Short-length Homologous Region exhaustive Search algorithm (SHRS): A primer design algorithm for differentiating bacteria at the species, subspecies, or strain level based on a whole genome sequence.

In fields such as the food industry, it is very important to identify target bacteria at the species level or lower for optimal product quality control. Bacteria identification at the subspecies or lower level requires time-consuming and high-cost analyses such as multi-locus sequence typing and amplified fragment length polymorphism analyses. Herein, we developed a primer design algorithm for precisely identifying bacteria based on a whole genome DNA sequence that is easy to apply. The algorithm designs primer sets that produce fragments from all input sequences and maximizes the differences in the amplicon size or amplicon sequence among input sequences. We demonstrate that the primer sets designed by the algorithm clearly classified six subspecies of Lactobacillus delbrueckii, and we observed that the resolution of the method is equal to that of a multi-locus sequence analysis. The algorithm allows the easy but precise identification of bacteria within a short time. (SHRS is available freely from PyPI under the MIT license.).

Modeling the sake brewing characteristics of rice from brown rice metabolites.

Sake, soy sauce, miso (Japanese bean paste), and beer are made from grains. The characteristics of the grain significantly affect the quality of the final product. Many studies have been performed to evaluate the sake-brewing characteristics of rice. However, current rice analysis methods are time and labor intensive and require large samples. We developed a novel method for predicting the brewing characteristics of sake rice using <1 g of sample. Brown rice extracts were analyzed by ultrahigh-performance liquid chromatography quadrupole time-of-flight mass spectrometry, and mass chromatogram data were used as explanatory variables. The objective variables were the physical and chemical properties of the rice, the enzymatic activity of the rice-koji, the fermentation properties of the sake mash, the standard analytical values of the sake, and the flavor component concentrations in the sake. Prediction models were developed using the orthogonal projections to latent structures method. The prediction performances of the models were verified, and 32 out of the 54 objective variables were used in well-performing models. In conclusion, we developed a method for predicting the rice properties and brewing characteristics from results acquired by analyzing <1 g of brown rice. The method is a powerful tool for breeding new sake rice cultivars for good brewing characteristics in early generations and will improve our understanding of fluctuations in the brewing characteristics of sake rice before each sake brewing season starts.

Muscarinic receptor binding activity in rat tissues by vibegron and prediction of its receptor occupancy levels in the human bladder.

OBJECTIVES: To examine the effects of vibegron, a selective ß3 -adrenoceptor agonist, used to treat overactive bladder, on muscarinic receptors in the rat bladder, and to predict the occupancy levels of muscarinic receptors by vibegron in the bladders of humans orally administered a clinical dose. METHODS: Muscarinic receptors in the rat bladder and other tissues were examined by a radioligand binding assay using [N-methyl-3 H]scopolamine chloride. The occupancy levels of muscarinic receptors by vibegron in bladders of humans after its oral administration were predicted from the estimation of unbound concentrations in human plasma and urine in the literature. RESULTS: Vibegron (0.1-100 µmol/L) inhibited specific [N-methyl-3 H]scopolamine chloride binding in the bladder and other tissues of rats in a concentration-dependent manner. The 50% inhibitory concentration value of vibegron in the bladder was approximately twofold higher than that in the heart, and approximately 315- and 3.5-fold lower than those in the submaxillary gland and brain, respectively. Therefore, the binding affinity of vibegron for muscarinic receptors was higher in the heart and bladder than in the submaxillary gland and brain. By using the rat bladder receptor binding affinity, occupancy levels of muscarinic receptors in the human bladder were predicted to be 51-91% until 24 h after its oral administration at 50 mg of vibegron. CONCLUSIONS: This is the first study to suggest that vibegron binds to muscarinic receptors in the rat bladder and other tissues, with a potentially higher affinity for the M2 subtype than the M1 and M3 subtypes. These results might be clinically relevant for pharmacotherapy with vibegron for overactive bladder.

Possible Involvement of Muscarinic Receptor Blockade in Mirabegron Therapy for Patients with Overactive Bladder.

