Airborne microplastics detected in the lungs of wild birds in Japan.

Microplastics (MPs) have been found in a wide range of animal species including humans. The detection of MPs in human lungs suggests that humans inhale airborne microplastics (AMPs). Although birds respire more efficiently than mammals and are therefore more susceptible to air pollution, little is known about their inhalation exposure to MPs. In this study, we analyzed samples isolated from the lungs of several species of wild birds in Japan by attenuated total reflection (ATR) imaging method of micro-Fourier transform infrared (µFTIR) spectroscopy to clear whether AMPs can be inhaled and accumulate within the lungs of wild birds. To isolate MPs from lung samples of rock doves (Columba livia), black kites (Milvus migrans), and barn swallows (Hirundo rustica) euthanized for pest control, digestion and density separation were performed. After each sample collected on an alumina filter was measured by ATR imaging method using µFTIR spectroscopy, the physical and chemical characteristics of the detected MPs were evaluated. Six MPs were detected in 3 of 22 lung samples. Polypropylene and polyethylene were found in rock doves and ethylene vinyl acetate was found in a barn swallow. Most MPs were fragments of 28.0-70.5 µm. Our results demonstrated that in addition to dietary sources, some wild birds are exposed to MPs by inhalation, and these MPs reach the lungs.

Relationship between intracellular protein denaturation and irreversible inactivation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles.

To clarify the relationship between irreversible inactivation and intracellular protein denaturation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles (CO2 MB) treatment, a storage test of S. pastorianus cells treated with CO2 MB was performed, and the effect on the intracellular protein was investigated. In the storage test, the S. pastorianus population, which decreased below the detection limit by CO2 MB treatment at a temperature of 45 and 50°C (MB45 and MB50), and thermal treatment at a temperature of 80°C (T80), remained undetectable during storage for 3 weeks at 25°C. However, 4.1 and 1.3-logs of the S. pastorianus populations, which survived after CO2 MB treatment at temperatures of 35 and 40°C (MB35 and MB40), increased gradually during storage for 3 weeks at 25°C. Insolubilization of intracellular proteins in S. pastorianus increased with increasing the temperature of CO2 MB treatment. Activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) identified as one of the insolubilized proteins increased at MB35 and MB40 than non-treatment but disappeared at MB45 and MB50, and T80. Therefore, it was revealed that S. pastorianus cells inactivated below the detection level by CO2 MB treatment did not regrow and that the denaturation of intracellular proteins of S. pastorianus was caused by CO2 MB and thermal treatments. Furthermore, it was suggested that denaturation of intracellular vital enzymes was an important factor for achieving irreversible inactivation of S. pastorianus by CO2 MB and thermal treatments.

Metabolite profiling in sphere-forming cells from canine mammary adenocarcinoma cell lines using gas chromatography-mass spectrometry.

Cancer consists of heterogeneous cells that contain a small population of cells that possess stem cell properties; these cells, referred to as cancer stem cells (CSCs) or tumor-initiating cells, are involved in tumor progression and metastasis. Using a sphere-forming assay, canine mammary CSCs were found to be similar to human breast CSCs. Metabolic reprogramming has been recognized as a hallmark of various cancers. However, the significance of cellular metabolism in CSCs remains unclear. The aim of this study was to define the metabolic characteristics of CSCs derived from canine mammary tumors and gain an understanding of the maintenance of stemness. We identified metabolite profiles of canine mammary adenocarcinoma cell lines using gas chromatography-mass spectrometry. Metabolites were extracted from both adherent and sphere-forming cells derived from three cell lines. Sphere-forming cells were separated from adherent cells using an orthogonal, partial least-squares discriminant analysis. Sphere-forming cells were found to contain high levels of the amino acids alanine, glycine and proline compared with adherent cells. They also had high levels of palmitoleate, palmitate and dihomo-gamma-linolenic acid compared with adherent cells. In a sphere-forming assay, palmitate increased the number of spheres for all cell lines. These results indicate that the sphere-forming cells derived from canine mammary adenocarcinoma cell lines have specific metabolic profiles that may be useful for the development of CSC-specific therapies targeting metabolic pathways and potential stemness biomarkers; these results also clarify the maintenance of stemness in canine mammary CSCs.

Mycobacterium shigaense sp. nov., a slow-growing, scotochromogenic species, is a member of the Mycobacterium simiae complex.

