An Organoid Biobank of Neuroendocrine Neoplasms Enables Genotype-Phenotype Mapping.

Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.

Divergent Routes toward Wnt and R-spondin Niche Independency during Human Gastric Carcinogenesis.

Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.

The iron chelator deferriferrichrysin induces paraptosis via extracellular signal-related kinase activation in cancer cells.

Cancer cells generally exhibit increased iron uptake, which contributes to their abnormal growth and metastatic ability. Iron chelators have thus recently attracted attention as potential anticancer agents. Here, we show that deferriferrichrysin (Dfcy), a natural product from Aspergillus oryzae acts as an iron chelator to induce paraptosis (a programmed cell death pathway characterized by ER dilation) in MCF-7 human breast cancer cells and H1299 human lung cancer cells. We first examined the anticancer efficacy of Dfcy in cancer cells and found that Dfcy induced ER dilation and reduced the number of viable cells. Extracellular signal-related kinase (ERK) was activated by Dfcy treatment, and the MEK inhibitor U0126, a small molecule commonly used to inhibit ERK activity, prevented the increase in ER dilation in Dfcy-treated cells. Concomitantly, the decrease in the number of viable cells upon treatment with Dfcy was attenuated by U0126. Taken together, these results demonstrate that the iron chelator Dfcy exhibits anticancer effects via induction of ERK-dependent paraptosis.

Denosumab-induced hypocalcemia in patients with solid tumors and renal dysfunction: a multicenter, retrospective, observational study.

BACKGROUND: Bone metastases are frequently observed in advanced cancer, and bone modifying agents are used to prevent or treat skeletal-related events. Zoledronic acid is contraindicated in patients with severe renal impairment (Ccr < 30 mL/min), but it is not completely known whether denosumab can be used in them. We aimed to determine the association between renal function and hypocalcemia development during denosumab treatment. METHODS: We included patients with solid cancer and bone metastases who started denosumab treatment between April 2017 and March 2019. They were classified into four groups based on creatinine clearance (Ccr; mL/min): normal (Ccr ≥ 80), mild (50 ≤ Ccr ˂80), moderate (30 ≤ Ccr ˂50), and severe (Ccr ˂30). Hypocalcemia was evaluated using the Common Terminology Criteria for Adverse Events (v5.0) based on the albumin-adjusted serum calcium levels; its incidence (stratified by renal function) and risk factors were investigated using a Chi-square test and logistic regression analysis. RESULTS: Of 524 patients (age: 69 ± 11 years; 303 men), 153 had a normal renal function and 222, 117, and 32 had mild, moderate, and severe renal dysfunction. The albumin-adjusted serum calcium level was higher than the measured (total) calcium level in most patients. The incidence of grade ≥ 1 hypocalcemia was 32.0% in the normal group and 37.4%, 29.9%, and 62.5% in the mild, moderate, and severe renal dysfunction groups, respectively. It was, therefore, higher in the severe renal dysfunction groups than in the normal group (P = 0.002). The incidence of grade ≥ 3 hypocalcemia did not differ significantly among the groups. Pre-treatment low serum calcium levels and severe renal dysfunction were risk factors for hypocalcemia. CONCLUSIONS: Evaluating denosumab-induced hypocalcemia required albumin adjustment, and its incidence was high among patients with severe renal dysfunction. Reduced serum calcium levels and severely impaired renal function were associated with an elevated hypocalcemia risk.

Diagnostic Performance of Near-Infrared Fluorescent Marking Clips in Laparoscopic Gastrectomy.

