Identification of four genes responsible for antimicrobial resistance of MEL-B against S. aureus.

There is increasing interest in the antimicrobial activity of mannosylerythritol lipids-B (MEL-B) against Gram-positive bacteria such as Staphylococcus aureus (S. aureus). However, the specific molecules involved in MEL-B's antimicrobial action against S. aureus have not been identified. This study utilized the Nebraska transposon mutant library (NTML), which contains 1920 mutants, each lacking three-quarters of the genes found in S. aureus. The NTML was screened to identify mutants resistant to MEL-B. Four mutants (Accession Number: SAUSA300_0904, SAUSA300_0752, SAUSA300_0387, and SAUSA300_2311) largely unaffected by incubation with MEL-B, indicating MEL-B resistance. Despite the strong binding of MEL-B to these mutants, the four molecules encoded by the deleted genes (yjbI, clpP, pbuX, or brpS) in each mutant were not directly recognized by MEL-B. Given that these molecules are not localized on the outer surface of S. aureus and that the antibacterial activity of MEL-B against S. aureus is facilitated by the effective transfer of two antibacterial fatty acids (caprylic acid and myristoleic acid) to S. aureus via ME, the deletion of each of the four molecules may alter the peptidoglycan structure, potentially inhibiting the effective transfer of these antimicrobial fatty acids into S. aureus.

Orphan gene expressed in flame cone cells uniquely found in seahorse epithelium.

The seahorse is one of the most unique teleost fishes in its morphology. The body is surrounded by bony plates and spines, and the male fish possess a brooding organ, called the brood pouch, on their tail. The surfaces of the brood pouch and the spines are surrounded by characteristic so-called flame cone cells. Based on our histological observations, flame cone cells are present in the seahorse Hippocampus abdominalis, but not in the barbed pipefish Urocampus nanus or the seaweed pipefish Syngnathus schlegeli, both of which belong to the same family as the seahorse. In the flame cone cells, we observed expression of an "orphan gene" lacking homologs in other lineages. This gene, which we named the proline-glycine rich (pgrich) gene, codes for an amino acid sequence composed of repetitive units. In situ hybridization and immunohistochemical analyses detected pgrich-positive signals from the flame cone cells. Based on a survey of the genome sequences of 15 teleost species, the pgrich gene is only found from some species of Syngnathiformes (namely, the genera Syngnathus and Hippocampus). The amino acid sequence of the seahorse PGrich is somewhat similar to the sequence deduced from the antisense strand of elastin. Furthermore, there are many transposable elements around the pgrich gene. These results suggest that the pgrich gene may have originated from the elastin gene with the involvement of transposable elements and obtained its novel function in the flame cone cells during the evolution of the seahorse.

Roles of mannosylerythritol lipid-B components in antimicrobial activity against bovine mastitis-causing Staphylococcus aureus.

Mannosylerythritol lipid-B (MEL-B), which comprises ester-bonded hydrophilic ME and hydrophobic fatty acids, is a bio-surfactant with various unique properties, including antimicrobial activity against most gram-positive bacteria. The gram-positive Staphylococcus aureus is a causative pathogen of dairy cattle mastitis, which results in considerable economic loss in the dairy industry. Here, we demonstrate the efficacy of MEL-B as a disinfectant against bovine-derived S. aureus and elucidate a mechanism of action of MEL-B in the inhibition of bacterial growth. The growth of bovine mastitis causative S. aureus BM1006 was inhibited when cultured with MEL-B above 10 ppm. The activity of MEL-B required fatty acids (i.e., caprylic and myristoleic acids) as ME, the component of MEL-B lacking fatty acids, did not inhibit the growth of S. aureus even at high concentrations. Importantly, ME-bound fatty acids effectively inhibited the growth of S. aureus when compared with free fatty acids. Specifically, the concentrations of ME-bound fatty acids and free caprylic and myristoleic acids required to inhibit the growth of S. aureus were 10, 1442, and 226 ppm, respectively. The involvement of ME in the antimicrobial activity of MEL-B was confirmed by digestion of MEL-B with alkali, which dissociated ME and fatty acids. These results indicated that a mechanism of action of MEL-B in inhibiting the growth of S. aureus could be explained by the effective transporting of antimicrobial fatty acids to the bacterial surface via hydrophilic ME.