The selective ß 3-adrenoceptor agonist mirabegron, an established alternative to antimuscarinic therapy for patients with overactive bladder, induces additional effects against receptors, transporters, and hepatic enzymes. The present study aimed to elucidate the effects of mirabegron on muscarinic receptors in the rat bladder using radioligand binding and functional assays. Mirabegron (0.1-100 µM) inhibited specific [N-methyl-3H]scopolamine methyl chloride binding in the bladder and other tissues of rats in a concentration-dependent manner. Binding affinity in the bladder was similar to that in the heart and significantly higher than those in the submaxillary gland and brain. Mirabegron induced the concentration-dependent relaxation of carbachol-induced contractions in the rat isolated bladder. Further analyses using a two-site model revealed that the relative quantities of high- and low-affinity components for mirabegron were 44.5% and 55.5%, respectively. Respective pEC50 values were 7.06 and 4.97. Based on the receptor binding affinity and pharmacokinetics of mirabegron, muscarinic receptor occupancy in the human bladder for 24 hours after the administration of a single oral dose of 50 mg mirabegron was 37%-76%. The present results demonstrate for the first time that mirabegron may relax the detrusor smooth muscle not only by ß 3-adrenoceptor activation but also muscarinic receptor blockade. SIGNIFICANCE STATEMENT: Mirabegron, the first selective ß 3-adrenoceptor agonist, represents an alternative to antimuscarinic agents for management of overactive bladder (OAB). The present study aimed to clarify whether mirabegron directly binds to muscarinic receptors and affects cholinergic agonist-induced contractions in rat urinary bladder and to predict muscarinic receptor occupancy in human bladder after oral administration of mirabegron. The results demonstrated that mirabegron therapy for patients with OAB may be due not only to ß 3-adrenoceptor activation but also muscarinic receptor blockade.

Changes in Bacterial and Chemical Components and Growth Prediction for Lactobacillus sakei during Kimoto-Style Fermentation Starter Preparation in Sake Brewing: a Comprehensive Analysis.

Kimoto-style seed mash is a traditional preparation method for sake that takes advantage of spontaneous lactic acid fermentation before the growth of yeast. Lactic acid helps decrease the pH in seed mash and control the growth of unfavorable microorganisms. In this study, we carried out a comprehensive analysis of the change in the bacterial community and chemical composition during the lactic acid fermentation stage in kimoto-style seed mash preparation. The bacterial transitions were diverse at five sake breweries, but they exhibited three patterns. Lactobacillus sakei was the dominant species in the later stage of lactic acid fermentation in all sake breweries. This species was found to be the most important bacterium for the accumulation of lactic acid, because its average production rate of lactic acid in seed mash reached 4.44 × 10-11 mg cell-1 h-1, which is 10 times higher than those of other species. As a result of specific growth rate analysis, it was revealed that the growth rate of L. sakei was influenced by the strain, pH, and temperature. The effects of pH and temperature were explained by the square root model, and the result indicates that the strains isolated in this study were incapable of growth below pH 3.9. The growth curve predicted using the growth model fit the actual cell density in two out of five sake breweries; however, our model did not work well for the remaining three sake breweries, and we presume that the error was caused by the strain or an unknown factor.IMPORTANCE It is important to produce lactic acid in kimoto-style seed mash; however, the bacterial transition is different depending on the sake brewery. The reason why there are diverse bacterial transitions during kimoto-style seed mash preparation for each sake brewery is unclear so far, and it causes difficulty in starting kimoto-style seed mash. Our findings indicate that the changes in pH caused by lactic acid bacteria grown prior to L. sakei in seed mash influence the growth of L. sakei and are related to the diversity of the bacterial transition. This study uses comprehensive analytical methods to reveal that there is a diversity of bacterial transition and chemical compositions in kimoto-style seed mash depending on the sake brewery and to explain the differences in bacterial transition depending on the characteristics of L. sakei.

Vasorelaxant effects of benzodiazepines, non-benzodiazepine sedative-hypnotics, and tandospirone on isolated rat arteries.

