Characterization and proposed spontaneous deletion mechanism of AVR-Pik locus in Pyricularia oryzae.

In phytopathogenic fungi, a mutation in the avirulence gene can lead to the breakdown of resistance in the host plant. The nucleotide sequences of the AVR-Pik locus in the strain Ina168 and its spontaneous mutant Ina168m95-5 of Pyricularia oryzae were determined. An AVR-Pik spontaneous deletion mechanism of Ina168m95-5, including multiple homologous recombination events involving repetitive transposable elements, is proposed.

Function of PoLAE2, a laeA homolog, in appressorium formation and cAMP signal transduction in Pyricularia oryzae.

A novel homolog of laeA, a global regulatory gene in filamentous fungi, was identified from Pyricularia oryzae. A deletion mutant of the homolog (PoLAE2) exhibited lowered intracellular cAMP levels, and decreased appressorium formation on non-host surface; the decrease was recovered using exogenous cAMP and IBMX, indicating that PoLAE2 deletion affected the cAMP signaling pathway.

Isolation of Previously Uncultured Slow-Growing Bacteria by Using a Simple Modification in the Preparation of Agar Media.

Most microorganisms living in the environment have yet to be cultured, owing at least in part to their slow and poor propagation properties and susceptibility to oxidative stress. Our previous studies demonstrated that a simple modification in the preparation of agar media, i.e., autoclaving the phosphate and agar separately (termed "PS" medium), can greatly improve the culturability of microorganisms by mitigating oxidative stress compared with the use of "PT" medium (autoclaving the phosphate and agar together). Here, we attempted to isolate phylogenetically novel bacteria by combining PS medium with prolonged cultivation. After inoculation with forest soil or pond sediment samples, significantly more colonies appeared on PS medium than on PT medium. A total of 98 and 74 colonies that emerged after more than 7 days of cultivation were isolated as slow growers from PS and PT media, respectively. Sequencing analysis of their 16S rRNA genes revealed that the slow growers recovered from PS medium included more phylogenetically novel bacteria than those from PT medium, including a strain that could be classified into a novel order in the class Alphaproteobacteria Further physiological analysis of representative strains showed that they were actually slow and poor growers and formed small but visible colonies only on PS medium. This study demonstrates that the culturability of previously uncultured bacteria can be improved by using an isolation strategy that combines a simple modification in medium preparation with an extended incubation time.IMPORTANCE Most microbial species inhabiting natural environments have not yet been isolated. One of the serious issues preventing their isolation is intrinsically slow and/or poor growth. Moreover, these slow and/or poor growers are likely to be highly sensitive to environmental stresses, especially oxidative stress. We reported previously that interaction between agar and phosphate during autoclave sterilization generates hydrogen peroxide, which adversely affects the culturability of environmental microorganisms, in particular, slow-growing organisms vulnerable to oxidative stress. In this study, we successfully isolated many slow-growing bacterial strains with phylogenetic novelty by simply modifying their cultivation on agar plates, i.e., autoclaving the phosphate and agar separately. The current limited repertoire of culture techniques still has room for improvement in the isolation of microorganisms previously considered unculturable.

Digital gene atlas of neonate common marmoset brain.

Interest in the common marmoset (Callithrix jacchus) as a primate model animal has grown recently, in part due to the successful demonstration of transgenic marmosets. However, there is some debate as to the suitability of marmosets, compared to more widely used animal models, such as the macaque monkey and mouse. Especially, the usage of marmoset for animal models of human cognition and mental disorders, is still yet to be fully explored. To examine the prospects of the marmoset model for neuroscience research, the Marmoset Gene Atlas (https://gene-atlas.bminds.brain.riken.jp/) provides a whole brain gene expression atlas in the common marmoset. We employ in situ hybridization (ISH) to systematically analyze gene expression in neonate marmoset brains, which allows us to compare expression with other model animals such as mouse. We anticipate that these data will provide sufficient information to develop tools that enable us to reveal marmoset brain structure, function, cellular and molecular organization for primate brain research.

