Heterologous biosynthesis of myxobacterial lanthipeptides melittapeptins.

The myxobacteria are an attractive bioresource for bioactive compounds since the large size genome contains many biosynthetic gene clusters of secondary metabolites. The genome of the myxobacterium Melittangium boletus contains three biosynthetic gene clusters for lanthipeptide production. One of the gene clusters includes genes coding lanthipeptide precursor (melA), class II lanthipeptide synthetase (melM), and transporter (melT). The amino acid sequence of melA indicated similarity with that of known lanthipeptides mersacidin and lichenicidin A1 by the alignment. To perform heterologous production of new lanthipeptides, the expression vector containing the essential genes (melA and melM) was constructed by utilizing codon-optimized synthetic genes. The co-expression of two genes in the host bacterial cells of Escherichia coli BL21 (DE3) afforded new lanthipeptides named melittapeptins A-C. The structures of melittapeptins A-C including lanthionine/methyllanthionine bridge pattern were proposed based on protease digestion and MS/MS experiments. The native strain of M. boletus did not produce melittapeptins A-C, so heterologous production using the biosynthetic gene cluster was effective in obtaining the lanthipeptides. Melittapeptins A-C showed specific and potent antibacterial activity to the Gram-positive bacterium Micrococcus luteus. To the best of our knowledge, this is the first report of antibacterial lanthipeptides derived from myxobacterial origin. KEY POINTS: • New lanthipeptides melittapeptins were heterologously produced in Escherichia coli. • Melittapeptins showed specific antibacterial activity against Micrococcus luteus. • Melittapeptins were the first antibacterial lanthipeptides of myxobacterial origin.

Effects of D-amino acids on sleep in Drosophila.

Sleep and metabolism are closely related and nutritional elements such as sugars and amino acids are known to regulate sleep differently. Here we comprehensively investigated the effects of D-amino acids fed in the diet on the sleep of Drosophila melanogaster. Among 19 amino acids examined, both D-serine (Ser) and D-glutamine (Gln) induced a significant increase in sleep amount and the effect of D-Ser was the largest at the same concentration of 1% of the food. The effects were proportional to its concentration and significant above 0.5% (about 50 mM). D-Ser is known to bind NR1 subunit of NMDA type glutamate receptor (NMDAR) and activate it. D-Ser did not increase the sleep of the NR1 hypomorphic mutant flies indicating its effects on sleep is mediated by NMDAR. In addition, hypomorphic mutants of D-amino acid oxidase (Daao1), which catabolizes D-amino acids and its disruption is known to increase D-Ser in the brain, showed increase in sleep. These results altogether suggested that D-Ser activated NMDAR in the brain thus increase sleep, and that D-Ser work physiologically to regulate sleep.

Unique Physiological and Genetic Features of Ofloxacin-Resistant Streptomyces Mutants.