A putative transporter gene PtMMF1-deleted strain produces mono-acylated mannosylerythritol lipids in Pseudozyma tsukubaensis.

Mannosylerythritol lipids (MELs) are glycolipid biosurfactants produced by various yeasts. Mmf1, a putative transporter of MELs, is conserved in the MEL biosynthesis gene clusters of diverse MEL producers, including the genera Ustilago, Pseudozyma, Moesziomyces, and Sporisorium. To clarify the function of Mmf1, we generated the gene-deleted strain of P. tsukubaensis ΔPtMMF1 and evaluated its MEL production. Using thin-layer chromatography analyses, we detected most MELs produced by ΔPtMMF1 in the culture supernatant. The spot size of diacylated MEL-B (the only product of the parental strain) was significantly smaller for strain ΔPtMMF1 than for the parental strain, and a mono-acylated MEL-D spot was detected. In addition, an unknown glycolipid was detected in the sample extracted from strain ΔPtMMF1. Liquid chromatography-mass spectrometry and nuclear magnetic resonance analyses revealed that the unknown glycolipid was a novel MEL homologue, mono-acylated MEL-B. KEY POINTS: • P. tsukubaensis is able to secrete MELs without PtMMF1p. • Strain ΔPtMMF1 mainly produced mono-acylated MELs.

Development and characterization of 19 novel microsatellite markers in the Pacific seaweed pipefish Syngnathus schlegeli using next-generation sequencing.

Syngnathids (pipefishes, seahorses and seadragons) are vulnerable to human-mediated habitat perturbation. The Pacific seaweed pipefish Syngnathus schlegeli has a large distribution in the northwestern Pacific, where deterioration, loss and fragmentation of its seagrass habitat are occurring through coastal development. So far, few studies have been conducted to access the genetic structure and conservation status of S. schlegeli because of the low number of genetic markers currently described. Nineteen polymorphic microsatellite markers were developed for S. schlegeli using next-generation sequencing, and characterized in 32 individuals. The mean number of alleles was 14, with 2-28 alleles per locus. The estimates of observed heterozygosity (HO) and expected heterozygosity (HE) varied depending on the locus, ranging from 0.063 to 1.000, and from 0.062 to 0.969, respectively. Seventeen of the 19 microsatellites conformed to Hardy-Weinberg equilibrium. These new microsatellite markers should provide a wealth of information for studies on conservation genetics and the behavioral ecology of S. schlegeli.

Synergetic binary organocatalyzed ring opening polymerization for the precision synthesis of polysiloxanes.

Organocatalytic ring-opening polymerization (ROP) is a versatile method for synthesizing well-defined polymers with controlled molecular weights, dispersities, and nonlinear macromolecular architectures. Despite spectacular advances in organocatalytic ROP, precision synthesis of polysiloxanes remains challenging due to the mismatch in polarity between highly polar initiators and nonpolar monomers and polymers and the difficulty in suppressing the formation of scrambling products via transetherification reactions during ROP of cyclic siloxanes. Here, we describe a binary organocatalytic ROP (BOROP) of hexamethylcyclotrisiloxane (D3) employing organic bases as catalysts and (thio)ureas as cocatalysts. The BOROP of D3 using triazabicyclodecene (TBD) and (thio)ureas generates polydimethylsiloxanes (PDMSs) with narrow dispersity (Mw/Mn < 1.1). Despite the similar basicities of TBD and 1,8-bis(tetramethylguanidino)naphthalene (TMGN), which is known as a proton sponge, a unitary organocatalytic system using TMGN was inactive for the ROP of D3. When the TMGN was paired with acidic urea, the BOROP of D3 yielded PDMSs with narrow dispersity (Mw/Mn < 1.1). Data suggest that the synergetic effect of TMGN and urea is results in an unprecedented activation-deactivation equilibrium between dormant and propagating species. The benefits of the present BOROP system are demonstrated by the formation of PDMS elastomers with more uniform network structures that are highly stretchy and have excellent mechanical properties.