INTRODUCTION: Accurate tumor localization and resection margin acquisition are essential in gastric cancer surgery. Preoperative placement of marking clips in laparoscopic gastrectomy as well as intraoperative gastroscopy can be used for gastric cancer surgery. However, these procedures are not available at all institutions. We conducted a prospective clinical trial to investigate the diagnostic performance of near-infrared fluorescent clips (ZEOCLIP FS) in laparoscopic gastrectomy. MATERIALS AND METHODS: Patients with gastric cancer or neuroendocrine tumor in whom laparoscopic distal, pylorus-preserving, or proximal gastrectomy was planned were enrolled (n = 20) in this study. Fluorescent clips were placed proximal and/or distal to the tumor via gastroscopy on the day before surgery. During surgery, the clips were detected using a fluorescent laparoscope, and suturing was performed where fluorescence was detected. The clip locations were then confirmed via gastroscopy, and the stomach was transected. The primary endpoint was the detection rate of the marking clips using fluorescence, and the secondary endpoints were complications and distance between the clips and stitches. RESULTS: Among the 20 patients enrolled, distal and pylorus-preserving gastrectomies were performed in 18 and 2 patients, respectively. All clips were detected in 15 patients, indicating a detection rate of 75.0% (90% confidence interval: 54.4%-89.6%). Furthermore, no complications related to the clips were observed. The median distance between the clips and stitches was 5 (range, 0-10) mm. CONCLUSIONS: We report the feasibility and safety of preoperative placement and intraoperative detection of near-infrared fluorescent marking clips in laparoscopic gastrectomy.

Improvement of valine and isobutanol production in sake yeast by Ala31Thr substitution in the regulatory subunit of acetohydroxy acid synthase.

The fruit-like aroma of two valine-derived volatiles, isobutanol and isobutyl acetate, has great impact on the flavour and taste of alcoholic beverages, including sake, a traditional Japanese alcoholic beverage. With the growing worldwide interest in sake, breeding of yeast strains with intracellular valine accumulation is a promising approach to meet a demand for sakes with a variety of flavour and taste by increasing the valine-derived aromas. We here isolated a valine-accumulating sake yeast mutant (K7-V7) and identified a novel amino acid substitution, Ala31Thr, on Ilv6, a regulatory subunit for acetohydroxy acid synthase. Expression of the Ala31Thr variant Ilv6 conferred valine accumulation on the laboratory yeast cells, leading to increased isobutanol production. Additionally, enzymatic analysis revealed that Ala31Thr substitution in Ilv6 decreased sensitivity to feedback inhibition by valine. This study demonstrated for the first time that an N-terminal arm conserved in the regulatory subunit of fungal acetohydroxy acid synthase is involved in the allosteric regulation by valine. Moreover, sake brewed with strain K7-V7 contained 1.5-fold higher levels of isobutanol and isobutyl acetate than sake brewed with the parental strain. Our findings will contribute to the brewing of distinctive sakes and the development of yeast strains with increased production of valine-derived compounds.

High-Level Production of Isoleucine and Fusel Alcohol by Expression of the Feedback Inhibition-Insensitive Threonine Deaminase in Saccharomyces cerevisiae.

A variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2 to 3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols. IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.

Development of sake yeast breeding and analysis of genes related to its various phenotypes.

Sake is a traditional Japanese alcoholic beverage made from rice and water, fermented by the filamentous fungi Aspergillus oryzae and the yeast Saccharomyces cerevisiae. Yeast strains, also called sake yeasts, with high alcohol yield and the ability to produce desired flavor compounds in the sake, have been isolated from the environment for more than a century. Furthermore, numerous methods to breed sake yeasts without genetic modification have been developed. The objectives of breeding include increasing the efficiency of production, improving the aroma and taste, enhancing safety, imparting functional properties, and altering the appearance of sake. With the recent development of molecular biology, the suitable sake brewing characteristics in sake yeasts, and the causes of acquisition of additional phenotypes in bred yeasts have been elucidated genetically. This mini-review summarizes the history and lineage of sake yeasts, their genetic characteristics, the major breeding methods used, and molecular biological analysis of the acquired strains. The data in this review on the metabolic mechanisms of sake yeasts and their genetic profiles will enable the development of future strains with superior phenotypes.

A novel milk-clotting enzyme from Aspergillus oryzae and A. luchuensis is an aspartic endopeptidase PepE presumed to be a vacuolar enzyme.