Fluorescent reporter analysis revealed the timing and localization of AVR-Pia expression, an avirulence effector of Magnaporthe oryzae.

In order to facilitate infection, the rice blast pathogen Magnaporthe oryzae secretes an abundance of proteins, including avirulence effectors, to diminish its host's defences. Avirulence effectors are recognized by host resistance proteins and trigger the host's hypersensitive response, which is a rapid and effective form of innate plant immunity. An understanding of the underlying molecular mechanisms of such interactions is crucial for the development of strategies to control disease. However, the expression and secretion of certain effector proteins, such as AVR-Pia, have yet to be reported. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that AVR-Pia was only expressed during infection. Fluorescently labelled AVR-Pia indicated that AVR-Pia expression was induced during appressorial differentiation in the cells of both rice and onion, as well as in a penetration-deficient (Δpls1) mutant capable of developing melanized appressoria, but unable to penetrate host cells, suggesting that AVR-Pia expression is independent of fungal penetration. Using live-cell imaging, we also documented the co-localization of green fluorescent protein (GFP)-labelled AVR-Pia and monomeric red fluorescent protein (mRFP)-labelled PWL2, which indicates that AVR-Pia accumulates in biotrophic interfacial complexes before being delivered to the plant cytosol. Together, these results suggest that AVR-Pia is a cytoplasmic effector that is expressed at the onset of appressorial differentiation and is translocated to the biotrophic interfacial complex, and then into the host's cytoplasm.

Diagnosis and Treatment of Obstructive Sleep Apnea Syndrome in Children.

Sleep is important for children pertaining to their physical and mental growth. Obstructive sleep apnea syndrome (OSAS) in children has been shown to have different effects as compared to OSAS in adults, including deficits in cognition and neuropsychological functions, hyperactivity, ADHD, behavior problems, aggressive behavior, learning problems and nocturnal enuresis. Hypertrophy of the adenoids and tonsils is a major cause of OSAS in children; therefore, adenotonsillectomy may decrease the effects of OSAS pertaining to physical and mental growth. It is important to accurately diagnose and appropriately treat OSAS in children to prevent OSAS in their adulthood.

Pneumolabyrinth with Labyrinthitis as the Suspected Cause.

Pneumolabyrinth is a rare condition with air bubbles existing in the vestibule and/or cochlea. We report a case of pneumolabyrinth without trauma that was suspected to be caused by labyrinthitis. A 65-year-old man presented with vertigo and hearing loss in the left ear after catching a cold. Computed tomography performed after there had been no improvement in the patient's symptoms showed the presence of air bubbles in the vestibule, semicircular canals and cochlea. The patient was transferred to our hospital with suspected perilymphatic fistula. Bacterial infection was suspected after the laboratory tests had indicated a severe inflammatory response, and the patient was treated with antibiotics. However, no bacteria were detected in a bacterial culture of the otorrhea. An exploratory tympanotomy was performed to improve the patient's staggering gait and to examine the middle ear, with no obvious fistula being observed. Subsequent fenestration of the round window revealed a white mass that appeared to contain bacteria which was collected from the cochlea and submitted for analysis and bacterial culture. However, no bacteria were detected and the mass contained white blood cells. We suspected pneumolabyrinth following labyrinth infection. However, the cause of air bubble formation remains unclear and needs to be validated with further research.

Radiological Assessment of the Anatomy of Frontal Recess Cells and the Anterior Ethmoidal Artery.

