant human IL11, which has been used extensively in mouse experiments to infer STAT3 activity downstream of IL11, increases pSTAT3 in Il11ra1 null mouse fibroblasts. Unexpectedly, inhibition of STAT3 was found to induce severe proteotoxic ER stress, generalized fibroblast dysfunction and cell death. In contrast, inhibition of ERK prevented fibroblast activation in the absence of ER stress. IL11 stimulated an axis of ERK/mTOR/P70RSK protein translation and its selectivity for Collagen 1 synthesis was ascribed to an EPRS-regulated, ribosome stalling mechanism. Surprisingly, the anti-fibrotic drug nintedanib caused dose-dependent ER stress and lesser pSTAT3 expression. Pirfenidone had no effect on ER stress whereas anti-IL11 specifically inhibited the ERK/mTOR axis while reducing ER stress. These studies define the translation-specific signaling pathways downstream of IL11, intersect immune and metabolic signaling and reveal unappreciated effects of nintedanib.

An EZH2-dependent transcriptional complex promotes aberrant epithelial remodelling after injury.

Unveiling the molecular mechanisms of tissue remodelling following injury is imperative to elucidate its regenerative capacity and aberrant repair in disease. Using different omics approaches, we identified enhancer of zester homolog 2 (EZH2) as a key regulator of fibrosis in injured lung epithelium. Epithelial injury drives an enrichment of nuclear transforming growth factor-ß-activated kinase 1 (TAK1) that mediates EZH2 phosphorylation to facilitate its liberation from polycomb repressive complex 2 (PRC2). This process results in the establishment of a transcriptional complex of EZH2, RNA-polymerase II (POL2) and nuclear actin, which orchestrates aberrant epithelial repair programmes. The liberation of EZH2 from PRC2 is accompanied by an EZH2-EZH1 switch to preserve H3K27me3 deposition at non-target genes. Loss of epithelial TAK1, EZH2 or blocking nuclear actin influx attenuates the fibrotic cascade and restores respiratory homeostasis. Accordingly, EZH2 inhibition significantly improves outcomes in a pulmonary fibrosis mouse model. Our results reveal an important non-canonical function of EZH2, paving the way for new therapeutic interventions in fibrotic lung diseases.

RORγt inhibitors block both IL-17 and IL-22 conferring a potential advantage over anti-IL-17 alone to treat severe asthma.

BACKGROUND: RORγt is a transcription factor that enables elaboration of Th17-associated cytokines (including IL-17 and IL-22) and is proposed as a pharmacological target for severe asthma. METHODS: IL-17 immunohistochemistry was performed in severe asthma bronchial biopsies (specificity confirmed with in situ hybridization). Primary human small airway epithelial cells in air liquid interface and primary bronchial smooth muscle cells were stimulated with recombinant human IL-17 and/or IL-22 and pro-inflammatory cytokines measured. Balb/c mice were challenged intratracheally with IL-17 and/or IL-22 and airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. Balb/c mice were sensitized intraperitoneally and challenged intratracheally with house dust mite extract and the effect of either a RORγt inhibitor (BIX119) or an anti-IL-11 antibody assessed on airway hyperreactivity, pro-inflammatory cytokines and airway neutrophilia measured. RESULTS: We confirmed in severe asthma bronchial biopsies both the presence of IL-17-positive lymphocytes and that an IL-17 transcriptome profile in a severe asthma patient sub-population. Both IL-17 and IL-22 stimulated the release of pro-inflammatory cytokine and chemokine release from primary human lung cells and in mice. Furthermore, IL-22 in combination with IL-17, but neither alone, elicits airway hyperresponsiveness (AHR) in naïve mice. A RORγt inhibitor specifically blocked both IL-17 and IL-22, AHR and neutrophilia in a mouse house dust mite model unlike other registered or advanced pipeline modes of action. Full efficacy versus these parameters was associated with 90% inhibition of IL-17 and 50% inhibition of IL-22. In contrast, anti-IL-17 also blocked IL-17, but not IL-22, AHR or neutrophilia. Moreover, the deregulated genes in the lungs from these mice correlated well with deregulated genes from severe asthma biopsies suggesting that this model recapitulates significant severe asthma-relevant biology. Furthermore, these genes were reversed upon RORγt inhibition in the HDM model. Cell deconvolution suggested that the responsible cells were corticosteroid insensitive γδ-T-cells. CONCLUSION: These data strongly suggest that both IL-17 and IL-22 are required for Th2-low endotype associated biology and that a RORγt inhibitor may provide improved clinical benefit in a severe asthma sub-population of patients by blocking both IL-17 and IL-22 biology compared with blocking IL-17 alone.

Therapeutic Targeting of IL-11 for Chronic Lung Disease.

Interleukin (IL)-11 was originally recognized as an immunomodulatory and hematopoiesis-inducing cytokine. However, although IL-11 is typically not found in healthy individuals, it is now becoming evident that IL-11 may play a role in diverse pulmonary conditions, including IPF, asthma, and lung cancer. Additionally, experimental strategies targeting IL-11, such as humanized antibodies, have recently been developed, revealing the therapeutic potential of IL-11. Thus, further insight into the underlying mechanisms of IL-11 in lung disease may lead to the ability to interfere with pathological conditions that have a clear need for disease-modifying treatments, such as IPF. In this review, we outline the effects, expression, signaling, and crosstalk of IL-11 and focus on its role in lung disease and its potential as a therapeutic target.