Complete mitochondrial genome of a subspecies of the great cormorant, Phalacrocorax carbo hanedae (Kuroda, 1925) (Suliformes: Phalacrocoracidae).

We determined the complete mitochondrial DNA sequence of a subspecies of the great cormorant, Phalacrocorax carbo hanedae (Kuroda, 1925) using long PCR and primer walking methods. The mitochondrial genome was 19,020 bp in length and contained 13 protein-coding genes (PCGs), two ribosomal RNA genes, 22 transfer RNA genes, and two control regions. It is basically consistent with the characteristics of the mitochondrial genomes of other Suliformes species. Phylogenetic analysis using 12 species of Suliformes based on the sequences of 13 concatenated protein-coding genes confirmed the monophyly of P. carbo ssp.

Complete mitochondrial genome of the Japanese Cormorant Phalacrocorax capillatus (Temminck & Schlegel, 1850) (Suliformes: Phalacrocoracidae).

The complete sequencing of mitochondrial DNA of the Japanese Cormorant Phalacrocorax capillatus was performed using long PCR and primer walking methods. The assembled genome was 19,105 bp in length. It contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, and two control regions. The phylogenetic analysis using the obtained sequence showed that P. capillatus is closest to P. carbo.

Improvement of Oil Degradation and MEL Production in a Yeast Strain, Pseudozyma tsukubaensis, by Translation Elongation Factor 1 Promoter-driven Expression of a Lipase.

The basidiomycetous yeast Pseudozyma tsukubaensis produces a mannosylerythritol lipid (MEL) homologue, a diastereomer type of MEL-B, from olive oil. In a previous study, MEL-B production was increased by the overexpression of lipase PaLIPAp in P. tsukubaensis 1E5, through the enhancement of oil consumption. In the present study, RNA sequence analysis was used to identify a promoter able to induce high-level PaLIPA expression. The recombinant strain, expressing PaLIPA via the translation elongation factor 1 alpha/Tu promoter, showed higher lipase activity, rates of oil degradation, and MEL-B production than the strain which generated in our previous study.

Brood pouch evolution in pipefish and seahorse based on histological observation.

INTRODUCTION: Fishes of the Syngnathidae family are rare in having male pregnancy: males receive eggs from females and egg development occurs in the male brood pouch that diverged during evolution. The family is divided into two subfamilies: Nerophinae and Syngnathinae. METHODS: We compared histologically five types of the brood pouch in Syngnathinae: an open pouch without skinfolds (alligator pipefish); an open pouch with skinfolds (messmate pipefish); a closed pouch with skinfolds (seaweed pipefish); and closed pouches with a sac-like pouch on the tail (pot-bellied seahorse) or within a body cavity (Japanese pygmy seahorse). RESULTS: Histological observations revealed that all the examined species possess vascular egg compartments during the brooding period. The present immunohistochemical study revealed that the pregnant egg compartment epithelium grows thin in both open and closed pouches. The placenta of open and closed pouches is composed of dermis and reticulin fibers, respectively. The closed pouch placenta is a flexible and moist tissue, suitable for substance transport between the father and embryos through the epithelium and blood vessels and responsible for supplying nutrition and removing waste. DISCUSSION: These results suggest that the basic egg incubation structures were established at an early stage of Syngnathinae evolution. On the other hand, it is likely that the innovation of tissue structure, where dermis was replaced with reticular fibers, occurred in closed brood pouches to regulate the pregnant pouch environment. The present study presents the morphological evolutionary pathway of the brood pouch in Syngnathinae, providing a basis for further molecular-level evolutionary studies.

Marine-dumped waste tyres cause the ghost fishing of hermit crabs.