Aspergillus oryzae RIB40 has 11 aspartic endopeptidase genes. We searched for milk-clotting enzymes based on the homology of the deduced amino acid sequence with chymosins. As a result, we identified a milk-clotting enzyme in A. oryzae. We expected other Aspergillus species to have a homologous enzyme with milk-clotting activity, and we found the most homologous aspartic endopeptidase from A. luchuensis had milk-clotting activity. Surprisingly, 2 enzymes were considered as vacuole enzymes according to a study on A. niger proteases. The 2 enzymes from A. oryzae and A. luchuensis cleaved a peptide between the 105Phe-106Met bond in κ-casein, similar to chymosin. Although both enzymes showed proteolytic activity using casein as a substrate, the optimum pH values for milk-clotting and proteolytic activities were different. Furthermore, the substrate specificities were highly restricted. Therefore, we expected that the Japanese traditional fermentation agent, koji, could be used as an enzyme source for cheese production.

Isolation and analysis of a sake yeast mutant with phenylalanine accumulation.

Sake is a traditional Japanese alcoholic beverage brewed by the yeast Saccharomyces cerevisiae. Since the consumption and connoisseurship of sake has spread around the world, the development of new sake yeast strains to meet the demand for unique sakes has been promoted. Phenylalanine is an essential amino acid that is used to produce proteins and important signaling molecules involved in feelings of pleasure. In addition, phenylalanine is a precursor of 2-phenylethanol, a high-value aromatic alcohol with a rose-like flavor. As such, adjusting the quantitative balance between phenylalanine and 2-phenylethanol may introduce value-added qualities to sake. Here, we isolated a sake yeast mutant (strain K9-F39) with phenylalanine accumulation and found a missense mutation on the ARO80 gene encoding the His309Gln variant of the transcriptional activator Aro80p involved in the biosynthesis of 2-phenylethanol from phenylalanine. We speculated that mutation of ARO80 would decrease transcriptional activity and suppress the phenylalanine catabolism, resulting in an increase of intracellular phenylalanine. Indeed, sake brewed with strain K9-F39 contained 60% increase in phenylalanine, but only 10% less 2-phenylethanol than sake brewed with the parent strain. Use of the ARO80 mutant in sake brewing may be promising for the production of distinctive new sake varieties. ONE-SENTENCE SUMMARY: The ARO80 mutant is appropriate for controlling the content of phenylalanine and 2-phenylethanol.

Retrospective multicentre study on the effectiveness of first-line direct-acting antivirals against hepatitis C virus genotype-1.

WHAT IS KNOWN AND OBJECTIVE: In Japan, ledipasvir/sofosbuvir, elbasvir/grazoprevir and glecaprevir/pibrentasvir are recommended as first-line treatments for patients with untreated hepatitis C virus genotype 1. Although they have demonstrated a high efficacy in clinical trials, there are no direct comparative studies. Clarification of their effectiveness and safety in real-world clinical practice is required. Therefore, we conducted a retrospective multicentre study on the effectiveness of these direct-acting antivirals in real-world clinical practice. METHODS: We retrospectively evaluated the clinical data of untreated patients with persistent HCV genotype 1 infection who started first-line treatment with ledipasvir/sofosbuvir, elbasvir/grazoprevir or glecaprevir/pibrentasvir between September 2015 and January 2019 at 11 medical institutions in Japan. The primary efficacy endpoint was a sustained virologic response after 12 weeks of treatment. The secondary endpoints included sustained virologic response after 24 weeks of treatment and end of treatment response. The safety endpoint was treatment completion rate. RESULTS AND DISCUSSION: During the study, 420 patients (median age, 70 years; 181 males) received ledipasvir/sofosbuvir, 48 (median age 72, years; 29 males) received elbasvir/grazoprevir and 63 (median age 66, years; 35 males) received glecaprevir/pibrentasvir. For ledipasvir/sofosbuvir, elbasvir/grazoprevir and glecaprevir/pibrentasvir, the sustained virologic response after 12 weeks of treatment was 98.6%, 97.9% and 100%; the sustained virologic response after 24 weeks of treatment was 99.0%, 97.7% and 100%; the end of treatment response was 99.8%, 97.9% and 98.4%; and the treatment completion rate was 98.3%, 91.7% and 100% respectively. WHAT IS NEW AND CONCLUSION: In real-world clinical practice, hepatitis C virus treatment with ledipasvir/sofosbuvir, elbasvir/grazoprevir and glecaprevir/pibrentasvir was effective with safety.