The novel TRPA1 antagonist BI01305834 inhibits ovalbumin-induced bronchoconstriction in guinea pigs.

BACKGROUND: Asthma is a chronic respiratory disease in which the nervous system plays a central role. Sensory nerve activation, amongst others via Transient Receptor Potential Ankyrin 1 (TRPA1) channels, contributes to asthma characteristics including cough, bronchoconstriction, mucus secretion, airway hyperresponsiveness (AHR) and inflammation. In the current study, we evaluated the efficacy of the novel TRPA1 antagonist BI01305834 against AHR and inflammation in guinea-pig models of asthma. METHODS: First, a pilot study was performed in a guinea-pig model of allergic asthma to find the optimal dose of BI01305834. Next, the effect of BI01305834 on (1) AHR to inhaled histamine after the early and late asthmatic reaction (EAR and LAR), (2) magnitude of EAR and LAR and (3) airway inflammation was assessed. Precision-cut lung slices and trachea strips were used to investigate the bronchoprotective and bronchodilating-effect of BI01305834. Statistical evaluation of differences of in vivo data was performed using a Mann-Whitney U test or One-way nonparametric Kruskal-Wallis ANOVA, for ex vivo data One- or Two-way ANOVA was used, all with Dunnett's post-hoc test where appropriate. RESULTS: A dose of 1 mg/kg BI01305834 was selected based on AHR and exposure data in blood samples from the pilot study. In the subsequent study, 1 mg/kg BI01305834 inhibited AHR after the EAR, and the development of EAR and LAR elicited by ovalbumin in ovalbumin-sensitized guinea pigs. BI01305834 did not inhibit allergen-induced total and differential cells in the lavage fluid and interleukin-13 gene expression in lung homogenates. Furthermore, BI01305834 was able to inhibit allergen and histamine-induced airway narrowing in guinea-pig lung slices, without affecting histamine release, and reverse allergen-induced bronchoconstriction in guinea-pig trachea strips. CONCLUSIONS: TRPA1 inhibition protects against AHR and the EAR and LAR in vivo and allergen and histamine-induced airway narrowing ex vivo, and reverses allergen-induced bronchoconstriction independently of inflammation. This effect was partially dependent upon histamine, suggesting a neuronal and possible non-neuronal role for TRPA1 in allergen-induced bronchoconstriction.

Concerning P450 Evolution: Structural Analyses Support Bacterial Origin of Sterol 14α-Demethylases.

Sterol biosynthesis, primarily associated with eukaryotic kingdoms of life, occurs as an abbreviated pathway in the bacterium Methylococcus capsulatus. Sterol 14α-demethylation is an essential step in this pathway and is catalyzed by cytochrome P450 51 (CYP51). In M. capsulatus, the enzyme consists of the P450 domain naturally fused to a ferredoxin domain at the C-terminus (CYP51fx). The structure of M. capsulatus CYP51fx was solved to 2.7 Å resolution and is the first structure of a bacterial sterol biosynthetic enzyme. The structure contained one P450 molecule per asymmetric unit with no electron density seen for ferredoxin. We connect this with the requirement of P450 substrate binding in order to activate productive ferredoxin binding. Further, the structure of the P450 domain with bound detergent (which replaced the substrate upon crystallization) was solved to 2.4 Å resolution. Comparison of these two structures to the CYP51s from human, fungi, and protozoa reveals strict conservation of the overall protein architecture. However, the structure of an "orphan" P450 from nonsterol-producing Mycobacterium tuberculosis that also has CYP51 activity reveals marked differences, suggesting that loss of function in vivo might have led to alterations in the structural constraints. Our results are consistent with the idea that eukaryotic and bacterial CYP51s evolved from a common cenancestor and that early eukaryotes may have recruited CYP51 from a bacterial source. The idea is supported by bioinformatic analysis, revealing the presence of CYP51 genes in >1,000 bacteria from nine different phyla, >50 of them being natural CYP51fx fusion proteins.

Relapse patterns after low-dose-rate prostate brachytherapy.

PURPOSE: When biochemical failure (BF) develops after low-dose-rate prostate brachytherapy, the relapse site is frequently not found. We set out to find whether prostate-specific membrane antigen positron emission tomography -CT (PSMA PET-CT) scanning has improved knowledge of relapse patterns. METHODS AND MATERIALS: A database was analyzed, which contained information and long-term followup on 903 men who had an iodine-125 seed implant as monotherapy for early-stage prostate cancer. There was a total of 68 BFs. RESULT: In 38 men developing BF before PSMA PET-CT scanning was available, the site of relapse was local in six, distant in twelve, and unknown in twenty. In 30 men developing BF more recently who had a PSMA PET-CT scan, the relapse site was demonstrated in all cases, and 19 (63%) men had relapsed at the prostate base. Radiation dosimetry of base relapses and paired controls demonstrated that implants routinely delivered a lower radiation dose to the base than to the rest of the prostate. Eight of seventeen cases found to have prostate relapse only underwent salvage prostatectomy. CONCLUSION: PSMA PET-CT scanning is highly effective in demonstrating the relapse site(s) when BF develops after low-dose-rate prostate brachytherapy. Knowledge of the relapse site increases management options for men developing BF.