Among non-tuberculous mycobacteria (NTM), the Mycobacterium simiae complex is one of the largest groups, consisting of 18 species of slow-growing mycobacteria. In 2009, a case of NTM-associated infectious skin disease was reported in Shiga Prefecture, Japan. The patient presented with scattered nodules on the chest, back and extremities, and an M. simiae-like organism was isolated from skin biopsy specimens obtained from one of these lesions. Based on several assessments, including multiple-gene analyses, biochemical characterization and drug susceptibility testing, we concluded that this isolate represented a novel species of NTM, and proposed the name 'Mycobacterium shigaense'. Since 2009, five more cases of NTM-associated infectious disease in which there was a suspected involvement of 'M. shigaense' have been reported. Interestingly, four of these six cases occurred in Shiga Prefecture. Here we performed multiple-gene phylogenetic analyses, physiological and biochemical characterization tests, drug susceptibility tests, and profiling of proteins, fatty acids and mycolic acids of eight clinical isolates from the six suspected 'M. shigaense' cases. The results confirmed that all of the clinical isolates were 'M. shigaense', a slow-growing, scotochromogenic species. Here M. shigaense is validly proposed as a new member of the M. simiae complex, with the type strain being UN-152T (=JCM 32072T=DSM 46748T).

Mycobacterium stephanolepidis sp. nov., a rapidly growing species related to Mycobacterium chelonae, isolated from marine teleost fish, Stephanolepis cirrhifer.

A previously undescribed rapidly growing, non-pigmented mycobacterium was identified based on biochemical and nucleic acid analyses, as well as growth characteristics. Seven isolates were cultured from samples collected from five thread-sail filefish (Stephanolepis cirrhifer) and two farmed black scraper (Thamnaconus modestus). Bacterial growth occurred at 15-35 °C on Middlebrook 7H11 agar. The bacteria were positive for catalase activity at 68 °C and urease activity, intermediate for iron uptake, and negative for Tween 80 hydrolysis, nitrate reduction, semi-quantitative catalase activity and arylsulfatase activity at day 3. No growth was observed on Middlebrook 7H11 agar supplemented with picric acid, and very little growth was observed in the presence of 5 % NaCl. α- and α'-mycolates were identified in the cell walls, and a unique profile of the fatty acid methyl esters and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) profiles of the protein and cell-wall lipids were acquired. Sequence analysis revealed that the seven isolates shared identical sequences for the 16S rRNA, rpoB, hsp65, recA and sodA genes. Phylogenetic analysis of the five gene sequences confirmed that the isolates were unique, but closely related to Mycobacterium chelonae. Antibiotic susceptibility testing revealed the minimum inhibitory concentration (MIC) of clarithromycin against this novel species was <0.25 µg ml-1, which was lower than that for Mycobacterium salmoniphilum. The hsp65 PCR restriction enzyme analysis pattern differed from those of M. chelonae and M. salmoniphilum. Based on these findings, the name Mycobacterium stephanolepidis sp. nov. is proposed for this novel species, with the type strain being NJB0901T (=JCM 31611T=KCTC 39843T).

Metabolic analysis of canine peripheral blood mononuclear cells treated ex vivo with dexamethasone.

In dogs, hyperadrenocorticism (HAC) is associated with insulin resistance and diabetes does progress with HAC. There are significant differences in the transcriptomic and proteomic patterns of activated T cells, which parallel the findings in muscle tissues. The aim of this study was to assess how glucocorticoids affect intracellular metabolites in canine peripheral blood mononuclear cells (CnPBMCs) using dexamethasone. A total of 96 metabolites were identified by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). After incubation with dexamethasone, the metabolites glucose 1-phosphate, glucose 6-phosphate, fructose 6-phosphate, sedoheptulose 7-phosphate and acetyl-CoA were significantly increased. However, ATP, CTP, dATP, pyruvic acid and NADP(+) were significantly decreased. These results show that a glucocorticoid reduces the catabolic reaction of glucose and accordingly decreases the glucose requirements of CnPBMCs.

Semiquantitative multi-analysis of plasma obtained from Romney lambs (Ovis aries) by inductively coupled plasma mass spectrometry, and the classification according to feed type.

The establishment of a classification system for domestic animals on consumed feed stuff is thought to be important from both a hygiene and market point of view. We collected plasma samples of Romney lambs (Ovis aries) which were fed one of the following: a herb-clover mix (n=10) which included chicory, red clover, white clover and plantain; a plant-grass mix (n=10) which included plantain, ryegrass and white clover; or a grass mix (n=10) which included ryegrass and white clover. A total of 20 elements in plasma samples obtained from the lambs were analyzed using inductively coupled plasma mass spectrometry. The data were then analyzed by principal component analysis. The lambs were divided into three groups on a score plot depending on the different feed conditions. Furthermore, discriminant analyses of the elements were examined, using linear discriminant analysis with forward stepwise regression. This discriminant function correctly classified the samples from each group. The accuracy of classification of each group, as shown by 10-fold cross-validation, proved the effectiveness of the established discriminant function. It is concluded that using linear discriminant analysis might be a useful tool for the validation of elements from plasma in lambs grown in different conditions.