New antimicrobial agents are urgently needed to combat the emergence and spread of multidrug-resistant bacteria. Activating the cryptic biosynthetic gene clusters for actinomycete secondary metabolites can provide essential clues for research into new antimicrobial agents. An effective method for this purpose is based on drug resistance selection. This report describes interesting results for drug resistance selection using antibiotics that target DNA replication and can effectively potentiate secondary metabolite production by actinomycetes. Ofloxacin-resistant mutants were isolated from five different streptomycetes. Ofloxacin is an antibiotic that binds to DNA complexes and type II topoisomerase, causing double-stranded breaks in bacterial chromosomes. Physiological and genetic characterization of the mutants revealed that the development of ofloxacin resistance in streptomycetes leads to the emergence of various types of secondary metabolite-overproducing strains. In Streptomyces coelicolor A3(2), ofloxacin-resistant mutants that overproduced actinorhodin, undecylprodigiosin, or carotenoid were identified. An ofloxacin-resistant mutant that overproduces methylenomycin A, whose biosynthetic gene cluster is located on the endogenous plasmid, SCP1, also was isolated. These observations indicate that ofloxacin resistance activates biosynthetic genes on both chromosomes and endogenous plasmids. We also identified the mutations that are probably involved in the phenotype of ofloxacin resistance and secondary metabolite overproduction in S. coelicolor A3(2). Furthermore, we observed an interesting phenomenon in which several ofloxacin-resistant mutants overproduced antibiotics in the presence of ofloxacin. Based on these results, we present the unique physiological and genetic characteristics of ofloxacin-resistant Streptomyces mutants and discuss the importance and potential development of the new findings. IMPORTANCE The abuse or overuse of antibacterial agents for therapy and animal husbandry has caused an increased population of antimicrobial-resistant bacteria in the environment. Consequently, fewer effective antimicrobials are now available. Due to the depleted antibiotic pipeline, pandemic outbreaks caused by antimicrobial-resistant bacteria are deeply concerning, and the development of new antibiotics is now an urgent issue. Promising sources of antimicrobial agents include cryptic biosynthetic gene clusters for secondary metabolites in streptomycetes and rare actinomycetes. This study's significance is the development of an unprecedented activation method to accelerate drug discovery research on a global scale. The technique developed in this study could allow for simultaneous drug discovery in different countries, maximizing the world's microbial resources.

Heterologous expression of a cryptic gene cluster from a marine proteobacterium Thalassomonas actiniarum affords new lanthipeptides thalassomonasins A and B.

AIMS: The aim of this study was to utilize a cryptic biosynthetic gene cluster (BGC) of a marine proteobacterium Thalassomonas actiniarum for production of new lanthipeptides by heterologous expression system. METHODS AND RESULTS: Based on genome mining, a new BGC of class I lanthipeptide was found in the genome sequence of a marine proteobacterium T. actiniarum. Molecular cloning was performed to construct an expression vector derived from commercially available plasmid pET-41a(+). Heterologous production of new lanthipeptides named thalassomonasins A and B was performed using the host Escherichia coli BL21(DE3) harbouring the expression vector. The structure of thalassomonasin A was determined by the interpretation of NMR and MS data. As a result, thalassomonasin A was determined to be a lanthipeptide with three units of lanthionine. The bridging pattern of the lanthionine rings in thalassomonasin A was determined by interpretation of NOESY data. The structure of thalassomonasin B was proposed by MS/MS experiment. CONCLUSIONS: We succeeded in heterologous production of new class I lanthipeptides using a BGC of a marine proteobacterium T. actiniarum. SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of heterologous production of lanthipeptides derived from proteobacterial origin. There are many cryptic biosynthetic gene clusters (BCGs) of this class of lanthipeptides in proteobacterial genomes. This study may lead to the production of new lanthipeptides by utilizing the BCGs.

Heterologous production of new lanthipeptides hazakensins A and B using a cryptic gene cluster of the thermophilic bacterium Thermosporothrix hazakensis.

The thermophilic bacterium Thermosporothrix hazakensis belongs to a class of Ktedonobacteria in the phylum Chloroflexota. Lanthipeptides are a naturally occurring peptide group that contains antibacterial compounds such as nisin. To find a new lanthipeptide that is a possible candidate for an antibacterial reagent, we performed genome-mining of T. hazakensis and heterologous expression experiments. Based on genome-mining, the presence of a total of ten putative biosynthetic gene clusters for class I and class II lanthipeptides was indicated from the genome sequence of T. hazakensis. New lanthipeptides named hazakensins A and B were produced by heterologous expression of a class I lanthipeptide biosynthetic gene cluster in the expression host Escherichia coli. Co-expression of the biosynthetic gene cluster with tRNA-Glu and glutamyl-tRNA synthetase coding genes derived from T. hazakensis increased the production yield of both lanthipeptides by about 4-6 times. The chemical structures of hazakensins A and B including the bridging pattern of lanthionine/methyllanthionine rings were determined by NMR and MS experiments. Since production of hazakensins A and B was not observed in the native strain T. hazakensis, heterologous production was an effective method to obtain the lanthipeptides derived from the biosynthetic gene cluster. This is the first report of heterologous production of class I lanthipeptides originating from the filamentous green non-sulfur bacteria, to the best of our knowledge. The success of heterologous production of hazakensins may lead to the discovery and development of new lanthipeptides derived from the origins of bacteria in the phylum Chloroflexota.