It is necessary for the surgeon to be familiar with frontal recess anatomy during an endoscopic approach to the frontal sinuses. The aim of this study was to evaluate the prevalence of frontal recess cells in Japanese adults as well as the association between the frontal recess and the location of the anterior ethmoidal artery (AEA). The frontal recess cells and the AEAs were retrospectively evaluated in CT scans of the nasal and paranasal sinuses for 89 patients. The prevalence of agger nasi cells was 90.7%. The frequency of frontal cell types 1, 2, 3 and 4 was 28.8, 0.6, 2.6 and 0%, respectively. Suprabullar cells (SBCs) and frontal bullar cells (FBCs) were identified in 78/96 sides (81.3%) and 24/96 sides (24%), respectively. The prevalence of the medial group of frontal recess cells (interfrontal sinus septal cells) was 12.4%. In 42/61 sides (68.9%), the AEAs were located within the posterior margin of the SBCs or the FBCs. Therefore, SBCs, FBCs and the vertical portion of the middle turbinate are reliable landmarks for the identification of AEAs.

Clinicopathological analysis of salivary gland tissue from patients with IgG4-related disease.

Conclusion The diagnosis of immunoglobulin G4-related disease (IgG4-RD) should be based on the morphology of tissue biopsy, and this study recommends a submandibular gland (SMG) biopsy for accurate diagnosis and to exclude malignant disease. Objective To clarify which type of biopsy specimen (SMG or labial salivary gland [LSG]) should be taken from patients with IgG4-RD. Methods This study included 33 patients with IgG4-RD (21 women; 12 men) who were subjected to both SMG and LSG biopsies at Sapporo Medical University between 2011-2015. Tissues obtained from the SMG and LSG specimens were evaluated. Results All SMG specimens satisfied the diagnostic criteria for IgG4-RD, whereas 19 (57.6%) LSG specimens satisfied the diagnostic criteria for IgG4-RD. Histological evaluation showed fibrosis in all the SMG specimens and in eight LSG specimens (24.2%). Obliterative phlebitis was seen in nine SMG specimens (27.3%), but it was absent in all the LSG specimens.

Interferon-gamma increased epithelial barrier function via upregulating claudin-7 expression in human submandibular gland duct epithelium.

Tight junctions (TJs) are necessary for salivary gland function and may serve as indicators of salivary gland epithelial dysfunction. IgG4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition which disrupts the TJ associated epithelial barrier. The salivary glands are one of the most frequently involved organs in IgG4-RD, however, changes of the TJ associated epithelial barrier in salivary gland duct epithelium is poorly understood. Here, we investigated the regulation and function of TJs in human submandibular gland ductal epithelial cells (HSDECs) in normal and IgG4-RD. We examined submandibular gland (SMG) tissue from eight control individuals and 22 patients with IgG4-RD and established an HSDEC culture system. Immunohistochemistry, immunocytochemistry, western blotting, and measurement of transepithelial electrical resistance (TER) were performed. Claudin-4, claudin-7, occludin, and JAM-A were expressed at the apical side of the duct epithelium in submandibular gland (SMG) tissue and at the cell borders in HSDECs of normal and IgG4-RD. The expression and distribution of TJs in SMG tissue were not different in control individuals and patients with IgG4-RD in vivo and in vitro. Although interferon-gamma (IFNγ) generally disrupts the integrity and function of TJs, as manifested by decreased epithelial barrier function, IFNγ markedly increased the epithelial barrier function of HSDECs via upregulation of claudin-7 expression in HSDECs from patients with IgG4-RD. This is the first report showing an IFNγ-dependent increase in epithelial barrier function in the salivary gland duct epithelium. Our results provide insights into the functional significance of TJs in salivary gland duct epithelium in physiological and pathological conditions, including IgG4-RD.

Clinical Evaluation of Sinonasal Lesions in Patients With Immunoglobulin G4-Related Disease.