Poorly managed waste tyres pose serious environmental and health risks, ranging from air pollution caused by fire, leaching of heavy metals and outbreaks of mosquitos, to destruction of vegetation and coral reefs. We report a previously unrecognized ecological risk to marine organisms from waste tyres. Over 1 year, we made monthly counts of hermit crabs (n = 1278) invading and/or being trapped within six tyres anchored to the seabed at 8 m depth in Mutsu Bay, Japan. A complementary aquarium experiment in which hermit crabs were released into a tyre confirmed that they could not escape. We report marine-dumped waste tyres to ghost fish in a manner analogous to discarded fishing gear. Because hermit crabs play important roles in coastal food webs as both prey and scavengers, declines in their numbers as a consequence of this ghost fishing might affect coastal ecosystems.

Complete mitochondrial genome of the Japanese field vole microtus montebelli (Milne-Edwards, 1872) (Rodentia: Arvicolinae).

The complete mitochondrial DNA sequence of the Japanese field vole Microtus montebelli was determined using Illumina MiSeq platform. The assembled genome was 16,307 bp in length and contained 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes. According to phylogenetic analysis of 13 protein-coding genes, M. montebelli and other Microtus species consist of paraphyletic clades and M. montebelli is most closely related to M. kikuchii, a species endemic to Taiwan.

Insulin-like growth factor binding protein-1 levels are increased in patients with IgA nephropathy.

The mechanisms underlying the pathogenesis of immunoglobulin A (IgA) nephropathy (IgAN) are not well understood. In this study, we examined gene expression profiles in kidneys obtained from mice with high serum IgA levels (HIGA mice), which exhibit features of human IgAN. Female inbred HIGA, established from the ddY line, were used in these experiments. Serum IgA levels, renal IgA deposition, mesangial proliferation, and glomerulosclerosis were increased in 32-week-old HIGA mice in comparison to ddY animals. By microarray analysis, five genes were observed to be increased by more than 2.5-fold in 32-week-old HIGA in comparison to 16-week-old HIGA; these same five genes were decreased more than 2.5-fold in 32-week-old ddY in comparison to 16-week-old ddY mice. Of these five genes, insulin-like growth factor (IGF) binding protein (IGFBP)-1 exhibited differential expression between these mouse lines, as confirmed by quantitative RT-PCR. In addition, serum IGFBP-1 levels were significantly higher in patients with IgAN than in healthy controls. In patients with IgAN, these levels correlated with measures of renal function, such as estimated glomerular filtration rate (eGFR), but not with sex, age, serum IgA, C3 levels, or IGF-1 levels. Pathologically, serum IGFBP-1 levels were significantly associated with the severity of renal injury, as assessed by mesangial cell proliferation and interstitial fibrosis. These results suggest that increased IGFBP-1 levels are associated with the severity of renal pathology in patients with IgAN.

Production of a novel glycolipid biosurfactant, mannosylmannitol lipid, by Pseudozyma parantarctica and its interfacial properties.

The development of a novel glycolipid biosurfactant was undertaken using the high-level producers of mannosylerythritol lipids (MELs) such as Pseudozyma parantarctica, Pseudozyma antarctica, and Pseudozyma rugulosa. Besides the conventional MELs (MEL-A, MEL-B, and MEL-C), these yeasts produced an unknown glycolipid when they were cultivated in a medium containing 4% (w/v) olive oil and 4% (w/w) mannitol as the carbon source. The unknown glycolipid extracted from the culture medium of P. parantarctica JCM 11752(T) displayed the spot with lower mobility than that of known MELs on TLC and provided mainly two peaks identical to mannose and mannitol on high-performance liquid chromatography after acid hydrolysis. Based on structural analysis by (1)H and (13)C nuclear magnetic resonance, the novel glycolipid was composed of mannose and mannitol as the hydrophilic sugar moiety and was identified as mannosylmannitol lipid (MML). Of the strains tested, P. parantarctica JCM 11752(T) gave the best yield of MML (18.2 g/L), which comprised approximately 35% of all glycolipids produced. We further investigated the interfacial properties of the MML, considering the unique hydrophilic structure. The observed critical micelle concentration (CMC) and the surface tension at CMC of the MML were 2.6 x 10(-6) M and 24.2 mN/m, respectively. In addition, on a water-penetration scan, the MML efficiently formed not only the lamella phase (Lalpha) but also the myelins at a wide range of concentrations, indicating its excellent self-assembling properties and high hydrophilicity. The present glycolipid should thus facilitate the application of biosurfactants as new functional materials.

