Interspecificity of Scab Fungus Isolated from Wild Syrian Pear (Pyrus syriaca).

Scab on pear is caused by two pathogens, Venturia pyrina on European pear and V. nashicola on Asian pear. Five races of V. pyrina and seven races of V. nashicola have been reported thus far and pathological specialization occurs in both species. Among them, the five race isolates of V. pyrina were previously found from wild Syrian pear. In this study, mating and morphological characteristics of Venturia isolates from Syrian pear were compared with those of isolates from European and Japanese pear cultivated in Japan. The results from mating experiments showed that Syrian pear isolates were compatible with European pear isolates of V. pyrina to produce ascospores but were sterile with V. nashicola isolates in culture. Interestingly, however, the size and shape of conidia collected from naturally infected leaves of Syrian pear resembled those of V. nashicola. This finding may open the way to study coevolution between pear hosts and Venturia spp. in the future.

An organ-derived extracellular matrix triggers in situ kidney regeneration in a preclinical model.

It has not been considered that nephrons regenerate in adult mammals. We present that an organ-derived extracellular matrix in situ induces nephron regeneration in a preclinical model. A porcine kidney-derived extracellular matrix was sutured onto the surface of partial nephrectomy (PN)-treated kidney. Twenty-eight days after implantation, glomeruli, vessels, and renal tubules, characteristic of nephrons, were histologically observed within the matrix. No fibrillogenesis was observed in the matrix nor the matrix-sutured kidney, although this occurred in a PN kidney without the matrix, indicating the structures were newly induced by the matrix. The expression of renal progenitor markers, including Sall1, Six2, and WT-1, within the matrix supported the induction of nephron regeneration by the matrix. Furthermore, active blood flow was observed inside the matrix using computed tomography. The matrix provides structural and functional foundations for the development of cell-free scaffolds with a remarkably low risk of immune rejection and cancerization.

Transplantation of bioengineered liver capable of extended function in a preclinical liver failure model.

Unlimited organ availability would represent a paradigm shift in transplantation. Long-term in vivo engraftment and function of scaled-up bioengineered liver grafts have not been previously reported. In this study, we describe a human-scale transplantable liver graft engineered on a porcine liver-derived scaffold. We repopulated the scaffold parenchyma with primary hepatocytes and the vascular system with endothelial cells. For in vivo functional testing, we performed auxiliary transplantation of the repopulated scaffold in pigs with induced liver failure. It was observed that the auxiliary bioengineered liver graft improved liver function for 28 days and exhibited upregulation of liver-specific genes. This study is the first of its kind to present 28 days of posttransplant evaluation of a bioengineered liver graft using a preclinical large animal model. Furthermore, it provides definitive evidence for the feasibility of engineering human-scale transplantable liver grafts for clinical applications.

DMI-Fungicide Resistance in Venturia nashicola, the Causal Agent of Asian Pear Scab-How Reliable Are Mycelial Growth Tests in Culture?

Scab, caused by Venturia nashicola, is among the most serious diseases of Asian pears and control of this disease largely relies on sterol demethylation inhibitor (DMI) fungicides. However, pear growers have complained about field performance of DMIs since the mid-2000s. In this study, to evaluate pathogen sensitivity, mycelial growth tests and inoculation tests were conducted using DMI-amended culture medium and fungicide-sprayed potted pear trees, respectively. Results confirmed distribution of isolates resistant to fenarimol, hexaconazole, and difenoconazole in the field populations. Importantly, results from tests in culture did not fully correlate with those from tests in planta. Due to phenotypic instability of resistance and poor sporulation of this pathogen in culture, resistance is generally assessed by laborious and time-consuming inoculation with conidia collected from a field. To improve the result interpretation from in vitro tests, the isolates were genotyped: the CYP51 gene which encodes the target sterol 14α-demethylase was sequenced and various mutations have been detected in the coding sequence of DMI-resistant isolates. In addition to the detected single nucleotide polymorphisms, alternative mechanisms, not based on changes in the structure of the target protein, may also increase DMI resistance. Development of molecular methods for the diagnosis of DMI resistance seems to be challenging in V. nashicola.

Pathogenic Specialization of Venturia nashicola, Causal Agent of Asian Pear Scab, and Resistance of Pear Cultivars Kinchaku and Xiangli.