OBJECTIVES: Immunoglobulin G4-related disease (IgG4-RD) is a systemic disease entity characterized by elevated serum IgG4 and extensive IgG4-positive plasma cell infiltration of various organs. Patients with IgG4-RD show nasal manifestations with chronic rhinosinusitis. The objective of this study was to evaluate the clinical characteristics of sinonasal lesions in patients with IgG4-RD. METHODS: We evaluated radiological findings of sinonasal lesions in 79 patients with IgG4-RD who were divided into 3 groups according to severity. We also compared serological findings, including serum IgG4 and IgE levels, and eosinophil counts. RESULTS: Rhinosinusitis was found in 41 patients (51.9%). Although there were no significant differences in the serum IgG4 and IgE levels of the groups, there was a significant increase in eosinophil counts (445 ± 311.9/mm³) in Group C. Furthermore, 14 of the 41 patients with rhinosinusitis (34.1%) showed improvement after prednisolone administration. Patients with IgG4-RD and serum eosinophilia tend to also have sinonasal lesions. CONCLUSIONS: Rhinosinusitis is common in patients with IgG4-RD, and its pathogenesis can be similar to eosinophilic chronic rhinosinusitis.

A case of progressively transformed germinal center-type IgG4-related lymphadenopathy.

Progressively transformed germinal centers (PTGC), a lymph node process unfamiliar to most otolaryngologists, is a morphological variant of reactive lymphofollicular hyperplasia of lymph nodes. Immunoglobulin (Ig)G4-related disease (IgG4-RD) is a newly identified condition, characterized by hyper-IgG4-γ-globulinemia and mass-forming or hypertrophic lesions associated with infiltration of IgG4(+) plasma cells in the affected organs. Recently, a case study of PTGC was reported that fulfilled the diagnostic criteria of IgG4-RD (IgG4(+) PTGC) [1]. A 68-year-old male was referred to our hospital with swelling in the left submandibular region. Palpation revealed swollen lymph nodes, the largest of which measured 5cm in diameter. (18)F-fluorodeoxyglucose (FDG)-positron emission tomography/computed tomography identified lymphadenopathy with high (18)F-FDG uptake in the left submandibular region. We strongly suspected malignant lymphoma, and excisional biopsy of the submandibular lymph node was performed under general anesthesia. Pathological findings were consistent with IgG4(+) PTGC, and serological examination demonstrated elevated levels of IgG4. These findings were consistent with IgG4-RD. The patient did not have systemic lesions; therefore, he has not undergone corticosteroid therapy. IgG4(+) PTGC should be considered as a differential diagnosis for cervical lymphadenopathy by otolaryngologists as well as pathologists.

Structural characterization reveals the keratinolytic activity of an arthrobacter nicotinovorans protease.

Elevated cadmium (Cd) concentrations in fishery byproducts are an environmental concern, that might be reduced by enzymatic removal and adsorption of the contaminants during recycling the byproducts as animal food. We cloned the gene for Arthrobacter nicotinovorans serine protease (ANISEP), which was isolated from the hepatopancreas of the Japanese scallop (Patiopecten yessoensis) and has been found to be an effective enzyme for Cd(II) removal. The gene is 993 bp in length and encodes 330 amino acids, including the pre (1-30) and pro (31-111) sequences. The catalytic triad consists of His, Asp, and Ser. Sequence similarities indicate that ANISEP is a extracellular serine protease. X-ray crystallography revealed structural similarities between ANISEP and the trypsin-like serine protease NAALP from Nesterenkonia sp. Site-directed mutagenesis identified Ser171 as catalytic residue. The keratinolytic activity of ANISEP was 10-fold greater than that of trypsin. ANISEP digested Cd(II)-bound recombinant metallothionein MT-10a from Laternula elliptica, but did not release Cd. These results further suggest ANISEP is a trypsin-like serine protease that can release Cd from the Japanese scallop hepatopancreas because of its strong keratinolytic activity.

The clinical characteristics of patients with IgG4-related disease with infiltration of the labial salivary gland by IgG4-positive cells.