Heterologous expression of a cryptic gene cluster from Marinomonas fungiae affords a novel tricyclic peptide marinomonasin.

The ω-ester-containing peptides (OEPs) are a group of ribosomally synthesized and post-translationally modified peptides (RiPPs). The biosynthetic gene clusters of ω-ester-containing peptides commonly include ATP-grasp ligase coding genes and are distributed over the genomes of a wide variety of bacteria. A new biosynthetic gene cluster of ω-ester-containing peptides was found in the genome sequence of the marine proteobacterium Marinomonas fungiae. Heterologous production of a new tricyclic peptide named marinomonasin was accomplished using the biosynthetic gene cluster in Escherichia coli expression host strain BL21(DE3). By ESI-MS and NMR experiments, the structure of marinomonasin was determined to be a tricyclic peptide 18 amino acids in length with one ester and two isopeptide bonds in the molecule. The bridging patterns of the three intramolecular bonds were determined by the interpretation of HMBC and NOESY data. The bridging pattern of marinomonasin was unprecedented in the ω-ester-containing peptide group. The results indicated that the ATP-grasp ligase for the production of marinomonasin was a novel enzyme possessing bifunctional activity to form one ester and two isopeptide bonds. KEY POINTS: • New tricyclic peptide marinomonasin was heterologously produced in Escherichia coli. • Marinomonasin contained one ester and two isopeptide bonds in the molecule. • The bridging pattern of intramolecular bonds was novel.

Isolation and structure determination of new linear azole-containing peptides spongiicolazolicins A and B from Streptomyces sp. CWH03.

Linear azole-containing peptides are a class of ribosomally synthesized and post-translationally modified peptides. We performed a chemical investigation on marine actinomycetes, and new linear azole-containing peptides named spongiicolazolicins A and B were found in the MeOH extracts of a newly isolated strain Streptomyces sp. CWH03 (NBRC 114659) and two strains of S. spongiicola (strain HNM0071T: DSM 103383T and strain 531S: NBRC 113560). The strain Streptomyces sp. CWH03 was indicated to be a new species closely related to S. spongiicola by phylogenetic analysis using the genome sequence. The new peptides named spongiicolazolicins A and B were isolated from the cell of Streptomyces sp. CWH03. The partial structure of spongiicolazolicin A was determined by 2D NMR experiments. Based on data of MS/MS experiments, the chemical structures of spongiicolazolicins A and B were proposed using the amino acid sequence deduced from the precursor-encoding gene, which was found from whole-genome sequence data of Streptomyces sp. CWH03. The biosynthetic gene cluster of spongiicolazolicins was proposed based on comparative analysis with that of a known linear azole peptide goadsporin. KEY POINTS: • Streptomyces sp. CWH03 was a new species isolated from marine sediment. • New linear azole-containing peptides named spongiicolazolicins A and B were isolated. • Biosynthetic pathway of spongiicolazolicins was proposed.

Heterologous production of new protease inhibitory peptide marinostatin E.

Bicyclic peptides, marinostatins, are protease inhibitors derived from the marine bacterium Algicola sagamiensis. The biosynthetic gene cluster of marinostatin was previously identified, although no heterologous production was reported. In this report, the biosynthetic gene cluster of marinostatin (mstA and mstB) was cloned into the expression vector pET-41a(+). As a result of the coexpression experiment, a new analogous peptide named marinostatin E was successfully produced using Escherichia coli BL21(DE3). The structure of marinostatin E was determined by a combination of chemical treatments and tandem mass spectrometry experiments. Marinostatin E exhibited inhibitory activities against chymotrypsin and subtilisin with an IC50 of 4.0 and 39.6 µm, respectively.