Scab caused by Venturia nashicola is one of the most serious diseases of Asian pears, including Japanese pear (Pyrus pyrifolia var. culta) and Chinese pears (P. bretschneideri and P. ussuriensis). Breeding scab-resistant pear cultivars is essential to minimize fungicide use and development of fungicide resistance. A survey of pathogenic specialization in V. nashicola is needed to ensure durable scab resistance in cultivated pears. V. nashicola race 1, 2, and 3 isolates, each differing in pathogenicity to Japanese pear cultivar Kousui and Asian pear strain Mamenashi 12, have been reported in Japan. In this study, isolates collected from scabbed pears in China and Taiwan were classified as V. nashicola based on conidial size and mating ability. However, various isolates had pathogenicity distinct from races 1, 2, and 3 according to tests on seven differential host genotypes of pear cultivars from Japan (Kousui and strain Mamenashi 12), China (Jingbaili, Yali, Linyuli, and Nanguoli), and Taiwan (Hengshanli). These new races were designated as races 4 to 7. Progenies characteristic of race 3 isolates were produced using a cross between race 1 and race 2 isolates, suggesting the possible role of sexual recombination in the emergence of novel races. Japanese pear cultivar Kinchaku and Chinese P. sinkiangensis 'Xiangli' (a Korla fragrant pear grown in China) did not show visible symptoms after inoculation with any of the seven races. Broad scab resistance in Kinchaku and Xiangli makes them a promising genetic resource for resistance breeding programs.

Type 1 and type 2 iodothyronine deiodinases in the thyroid gland of patients with huge goitrous Hashimoto's thyroiditis.

PURPOSE: The serum free triiodothyronine (FT3)/free thyroxine (FT4) ratio in patients with huge goitrous Hashimoto's thyroiditis (HG-HT) is relatively high. We investigated the cause of high FT3/FT4 ratios. METHODS: We measured the serum FT3, FT4, and thyrotropin (TSH) levels of seven patients with HG-HT who had undergone a total thyroidectomy. Eleven patients with papillary thyroid carcinoma served as controls. The activities and mRNA levels of type 1 and type 2 iodothyronine deiodinases (D1 and D2, respectively) were measured in the thyroid tissues of HG-HT and perinodular thyroid tissues of papillary thyroid carcinoma. RESULTS: The TSH levels in the HG-HT group were not significantly different from those of the controls. The FT4 levels in the HG-HT group were significantly lower than those of the controls, whereas the FT3 levels and FT3/FT4 ratios were significantly higher in the HG-HT group. The FT3/FT4 ratios in the HG-HT group who had undergone total thyroidectomy and received levothyroxine therapy decreased significantly to normal values. Both the D1 and D2 activities in the thyroid tissues of the HG-HT patients were significantly higher than those of the controls. However, the mRNA levels of both D1 and D2 in the HG-HT patients' thyroid tissues were comparable to those of the controls. Interestingly, there were significant correlations between the HG-HT patients' D1 and D2 activities, and their thyroid gland volume or their FT3/FT4 ratios. CONCLUSIONS: Our results indicate that increased thyroidal D1 and D2 activities may be responsible for the higher serum FT3/FT4 ratio in patients with HG-HT.

Systemic resistance inducer acibenzolar-S-methyl (ASM) and its microencapsulated formulations: their long-lasting control efficacy against cucumber diseases and mitigation of phytotoxicity.

BACKGROUND: The development of fungicide resistance by pathogens is a major limiting factor for the control of plant diseases. To combat resistance development, the use of broad-spectrum but nonfungitoxic resistance inducers such as acibenzolar-S-methyl (ASM) is a promising approach because the orchestrated mechanisms underlying systemic acquired resistance induced by ASM are less likely to be overcome easily by pathogens. However, phytotoxicity is the main limiting factor of ASM. RESULTS: ASM was highly active at inducing systemic resistance against powdery and downy mildews, the two major cucumber diseases. Based on the duration of the control, ASM effectiveness against these diseases was maintained well in plastic greenhouses and the number of sprays could be reduced. Control efficacy after seed treatment with ASM and the applications of microencapsulated ASM was also high against mildews in pots as well as in greenhouse experiments, with no problematic phytotoxicity. CONCLUSION: The use of ASM is a potential integrated pest management-based tactic to control cucumber powdery and downy mildews because its long-lasting efficacy allows the application of typical fungicides to be reduced. The risk for resistance development in mildew pathogens will also be reduced. ASM seed treatment as well as soil amendment with microencapsulated ASM is effective in lowering the risk for the phytotoxicity of this compound. © 2018 Society of Chemical Industry.