OBJECTIVES: Mikulicz's disease (MD) is an immunoglobulin (Ig) G4-related disease with systemic symptoms. Submandibular gland (SMG) biopsy is recommended for patients with possible IgG4-related MD for accurate differential diagnosis; however, it is difficult for certain patients to undergo this procedure. In contrast, labial salivary gland (LSG) biopsy is more convenient. Here we present an analysis of patients with IgG4-related MD whose LSG specimens were infiltrated with abundant IgG4-positive plasma cells. METHODS: Sixteen patients diagnosed with IgG4-related MD underwent simultaneous SMG and LSG biopsies. We evaluated patients' serological and (18)F-fluoro-2-deoxyglucose-positron emission tomography/computed tomography ((18)F-FDG-PET/CT) and grouped them as LSG+ (LSG specimens with > 40% IgG4-positive plasma cells/IgG-positive plasma cells, 11 patients) or LSG- (LSG specimens with < 40% IgG4-positive plasma cells/IgG-positive plasma cells, 6 patients). RESULTS: There were not significant differences in serum IgG and IgG4 levels between the two groups; however, serum concentrations of soluble interleuikin-2 receptor (sIL-2R) were significantly higher in the LSG+ group. All patients with increased (18)F-FDG uptake in their parotid glands were a part of the LSG+ group. CONCLUSIONS: When a SMG biopsy is not possible, the serum concentration of sIL-2R and (18)F-FDG-PET/CT findings may predict whether LSG biopsy will facilitate the diagnosis of IgG4-related MD.

Evaluation of submandibular versus labial salivary gland fibrosis in IgG4-related disease.

The newly comprehensive diagnostic criteria in 2011 emphasize the importance of IgG4-positive plasmacyte infiltration along with storiform or swirling fibrosis and obliterative phlebitis in diagnosing IgG4-related disease(RD). Although labial salivary gland (LSG) biopsy is a minimally invasive and convenient procedure for obtaining tissues, LSG fibrosis is thought to be inconspicuous or absent in IgG4-RD cases. In this study we evaluated 15 patients with IgG4-RD, in whom both submandibular gland (SMG) and LSG biopsies were performed at the same time. Histological evaluation revealed fibrosis in all the SMG specimens but in only one LSG specimen (6.7%). The diagnosis of IgG4-RD is primarily based on its morphological appearance on biopsy. The results of this study demonstrated that although more invasive than LSG biopsy, SMG biopsy is recommended for accurate diagnosis of IgG4-related MD and to exclude malignant diseases.

Nonomuraea sp. ID06-A0189 inulin fructotransferase (DFA III-forming): gene cloning, characterization and conservation among other Nonomuraea species.

The inulin fructotransferase (DFA III-forming)(EC 4.2.2.18) gene in Nonomuraea sp. ID06-A0189 was amplified from genomic DNA, sequenced and expressed in Escherichia coli. The 1326-bp gene, designated as Nsp-ift, encodes a protein composed of a putative 37-amino-acid signal peptide and 404-amino-acid mature protein. A putative ribosomal binding sequence was identified 12 bases upstream from the start codon. However, a typical bacterial promoter could not be found by in silico analysis. The deduced amino-acid sequence of the enzyme was most similar to that of inulin fructotransferase (DFA I-forming) in Frankia sp. EAN1pec. Phylogenetic analysis of deduced amino-acid sequences indicated that Nonomuraea sp. ID06-A0189 and Frankia sp. EAN1pec inulin fructotransferases formed a distinct clade from those from Arthrobacter sp. H65-7, A. globiformis and Bacillus sp. snu-7 that showed 57, 56 and 56% identity to that of Nsp-ift, respectively. The Nsp-ift without a putative signal peptide was successfully expressed in E. coli and partially purified using His-tag affinity chromatography. The recombinant enzyme displayed optimum temperature between 65 and 70 °C, optimum pH between 5.5 and 6.0 and remained stable up to 70 °C. The properties were identical to those of the original enzyme. Of 10 Nonomuraea species tested by Southern hybridization, enzyme activity measurements and PCR, only Nonomuraea sp. ID06-A0189 has the Nsp-ift gene, suggesting that Nsp-ift is not highly conserved in this genus.

A study of infraorbital nerve swelling associated with immunoglobulin G4 Mikulicz's disease.