Validation of the FIGO 2018 staging system of cervical cancer: Retrospective analysis of FIGO 2009 stage IB1 cervical cancer with tumor under 2 cm.

AIM: The International Federation of Gynecology and Obstetrics (FIGO) revised the cervical cancer staging system in 2018. This study aims to validate the revised staging system in patients with tumors <2 cm in size who were classified as FIGO 2009 stage IB1. METHODS: We evaluated 62 women with stage IB1 cervical cancer (FIGO 2009) who underwent radical hysterectomy as the initial treatment between November 2004 and August 2018 in our institution. The patients with FIGO 2009 stage IB1 and tumors <2 cm in size were enrolled. We reclassified their stage according to the FIGO 2018 staging system and analyzed their clinicopathological data retrospectively. RESULTS: Twenty-five patients met the inclusion criteria. According to the FIGO 2018 classification, 9 (36.0%) patients were classified as stage IA, 13 (52.0%) as stage IB1, and 3 (12.0%) as stage IIIC, respectively. One (11.1%), six (46.2%), and three (100%) patients with lymphovascular space invasion were classified as stage IA, IB1, and IIIC, respectively. No significant differences were found in the 5-year overall survival or progression-free survival among the three stages. CONCLUSIONS: As many as 36.0% of patients classified as FIGO 2009 stage IB1 with a tumor <2 cm in size were classified as stage IA in the FIGO 2018 classification. For these cases, a treatment less invasive than radical hysterectomy or radiotherapy might be sufficient. Our results suggest that cervical cancer patients with tumors <2 cm should be carefully diagnosed by performing cervical conization and assessed the pathological findings before hysterectomy.

Heterologous expression of a cryptic gene cluster from Grimontia marina affords a novel tricyclic peptide grimoviridin.

Microviridins are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) that have been isolated from a wide variety of cyanobacterial strains. There are similar gene clusters of RiPPs distributed in the genomes of bacteria belonging to the phyla Proteobacteria and Bacteroidetes. A cryptic gene cluster for the production of microviridin-type peptide was found in the genome of the marine γ-Proteobacterium Grimontia marina. Heterologous production of new microviridin-type peptide named grimoviridin was accomplished in Escherichia coli using the biosynthetic gene cluster of G. marina. The structure of grimoviridin was determined by analysis of MS and NMR data. Grimoviridin contained one isopeptide and two ester bonds, which had exactly the same bridging pattern as other microviridin-type peptides. The absolute stereochemistries of constituent amino acids were determined to be all L-forms by modified Marfey's method. Grimoviridin showed potent inhibitory activity against trypsin with an IC50 value of 238 nM. This is the first report of heterologous production of microviridin-type peptide using a biosynthetic gene cluster from a Proteobacterium. Key points • Heterologous production afforded new microviridin-type peptide named grimoviridin. • This is the first report of microviridin-type peptide from proteobacterial origin. • Grimoviridin showed potent inhibitory activity against trypsin.

Detection and Determination of Fumonisins B1, B2, and B3 Contaminating Japanese Domestic Wine by Liquid Chromatography Coupled to Tandem Mass Spectrometry (LC-MS/MS).

Nine domestic wine samples collected from a Japanese winery were examined for the presence of fumonisin B1 (FB1), fumonisin B2 (FB2), and fumonisin B3 (FB3), as well as ochratoxin A (OTA) and ochratoxin B (OTB). Wine samples spiked with 13C-labeled internal standards (13C34-FB1 and 13C20-OTA) were diluted with phosphate buffered saline (PBS) buffer, loaded on immunoaffinity cartridges to purify of fumonisins and ochratoxins, and subjected to liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis. The data revealed that the domestic wine samples were possibly contaminated with FB1 and FB3, in addition to FB2, whereas none of the tested wine samples were contaminated with OTA and OTB. These results suggest that Fusarium fungi can be associated with the fumonisin contamination of Japanese domestic wine, whereas Aspergillus niger seems to be frequently reported as the major causal fungus of fumonisin contamination of wine in Europe. Analysis of the intermediate samples during the wine brewing indicated that fumonisin concentrations did not increase during wine production, suggesting that fumonisin contamination did not occur during the brewing process, but was derived from the raw materials of grape berries.

verA Gene is Involved in the Step to Make the Xanthone Structure of Demethylsterigmatocystin in Aflatoxin Biosynthesis.