Type 3 iodothyronine deiodinase is expressed in human induced pluripotent stem cell derived cardiomyocytes.

AIMS: Type 3 iodothyronine deiodinase (D3), which converts thyroxine (T4) and 3,5,3'-triiodothyronine (T3) to 3,3',5'-triiodothyronine (rT3) and 3,3'-diiodothyronine (T2), respectively, inactivates thyroid hormones. We investigated the expression and regulation of D3 in human cardiomyocytes which were differentiated from human induced pluripotent stem cells (hiPSCs). MAIN METHODS: We characterized D3 activity using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. D3, myosine heavy chain α and ß (MHCα and ß, respectively), sarcoplasmic reticulum calcium ATPase (SERCA), and phospholamban (PLB) mRNA levels were analyzed by quantitative real-time PCR (qPCR) in hiPSC-derived cardiomyocytes (hiPS-CMs). KEY FINDINGS: We identified enzyme activity that catalyzes the conversion of T3 to T2 in both hiPS-CMs and hiPSCs, which showed characteristics compatible with those for D3. D3 mRNA was identified in these cells using qPCR analysis. T3 and hypoxia mimetics such as CoCl2 and DFO, increased the D3 mRNA level in both hiPS-CMs and hiPSCs. Addition of iopanoic acid, a competitive inhibitor of iodothyronine deiodination, in the culture medium of hiPS-CMs, increased the mRNA levels such as MHCα and ß, SERCA, and PLB induced by T3. SIGNIFICANTS: Our findings indicate that D3 is expressed in hiPS-CMs, and may decrease the intracellular T3 concentration, and may decrease the expression of cardiac genes such as MHCα and ß, SERCA, and PLB in hiPS-CMs.

Type 2 iodothyronine deiodinase is expressed in human preadipocytes.

BACKGROUND: Type 2 iodothyronine deiodinase (D2) is an enzyme that catalyzes the production of triiodothyronine (T3) from thyroxine (T4) and plays a critical role in providing the local intracellular T3. Although D2 is highly expressed in brown adipose tissue, it was thought that D2 is hardly expressed in white adipose tissue. In the present study, we examined whether D2 is expressed in human preadipocytes, using human mesenteric and subcutaneous preadipocytes (HMPA and HSCPA, respectively). METHODS: HMPA and HSCPA were purchased and cultured in the preadipocyte medium containing 10% fetal bovine serum. We measured D2 activity and mRNA level in HMPA and HSCPA incubated with or without dibutyryl cyclic adenosine monophosphate [(Bu)2cAMP]. RESULTS: D2 activity and mRNA were detectable in human HMPA and HSCPA. The apparent Michaelis-Menten constant (K(m)) value for T4 in HMPA was 2.1 ± 0.2 nM, and the maximum velocity (V(max)) value was 333.3 ± 28.0 femtomols of I⁻ released/mg protein/hour, respectively. On the other hand, the apparent K(m) value for T4 in HSCPA was 2.0 ± 0.2 nM and the V(max) value was 91.2 ± 8.7 femtomols of I⁻ released/mg protein/hour, respectively. D2 activities in HMPA and HSCPA incubated with 1 mM (Bu)2cAMP for 24 hours were 7-fold (HMPA) and 3-fold (HSCPA) higher than those without (Bu)2cAMP, respectively. D2 mRNA levels in HMPA and HSCPA incubated with 1 mM (Bu)2cAMP for 3 hours were 10-fold (HMPA) and 5-fold (HSCPA) higher than those without (Bu)2cAMP, respectively. CONCLUSIONS: In the present study, we have clearly demonstrated that D2 activity and mRNA are present in the human preadipocytes from both mesenteric and subcutaneous adipose tissue. Our experiments are the first ones that identify human preadipocytes as one of the sources of T3 production.

Site-directed mutagenesis of the cytochrome b gene and development of diagnostic methods for identifying QoI resistance of rice blast fungus.