Crystal structure of a lactosucrose-producing enzyme, Arthrobacter sp. K-1 ß-fructofuranosidase.

Arthrobacter sp. K-1 ß-fructofuranosidase (ArFFase), a glycoside hydrolase family 68 enzyme, catalyzes the hydrolysis and transfructosylation of sucrose. ArFFase is useful for producing a sweetener, lactosucrose (4(G)-ß-D-galactosylsucrose). The primary structure of ArFFase is homologous to those of levansucrases, although ArFFase catalyzes mostly hydrolysis when incubated with sucrose alone, even at high concentration. Here, we determined the crystal structure of ArFFase in unliganded form and complexed with fructose. ArFFase consisted of a five-bladed ß-propeller fold as observed in levansucrases. The structure of ArFFase was most similar to that of Gluconacetobacter diazotrophicus levansucrase (GdLev). The structure of the catalytic cleft of ArFFase was also highly homologous to that of GdLev. However, two amino acid residues, Tyr232 and Pro442 in ArFFase, were not conserved between them. A tunnel observed at the bottom of the catalytic cleft of ArFFase may serve as a water drain or its reservoir.

Purification and N-terminal amino acid sequence of solanapyrone synthase, a natural Diels-Alderase from Alternaria solani.

The first natural Diels-Alderase, solanapyrone synthase, was purified 1,630-fold from a crude extract. The 41-kDa protein on SDS-polyacrylamide gel electrophoresis was identified as truncated solanapyrone synthase, and its N-terminal amino acid sequence was found to be QETQNLNNFLESNAINP.

Parvitexins A-E, clerodane-type diterpenes isolated from the in vitro-cultured liverwort, Scapania parvitexta.

New clerodane-type diterpenes, designated as parvitexins A (1)-E (5), were isolated from the in vitro-cultured liverwort, Scapania parvitexta. These compounds were determined to be monoacetylated clerodane-type diterpenes based on spectroscopic evidence.

Structural and functional analysis of tetracenomycin F2 cyclase from Streptomyces glaucescens. A type II polyketide cyclase.

Tetracenomycin F2 cyclase (tcmI gene product), catalyzes an aromatic rearrangement in the biosynthetic pathway for tetracenomycin C in Streptomyces glaucescens. The x-ray structure of this small enzyme has been determined to 1.9-A resolution together with an analysis of site-directed mutants of potential catalytic residues. The protein exhibits a dimeric betaalphabeta ferredoxin-like fold that utilizes strand swapping between subunits in its assembly. The fold is dominated by four strands of antiparallel sheet and a layer of alpha-helices, which creates a cavity that is proposed to be the active site. This type of secondary structural arrangement has been previously observed in polyketide monooxygenases and suggests an evolutionary relationship between enzymes that catalyze adjacent steps in these biosynthetic pathways. Mutational analysis of all of the obvious catalytic bases within the active site suggests that the enzyme functions to steer the chemical outcome of the cyclization rather than providing a specific catalytic group. Together, the structure and functional analysis provide insight into the structural framework necessary to perform the complex rearrangements catalyzed by this class of polyketide cyclases.

Cloning, sequencing, and functional analysis of the biosynthetic gene cluster of macrolactam antibiotic vicenistatin in Streptomyces halstedii.

Vicenistatin, an antitumor antibiotic isolated from Streptomyces halstedii, is a unique 20-membered macrocyclic lactam with a novel aminosugar vicenisamine. The vicenistatin biosynthetic gene cluster (vin) spanning approximately 64 kbp was cloned and sequenced. The cluster contains putative genes for the aglycon biosynthesis including four modular polyketide synthases (PKSs), glutamate mutase, acyl CoA-ligase, and AMP-ligase. Also found in the cluster are genes of NDP-hexose 4,6-dehydratase and aminotransferase for vicenisamine biosynthesis. For the functional confirmation of the cluster, a putative glycosyltransferase gene product, VinC, was heterologously expressed, and the vicenisamine transfer reaction to the aglycon was chemically proved. A unique feature of the vicenistatin PKS is that the loading module contains only an acyl carrier protein domain, in contrast to other known PKS-loading modules containing certain activation domains. Activation of the starter acyl group by separate polypeptides is postulated as well.