In the biosynthesis of aflatoxin, verA, ver-1, ordB, and hypA genes of the aflatoxin gene cluster are involved in the pathway from versicolorin A (VA) to demethylsterigmatocystin (DMST). We herein isolated each disruptant of these four genes to determine their functions in more detail. Disruptants of ver-1, ordB, and hypA genes commonly accumulated VA in their mycelia. In contrast, the verA gene disruptant accumulated a novel yellow fluorescent substance (which we named HAMA) in the mycelia as well as culture medium. Feeding HAMA to the other disruptants commonly caused the production of aflatoxins B1 (AFB1) and G1 (AFG1). These results indicate that HAMA pigment is a novel aflatoxin precursor which is involved at a certain step after those of ver-1, ordB, and hypA genes between VA and DMST. HAMA was found to be an unstable substance to easily convert to DMST and sterigmatin. A liquid chromatography-mass spectrometry (LC-MS) analysis showed that the molecular mass of HAMA was 374, and HAMA gave two close major peaks in the LC chromatogram in some LC conditions. We suggest that these peaks correspond to the two conformers of HAMA; one of them would be selectively bound on the substrate binding site of VerA enzyme and then converted to DMST. VerA enzyme may work as a key enzyme in the creation of the xanthone structure of DMST from HAMA.

Genetic Differentiation Associated with Fumonisin and Gibberellin Production in Japanese Fusarium fujikuroi.

Fusarium fujikuroi is a pathogenic fungus that infects rice. It produces several important mycotoxins, such as fumonisins. Fumonisin production has been detected in strains of maize, strawberry, and wheat, whereas it has not been detected in strains from rice seedlings infested with bakanae disease in Japan. We investigated the genetic relationships, pathogenicity, and resistance to a fungicide, thiophanate-methyl (TM), in 51 fumonisin-producing strains and 44 nonproducing strains. Phylogenetic analyses based on amplified fragment length polymorphism (AFLP) markers and two specific genes (a combined sequence of translation elongation factor 1α [TEF1α] and RNA polymerase II second-largest subunit [RPB2]) indicated differential clustering between the fumonisin-producing and -nonproducing strains. One of the AFLP markers, EATMCAY107, was specifically present in the fumonisin-producing strains. A specific single nucleotide polymorphism (SNP) between the fumonisin-producing and nonproducing strains was also detected in RPB2, in addition to an SNP previously found in TEF1α. Gibberellin production was higher in the nonproducing than in the producing strains according to an in vitro assay, and the nonproducing strains had the strongest pathogenicity with regard to rice seedlings. TM resistance was closely correlated with the cluster of fumonisin-nonproducing strains. The results indicate that intraspecific evolution in Japanese F. fujikuroi is associated with fumonisin production and pathogenicity. Two subgroups of Japanese F. fujikuroi, designated G group and F group, were distinguished based on phylogenetic differences and the high production of gibberellin and fumonisin, respectively.IMPORTANCEFusarium fujikuroi is a pathogenic fungus that causes rice bakanae disease. Historically, this pathogen has been known as Fusarium moniliforme, along with many other species based on a broad species concept. Gibberellin, which is currently known as a plant hormone, is a virulence factor of F. fujikuroi Fumonisin is a carcinogenic mycotoxin posing a serious threat to food and feed safety. Although it has been confirmed that F. fujikuroi produces gibberellin and fumonisin, production varies among strains, and individual production has been obscured by the traditional appellation of F. moniliforme, difficulties in species identification, and variation in the assays used to determine the production of these secondary metabolites. In this study, we discovered two phylogenetic subgroups associated with fumonisin and gibberellin production in Japanese F. fujikuroi.