OBJECTIVES: Recent studies revealed that Mikulicz's disease (MD) should be considered as an immunoglobulin (Ig) G4-related disease with aspects of systemic disorders involving the orbit. This study aimed to analyze the relationship between Immunoglobulin G4 (IgG4)-related MD and infraorbital nerve thickness. METHODS: We measured infraorbital nerve thickness in 68 patients diagnosed as IgG4-related MD at our hospital and performed computed tomography scans of the head and neck region before treatment and compared these scans with those of the control group. RESULTS: The mean infraorbital nerve diameter (± standard deviation) was 3.2 ± 1.2 mm in patients with IgG4-related MD, and 2.6 ± 0.3 mm in the control group. Infraorbital nerves were significantly thicker in patients with IgG4-related MD. Nerve swelling was found in 20 of 68 patients (29.4%) with a cutoff value of 3.3 mm because this value was obtained from diameter of nerves in the control group + 2 standard deviation mm. No patients in the control group showed nerve swelling. In addition, we found significant correlations between infraorbital nerve swelling and serum IgG4 levels as well as the existence of multiple organ lesions. CONCLUSIONS: We found that significant infraorbital nerve swelling occurred in patients with IgG4-related MD and was thought to be a part of IgG4-related disease.

Homologous recombination causes the spontaneous deletion of AVR-Pia in Magnaporthe oryzae.

AVR-Pia, an avirulence gene in the genome of the rice blast fungus Magnaporthe oryzae, triggers a hypersensitive reaction in rice cultivars harbouring the resistance gene Pia. The copy number of AVR-Pia was revealed to vary from one to three among M. oryzae isolates avirulent to Pia rice, and three copies of the gene were located on a single chromosome in strain Ina168, from which the gene was originally cloned. The spontaneous avr-Pia mutant originated from Ina168, named Ina168m95-1, which lacks the AVR-Pia gene, and was therefore used to elucidate the molecular mechanism of the deletion of all three copies of AVR-Pia. Screening and analysis of cosmid clones indicated that two copies of the DNA-type transposon Occan (Occan(9E12) and Occan(3A3) ) were located on the same chromosome, and three copies of AVR-Pia were located in between the two Occan elements. Ina168m95-1 contains a conserved Occan element, named Occan(m95-1) , between sequences homologous to the 5'-flanking region of Occan(3A3) and the 3'-flanking region of Occan(9E12) . In addition, sequence polymorphisms indicated a homologous recombination between Occan(3A3) and Occan(9E12) , which resulted in Occan(m95-1) . Based on these observations, we propose the hypothesis that homologous recombination in the two Occan elements leads to the deletion of AVR-Pia in Ina168m95-1.

Purification and characterization of phytase from Klebsiella pneumoniae 9-3B.

Phytase, an enzyme that catalyzes the hydrolysis of phytate, was purified from Klebsiella pneumoniae 9-3B. The isolate was preferentially selected in a medium which contains phytate as a sole carbon and phosphate source. Phytic acid was utilized for growth and consequently stimulated phytase production. Phytase production was detected throughout growth and the highest phytase production was observed at the onset of stationary phase. The purification scheme including ion exchange chromatography and gel filtration resulted in a 240 and 2077 fold purification of the enzyme with 2% and 15% recovery of the total activity for liberation of inorganic phosphate and inositol, respectively. The purified phytase was a monomeric protein with an estimated molecular weight of 45kDa based on size exclusion chromatography and SDS-PAGE analyses. The phytase has an optimum pH of 4.0 and optimum temperature of 50°C. The phytase activity was slightly stimulated by Ca(2+) and EDTA and inhibited by Zn(2+) and Fe(2+). The phytase exhibited broad substrate specificity and the K(m) value for phytate was 0.04mM. The enzyme completely hydrolyzed myo-inositol hexakisphosphate (phytate) to myo-inositol and inorganic phosphate. The properties of the enzyme prove that it is a good candidate for the hydrolysis of phytate for industrial applications.