Oryzapsins, the orthologs of yeast yapsin in Aspergillus oryzae, affect ergosterol synthesis.

The oryzapsin genes opsA and opsB in Aspergillus oryzae encoding glycosylphosphatidylinositol (GPI)-anchored aspartic endopeptidase are homologs of Saccharomyces cerevisiae yapsins. We recently found another homolog, opsC, in the A. oryzae genome database, which was suggested to be a pseudogene. However, the profiles and roles of the proteins encoded by these genes have not yet been clarified. Toward this end, we first produced opsA- and opsB-overexpression strains and performed enzymatic analyses, revealing that OpsA and OpsB can attack sites other than the carboxyl-terminal peptide bonds of basic amino acids. Moreover, OpsA and OpsB were confirmed to bind to the cell membrane with a GPI anchor. Second, opsA and opsB single-deletion and double-deletion strains (ΔopsA, ΔopsB, and ΔopsAΔopsB) were constructed to explore the expected roles of oryzapsins in cell wall synthesis, similar to the role of yapsins. The transcription level of mpkA in the cell wall integrity pathway was increased in ΔopsB and ΔopsAΔopsB strains, suggesting that OpsB might be involved in processing cell wall synthesis-related proteins. Treatment with an ergosterol biosynthesis inhibitor reduced the growth of the ΔopsAΔopsB strain. Moreover, the mRNA levels of Aoerg1, Aoerg3-1, Aoerg3-2, Aoerg7b, Aoerg11, and Aohmg1,2 showed a decreasing tendency in the ΔopsAΔopsB strain, and the ergosterol content in the membrane was reduced in the ΔopsAΔopsB strain. These results suggest that oryzapsins exist in the cell membrane and play roles in the formation of cell membranes. This is the first report of the involvement of GPI-anchored aspartic endopeptidases in ergosterol biosynthesis.Key points• The oryzapsins have wider substrate specificity than yaspins in S. cerevisiae.• Unlike the yapsins, the oryzapsins might not be involved in the main structure synthesis of the cell wall.• The oryzapsins would be involved in ergosterol biosynthesis.

Mutation in gene coding for glucose-induced degradation-deficient protein contributes to high malate production in yeast strain No. 28 and No. 77 used for industrial brewing of sake.

Saccharomyces cerevisiae produces organic acids including malate during alcohol fermentation. Since malate contributes to the pleasant flavor of sake, high-malate-producing yeast strain No. 28 and No. 77 have been developed by the Brewing Society of Japan. In this study, the genes responsible for the high malate phenotype in these strains were investigated. We had previously found that the deletion of components of the glucose-induced degradation-deficient (GID) complex led to high malate production in yeast. Upon examining GID protein-coding genes in yeast strain No. 28 and No. 77, a nonsense homozygous mutation of GID4 in strain No. 28 and of GID2 in strain No. 77 were identified as the cause of high malate production. Furthermore, complementary tests of these mutations indicated that the heterozygous nonsense mutation in GID2 was recessive. In contrast, the heterozygous nonsense mutation in GID4 was considered semidominant.

Effects of mutations of GID protein-coding genes on malate production and enzyme expression profiles in Saccharomyces cerevisiae.

During alcohol fermentation, Saccharomyces cerevisiae produces organic acids, including succinate, acetate, and malate. Since malate contributes to the pleasant flavor of sake (a Japanese alcoholic beverage), various methods for breeding high-malate-producing yeast have been developed. We previously isolated a high-malate-producing strain and found that a missense mutation in GID4 was responsible for the high-malate-producing phenotype. Gid4 is a component of the GID (glucose-induced degradation-deficient) complex and stimulates the catabolic degradation of gluconeogenic enzymes. In this study, the mechanism by which this mutation led to high malate production in yeast cells was investigated. The evaluation of disruptants and mutants of gluconeogenic enzymes revealed that cytosolic malate dehydrogenase (Mdh2) participated in the malate production. Furthermore, target proteome analysis indicated that an increase in malate production resulted from the accumulation of Mdh2 in gid4 disruptant due to the loss of GID complex-mediated degradation. Next, we investigated the effects of GID protein-coding genes (GID1-GID9) on organic acid production and enzyme expression profiles in yeast. The disruptants of GID1, 2, 3, 4, 5, 8, and 9 exhibited high malate production. Comparison of protein abundance among the GID disruptants revealed variations in protein expression profiles, including in glycolysis and tricarboxylic acid cycle-related enzymes. The high-malate-producing disruptants showed the activation of several glycolytic enzymes and a reduction in enzymes involved in the conversion of pyruvate to ethanol. Our results suggest that high-malate-producing disruptants adapt their metabolism to produce malate in excess via the regulation of protein expression in glucose assimilation and ethanol fermentation. KEY POINTS: An increase in malate level of GID4 mutant resulted from the accumulation of Mdh2. The disruptants of GID1, 2, 3, 4, 5, 8, and 9 showed high malate production. The protein expression profiles in the GID disruptants differed from one another.