[Case of multiple nodular lesions of bilateral kidneys with IgG4-related systemic disease].

A 59-year-old woman was admitted to our hospital because of a pancreatic mass lesion. Serum gamma-globulin and IgG4 levels were elevated to 2.2 g/dL and 1,310 mg/dL, respectively. Computed tomography examination revealed multiple low-density areas without enhancement by contrast in the pancreatic body and bilateral kidneys. Endoscopic retrograde cholangiopancreatography images demonstrated diffuse narrowing of the main pancreatic duct with an irregular wall from the body to the tail of the pancreas. Positron emission tomography examination revealed intense 18F-fluorodeoxyglucose uptake by the pancreas and kidneys. Accordingly, the patient was diagnosed as having IgG4-related autoimmune pancreatitis. In addition, the findings of a renal tissue specimen obtained by biopsy demonstrated IgG4-positive plasma cell infiltration in both abnormal mass lesions and normal regions by imaging, leading to the final diagnosis of IgG4-related sclerotic disease. The patient was treated with prednisolone (30 mg/day), and the size of the pancreatic and renal lesions markedly decreased four weeks later. We report here a rare case of IgG4-related autoimmune pancreatitis with multiple renal lesions, which were confirmed by renal biopsy.

Sex-role reversal of a monogamous pipefish without higher potential reproductive rate in females.

In monogamous animals, males are usually the predominant competitors for mates. However, a strictly monogamous pipefish Corythoichthys haematopterus exceptionally exhibits a reversed sex role. To understand why its sex role is reversed, we measured the adult sex ratio and the potential reproductive rate (PRR), two principal factors influencing the operational sex ratio (OSR), in a natural population of southern Japan. The adult sex ratio was biased towards females throughout the breeding season, but the PRR, which increased with water temperature, did not show sexual difference. We found that an alternative index of the OSR (Sf/Sm: sex ratio of 'time in') calculated from the monthly data was consistently biased towards females. The female-biased OSR associated with sex-role reversal has been reported in some polyandrous or promiscuous pipefish, but factors biasing the OSR differed between these pipefish and C. haematopterus. We concluded that the similar PRR between the sexes in C. haematopterus does not confer reproductive benefit of polygamous mating on either sex, resulting in strict monogamous mating, and its female-biased adult sex ratio promotes female-female competition for a mate, resulting in sex-role reversal.

Application of stable isotope analysis for detecting filial cannibalism.

A novel type of filial cannibalism has been reported in pipefishes, in which the eggs are absorbed through the male's brood-pouch epithelium. The present study explored the applicability of stable isotope analysis for the detection of paternal brood cannibalism in the seaweed pipefish Syngnathus schlegeli. As expected, the δ15N values for liver, which conveys short-term dietary information about the recent reproductive season, were higher in males than in females. In contrast, the δ15N values for muscle, which reflects longer-term feeding habits that span both the reproductive and non-reproductive seasons, did not significantly differ between the sexes. This finding indicates that males occupy a higher trophic position than females only during the reproductive season, and it is probable that this difference is a result of paternal uptake of nutrients from embryos in the brood pouch.

Apolipoprotein E regulates primary cultured human mesangial cell proliferation.