Diverse stochasticity leads a colony of ants to optimal foraging.

A mathematical model of garden ants (Lasius japonicus) is introduced herein to investigate the relationship between the distribution of the degree of stochasticity in following pheromone trails and the group foraging efficiency. Numerical simulations of the model indicate that depending on the systematic change of the feeding environment, the optimal distribution of stochasticity shifts from a mixture of almost deterministic and mildly stochastic ants to a contrasted mixture of almost deterministic ants and highly stochastic ants. In addition, the interaction between the stochasticity and the pheromone path regulates the dynamics of the foraging efficiency optimization. Stochasticity could strengthen the collective efficiency when the variance in the sensitivity to pheromone for ants is introduced in the model.

Carotid Artery Stenting Using a Closed-Cell Stent-in-Stent Technique for Unstable Plaque.

Purpose: To examine whether carotid artery stenting (CAS) of stenoses with unstable plaque using a closed-cell stent-in-stent technique prevents plaque protrusion. Materials and Methods: Between December 2014 and August 2018, 35 consecutive patients (mean age 75.8 years; 29 men) with carotid artery stenosis (20 symptomatic) and unstable plaque diagnosed by magnetic resonance imaging were prospectively analyzed. Mean diameter stenosis was 83.5%. All CAS procedures were performed with stent-in-stent placement of Carotid Wallstents using an embolic protection device and conservative postdilation. The technical success rate, incidence of plaque protrusion, ischemic stroke rate within 30 days, and new ipsilateral ischemic lesions on diffusion-weighted imaging (DWI) within 48 hours after CAS were prospectively assessed. Follow-up outcomes included the incidences of ipsilateral stroke and restenosis. Results: The technical success rate was 100%. No plaque protrusion or stroke occurred in any patient. New ischemic lesions were observed on DWI in 10 (29%) patients. During the mean 11.6-month follow-up, no ipsilateral strokes occurred. Two (6%) patients developed asymptomatic restenosis recorded as 53% lumen narrowing and occlusion, respectively. Conclusion: CAS using a closed-cell stent-in-stent technique for unstable plaque may be useful for preventing plaque protrusion and ischemic complications.

In Vitro Differentiation of Chicken Astrocytes: Growth, Morphology, and Protein Expression of Astrocytes in Primary Cultures.

Astrocytes regulate synaptic transmission in the central nervous system. Astrocytes in vivo have "stems" that express glial fibrillary acidic protein (GFAP), intermediate filaments, and peripheral astrocyte processes (PAPs), which contain actin-rich cytoskeletal structures. At the PAPs, the perisynaptic glia contacts and enwraps synapses, and modulates glia-neuronal communication. Cultured astrocytes have been an invaluable tool for studying roles of astrocytes; however, the morphology of mammalian primary astrocytes cultured in conventional medium containing fetal bovine serum (FBS) was similar to that of fibroblasts, and many culture conditions have been developed to generate stellate astrocytes observed in vivo. Avian astrocytes have been prepared from embryonic chick forebrain and maintained at a high cell density in conventional FBS-containing medium as mammalian astrocytes, thus the morphological analysis of chicken astrocytes has not yet been performed. In the present study, we report that the morphology of astrocytes freshly harvested from the forebrain of a chicken embryo in serum-free Neurobasal medium with B-27 supplement and basic fibroblast growth factor (bFGF) is similar to that of the astrocyte morphology in vivo. We also find that astrocytes in this medium express similar levels of GFAP and two actin-binding proteins as astrocytes in conventional FBS-containing medium, although they have different morphologies. Furthermore, we confirmed that cryopreserved astrocytes differentiate faster than freshly harvested astrocytes.

Plasma quercetin metabolites are affected by intestinal microbiota of human microbiota-associated mice fed with a quercetin-containing diet.