Effects of a novel variant of the yeast γ-glutamyl kinase Pro1 on its enzymatic activity and sake brewing.

Sake is a traditional Japanese alcoholic beverage brewed with the yeast Saccharomyces cerevisiae. Sake taste is affected by sugars, organic acids, and amino acids. We previously isolated mutants resistant to the proline analogue azetidine-2-carboxylate derived from a diploid sake yeast strain. Some of the mutants produced a greater amount of proline in the brewed sake. One of them (strain K-9-AZC) carried a novel mutation in the PRO1 gene encoding the Gln79His variant of the γ-glutamyl kinase Pro1, a key enzyme in proline biosynthesis in S. cerevisiae. This mutation resulted in extreme desensitization to feedback inhibition by proline, leading to proline overproduction. Interestingly, sake brewed with K-9-AZC contained 3.7-fold more proline, but only 25% less succinate than sake brewed with the parent strain. Metabolome analysis suggests that the decrease in succinate was attributable to a lower level of 2-oxoglutarate, which is converted into glutamate. The approach here could be a practical method for breeding of yeast strains involved in the diversity of sake taste.

Three extracellular dipeptidyl peptidases found in Aspergillus oryzae show varying substrate specificities.

Three extracellular dipeptidyl peptidase genes, dppB, dppE, and dppF, were unveiled by sequence analysis of the Aspergillus oryzae genome. We investigated their differential enzymatic profiles, in order to gain an understanding of the diversity of these genes. The three dipeptidyl peptidases were expressed using Aspergillus nidulans as the host. Each recombinant enzyme was purified and subsequently characterized. The enzymes displayed similar optimum pH values, but optimum temperatures, pH stabilities, and substrate specificities varied. DppB was identified as a Xaa-Prolyl dipeptidyl peptidase, while DppE scissile substrates were similar to the substrates for Aspergillus fumigatus DPPV (AfDPPV). DppF was found to be a novel enzyme that could digest both substrates for A. fumigatus DPPIV and AfDPPV. Semi-quantitative PCR revealed that the transcription of dppB in A. oryzae was induced by protein substrates and repressed by the addition of an inorganic nitrogen source, despite the presence of protein substrates. The transcription of dppE depended on its growth time, while the transcription of dppF was not affected by the type of the nitrogen source in the medium, and it started during the early stage of the fungal growth. Based on these results, we conclude that these enzymes may represent the nutrition acquisition enzymes. Additionally, DppF may be one of the sensor peptidases responsible for the detection of the protein substrates in A. oryzae environment. DppB may be involved in nitrogen assimilation control, since the transcription of dppB was repressed by NaNO3, despite the presence of protein substrates.

A novel non-thermostable deuterolysin from Aspergillus oryzae.

Three putative deuterolysin (EC 3.4.24.29) genes (deuA, deuB, and deuC) were found in the Aspergillus oryzae genome database ( http://www.bio.nite.go.jp/dogan/project/view/AO ). One of these genes, deuA, was corresponding to NpII gene, previously reported. DeuA and DeuB were overexpressed by recombinant A. oryzae and were purified. The degradation profiles against protein substrates of both enzymes were similar, but DeuB showed wider substrate specificity against peptidyl MCA-substrates compared with DeuA. Enzymatic profiles of DeuB except for thermostability also resembled those of DeuA. DeuB was inactivated by heat treatment above 80° C, different from thermostable DeuA. Transcription analysis in wild type A. oryzae showed only deuB was expressed in liquid culture, and the addition of the proteinous substrate upregulated the transcription. Furthermore, the NaNO3 addition seems to eliminate the effect of proteinous substrate for the transcription of deuB.