BACKGROUND: The role of apolipoprotein (apo) E in kidney disease is still unclear. Animal studies have been performed, but it is doubtful if the conclusions are applicable to human beings. The objective of this study was to determine how apo E acts on human kidneys using primary cultured normal human mesangial cells (NHMCs) rather than animals used in previous studies. METHODS: apo E and its isoforms E2, E3 and E4, or combinations with apo B were cocultured with primary NHMCs in serum-free medium. Premix WST-1 Cell Proliferation Assay System and DNA-Prep Reagent System were used to measure the proliferation and apoptosis of NHMCs, respectively. RESULTS: (1) apo E itself increased NHMC proliferation at 24 h of culture, while it inhibited this proliferation after 48 h. (2) At 72 h of culture, apo E alone inhibited NHMC proliferation at concentrations higher than 0.78 microg/ml in concentration-dependent manner. (3) When co-cultured with both apo E and apo B, NHMC proliferation was higher than that with apo E alone and lower than that with apo B alone. (4) At 72 h of culture, apo E2, E3 and E4 inhibited NHMC proliferation at different intensities, with no proliferative effect observed. (5) Neither apo E nor apo B caused NHMC apoptosis. CONCLUSION: apo E regulates primary NHMC proliferation by (1) inhibiting NHMC proliferation or reducing NHMC proliferation induced by apo B, which implies that apo E has a protective effect on the kidney, and (2) increasing the proliferation under certain conditions.

Within species support for the expensive tissue hypothesis: a negative association between brain size and visceral fat storage in females of the Pacific seaweed pipefish.

The brain is one of the most energetically expensive organs in the vertebrate body. Consequently, the high cost of brain development and maintenance is predicted to constrain adaptive brain size evolution (the expensive tissue hypothesis, ETH). Here, we test the ETH in a teleost fish with predominant female mating competition (reversed sex roles) and male pregnancy, the pacific seaweed pipefish Syngnathus schlegeli. The relative size of the brain and other energetically expensive organs (kidney, liver, heart, gut, visceral fat, and ovary/testis) was compared among three groups: pregnant males, nonpregnant males and egg producing females. Brood size in pregnant males was unrelated to brain size or the size of any other organ, whereas positive relationships were found between ovary size, kidney size, and liver size in females. Moreover, we found that the size of energetically expensive organs (brain, heart, gut, kidney, and liver) as well as the amount of visceral fat did not differ between pregnant and nonpregnant males. However, we found marked differences in relative size of the expensive organs between sexes. Females had larger liver and kidney than males, whereas males stored more visceral fat than females. Furthermore, in females we found a negative correlation between brain size and the amount of visceral fat, whereas in males, a positive trend between brain size and both liver and heart size was found. These results suggest that, while the majority of variation in the size of various expensive organs in this species likely reflects that individuals in good condition can afford to allocate resources to several organs, the cost of the expensive brain was visible in the visceral fat content of females, possibly due to the high costs associated with female egg production.

Epistasis effects of multiple ancestral-consensus amino acid substitutions on the thermal stability of glycerol kinase from Cellulomonas sp. NT3060.

Thermostable variants of the Cellulomonas sp. NT3060 glycerol kinase have been constructed by through the introduction of ancestral-consensus mutations. We produced seven mutants, each having an ancestral-consensus amino acid residue that might be present in the common ancestors of both bacteria and of archaea, and that appeared most frequently at the position of 17 glycerol kinase sequences in the multiple sequence alignment. The thermal stabilities of the resulting mutants were assessed by determining their melting temperatures (Tm), which was defined as the temperature at which 50% of the initial catalytic activity is lost after 15 min of incubation, as well as when the half-life of the catalytic activity occurs at a temperature of 60°C (t1/2). Three mutants showed increased stabilities compared to the wild-type protein. We then produced five more mutants with multiple amino acid substitutions. Some of the resulting mutants showed thermal stabilities much greater than those expected given the stabilities of the respective mutants with single mutations. Therefore, the effects of mutations are not always simply additive and some amino acid substitutions, which do not affect or only slightly improve stability when individually introduced into the protein, show substantial stabilizing effects in combination with other mutations.