BACKGROUND: It is possible that a single nucleotide polymorphism (SNP) (G143A mutation) in the cytochrome b gene could confer resistance to quinone outside inhibiting (QoI) fungicides (strobilurins) in rice blast fungus because this mutation caused a high level of resistance to fungicides such as azoxystrobin in Pyricularia grisea Sacc. and other fungal plant pathogens. The aim of this study was to survey Magnaporthe oryzae B Couch sp. nov. isolates in Japan for resistance to QoIs, and to try to develop molecular detection methods for QoI resistance. RESULTS: A survey on the QoI resistance among M. oryzae isolates from rice was conducted in Japan. A total of 813 single-spore isolates of M. oryzae were tested for their sensitivity to azoxystrobin using a mycelial growth test on PDA. QoI fungicide resistance was not found among these isolates. The introduction of G143A mutation into a plasmid containing the cytochrome b gene sequence of rice blast fungus was achieved by site-directed mutagenesis. Molecular diagnostic methods were developed for identifying QoI resistance in rice blast fungus using the plasmid construct. CONCLUSION: As the management of rice blast disease is often dependent on chemicals, the rational design of control programmes requires a proper understanding of the fungicide resistance phenomenon in field populations of the pathogen. Mutation of the cytochrome b gene of rice blast fungus would be specifically detected from diseased leaves and seeds using the molecular methods developed in this study.

Thyroid hormone activation in vascular smooth muscle cells is negatively regulated by glucocorticoid.

BACKGROUND: Type 2 iodothyronine deiodinase (D2) catalyzes the production of triiodothyronine from thyroxine. D2 is present in rat aorta media, and there is a circadian variation in the D2 expression. In rat aorta media, the D2 activity exhibited the maximal value at 1200 hour and low value between 1800 and 2400 hour. To understand the mechanisms that induce the circadian variation in the D2 expression, we examined the effects of glucocorticoid on the D2 activity and mRNA in rat aorta media and cultured vascular smooth muscle cells (VSMCs). METHODS: The effects of intrinsic and extrinsic glucocorticoid on the D2 activity and mRNA in rat aorta media were studied using metyrapone, a corticosterone synthesis inhibitor, and dexamethasone (DEX). Further, the effects of DEX on D2 expression were studied using the cultured rat VSMCs. RESULTS: The trough values of D2 activity and mRNA at 2100 hour were increased by the treatment with metyrapone. On the other hand, the peak values of D2 activity and mRNA were decreased by the treatment with DEX. D2 activity and mRNA in cultured rat VSMCs were increased by the addition of 10(-3) M dibutyryl cyclic adenosine monophosphate [(Bu)(2)cAMP]. The increments were reduced by coincubation with 10(-6) M DEX. CONCLUSIONS: These results suggest that glucocorticoids might directly suppress the D2 expression in rat VSMCs induced by a cAMP-dependent mechanism.

Characterisation of QoI-resistant field isolates of Botrytis cinerea from citrus and strawberry.

BACKGROUND: In 2004, field isolates of Botrytis cinerea Pers. ex Fr., resistant to strobilurin fungicides (QoIs), were first found in commercial citrus orchards in Wakayama Prefecture, Japan. Subsequently, QoI-resistant isolates of this fungus were also detected in plastic strawberry greenhouses in Saga, Ibaraki and Chiba prefectures, Japan. Biological and molecular characterisation of resistant isolates was conducted in this study. RESULTS: QoI-resistant isolates of B. cinerea grew well on PDA plates containing kresoxim-methyl or azoxystrobin at 1 mg L(-1), supplemented with 1 mM of n-propyl gallate, an inhibitor of alternative oxidase, whereas the growth of sensitive isolates was strongly suppressed. Results from this in vitro test were in good agreement with those of fungus inoculation tests in vivo. In resistant isolates, the mutation at amino acid position 143 of the cytochrome b gene, known to be the cause of high QoI resistance in various fungal pathogens, was found, but only occasionally. The heteroplasmy of cytochrome b gene was confirmed, and the wild-type sequence often present in the majority of resistant isolates, indicating that the proportion of mutated cytochrome b gene was very low. CONCLUSION: The conventional RFLP and sequence analyses of PCR-amplified cytochrome b gene are insufficient for molecular identification of QoI resistance in B. cinerea.

Two new species of Kazachstania that form ascospores connected by a belt-like intersporal body: Kazachstania zonata and Kazachstania gamospora.