A wheat homolog of MOTHER OF FT AND TFL1 acts in the regulation of germination.

Seed dormancy is an adaptive mechanism and an important agronomic trait. Temperature during seed development strongly affects seed dormancy in wheat (Triticum aestivum) with lower temperatures producing higher levels of seed dormancy. To identify genes important for seed dormancy, we used a wheat microarray to analyze gene expression in embryos from mature seeds grown at lower and higher temperatures. We found that a wheat homolog of MOTHER OF FT AND TFL1 (MFT) was upregulated after physiological maturity in dormant seeds grown at the lower temperature. In situ hybridization analysis indicated that MFT was exclusively expressed in the scutellum and coleorhiza. Mapping analysis showed that MFT on chromosome 3A (MFT-3A) colocalized with the seed dormancy quantitative trait locus (QTL) QPhs.ocs-3A.1. MFT-3A expression levels in a dormant cultivar used for the detection of the QTL were higher after physiological maturity; this increased expression correlated with a single nucleotide polymorphism in the promoter region. In a complementation analysis, high levels of MFT expression were correlated with a low germination index in T1 seeds. Furthermore, precocious germination of isolated immature embryos was suppressed by transient introduction of MFT driven by the maize (Zea mays) ubiquitin promoter. Taken together, these results suggest that MFT plays an important role in the regulation of germination in wheat.

Effect of yeast chromosome II aneuploidy on malate production in sake brewing.

Chromosome aneuploidy is a common phenomenon in industrial yeast. Aneuploidy is considered one of the strategies to enhance the industrial properties of Saccharomyces cerevisiae strains. However, the effects of chromosomal aneuploidy on the brewing properties of sake have not been extensively studied. In this study, sake brewing was performed using a series of genome-wide segmental duplicated laboratory S. cerevisiae strains, and the effects of each segmentally duplicated region on sake brewing were investigated. We found that the duplication of specific chromosomal regions affected the production of organic acids and aromatic compounds in sake brewing. As organic acids significantly influence the taste of sake, we focused on the segmental duplication of chromosome II that alters malate levels. Sake yeast Kyokai No. 901 strains with segmental chromosome II duplication were constructed using a polymerase chain reaction-mediated chromosomal duplication method, and sake was brewed using the resultant aneuploid sake yeast strains. The results showed the possibility of developing sake yeast strains exhibiting low malate production without affecting ethanol production capacity. Our study revealed that aneuploidy in yeast alters the brewing properties; in particular, the aneuploidy of chromosome II alters malate production in sake brewing. In conclusion, aneuploidization can be a novel and useful tool to breed sake yeast strains with improved traits, possessing industrial significance.

High production of llama variable heavy-chain antibody fragment (VHH) fused to various reader proteins by Aspergillus oryzae.

Llama variable heavy-chain antibody fragment (VHH) fused to four different reader proteins was produced and secreted in culture medium by Aspergillus oryzae. These fusion proteins consisted of N-terminal reader proteins, VHH, and a C-terminal his-tag sequence which facilitated purification using one-step his-tag affinity chromatography. SDS-PAGE analysis of the deglycosylated purified fusion proteins confirmed that the molecular weight of each corresponded to the expected sum of VHH and the respective reader proteins. The apparent high molecular weight reader protein glucoamylase (GlaB) was found to be suitable for efficient VHH production. The GlaB-VHH-His protein bound its antigen, human chorionic gonadotropin, and was detectable by a new ELISA-based method using a coupled assay with glucoamylase, glucose oxidase, peroxidase, maltose, and 3,3',5,5'-tetramethylbenzidine as substrates. Addition of potassium phosphate to the culture medium induced secretion of 0.61 mg GlaB-VHH-His protein/ml culture medium in 5 days.