Benzodiazepines (BDZs) and non-BDZ sedative-hypnotics are effective for the management of chronic insomnia; however, they are associated with adverse effects such as headache, dizziness, and palpitations. Furthermore, long-term use of these medications is associated with decreased blood pressure (BP) or depressed baroreflex function. Therefore, here, we assessed whether BDZs and non-BDZs cause vasorelaxation directly. Vasorelaxation in response to 22 BDZs, 2 non-BDZs, and tandospirone was determined by myograph methods using isolated Wistar rat thoracic aortas. All the drugs relaxed phenylephrine-contracted rat aortas in a concentration-dependent manner. Zolpidem and tandospirone caused over 80% relaxation at a concentration of 10 µM; diazepam, estazolam, etizolam, and tofisopam caused 60-70% relaxation; whereas 18 other BDZs (alprazolam, bromazepam, brotizolam, chlordiazepoxide, clobazam, clonazepam, clorazepate, ethyl loflazepate, flunitrazepam, flurazepam, lorazepam, lormetazepam, midazolam, nimetazepam, nitrazepam, oxazepam, temazepam, and triazolam) and zaleplon caused less than 50% relaxation. The relaxation was partially but significantly inhibited to the same extent by a nitric oxide (NO) synthase antagonist and after endothelium removal. Binding assay of gamma-aminobutyric acid type A receptors was performed using [3H]flunitrazepam. No correlation was observed between vasorelaxation at a concentration of 10 µM and the binding affinities for 23 drugs. The study demonstrated that zaleplon, zolpidem, tandospirone, and many BDZs cause vasorelaxation to different extents via endothelial NO-dependent and endothelium-independent pathways. In conclusion, the direct vasodilatory effects of these drugs may be involved in the mechanisms underlying their adverse effects. Additionally, the decreased BP observed in persons who take BDZs or non-BDZs may be partly due to direct vasodilation.

5-Aminolevulinic acid fermentation using engineered Saccharomyces cerevisiae.

BACKGROUND: 5'-Aminolevulinic acid (ALA) is widely used in the pharmaceutical industry, healthcare, and food production, and is a substrate for the biosynthesis of heme, which is required for respiration and photosynthesis. Enhancement of ALA biosynthesis has never been developed in Saccharomyces cerevisiae, which is a well-known model microorganism used for bioproduction of many value-added compounds. RESULTS: We demonstrated that metabolic engineering significantly improved ALA production in S. cerevisiae. First, we found that overexpression of HEM1, which encodes ALA synthetase, increased ALA production. Furthermore, addition of an optimal amount of glycine, a substrate for ALA biosynthesis, or levulinic acid, an inhibitor of ALA dehydrogenase, effectively increased ALA production. Next, we developed an assay for multiple metabolites including ALA and found that aconitase, encoded by ACO1 and ACO2, is the rate-limiting enzyme of ALA biosynthesis when sufficient glycine is supplied. Overexpression of ACO2 further enhanced ALA production in S. cerevisiae overexpressing HEM1. CONCLUSIONS: In this study, ALA production in S. cerevisiae was enhanced by metabolic engineering. This study also shows a strategy to identify the rate-limiting step of a target synthetic pathway by assay for multiple metabolites alongside the target product. This strategy can be applied to improve production of other valuable products in the well-studied and well-industrialized microorganism S. cerevisiae.

Evaluation of genes involved in oxidative phosphorylation in yeast by developing a simple and rapid method to measure mitochondrial ATP synthetic activity.

BACKGROUND: Measurement of mitochondrial ATP synthesis is a critical way to compare cellular energetic performance. However, fractionation of mitochondria requires large amounts of cells, lengthy purification procedures, and an extreme caution to avoid damaging intact mitochondria, making it the highest barrier to high-throughput studies of mitochondrial function. To evaluate 45 genes involved in oxidative phosphorylation in Saccharomyces cerevisiae, we aimed to develop a simple and rapid method to measure mitochondrial ATP synthesis. RESULTS: To obtain functional mitochondria, S. cerevisiae cells were lysed with zymolyase followed by two-step, low- then high-speed centrifugation. Using a firefly luciferin-luciferase assay, the ATP synthetic activity of the mitochondria was determined. Decreasing the ATP synthesis in the presence of mitochondrial inhibitors confirmed functionality of the isolated crude mitochondria. Deletion of genes encoding mitochondrial ATP synthesis-related protein showed their dependency on the oxidative phosphorylation in S. cerevisiae. CONCLUSIONS: Compared with conventional procedures, this measurement method for S. cerevisiae Mitochondrial ATP Synthetic activity in High-throughput (MASH method) is simple and requires a small amount of cells, making it suitable for high-throughput analyses. To our knowledge, this is the first report on a rapid purification process for yeast mitochondria suitable for high-throughput screening.