Four strains of ascomycetous yeasts were isolated from samples collected at two locations in southern Japan. The strains formed two warty ascospores that were joined together by an intersporal body appearing as a belt. Phylogenetic analysis of rRNA gene nucleotide sequences indicated that the strains represented two new and closely related species of the genus Kazachstania. Isolates of one of the species were from Miyazaki Prefecture and those of the other species were from the Iriomote Islands. Genetic separation of the two species was further confirmed by DNA-DNA reassociation, which gave values of 63.3-78.1%, and from interspecific crosses, which gave nonviable ascospores. On the basis of these data, the isolates from Miyazaki Prefecture are described as Kazachstania zonata sp. nov. [type strain NBRC 100504=CBS 10326, mating types NBRC 101821 (+), NBRC 101822 (-)], and the isolates from the Iriomote Islands are described as Kazachstania gamospora sp. nov. [type strain NBRC 11056=CBS 10328, mating types NBRC 101825 (+), NBRC 101826 (-)].

Fungicide Sensitivity and Phylogenetic Relationship of Anthracnose Fungi Isolated from Various Fruit Crops in Japan.

Anthracnose diseases of fruit crops are mainly caused by Colletotrichum gloeosporioides and C. acutatum. In these Colletotrichum species, intra- and interspecific variation in fungicide sensitivity has been reported; however, the relationship between fungicide sensitivity and molecular phylogeny has not been analyzed. Fifty-one isolates from 10 fruit crops, acacia, and tea were tested for their sensitivities to thiophanate-methyl, diethofencarb, and iminoctadine-triacetate, and their internal transcribed spacer (ITS) and 5.8S regions of rDNA were analyzed. C. gloeosporioides isolates were divided into sensitive, less sensitive, intermediate resistant, or resistant to the three fungicides. In contrast, C. acutatum isolates were all less sensitive. In molecular phylogenetic analyses, C. gloeosporioides isolates fell into the same genetic group, whereas C. acutatum isolates were placed into two genetic groups. Although phylogenetic relationship was not closely related to fungicide sensitivity, the isolates of C. gloeosporioides most resistant to iminoctadine-triacetate were found in the same phylogenetic subgroup.

Species distinction of the ascomycetous heterothallic yeast-like fungus Stephanoascus ciferrii complex: description of Candida allociferrii sp. nov. and reinstatement of Candida mucifera Kocková-Kratochvílová et Sláviková.

The nucleotide sequences of the 18S rRNA gene (rDNA) from nine strains of the heterothallic ascomycetous Stephanoascus ciferrii complex were determined and the strains were separated into three groups according to their sequences. 18S rDNA sequences were identical within the same group. In group A the 18S rDNA sequences had no introns; in group B there was one group I intron, Sc1506-1 at position 1506; and in group C there were two group I introns, Sc943 at position 943 and Sc1506-2 at position 1506. Sc1506-1 and Sc1506-2 at position 1506 exhibited 19 base differences but were very similar. Therefore, it is suggested that these introns existed in the common ancestor of groups B and C, and that they were vertically inherited. DNA similarity values showed that the strains within the same group were of identical species. Group B included the isotype strains of Stephanoascus ciferrii and the type strains of Candida ciferrii and Sporothrix catenata; this confirmed that group B strains correspond to Stephanoascus ciferrii and that Candida ciferrii and Sporothrix catenata are synonyms of Stephanoascus ciferrii. The single member of group C, strain IFO 10918T, corresponds to the type strain of Candida mucifera and was independent of the other tested strains. Thus, Candida mucifera should be regarded as an independent species from Stephanoascus ciferrii. It is suggested that group A strains might comprise a new Stephanoascus species, but since group A strains could not form asci by themselves in this study they are described as a new species for which the name Candida allociferrii sp. nov. (type strain IFO 10194T) is proposed.

Two distinct 18S rRNA secondary structures in Dipodascus (Hemiascomycetes).

The nucleotide sequences of the 18S rRNA gene from ascomycetous yeast-like fungi in the genera Dipodascus, Galactomyces and Geotrichum were determined and the tested strains were separated into two groups by sequence length. In group 1, the length and secondary structure of 18S rRNA corresponded to those of typical eukaryotes. In group 2, the 18S rRNA gene sequences were about 150 nt shorter than those of most other eukaryotes and the predicted secondary structure lacked helices 10 and E21-5. Many substitutions and some deletions in group 2 18S rRNA gene were not only found in variable regions, but also in regions that are highly conserved among ascomycetes. Despite the considerable differences in 18S rRNA gene sequence and secondary structure between group 2 and other fungi, including group 1, phylogenetic analysis revealed that groups 1 and 2 are closely related. These findings suggest that a number of deletions occurred in the 18S rRNA of the common ancestor of group 2 strains.