Protective effect of quercetin on high-fat diet-induced non-alcoholic fatty liver disease in mice has been reported. Recent research has revealed that several intestinal bacteria metabolize quercetin. We hypothesize that the difference in composition of intestinal microbiota affects quercetin absorption from the intestine. Germ-free BALB/cA female mice (18 weeks old) were randomly divided into four groups and orally administered with fecal suspension from four human individuals (HF1, HF2, HF3, HF4) to produce the human microbiota-associated mice. All mice were fed the 0.05% quercetin-containing pelleted diet for four weeks. Significant differences were observed in plasma total cholesterol and cecal microbiota among the four groups. Plasma quercetin concentration was significantly higher in the HF3 group than in the HF1 group. The plasma isorhamnetin/quercetin ratio showed significant negative correlation with visceral fat levels (r = -0.544, p = 0.013). Positive correlation was observed between the Log10 Enterobacteriaceae count and the plasma quercetin metabolites. Principal component analysis showed that all groups were distributed in different regions by using the correlation diagram with the second and third principal component. This study indicates that intestinal microbiota of human microbiota-associated mice inoculated with different fecal suspensions react to dietary quercetin in different ways.

Biosynthesis and in vitro enzymatic synthesis of the isoleucine conjugate of 12-oxo-phytodienoic acid from the isoleucine conjugate of α-linolenic acid.

The isoleucine conjugate of 12-oxo-phytodienoic acid (OPDA-Ile), a new member of the jasmonate family, was recently identified in Arabidopsis thaliana and might be a signaling molecule in plants. However, the biosynthesis and function of OPDA-Ile remains elusive. This study reports an in vitro enzymatic method for synthesizing OPDA-Ile, which is catalyzed by reactions of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) using isoleucine conjugates of α -linolenic acid (LA-Ile) as the substrate. A. thaliana fed LA-Ile exhibited a marked increase in the OPDA-Ile concentration. LA-Ile was also detected in A. thaliana. Furthermore, stable isotope labelled LA-Ile was incorporated into OPDA-Ile. Thus, OPDA-Ile is biosynthesized via the cyclization of LA-Ile in A. thaliana.

Successful transcatheter arterial antimicrobial and steroid therapy for refractory liver abscess in chronic granulomatous disease: A case report and review of literature.

Hepatic abscess in chronic granulomatous disease (CGD) is very refractory and frequently requires multiple surgeries with frequent morbidities. Although surgical interventions are often required, patients are often not able to have surgery for various reasons. We present the case of a 21-year-old man with recurrent hepatic abscess in CGD. We could not provide surgical interventions due to the lack of a fluid cavity and the patient's refusal, and therefore we administered transcatheter arterial antimicrobial and steroid therapy. He did not have any exacerbation for more than 18 months after the final transcatheter treatment. This is the first reported case of successful transcatheter arterial antimicrobial and steroid therapy for refractory hepatic abscess in CGD. Although the patient's burden and medical cost were not inconsequential, this case shows that the transcatheter arterial antimicrobial and steroid therapy may be a treatment option for patients who are not candidates for surgical interventions.

Heterologous production of a new lasso peptide brevunsin in Sphingomonas subterranea.

A shuttle vector pHSG396Sp was constructed to perform gene expression using Sphingomonas subterranea as a host. A new lasso peptide biosynthetic gene cluster, derived from Brevundimonas diminuta, was amplified by PCR and integrated to afford a expression vector pHSG396Sp-12697L. The new lasso peptide brevunsin was successfully produced by S. subterranea, harboring the expression vector, with a high production yield (10.2 mg from 1 L culture). The chemical structure of brevunsin was established by NMR and MS/MS experiments. Based on the information obtained from the NOE experiment, the three-dimensional structure of brevunsin was determined, which indicated that brevunsin possessed a typical lasso structure. This expression vector system provides a new heterologous production method for unexplored lasso peptides that are encoded by bacterial genomes.